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Florističko-ekološka analiza silikatnih algi (Bacillariophyta) reke Nišave i pritoka Jerme i Temske | Floristic-ecological analysis of (Bacillariophyta) from the Nišava river and tributaries Jerma and Temska rivers | Florističko-ekološka analiza silikatnih algi reke Nišave i pritoka Jerme i Temske
realizovana je sa ciljem da se utvrdi floristički sastav i sezonska dinamika zajednice
silikatnih algi i da se proceni kvalitet vode ovih reka na osnovu TDI (Trophic Diatom
Index).
Algološki materijal je sakupljan od maja 2008. godine do maja 2009. godine, na 11
lokaliteta u reci Nišavi i po jednom lokalitetu u rekama Jermi i Temskoj. Osnovni
fizičko-hemijski parametri vode su mereni na svakom lokalitetu. Uzorci silikatnih algi
su u laboratoriji tretirani jakim kiselinama, zatim zatopljeni u sintetičku smolu i
posmatrani pod svetlosnim mikroskopom.
Fizičko-hemijske analize vode reke Nišave ukazuju na povećanje koncentracije
nutrijenata (amonijum jona, nitrata, nitrita) na lokalitetima posle Dimitrovgrada, Bele
Palanke, Pirota i Niša. Izmerena vrednost pH vode na većini lokaliteta je u granici od 6
do 7. Ove vrednosti su niže od očekivanog slabo baznog karaktera vode za slivno
područje Nišave.
Analizom florističkog sastava u sve tri reke utvrđena su ukupno 194 taksona,
svrstanih u 58 rodova. Taksonima najbrojniji rodovi su Navicula (24), Nitzschia (17) i
Gomphonema (13). U reci Nišavi utvrđeno je 177 taksona, u Jermi 115 i u Temskoj 93.
Dva taksona (Navicula novaesiberica i Neidiomorpha binodiformis) predstavljaju nove
nalaze za floru silikatnih algi Srbije.
Sezonska dinamika silikatnih algi u Nišavi, Jermi i Temskoj i njihova procentualna
zastupljenost u epilitnoj zajednici u ispitivanom periodu, zavise od koncentracije
nutrijenata i temperature vode. Zapažamo smenu zajednica silikatnih algi. U prolećnoj
sezoni dominiraju predstavnici roda Achnanthidium.
U letnjoj sezoni zajednicu silikatnih algi u Nišavi, Jermi i Temskoj uglavnom grade
dva ili više subdominantna taksona (Amphora, Cymbella, Navicula, Nitzschia,
Diatoma). U jesenjoj sezoni dominiraju predstavnici roda Navicula i Nitzschia, a u
zimskoj predstavnici roda Diatoma i Gomphonema.
Na osnovu izračunatih srednjih vrednosti i standardnih devijacija za TDI vrednosti,
Temska se izdvaja kao reka sa najčistijom vodom u ispitivanom periodu (oligo-
mezotrofna do mezotrofna zona), dok poslednji lokalitet u reci Nišavi (posle Niša) ima
najnekvalitetniju vodu (hipertrofna zona). Dobijene vrednosti TDI imaju statistički
značajne veze sa fosfatima, nitritima i amonijum jonom. Ove korelacije su generalno
pozitivne i slabe. | Floristic and ecological analysis of the diatoms from the Nišava River and tributaries
Jerma and Temska Rivers was conducted to determine the floristic composition and
seasonal dynamics of diatom communities as well as to evaluate the water quality of
these rivers based on the TDI (Trophic Diatom Index).
Algological material was collected from May 2008 until May 2009 at 11 sampling
sites in the Nišava River and one site in the Jerma and Temska Rivers. Basic physico-
chemical properties of the water were measured at each sampling site. In the laboratory
diatom samples were treated with strong acids, then mounted in a synthetic medium and
examined with a light microscope.
Physico-chemical analysis of the water of the Nišava River indicated an increase in
nutrient concentration (ammonium ions, nitrate, nitrite) at sites located downstream of
Dimitrovgrad, Bela Palanka, Pirot and Niš cities. The majority of sampling sites had
pHs that ranged from 6 to 7. These pHs were lower than the weakly alkaline values
expected for the Nišava River catchment area.
Analysis of the floristic composition of the three rivers determined a total of 194 taxa
grouped in 58 genera. The genera with the most numerous taxa were Navicula (24),
Nitzschia (17) and Gomphonema (13). In total, 177 taxa were identified in the Nišava
River, 115 in the Jerma River and 93 taxa in the Temska River. Two taxa (Navicula
novaesiberica and Neidiomorpha binodiformis) were new findings for the Serbian
diatom flora.
The seasonal dynamics of diatoms in the Nišava, Jerma and Temska Rivers and their
proportional representations in epilithic communities depended on the concentration of
nutrients and water temperature. A seasonal shift of diatom communities was noticed.
The spring season was dominated by taxa from the genus Achnanthidium.
In the summer season the diatom community in the Nišava, Jerma and Temska
Rivers was mainly composed of two or more subdominant taxa (Amphora, Cymbella,
Navicula, Nitzschia, Diatoma). The autumn season was dominated by taxa from the
genus Navicula and Nitzschia, and the winter season was dominated by taxa from the
genus Gomphonema.
Based on the means and standard deviations for TDI values, the Temska River stood
out as the river with the cleanest water during the study period (oligo-mesotrophic to
mesotrophic zone), while the last locality in the river Nišava (after Niš city) had the
poorest water quality (hypertrophic zone). These TDI values were significantly
positively associated with phosphates, nitrites and ammonium ions, though the
correlations were generally weak. | true |
Alelopatski efekti transformisanih korenova Chenopodium murale L. in vitro | Allelopathic effects of Chenopodium murale L. hairy roots in vitro | Korenov sistem C. murale produkuje i luči hemijske materije koje imaju
alelopatsku aktivnost. Proučavanje efekata na druge organizme, kao i izolovanje i
karakterizacija metabolita koji su izlučeni iz korenova u zemljište je veoma kompleksan
proces. Zbog toga je cilj ovog istraživanja bio uspostavljanje kulture transformisanih
korenova C. murale in vitro, kao novog sistema pogodnog za izučavanje alelopatskog
efekta.
Transgeni korenovi su indukovani pomoću Agrobacterium rhizogenes A4M70GUS
na eksplantatima korenova, kotiledona, listova i internodija klijanaca C. murale. Eksplantati
korenova su imali najveću efikasnost transformacije (11,1%). Uspostavljeni su klonovi
transformisanih korenova različite morfologije i potencijala rasta koji se gajenjem
(subkultivisanjem) u tečnom MS medijumu održavaju više od dve godine. Potvrda
transformacije ovih klonova je urađena pomoću PCR, RT–PCR i histohemijske GUS
analize. Nije ustanovljena razlika u prisustvu rol gena među klonovima.
Ispitivan je alelopatski efekat tečnih medijuma u kojima su rasli transformisani i
netransformisani korenovi C. murale (fitotoksični medijum) na klijanje i rast klijanaca test
biljaka arabidopsisa (Arabidopsis thaliana (L.) Heynh), pšenice (Triticum aestivum L.) i
salate (Lactuca sativa L.). Kod sve tri test biljke ustanovljen je inhibitorni efekat na klijanje
semena i rast klijanaca. Klonovi transformisanih klonova: R1 i R8, imali su najveći
alelopatski potencijal.
Kod test biljaka arabidopsisa i pšenice, koje su bile tretirane fitotoksičnim
medijumima, praćena je ukupna aktivnost antioksidativnih enzima – katalaza, peroksidaza i
superoksid dismutaza (spektrofotometrijski), kao i zastupljenost i aktivnost pojedinih
izoformi navedenih enzima (elektroforetski). Aktivnost ovih enzima (izuzev katalaza kod
arabidopsisa) bila je povećana kod klijanaca isklijavanih i gajenih na fitotoksičnim
medijumima, što je u skladu sa aktiviranjem odbrambenih mehanizama u biljkama kada se
one bile izlažu stresu (fitotoksinima, tj. alelohemikalijama). Fitotoksični medijumi različitih
klonova transformisanih korenova dovodili su do različitog nivoa povećanja aktivnosti
katalaza, peroksidaza i superoksid dismutaza kod različitih test biljaka.
Pošto su fitotoksični medijumi dovodili do inhibicije rasta i razvoja klijanaca, kod
tretiranih test biljaka ispitivana je i ekspresija gena regulatora ćelijskog ciklusa. Metodom
qPCR–a je pokazano statistički značajno smanjenje ekspresije gena koji regulišu prelaz iz
G1 u S fazu kao i prelaz iz G2 u M fazu, što ukazuje na potencijalnu inhibiciju procesa
DNK sinteze, kao i same mitoze. Homologi geni arabidopsisa i pšenice (CDKA;1 / CDC2
kao i CYCD2;1 / CYCD2) su pokazivali sličan profil ekspresije.
HPLC analizom fitotoksičnih medijuma utvrđeno je prisustvo fenolnih kiselina,
kafeinske, ferulinske i p–kumarinske kiseline u koncentraciji od 0,07 – 2,85 µmol/l. Sve tri
fenolne kiseline, komercijalno proizvedene, primenjene pojedinačno ili u binarnim ili
tercijarnim smešama, u koncentraciji od 5 mmol/l za pšenicu i salatu i 0,5 mmol/l za
arabidopsis, pokazale su sličan inhibitorni efekat na klijanje semena i rast klijanaca test
biljaka, kao i u slučaju primene fitotoksičnih medijuma. Međutim, pošto su koncentracije
čistih supstanci fenolnih kiselina koje izazivaju inhibitorni efekat bile znatno veće od
koncentracija
ovih
fenolnih
kiselina
određenih
u
fitotoksičnim
medijumima,
pretpostavljamo da alelopatsko svojstvo fitotoksičnih medijuma potiče od nekih drugih
hemijskih jedinjenja ili njihovih interakcija, što će biti predmet daljih istraživanja.
Rezultati ovog istraživanja predstavljaju osnovu za rasvetljavanje hemijske prirode i
mehanizma alelopatske interferencije biljaka C. murale. | Root system produces and releases chemical compounds with allelopathic activity.
Isolation and characterization of these metabolites, as well as investigation of their effects
on other organisms, are very complicated processes. The aim of the present study was to
initiate and establish Chenopodium murale L. transgenic hairy root in vitro culture system,
as a new tool for allelopathic assays.
Transgenic hairy roots were induced by Agrobacterium rhizogenes A4M70GUS
from roots, cotyledons, leaves and internodes of C. murale seedlings. Roots were found to
be the best target explants, providing transformation efficiency of up to 11.1%. Established
13 hairy root clones differed in their morphology and growth potential. The liquid culture
system of characterized hairy root clones was maintained for over two years. Molecular
characterization of these clones was carried out by PCR, RT-PCR and histochemical GUS
analyses. No differences in rol gene presence were observed.
We investigated allelopathic effect of wild-type and hairy root growth media
(phytotoxic media) against germination and seedling development of Arabidopsis thaliana
(L.) Heynh, wheat (Triticum aestivum L.) and lettuce (Lactuca sativa L.) test plants. The
inhibitory potential varied depending on the test plant and hairy root clone. R1 and R8
clones had strong allelopathic effects. These results revealed that hairy roots as an
independent system synthesize some bioactive substances with allelopathic activity and
exude them into the growth medium.
In test plants A. thaliana and wheat, after treatment with phytotoxic media, activity
of different antioxidative enzymes (catalase, peroxidase and superoxide dismutase) were
analyzed spectrophotometrically and electrophoretically. Activities of these antioxidative
enzymes were higher in comparison to the control, except the catalase activity in A.
thaliana, which was the same or lower. Phytotoxic media from different hairy root clones
cause different changes in activities of catalases, peroxidases and superoxide dismutases in
test plants.
Allelopathic interactions implicate the inhibition of cell division by allelochemicals.
We investigated expression of cell cycle-related genes (CDK and CYC) in test plant A.
thaliana and wheat, after phytotoxic medium treatment of clone R8. We demonstrated, by
q-PCR method, that expression of genes involved in G1-to-S and G2-to-M transition were
significantly down-regulated by phytotoxic medium of clone R8 in root and shoot apexes of
test plants, indicating inhibition of DNK synthesis and mitosis. Homologous genes of A.
thaliana and wheat (CDKA;1/CDC2 and CYCD2;1/CYCD2) had similar expression
profiles, after the treatment with phytotoxic medium.
Concentrations of caffeic, ferulic and p-coumaric acids (0.07 – 2.85 µmol/l)
identified and quantified by HPLC analyses in the growth media, were significantly lower
than the inhibitory active concentration of pure grade phenolic acids when applied to A.
thaliana (0.5 mmol/l) or wheat and lettuce (5.0 mmol/l, both), suggesting that phenolic
acids have a limited role in the allelopathic phenomena of C. murale. The presented hairy
root system appears to be a suitable tool for further investigation of the potential and nature
of root-mediated allelopathic interference of C. murale. | true |
Uticaj elektromagnetnog polja (0,5 mT, 50 Hz) na motorno ponašanje, parametre oksidativnog stresa i morfološke karakteristike mozga nakon eksperimentalno izazvane moždane ishemije na modelu pustinjskog miša | Influence of electromagnetic field (0,5mT, 50 Hz) on motor behaviour, oxidative stress parameters and morphological characteristicsof brain after global cerebral ischemia in mongolian gerbils | današnje vreme, jedna od najčešćih bolesti koja pogaña celokupno
društvo je moždani udar, koji može biti uzrokovan moždanom ishemijom.
Osim što u velikom procentu dovodi do smrti, posledice moždanog udara su
najčešće trajne i za njih ne postoji efikasna terapija. Još jedna karakteristika
modernog doba je razvitak elektronske industrije i primena sve raznovrsnijih
električnih
ureñaja,
a
samim
tim
i
povećanje
stepena
zagañenja
“elektromagnetnim
smogom”.
Povećano
prisustvo
niskofrekventnih
elektromagnetnih polja (EMP) je privuklo interesovanje velikog broja naučnika
te su ispitivani njegovi efekti, izmeñu ostalog i na funkcionalne karakteristike
centralnog nervnog sistema. Na osnovu poznatih činjenica o njegovom dejstvu
možemo pretpostaviti da ovo polje utiče i na razvoj patofizioloških dogañaja i
oporavak u uslovima moždane ishemije.
Cilj naše studije je bio da ispitamo da li elektromagnetno polje (0,5 mT,
50 Hz, u trajanju od 7 dana) utiče na hipermotorno ponašanje pustinjskih
miševa izazvano 10-min globalnom moždanom ishemijom i to 1., 2., 4. i 7. dana
(neposredan efekat EMP), kao i 14. dana nakon reperfuzije (odloženi efekat
EMP). Takoñe, na modelu moždane ishemije smo ispitali da li primenjeno EMP
utiče na parametre oksidativnog stresa, koji su inače povišeni nakon moždane
ishemije, u pojedinim regionima mozga 7. i 14. dana nakon reperfuzije. Praćen
je i uticaj EMP na morfološke promene u najosetljivijim strukturama mozga
(hipokampus, striatum i kora velikog mozga) izazvane ishemijom 7. i 14. dana
nakon reperfuzije i morfometrijskom analizom su kvantifikovane promene na
neuronima i glijalnim ćelijama.
Ponašanje eksperimentalnih životinja je registrovano u trajanju od 60
minuta u testu otvorenog polja - “open field”-u. Pokazano je da 10-min ishemija
indukuje značajno povećanje motorne aktivnosti (lokomocija, stereotipija i
rotacije) do 4. dana nakon reperfuzije. Izlaganje eksperimentalnih životinja EMP
je inhibiralo razvitak hipermotorne aktivnosti izazvane ishemijom i to značajno
u prva dva dana nakon reperfuzije kada je i postishemijska hiperaktivnost
najuočljivija. Motorna aktivnost ovih pustinjskih miševa je bila značajno
povećana u poreñenju sa kontrolnom grupom samo prvog dana nakon
reperfuzije.
Biohemijski su odreñeni parametri oksidativnog stresa (NO, O2-, SOD,
ILP) u hipokampusu, striatumu i kori prednjeg mozga. Ishemija dovodi do
povećanja oksidativnog stresa u svim ispitivanim strukturama mozga i to kako
7. tako i 14. dana nakon reperfuzije, a vrednosti za SOD ostaju na kontrolnom
nivou. EMP takoñe povećava produkciju slobodnoradikalskih vrsta 7. dana
(neposredni efekat EMP) ali u značajno većoj meri nego ishemija, kao i
aktivnost SOD. Sedam dana nakon završetka izlaganja (odloženi efekat EMP)
izmerene vrednosti parametara oksidativnog stresa su na nivou kontrolnih.
Ishemični pustinjski miševi izloženi EMP imaju povećane vrednosti merenih
parametara oksidativnog stresa 7. dana nakon reperfuzije (neposredni efekat
EMP), ali u znatno manjoj meri nego jedinke ishemične grupe ili grupe izložene
EMP. Parametri oksidativnog stresa u mozgu ishemičnih pustinjskih miševa
izloženih EMP su na nivou kontrolnih 14. dana nakon reperfuzije.
Primenom stereoloških metoda, utvrñeni su volumeni striatuma i
hipokampusa, volumenska gustina oštećenih neurona i astrocita, kao i broj
mikroglijalnih ćelija po jedinici površine u hipokampusu. Izlaganje EMP nije
dovelo do morfoloških promena u ispitivanim strukturama, dok je 10-min
globalna moždana ishemija dovela do ćelijske smrti u najvećoj meri u CA1
regionu hipokampusa. Ishemični pustinjski miševi izloženi EMP su imali
znatno niži stepen propalih ćelija i veći odgovor astrocita i mikroglijalnih ćelija
nego postishemijski pustinjski miševi bez izlaganja 7. dana nakon reperfuzije
(neposredni efekat EMP). Sličan odgovor je zapažen i 14. dana nakon
reperfuzije (odloženi efekat EMP).
Rezultati ovih istraživanja su pokazali da primenjeno EMP (0,5 mT, 50
Hz) smanjuje motornu hiperaktivnost izazvanu 10-min globalnom moždanom
ishemijom modulacijom procesa koji leže u osnovi ovakvog ponašanja. Takoñe,
EMP smanjuje oksidativni stres izazvan globalnom moždanom ishemijom i na
taj način smanjuje moguće negativne posledice koje slobodnoradikalske vrste
imaju u mozgu. EMP pokazuje i neuroprotektivnu ulogu u hipokampusu,
najosetljivijoj strukturi na ishemijsko oštećenje, redukujući broj oštećenih
neurona i aktivirajući astrocite i mikroglijalne ćelije. | Nowadays, one of the most common diseases, which affect the whole
society, is brain stroke, which can be caused by cerebral ischemia. Beside high
mortality rate, consequences of brain stroke are often permanent without
appropriate treatment. Another hallmark of modern society is development of
electronic industry and usage of diverse electron devices, therefore increasing
degree of pollution with “electromagnetic smog”. The increased presence of
extremely low frequency magnetic fields (ELF-MF) has attracted the interest of
many scientists to explore its effects, among others, on functional properties of
central nervous system. Based on the known facts of its action, we can propose
that this field affects development of pathophysiological events and recovery in
conditions like cerebral ischemia.
The purpose of this study was to determine whether ELF-MF (0.5 mT, 50
Hz, continuous exposue during 7 days) affects hypermotor activity of gerbils,
induced by 10-min global cerebral ischemia on the 1st, 2nd, 4th and 7th day
(immediate effect) as well as on the 14th day after reperfusion (delayed effect).
Also, on this model we examined whether applied ELF-MF affect oxidative
stress parameters, otherwise increased after cerebral ischemia, in some brain
regions on the 7th and 14th reperfusion day. Also, we examined effect of ELF-MF
on morphological changes in the most vulnerable structures of brain
(hippocampus, striatum and forebrain cortex) induced by ischemia on the 7th
and 14th reperfusion day, as well as performed quantification of this changes in
neurons and glial cells.
Animal behaviour was monitored for 60 min in the open field. It was
shown that the 10-min global cerebral ischemia per se induced a significant
motor
activity
increase
(locomotion,
stereotypy
and
rotations),
and
consequently immobility decrease until day 4 after reperfusion, compared to
control gerbils. Exposure to ELF-MF inhibited development of ischemia-
induced motor hyperactivity during the whole period of registration, but
significantly in the first 2 days after reperfusion, when the postischemic
hyperactivity was most evident. Motor activity of these gerbils was still
significantly increased compared to control ones, but only on the 1st day after
reperfusion.
Parameters of oxidative stress (NO, O2-, SOD, ILP) were determined in
hippocampus, striatum and forebrain cortex applying biochemical assays.
Ischemia per se caused increasing of oxidative stress in all examined structures
on the 7th and also 14th reperfusion day with SOD values on the control level.
ELF-MF also increased production of free radical species on the 7th day
(immediate effect of ELF-MF) but in higher degree then ischemia, as well as,
activity of SOD. 7 days after cessation of exposure (delayed effect of ELF-MF)
measured values for all parameters were at the control levels. Ischemic gerbils
exposed to ELF-MF had increased values of parameters of oxidative stress on
the 7th reperfusion day (immediate effect of ELF-MF) but to a lesser extent than
ischemic animals or animals exposed to ELF-MF. On the 14th reperfusion day,
parameters of oxidative stress in the brain of ischemic gerbils exposed to ELF-
MF were at the control levels.
Applying stereological methods, histological evaluation of changes in the
hippocampus was done determining its volume, volume density of
degenerating neurons and astrocytes, as well as the number of microglial cells
per unit area. ELF-MF per se did not induce any morphological changes in the
examined brain structures, while 10-min global cerebral ischemia led to
neuronal death, especially in CA1 region of the hippocampus, as expected.
Ischemic gerbils exposed to ELF-MF had significantly lower degree of cell loss
in the examined structure and greater response of astrocytes and microglial
cells than postischemic gerbils without exposure on the 7th day after reperfusion
(immediate effect of ELF-MF). Similar response was observed on the 14th day
after reperfusion (delayed effect of ELF-MF), however differences in measured
parameters were not significant.
Results of this study revealed that the applied ELF-MF (0.5 mT, 50 Hz)
decreased motor hyperactivity induced by the 10-min global cerebral ischemia,
via modulation of the processes that underlie this behavioural response. Also,
ELF-MF decreased oxidative stress induced by global cerebral ischemia thus
decreasing possible negative consequences which free radical species have in
the brain. Applied ELF-MF has possible neuroprotective function in the
hippocampus, the most sensitive brain structure in the model of global cerebral
ischemia, through reduction of neuronal death and activation of astrocytes and
microglial cells. | true |
Molekularna detekcija i genotipizacija sojeva parazita Toxoplasma gondii izolovanih u Srbiji | Molecular detection and genotyping of Toxooplasma gondiistrains isolated in Serbia | ii
Molekularna detekcija i genotipizacija sojeva parazita
Toxoplasma gondii izolovanih u Srbiji
REZIME
Toksoplazmoza, oboljenje koje izaziva parazit Toxoplasma gondii, jedna je
od najrasprostranjenijih zoonoza u svetu, i procenjuje se da je čak jedna trećina
čovečanstva inficirana ovim parazitom. Iako serološki testovi predstavljaju
primarnu metodu u rutinskoj dijagnostici toksoplazmoze, interpretacija
rezultata seroloških analiza u nekim kliničkim slučajevima može biti
komplikovana i dugotrajna, a neretko i nedovoljno informativna. Zbog toga je
uvođenje molekularnih metoda koje inače odlikuje velika osetljivost i pomoću
kojih je moguće dobiti rezultate u kratkom vremenskom periodu, od velikog
značaja za dijagnostiku toksoplazmoze. Otuda je cilj ovog istraživanja bio
uvođenje molekularne dijagnostike toksoplazmoze po prvi put u Srbiji, kao i
genotipizacija izolovanih sojeva T. gondii.
Ovom studijom je obuhvaćeno 160 uzoraka poreklom od 127 pacijenata
serološki suspektnih na toksoplazmozu, kod kojih je prisustvo T. gondii
analizirano PCR-om u realnom vremenu i biološkim ogledom. Pokazano je da
uspešnost dokazivanja DNK T. gondii u materijalu humanog porekla zavisi kako
od tipa materijala tako i od ukupne zapremine materijala od kojeg se uzima
uzorak. Tako je PCR u realnom vremenu bio pozitivan u 30,8% uzoraka periferne
krvi, u 33,3% uzoraka krvi iz pupčanika, u 35,7% uzoraka plodovih voda, u 57,1%
uzoraka likvora i u 60% uzoraka očnih vodica. U odnosu na izolaciju parazita
biološkim ogledom, pokazana je znatno viša stopa detekcije DNK T. gondii, i to
i u uzorcima krvi od pacijenata suspektnih na akutnu toksoplazmozu tako i u
onima poreklom od fetusa i novorođenčadi suspektnih na kongenitalnu
toksoplazmozu. Dobijeni rezultati ukazuju pre svega na veliki potencijal i
praktični značaj molekularne dijagnostike kao i na potrebu uvođenja ove metode
kao dela standardnog protokola za dijagnostiku toksoplazmoze.
iii
T. gondii ima veoma širok spektar domaćina koji uključuje sve toplokrvne
životinje, te su u drugom delu ovog istraživanja molekularne metode za
detekciju toksoplazmoze primenjene na materijalu životinjskog porekla, i to
konkretno od glodara. Zbog njihove potencijalno značajne uloge kao karike u
epidemiološkom lancu toksoplazmoze u okviru ove studije ispitano je i 156
uzoraka poreklom od 144 braon pacova (Rattus norvegicus) i 12 kućnih miševa
(Mus musculus). DNK T. gondii je detektovana u mozgu 10,4% pacova i kod čak
83,3% kućnih miševa. Relativno visoka stopa pozitivnih nalaza kod pacova i
miševa sakupljenih u Beogradu ukazuje na kontaminiranost naše sredine
parazitom T. gondii koja predstavlja rizik za javno zdravlje.
Poslednji deo ovog rada se odnosi na prva i poptuno originalna
filogenetska istraživanja populacione strukture T. gondii. Naime, skorašnja
istraživanja u svetu ukazuju na postojanje klonske populacione strukture koju
karakterišu tri široko rasprostranjena glavna klonska tipa (I, II i III), od kojih
su tipovi I i II predominantni u Evropi i Severnoj Americi. Postoje takođe i
rekombinantni i atipični sojevi koji su uglavnom prisutni u Južnoj Americi i
Africi. U ovom istraživanju određivanje klonskog tipa, čime su dobijeni i
prvi podaci o populacionoj strukturi T. gondii u Srbiji, vršeno je korišćenjem
PCR-RFLP metode. Analizirano je ukupno 22 uzorka i to 19 humanog porekla i 3
poreklom od pacova. Uspešno je genotipizirano pet uzoraka poreklom od ljudi,
za koje se pokazalo da četiri pripadaju tipu II a jedan tipu I. Sojevi tipa II su
izolovani iz kliničkih uzoraka bolesnika sa kongenitalnom toksoplazmozom,
toksoplazmozom stečenom u trudnoći i reaktivacijom infekcije usled post-
transplantacione imunosupresije, dok je soj tipa I izolovan iz jednog uzorka
poreklom od kongenitalno inficiranog novorođenčeta. Ovi rezultati, prvi za
čitavo područje Jugoistočne Evrope, pokazuju da je i u našoj zemlji, kao i u
većem delu Evrope, predominantan pre svega genotip II. | v
SUMMARY
Toxoplasmosis, infection caused by the parasite Toxoplasma gondii, is one of
the most widespread zoonoses in the world and it has been estimated that up to one third
of the global human population is infected with this parasite. Although serological tests
are the primary method in the routine diagnosis of toxoplasmosis, interpretation of the
results can be not only difficult and time-consuming, but they can be inconclusive.
Therefore, the introduction of highly sensitive molecular methods, by which results can
be obtained within a day, into the diagnosis of toxoplasmosis is of great importance.
The aim of this research was the introduction of molecular methods into the diagnosis of
toxoplasmosis for the first time in Serbia, as well as genotypization of the isolated T.
gondii strains.
This study included 160 human biological samples from 127 patients
serologically suspected of toxoplasmosis in which the presence of T. gondii was
analyzed by PCR and by bioassay in mice. It was found that the detection of T. gondii
DNA in human biological materials depends on the type of sample as well as on the
initial volume. Real-time PCR was positive in 30.8% of peripheral blood samples,
33.3% of cord blood samples, 35.7% of amniotic fluid samples, 57.1% of CSF samples
and 60% of humor aqueous samples. The detection rate of T. gondii by molecular
methods was higher than by bioassay, in both blood samples from adults suspected of
acute toxoplasmosis and samples from fetuses and newborns suspected of congenital
toxoplasmosis. The results of this study emphasize the huge practical importance and
potential of molecular diagnostics and show the benefit of introducing this method as
part of the standard protocol for the diagnosis of toxoplasmosis.
T. gondii has a wide range of host organisms, including all warm-blooded
animals, so that in the second part of this research molecular methods for the detection
of this parasite were applied in animal material, rodents specifically. Rodents were
chosen because of their potentially important role as a link in the transmission chain of
T. gondii infection. A total of 156 brain samples from 144 brown rats (Rattus
norvegicus) and 12 house mice (Mus musculus) were analyzed. T. gondii DNA was
vi
detected in 10.4% of rats and in as many as 83.3% of house mice. The rate of positive
findings in rats and mice collected in Belgrade is an indicator of the degree of
contamination of the urban environment with T. gondii, reflecting a considerable public
health risk.
Finally, this research provides the very first and original phylogenetic data on
T. gondii population structure in Serbia. Recent studies performed world-wide indicate
the existence of a clonal population structure characterized by three main, widespread
clonal lineages (I, II and III), of which types I and II are predominant in Europe and
North America. There are also recombinant and atypical strains that are mainly present
in South America and Africa. In this study, the identification of the T. gondii clonal type
was performed in 22 samples, 19 from humans and three from rats, using the PCR-
RFLP method. Genotyping was successful in five human samples of which four isolates
originating from cases of congenital toxoplasmosis, toxoplasmosis in pregnancy and
bone marrow transplantation were type II, while one isolate from a case of congenital
toxoplasmosis was type I. These results, first for the Southeastern Europe, indicate the
predominance of genotype II in our country, as well as in most of the Europe. | true |
Uticaj fulerenola na učestalost hromozomskih aberacija i mikronukleusa u ćelijskoj liniji CHO-K1 ovarijuma hrčka i u kulturi limfocita periferne krvi čoveka | The influence of fullerenol on the frequency of chromosome aberrations and micronuclei in ovarian hamster cell line CH0-K1 and in human peripheral blood lymphocites cell culture | Nanomaterijali zahvaljujući svojim malim dimenzijama i raznovrsnim fizičkohemijskim osobinama imaju velik potencijal za primenu u različitim oblastima života. Među nanočestice sa širokom upotrebom u biomedicini ubraja se fuleren C60. Njegova slaba vodorastvorljivost može se prevazići derivatizacijom, pri čemu fuleren stiče nova svojstva koja utiču na njegovu funkcionalnost. Najpoznatiji vodorastvorni derivati C60 su fulerenoli (S60(ON)n, n=2-40), koji imaju velik potencijal za primenu u medicini. Taj potencijal je baziran na njihovoj sposobosti da u biološkim sistemima skupljaju slobodne radikale nastale u oksidativnom stresu. Međutim, sa druge strane, fulerenoli imaju sposobnost da pod određenim uslovima deluju prooksidativno. Zbog toga je neophodno definisanje koncentracija i uslova pod kojima fulerenol i njegovi derivati deluju kao antioksidanti odnosno prooksidanti. Taj aspekt je veoma važan zbog uticaja ovih nanočestica na DNK i određuje u velikoj meri njihova genotoksična odnosno antigenotoksična svojstva. Pored uticaja koncentracije nanočestica na oksidativni status, na genotoksičnost utiču i fizičkohemijske karakteristike samih nanočestica kao što su: veličina, oblik, površinsko naelektrisanje, čistoća, rastvorljivost, površinske modifikacije i agregacija, koje je takođe potrebno precizno definisati. Konačno, ispitivanje uticaja novosintentisanih supstanci sa potencijalom za medicinsku aplikaciju na DNK, predstavlja neophodan korak pre faze kliničkih studija. Zbog svega navedenog, primarni cilj ovog rada jeste ispitivanje genotoksičnih odnosno antigenotoksičnih svojstava vodorastvornog fulerenskog derivata fulerenola C60(OH)24, na dva različita sisarska modela. Prvi model je ćelijska kultura ovarijuma kineskog hrčka CHO-K1, a drugi primarna ćelijska kultura humanih limfocita. U radu su evaluirane fizičkohemijske karakteristike nanočestice fulerenola C60(OH)24 u medijumu za ćelijske kulture sa serumom i to u uslovima koji odgovaraju 6 eksperimentalnim uslovima u testovima mikronukleusa i hromozomskih aberacija. Zatim je ispitan uticaj fulerenola na preživljavanje ćelija u cilju utvrđivanja subcitotoksičnih koncentracija fulerenola, potrebnih za ispitivanje genotoksičnosti i antigenotoksičnosti fulerenola. Nakon toga je izvršena evaluacija incidencije biomarkera oštećenja DNK radi utvrđivanja genotoksičnog potencijala fulerenola. U poslednjem segmentu rada je evaluacijom istih biomarkera analiziran i antigenotoksičan potencijal fulerenola, na ćelijskim kulturama koje su prethodno oštećene poznatim citostatskim agensom mitomicinom C. Rezultati analiza su pokazali da je C60(OH)24 dimenzija od 40-180 nm u zavisnosti od kosolventa, kao i da gradi stabilne nanočestice u medijumu za ćelijsku kulturu sa fetalnim goveđim serumom. Citotoksičnost fulerenola kreće se u opsegu koncentracija od 88,6 μM-443,2 μM. Analizom genotoksičnosti fulerenola testom mikronukleusa i hromozomskih aberacija pokazano je da fulerenol nije bio genotoksičan u ispitivanim ćelijskim kulturama, pri ispitivanim subcitotoksičnim koncentracijama i pri datim vremenskim tretmanima. Ispitivanje antigenotoksičnog potencijala je pokazalo da fulerenol utiče na snižavanje ispitivanih biomarkera oštećenja DNK. | Nanomaterials, due to their small dimensions and manifold physicochemical characteristics have great potential for application in various areas of life. One of nanoparticles with wide implementation in biomedicine is fullerene C60. Its weak water solubility can be overcome by derivatization after which fullerene acquires new characteristics, which affect its functionality. The most famous water soluble C60 derivatives are fullerenols (С60(ОН)n, n=2-40), which have great potential for implementation in medicine. This potential is based on their ability to collect free radicals occurring in oxidative stress in biological systems. However, on the other hand, fullerenols are able to act prooxidatively under certain conditions. Thus, it is necessary to define concentrations and conditions under which fullerenol and its derivatives act as antioxidants, i.e. prooxidants. This aspect is very important because of the impact of these nanoparticles to the DNA, which largely defines their genotoxic i.e. antigenotoxic characteristics. Besides the impact of nanoparticles’ concentration to oxidative status, genotoxicity is also affected by physicochemical characteristics of nanoparticles themselves: their size, shape, surface charge, purity, solubility, surface modifications and aggregation, which are all necessary to be precisely defined. Finally, the testing of the impact of newly synthesized substances with potential for medical application to DNA, represents a necessary step before the phase of clinical trials. Due to all the above state, the primary aim of this paper was to test genotoxic i.e. antigenotoxic characteristic of water-soluble fullerene derivative fullerenol C60(OH)24, in two different mammalian models. The first model was an ovarian cell culture of a Chinese hamster CHO-K1, and the other was a primary humane lymphocyte cell culture. The paper evaluated physicochemical characteristics of fullerenol nanoparticle C60(OH)24 in the cell culture medium with serum under the conditions which were in accordance with the experimental conditions in micronuclei and chromosomal 8 aberrations tests. Furthermore, the affect of fullerenol to cell survival was tested with the aim of determination of fullerenol subcytotoxic concentrations, necessary for testing of fullerenol genotoxicity and antigenotoxicity. After that, the DNA damage biomarkers incidence evaluation was performed for determination of fullerenol genotoxicity potential. In the last segment of the paper, the antigenotoxic potential of fullerenol was analyzed by evaluation of the same biomarkers, in cell cultures, which were previously damaged by the known cytostatic agent mitomycin C. The results of the analysis showed that C60(OH)24 dimensions of 40-180nm depended on the cosolvent, and that it crated stable nanoparticles in the cell culture medium with fetal bovine serum. Cytotoxicity of fullerenol varied in the range of concentrations from 88.6 μM-443.2 μM. The analysis of fullerenol genotoxicity by the micronuclei test and chromosomal aberrations test showed that fullerenol was not genotoxic in the tested cell cultures, at given subcytotoxic concentrations and treatment duration. Testing of antigenotoxic potential showed that fullerenol affected the decrease of the tested DNA damage biomarkers. | false |
Ispitivanje uticaja aloe emodina na ćelije melanoma B16 i A375 i njihovu osetljivost na citostatike i imunski odgovor | Evaluation of aloe emodin effect on B16 and A375 melanoma cells and their sensitivity to anticancer drugs and immune response | Aloe emodin (AE) spada u biljne molekule antraciklinskog tipa koji zahvaljujući
reaktivnim grupama na atomima ugljenika benzenovih prstenova pokazuje izrazitu
reaktivnost i posledično široki spektar biološke aktivnosti poput baktericidnog,
fungicidnog,
viricidnog,
imunosupresivnog,
antiinflamatornog, hepatoprotektivnog,
laksativnog i vazorelaksirajućeg delovanja. Istraživanja novijeg datuma stavljaju u žižu
interesovanja njegova antitumorska dejstva.
Ciljevi koji su postavljeni u ovoj tezi podrazumevali su utvrđivanje tumoricidnog delovanja
AE-a na ćelije mišjeg, B16 i humanog, A375 melanoma koje se inicijalno razlikuju u
redoks statusu i aktivnosti signalnih puteva uključenih u kancerogenezu. Uz uporednu
analizu unutarćelijskog odgovora pomenutih linija na tretman AE-om, ispitivan je i uticaja
AE-a na osetljivost melanoma na imunski odgovor usmeren protiv transformisane ćelije
kao i na efikasnost konvencionalne citostatske terapije.
AE je snažno inhibirao rast obe ćelijske linije posredstvom različitih mehanizama. U
slučaju A375 ćelija, AE je doveo do indukcije apoptoze zavisne od aktivnosti kaspaza uz
smanjenje nivoa antiapoptotskih proteina XIAP i Bcl-2. Suprotno, tretman AE-om je
indukovao ireverzibilnu diferencijaciju B16 ćelija u smeru primarnog fenotipa
manifestovanog povećanom ekspresijom enzima tirozinaze i naknadnom produkcijom
melanina. Opisana promena B16 ćelija bila je praćena brzom akumulacijom p53, ciklina D1
i D3. Ključni događaj u diferencijaciji B16 ćelija je, najverovatnije, povećana produkcija
H2O2 indukovana AE-om. U osnovi heterogenih ishoda tretmana istovetnim agensom je
recipročna regulacija aktivnosti ERK1/2 i Akt. Poseban značaj ima podatak da se apoptoza
pokrenuta AE-om u A375 ćelijama odvija u uslovima prekomerne aktivnosti Akt. Analiza
paralelnog
tretmana ćelija
melanoma
AE-om
i
konvencionalnim citostaticima
(doksorubicin i paklitaksel) pokazala je da pomenuta supstanca umanjuje efikasnost
hemioterapije. Nekonzistentnost ishoda ko-tretmana AE-a i medijatora imunskog odgovora
iz familije TNF molekula ukazala je na kompleksnost uticaja mikrosredine na učinak AE-a.
U celini, rezultati ove doktorske disertacije pokazuju kako specifičnost ćelija melanoma
definiše mehanizam delovanja AE-a iako ne ugrožava njegov antitumorski potencijal. S
druge strane, opisani rezultati upozoravaju na opasnost kombinovanja biljnih preparata sa
konvencionalnim terapeuticima usled mogućeg neutrališućeg efekta. | Aloe emodin is an herbal antraquinone that, due to reactive groups on the carbon atoms of
benzene rings, posseses a strong reactivity and consequently shows a wide range of
biological
activities
such
as
antibacterial,
antifungal,
antiviral,
diuretic,
immunosuppressive, anti-inflammatory, hepatoprotective, laxative and vasorelaxant.
Recent studies put the focus on its antitumor effects. The aim of this thesis was to
determine and evaluate antitumor activity of AE on two melanoma cell lines that initially
differs in redox status and activities of signaling pathways involved in carcinogenesis:
mouse melanoma B16, and human melanoma A375 cells. Together with comparative
analysis of the intracellular responses of two lines to the AE treatment, the impact of AE on
the sensitivity of melanoma to antitumor immune response as well as the efficiency of
conventional cytostatic therapy, were examined.
AE decreased the growth of both cell lines through different mechanisms. In the case of
A375 cells, AE inducted caspase dependent apoptosis by reducing protein levels of
antiapoptotic molecules XIAP and Bcl-2. In contrast, treatment with AE promoted
irreversible differentiation of B16 cells towords its primary phenotype manifested through
increased expression of the tyrosinase and the subsequent production of melanin. Described
change in B16 cells was accompanied by a rapid accumulation of p53, cyclin D1 and D3.
Increased production of H2O2 triggered by AE is probably first in a line of events
responsible for induction of differentiation of B16 cells. Opposite regulation of two main
signaling pathways involved in cell proliferation, differentiation and death- MEK-ERK1/2
and PI3K-Akt in tested cell lines could be essential for different outcome of the treatment
with single agent. Furthermore, analysis of the parallel treatment of melanoma cells with
the AE and conventional cytostatics (doxorubicin and paclitaxel) showed that introduced
substance decreases the effectiveness of chemotherapy. Incoherent outcome of co-treatment
of AE and TNF, FasL or TRAIL, the most relevant mediators of nonspecific immune
response aginst tumor cells, points to the complexity of the influence of
microenvironmental factors to the effect of AE. In general, the results of this PhD thesis
show that cell specificity of melanomas defines the mechanism of AE action, while it does
not jeopardize its anticancer potential. On the other hand, presented results point to a
danger of combining herbal medicines with conventional therapeutics due to their possible
neutralizing effects. | true |
Histološke i ultrastrukturne promene hipofize i nadbubrežnih žlezda pacova u uslovima akutnog toplotnog stresa | Histological and ultrastructural changes in the pituitary and adrenal glands of rats under acute heat stress | Ispitivan je uticaj akutnog toplotnog stresa na histološke, imunohistohemijske,
ultrastrukturne, stereološke i biohemijske karakteristike adrenokortikotropnih ćelija
hipofize i ćelija nadbubrežnih žlezda pacova kao ciljnih organa hipotalamo-hipofizne-
adrenalne osovine u odgovoru na dejstvo stresora. Pacovi mužjaci soja Wistar izlagani su
temperaturi od 38C u komori u trajanju od 60 minuta. Životinje su nakon tretmana
žrtvovane dekapitacijom u prepodnevnim časovima, u približno isto vreme, da bi se
izbegao uticaj cirkadijalnog ritma na proučavane parametre. Kortikotropne ćelije
adenohipofize su analizirane na nivou svetlosne i transmisione elektronske mikroskopije
primenom imunohistohemijskih i stereoloških metoda. U nadbubrežnim žlezdama
ispitivane su histološke i ultrastrukturne karakteristike ćelija kore i srži. Kvantitativne
odlike nadbubrežnih žlezda određivane su primenom stereoloških i morfometrijskih metoda
na nivou svetlosne i elektronske mikroskopije. Peptidergički neuroni (VIP, SP i NPY) u
kori i srži nadbubrežnih žlezda procenjivani su imunohistohemijskim metodama. Za
prikazivanje protoka krvi u nadbubrežnim žlezdama korišćena je Noveli histološka metoda
bojenja. Ćelije u proliferaciji procenjivane su preko Ki-67 antigena, dok je za detekciju
apoptotskih
ćelija nadbubrežnih
žlezdi
primenjeno
bojenje propidijum-jodidom.
Određivane su i koncentracije adrenokortikotropnog hormona hipofize (ACTH), kao i
kortikosterona, aldosterona, adrenalina i noradrenalina nadbubrežnih žlezda pacova u
cirkulaciji.
U hipofizi, primenjeni tretman doveo je do povećanog izlučivanja ACTH iz
kortikotropnih ćelija. Ovo se na nivou svetlosne mikroskopije manifestovalo smanjenjem
broja ćelija imunopozitivnih na ACTH, a na nivou elektronske mikroskopije smanjenjem
broja granula u ACTH ćelijama. U skladu sa tim, biohemijski podaci pokazali su da je
koncentracija ACTH u cirkulaciji povećana. U krvnim sudovima hipofize prisutna je
agregacija krvnih pločica.
U nadbubrežnim žlezdama promene su bile izraženije kako na nivou svetlosne
tako i na nivou elektronske mikroskopije.
U ZG kore nadbubrežnih žlezda došlo je do povećane sinteze i sekrecije aldosterona
na šta su ukazali značajno smanjenje broja lipidnih kapljica, uvećanje broja mitohondrija i
visok nivo aldosterona u cirkulaciji. Porast koncentracije aldosterona u cirkulaciji
predstavlja odgovor organizma na dehidrataciju izazvanu izlaganjem životinja visokoj
temperaturi spoljne sredine.
U ZF zapaženo je značajno povećanje površine profila i volumena ćelija, posebno
citoplazmatične komponente, zajedno sa smanjenjem broja lipidnih kapi, porastom broja
mitohondrija, uz značajno povećanje nivoa kortikosterona u krvi. Uvećanje ćelija
uključenih u sintezu i sekreciju hormona stresa, kortikosterona, promena relativne
zastupljenosti subćelijskih komponenata u pravcu koji ukazuje na veću angažovanost u
sintezi hormona, kao i biohemijski podaci o povišenju nivoa kortikosterona u cirkulaciji
ukazuju na aktivno učešće ćelija ZF u odgovoru na stres izazvan akutnim izlaganjem
pacova povišenoj temperaturi spoljne sredine.
U ZR je nađeno izuzetno veliko nagomilavanje lipidnih kapljica što bi ukazivalo na
zastoj u sintezi i sekreciji hormona ove zone.
U svim zonama kore nadbubrežnih žlezda, posle akutnog izlaganja toplotnom
stresu, nađeni su uvećanje broja apoptotskih ćelija, proliferacija vlakana vezivnog tkiva,
smanjenje NPY,VIP i SP imunoreaktivnosti nervnih vlakana i primetna agregacija krvnih
pločica u kapilarima. Sve ovo upućuje na zaključak da je akutni toplotni stres, pored toga
što je podstakao sintetsku i sekretnu aktivnost parenhimskih ćelija ZG i ZF, imao opšte
nepovoljno dejstvo na koru nadbubrežnih žlezda. Agregacija krvnih pločica verovatno je
posledica dehidratacije, dok pojava apoptoze i proliferacija vezivnog tkiva mogu biti
prethodnice fibrozi parenhima.
U srži nadbubrežnih žlezda najveće promene nađene su na adrenalinskim ćelijama
čija je površina profila bila značajno smanjena, uz smanjenje citoplazmatičnog i jedarnog
dela ćelije. Značajno smanjenje punih granula, uz istovremeno značajno uvećanje druge
dve kategorije mobilisanih granula (izmenjenih i praznih) i prisustvo celih adrenalinskih
granula u kapilarima ukazuju na snažno izbacivanje adrenalina, što je potvrđeno njegovim
visokim nivoom u cirkulaciji. U noradrenalinskim ćelijama srži nadbubrežnih žlezda došlo
je do preraspodele u zastupljenosti različitih tipova granula, smanjen je broj punih granula,
a povećan broj druga dva tipa granula. Koncentracija noradrenalina u sistemskoj cirkulaciji
značajno je uvećana. S obzirom na to da su adrenalin i noradrenalin direktno uključeni u
reakciju "bori se ili beži", navedeni podaci upotpunjuju sliku o stresogenom efektu
primenjenog eksperimentalnog tretmana.
U različitim ganglijskim, nervnim i kateholaminskim ćelijama srži nadbubrežnih
žlezda, ali i u regionu kore, nađena je smanjena NPY, VIP i SP imunoreaktivnost.
Na osnovu svega navedenog, jasno je da je akutno izlaganje pacova povišenoj
temperaturi spoljašnje sredine, u trajanju od 60 minuta bilo dovoljno da se pokrenu
mehanizmi uključeni u odgovor na stres, i to na biohemijskom i ćelijskom nivou. Pored
povećanja nivoa hormona u cirkulaciji koje može da se detektuje praktično neposredno po
otpočinjanju delovanja stresogenog stimulusa, uočljivo je i prilagođavanje fine strukture
ćelija koje zahteva više vremena i počiva na složenijim mehanizmima. Pored toga, na
osnovu dobijenih rezultata izgleda da su nadbubrežne žlezde i posle ovako kratkotrajnog
tretmana usmerene ka kompleksnijim histomorfološkim alteracijama. U okviru toga ne
smeju se zanemariti moguće ozbiljne patološke posledice kao što je embolija krvnih sudova
nastala usled povećane agregacije krvnih pločica. | The aim of the study was the effect of acute heat stress on the histological,
immunohistochemical, ultrastructural, stereological and biochemical characteristics of
corticotroph pituitary cells and cells of the adrenal glands of rats as a target organ of the
hypothalamic-pituitary-adrenal axis in response to the effects of stressors. Wistar male rats
were exposed to a temperature of 38C in the chamber for 60 minutes. Animals were
sacrificed by decapitation after treatment in the morning, at about the same time, in order to
avoid the influence of circadian rhythm on the studied parameters. Corticotroph pituitary
cells were analyzed at the level of light and by transmission electron microscopy using
immunohistochemical and stereological methods. The adrenal glands were studied by
histological and ultrastructural characteristics of the cells of cortex and medulla.
Quantitative characteristics of the adrenal glands were determined using stereological and
morphometric methods at light and electron microscopy. Peptide neurons (VIP, SP and
NPY) in the cortex and medulla of the adrenal glands were assessed by
immunohistochemical methods. To show the flow of blood to the adrenal glands a Novelli
histological method of staining was used. The cells in proliferation were assessed by Ki-67
antigen, whereas the detection of apoptotic cells of the adrenal gland was enabled by
propidium-iodide staining. The concentration of pituitary adrenocorticotropic hormone
(ACTH) and corticosterone, aldosterone, adrenaline and noradrenaline in the rat adrenal
gland circulation were determined.
In the pituitary, the applied treatment resulted in increased secretion of ACTH
from corticotroph cells. On the level of light microscopy, it was demonstrated by decreased
number of immunopositive ACTH cells, and at the level of electron microscopy by reduced
number of granules in the ACTH cells. Accordingly, biochemical data showed that the
concentration of ACTH in the circulation increased. The blood vessels of the pituitary
gland in platelet aggregation were present.
In the adrenal glands changes were evident at both light and electron
microscopy level.
In the ZG, there was an increased synthesis and secretion of aldosterone what was
showed by significant reduction in the number of lipid droplets, increasing the number of
mitochondria and high levels of circulating aldosterone. The increase in concentration of
circulating aldosterone is a response to dehydration caused by the exposure of animals to
high temperature of the external environment.
In the ZF a significant increase in the surface profile and volume of the cells were
observed, particularly in cytoplasmic components, together with reduction in the number of
lipid droplets, increasing number of mitochondria, and significant increase in corticosterone
levels in the blood at the same time. Enlargement of cells involved in the synthesis and
secretion of the stress hormone corticosterone, changing of relative abundance of
components in the cells in a direction which indicates a greater involvement in the synthesis
of hormones, and biochemical data on the increase in circulating corticosterone levels
suggested an active participation of ZF cells in response to stress caused by acute exposure
of rats to elevated environmental temperatures.
In the ZR an extremely high accumulation of lipid droplets was found, which
would point to a slowdown in the synthesis of hormones and secretion of this zone.
In all zones of adrenal cortex, after acute exposure to the heat stress, the increase in
the number of apoptotic cells, proliferation of connective tissue fibers, the reduction of
NPY, VIP and SP immunoreactive nerve fibers and the apparent aggregation of platelets in
the capillaries were found. All this suggests that the acute heat stress, beside inspiring
synthetic activity of parenchyma cells of ZG and ZF, had a general negative effect on the
crust of the adrenal glands. Aggregation of platelets is probably the result of dehydration,
while the occurrence of apoptosis and proliferation of connective tissue may be the
predecessor of parenchymal fibrosis.
In the adrenal medulla the largest changes were found in the adrenaline cells of
which the profile surface was significantly reduced with the decrease of cytoplasm and
nuclei of cells. A significant reduction of full granules, while significantly increase in the
other two categories of mobilized granules (altered and empty) and the presence of whole
adrenaline granules in capillaries indicate strong ejection of adrenaline, which was
confirmed by its high levels in the circulation. In noradrenaline cells of the adrenal gland
there was redistribution in the presence of different types of granules, the decrease of full
granules, and the increase of the number of the other two types of granules. The
concentration of noradrenaline in the systemic circulation was significantly increased.
Given the fact that adrenaline and noradrenaline are directly involved in the reaction of
"fight or flight", the above data complete the picture of the applied stress-inducing effect of
experimental treatment.
In various ganglionic, neural and catecholamine cells of the adrenal glands, as well
as in the region of the cortex, reduced NPY, VIP and SP immunoreactivity were found.
Based on the foregoing, it is clear that the acute exposure of rats to elevated environmental
temperature for 60 minutes, it was enough to trigger the mechanisms involved in the stress
response, both on the biochemical and cellular level. In addition to increased levels of
circulating hormones, which can be detected almost immediately after the commencement
of stress acting stimuli, adjustment of the fine structure of cells is evident, requiring more
time and basing itself on more complex mechanisms. In addition, according to the obtained
results it appears that the adrenal glands, even after this acute treatment aim at complex
hystomorphologic alterations. In that case, it may not be possible to ignore the serious
pathological consequences such as embolism of blood vessels caused by increased platelet
aggregation. | true |
Povezanost polimorfizama VDR, CYP27B1 i CYP24A1 gena sa etiološkim faktorima i ishodom bolesti kod pacijenata sa oralnim skvamocelularnim karcinomom | Association of polymorphisms in VDR, CYP27B1 and CYP24A1 genes with etiological factors and disease outcomes in patients with oral squamous cell carcinoma | Oralni skvamocelularni karcinomi (Oral Squamous Cell Carcinoma- OSCC) se
karakterišu visokom stopom mortaliteta, lošom prognozom i porastom incidence
među osobama mlađe populacije. I pored uloženih napora na polju prevencije,
petogodišnje preživljavanje je ostalo nepromenjeno poslednjih nekoliko decenija,
što nameće potrebu za nove, molekularne vidove karakterizacije
oralnog
karcinoma. Poznati faktori rizika za nastanak oralnog karcinoma su: pušenje i
žvakanje duvana, konzumiranja alkohola, loša oralna higijena
i infekcije
visokorizičnim tipovima humanog papiloma virusa. Činjenica da samo izvesni
procenat osoba izloženih navedenim faktorima rizika zaista i razvije OSCC, ukazuje
na značaj genetičke osnove u procesu oralne kancerogeneze.
Mnogobrojnim studijama je pokazano da vitamin D ima antikancerogeni
efekat, koji se ogleda u zaustavljanju ćelijskog ciklusa,
inicijaciji ćelijske
diferencijacije,
indukciji apoptoze, sprečavanju invazije malignih ćelija i
antiangiogenetskom dejstvu. Vitamin D ispoljava svoju fiziološki aktivnu ulogu
nakon vezivanja za receptor za vitamin D (engl. Vitamin D Receptor- VDR) koji je
kodiran VDR genom. U VDR genu je identifikovan veliki broj polimorfizama
nukleotidne sekvence (engl. Single Nucleotide Polymorphisms- SNP), od kojih
samo mali broj ima funkcionalni efekat. U metabolizam vitamina D su uključeni
članovi velike familije proteina citohroma P450 i to: 1α-hidroksilaza uključena u
proces anabolizma (kodirana CYP27B1 genom), odnosno 24-hidroksilaza koja ima
ulogu u katabolizmu (kodirana CYP24A1 genom). Obzirom na značaj proučavanja
SNP-ova u genetičkim studijama asocijacije, postavlja se pitanje da li postojanje
izvesnih genetičkih varijanti u genima uključenim u funkcionisanje i metabolizam
vitamina D može biti asocirano sa rizikom nastanka oralnog karcinoma, kliničko
patološkim karakteristikama i preživljavanjem pacijenata.
Cilj. Cilj ove doktorske disertacije je bilo utvrđivanje postojanja asocijacije između
polimorfizama EcoRV, FokI, TaqI, ApaI, BsmI i rs11574085 u VDR genu, rs4646536
u CYP27B1 i rs2296241 polimorfizma u CYP24A1 genu sa rizikom za nastanak
oralnog karcinoma, etiološkim faktorima i ukupnim preživljavanjem pacijenata.
Pored navedenog, cilj je bilo i utvrđivanje postojanja haplotipskih blokova u
studijskoj grupi i asocijacije između registrovanih haplotipova sa rizikom za
nastanak oralnog karcinoma i preživljavanjem.
Materijal i metode.
Studijska grupa
je obuhvatala
110 pacijenata sa
dijagnostikovanim oralnim skvamocelularnim karcinomom i 122 zdrave osobe
kontrolne grupe odgovarajuće polne i starosne distribucije. DNK je izolovana iz
uzoraka primarnih tumora u grupi pacijenata i periferne krvi kontrolne grupe.
Genotipovi analiziranih polimorfizama su određivani PCR-RFLP metodom u slučaju
VDR EcoRV, FokI, TaqI i ApaI polimorfizama, odnosno Real Time PCR metodom za
rs11574085 polimorfizam u VDR genu i polimorfizme u CYP27B1 i CYP24A1
genima. Dobijeni rezultati su obrađeni statističkim programom SPSS. Vrednosti
neravnoteže vezanosti kao i učestalosti haplotipova su računate pomoću programa
Haploview,
dok
je
programom
Thesias
analizirana
povezanost
između
registrovanih haplotipova i preživljavanja. Asocijacije su smatrane statistički
značajnim ukoliko je p vrednost bila manja od 0.05.
Rezultati.
Zabeležene
su
značajne razlike u distribuciji genotipova VDR
polimorfizama EcoRV (p=0.019, χ2 test), ApaI (p=0.013, χ2 test) i BsmI (p=0.025, χ2
test), kao i CYP24A1 rs2296241 polimorfizma (p=0.025, Fisher test) između OSCC i
kontrolne grupe. Statistički značajna razlika u distribuciji alela je zabeležena u
slučaju EcoRV (p=0.019, χ2 test) i ApaI polimorfizma (p=0.019, χ2 test). Asocijacija
sa polom je zabeležena za VDR TaqI (p=0.006, χ2 test), BsmI (p=0.002, χ2 test) i
CYP27B1 rs4646536 (p=0.014, χ2 test), dok je značajna veza uočena između godina
i ApaI SNP-a (p=0.049, χ2 test). Pušenje je bilo u asocijaciji sa TaqI polimorfizmom
(p=0.039, χ2 test), dok je analizirani SNP u CYP27B1 genu bio asociran sa
konzumacijom alkohola (p=0.012, χ2 test). Kada je reč o kliničko patološkim
odlikama bolesti, zabeleženo je postojanje statistički značajne veze između TaqI
polimorfizma i recidiva bolesti (p=0.008, χ2 test).
Uočeno je statistički značajno smanjenje rizika za razvoj oralnog karcinoma
kod heterozigota AG analiziranog polimorfizma CYP24A1 gena, u odnosu na divlji
AA genotip (OR=0.281, p=0.000).
Pacijenti FokI ff genotipa su imali lošije preživljavanje (p=0.012, log-rank
test) u poređenju sa heterozigotnim i mutiranim genotipom zajedno. Stratifikovana
analiza prema nodalnom statusu i stadijumu tumora je pokazala da je ff genotip
asociran sa lošijim preživljavanjem u grupi sa negativnim i pozitivnim nodalnim
statusom (redom: p=0.025, p=0.040, log-rank test) i u III stadijumu tumora
(p=0.026, log-rank test). Multivarijantna Cox-regresiona analiza je pokazala da se
VDR FokI polimorfizam može koristiti kao nezavisni prognostički faktor.
Zaključak. Analiza polimorfizama gena uključenih u funkcionisanje i metabolizam
vitamina D može dati važne informacije o razvoju i progresiji oralnog karcinoma.
Polimorfizam u CYP24A1 genu je značajno asociran sa sniženim rizikom za razvoj
oralnog karcinoma, što je najverovatnije rezultat njegovog funkcionalnog efekta ili
neravnoteže vezanosti sa drugim polimorfizmima unutar CYP24A1 gena ili čak sa
polimorfizmima blisko lociranih susednih gena. Pored toga, ovakav nalaz bi mogao
ukazati na potencijalno korišćenje datog polimorfizma CYP24A1 gena u
prediktivne svrhe. VDR FokI polimorfizam može biti razmatran kao potencijalni
molekularni marker za preživljavanje, to jest može se koristiti kao nezavisni
prognostički indikator. Ovakav nalaz je najverovatnije posledica postojanja
funkcionalnog efekta FokI polimorfizma. | Oral Squamous Cell Carcinoma (OSCC) is characterized with a high
mortality, low survival rate and rising incidences among members of younger age
groups. Despite efforts in prevention, the survival rate remains unchanged for the
last few decades, which indicates
the need for
oral cancer
molecular
characterization. The well-known risk factors are tobacco smoking and chewing,
alcohol consumption, poor oral hygiene and infection with high risk human
papilloma virus types. The fact that only a small percentage of people exposed to
the risk factors really develops OSCC indicates the importance of genetic
background in the process of oral cancerogenesis.
Numerous studies have demonstrated the anticancerogen effects of vitamin
D, which is reflected in cell cycle arrest, induction of cell differentiation, apoptosis
induction, inhibition of malignant cell invasion and antiangiogenic effect. Vitamin D
exerts it’s physiological function after binding to the Vitamin D Receptor (VDR),
which is encoded by the VDR gene. A great number of Single Nucleotide
Polymorphisms (SNPs) have been identified in the VDR gene, among which a small
number have functional effects. Members of the huge protein cytochrome P450
family are involved in vitamin D metabolism: 1α-hydroxylase is involved in
anabolism (encoded by the CYP27B1 gene) and 24-hydroxylase is involved in
catabolism (encoded by the CYP24A1 gene). Since SNPs investigation is of great
importance in the genetic association studies, it is interesting to consider whether
genetic variants in genes, involved in vitamin D functioning and metabolism, could
be associated with oral cancer risk, clinicopathological characteristics and patient
survival.
Aim. The aim of the doctoral dissertation was to determine the association
between polymorphisms EcoRV, FokI, TaqI, ApaI, BsmI and rs11574085 in VDR
gene, rs4646536 in CYP27B1 and rs2296241 polymorphisms in CYP24A1 gene
with oral cancer risk, etiological factors and overall patients’ survival. Besides that,
the aim was to determine the existence of haplotype blocks in the study group and
to determine the association between detected haplotypes with risk of oral cancer
development and survival.
Material and methods. The study group consisted of 110 patients with diagnosed
oral squamous cell carcinoma and 122 healthy individuals in the control group,
matched in gender and age. DNA was isolated from the tumour tissue samples in
the patients group and from peripheral blood samples, in the control group. VDR
gene polymorphisms EcoRV, FokI, TaqI and ApaI were determined using the PCR-
RFLP method and PCR Real Time method in the case of rs11574085 in VDR and
polymorphisms in CYP27B1 and CYP24A1 genes. Obtained results were analysed
by the statistical program SPSS. Linkage disequilibrium values as well as haplotype
frequencies were calculated using the Haploview program, while association
between detected haplotypes and survival were performed by the program
Thesias. Associations were considered as significant when p values were less than
0.05.
Results. The statistically significant differences in the frequencies of the VDR
genotypes were observed between the OSCC and control group for the EcoRV
(p=0.019, χ2 test), ApaI (p=0.013, χ2 test) and BsmI (p=0.025, χ2 test)
polymorphisms, as well for CYP24A1 rs2296241 polymorphism (p=0.025, Fisher
test). The statistically significant differences in the allele distribution was noticed
for the EcoRV (p=0.019, χ2 test) and ApaI polymorphism (p=0.019, χ2 test). The
association with gender was observed for VDR TaqI (p=0.006, χ2 test), BsmI
(p=0.002, χ2 test) and CYP27B1 rs4646536 (p=0.014, χ2 test), while significant
association with age was observed for ApaI SNP (p=0.049, χ2 test). Smoking was
associated with TaqI polymorphism (p=0.039, χ2 test), while analysed SNP in
CYP27B1 gene was associated with alcohol consumption (p=0.012, χ2 test). In the
case of clinicopathological variables, statistically significant association was
detected between TaqI polymorphism and recurrence (p=0.008, χ2 test).
The statistically significant decrease of oral cancer risk was observed in
heterozygote AG of analysed polymorphism in CYP24A1 gene, in comparison with
wild type AA genotype (OR=0.281, p=0.000).
Patients with FokI ff genotype had a lower survival rate (p=0.012, log-rank
test) compared with heterozygous and mutated genotype combined. A stratified
analysis by the lymph node involvement and tumours stage showed that ff
genotype is associated with poor survival in groups with and without lymph node
involvement (p=0.025, p=0.040, respectively, log-rank test) and in stage III
tumours (p=0.026, log-rank test). Multivariate Cox-regression analysis revealed
that VDR FokI polymorphism could be considered as an independent prognostic
factor.
Conclusions. Analysis of gene polymorphisms involved in vitamin D functioning
and metabolism could provide important information on oral cancer development
and progression. Polymorphism in CYP24A1 gene was significantly associated with
oral cancer development, which could be ca onsequence of its functional effect or
linkage disequilibrium with other polymorphisms in CYP24A1 gene or even with
polymorphisms in the nearby different genes. Besides, this finding indicates that
analysed polymorphism in CYP24A1 gene could be used for predictive purposes.
VDR FokI polymorphism could be considered as a potential molecular marker of
survival and could be used as an independent prognostic indicator. These findings
most probably represent the functional effect of FokI polymorphism. | true |
Uloga humoralnog imunskog odgovora u etiopatogenezi amiotrofične lateralne skleroze | The role of humoral immune response in the aetiopathogenesis of amyotrophic lateral sclerosis | Amiotrofična lateralna skleroza (ALS) je fatalna neurodegenerativna bolest sa
početkom u adultnom dobu koja se karakteriše progresivnim gubitkom gornjih i donjih
motoneurona, što dovodi do atrofije muskulature, mišićne paralize i smrti usled slabosti
respiratorne muskulature. Oko 5-10% ALS slučajeva su sa familijarnom istorijom bolesti
(fALS), dok su preostali slučajevi sporadični (sALS) sa nepoznatim uzrokom bolesti. Cilj
ove disertacije bio je da proceni efekat imunoglobulina G (IgG) izolovanog iz sALS
bolesnika (ALS IgG) na homeostazu kalcijuma i mobilnost endozoma/lizozoma kod
pacovskih kortikalnih astrocita u kulturi. ALS IgG (0.1 mg/ml) poreklom od 7 ALS
pacijenata, za razliku od IgG iz 3 kontrolne osobe, izazvao je prolazne promene
unutarćelijske koncentracije kalcijuma (Ca2+-talas) u astrocitima. Povećana aktivnost
kalcijuma detektovana je kod oko polovine astrocita nakon tretmana sa ALS IgG, bez
obzira na prisustvo vanćelijskog kalcijuma (47,5 ± 12,9 %, 2 mM Ca2+ naspram 48,2 ± 13,6
%, 0 mM Ca2+). U prisustvu vanćelijskog kalcijuma, maksimalna vrednost promene
dostignuta je ~3x brže (19,7 ± 2,8 s, 2 mM Ca2+ naspram 56,3 ± 6,8 s, 0 mM Ca2+) i ukupno
povećanje kalcijuma (površina pod Ca2+-talasom) bilo je ~1.7x veće (26,0 ± 1,6 F/F0 *s, 2
mM Ca2+ naspram 15,6 ± 1.1 F/F0 *s, 0 mM Ca2+), ali vanćelijski kalcijum nije uticao na
maksimum amplitude kalcijumovog talasa. Primena farmakoloških inhibitora otkrila je da
je aktivacija inozotol 1,4,5-trifosfatnog (IP3), za razliku od rianodinskog receptora
neophodna i dovoljna da inicira povećanu aktivnost, a ulazak kalcijuma kroz TRPC kanale
produžava
odgovor.
Inhibicija fosfolipaze
C
(PLC)
umanjuje,
dok inhibicija
fosfatidilinozitol-3-kinaze (PI3K) u potpunosti sprečava tipičan ALS IgG-izazvan
kalcijumski odgovor. Populacija vezikula obeleženih lizotrekerom koji boji endozome i
lizozome, sastojala se od stacionarnih vezikula (6,1%) sa prosečnom brzinom koja nije
prelazila 67 nm/s i mobilnih vezikula (93.9%) čija je ukupna dužina putanje (TL) pređene
za 15 s iznosila u proseku 3,03±0,01 µm. ALS IgG (0.1 mg/ml) poreklom iz 12 od 13 ALS
bolesnika povećao je TL mobilnih vezikula za ~24% i maksimalni pomeraj (MD) za ~26%
tokom praćenih 4 min, za razliku od kontrolnih IgG (poreklom od 4 osobe) koji nisu uticali
na mobilnost vezikula. ALS IgG-izazvano povećanje mobilnosti bilo je manje izraženo u
rastvoru bez Ca2+, što ukazuje da su promene u homeostazi kalcijuma uključene u proces
kojim ALS IgG povećava mobilnost vezikula. Interesantno, ALS IgG kao i ATP (1 mM)
nisu uticali na oslobađanje sadržaja vezikula, što znači da je brza i kompletna egzocitoza
malo verovatan događaj kod astrocita u kulturi u našim eksperimentalnim uslovima.
Sumarno, ALS IgG utiče na kalcijumovu homeostazu astrocita preko IP3-posredovanog
oslobađanja kalcijuma iz endoplazmatičnog retikuluma i ulaska kalcijuma kroz TRPC
kanale, uz aktivaciju PI3K uzvodno od PLC. ALS IgG povećavaju mobilnost endozoma i
lizozoma, a ovaj efekat delimično zavisi od vanćelijskog kalcijuma. Ova studija je otkrila
molekulske mehanizme kojima astrociti postaju meta humoralnog imunskog odgovora u
ALS, što može da doprinese etiopatogenezi bolesti. | Amyotrophic lateral sclerosis (ALS) is an adult onset fatal neurodegenerative
disease characterized mainly by the progressive loss of upper and lower motor neurons
resulting in wasting, paresis and death from respiratory failure. Approximately 5-10% of
ALS cases are familial (fALS), while the rest are sporadic (sALS) with unknown cause of
disease. The objective of this dissertation was to evaluate the effect of immunoglobulin G
(IgG) isolated from sALS patients (ALS IgG) on calcium homeostasis and mobility of
endosomes/lysosomes in cultured rat cortical astrocytes. ALS IgG (0.1 mg/ml) from 7 ALS
patients, but not IgG from 3 control individuals evoked calcium transients (Ca2+-waves) in
astrocytes. About half of the tested astrocytes responded with elevated calcium activity
regardless of the presence of extracellular calcium (47.5 ± 12.9 %, 2 mM Ca2+ vs. 48.2 ±
13.6 %, 0 mM Ca2+). In presence of extracellular calcium, the peak amplitude developed
~3x faster (19.7 ± 2.8 s, 2 mM Ca2+ vs. 56.3 ± 6.8 s, 0 mM Ca2+) and the overall magnitude
of calcium rise (area under the Ca2+-wave) was ~1.7x larger (26.0 ± 1.6 F/F0 *s, 2 mM Ca2+
vs. 15.6 ± 1.1 F/F0 *s, 0 mM Ca2+), while the peak amplitude itself was not affected.
Application of pharmacological inhibitors revealed that activation of inositol 1,4,5-
triphosphate (IP3), but not of ryanodine receptors is necessary and sufficient to initiate
elevated activity, while the influx of extracellular calcium through TRPC channels prolongs
the responses. Inhibition of phospholipase C (PLC) diminishes, while the inhibition of
phosphatidilinositol-3-kinase (PI3K) completely prevents typical ALS IgG evoked calcium
response. The population of vesicles labeled with lysotracker that mainly stains endosomes
and lysosomes, consisted of non-mobile (6.1%) vesicles with the average speed of <67
nm/s and mobile vesicles (93.9%) with total track length (TL) in 15 s averaging at
3.03±0.01 µm. ALS IgG (0.1 mg/ml) from 12 of 13 patients increased the TL of mobile
vesicles by ~24% and the maximal displacement (MD) by ~26% within 4 min, while IgG
from control group (n=4) did not alter the vesicle mobility. The mobility enhancement by
ALS IgG was less pronounced in Ca2+-free extracellular solution, indicating that ALS IgG
vesicle mobility enhancement involves changes in Ca2+ homeostasis. Interestingly, neither
ALS IgG nor ATP (1 mM) triggered the release of vesicular cargo, making rapid and
complete exocytosis unlikely in cultured astrocytes in our experimental conditions. In
conclusion, ALS IgG affect calcium homeostatic system in astrocytes by IP3 mediated
calcium release from the endoplasmic reticulum and entry of extracellular calcium through
TRPC channels, with the activation of PI3K upstream of PLC. ALS IgG enhance the
mobility of endosomes and lysosomes, and this effect is partialy dependent on extracellular
calcium. This study revealed the molecular mechanisms by which astrocytes become
targeted cells for humoral immune response in ALS that could contribute to the
aetiopathogenesis of the disease. | true |
Efekti pola i heterogenih uslova svetlosti u populacijama dvodome vrste Mercurialis perennis L. na diferencijalnu herbivoriju | Effects of plant sex and heterogeneous habitat light conditions in populations of dioecious Mercurialis perennis L. on differential herbivory | U ovom istraživanju ispitivano je prisustvo polno-specifične i diferencijalne
herbivorije u odnosu na svetlosne uslove staništa kod dvodome vrste Mercurialis
perennis L. (Euphorbiaceae). Testirani su efekti pola, svetlosnih uslova staništa i
ontogenetskog stadijuma na osobine veličine biljaka (visinu, ukupnu površinu listova) i
listova (dužinu drške i centralnog nerva, širinu i površinu lisne ploče), nutritivne
(sadržaj vode, ukupnih solubilnih proteina i nestrukturnih ugljenih hidrata) i defanzivne
osobine listova (sadržaj fenola i kondenzovanih tanina), specifičnu lisnu površinu i
razvojnu nestabilnost (procenjenu preko indeksa fluktuirajuće asimetrije – FA).
Ispitivano je koje od analiziranih osobina biljaka imaju ulogu u interakcijama M.
perennis sa herbivorima u zavisnosti od svetlosnih uslova staništa i ontogenetskog
stadijuma. Analizom korelacionih odnosa istraživana je povezanost između stepena
herbivorije, razvojne nestabilnosti i ispitivanih osobina biljaka u odnosu na pol i
svetlosne uslove staništa.
Mercurialis perennis ili šumski prosinac je višegodišnja, zeljasta, dvodoma biljka
koja je najčešće član biljnih zajednica prizemnog sprata nenarušenih šumskih
ekosistema. Ređe se može naći na staništima sa otvorenim vegetacijskim sklopom. Kod
dvodomih vrsta polovi se mogu razlikovati kako u vegetativnim osobinama tako i u
stepenu oštećenja listova izazvanih aktivnošću herbivora. Iz navedenih razloga M.
perennis predstavlja izuzetno pogodan model sistem za istovremeno proučavanje uticaja
pola i svetlosnih uslova staništa na stepen herbivorije, osobine biljaka i razvojnu
nestabilnost.
U ovo istraživanje uključene su populacije M. perennis sa lokaliteta Bele stene
(Kopaonik) i Beograd. Na lokalitetu Bele stene biljke su uzorkovane tokom dve
uzastopne godine sa dva susedna staniša. Jedno stanište je bilo izloženo punoj dnevnoj
svetlosti u sastavu rudine, dok se drugo nalazilo pod vegetacijskom senkom unutar
četinarske šume. Razlike u svetlosnim uslovima na staništu kvantifikovane su
merenjem razlika u pokrovnosti staništa korišćenjem hemisfernih fotografija. Na uzorku
sa lokaliteta Bele stene iz 2015. godine testirani su efekti pola i svetlosnih uslova
staništa na stepen herbivorije i analizirane osobine biljaka, dok su na uzorku 2016.
godine, pored već navedenog, ispitivani i uticaji pomenutih faktora na osobine veličine
lista i fluktuirajuću asimetriju kao pokazatelj razvojne nestabilnosti. Razvojna
nestabilnost procenjena je preko indeksa fluktirajuće asimetrije za četiri osobine lista:
dužinu drške, dužinu centralnog nerva, širinu i površinu lisne ploče. Za opisivanje
vrednosti fluktuirajuće asimetrije na nivou više osobina korišćen je multivarijantni
indeks fluktuirajuće asimetrije. Biljke sa lokaliteta Beograd uzorkovane su dva puta u
toku jedne vegetacijske sezone kako bi se proučio efekat ontogenetskog stadijuma
(reproduktivnog i post-reproduktivnog) i pola na stepen herbivorije i analizirane
osobine biljaka.
Rezultati ovog istraživanja su pokazali prisustvo diferencijalne herbivorije kod M.
perennis u odnosu na svetlosne uslove staništa. Biljke na staništu izloženom punoj
dnevnoj svetlosti su bile više oštećene aktivnošću herbivora, pri čemu su bile značajno
manje (sa manjom visinom i ukupnom površinom listova), njihovi listovi imali nižu
nutritivnu vrednost (manji sadržaj vode i solubilnih proteina) kao i manju specifičnu
lisnu površinu. Na oba staništa stepen oštećenja je bio negativno povezan sa
specifičnom lisnom površinom. Na staništu izloženom punoj dnevnoj svetlosti stepen
herbivorije je bio negativno povezan sa sadržajem vode, a na staništu pod
vegetacijskom senkom sa visinom biljaka i sadržajem solubilnih proteina. Navedeni
rezultati ukazuju na prisustvo fenomena kompenzatorne ishrane tkivom nižeg
nutritivnog kvaliteta.
Biljke u post-reproduktivnoj fazi bile su više oštećene u odnosu na biljke u
reproduktivnoj fazi, pri čemu su njihovi listovi imali nižu nutritivnu vrednost (sa
manjim sadržajem vode i solubilnih proteina) i niže nivoe odbrane (sa nižim sadržajem
ukupnih fenola i kondenzovanih tanina). Zabeleženo smanjenje sadržaja defanzivnih
sekundarnih metabolita u listovima M. perennis u post-reproduktivnoj fazi je u
saglasnosti sa osnovnim pretpostavkama hipoteze optimalne odbrane. Na stepen
oštećenja kod biljaka u reproduktivnoj fazi negativno je uticao sadržaj ukupnih
nestrukturnih ugljenih hidrata, a kod biljaka u post-reproduktivnoj fazi sadržaj
kondenzovanih tanina. U post-reproduktivnoj fazi stepen herbivorije je bio pozitivno
povezan sa veličinom biljaka (visinom i ukupnom površinom listova) što je u
saglasnosti sa hipotezom biljne vitalnosti.
Konzistentne razlike u stepenu herbivorije između muških i ženskih biljaka M.
perennis nisu pronađene. Polno-specifična herbivorija zabeležena je u uzorku sa
lokaliteta Bele stene iz 2016. godine gde su muške biljke bile više oštećene. U uzorku
sa lokaliteta Beograd u post-reproduktivnom stadijumu su takođe bile prisutne razlike
između polova u stepenu herbivorije pri čemu su ženske biljke bile pod većim pritiskom
herbivora. Navedeni rezultati ukazuju na varijabilne obrasce razlika između polova u
stepenu oštećenja.
Svetlosni uslovi na staništu su značajno uticali na obrasce polnog dimorfizma u
sadržaju solubilnih proteina, ukupnih fenola i kondenzovanih tanina, dok je
ontogenetski stadijum imao efekat na prisustvo polnog dimorfozma u ukupnoj površini
listova, sadržaju solubilnih proteina i ukupnih fenola. Opisani obrasci mogu biti
interpretirani u kontekstu razlika između polova u fenologiji procesa reprodukcije kao i
reproduktivnoj alokaciji.
Zabeležene su značajne razlike između staništa sa različitim svetlosnim uslovima
u indeksu fluktuirajuće asimetrije lisne drške i kompozitnog indeksa, pri čemu su
njihove vrednosti bile više na staništu izloženom punoj dnevnoj svetlosti. Ostali
kvantifikovani indeksi nisu se razlikovali značajno između ispitivanih staništa. Ženske
biljke M. perennis su imale duži centralni nerv lista, šire listove sa većom površinom
lisne ploče, međutim razlike među polovima nisu bile zabeležene u vrednostima FA
indeksa.
Uočeni obrasci korelacija između stepena herbivorije, indeksa fluktuirajuće
asimetrije i analiziranih osobina biljaka značajno su se razlikovali između staništa, pri
čemu je zabeleženo postojanje većeg broja značajnih korelacija na staništu pod
vegetacijskom senkom. Indeksi fluktuirajuće asimetrije bili su značajno korelisani
međusobno, međutim nisu bili povezani sa ukupnom površinom svih listova,
nutritivnim i defanzivnim osobinama lista na oba staništa. Stepen herbivorije bio je u
negativnoj korelaciji sa indeksom fluktuirajuće asimetrije površine lisne ploče kod
muških biljaka sa staništa izloženog punoj dnevnoj svetlosti, i u pozitivnoj korelaciji sa
indeksom fluktuirajuće asimetrije dužine centralnog nerva kod muških biljaka sa
staništa pod vegetacijkom senkom, dok nije bio značajno povezan sa ostalim indeksima
FA. Ovi nalazi se mogu protumačiti u kontekstu manjeg uticaja stresa na nivoe razvojne
nestabilnosti analiziranih osobina M. perennis. | This study investigated the presence of sex – biased and light differential
herbivory in dioecious Mercurialis perennis L. (Euphorbiaceae). It was tested if plant
sex, light conditions and the ontogenetic stage had significant effect on plant size
(height, total leaf area) and leaf size (petiole and midvein length, width and leaf blade
surface), leaf nutritional (water, total soluble protein and nonstructural carbohydrate
content) and defensive traits (total phenolic and condensed tannin content), specific leaf
area and developmental instability (estimated as fluctuating asymmetry). It was
examined which of the analyzed plant traits mediated interactions between M. perennis
and herbivores depending on habitat light conditions and plant ontogenetic stage. By
using correlation analyses the connection between herbivory, developmental instability
and analyzed plant traits was investigated in relation to habitat light conditions.
Mercurialis perennis or dogs' mercury is a perennial, dioecious herb that is a
common member of the ground level communities of undisturbed forests. Rarely this
species can be found in completely open field. In dioecious plants, the sexes can differ
in vegetative traits as well as in herbivory rates. Therefore, M. perennis represents a
suitable model system for exploring simultaneously the effects of plant sex and habitat
light conditions on herbivory, plant traits and developmental instability.
This research included populations of M. perennis from two sites: Bele stene
(Kopaonik) and Belgrade. At Bele stene site the plants were sampled during two
consecutive years from two neighboring habitats. One habitat was exposed to full
sunlight in an open field, while the other was inside of an evergreen forest. Differences
in light conditions between the habitats (canopy openness) were quantified by means of
hemispherical photography. The sample from 2015. was used to test the effects of plant
sex and habitat light conditions on herbivory and analyzed plant traits, while the sample
from 2016. apart from already stated, was also used to test the mentioned effects on leaf
size traits and fluctuating asymmetry as an indicator of developmental instability. Four
fluctuating asymmetry indices were quantified for leaf petiole length, midvein length,
leaf blade width and leaf blade surface area. To quantify developmental instability for
multiple traits simultaneously, multivariate index of fluctuating asymmetry was used.
At Belgrade site the plants were sampled twice in one year to investigate the effect of
ontogenetic stage (reproductive and post-reproductive) and sex on herbivory and
analyzed plant traits.
The results of this research showed presence of light differential herbivory in M.
perennis. Plants at sun expose site were more damaged althoug they were smaller
(lower and with smaller total leaf area), their leaves had lower nutritive value (lower
water and soluble protein content) and smaller specific leaf area. At both habitats
herbivory was negatively correlated with specific leaf area. At sun exposed habitat
herbivory levels were negatively affected by leaf water content and in the shade by
plant height and leaf soluble protein content. These results indicate presence of
compensatory consumption of lower quality food.
Plants in post-reproductive stage were more damaged compared to the plants in
reproductive stage, while their leaves had lower nutritional value (lower water and
soluble protein content) and lower defense levels (lower total phenolic and condensed
tannin content). The decline in defensive levels during post-reproductive stage is in
concordance with basic assumptions of optimal defense hypothesis. Levels of damage
were negatively affected by total nonstructural carbohydrate content in reproductive
stage, while in post-reproductive stage by the total condensed tannin content. In post-
reproductive stage plant size (height and total leaf area) positively affected herbivory
rates, which is in accordance with plant vigor hypothesis.
No consistent sex bias in herbivory rates was found in M. perennis. In Bele stene
sample form one year the males were more damaged, while in the Belgrade sample
females were under higher herbivore pressure during post-reproductive stage. These
results indicate variable pattern of sex bias of herbivory rates in M. perennis.
Habitat light conditions significantly affected patterns of sexual dimorphism in
soluble protein, total phenolic and condensed tannin content, while ontogenetic stage
had effect on presence of intersexual differences in total leaf area, soluble protein and
total phenolic content. These patterns could be interpreted in context of intersexual
differences in reproductive phenology and allocation.
Significant differences between habitats with different light conditions were
recorded in fluctuating asymmetry index of petiole length and multivariate index, with
FA values being higher at sun exposed habitat. Other quantified indices of fluctuating
asymmetry did not differ significantly between examined habitats. Female plants had
longer petioles and midveins, wider leaves with larger blade surface areas; however
there were no significant intersexual differences in FA indices.
Correlation patterns between herbivory, fluctuating asymmetry indices and
analyzed plant traits differed between the examined habitats, with higher number of
statistically significant correlations being present in the sample from the shade habitat.
Fluctuating asymmetry indices were mutually significantly correlated; however they
were not related to total leaf area, nutritional or defensive leaf traits at either habitat.
Herbivory levels were negatively correlated with index of fluctuating asymmetry of leaf
blade surface area in male plants from the sun exposed habitat, and positively correlated
with index of fluctuating asymmetry of leaf midvein length in male plants from the
shade, while thez were not significantly related to the rest of the quantified FA indices.
These findings could be interpreted in context of lower impact of stress to levels of
developmental instability of analyzed traits in M. perennis. | true |
Mitotička i mejotička nestabilnost DMPK ekspanzija sa varijantnim ponovcima kao genetički modifikator fenotipa miotonične distrofije tipa 1 | Mitotic and meiotic instability of DMPK expansions with variant repeats as a genetic modifier of myotonic dystrophy type 1 phenotype. | otonična distrofija tip 1 (DM1) je najčešća nasledna mišićna distrofija odraslih osoba i
praćena je multisistemskim simptomima. Uzrokovana je ekspanzijama (povećanjem broja)
CTG ponovaka u genu DMPK, koje se odlikuju mejotičkom i mitotičkom nestabilnošću sa
težnjom ka daljem povećanju broja ponovaka. DM1 je jedna od fenotipski najvarijabilnijih
monogenskih bolesti. Broj CTG ponovaka je glavni faktor koji utiče na uzrast početka
bolesti i težinu kliničke slike, a pretpostavlja se da različiti genetički, epigenetički i/ili
sredinski faktori modifikuju njegov efekat. Manje od 5% DM1 bolesnika poseduje
varijantne ponovke (CCG, CTC, GGC ili CAG) rasute među CTG ponovcima, i može
ispoljiti neobične i blaže simptome, nego što se očekuje na osnovu njihovog broja
ponovaka. Predmet ove doktorske teze bila je genetička i epigenetička karakterizacija
varijantnih ponovaka kod DM1 bolesnika i njihov modifikujući efekat na fenotip.
Varijantni ponovci su detektovani samo na 3′ kraju ekspanzija kod 9 od 243 DM1
bolesnika (3,7%) iz 5 od 174 porodice (2,9%), primenom repeat-primed PCR-a (RP-PCR).
Sangerovim sekvenciranjem RP-PCR proizvoda, CCG ponovci, organizovani kao
pojedinačni, kratki ili dugi perfektni nizovi, ili kao pojedinačni ili pravilni nizovi CCGCTG
heksamera, identifikovani su kod osam bolesnika. Kod preostalog bolesnika, prvi put je
opisana DMPK ekspanzija sa de novo nastalim CTC ponovkom. Praćenjem četiri
međugeneracijska prenošenja varijantnih DMPK ekspanzija, primenom longe-range PCR-a
i Southern blota, zapažena su stabilna prenošenja ili kontrakcije (smanjenje broja
ponovaka). Korišćenjem podataka iz ove i publikovanih studija, pokazano je da se
varijantne DMPK ekspanzije, suprotno čistim (sastavljenim iz perfektnih CTG ponovaka),
međugeneracijski češće stabilno prenose ili kontrahuju, posebno kada je prenosilac majka.
Somatska nestabilnost DMPK ekspanzija kvantifikovana je single-molecule small-pool
PCR-om. Umnoženo je ~5700 alela, sa preko 200 alela po analiziranom uzorku krvi i
bukalnog brisa. Somatska nestabilnost varijantnih DMPK ekspanzija, slično čistim,
odlikovala se tkivnom specifičnošću i težnjom ka povećanju broja ponovaka tokom života
bolesnika. Međutim, matematičkim modelovanjem pokazano je da se varijantne DMPK
ekspanzije karakterišu nižim nivoom somatske nestabilnosti, koja je praćena manjim
povećanjem broja ponovaka tokom vremena. RP-PCR profili su ukazali na to da su
varijantni ponovci stabilni u ćelijama krvi tokom vremena i između ćelija krvi i bukalne
sluznice. Navedeni rezultati pružaju dokaze da različiti tipovi i obrasci varijantnih
ponovaka doprinose stabilnosti DMPK ekspanzija u somatskim i polnim ćelijama.
Primenom metil-specifičnog RP-PCR-a na bisulfitno-konvertovanoj genomskoj DNK i
klasičnog RP-PCR-a na genomskoj DNK digeriranoj enzimom SsiI, prvi put je pokazano da
su sami CCG ponovci u DMPK ekspanzijama heterogeno metilovani. Bisulfitnim
sekvenciranjem uočena je i heterogena metilacija CpG ostrvaca uzvodno i nizvodno od
ponovaka. Na osnovu zapažanja da relativni stepen metilacije samih CCG ponovaka i
okolnih CpG ostrvaca zavisi od zastupljenosti i obrasca CCG ponovaka, pretpostavljeno je
da se metilacija inicira na samim CCG ponovcima i lokalno širi na okolna CpG ostrvca.
Neki od bolesnika sa varijantnim DMPK ekspanzijama odlikovali su se atipičnim
simptomima, kliničkom prezentacijom koja je pre ukazivala na miotoničnu distrofiju tip 2,
kao i relativno kasnim uzrastom početka bolesti u odnosu na očekivani prema broju
ponovaka. Matematičko modelovanje je pokazalo da individualno-specifične razlike u
nivou somatske nestabilnosti imaju veći uticaj na uzrast početka simptoma kod bolesnika sa
varijantnim ekspanzijama u odnosu na one sa čistim. Posredno, ovaj rezultat ukazuje na to
da je mehanizam kojim varijantni ponovci doprinose kasnijem ispoljavanju početka bolesti
vezan za stabilizaciju DMPK lokusa u somatskim ćelijama.
Rezultati ove doktorske teze pružaju dokaze o ulozi varijantnih ponovaka kao
modifikatora DM1 fenotipa, ističući njihovu kliničku relevantnost. Aplikativni značaj
rezultata bi mogao da se ogleda u doprinosu oko postizanja konsenzusa vezanog za
genetičko savetovanje porodica sa varijantnim ponovcima, adekvatnom regrutovanju
bolesnika za kliničke studije i dizajnu novih terapeutika, koji bi efekat DMPK ekspanzija
modifikovali kroz suprimiranje njihove somatske nestabilnosti. Sa druge strane, otkriće
metilacije CCG ponovaka u DMPK ekspanzijama otvara bazična pitanja vezana za ulogu
epigenetičkih mehanizama u stabilizaciji DMPK lokusa. | adults, characterized by multisistem features. It is caused by expansions (an increase in the
number) of CTG repeats in the DMPK gene, which are meiotically and mitotically unstable
and tend to expand further. DM1 is phenotypically one of the most variable monogenic
diseases. The number of CTG repeats is the main factor influencing age at onset and
severity of disease. The effect of the causing mutation is assumed to be modified by
different genetic, epigenetic and/or environmental factors. Less than 5% of DM1 patients
carry variant repeats (CCG, CTC, GGC or CAG) scattered among CTG repeats, and they
can express some unusual and milder symptoms than expected based on their number of
repeats. The subject of this doctoral thesis was the genetic and epigenetic characterization
of variant repeats in DM1 patients and their modyfing effect on the phenotype.
Variant repeats were detected only at the 3′ end of expansions in 9 out of 243 DM1
patients (3,7%) from 5 out of 174 families (2,9%), by repeat-primed PCR (RP-PCR).
Sanger sequencing of RP-PCR products identified the CCG repeats in 8 patients, which
were present as individual repeats, short or long pure tracts, or individual or short tracts of
CCGCTG hexamers. In the remaining patient, a DMPK expansion with a de novo CTC
variant repeat has been described for the first time. The analysis of four intergenerational
transmissions of variant DMPK expansions by longe-range PCR and Southern blot showed
stable transmissions or even contractions (a decrease in the number). By combining data
from this and previously published studies, it was shown that variant DMPK expansions, in
contrast to the pure ones (carrying only CTG repeats), were more often stably transmitted
or contracted, particularly when the transmitting parent was mother. Somatic instability of
DMPK expansions was quantified by single-molecule small-pool PCR. A total of ~5700
alleles were amplified, with more than 200 alleles per analyzed blood and buccal swab
sample. The somatic instability of variant DMPK expansions had characteristics similar to
expansions consisting of pure CTG repeats: tissue specificity and a bias toward further
expansions throughout the patient’s life. However, mathematical modeling demonstrated
that variant DMPK expansions were characterized by a lower level of somatic instability,
which was accompanied by the lower expansion size increment in blood cells over time.
RP-PCR profiles indicated that variant repeats were stable at the 3 end of DMPK
expansions in blood and buccal cells, and in blood cells over time. These results provide
evidence that different types and patterns of variant repeats contribute to the stability of
DMPK expansions in somatic and germ cells. Using methyl-specific RP-PCR on a
bisulfite-converted genomic DNA and classical RP-PCR on genomic DNA digested by SsiI
enzyme, it was discovered that variant CCG repeats were heterogeneously methylated
within DMPK expansions. Bisulfite sequencing revealed heterogenic methylation of CpG
islands upstream and downstream of repeat tract. Based on the observation that the relative
degree of methylation of CCG repeats and CpG islands depends on quantity and patterns of
CCG repeats, it is hypothesized that methylation is initiated on CCG repeats and spreads
locally on the surrounding CpG islands. Some patients with variant DMPK expansions
were characterized by atypical symptoms, e.g. clinical presentation indicative of myotonic
dystrophy type 2, as well as relatively later age at onset than expected based on the number
of repeats. Mathematical modeling demonstrated that individual specific differences in the
level of somatic instability had a greater impact on age at onset in patients with variant
expansions compared to those with pure expansions. Indirectly, this result implies that
variant repeats contribute to the later age at onset by the mechanism related to stabilization
of DMPK locus in somatic cells.
Results of this doctoral thesis provide evidence for the role of variant repeats as
modifiers of DM1 phenotype, emphasizing their clinical relevance. The applicative
significance of the results could be reflected in the contribution to reaching a consensus on
genetic counseling for families with variant repeats, adequate recruitment of patients for
clinical studies and the design of new therapies, which would modify the effect of DMPK
expansions by suppressing their somatic instability. On the other hand, the discovery of
methylation of CCG repeats in DMPK expansions opens questions about the role of
epigenetic mechanisms in the stabilization of DMPK locus. | true |
Procena kvaliteta površinske vode na osnovu mikrobioloških parametara i ekogenotoksikoloških i histopatoloških analiza tkiva deverike Abramis brama (L., 1758) , krupatice Blicca bjoerkna (L., 1758) i crnooke deverike Ballerus sapa (P., 1814) | Assessment of surface water quality based on microbiological parameters and ecogenotoxicological and histopathological analysis of common bream Abramis brama (L., 1758), white bream Blicca bjoerkna (L., 1758) and white-eye bream Ballerus sapa (P., 1814) tissues | Biomonitoring predstavlja neophodnu komponentu tradicionalnih tehnika monitoringa, sa
ciljem da se uspostavi veza izmeŤu spoljašnjih koncentracija zagaŤivaţa, koncentracija
zagaŤivaţa u tkivima bioindikatora i ranih štetnih efekata po ispitivane organizme. Ribe su
korisni bioindikatori i ţesto se primenjuju u ekogenotoksikološkim ispitivanjima. U ovoj
studiji, procena stanja kvaliteta površinske vode vršena je na reci Savi na lokalitetu Duboko
i na reci Dunav na lokalitetu Višnjica tokom 2014. godine, kako bi se procenio uticaj
razliţitih sezona na variranje parametara kvaliteta i odgovor biomarkera deverike, krupatice
i crnooke deverike. Procena kvaliteta vode vršena je merenjem fiziţko-hemijskih i
mikrobioloških parametara fekalnog i organskog zagaŤenja. Procena genotoksiţnog
potencijala vršena je primenom alkalnog komet testa za kvantifikaciju DNK oštešenja u
šelijama krvi, jetre i škrga riba, kao biomarkera izlaganja. Histopatološke promene u jetri i
škrgama prašene su kao biomarker efekta. Paralelno, prašene su koncentracije metala i
metaloida u jetri, škrgama, gonadama i mišišu, primenom metode ICP-OES. Na lokalitetu
Višnjica zabeleţen je viši nivo fekalnog i organskog zagaŤenja. Na lokalitetu Duboko
zabeleţene su više koncentracije ispitivanih elemenata u sva ţetiri tkiva, u poreŤenju sa
lokalitetom Višnjica. Na oba lokaliteta škrge su akumulirale najviše koncentracije metala i
metaloida, a mišiš najniţe. Na oba lokaliteta, krv je bila tkivo sa najvišim nivoom DNK
oštešenja, dok je jetra tokom vešine meseci imala najniţe DNK oštešenje. Na lokalitetu
Višnjica zabeleţene su više vrednosti DNK oštešenja krvi, na lokalitetu Duboko više
vrednosti DNK oštešenja škrga, dok je nivo DNK oštešenja u šelijama jetre na oba
lokaliteta bio pribliţno jednak. Na lokalitetu Duboko pri najvešim vrednostima
histopatološkog indeksa škrga zabeleţeno je blago do umereno oštešenje, a na lokalitetu
Višnjica umereno do teško oštešenje škrga. Na oba lokaliteta, jetra je bila organ sa vešim
obimom histopatoloških promena u odnosu na škrge. Sveukupno, škrge kao prvi organ u
direktnom kontaktu sa zagaŤivaţima iz vode pokazale su vešu akumulaciju ispitivanih
elemenata i viši nivo DNK oštešenja, dok je jetra kao glavni organ za procesuiranje
zagaŤivaţa iz vode i hrane pokazala viši nivo histopatoloških promena. Odabrane vrste
pokazale su se kao pogodni bioindikatori za in situ ispitivanja efekata zagaŤenja. | Biomonitoring represents an essential part of traditional monitoring techniques with the aim
to establish relationship between external concentrations of pollutants, concentrations of
pollutants in the tissues of bioindicators and early adverse effects in examined organisms.
Fish are useful bioindicators often used in ecogenotoxicological studies. In this study,
assessment of the surface water quality was performed on the Sava River locality Duboko
and on the Danube River locality Višnjica during 2014 in order to examine the impact of
different seasons on the variation of the quality parameters and biomarker response in
common bream, white bream and white-eye bream. Assessment of the water quality was
performed based on the physico-chemical parameters and microbiological parameters of
fecal and organic pollution. Genotoxic potential was assessed by using the alkaline comet
assay in order to quantify DNA damage level, in blood, liver and gill cells, as a biomarker
of exposure. Histopathological analyses of liver and gill tissue were monitored as a
biomarker of effect. Additionally, concentrations of metals and metalloids in liver, gills,
gonads and muscle were performed by the ICP-OES method. The higher level of fecal and
organic pollution was present on the locality Višnjica. The higher concentrations of
examined elements in all four tissues were present in fish from the locality Duboko, in
comparison to the locality Višnjica. At both sites gills accumulated the highest
concentrations of metals and metalloids, while muscle accumulated the lowest
concentrations. Blood was the tissue with the highest level of DNA damage, while liver had
the lowest level of DNA damage during most of the months at both sites. Higher DNA
damage level in blood cells was observed at the locality Višnjica, in gill cells at the locality
Duboko, while the level of DNA damage in liver was approximately the same at both
localities. Based on the highest level of histopathological index of gills at the locality
Duboko gills were slightly to moderately damaged, while at the locality Višnjica gills were
moderately to heavily damaged. At both localities liver had higher level of
histopathological alterations in comparison to gills. Overall, gills as the first organ in direct
contact with contaminants form water showed higher level of examined elements and DNA
damage, while the liver as the main organ for processing contaminants from water and food
showed higher level of histopathological alterations. Examined fish species proved to be
useful bioindicators for in situ assessment of the pollution effects. | true |
Morfološka studija pigidijalnih žlezda i analiza hemijskog sastava njihovih sekreta kod odabranih vrsta trčuljaka (Insecta: Coleoptera: Carabidae) | Morphological study of the pygidial glands and analysis of the chemical composition of the secretions of selected ground beetle species (Insecta: Coleoptera: Carabidae) | Kao odgovor na terestrični način života, zglavkari, a naročito insekti, su razvili
niz adaptacija koje su omogućile najpre opstanak, a potom i kolonizaciju gotovo svih
ekoloških niša u novoj sredini. Insekti su ekstremno diverzifikovana grupa organizama
zahvaljujući svakako i činjenici da poseduju veliki biohemijski potencijal. Sekreti koje
produkuju insekti imaju najrazličitije namene: u pitanju su iritanti i/ili repelenti protiv
predatora, otrovi za imobilizaciju i ubijanje plena, feromoni, itd. Svi pomenuti hemijski
produkti, sa ulogom u intra- i interspecijskim interakcijama su najčešće sekundarni
metaboliti, tj. produkti poreklom od jedinjenja koja učestvuju u primarnim
biohemijskim procesima nastalim različitim hemijskim transformacijama.
Veliki asortiman antipredatorske odbrane trčuljaka podrazumeva morfološke,
ponašajne i biohemijske strategije. Hemijska odbrana je izuzetno efikasna kod adultnih
trčuljaka i podrazumeva izbacivanje produkata pigidijalnih žlezda u vidu odbrambenih
sekreta. Pigidijalne žlezde kao parne dorzalne invaginacije telesnog zida u posteriornom
delu abdomena su univerzalno prisutne kod Carabidae i ostalih pripadnika nadporodice
Caraboidea. Svaka žlezda se sastoji od agregacije sferičnih ili manje-više izduženih
sekretornih lobusa (acinusa), u kojima su sekretorne ćelije raspoređene oko centralnog
(aksijalnog) sabirnog lumena. Sekreti se dalje sprovode dugim, glavnim sabirnim
kanalom do rezervoara za skladištenje. Zidovi rezervoara su obloženi glatkom
muskulaturom i promenljive su debljine. Rezervoar se sužava u cilindričnu cev
(eferentni kanal), koja se otvara u spoljašnju sredinu.
Komponente sekreta detektovane hemijskim analizama moguće je grupisati u
deset glavnih kategorija: (1) hinoni, (2) aldehidi, (3) alkoholi, (4) estri, (5) fenoli, (6)
karboksilne kiseline, (7) ketoni, (8) monoterpeni, (9) nitrili i (10) ugljovodonici.
Ciljevi ove doktorske disertacije usmereni su na dopunjavanje i proširivanje
znanja o građi egzokrinih pigidijalnih žlezda i hemijskom sastavu njihovih sekreta kod
trčuljaka.
U ovoj studiji su analizirane adultne jedinke 27 vrsta trčuljaka iz 13 rodova i pet
potporodica sakupljene na teritorijama Srbije i Crne Gore. Isključivo morfološka
vi
karakterizacija pigidijalnih žlezda vršena je kod 15 vrsta, a analizan hemijski sastav
sekreta kod četiri vrste, dok su obe analize sprovedene kod osam vrsta.
Morfološki deo studije urađen je pomoću svetlosne mikroskopije, dok je za
hemijske analize korišćena hibridna tehnika gasna hromatografija sa masenom
spektrometrijom (GC-MS). Analizirana je morfo-anatomija pigidijalnih žlezda, kao i
njihova histološka struktura. Neke vrste su po prvi put analizirane u ovom kontekstu,
dok su za druge vrste koje su ranije hemijski analizirane po prvi put dati prikazi
pigidijalnih žlezda.
Analizom hemijskog sastava sekreta pigidijalnih žlezda 12 odabranih vrsta
trčuljaka iz šest rodova i četiri potporodice, detektovano je ukupno 52 različita
jedinjenja (51 identifikovano i jedno neidentifikovano). Najjednostavnija smeša
jedinjenja u sekretu konstatovana je kod vrste Carabus montivagus Palliardi, 1825 (dve
komponente), dok je najsloženija smeša jedinjenja detektovana u sekretu
visokoevoluirane troglobiontne vrste Pheggomisetes globiceps Buresch, 1925 (čak 32
komponente).
Naposletku, koristeći podatke iz literaturnih izvora, uključujući i podatke iz ove
disertacije, dato je sumiranje svih analiza hemijskog sastava sekreta pigidijalnih žlezda
adultnih trčuljaka (494 vrste iz 179 rodova i 24 potporodice).
Budući da je na svetskom nivou hemijski okarakterisan sekret tek 1,24% vrsta
trčuljaka, a još je manji postotak onih vrsta kod kojih su žlezde morfološki
okarakterisane, svakako je na budućim istraživanjima taj kolosalni zadatak da se
značajno unapredi naše poznavanje hemijskog arsenala trčuljaka, kao i morfo-
funkcionalne osnove koja stoji iza njega. | In response to the terrestrial lifestyle, arthropods (especially insects) developed a series
of adaptations that firstly enabled the survival, and then the colonization of almost all
ecological niches in the new environment. Insects are an extremely diversified group
also due to the fact that they have a great biochemical potential. Secretions produced by
insects have a variety of uses: irritants and/or repellents against predators,
immobilization and killing of pray, pheromones, etc. All of the mentioned chemical
products with the role in intra- and interspecfic interactions are usually secondary
metabolites, i.e., products originated from compounds that participate in primary
biochemical processes by different chemical transformations.
A large assortment of antipredator defense of ground beetles involves
morphological, behavioral and biochemical strategies. An extremely effective way of
defending against predators in adult beetles is via chemicals which are discharged as
pygidial gland products in the form of defense secretions. Pygidial defensive glands are
dorsal body wall invaginations in the posterior part of the abdomen, and are universally
present in Carabidae and other members of the Caraboidea superfamily. Each gland
consists of an aggregation of spherical or more or less elongate secretory lobes (acini),
in which secretory cells are arranged around the central (axial) collecting lumen.
Secretions are further transported by a long main collecting canal to a storage reservoir.
The reservoir walls are coated with smooth muscles of variable thicknesses. The
reservoirs are continuing into cylindrical tubes (efferent ducts) which open into the
environment.
Chemical components of the pygidial secretions in Carabidae can be grouped
into ten main categories: (1) quinones, (2) aldehydes, (3) alcohols, (4) esters, (5)
phenols, (6) carboxylic acids, (7) ketones, (8) monoterpenes, (9) nitriles, and (10)
hydrocarbons.
The goals of this doctoral dissertation are focused on complementing and
expanding knowledge on the morphological structure of exocrine pygidial glands and
the chemical composition of their secretions in ground beetles.
viii
In this study, adult individuals of 27 species and 13 genera from five subfamilies
of ground beetles collected on the territories of Serbia and Montenegro were analyzed.
Only morphological characterization of the pygidial glands was performed for 15
species, the chemical composition of the secretions in four species is analyzed, while
both analyzes were carried out for eight species.
The morphological part of the study was done using light microscopy, while for
chemical analyzes a hybrid technique was used – gas chromatography with mass
spectrometry (GC-MS). Pygidal glands were analyzed both morpho-anatomically and
histologically. Some species have been analyzed for the first time in this context, while
for other species that have previously been chemically analyzed only pygidial glands
have been shown for the first time.
By analyzing the chemical composition of the secretions of the pygidial glands,
a total of 52 different compounds (51 identified and one unidentified) were detected in
12 selected species of six genera and four subfamilies of ground beetles. The simplest
mixture of the compounds in the secretion was found in the species Carabus
montivagus Palliardi, 1825 (two components), while the most complex is detected in the
highly evolved troglobitic species Pheggomisetes globiceps Buresch, 1925 (as much as
32 components).
At the end, using data from literature sources and including data from this
dissertation, a summary of all analyzes of the secretions of pygidial glands of adult
ground beetles is given (a total of 494 species from 179 genera and 24 subfamilies).
Since pygidial gland secretions of ground beetles are chemically characterized in
only 1.24% of species, and even smaller is the percentage of the species whose glands
are morphologically characterized, the colossal task to significantly improve our
knowledge on the chemical arsenal of ground beetles as well as the morpho-functional
basis behind is definitely on the future studies. | true |
Uticaj N-acetil-L-cisteina in vitro na proliferaciju i diferencijaciju matičnih ćelija zubne pulpe mlečnih zuba dece | Effect on N-acetil-L-cysterine in vitro on proliferation and differentiation of children deciduous teeth dental pulp stem cells | Zubna pulpa vodi poreklo od embrionalnog ektomezenhima i potencijalno je vaţan izvor mezenhimalnih matičnih ćelija (MMĆ) za regeneraciju svih tkiva kraniofacijalne regije koja su, takođe, poreklom od ektomezenhima. N-acetil-L-cistein (NAC) je antioksidant koji moţe da ima uticaj na terapijsku primenu MMĆ. Cilj ovog rada je bio da utvrdi da li ćelije izolovane iz zubne pulpe mlečnih zuba dece i ekspandirane in vitro, imaju karakteristike MMĆ i da ispita dejstvo NAC-a na njihovu proliferaciju i diferencijaciju, kao i da se utvrdi da li NAC utiče na aktivnost enzima antioksidativne zaštite, oksidativno oštećenje različitih ćelijskih struktura i metabolizam glukoze. Metodom tkivnog eksplanta, iz šest pulpi mlečnih zuba dece, je dobijena početna populacija adheretnih ćelija. Za procenu broja CFU-F (eng. Colony Forming Unit – Fibroblast), ćelije su zasejavane u maloj gustini. Proliferativni potencijal i vreme udvajanja broja ćelija u kulturi je praćeno brojanjem ćelija posle tripsinizacije subkonfluentnih kultura, svaka 4 dana, tokom 40 dana. Protočna citometrija je korišćena za imunofenotipizaciju ex vivo umnoţenih ćelija, određivanje aktivnosti aldehid- dehidrogenaze (ALDH), brzinu ulaska ćelija u sintetsku (S) fazu ćelijskog ciklusa, određivanje broja ćelijskih deoba i detekciju apoptoze. Aktivnost -galaktozidaze (SA-β-Gal), određivana je korišćenjem citohemije. Diferencijacija ekspandiranih ćelija u smeru osteogeneze, hondrogeneze i adipogeneze izvedena je korišćenjem komercijalnih medijuma. Promene na ćelijama tokom osteogene diferencijacije su praćene preko aktivnosti alkalne fosfataze spektrofotometrijski, deponovanja kalcijuma u ekstracelularni matriks (bojenje alizarin crvenim) i pojave osteokalcina (imunocitohemija). Hondrogena diferencijacija u peletama je praćena određivanjem kolagena tip I (in situ hibridizacija), kolagena tip 2 (imunohistohemija) i određivanjem koncentracije glikozaminoglikana (spektrofotometrija). Adipogena diferencijacija je ispitana vizuelizacijom masnih kapljica (Oil red O bojenje). Aktivnost katalaze i superoksid-dismutaze (SOD) u ćelijskom lizatu je određena spektrofotometrijski. Koncentracija malondialdehida (MDA) je određena reakcijom sa tiobarbiturnom kiselinom (TBA). Karbonilni derivati proteina određivani su reakcijom sa 2,4 -dinitrofenilhidrazinom. Posle ekstrakcije ukupnih lipida i metilovanja masnih kiselina, metil-estri masnih kiselina su razdvajani gasno-tečnom hromatografijom (GLC), a koncentracija zasićenih (SFA), mononezasićenih (MUFA) i i polinezasićenih masnih kiselina (PUFA) je izraţena procentualno u odnosu na ukupne masne kiseline. Izoenzimski oblici laktat-dehidrogenaze (LDH1, LDH2, LDH3, LDH4 i LDH5) su određivani vertikalnom elektroforezom. Sva navedena merenja su izvedena bez i sa različitim koncentracijama NAC-a (0,1 mM, 1 mM i 2 mM). Naši rezultati su pokazali da je iz zubne pulpe mlečnih zuba dece dobijena populacija ćelija koja formira značajan broj CFU-F (4% izolovanih ćelija) i kontinuirano proliferiše tokom 40 dana, bez opadanja vremena potrebnog za udvajanje njihovog broja u kulturi. Ipak, dobijena populacija ćelija je bila heterogena po brzini deoba. Oko 12% ćelija je posle tri dana kultivacije bilo podeljeno manje od četiri puta, dok se ostatak ćelija podelio više od četiri puta. Vijabilitet ćelija je bio u proseku oko 95%, a svega 3% ćelija je bilo pozitivno na β-galaktozidazu. Tokom čitavog vremena kultivacije kapacitet za diferencijaciju u osteoblaste, adipocite i hondrocite se nije menjao. Gotovo sve ćelije su u pasaţi četiri eksprimirale CD44, CD73, kolagen tipa I, CD29, CD90 i osteonektin. Deo ćelija je eksprimirao STRO-1, CD146 i CD106. Marker matičnih ćelija hematopoeze nije detektovan u našem sistemu kultivacije. Opseg u kome se kretao procenat ćelija koje pokazuju povišenu aktivnost ALDH je iznosio od 4% do 24% i bio je u negativnoj korelaciji sa vremenom udvajanja ćelija u kulturi... | Dental pulp originates from the embryonic ectomesenchyme and represents potentially important source of mesenchymal stem cells (MSCs) with the ability to regenerate all tissues of the craniofacial region, originating from the ectomesenchyme, too. N-acetyl-L-cysteine (NAC) is an antioxidant that may have an impact on the therapeutic use of MSCs. The aim of this study was to determine whether the cells isolated from the dental pulp of children deciduous teeth of children and expanded in vitro, have the characteristics of MSC, to examine the effect of NAC on their proliferation and differentiation and to determine whether NAC influences the metabolism of glucose, the activity of antioxidant enzymes and whether it reduces oxidative damage of various cellular macromolecules. The initial cell population was obtained from six pulps of deciduous teeth using ex vivo tissue explants method. The number of colonies (Colony Forming Unit – Fibroblast; CFU-F) was determined by seeding the low density cell culture. Proliferative potential and population doubling time of the cells in culture were followed by counting the cells after tripsinization of subconfluent cultures, every 4 days, respectively, during 40 days of cultivation. Flow cytometry was used for cell immunophenotypisation, aldehyde dehydrogenase (ALDH) activity determination, number of cells in different phases of cell cycle, number of cell divisions and percentage of cells in apoptosis. Activity of β-galactosidase (SA-β-Gal), was determined using cytochemistry. Osteogenesis, chondrogenesis and adipogenesis were induced using complete commercial mediums. Osteogenic differentiation was monitored via alkaline phosphatase activity (spectrophotometry), deposition of calcium in the extracellular matrix (Alizarin red staining) and the appearance of osteocalcin (immunocytochemistry). Chondrogenic differentiation was followed by measuring collagen type I (in situ hybridization), collagen type 2 (immunohistochemistry) and the determination of the concentration of glycosaminoglycans (spectrophotometry). Adipogenic differentiation was examined by visualization of fat droplets (Oil Red O staining). Activity of catalase and superoxide dismutase (SOD) in cell lysates was determined spectrophotometrically. Malondialdehyde (MDA) was determined by reaction with thiobarbituric acid (TBA). Carbonyl derivatives of proteins were determined by reaction with 2,4-dinitrophenylhydrazine. After total lipid extraction and methylation of fatty acids, fatty acid methyl esters were separated by gas-liquid chromatography, and the concentration of saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids was expressed as percentage of total fatty acids detected in the sample. Isoenzyme forms of lactate dehydrogenase (LDH1, LDH2, LDH3, LDH4 and LDH5) were determined by vertical electrophoresis. All the above measurements were made with and without different concentrations of NAC (0,1 mM, 1 mM and 2 mM). Our results showed that the dental pulp of deciduous teeth of children had population of cells that formed a significant number of CFU-F (4% of the isolated cells) and continually proliferated for 40 days without decrease in the population doubling time. However, the resulting cell population was heterogeneous in terms of the division velocity. After three days of cultivation, around 12% of the cells were divided less than four times, while the remaining cells divided more than four times. Viability of cells was in average 95%, and only 3% of the cells were positive for β-galactosidase. During the entire time of the cultivation, expanded cells retained the ability to differentiate into osteoblasts, adipocytes and chondrocytes. Almost all cells expressed CD44, CD73, collagen type I, CD29, CD90 and osteonectin. Part of the cells expressed STRO-1, CD146 and CD106. Marker of hematopoietic stem cells have not been detected. Elevated ALDH activity ranged from 4% to 24% and was negatively correlated with the population doubling time... | false |
Analiza učestalosti i tipova mutacija KRAS i BRAF gena u karcinomima kolorektuma u populaciji Srbije | Frequency and mutation type analysis of KRAS and BRAF genes in colorectal carcinomas in Serbian population | Uvod: Kolorektalni kancer (CRC) se po svojoj učestalosti i smrtnosti nalazi u samom
vrhu svih kancera kod oba pola, kako u svetu, tako i u Srbiji. Za ranu kancerogenezu
ovog maligniteta karakteristične su somatske genske promene KRAS i BRAF
protoonkogena. Mutacije ovih gena predstavljaju molekularne biomarkere odgovora na
ciljanu terapiju, dok su rezultati o njihovoj ulozi kao prognostičkih i prediktivnih
faktora kontradiktorni. Podaci o učestalosti i tipu pojedinačnih KRAS i BRAF mutacija
kod CRC-a u našoj zemlji su malobrojni, a njihova veza sa kliničko-histopatološkim
karakteristikama tumora, karakteristikama bolesnika i tokom bolesti nije dovoljno
ispitana.
Cilj: Utvrđivanje prisustva, učestalosti i tipa KRAS mutacija i specifične BRAF p.V600E
mutacije kod bolesnika sa CRC-om u populaciji Srbije i povezanost prisustva i tipa ovih
mutacija sa kliničko-histopatološkim karakteristikama tumora i karakteristikama
bolesnika. Takođe, ispitivanje navedenih mutacija kao prediktivnih biomarkera
adjuvantne i EGFR-ciljane terapije.
Materijal i metode: U radu je analizirano 188 parafinskih uzoraka karcinoma
kolorektuma. DNK je izolovana komercijalnim kitom za izolaciju genomske DNK iz
parafinskih tkiva (QIAamp® DNA FFPE Tissue kit, QIAGEN). Detekcija prisustva i
tipa mutacija kodona 12 i 13 KRAS gena urađena je pomoću dva dijagnostički validirana
testa bazirana na eseju lančane reakcije polimeraze (PCR) u realnom vremenu (DxS
TheraScreen K-RAS PCR kit, QIAGEN) i PCR reakciji praćenoj reverznom
hibridizacijom (KRAS StripAssayTM, ViennaLab Diagnostics). Detekcija p.V600E
mutacije u BRAF genu urađena je analizom krive topljenja (HRM) PCR produkta i
metodom direktnog sekvenciranja. Za statističku obradu podataka korišćeni su Fišerov
egzaktni, χ2 i Log-Rank testovi.
Rezultati: Tačkaste mutacije u kodonima 12 i 13 KRAS gena su bile prisutne u 35,1%
analiziranih pacijenata sa CRC-om. Distribucija detektovanih mutacija je bila sledeća:
p.G12D 42,4%, p.G12V 21,2%, p.G12A 10,6%, p.G12C 7,6%, p.G12S 6,1%, p.G12R
1,5% i p.G13D 10,6 %. U grupi pacijenata sa wild-type (wt) KRAS statusom gena
analiziranih na prisustvo p.V600E BRAF mutacije (n=101), samo jedan pacijent je bio
heterozigot za datu mutaciju, dok su ostali bili wt.
Od testiranih kliničko-histolopatoloških osobina tumora i osobina bolesnika, KRAS
mutacije su se statistički značajno češće javljale kod muškaraca nego kod žena, kao i
kod pacijenata sa lošijim performans statusom u odnosu na one sa boljim. Analizirajući
povezanost pojedinih KRAS mutacija sa ispitivanim parametrima, zapazili smo
statistički višu zastupljenost p.G12V mutacije kod tumora koji ne prodiru u dublje
slojeve zida kolona i rektuma, u odnosu na tumore koji ih zahvataju ili probijaju serozu.
Iako nije pokazana statistička značajnost, učestalost KRAS mutacija raste sa porastom
veličine tumora, prisustvom i brojem metastaza, i oko dva puta je veća kod bolesnika sa
raširenom u odnosu na lokalizovanu bolest. Povezanost BRAF mutacije p.V600E sa
pomenutim parametrima nismo mogli da ispitamo jer je samo jedan bolesnik bio nosilac
ove mutacije.
U podgrupi od 49 bolesnika koji nisu imali inicijalne metastaze i koji su primili
adjuvantnu terapiju, nije postojala statistički značajna razlika u vremenu do pojave
metastaza u odnosu na KRAS mutacioni status. Ipak, može se zapaziti trend boljeg
preživljavanja bez metastaza kod pacijenata sa mutacijama KRAS-a (medijana 19,5
meseci) u odnosu na wt KRAS grupu (medijana 13,2 meseci).
U grupi pacijenata sa wt KRAS genom koji su primali kombinovanu EGFR-ciljanu
terapiju (n=72), 15,3% je postiglo terapijski odgovor.
Zaključak: Na osnovu dobijenih rezultata značajna povezanost mutacionog statusa
KRAS gena sa kliničko-histopatološkim karakteristikama CRC-a i karakteristikama
obolelih nije utvrđena. Zastupljenost i distribucija mutacija u ovom genu su u skladu sa
rezultatima drugih studija u svetu. Prediktivni značaj KRAS gena u odgovoru na
adjuvantnu terapiju nije pokazan, ali je zapažen trend boljeg preživljavanja bez
metastaza kod nosilaca KRAS mutacija. Zbog malog procenta BRAF p.V600E mutacije
u našoj grupi nismo mogli da proverimo njen značaj kao biomarkera ispitivanog
maligniteta. Određivanje i drugih, dodatnih genskih alteracija bi bilo od velike koristi za
preciznije definisanje genskog profila kolorektalnog tumora i individualizaciju
terapijskog pristupa. | Introduction: Colorectal cancer (CRC) incidence and mortality rates are among the
highest in both sexes worldwide, as well as in Serbia. The early carcinogenesis of this
malignancy is characterized by somatic gene alterations of KRAS and BRAF
protooncogenes. Mutations of these genes represent molecular biomarkers of response
to targeted therapy, while their roles as prognostic and predictive factors are still
contradictory. There are few data about the frequency and types of single KRAS and
BRAF
mutations
in
CRC
in
our
country,
and
their
correlation
with
clinicohistopathological characteristics of tumor, characteristics of patients and the
course of disease is still to be established.
Aim: Determination of presence, frequency and types of KRAS mutations and specific
BRAF p.V600E mutation in CRC patients in Serbian population and correlation of
presence and types of these mutations with tumor clinicohistopathological
characteristics and patient characteristics. Also, the examination of specified mutations
as potential predictive biomarkers of adjuvant and EGFR-targeted therapy.
Material and methods: In this study 188 CRC paraffin samples were analyzed. DNA
was extracted using the commercial kit for genomic DNA isolation from paraffin tissues
(QIAamp® DNA FFPE Tissue kit, QIAGEN). Detection of presence and types of KRAS
codon 12 and 13 mutations was performed with two validated diagnostic tests based on
real-time polymerase chain reaction (PCR) assay (DxS TheraScreen K-RAS PCR kit,
QIAGEN) and PCR reaction followed by reverse hybridization (KRAS StripAssayTM,
ViennaLab Diagnostics). BRAF p.V600E mutation was assessed by high resolution
melting (HRM) analysis of PCR products and automatic DNA sequencing. Fisher exact,
χ2 and Log-Rank tests were used for statistical analysis.
Results: KRAS point mutations in codons 12 and 13 were present in 35.1% of analyzed
CRC patients. The frequency distribution of detected mutations was as follows: p.G12D
42.4%, p.G12V 21.2%, p.G12A 10.6%, p.G12C 7.6%, p.G12S 6.1%, p.G12R 1.5% and
p.G13D 10.6 %. In the group of samples with wild-type (wt) KRAS gene status analized
for the presence of the BRAF p.V600E mutation (n=101), only one patient was
heterozygote for the specified mutation, while all the rest were wt.
Among tested clinicohistopathological characteristics of tumor and characteristics of
patients, KRAS mutations were significantly more common in males than in females, as
well as in patients with worse performance status than in those with better one.
Analyzing the association of single KRAS mutations with the investigated parameters,
we noticed that the frequency of p.G12V mutation was significantly higher in tumors
which do not infiltrate deeper the wall of colon and rectum, in comparison with those
that penetrate in deeper layers or go through serosa. Although the statistical significance
was not shown, the percentage of the KRAS mutations increases with the growth of
tumor size, the presence and number of metastases, and it is two times higher in patients
with the widespread disease in comparison with the localized one. The correlation of the
BRAF p.V600E mutation with the mentioned parameters was not performed because
only one patient carried this mutation.
In the subgroup of 49 patients with no initial metastases, who received adjuvant therapy,
there was no statistically significant difference in time to metastases appearance in
regard to the KRAS mutational status. However, the tendency of better survival without
metastases in patients with the KRAS mutations (median 19.5 months) in comparison
with those without them (median 13.2 months) was noticeable.
In the wt KRAS gene group of patients who were treated with combined EGFR-targeted
therapy (n=72), 15.3% achieved the therapeutic response.
Conclusion: According to the obtained results, no significant connection between the
mutational status of the KRAS gene and clinicohistopathological characteristics of CRC
and characteristics of patients was determined. KRAS mutation frequency and
distribution are in accordance with the results of other studies in the world. KRAS gene
predictive significance for the response to adjuvant therapy has not been shown,
although the trend of better survival without metastasis was observed in patients with
the KRAS mutations. Owing to the small percentage of the BRAF p.V600E mutation in
our group, we could not test its importance as a biomarker of the examined malignancy.
Determination of other, additional gene alterations would be of great importance for
more precised difining of CRC gene profile and for individual therapeutic approach. | true |
Transkripciona regulacija ekspresije humanog SOX18 gena | Transcriptional regulation of the human SOX18 gene expression | Humani SOX18 gen pripada familiji SOX gena koji kodiraju DNK-vezujuće
proteine koji imaju ulogu transkripcionih faktora i arhitektonskih komponenti hromatina.
SOX18 gen ima važnu ulogu u regulaciji vaskularnog razvića, i učestvuje u specifikaciji i
diferencijaciji endotelijalnih ćelija, angiogenezi i limfangiogenezi. Mutacije u SOX18 genu
kod čoveka su povezane sa sidromom Hipotrihoza-Limfedem-Talengiektazija (eng.
Hypotrichosis-Lymphedema-Talengiectasia) čije su karakteristike poremećaji u razviću
dlake, vaskularnog i limfnog sistema. Iako do danas ima dosta podataka o ulozi SOX18
gena u procesima vaskularogeneze, angiogeneze i limfangiogeneze, još uvek se malo zna o
molekularnim mehanizmima uključenim u regulaciju ekspresije ovog gena.
Osnovni ciljevi, istraživanja predstavljenog u ovoj tezi, bili su analiza
transkripcione regulacije ekspresije humanog SOX18 gena, kao i analiza uticaja pro-
angiogenetskih faktora i inhibitora angiogeneze na ekspresiju ovog gena u endotelijalnim
ćelijama. Ispitivanja transkripcione regulacije su obuhvatala analizu uloge određenih
transkripcionih faktora u regulaciji aktivnosti SOX18 promotora, kao i u regulaciji
endogene ekspresije SOX18 gena. Transkripciona regulacija je ispitivana u dva model
sistema: HeLa ćelijama, koje su korišćena kao tumorski model sistem i EA.hy926 ćelijama,
koje su korišćene kao endotelijalni model sistem. In silico analizom su identifikovana
potencijalna vezivna mesta za različite transkripcione faktore koji mogu biti uključeni u
regulaciju ekspresije SOX18 gena. Za dalju funkcionalnu analizu odabrani su transkripcioni
faktori Sp3, ZBP-89, NF-Y i EGR1. Na osnovu eksperimenata smanjene elektroforetske
pokretljivosti, funkcionalnih/mutacionih analiza, i analiza ekspresije u nativnom kontekstu,
pokazano je da su transkripcioni faktori Sp3 i ZBP-89 negativni, a NF-Y i EGR1 pozitivni
regulatori transkripcije humanog SOX18 gena. Na ovaj način pokazana je funkcionalna
veza između transkripcionih faktora Sp3, ZBP-89, NF-Y i EGR1 i SOX18 gena i
omogućeno je bolje razumevanje, dela, transkripcione kontrole ekpresije ovog gena.
Polazeći od predpostavke da su procesi vaskularogeneze i angiogeneze kontrolisani
velikim brojem različitih faktora, uključujući faktore rasta, citokine, adhezione molekule i
endogene ihibitore, deo istraživanja se odnosio na analizu uticaja odabranih pro- i anti-
angiogenetskih faktora na ekspresiju SOX18 proteina u endotelijalnom model sistemu.
Tretman endotelijalnih ćelija sa određenim faktorom, a potom analiza uticaja na nivou
SOX18 proteina, pokazao je da su vaskularni endotelijalni faktor rasta (eng. vascular
endothelial growth factor-VEGF ) i tumorski faktor nekroze (eng. tumor necrosis factor-
TNF) odgovorni za povećanje nivoa SOX18 proteina u endotelijalnim ćelijama. Na ovaj
način je pokazano da je SOX18 pod uticajem VEGF i TNF signalnih puteva, koji stimulišu
angiogenezu. Sa druge strane, nesteroidni antiinflamatorni lekovi (eng. nonsteroidal
antiinflamatory drugs-NSAID) dovode do izraženog smanjenja u nivou SOX18 proteina u
endotelijalnim ćelijama. Dobijeni rezultati otvarajući mogućnost za potencijalnu
farmakološku manipulaciju ekspresijom SOX18 gena. | Human SOX18 gene belongs to the family of SOX genes that encode DNA-binding
proteins, which display properties of both transcription factors and architectural
components of chromatin. SOX18 gene plays important role in vascular development,
endothelial cell specification and differentiation, angiogenesis and lymphangiogenesis.
Mutations in human SOX18 gene are associated with Hypotrichosis-Lymphedema-
Talengiectasia syndrome, characterized by defects in hair, vascular and lymphatic
development. Despite the mounting evidence that SOX18 gene is an important player in
vasculogenesis, angiogenesis and lymphangiogenesis, little is known about molecular
mechanisms involved in the regulation of its expression.
The aim of this study was to investigate transcriptional regulation of the human
SOX18 gene expression, as well as the effecs of pro- and anti-angiogenic factors on SOX18
expression in endothelial cells. Analyses of transcriptional regulation included
identification of transcription factors that are involved in regulation of SOX18 promoter
activity, as well as in regulation of endogenous SOX18 expression. Two model systems
were used: HeLa cells, as a tumor model system, and EA.hy926 cells, as an endothelial
model system. Several putative transcription factor binding sites were identified by in silico
analysis of the SOX18 promoter sequence. Transcription factors Sp3, ZBP-89, NF-Y and
EGR1 were selected for further functional analysis. By in vitro binding assays,
functional/mutagenesis assays and analyses of endogenous SOX18, it has been shown that
transcription factors Sp3 and ZBP-89 act as negative regulators, while NF-Y and
EGR1operate as positive regulators of SOX18 gene expression. These results gave first
functional link between Sp3, ZBP-89, NF-Y and EGR1 transcription factors and SOX18
gene, thus providing better understanding of transcriptional regulation of SOX18 gene
expression.
Considering that processes of vasculogenesis and angiogenesis are under control of
many various factors including growth factors, cytokines, adhesion molecules and
endogenous inhibitors , further investigation included analysis of the effect of selected pro-
and anti-angiogenic factors on SOX18 expression in endothelial model system. Treatment
of endothelial cells with selected factors, followed by Western blot analyses revealed that
vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF), increases the
level of SOX18 protein. By this it has been shown that SOX18 expression is under control
of VEGF and TNF signaling pathways. On the other hand, treatment with selected non-
steroidal anti-inflamatory drugs (NSAID), led to strong decrease in SOX18 protein level.
Taken together, these results open the possibility of pharmacological manipulation of
SOX18 gene expression. | true |
Korelacija prisustva bakteriocinskih gena i proizvodnje bakteriocina prirodnih izolata Lactobacillus casei/paracasei grupe | Correlation between the presence of bacteriocin genes and bacteriocin production in natural isolates of Lactobacillus casei/paracasei group | Veliki broj vrsta roda Lactobacillus zauzima ključno mesto u proizvodnji
fermentisanih mlečnih i mesnih proizvoda, prisutni su i u fermentisanom povrću i
proizvodima od žitarica. Laktobacili se takođe koriste i u medicini usled pozitivnih
efekata koje pojedini sojevi imaju na zdravlje domaćina. Pojedini sojevi laktobacila
imaju
i
probiotski
potencijal
zahvaljujući
sposobnosti
produkcije
različitih
antimikrobnih jedinjenja, egzopolisaharida (EPS), proteinaza i dr.
Vrste roda Lactobacillus poseduju potencijal da inhibiraju rast konkurentskih
mikroorganizama u ekološkim nišama koje naseljavaju, korišćenjem širokog spektra
različitih odbrambenih mehanizama uključujući proizvodnju velikog broja metaboličkih
nusproizvoda, kao i mnogih biološki aktivnih agenasa proteinske prirode kao što
bakteriocini. Sojevi koji proizvode više od jednog bakteriocina sa uskim, ali različitim
inhibitornim spektrom, imaju veće šanse za opstanak u okruženju koje dele sa blisko
srodnim vrstama sa kojima su u kompeticiji za iste nutrijente.
Veliki broj sojeva proizvođača bakteriocina objašnjava se činjenicom da su
bakteriocinske genetičke determinante često locirane na mobilnim genetičkim
elementima, kao što su konjugativni plazmidi ili transpozoni, što omogućava njihovu
široku distribuciju putem horizontalnog transfera gena.
Cilj ove doktorske teze bio je da se utvrdi antimikrobni potencijal 52 soja
Lactobacillus
casei/paracasei
grupe,
zatim
da
se
utvrdi
rasprostranjenost
bakteriocinskih gena neophodnih za produkciju bakteriocina BacSJ i acidocina 8912, a
potom i korelacija prisustva bakteriocinskih gena i proizvodnje bakteriocina kod
analiziranih sojeva. Ovakav pristup omogućio je identifikaciju potencijalnih
bakteriocinskih gena kao i novih bakteriocina nezavisno od toga da li je analizirani
bakterijski soj identifikovan kao proizvođač bakteriocina.
Sojevi Lb. casei/paracasei grupe analizirani u ovom radu izolovani su iz
fermentisanih mlečnih proizvoda (različite vrste sireva i kajmak) proizvedenih u
domaćinstvima, determinisani su putem mikrobiološko-biohemijskih testova i
korišćenjem metoda molekularne determinacije [PCR reakcije sa (GTG)5 prajmerom i
sekvenciranjem gena za 16S rRNK].
Detekcija antimikrobne aktivnosti sojeva Lb. casei/paracasei grupe urađena je
primenom bakteriocinskih testova sa senzitivnim sojevima laktobacila i laktokoka kao
indikatorima, kao i testovima unakrsne inhibicije između sojeva proizvođača
bakteriocina. Za utvrđivanje distribucije gena koji kodiraju bakteriocin BacSJ, acidocin
8912, ABC-transporter i pomoćni protein (Acc), korišćena je PCR metoda sa
specifičnim prajmerima za navedene gene.
Za restauraciju sinteze bakteriocina BacSJ u soju BGNK1-62, kod koga je
identifikovano
prisustvo
potencijalnog
bacSJ2-8/bacSJ2-8i operona, korišćen je
plazmid pA2A. Plazmid pA2A je dobijen kloniranjem PCR fragmenta koji nosi abcT i
acc
gene,
uključene
u
transport
bakteriocina
BacSJ,
u
pA13
laktobaciusni/laktokokalni/E. coli vektor.
Biohemijska karakterizacija dva nova bakteriocina BacUB9 koji sintetiše soj Lb.
paracasei subsp. paracasei BGUB9 i BacGR koji sintetiše soj Lb. paracasei subsp.
paracasei BGGR2-66, rađena je praćenjem uticaja proteolitičkih enzima, različitih pH
vrednosti i temperatura na njihovu aktivnost. Izolovanje bakteriocina BacUB9 iz
supernatanta prekonoćne kulture, je rađeno na dva načina: amonijum sulfatnom
precipitacijom i precipitacijom acetonom. Uzorak dobijen amonijum sulfatnom
precipitacijom je prečišćen reverzno-faznom hromatografijom sa nelinearnim
gradijentom elucije, a zatim je analiziran na Tricin SDS-poliakrilamidnom gelu. Iz
uzorka dobijenog acetonskom precipitacijom zatim je određivana molekulska masa
bakteriocina korišćenjem masene spektrometrije LC/MS. Izolovanje bakteriocina
BacGR, adsorbovanog na površini ćelija, rađeno je hloroformskom ekstrakcijom nakon
gajenja soja BGGR2-66 na čvrstom MRS medijumu, 48 sati. Uzorak dobijen
hloroformskom ekstrakcijom zatim je analiziran na dva načina. Reverzno-faznom
tečnom hromatografijom visokih performansi (HPLC) dobijen je uzorak iz koga je
određivana molekulska masa bakteriocina BacGR, kombinovanom tehnikom tečne
hromatografije i masene spektrometrije (ESI/MS). Uzorak dobijen hloroformskom
ekstrakcijom takođe je analiziran na diskontinuiranom Tricin SDS-poliakrilamidnom
gelu, nakon čega je prenesen na PVDF membranu i poslat na N-terminalno
sekvenciranje.
Analizom
antimikrobne
aktivnosti
52
soja
Lb.
casei/paracasei
grupe
identifikovano je 17 sojeva koji proizvode bakteriocine. Na osnovu sličnosti
inhibitornog spektra definisano je pet grupa sojeva (A-E). U grupi A su sojevi koji
imaju sličan antimikrobni spektar kao okarakterisani proizvođač bakteriocina soj
BGSJ2-8, a u grupi C sojevi sa sličnim spektrom kao okarakterisani proizvođač
bakteriocina soj BGBUK2-16.
Usled toga što upotreba klasičnih metoda za detekciju sojeva proizvođača
bakteriocina može da dovede do značajnog potcenjivanja potencijala za proizvodnju
bakteriocina testiranih sojeva, sproveden je PCR skrining rasprostranjenosti relevantnih
bakteriocinskih gena, koji je obuhvatio sva 52 soja Lb. casei/paracasei grupe, a ne samo
sojeve koji su pokazali sličan antimikrobni spektar kao soj BGSJ2-8. Pokazano je da su
genetičke determinante neophodne za proizvodnju bakteriocina BacSJ (bacSJ2-
8/bacSJ2-8i operon) i/ili acidocina 8912 (acdT gen) veoma rasprostranjene u okviru Lb.
casei/paracasei grupe sa učestalošću od čak 96,15%. Na osnovu toga da li proizvode
bakteriocine, odnosno da li poseduju ili ne poseduju analizirane bakteriocinske gene
sojevi Lb. casei/paracasei grupe su podeljeni na dve glavne grupe: sojeve proizvođače
bakteriocina (P1-P7) i sojeve kod kojih nije detektovana bakteriocinska aktivnost (N1-
N8). Kod svih sojeva proizvođača bakteriocina identifikovano je prisustvo oba ili bar
jednog strukturnog bakteriocinskog gena (bacSJ2-8/bacSJ2-8i operon i/ili acdT gen).
Zajednička karakteristika sojeva P3 grupe, koja ih razlikuje od sojeva P1 grupe, je
odsustvo abcT gena. Uži spektar delovanja tri od pet sojeva P3 grupe (BGGR2-64,
BGGR2-66 i BGBUK2-16), može se objasniti nemogućnošću da se kompenzuje
odsustvo abcT gena, kao i produkcijom potencijalno novog bakteriocina. Zajednička
karakteristika sojeva iz grupa P2 i P4-P7 je odsustvo acc gena, čiji se uzak spektar
delovanja može objasniti gubitkom acc gena što uslovljava njihovu slabu bakteriocinsku
aktivnost ili produkcijom novog bakteriocina za čiju aktivnost nije neophodno prisustvo
pomoćnog proteina.
Interesantno je da je u genomima 33 soja Lb. casei/paracasei grupe koji ne
proizvode bakteriocine identifikovano prisustvo jednog ili oba potencijalna strukturna
gena za bakteriocin BacSJ i acidocin 8912. Odsustvo proizvodnje bakteriocina, moguće
je objasniti prisustvom mutacija u genima ili regulatornim sekvencama gena koji
kodiraju komponente uključene u sintezu ili sekreciju bakteriocina. Takođe je moguće
da se strukturni bakteriocinski geni eksprimiraju, ali da gubitak abcT gena (N3 i N6)
kao i nedostatak acc gena (N2, N4, N5 i N7) uslovljavaju izostanak sekrecije
sintetisanog bakteriocina.
Restauracijom
bakteriocinskog
fenotipa
soja
BGNK1-62
heterolognom
ekspresijom abcT i acc gena ukazano je na važnost identifikacije potencijalnih
bakteriocinskih gena prilikom analize antimikrobnog potencijala bakterijskih sojeva.
Sprovedena studija je pokazala da sojevi BGUB9 i BGGR2-66 poseduju
genetički potencijal za produkciju tri bakteriocina, ali da je njihova bakteriocinska
aktivnost
najverovatnije
posledica
delovanja
novog
do
sada
neokarakterisanog
bakteriocina BacUB9 odnosno bakteriocina BacGR. Korišćenjem DNK-DNK
hibridizacije pokazano je da su kod sojeva BGUB9 i BGGR2-66 potencijalni geni
uključeni u proizvodnju bakteriocina BacSJ i acidocina 8912 plazmidno lokalizovani,
takođe čišćenjem plazmida iz ovih sojeva dobijeni su Bac- derivati, što je ukazalo da se i
genetičke determinante neophodne za proizvodnju bakteriocina BacUB9 i BacGR
nalaze na plazmidima.
Bakteriocin BacUB9 je mali (3758 Da) umereno termostabilan molekul, aktivan
u širokom opsegu pH vrednosti, sa bakteriostatičkim načinom delovanja, osetljiv na
delovanje proteolitičkih enzima, koji pored blisko srodnih vrsta inhibira i rast soja
Listeria innocua ATCC 33090T, na osnovu čega je svrstan u klasu IIa bakteriocina.
Bakteriocin BacGR je takođe mali (3218 Da) umereno termostabilan molekul,
aktivan u širokom opsegu pH vrednosti, osetljiv na delovanje proteolitičkih enzima, ali
uskog spektra delovanja ograničenog na blisko srodne vrste. Na osnovu molekulske
mase, biohemijske analize i N-terminalne sekvence bakteriocin BacGR pripada
bakteriocinima klase IId. | A large number of Lactobacillus species play an important role in the production
of fermented dairy products and meat products, and they are also present in fermented
vegetables and grain products. Lactobacilli are used in medicine due to the positive
effects that some strains have on the health of the host. Some Lactobacillus strains have
a probiotic potential due to their ability to produce various antimicrobial compounds,
exopolysaccharides (EPS), proteinases and other.
Species of the Lactobacillus genus have the potential to inhibit the growth of
competing microorganisms in the ecological niches they inhabit, using a wide range of
various defense mechanisms including the production of a large number of secondary
metabolic products, as well as many biologically active proteinaceous agents, such as
bacteriocins. Strains that produce more than one bacteriocin with a narrow but different
inhibitory spectrum have a better chance to survive in an environment shared with
closely related species with which they are in competition for the same nutrients.
A presence of huge number of bacteriocin producing strains could be explained
by the fact that the bacteriocin genes were often located on mobile genetic elements
such as conjugative plasmids or transposons, allowing their wide distribution via
horizontal gene transfer.
The aim of this doctoral dissertation was to determine the antimicrobial potential
of 52 strains of Lactobacillus casei/paracasei group, followed by a comparative
screening of the bacterial genomes in order to determine the distribution of BacSJ and
acidocin 8912 encoding genes, and also to determine the correlation of presence of
bacteriocin genes and bacteriocin production in the analyzed strains. This approach
enabled identification of potentially new bacteriocins and putative bacteriocin genes
regardless of whether the analyzed bacterial strains produced bacteriocin.
Strains of Lb. casei/paracasei group analyzed in this study, isolates from
fermented dairy products (different types of cheese and kajmak) produced in
households, were determined through microbiological and biochemical tests and using
molecular methods [PCR reactions with (GTG)5 primer and sequencing of 16S rRNA
gene].
Detection of antimicrobial activity of strains from Lb. casei/paracasei group was
performed by bacteriocin activity assay using sensitive Lactobacillus and Lactococcus
indicator strains and cross-inhibition tests among bacteriocin producer strains. To
determine the distribution of genes encoding bacteriocin BacSJ, acidocin 8912, ABC-
transporter and accessory protein (Acc), PCR analysis with specific primers for those
genes was used.
For the re-establishment of the ability to produce BacSJ in BGNK1-62 strain, in
which the presence of a potential operon bacSJ2-8/bacSJ2-8i was identified, plasmid
pA2A was used. Plasmid pA2A was obtained by cloning of a PCR fragment carrying
abcT
and
acc
genes,
involved
in
transport
of
bacteriocin
BacSJ,
in
lactobacilli/lactococci/E. coli shuttle cloning vector pA13.
Biochemical characterization of two new bacteriocins BacUB9 produced by
strain Lb. paracasei subsp. paracasei BGUB9 and BacGR produced by strain Lb.
paracasei subsp. paracasei BGGR2-66, was done by following the effects of
proteolytic enzymes, different pH values and temperature on their activity. Isolation of
bacteriocin BacUB9 from supernatant of the overnight culture was done in two ways:
ammonium sulphate and acetone precipitation. The sample obtained by ammonium
sulphate precipitation was purified by reversed-phase chromatography with a "step"
gradient elution, and then analyzed on Tricin SDS-polyacrylamide gel. Sample obtained
from acetone precipitation was used for determination of bacteriocin molecular mass by
using mass spectrometry LC/MS. Isolation of bacteriocin BacGR, adsorbed on the
surface of cells, was done with chloroform extraction after cultivation of BGGR2-66
strain on solid MRS medium for 48 hours. The sample obtained by chloroform
extraction was then analyzed in two ways. The sample obtained from reverse-phase
high performance liquid chromatography (HPLC) was used to determine the molecular
weight of bacteriocin BacGR, by using mass spectrometry (ESI/MS). Also, a sample
obtained from chloroform extraction was analyzed on discontinuous Tricin SDS-
polyacrylamide gel, after which the sample was transferred to PVDF membrane and
sent to N-terminal amino acid sequencing.
The analysis of antimicrobial activity showed that 17 of 52 tested Lactobacillus
casei/paracasei strains produced bacteriocins. Based on the similarity of the inhibitory
spectrum, five groups of strains we defined (A-E). Group A includes strains that have a
similar antimicrobial spectrum as previously characterized bacteriocin producer strain
BGSJ2-8, and group C includes strains that have a similar spectrum as characterized
bacteriocin producer strain BGBUK2-16.
Because the use of classical methods for detection of bacteriocin producers may
underestimate the bacteriocinogenic potential of tested strains, all 52 strains of Lb.
casei/paracasei group, not only the strains with a similar antimicrobial spectrum as
strain BGSJ2-8, were screened by PCR amplification for presence of relevant
bacteriocin genes. It was shown that the genetic determinants required for the
production of bacteriocins BacSJ (bacSJ2-8/bacSJ2-8i operon) and/or acidocin 8912
(acdT gene) were widespread within the Lb. casei/paracasei group with a frequency of
96.15%. On the basis of whether they produce bacteriocins, and whether they have or
do not possess the analyzed bacteriocin genes, strains of Lb. casei/paracasei group were
divided into two main groups: bacteriocin producer strains (P1-P7) and strains in which
the bacteriocin activity was not detected (N1-N8). In all bacteriocin producers the
presence of both or at least one structural bacteriocin gene (bacSJ2-8/bacSJ2-8i operon
and/or acdT gene) was identified. A common feature of strains from P3 group, which
distinguishes them from strains from P1 group, is the absence of abcT gene. The narrow
spectrum of activity of three of the five strains of P3 group (BGGR2-64, BGGR2-66
and BGBUK2-16) can be explained by the inability to compensate for the absence of
abcT gene, as well as with production of potentially new bacteriocin. The common
characteristic of strains belonging to groups P2 and P4-P7 is a lack of acc gene, which
narrow spectrum of activity can be explained by the loss of acc gene, which causes their
poor bacteriocin activity, or by production of new bacteriocin, which activity does not
require the presence of accessory protein.
Interestingly, in the genomes of 33 strains of Lb. casei/paracasei group, that do
not produce bacteriocins, the presence of one or both putative structural genes for
production of bacteriocin BacSJ and acidocin 8912 were identified. Absence of ability
to produce bacteriocins can be explained by the presence of mutations in the genes or
regulatory sequences of genes encoding components involved in the synthesis and
secretion of bacteriocins. It is also possible that the structural bacteriocin genes were
expressed, but that the loss abcT gene (N3 and N6) and the lack of acc gene (N2, N4,
N5 and N7) caused the absence of secretion of the synthesized bacteriocin.
Re-establishment of the ability of strain BGNK1-62 to produce bacteriocin by in
trans expression of heterologous abcT and acc genes illustrated the importance of
identifying putative bacteriocin genes in the analysis of antimicrobial potential of
bacterial strains of interest.
This study showed that both strains BGUB9 and BGGR2-66 possess genetic
potential to produce three bacteriocins, but that their antimicrobial activities probably
were result of production of new, until now not characterized bacteriocins BacUB9 and
BacGR. DNA-DNA hybridization showed plasmid localization of putative genes
involved in production of bacteriocin BacSJ and acidocin 8912 in genome of the
BGUB9 and BGGR2-66 strains, and also Bac-derivatives of these strains, obtained after
plasmid curing experiments, indicated that the genetic determinants necessary for
production of bacteriocins BacUB9 and BacGR were plasmid-encoded.
BacUB9 is a relatively heat-stable, low-molecular-weight peptide (3758 Da),
active within a broad pH range, with a bacteriostatic mode of action, sensitive to
proteolytic enzymes, which beside closely related species also inhibits the growth of
Listeria innocua ATCC 33090T strain. Based on these characteristics it appears that
BacUB9 belongs to the class IIa bacteriocins.
Bacteriocin BacGR is also small (3218 Da) and relatively heat-stable peptide,
active in a wide range of pH values, inactivated by proteolytic enzymes, but with a
narrow spectrum of activity limited to closely related species. Based on the molecular
size, biochemical characterization and obtained N-terminal sequence, bacteriocin
BacGR probably belongs to the class IId bacteriocins. | true |
Modelovanje ekoloških niša Podarcis sicula i P. melisellensis (Sauria, Lacertidae) u eumediteranu i submediteranu istočnog Jadrana | Ecological niche modelling of Podarcis sicula and P. melisellensis (Sauria, Lacertidae) in the eumediterranean and submediterranean of the eeastern Adriatic sea | Rad obuhvata modelovanje ekoloških niša dve vrste guštera Podarcis sicula i
P. melisellensis na dva područja kopnene oblasti istočnog Jadrana povezane sa analizom
rizika izumiranja metapoulacionih demografskih modela formiranih na osnovu prostornih
niša. Obe vrste su su od opisivanja u XIX veku bile epizodično predmet zoogeografskih,
ekoloških i evolucionih istraživanja u Jadranskoj oblasti. Brojni dosad prikupljeni bionomski
i ekološki podaci o obe vrste na istraživanom području, međutim, uglavnom nisu stavljeni u
celovit kvantitativni koncept, velikim delom ostajući na nivou manje ili više detaljnog
kvalitativnog opisa. Postojeći kvantitativni podaci, prikazani su kao “lateralni” ili
“akcesorni” rezultati istraživanja čiji je osnovni cilj bio sasvim drugačiji te je njihova
upotrebna vrednost u potpunosti podređena kontekstu realizovanih istraživanja. Posebno se to
odnosi na autekološka i populaciono-ekološka istraživanja. Ostrvske populacije su znatno
bolje istražene od kopnenih. Realizujući kvantitativno modeliranje niša kopnenih populacija,
pokušali smo da makar malo taj problem ublažimo.
U modelovanju ekoloških niša korišćeno je 47 kvantitativnih i 9 kvalitativnih EGV, u
zavisnosti od metode, koje su formirane na osnovu globalnih baza podataka. Obuhvaćena
područja, oko 100 km2 po površini, nalazila su se u Poljicama (okolina Splita, Hrvatska) i
šireg regiona Boke Kotorske (Crna Gora) na kojoj su detaljno mapirane obe vrste. Korišćene
su tri metode sa prisustvom vrsta (P/O, ENFA, MaxEnt i GARP), četiri koje zahtevaju
prisustva/odsustva (P/A, SpFA, PLS, GRM, DA), i tri koje mogu koristiti oba tipa podataka
(P/O ili P/A, GAM, MARS, BRT). Na osnovu profila stanišne povoljnosti formirane su
detaljne karte prostornih niša. Sve metode su identifikovale centre povoljnostog staništa istog
ili sličnog položaja i obuhvata. P/A metode su identifikovale mnogo šire zone suboptimalnih
staništa za obe vrste na oba područja. EGV korišćene za analizu su ocenjene po čestoći
odabira u modelima i minimalni skup promenljivih koji daju pouzdane predikcije je: ALT,
*OVER1, *CONVEX1, SLOPE10, BIO1, *4, *5, *7, *8, *9, *10, *11, *12, *19, WB, PET, TOWNSFQ1, *TOPODST,
*BLENGTH, AGRIFQ1, *TOPODST, *BLENGTH, OPENFQ1, *TOPODST, *BLENGTH, SPARSEFQ1, *TOPODST,
*BLENGTH. Svi modeli niša su bili statistički značajni, a po uspešnosti predikciji ističu se
MARS, PLS, GRM. Za područje Hrvatske, sve metode imaju lošiju uspešnost nego za
područje Crne Gore. Modeli niša bili su uspešniji za Podarcis sicula na oba područja. Modeli
ekoloških niša nisu transportabilni po područjima, zbog suviše velikih razlika u intenzitetu
delovanja i prostornom rasporedu ekoloških faktora.
Utvrđene su demografske karakteristike obe vrste: izrazito konkavna preživljavanja je
izrazito konkavna, prosečna očekivane dužine života se kreću od 3-4 godina u predadultnom;
uzrasno-specifični fekunditeti rastu tokom prve trećine života, stabilizuju se na oko 16-20 jaja
po ženki i potom lagano opadaju do kraja životnog ciklusa; prosečno vreme generacije iznosi
4-5 godina sa godišnjim stopama rasta 6-13 %. PVA pokazuje da vrednosti kapaciteta
500<K<1000 za obe vrste daju rizik izumiranja manji od 10 % za demografski i sredinski stohasticitet
do 20%.
Na osnovu ENFA metode, identifikovano je 7 dema za P. melisellensis prosečne
stanišne povoljnosti od 75% i 11 za P. sicula u Hrvatskoj prosečne stanišne povoljnosti od
74-84%, odnosno 3 za P. melisellensis (HS=70%) i dve za P. sicula u Crnoj Gori (HS=72%).
PVA analiza metapopulacija, u scenariju uticaja HS samo na kapacitet sredine, pokazala su
da na području Hrvatske u vremenskom horizontu od 100 godina ne postoji opšti rizik
izumiranja istraživanih vrsta, čak i u uslovima visokog sredinskog stohasticiteta. Minimalne
vijabilne populacije brojnije od 1000 jedinki a realizovani kapaciteti veći od 3000 jedinki (u
najgorem slučaju 50-60 % kapaciteta). Specifični parametri rizika pokazuju, na visokom
nivou sredinske varijabilnosti, osetljivost obe populacije na variranje kapaciteta, sa rizicima
da ukupne populacije padnu ispod 1000-2000 jedinki i sa verovatnoćom većom od 10 % da
lokalne populacije padnu ispod brojnosti od 100-200 jedinki. Kraški gušter je osetljiviji na ta
variranja. Na području Crne Gore, po osnovnom scenariju nema rizika izumiranja a MVP su
iznad 300 jedinki a dostignute brojnosti su na više od 50 % kapaciteta. U uslovima 30%
stohasticiteta, kod obe vrste je uočen opšti rizik izumiranja, nizak i prihvatljiv za P.
melisellensis, a na granici prihvatljivosti od 5 % za P. sicula, sa relativno niskim MVP i
dostignutim kapacitetima na granici ili ispod 50 %. Obe vrste su na području Crne Gore, pre
svega zbog nižih procena kapaciteta sredine, osetljive na visoko variranje sredinskih faktora.
Očekivano, P. sicula je osetljiviji na izumiranje zbog malog prostornog obuhvata i
izolovanosti Kotorske populacije. Kada povoljnost staništa utiče i na demografske
karakteristike, pokazuje se da obe vrste, na oba područja, nemaju demografski kapacitet za
preživljavanje. | Ecological niche models were generated for two lizard species, Podarcis sicula i
P. melisellensis, in two mainland areas of the eastern Adriatic, and coupled with spatially-
explicit PVA based on habitat suitabilty profiles generated by ENM. Both species have, since
their description in the XIX century, been episodically studied from zoogeographical,
ecological, and evolutionary aspects. However, a wealth of bionomic data collected by
investigators was qualitative, lacking a comprehensive quantitative framewok, and as such
are of limited value. The available bionomic and ecological data was largely presented as
"lateral" or "accessory" data collected for a different scientific objective, and their utility is
largely determined by the context of that research. Specifically, that problem pertains to the
utility of reported autecological and population-ecological data. Mainland populations of both
species were also much less studied than the island populations in the Adriatic. We addressed
this problem by conducting quantitative niche-modelling research based on the mainland.
For ecological niche modelling we used 47 quantitative and 9 qualitative EGV's,
depending on the method, which were generated from global datasets. Both regions were cc.
100 km2 in area, and were located in Poljice (Split, Croatia) and the general region of Boka
Kotorska (Montenegro) for we had which detailed distribution data. Three methods requiring
presence/only data were used (P/O, ENFA, MaxEnt i GARP), four requiring
presence/absence (P/A, SpFA, PLS, GRM, DA), and three which could use both (P/O or P/A,
GAM, MARS, BRT). Habitat suitability profiles generated by these methods were used to
map the spatial niches. All methods identified identical or very similar core areas of HS. P/A
methods were much more liberal in assigning adjacent suboptimal HS zones for both species
in both areas. EGV's selected by the methods were scored by frequency of selection in
models, and a minimal subset generating reliable predictions was identified: ALT, *OVER1,
*CONVEX1, SLOPE10, BIO1, *4, *5, *7, *8, *9, *10, *11, *12, *19, WB, PET, TOWNSFQ1, *TOPODST, *BLENGTH,
AGRIFQ1, *TOPODST, *BLENGTH, OPENFQ1, *TOPODST, *BLENGTH, SPARSEFQ1, *TOPODST, *BLENGTH.
All niche models were satistically significant, with MARS, PLS, GRM generally preforming
better than the rest. For Croatia, all methods had lower performance than for Montenegro.
Niche models were more reliable for Podarcis sicula in both areas. Large differences in
context due to differences in intensity and spatial distibution of EGV's precluded model
transportability over areas.
A general demographic model for both species was constructed: a very concave
survivorship curve, 3-4 years life expectancy, continous fecundity at 16-20 eggs per female in
over a third of their life span, generation time 4-5 years, and yearly growth rates of 6-13 %.
PVA analysis showed that carrying capacity values of 500<K<1000 for both species give a less
than 10% extincinction risk for environmental and demographic stochasticity levels of up to 20%.
The habitat maps generated by ENFA identified 7 local populations of P.
melisellensis with average HS of 75% and 11 of P. sicula in Croatia with average HS
between 74% and 84%. In Montenegro, 3 local populations were identified for P.
melisellensis (HS=70%) and two for P. sicula (HS=72%). A spatially explicit PVA of local
populations connected by dispersal with HS affecting K and demographic stochasticity,
showed that, for Croatia there is no general extinction risk in a 100 year time-frame, even
with high levels of environmental stochasticity. Minimum viable population sizes were larger
than 1000 individuals and effective total K's for each region were larger than 3000 (worst
case: 50-60 % K). Specific extinction risk estimates, only with 30%, showed that both species
are susceptible to decline, with effective K's of 1000-2000 and a >10 % chance that local
populations fall below 100-200 individuals. P. melisellensis is more susceptible to such risks.
For Montenegro, MVP's are > 300 individuals, with efective K's > 50%. With high levels of
environmental stochasticity (30%), both species show a rise in general extinction risk,
ER<5% for P. melisellensis, around 5 % for P. sicula, with low MVP's and effective K's
around 50 %. Lower estimates of overall K for Montenegro are responsible for such trends.
As expected, P. sicula is more susceptible to local extinction due due small its small
distribution and isolation of the Kotor population. Neither species has the demographic
capacity for survival if HS is linked to demographic properties as well as to K. | true |
Kateholamini kao medijatori delovanja muških polnih hormona na timopoezu : | Catecholamines as mediators of male gonadal hormone action on thymopoiesis | Danas je široko prihvaćeno mišljenje da je progresivno smanjenje efikasnosti
timopoeze, koje počinje u pubertetu, kauzalno povezano sa porastom koncentracije
polnih hormona. Pored toga, i koncentracija kateholamina, toničnih inhibitora
timopoeze posredstvom β-adrenergičih receptora, se u timusu povećava, počevši od
pubertetnog perioda. Poznato je da zrele ćelije imunskog sistema (T-limfociti,
makrofage i dendritske ćelije), i različiti tipovi epitelnih ćelija, imaju sposobnost sinteze
kateholamina. Konačno, u različitim tkivima, muški polni hormoni utiču na sintezu
kateholamina i ispoljavanje β-adrenergičkih receptora. Ova saznanja su nužno
nametnula pitanje da li timociti i/ili ćelije strome timusa poseduju sposobnost sinteze
kateholamina, i, ukoliko je odgovor potvrdan, da li i u kojim tipovima ćelija timusa
hormoni testisa utiču na sintezu kateholamina i/ili ispoljavanje β2-adrenergičkog
receptora i time, moguće, na modulatorno delovanje kateholamina na proces timopoeze.
Imajući u vidu sve prethodno navedene nalaze, postavljeni su ciljevi ove
disertacije: 1) da se utvrdi da li timociti i/ili ćelije strome timusa odraslih mužjaka
pacova sintetišu kateholamine, i da se precizno definišu tipovi ćelija u ovom organu koji
ispoljavaju β2-adrenergički receptor, 2) da se ispita da li orhidektomija u uzrastu od 30
dana, kada je timus pacova najosetljiviji na efekte kastracije, utiče na koncentraciju
kateholamina i ispoljavanje β2-adrenergičkog receptora u timusu odraslih pacova, i, ako
je odgovor potvrdan, 3) da se utvrdi da li se modulatorno delovanje kateholamina na
timopoezu razlikuje kod orhidektomisanih i neorhidektomisanih životinja.
Nađeno je da timociti, timusne epitelne ćelije i makrofage sadrže i sintetišu
kateholamine. Osim toga, subpopulacije ovih tipova ćelija, takođe, ispoljavaju β2-
adrenergički receptor.
Orhidektomija u uzrastu od 30 dana je smanjila koncentraciju noradrenalina u
timusu odraslih životinja, usled smanjenja gustine noradrenergičkih nervnih vlakana i
ćelija strome timusa koje sadrže kateholamine i sadržaja noradrenalina u njima. Pored
toga, u timusu ovih životinja je detektovano i smanjenje ekspresije iRNK za β2-
adrenergički receptor, kao i površinske gustine ovog receptora na membrani timocita.
Orhidektomija je dovela do povećanja ukupnog broja timocita, kao i efikasnosti
diferencijacije/sazrevanja timocita, a posebno nastanka CD4+ T-ćelija, sudeći prema
povećanju procentualne zastupljenosti najzrelijih CD4+CD8- timocita, sa visokom
površinskom gustinom TCRαβ (TCRαβ++), i svežih timusnih emigranata u okviru
populacije CD4+ T-ćelija periferne krvi. Opisane promene su, najverovatnije,
odražavale intenziviran ulazak prekursora timocita, efikasnije odvijanje njihovog
početnog razvoja, efikasniju β-selekciju timocita, kao i smanjenu efikasnost negativne
selekcije (najverovatnije usled veće gustine CD90 molekula na membrani timocita).
Takođe, u timusu odraslih orhidektomisanih životinja, došlo je i do promena u
sazrevanju regulatornih ćelija, sudeći prema smanjenju procentualne zastupljenosti
CD4+CD25+Foxp3+
timocita
i
povećanju
procentualne
zastupljenosti
CD8+CD161+TCRαβ+ ćelija.
Da bi se procenio doprinos mogućih promena u delovanju kateholamina
posredstvom β-adrenergičkih receptora povećanoj efikasnosti timopoeze kod
orhidektomisanih
životinja,
ispitivani
su
efekti
četrnaestodnevnog
tretmana
antagonistom β-adrenergičkih receptora, propranololom, na diferencijaciju/sazrevanje
timocita, kod odraslih neorhidektomisanih i orhidektomisanih pacova. Kod odraslih
neorhidektomisanih
pacova,
propranolol
je
povećao
efikasnost
diferencijacije/sazrevanja timocita, povećavajući efiksanost ranih faza razvoja CD4-
CD8-TCRαβ- timocita i, najverovatnije, efikasnost β-selekcije, a smanjujući efikasnost
negativne selekcije (najverovatnije usled veće gustine CD90 molekula na membrani
timocita). Ovakve promene u timopoezi konačno su dovele do povećanja procentualne
zastupljenosti najzrelijih CD4+CD8-TCRαβ++ i, posebno, CD4-CD8+TCRαβ++
timocita, i odgovarajućeg povećanja procentualne zastupljenosti svežih timusnih
emigranata u okviru populacija CD4+ i CD8+ T-ćelija periferne krvi. Uz sve navedeno,
davanje
propranolola
neorhidektomisanim
pacovima
je
povećalo
stvaranje
CD4+CD25+Foxp3+ i CD8+CD161+TCRαβ+ regulatornih ćelija u timusu, sudeći
prema povećanju njihove procentualne zastupljenosti u ovom organu. Prethodno
navedeni nalazi su ukazali na postojanje toničnog inhibitornog delovanja kateholamina,
putem
β-adrenergičkih
receptora,
na
proces
timopoeze.
Za
razliku
od
neorhidektomisanih
životinja,
tretman
propranololom
kod
odraslih
životinja
orhidektomisanih 30. dana života, nije se pokazao efikasnim u modulaciji timopoeze.
Imajući u vidu da: 1) u timusu, orhidektomija u uzrastu od 30 dana smanjuje
koncentraciju
noradrenalina
(delujući
na
sintezu
i
sadržaj
ovog
hormona/neurotransmitera u nervnim vlaknima i ćelijama timusa koje sadrže
kateholamine), kao i ekspresiju gena za β2-adrenergički receptor i time, moguće,
efikasnost delovanja kateholamina, i 2) orhidektomija i tretman propranololom kod
neorhidektomisanih pacova dovode do sličnih promena, bar u ispoljavanju CD90
molekula na membrani timocita (koje je regulisano delovanjem noradrenalina
posredstvom β-adrenergičkih receptora) i posledično u negativnoj selekciji ovih ćelija,
može se pretpostaviti da je povećanje efikasnosti timopoeze koje je uočeno kod odraslih
pacova koji su orhidektomisani u uzrastu od 30 dana, jednim delom, odražavalo manju
efikasnost delovanja kateholamina na timopoezu putem β-adrenergičkih receptora. | It is widely accepted that the progressive decline in the efficiency of
thymopoiesis, starting at puberty, is causally related to increasing concentrations of
gonadal hormones. In addition, thymic level of catecholamines, which act, via β-
adrenoceptors, as tonic inhibitors of thymopoiesis, increases around puberty. There is a
pile of evidence that mature immune cells (T-lymphocytes, macrophages and dendritic
cells) as well as different types of epithelial cells, synthesize catecholamines.
Furthermore, it has been shown that in various tissues male gonadal hormones affect
catecholamine synthesis and β-adrenoceptor expression. These findings imposed the
question whether thymocytes and/or thymic stromal cells synthesize catecholamines,
and, if so, whether testicular hormones influence the synthesis of catecholamines and/or
expression of β2-adrenoceptors, and possibly, consequently, the modulatory action of
catecholamines on thymopoiesis.
Considering all the aforementioned, the dissertation was aimed to: 1) explore
whether thymocytes and/or thymic stromal cells in adult male rats synthesize
catecholamines, and to define the types of thymic cells expressing β2-adrenoceptor; 2)
examine whether orchidectomy at the age of 30 days, when rat thymus is the most
sensitive to testicular hormone deprivation, affects catecholamine levels and β2-
adrenoceptor expression in the thymus of adult rats, and, if so, 3) determine whether the
modulatory effects of catecholamines on thymopoiesis differ in orchidectomized and
non-orchidectomized animals.
It has been found that thymocytes, thymic epithelial cells and macrophages in
adult rats contain and synthesize catecholamines. In addition, it was shown that subsets
of these cells also express the β2-adrenoceptors.
Orchidectomy at the age of 30 days decreased the concentration of noradrenaline
in the thymus of adult animals by diminishing the density of noradrenergic nerve fibers
and catecholamine-containing thymic stromal cells and their noradrenaline content. In
addition, in thymi of these animals the decreased β2-adrenoceptor mRNA expression
and thymocyte β2-adrenoceptor surface density were found. Moreover, orchidectomy
increased the overall number of thymocytes and efficiency of thymopoiesis, in
particular generation of CD4+ T cells, judging by the increased proportion of the most
mature CD4+CD8- thymocytes, expressing TCRαβ at high level (TCRαβ++) and recent
thymic emigrants among CD4+ T cells in peripheral blood. This most likely reflected
enhanced entry of thymocyte precursors, more efficient stages of their initial
differentiation, enhanced thymocyte β-selection, and diminished efficiency of negative
selection (most likely due to increased CD90 surface density). Moreover, in thymi of
adult orchidectomized animals, differentiation/maturation of regulatory cells was also
altered, judging by the reduced and increased percentage of CD4+CD25+Foxp3+ and
CD8+CD161+TCRαβ+ cells, respectively.
Next, to assess the contribution of putative changes in β-adrenoceptor–mediated
catecholamine action to the augmented thymopoietic efficiency in orchidectomized rats,
the effects of 14-day-long treatment with propranolol, a β-adrenoceptor antagonist, on
thymocyte differentiation/maturation in non-orchidectomized and orchidectomized adult
rats were examined. Propranolol, in non-orchidectomized adult rats, increased
efficiency of thymocyte differentiation/maturation, by enhancing efficiency of the early
steps of CD4-CD8-TCRαβ- thymocyte development and, most likely, thymocyte β-
selection and diminishing efficiency of negative selection (most likely due to increased
CD90 surface density). These changes ultimately led to the increased proportion of the
most mature CD4+CD8-TCRαβ++ and, in particular, CD4-CD8+TCRαβ++ thymocytes
in thymus, and, consequently, recent thymic emigrants within both CD4+ and CD8+
peripheral blood T-cell populations. In addition, propranolol treatment in non-
orchidectomized rats augmented thymic generation of both CD4+CD25+Foxp3+ and
CD8+CD161+TCRαβ+ regulatory cells, judging by their increased proportions in the
thymus. These findings suggested a tonic β-adrenoceptor–mediated inhibitory action of
catecholamines on thymopoiesis. However, differently from non-orchidectomized rats,
in orchidectomized rats propranolol treatment was inefficient in modulating
thymopoiesis.
Considering that: 1) orchidectomy at the age of 30 days diminished thymic
noradrenaline concentration (through affecting its synthesis and content in nerve fibers
and catecholamine-containing thymic cells), and thymic β2-adrenoceptor gene
expression, and thereby, possibly, efficiency of catecholamine action, and 2)
orchidectomy and propranolol treatment, in non-orchidectomized rats, led to similar
changes, at least, in thymocyte CD90 expression (that is shown to be regulated by β-
adrenoceptor–mediated noradrenaline action) and consequently thymocyte negative
selection, it may be assumed that the increase in thymopoietic efficiency in adult rats,
orchidectomized at the age of 30 days, partly reflected less efficient β-adrenoceptor–
mediated influence of catecholamines on thymopoiesis. | true |
Molekularna osnova hronične limfocitne leukemije : korelacija između mutacionog statusa teških lanaca imunoglobulina i ekspresije apoptotskih gena | Molecular basis of chronic lymphocytic leukemia: correlation between the immunoglobulin heavy chain mutational status and the expression of apoptotic genes | Hronična limfocitna leukemija (HLL), najčešći tip leukemije u Evropi i Severnoj Americi, se
manifestuje kao klonska ekspanzija zrelih CD5+ CD19+ CD23+ sIgM+/- B limfocita i karakteriše
se izuzetno heterogenim kliničkim tokom.
Leukemični, kao i normalni B limfociti, na svojoj površini eksprimiraju imunoglobulinski
antigenski receptor. Struktura varijabilnog regiona njegovog teškog lanca (IGH) je determinisana
specifičnim rearanžmanima između IGHV, IGHD i IGHJ gena, koji se odvijaju tokom
diferencijacije B ćelija. Nakon susreta sa antigenom, B limfociti ulaze u proces afinitetnog
sazrevanja u germinalnim centrima sekundarnih limfnih folikula, tokom koga dolazi do
akumulacije somatskih hipermutacija u IGHV-IGHD-IGHJ rearanžmanima.
Pokazano je da je mutacioni status rearanžiranih gena za varijabilni region teških lanaca
imunoglobulina (IGHV) najpouzdaniji molekularni marker u HLL, koji definiše dva podtipa
bolesti: M-HLL i N-HLL. Pacijenti sa malim procentom ili bez IGHV mutacija (N-HLL) obično
imaju mnogo agresivniji tok bolesti i lošiju prognozu od pacijenata sa mutiranim IGH
rearanžmanima (M-HLL). Razlike u IGHV genskom repertoaru između M-HLL i N-HLL
klonova, populacione varijacije u učestalosti određenih IGHV gena u HLL rearanžmanima kao i
ekspresija visoko homologih, ˝stereotipnih˝ rearanžmana, ukazuju na ulogu antigenske
stimulacije u patogenezi HLL.
HLL se smatra tipičnim primerom maligniteta uzrokovanog poremećajima u procesu apoptoze. U
HLL su detektovane genetičke promene i aberantna ekspresija brojnih proteina regulatora
apoptoze, koji su uključeni kako u spoljašnji tako i u unutrašnji put aktivacije ovog procesa.
Smatra se da je smanjeni apoptotski potencijal HLL klonova uzrokovan, između ostalog, i
poremećajima u ekspresiji proteina Bcl2 familije.
U ovom radu je analiziran IGHV mutacioni status i genski repertoar IGHV-IGHD-IGHJ
rearanžmana kod pacijenata obolelih od HLL. Pored toga, analizirana je ekspresija gena Bcl2
familije, kao osnovnih regulatora unutrašnjeg puta aktivacije apoptoze, u cilju utvrđivanja
njihove uloge u apoptotskoj rezistenciji HLL B limfocita. qRT-PCR metodom je merena
ekspresija Bcl2, Bax i Bcl2L12 gena, i ispitivana asocijacija nivoa njihove ekspresije sa
odabranim kliničkim i molekularnim prognostičkim faktorima (IGHV mutacionim statusom,
ekspresijom CD38 i lipoprotein lipaze).
Bcl2L12 je novi član Bcl2 familije apoptotskih proteina čija pro- ili anti-apoptotska funkcija još
uvek nije razjašnjena. Pored analize ekspresije Bcl2L12 gena u HLL, jedan od ciljeva ovog rada
je bilo i definisanje promotorskog regiona i mesta starta transkripcije Bcl2L12, što je neophodno
za proučavanje mehanizama njegove transkripcione regulacije.
Određivanjem IGHV mutacionog statusa je pokazano da 55.3% analiziranih pacijenata pripada
M-HLL, a 44.7% N-HLL podtipu, kao i da u N-HLL preovlađuju pacijenti sa progresivnim
oblikom bolesti. U analiziranim rearanžmanima su sa najvećom frekvencom bili zastupljeni geni
IGHV3 familije (55.7%), a zatim IGHV1 (27.3%), IGHV4 (12.5%), IGHV5 (2.3%), IGHV2
(1.1%) i IGHV6 (1.1%) geni. Pokazano je prisustvo tzv. ˝stereotipnih˝ rearanžmana kod 15.3%
pacijenata, predominantno u N-HLL podtipu. Na osnovu određenih frekvenci IGHV, IGHD i
IGHJ gena i genskih familija je zaključeno da je IGH genski repertoar leukemičnih klonova kod
pacijenata iz Srbije veoma sličan repertoaru detektovanom kod pacijenata iz zemalja
mediteranskog područja, sa izuzetkom gena IGHV4 familije koji su kod pacijenata u ovoj studiji
zastupljeni sa manjom učestalošću.
Analizom ekspresije Bcl2, Bax i Bcl2L12 su detektovani znatno viši nivoi ekspresije sva tri gena
kod HLL pacijenata u odnosu na zdrave kontrole, pri čemu je povećanje ekspresije bilo
najizraženije u slučaju Bcl2 gena. Povišena ekspresija Bcl2 je pokazala asocijaciju sa
nepovoljnim prognostičkim parametrima: progresivnim tipom bolesti, visokim nivoom
serumskog β2-mikroglobulina i povišenim nivoom ekspresije gena za lipoprotein lipazu (LPL).
Ekspresija Bax je pokazala korelaciju samo sa ekspresijom LPL, dok je ekspresija Bcl2L12 bila
relativno homogena među HLL pacijentima i, kao takva, nije ispoljila značajnu asocijaciju sa
većinom kliničkih i molekularnih prognostičkih faktora. Ekspresija Bcl2, Bax i Bcl2L12 je bila
viša u grupi N-HLL pacijenata u odnosu na M-HLL pacijente, ali ova razlika nije dostigla
statističku značajnost.
Kloniranjem fragmenata 5' kraja Bcl2L12 gena i funkcionalnom analizom reporterskih
konstrukata, detektovan je region koji ispoljava jaku promotorsku aktivnost. Ovaj region se
prostire od 338 nukleotida uzvodno, do 148 nizvodno od početka kodirajućeg dela egzona 1, a
esejom usporene elektroforetske pokretljivosti (˝EMSA˝) je pokazano da se za njega vezuju Sp1 i
GATA-1 transkripcioni faktori. Metodom elongacije reverznog prajmera (˝primer extension˝) je
određen položaj starta transkripcije Bcl2L12 gena, 33 nukleotida uzvodno od translacionog start
kodona. | Chronic lymphocytic leukemia (CLL), the most common type of leukemia in Western countries,
manifests as clonal expansion of mature CD5+ CD19+ CD23+ sIgMlow B lymphocytes and it is
characterized by an extremely heterogeneous clinical course.
Leukemic, as well as normal B lymphocytes, express immunoglobulin antigenic receptor at their
surface. The structure of its heavy chain (IGH) variable region is being formed during B-cell
differentiation, through rearrangements between IGHV, IGHD and IGHJ genes. After antigen
encouner, B lymphocytes undergo the process of affinity maturation in germinal centers of
secondary lymphoid follicles, durring which IGHV-IGHD-IGHJ rearrangements accumulate
somatic hypermutations.
It has been shown that the mutational status of rearranged immunoglobulin heavy variable genes
(IGHV) represents the most reliable molecular marker in CLL, which defines two CLL subsets:
M-CLL and U-CLL. The patients without or with a small percentage of IGHV mutations (U-
CLL) usually have more agressive disease and inferior prognosis in comparison to patients
expressing mutated IGH rearrangements (M-CLL). Biased IGHV gene repertoire between M-
CLL and U-CLL clones, population differences in IGHV gene usage, as well as the expression of
highly homologous, ˝stereotyped˝ rearrangements, strongly imply the role of antigenic
stimulation in pathogenesis of CLL.
CLL typifies the malignancy caused by defective apoptosis. Genetic alterations and aberrant
expression of numerous proteins involved in extrinsic and intrinsic pathways of apoptosis
regulation have been described in CLL. Reduced apoptotic capacity of CLL clones is, in part,
caused by disturbances in the expression of Bcl2 family proteins.
In this study, we analized IGHV mutational status and gene repertoire of IGHV-IGHD-IGHJ
rearrangements in CLL patients. Furthermore, the expression of Bcl2 family genes, the key
regulators of intrinsic apoptotic pathway, has been studied, in order to elucidate their role in
resistance of CLL B lymphocytes to apoptosis. The expression levels of Bcl2, Bax and Bcl2L12
genes were measured by qRT-PCR, and association of their expression with clinical and
molecular prognostic factors (IGHV mutational status, expression of CD38 and lipoprotein
lipase) was analized.
Bcl2L12 is a novel member of Bcl2 family of apoptotic proteins, whose pro- or anti-apoptotic
function has not been elucidated yet. Besides the expression analysis of Bcl2L12 gene in CLL,
one of the aims of this dissertation was defining the promoter region and transcription start site of
Bcl2L12, which is essential for the study of mechanisms involved in its transcriptional regulation.
The analysis of IGHV mutational status showed that 55.3% of patients enrolled in this study
belonged to M-CLL, and 44.7% to U-CLL subset, and that in U-CLL predominated patients with
the progressive form of the disease. The most frequently expressed genes were those belonging to
IGHV3 family (55.7%), followed by IGHV1 (27.3%), IGHV4 (12.5%), IGHV5 (2.3%), IGHV2
(1.1%) i IGHV6 (1.1%) genes. The presence of ˝stereotyped˝ rearrangements was detected in
15.3% of patients, predominantly in U-CLL subset. Based on the determined frequencies of
IGHV, IGHD and IGHJ genes and gene families, we concluded that the IGH gene repertoire of
leukemic clones of Serbian CLL patients closely resembles the repertoire observed in patients
from Mediterranean countries, with the exception of IGHV4 family, which was underrepresented
in our cohort.
The expression analysis of Bcl2, Bax and Bcl2L12 showed that those three genes were
overexpressed in CLL patients compared to healthy controls, and that elevation of expression
level was the most prominent in the case of Bcl2 gene. High expression levels of Bcl2 were
associated with unfavorable prognostic parameters: progressive form of the disease, elevated β2-
microglobulin and high expression of lipoprotein lipase gene (LPL). The expression of Bax was
correlated only with the expression of LPL, while the expression of Bcl2L12 turned out to be
relatively homogenous among CLL patients and, as such, failed to show association with the
majority of clinical and molecular prognostic factors. Expression levels of Bcl2, Bax and Bcl2L12
were higher in U-CLL in comparison to M-CLL group of patients, but this difference did not
reach statistical significance.
Cloning of fragments corresponding to 5' end of Bcl2L12 gene and functional analysis of reporter
constructs, led to identification of a region which exerts high promoter activity. This region
extends from 338 nucleotides upstream, to 148 nucleotides downstream relative to the beginning
of exon 1 coding sequence, and electromobility shift assay (˝EMSA˝) showed that it binds Sp1
and GATA-1 transcription factors. Transcription start site of Bcl2L12 gene was determined by
primer extension assay, which showed that it is located 33 nucleotides upstream of translation
start codon. | true |
Ispitivanje elemenata respiratornog lanca gljive Phycomyces blakesleeanus burgeff: veza sa metabolizmom fosfatnih jedinjenja | Investigation of respiratory chain elements in fungus Phycomyces blakesleeanus: connecton with metabolism of phosphate compounds | U ovom radu je ispitivano postojanje i aktivnost alternativnih komponenata
elektron transportnog lanca kod gljive Phycomyces blakesleeanus, sa akcentom na
enzim alternativna oksidaza (AOX), kao i ponašanje respiratornog sistema u uslovima
smanjene koncentracije kiseonika. Odgovor respiratornog sistema na ovaj stres je
upoređen sa odgovorom druge komponente energetskog metabolizma, polifosfata
(PPn), sa namerom da se utvrdi da li su, i na koji način, ova dva metabolička sistema
povezana.
Ispitivanjima izvedenim pomoću kiseonične elektrode tipa Klark je utvrđeno
postojanje aktivnosti alternativnih elemenata respiratornog lanca kod P. blakesleeanus,
a za zabeleženu aktivnost su odgovorni AOX, zatim enzim ili enzimski kompleks koji
se indukuje u uslovima dugotrajne inhibicije Kompleksa III i nazvan je Kompleks
IIIPAR, a vrlo je verovatno da postoji i spoljašnja alternativna NADH:dehidrogenaza
(NDE). Utvrđeno je da se AOX sintetiše u citoplazmi nakon čega se enzim unosi u
mitohondriju energetski zavisnim transportom.
U uslovima smanjene koncentracije kiseonika kapacitet AOX se ne menja, ali
učešće enzima u respiraciji značajno raste zbog inhibicije citohrom c oksidaze (COX) u
ovim eksperimentalnim uslovima. Ovakav odgovor respiratornog sistema P.
blakesleeanus se može pripisati potrebi za brzom odbranom od nastanka reaktivnih
kiseoničnih vrsta (ROS) prilikom reoksigenacije koja dovodi do velikog porasta protoka
elektrona kroz citohromski respiratorni put.
31P NMR spektroskopija je pokazala da u istim eksperimentalnim uslovima
odnos intenziteta centralnog signala PPn i unutarćelijskog neorganskog fosfata (PPc/Pi),
koji je dobar pokazatelj energetskog stanja ćelije, opada, ali iznenađujuće, dolazi do
rasta nivoa ATP-a. Analiza glavnih komponenti (PCA), je pokazala snažnu negativnu
korelaciju između ova dva parametra, ali i negativnu korelaciju između odnosa PPc/Pi i
učešća alternativne u ukupnoj respiraciji. Osim toga, dodatak azida, snažnog inhibitora
COX, nije dodatno smanjio odnos PPc/Pi u uzorcima micelijuma koji su bili izloženi
tretmanu smanjene koncentracije kiseonika. Ovi rezultati ukazuju na povezanost ove
dve komponente energetskog metabolizma, odnosno na mogućnost da se hidrolizom
PPn nadoknadi manjak ATP-a u ćelijama izazvan inhibicijom citohromskog lanca.
Dodatnu potvrdu ovog zaključka je pružio set eksperimenata u kojima je
micelijum P. blakesleeanus bio izložen dugotrajnom dejstvu cijanida (5 h). Nakon 100
min, merenja na kiseoničnoj elektrodi su pokazala početak oporavka aktivnosti COX
koji je do kraja inkubacionog perioda bio potpun. Ove promene u aktivnosti COX su
praćene promenama PPc/Pi odnosa, ali do oporavka PPc/Pi odnosa nije došlo prilikom
inkubacije micelijuma u antimicinu A i azidu, gde ne dolazi do oporavka COX funkcije.
Svi ovi podaci ukazuju da je za održavanje nivoa PPn u ćelijama P. blakesleeanus
neophodan funkcionalan citohromski respiratorni lanac koji ima sposobnost formiranja
transmembranskog protonskog gradijenta i sinteze ATP-a. | The existence and activity of the alternative components of electron transport
chain in fungus Phycomyces blakesleeanus, especially alternative oxidase (AOX), as
well as respiratory chain behavior in the conditions of oxygen deprivation, was
investigated in this work. Response of respiratory system to these stress conditions was
compared to response of another component of energy metabolism, polyphosphates
(PPn), with the intention of determining whether, and in what way, are these two
metabolic systems connected.
Activity of the alternative elements of respiratory chain in P. blakesleeanus was
recorded by means of Clark type oxygen electrode, and the elements responsible for this
activity were AOX and an enzyme or enzymatic complex induced by long-term
inhibition of Complex III which was named Complex IIIPAR. Also, there is a strong
possibility of external alternative NADH:dehydrogenase (NDE) existence. It was
established that synthesis of AOX takes place in the cytosol, and the enzyme is then
imported into the mitochondrion by energy dependent transport.
In the conditions of oxygen deprivation AOX capacity is not affected, but its
engagement in total respiration increases significantly due to the cytochrome c oxidase
(COX) inhibition. This type of respiratory chain response in P. blakesleeanus can be
attributed to a need for fast defense from reactive oxygen species (ROS) formed during
reoxygenation, which leads to the increase in electron flow through the cytochrome
respiratory path.
31P NMR spectroscopy revealed that the intensity ratio of core PPn to
intracellular inorganic phosphate signal (PPc/Pi), which is a good indicator of overall
cellular energy metabolism, decreases in these experimental conditions, but
surprisingly, ATP level raises. Principal component analysis (PCA), has shown strong
negative correlation between these two parameters, and also between PPc/Pi ratio and
participation of alternative in total respiration. Apart from that, addition of azide, a
strong COX inhibitor, did not additionally decrease PPc/Pi ratio in mycelium samples
exposed to oxygen deprivation. These results indicate that there is a connection between
these two components of energy metabolism, i.e., there is a possibility that the decrease
in cellular ATP content caused by cytochrome chain inhibition can be compensated by
PPn hydrolysis.
This conclusion was corroborated by the additional set of experiments where P.
blakesleanus mycelium was exposed to cyanide for 5 h. After 100 min, oxygen
electrode measurements have shown the beginning of COX activity recovery, which
was complete by the end of the incubation period. These changes in COX activity were
accompanied by changes in PPc/Pi ratio, but there was no PPc/Pi ratio recovery when
mycelium was incubated in antimycin A or azide, where there was also no recovery of
COX activity. These data indicate that, in P. blakesleeanus cells, PPn level preservation
requires existence
of
functional
cytochrome respiratory
chain capable
of
transmembrane proton gradient formation and ATP synthesis. | true |
Proučavanje razvoja fitoekologije i fitogeografije u jugoslovenskim zemljama u periodu 1759-1988 : bibliometrijska studija | Research of the development of ecology and geography of plants in southslavonic lands between 1759. and 1988. : a bibliometric study | Ova disertacija predstavlja pokušaj analize informacionih masiva, kao odraza strukture nauke, primenom naukometrijskih, posebno bibliometrijskih, metoda. Prva istraživanja iz fitoekologije i fitogeografije vršili su uglavnom stranci. U drugoj polovini 19. veka osnivaju se prve domaće naučnoistraživačke institucije. Između dva svetska rata dolazi do njihovog jačanja, a posle Drugog svetskog rata do naglog širenja mreže institucija i udesetostručenja broja istraživača. Sedamdesetih godina 20. veka rast se usporava, a krajem osamdesetih dolazi do opadanja. Osnovu za bibliometrijsku studiju činila je bibliografija o ekologiji, rasprostranjenju i zaštiti flore i vegetacije u jugoslovenskim zemljama 1759-1988, koja je urađena kao prilog uz ovu disertaciju. Ona obuhvata 391 monografsku publikaciju, 226 doktorskih disertacjia i 6.012 članaka iz časopisa i zbornika. Analizirana je vremenska raspodela bibliografskih jedinica, tematska i jezička struktura bibliografije, raspodela autora i časopisa po produktivnosti i uključenost časopisa u svetski sistem protoka informacija. Utvrđeno je da je: moguće izdvojiti 3 osnovna perioda razvoja; razvoj posmatranih disciplina pratio je razvoj u svetu; 17% radova je objavljeno na stranim jezicima, a 53% članaka ima sažetke na svetskim jezicima; preko 50%članaka objavljenih 1980-1988. nalazili su se u časopisima koji su uključeni u svetske baze podataka za odgovarajuće oblasti. Bibliografija sadrži radove 1.859 autora, čija se raspodela po produktivnosti ponaša po bibliometrijskim zakonima, a parametar rasopodele, koji je i osnovni pokazatelj strukture posmatranog sistema, iznosi 0,67. Bibliografijom je obuhvaćeno 5.863 članaka iz 556 časopisa, čija se raspodela po časopisima ponašala po bibliometrijskim zakonima, a parametar raspodele iznosi 0,49. Vrednosti parametra raspodele manje od 1 ukazuju na negausovsku Cipfovu raspodelu sa jako izraženom stratifikacijom i povećanim udelom visokoproduktivnih nosilaca posmatranih pojava, što je karakteristično za razvoj nauke u nepovoljnim uslovima. Činjenica da je od 150 godina, koliko traje redovno objavljivanje naučnih rezultata iz posmatranih oblasti, 40 godina bilo sa eksponencijalnim rastom, 40 godina sa trendom opadanja, a 70 godina je proteklo u povratku na nivo pre početka negativnog trenda, najbolje ukazuje na suštinu problema razvoja nauke na ovim prostorima. | This dissertation is an attempt to present the massifs of information, which reflect the structure of science, bу using scientometric and specially bibliometric methods. First researches on plant ecology and geography were done mostly by foreigners. the first southslavonic scientific institutions were founded in the second half of 19th the century. Between the world wars the institutions grew, after the second world war the network of scientific institutions spread fast, and the number of researchers was ten times multiplied. In the seventies of the 20th century the growth was slowing down, and in the eighties the falling started. Basis for this bibliometric study was the Bibliography of ecology, dispersion and protection of flora and vegetation in southslavonic lands 1759-1988, which was compiled as an appendix to this dissertation. It includes 391 monographs, 226 doctoral dissertations and 6.012 articles from journals and collections. Temporal distribution of publications, thematic and linguistic structure of the bibliography, dispersion of authors and journals according to productivity and presence of journals in world system of scientific information are analyzed. It is established that: it is possible to divide the development in three basic periods; development of those disciplines followed the development in the world; 17% of papers were published in foreign languages and 53% of articles had summaries in foreign languages; more then 50% of articles published between 1980. and 1988. were in journals included in world databases for adequate disciplines. Bibliography includes papers of 1.859 authors, whose productivity is dispersed in accordance with bibliometric laws. Numeric value of bibliometric parameter, which is the indicator for the structure of examined system of science, was 0,67. Bibliography included 5.863 articles from 556 journals, whose dispersion was in accordance with bibliometric laws, with parameter of dispersion 0,49. Parameter values less then 1 indicate that the dispersion is nongaussian, Zipfs, with highly expressed stratification and with greater part of highly productive sources. That is the characteristic for the development of science in unpleasant environment. The fact that in period of 150 years of continual publishing of scientific results in plant ecology and geography 40 years had exponential growth of the number of publications, 40 years had falling, and 70 years passed in reaching the level before negative trend started, is the best indicator of the essential problems for the development of science in this part of the world. | false |
Uticaj abiotičkih stresora na stabilnost razvića i morfološku varijabilnost Iris pumila u prirodnim i eksperimentalnim populacijama | The influence of abiotic stressors on the development stability and morphological variability of Iris pumila in natural and experimental populations | Uticaj različitog inteziteta svetlosti, kao jednog od abiotičkih faktora životne sredine, na nivo stabilnosti razvića i morfološku varijabilnost osobina cveta I. pumila ispitivan je na jedinkama populacija koje naseljavaju dva prirodna staništa različitiog inteziteta i kvaliteta raspoložive svetlosti (otvoreno i zasenčeno stanište), kao i na jedinkama populacija poreklom sa otvorenog i zasenčenog staništa, koje su bile izložene tretmanima visokog i niskog svetlosnog inteziteta svetlosti u eksperimentalnim uslovima. Osnovna hipoteza je bila da pod uticajem suboptimalnog intenziteta svetlosti dolazi do smanjenja stabilnosti razvića cvetnih organa što se može utvrditi na osnovu stepena asimetrije radijalno i bilateralno simetričnih cvetnih organa. Stepen fluktuirajuće asimetrije i radijalne asimetrije kao mere nestabilnosti razvića jedinke ili populacije, zavisi od intenziteta delovanja biotičkih i abiotičkih stresora, a pošto je stepen asimetrije negativno korelisan sa stabilnošću razvića, a pozitivno sa razvojnim šumom, na osnovu toga treba očekivati veće vrednosti indeksa asimetrije, tj. veću nestabilnost razvića u stresnijim sredinskim uslovima. Veličina i obrazac stabilnosti razvića osobina cveta I.pumila su se razlikovali u zavisnosti od analizirane osobine, kao i od evolucione istorije istraživanih populacija. Prosečne vrednosti indeksa radijalne asimetrije su bile više u zasenčenom nego u otvorenom staništu, ukazujući da je zasenčeno stanište verovatno stresnija sredina. Populacije poreklom sa otvorenog i zasenčenog staništa su se razlikovale u srednjoj vrednosti indeksa radijalne asimetrije osobina cveta u okviru svakog od svetlosnih tretmana. Fluktuirajuća asimetrija, kao mera bilateralne simetrije osobina cveta I.pumila, imala je veće vrednosti u prirodnim staništima sa visokim svetlosnim intezitetom u poređenju sa niskim, na osnovu čega bi se moglo zaključiti da je otvoreno stanište stresnije za ovaj tip osobina cveta. Aktuelni svetlosni uslovi u alternativnim svetlosnim staništima su indukovali statistički značajne razlike u strukturi fenotipskih i genetičkih korelacionih matrica morfoloških osobina cveta I.pumila. Jaka korelativna veza između veličine fola i standarda na unutar-individualnom nivou u sredinama različitog svetlosnog inteziteta može da ukazuje na bliske veze procesa razvića pomenutih osobina. | The main goal of thesis was to examine the influence of an abiotic environmental factor, such as light intensity, on the developmental stability level and morphological variability of I. pumila floral traits. The individuals, from natural habitat populations which located different light condition habitats (exposed and shaded one), as well as the individuals of populations derived from natural habitat but raised in experimental light conditions of low and high intensity, were used. The basic hypothesis was whether suboptimal light intensity decreased developmental stability of floral traits. Since the level of fluctuating and radial asymmetry is in negative correlation with developmental stability, but is positively correlated with developmental noise, the larger values of asymmetry index are to be expected in more stressful conditions. The developmental stability size and pattern of I. pumila floral traits differed depending on analyzed trait, as well as on the evolutionary history of analyzed populations. The radial asymmetry index showed higher average values in shaded habitat comparing to the exposed one, meaning that the exposed location is more stressful environment for development of the identical replicas of different floral organs. Populations derived from open and shaded habitat differed in an average value of radial asymmetry index of floral traits inside each light intensity treatments, but didn’t differ in fluctuating asymmetry level.Statistically significant interaction between treatment and population, that we got for the radial asymmetry of the standard length and all the indices of fluctuating asymmetry, indicate that the effect of the light intensity on the sensitivity of developmental stability of Iris pumila floral traits depends on the population origin: whether they originated from an open or a shaded habitat. The actual light regimes in different habitats create statistically significant differences in phenotypic and genetic correlation matrices’ structures of morphological Iris pumila floral traits. The correlation matrices of morphological Iris pumila floral traits coincided within and between populations derived from alternative light intensity treatments. | false |
Molekulski mehanizmi metaboličke regulacije u hibernaciji i tokom aklimacije na hladnoću kod tekunice (Spermophilus citellus) | Molecular mechanisms of metabolic regulation in hibernation and during cold-acclimation in European ground squirrel (Spermophilus citellus) | Fenomen hibernacije predstavlja fascinantan primer plastičnosti kod sisara. Hibernirajući sisari podliježu složenom nizu biohemijskih, fizioloških i bihevioralnih promjena u odgovoru na senzonske energetski-zahtijevne periode kuplovane sa redukovanom dopstupnošću hrane. Cilj disertacije je da se ispitaju promjene ćelijskog metabolizma u tkivima i organima tekunica, ključnim za održanje ukupne energetske homeostaze - mrko i bijelo masno tkivo (engl. brown adipose tissue, BAT i white adipose tissue, WAT), mišići i jetra, tokom perioda aklimacije na nisku temperaturu, kao i u fazi hibernacije. Efekat aklimacije/hibernacije na metaboličko remodeliranje u tkivima i organima tekunica praćen je određivanjem: mitohondrijalnog kapaciteta (genska i/ili proteinska ekspresije komponenti respiratornog lanca i ATP sintaze); termogenog kapaciteta (ekspresija dekuplujućeg proteina 1); ekspresionih profila enzima ključnih metaboličkih puteva: glikolize, β-oksidacije, Krebsovog ciklusa, metabolizama triacilglicerola i glikogena. Ispitivani su i transkripcioni faktori uključeni u metaboličku regulaciju, kao i enzimi antioksidativne odbrane. Takođe, ispitivane su promjene u BAT i depoima WAT na strukturnom i ultrastrukturnom nivou. Mužjaci evropske tekunice Spermophilus citellus su početkom septembra podijeljeni u dvije grupe: kontrolnu, koja je boravila na sobnoj temeraturi (22 ± 1 ºC) i grupu aklimiranu na nisku temperaturu (4 ± 1 ºC). Aktivne, eutermične tekunice, koje nisu ušle u duboku hibernaciju tokom aklimacije, žrtvovane su nakon 1, 3, 7, 12, odnosno 21 dan. Tekunice koje su ušle u hibernaciju žrtvovane su nakon 2-5 dana trajanja hibernacije (kontinuirana rektalna temperatura 4 ºC). BAT, subkutano, retroperitonealno i epididimalno WAT (sWAT, rWAT i eWAT), skeletni mišić (musculus quadriceps) i jetra su uzorkovani odmah po žrtvovanju. Kako bi se ispitali mehanizmi metaboličke regulacije u BAT, specifični za hibernatore, paralelno je rađena komparativna studija aklimacije pacova na nisku temperaturu... | The phenomenon of hibernation is a fascinating example of plasticity in mammals. Hibernating mammals are subjected to a complex series of biochemical, physiological and behavioral changes in response to seasonal energy-demanding periods coupled with reduced food availability. The aim of the thesis was to investigate metabolic changes in the key tissues and organs of the ground squirrel, responsible for maintaining overall energy homeostasis - brown and white adipose tissue (BAT and WAT), skeletal muscle and liver, during the acclimation to low temperature, as well as in the hibernation. Effects of acclimation/hibernation on metabolic remodeling in the tissues and organs of the ground squirrel were determined by: mitochondrial capacity (gene and/or protein expression of the components of the respiratory chain and ATP synthase); thermogenic capacity (uncoupling protein 1 content), the expression profile of the key metabolic enzymes involved in: glycolysis, β-oxidation, Krebs cycle, glycogen and triglycerides metabolism. Furthermore, transcription factors involved in the metabolic regulation, as well as antioxidant enzymes, were examined. In parallel, we examined the changes in BAT and WAT depots on the structural and ultrastructural level. Males of the European ground squirrel Spermophilus citellus were divided into two groups in early September: the control group, kept at room temperature (22 ± 1 º C) and a group acclimated to low temperature (4 ± 1 ºC). Active, euthermic ground squirrels, which did not enter into deep hibernation during acclimation, were sacrificed after 1, 3, 7, 12, or 21 days. Ground squirrels that entered hibernation were sacrificed after 2-5 days of hibernation (continuous rectal temperature of 4 ºC). BAT, subcutaneous, retroperitoneal and epididimal WAT (sWAT, rWAT and eWAT), skeletal muscle (musculus quadriceps) and liver were sampled. Parallel, in order to examine the mechanisms of metabolic regulation in the BAT specific for hibernation, comparative study with rats acclimated to low temperatures, was made. Results show that, when hibernating animals are exposed to low temperature, the cellular metabolism in all examined tissues/organs is initially subordinated to maintaining euthermia, i.e. thermogenic process. In the early period of the cold-exposure, shivering and nonshivering thermogenesis is induced in the muscle and BAT, respectively... | false |
Varijabilnost gena uključenih u inflamatorne, imunomodulatorne i apoptotske procese kao faktor rizika za nastanak bazocelularnog karcinoma glave i vrata | Variability of genes involved in inflammatory, immunomodulatory and apoptotic process as a risk factor for head and neck basal cell carcinomas | Bazocelularni karcinom (BCK), poznat kao i bazaliom, je najčešći oblik
kancera u populaciji Kavkazijanaca. BCK čini oko 75% svih karcinoma kože.
Metastazira veoma retko, ali sa porastom veličine tumora povećava se i incidenca
metastaza. U najvećem broju slučajeva ovaj karcinom se javlja na delovima kože
izloženim Sunčevom (UV) zračenju, ali faktori kao što su starost, postojanje ranijih
opekotina od Sunca mogu doprineti povećanom riziku za razvoj BCK-a. Precizni
mehanizmi nastanka BCK-a još uvek nisu poznati, ali se pretpostavlja da
patogeneza BCK-a najčešće podrazumeva apsorpciju UVB zraka od strane DNK
molekula, što dalje dovodi do mutacija u različitim genima, posebno u genima
uključenim u proces ćelijske proliferacije, diferencijacije i smrti, kao što su survivin
i TP53. Poznato je i da keratinocite pod dejstvom UV zračenja oslobađaju i
prostaglandine i citokine, između ostalog i TNF-alpha koji ostvaruje svoje biološke
efekte u ćelijama preko molekula koji su svrstani u porodicu receptora za TNF,
među kojima su najznačajnija dva TNF-R1 i TNF-R2. Produkcija citokina je blisko
povezana sa produkcijom matriks metaloproteinaza (MMP) koje su odgovorne za
lokalnu tkivnu destrukciju. Na funkciju i stabilnost ovih proteina, mogu da utiču
različiti polimorfizmi prisutni u kodirajućim i nekodirajućim sekvencama njihovih
gena. Bazocelularni karcinomi, se javljaju kao posledica različitih štetnih uticaja
spoljašnje sredine, ali isto tako pokazuju i izraženu naslednu predispoziciju, pa se u
poslednje vreme sve više pažnje usmerava upravo na proučavanje doprinosa
genskih polimorfizama kao modulatora predispozicije za malignu transformaciju.
Cilj: Cilj ove doktorske disertacije je analiza funkcionalnih polimorfizama u genima
za TNF-alpha, TNF-R1, TNF-R2, MMP-9, survivin i TP53, kao i analiza njihove
povezanosti sa patološkim karakteristikama BCK-a.
Materijal i metode:
Studijska grupa je obuhvatala 80 pacijenata sa
dijagnostikovanim BCK-om i 80 zdravih osoba, kontrolne grupe, koji prema
starosti i polu odgovaraju grupi ispitanika, svi su srpske nacionalnosti. DNK je
izolovana iz tkiva negativnih margina tumora u grupi pacijenata i periferne krvi
kontrolne grupe. Restrikcionom digestijom PCR produkata i analizom dužine
restrikcionih fragmenata (PCR-RFLP) analizirani su polimorfizmi u genima za TNF-
alpha (-308G/A), TNF-R1 (+36A/G), TNF-R2 (+676T/G), MMP-9 (-1562C/T), Survivin
(-31G/C), TP53 (Pro47Ser), kao i insercioni polimorfizam (TP53 PIN3 Ins 16bp)
kako bi se utvrdila distribucija učestalosti genotipovai alela u grupi pacijenata i
kontrolnoj grupi. Hi-kvadrat i Fišerov egzaktni test su korišćeni za utvrđivanje
razlika u distribuciji učestalosti različitih genotipova i alela. Logističkom
regresionom analizom utvrđivan je rizik za oboljevanje od BCK-a. Sve vrednosti p
smatrale su se statistički značajnim ako su bile manje od 0.05. Dobijeni rezultati su
obrađeni statističkim programom SPSS 13.0.
Rezultati: Najveći broj obolelih od BCK-a su osobe starije od 60 godina (84%), što
potvrđuje ranije podatke da je BCK češći u starijoj populaciji, kao i da se incidence
njegovog nastanka povećava sa godinama života. Što se tiče lokacije BCK-a,
najzastupljeniji je u predelu nosa, kao najisturenijem delu lica. Zabeležene su
značajne razlike u distribuciji genotipova u analizi polimorfizma +676T/G TNF-R2
gena, (p=0.001, χ2 test) i PIN3 Ins 16bp TP53 gena (p=0.043, χ2 test). Statistički
značajna razlika u distribuciji alela je zabeležena samo u slučaju polimorfizma
+676T/G TNF-R2 gena, (p=0.011, χ2 test). Značajno povećanje rizika za razvoj BCK-
a utvrđeno je kod heterozigota TG u analizi polimorfizma +676T/G TNF-R2 gena,
(OR=4.86, p‹0.001) u poređenju sa wild type TT genotipom, kao i kod heterozigota
A1/A2 u analizi polimorfizma PIN3 Ins 16bp TP53 gena (OR=2.40, p=0.027) u
poređenju sa wild type A1/A1 genotipom. Interesantno je da polimorfizam -31G/C
gena za survivin je pokazao značajno smanjenje rizika za razvoj BCK-a tek nakon
što je računat Adjusted OR, za heterozigote GC genotipa (OR=0.46, p=0.047) u
odnosu na homozigote GG genotipa. Ostali polimorfizmi u genima za TNF-alpha (-
308G/A), TNF-R1 (+36A/G), MMP-9 (-1562C/T), TP53 (Pro47Ser) nisu pokazali
postojanje značajne razlike u distribuciji učestalosti genotipova i alela između
grupe pacijenata i kontrolne grupe.
Zaključak: Ovo istraživanje bazirano na analizi polimorfizama nekoliko odabranih
gena, pokazalo je da su polimorfizmi nukleotidne sekvence
+676T/G u TNF-R2
genu i -31G/C u promotoru gena za survivin, kao i insercioni polimorfizam PIN3 Ins
16bp u TP53 genu modulatori rizika za razvoj BCK-a u našoj populaciji. | Introduction: Basal-cell carcinoma (BCC), also known as basalioma, is the most
frequent type of cancer among the Caucasus. BCC accounts for approximately 75%
of all skin cancers. It metastasizes extremely rarely, however its growth increases
the risk and incidence of metastases. In the majority of cases, this cancer appears
on the skin areas exposed to the sun rays (UV), but factors including age and the
existence of earlier sunburn may considerably enhance the risk of BCC
development. The precise mechanisms of BCC development are yet unknown, but
BCC pathogenesis most commonly relates to the DNA molecule absorption of UVB
rays, which furthermore, leads to mutations in various genes, especially in those
included in the process of cell proliferation, differentiation, and death, like survivin
and TP53. It is also known that keratinocytes under influence of UV rays release
prostaglandins and cytokines and, among others, TNF-alpha which exerts its
effects in cells via molecules belonging to the family of TNF receptors, the most
significant of which are TNF-R1 and TNF-R2. The production of cytokines is closely
connected to the production of matrix metalloproteinases (MMP) that are
responsible for the local tissue destruction. Different polymorphisms can affect the
function and stability of these proteins, which are present in the coding and
noncoding sequences of their genes. Basal cellcarcinomas occuras a result of
various adverse environmental impacts, but also show an expressed hereditary
predisposition, so more attention is recently directed precisely to the study of the
contribution of genetic polymorphisms as modulators of predisposition to
malignant transformation.
Aim: The aim of this doctoral dissertation is the analysis of functional
polymorphisms in genes for TNF-alpha, TNF-R1, TNF-R2, MMP-9, survivin and TP53,
but also the analysis of their association with pathological features of BCC.
Material and methods: The study group included 80 patients diagnosed with
basal cell carcinoma and 80 healthy individuals, the control group, matched in age
and gender with the examinees, all of them of Serbian nationality. In the patients
group, DNA was isolated from the tissue of histologically negative tumor margins,
and in the control group from peripheral blood samples. The polymorphisms in
TNF-alpha gene (-308G/A), TNF-R1 (+36A/G), TNF-R2 (+676T/G), MMP-9 (-
1562C/T), survivin (-31G/C), TP53 (Pro47Ser), and insertion polymorphism (TP53
PIN3 Ins 16bp) were analyzed by restriction digestion of PCR products and by
analysis of restriction fragment length, in order to determine the distribution
frequency of genotype and allele in the patient and control group. Chi square test
and Fisher exact test were used for determination of differences in the genotype
and allele distribution frequencies. Logistic regression was used for BCC risk
assessment. If amounting to less than 0.05, all the p values were considered
statistically significant. Calculations were performed by using SPSS 13.0 statistical
software.
Results: The largest number of patients with BCC is among patients over 60 years
(84%), which confirms earlier reports that BCC is more common in the older
population, and that its incidence increases with age. As for the location of BCC, it
affects most commonly the nose area, as the most protruding part of the face.
Statistically significant differences in the distribution of alleles and genotypes of
the polymorphisms+6 76T/G TNF-R2 (p=0.001, χ2 test) and PIN3 Ins 16bp TP53
gene (p=0.043, χ2 test) were observed. A significant increase of BCC development
risk was observed in TG heterozygotes for the polymorphism +676T/G TNF-R2 in
comparison to wild type TT genotypes (OR=4.86, p‹0.001), and also in A1/A2
heterozygotes of PIN3 Ins 16bp analyzed polymorphism in TP53 gene in
comparison to the wild type A1/A1 genotypes (OR=2.40, p=0.027). Interestingly,
GC heterozygotes for the polymorphism -31G/C in the survivin gene showed a
significant decrease of BCC development risk after calculating the adjusted OR in
comparison to GG genotypes (OR=0.46, p=0.047). The remaining polymorphisms
in TNF-alpha gene (-308G/A), TNF-R1 (+36A/G), MMP-9 (-1562C/T), TP53
(Pro47Ser) did not show significant difference in the frequencies of genotypes and
alleles between patients and control group.
Conclusion: This research, based on the analysis of polymorphisms in several
selected genes, showed that the polymorphisms +676T/G of TNF-R2 gene, PIN3
Ins 16bp of TP53 gene, and -31G/C of survivin gene are modulators of the risk for
BCC development in Serbian population. | true |
Dinamika zajednica zooplanktona u Bokokotorskom zalivu | Zooplankton community dynamics in the Bay of Boka Kotorska | Istraživanje zooplanktona obuhvatilo je analizu kvalitativnog i kvantitativnog sastava kao i određivanje vremenske i prostorne distribucije zooplanktona, pojedinih grupa i njihovih vrsta na području Bokokotorskog zaliva. Istraživanje je obavljeno na tri lokaliteta u Kotorskom, dva u Tivatskom i jednom lokalitetu u Hercegnovskom zalivu. Uzorci su sakupljeni planktonskom mrežom promjera okaca 125μm, jednim potezom od dna do površine. Uzorkovanje je vršeno sedmično, 15-dnevno i mjesečno, sa ciljem da se objasni velika brzina promjene brojnosti i distribucije zooplanktona u plitkim oblastima. Tokom 16-mjesečnog istraživanja mezozooplanktona Bokokotorskog zaliva određen je 81 takson iz ukupno 7 filuma. U Kotorskom zalivu je nađeno 69 taksona, u Tivatskom 70, dok su u Hercegnovskom zalivu zabilježena 72 taksona. Godišnja dinamika ukupne gustine na sva tri područja (Kotorski, Tivatski i Hercegnovski zaliv) pokazala je povećanje u toplijem dijelu godine. Uzrok visokih vrijednosti u ljetnjim mjesecima na području Kotorskog zaliva bila je pojava protozoe Noctiluca scintillans sa velikim gustinama populacije, kao i razvoj kladocera. Razvoj kladocera bio je i razlog ljetnjeg maksimuma u Tivatskom i Hercegnovskom zalivu, dok se zimski maksimum u Hercegnovskom zalivu javio kao posljedica velike gustine populacija kopepoda. Dominantna grupa zooplanktona bile su kopepode. Srednja procentualna zastupljenost iznosila je 67% u Kotorskom, 73% u Tivatskom i 81% u Hercegnovskom zalivu. Takosoni Oncaeidae i Oithona nana su dominirali u sastavu kopepoda na svim lokalitetima u istraživanom periodu. Maksimalnim gustinama kopepoda prethodile su visoke vrijednosti gustine fitoplanktona. Pojava velike gustine populacije ktenofore Bolinopsis vitrea u Kotorskom zalivu, predstavlja prvi takav nalaz u južnom Jadranu, kao i prvu masovnu pojavu ove vrste u Mediteranu. Visoka negativna korelacija između B. vitrea i kopepoda potvrđuje činjenicu da su ktenofore vrlo bitni predatori čija aktivnost za posljedicu može imati značajan uticaj na zooplanktonske zajednice. VII Rezultati ovog istraživanja su pokazali da je u obalnim područjima korišćenje planktonske mreže promjera okaca 125 μm neophodno za dobijanje preciznijih vrijednosti ukupnog zooplanktona, jer najveći udio predstavljaju rani razvojni stadijumi malih kalanoida i ciklopoida koji prolaze kroz mreže grubljeg tkanja. Velike varijacije vrijednosti u svim sezonama, posebno kopepoda, sugerišu da je za bolje razumijevanje procesa u ovako produktivnim sistemima potrebno uzimati uzorke češće od jednom mjesečno. Istraživanja ukazuju i na to da brze promjene hidrografskih i produkcijskih parametara u zatvorenom i eutrofnom Bokokotorskom zalivu značajno utiču na gustine populacija zooplanktona. | The study on zooplankton included the analysis of qualitative and quantitative composition, as well as the analysis of spatial and temporal distribution of certain zooplankton groups and species in the Bay of Boka Kotorska. The research was realised at 3 locations within the Bay of Kotor, 2 locations within the Bay of Tivat, and 1 location within the Bay of Herceg Novi. Samples were collected using a plankton net with 125μm mesh size, with one vertical haul from the sea bottom to the water surface. Sampling was done either weekly, every 15 days, or once a month, with the aim to explain the high turnover rate of the abundance and distribution of zooplankton in shallow areas. During the 16-month research on zooplankton in the Bay of Boka Kotorska, we determined 81 taxa from 7 phyla. In the Bay of Kotor, we found 69 taxa, in the Bay of Tivat 70 taxa, and in the Bay of Herceg Novi 72 taxa. The annual density dynamics in all the three areas (within the three sub-bays, of Kotor, Tivat, and Herceg Novi) showed an increase in the warmer period of the year. The cause of high density values during the summer months in the Bay of Kotor was the appearance of the protozoan Noctiluca scintillans with a high population density, as well as the development of cladocerans. The development of cladocerans was the reason for the summer maximum in the Bay of Tivat and Herceg Novi as well, while the winter maximum in the Bay of Herceg Novi was the consequence of the high population density of copepods. The dominant group of zooplankton were copepods, with average percentages of 67% in the Bay of Kotor, 73% in the Bay of Tivat, and 81% in the Bay of Herceg Novi. Oncaeidae and Oithona nana were the dominant copepod taxa at all locations during the research. Maximum density of copepods was preceded by high densities of phytoplankton. The occurrence of a high population density of the ctenophore Bolinopsis vitrea in the Bay of Kotor is the first such occurrence recorded in the South Adriatic Sea, as well as the first mass occurrence of this species in the Mediterranean Sea. A high negative correlation between B. vitrea and copepods confirms the fact that ctenophores IX are very important predators, the activity of which might have a significant impact on the zooplankton communities. The results of this research indicate that it is necessary to use a plankton net with 125μm mesh size in order to obtain more precise values of the total zooplankton, because early stadiums of small calanoids and cyclopoids, which contribute the most to the overall density of zooplankton, pass through the plankton nets with higher mesh sizes. Large variability of densities, particularly of copepods, during all seasons, suggest that, for a better understanding of the processes in these productive systems, it is necessary to take samples more often than once a month. The research also indicates that rapid changes of hydrographic and productivity parameters in a closed and eutrophicated bay, which is the case with the Bay of Boka Kotorska, have a significant influence on the population densities of zooplankton. | false |
Molekularni mehanizmi patogeneze mijeloproliferativnih neoplazija : poremećaj ekspresije gena uključenih u proliferaciju i apoptozu | Molecular mechanisms of pathogenesis of myeloproliferative neoplasms: deregulation of genes involved in cell proliferation and apoptosis | Uvod Mijeloproliferativne neoplazije (MPN) su hronični hematološki maligniteti koji se odlikuju
autonomnom proliferacijom opredeljenih progenitora hematopoeze i aberantnom aktivacijom
tirozin kinaznih signalnih puteva u kombinaciji sa snažnim odgovorom na citokine i faktore rasta.
Tri bolesti predstavljaju MPN u užem smislu: policitemija vera (PV), esencijalna trombocitemija
(ET) i mijelofibroza (MF). Jedna od komplikacija ovih oboljenja je njihova kasna evolucija u
akutnu mijeloidnu leukemiju (AML).
Važno obeležje ovih bolesti ja prisustvo „missense“ JAK2-V617F mutacije u sve tri bolesti,
a procenat zastupljenosti mutacije po bolestima je različit. TakoĎe pokazano je da kod ovih
pacijenata postoji tzv. efekat “doze gena”, odnosno da različit nivo V617F alela utiče kliničku sliku
bolesti.
JAK2-V617F mutacija dogaĎa se u 80% slučajeva na specifičnom haplotipu koji je nazvan
46/1 haplotip. Na koji način ovaj niz SNP-ova, koji se nalaze u JAK2 genu, predisponira nastajanje
mutacije kao i njen uticaj na fenotip MPN, još nije utvrĎeno. Jedan od mogućih mehanizama je
uticaj ovog haplotipa na transkripciju. Posebnu pažnju u okviru 46/1 haplotipa je privukao SNP
rs12343867, koji u potpunosti asocira sa MPN.
Proces apoptoze je deregulisan u hematološkim malignitetima, što dovodi do rezistencije
malignih ćelija na signale smrti i obezbeĎuje im duži život u odnosu na normalne ćelije. Proces
apoptoze nije detaljno izučen kod MPN, mada se zna da je direktno pogoĎen JAK2-V617F
mutacijom. Naime, glavni signalni put preko STAT5 (Signal Transducers and Activators of
Transcription) proteina direktno aktivira anti- apoptotski BCL2-xL protein, čime se smanjuje
apoptoza. Deregulacija ostalih apoptotskih puteva u MPN nije u potpunosti rasvetljena.
Ciljevi istraživanja i metode
Cilj našeg istraživanja je bio ispitivanje markera koji učestvuju u
procesima proliferacije i apoptoze kod pacijenata sa MPN. Stoga je istraživanje obuhvatilo
proučavanje mutacija JAK2-V617F i FLT3-ITD, molekularnih markera uključenih u proces
proliferacije. Ove mutacije detektovane su metodama baziranim na PCR. Metodom qPCR odreĎen
je udeo zastupljenosti mutiranog V617F alela. Metodom sekvenciranja je analiziran DNK region u
intronu 14 JAK2 gena u kome se nalazi SNP rs12343867. Okarakterisana je njegova funkcija in
vitro i in vivo i analiziran njegov uticaj na transkripciju JAK2 gena. U ovom segmentu istraživanja
korišćene su metode rekombinantne DNK, tranzijentna transfekcija K562 ćelija, funkcionalni CAT
esej i EMSA esej. Metodom RT-qPCR je proučavana rezistencija granulocita pacijenata sa MPN na
apoptozu, praćenjem ekspresije markera unutrašnjeg (BCL2, BAX I BCL2L12) i spoljašnjeg (FASR)
apoptotskog puta.
Rezultati, diskusija i zaključci Dobijeni rezultati pokazuju da je, od ukupnog broja od 199
analiziranih pacijenta sa MPN, JAK2-V617F mutacija detektovana kod 53% pacijenta sa ET, 78%
pacijenata sa PV i 38% pacijenata sa MF. Kada je u pitanju zastupljenost V617F alela, pokazano je
da je kod pacijenata sa ET prisutan nizak nivo mutiranog alela ( 27%), kod pacijenata sa PV prisutni
su veće “doze” mutiranog alela (44%), dok je kod pacijenata sa MF mutirani V617F alel
najzastupljeniji (81%). Proučavanjem prisustva FLT3-ITD mutacije kod pacijenata sa MPN
konstatovano je da ova mutacije nije prisutna ni kod JAK2-V617F- negativnih (15/33), ni kod
JAK2-V617F- pozitivnih (18/33) ispitanika. Naši rezultati pokazuju da, u početnom stadijumu
MPN, FLT3-ITD ne predstavlja mutacioni dogaĎaj koji utiče na proliferativni kapacitet malignog
klona.
Ispitivanje DNK regiona u intronu 14 JAK2 gena koji sadrži SNP rs12343867 je pokazalo
da ovaj region ima snažan represorski transkripcioni potencijal. U ovom radu je prvi put pokazano
za neki intronski region u genu za JAK2 da je transkripcioni regulatorni element. UtvrĎeno je da
proteinski kompleks koji interaguje sa ovim regulatornim regionom, sadrži transkripcioni faktor
MEIS1/2, i to specifično vezan kada je nukleotid C prisutan u ovom regionu. Ispitivanje ekspresije
JAK2 gena pokazalo je da je ekspresija pod snažnim uticajem JAK2-V617F mutacije, a da haplotip
nema značajnu ulogu u regulaciji proliferacije i apoptoze.
Ispitivanje apoptotskih markera kod pacijenata sa MPN je pokazalo da je izmenjena
ekspresija kako gena uključenih u spoljašnji, tako i gena uključenih u unutrašnji apoptotski put.
TakoĎe je primećeno da JAK2-V617F mutacija nema presudan uticaj na nivo ekspresije ovih
apoptotskih markera. Ovim je pokazano da je apoptoza deregulisana kod pacijenata sa MPN i
predstavlja važan mehanizam akumulacije malignog klona.
Rezultati ovog istraživanja doprineli su boljem razumevanju molekularne osnove MPN i
bacili novo svetlo na transkripcionu regulaciju JAK2 gena, koja predstavlja još jedan od mogućih
načina na koji JAK2 gen utiče na genezu ovih bolesti. | Introduction Myeloproliferative
neoplasms
(MPN)
are
chronic
hematological
malignancies that are characterized by autonomous proliferation of committed
hematopoietic progenitors and aberrant activation of tyrosine kinase signaling pathways, in
combination with a strong response to cytokines and growth factors. Three major entities
constitute MPN: polycythaemia vera (PV), essential thrombocythemia (ET) and primary
myelofibrosis (PMF). One of the complications of these diseases is their late evolution into
acute myeloid leukemia.
Important feature of these diseases is the presence of missense mutation JAK2-
V617F and its variable representation among MPN entities. It is also shown that there is so-
called effect of "gene dosage" in these patients, meaning that a different level of V617F
alleles influences the clinical picture of the disorders.
JAK2-V617F mutation occurs in 80% of cases on a specific haplotype, called 46/1
haplotype. Exact mechanism of action of this set of SNPs, that are located within the JAK2
gene, has not been determined yet. One of the possible mechanisms could be that it effects
transcription. Among eight SNPs, included in this haplotype, SNP rs12343867 has drawn
special attention because of its strong association with the MPN.
The process of apoptosis is deregulated in hematological malignancies, leading to
resistance of cancer cells to death signals, thus providing them a longer life span compared
to normal cells. The process of apoptosis has not been extensively studied in MPN,
although it is known that it is directly affected by the JAK2-V617F mutation. Specifically,
the main signaling pathway through STAT5 protein directly activates anti-apoptotic BCL2-
xL protein, thereby reducing apoptosis. Deregulation of other apoptotic pathways in MPN
is not fully understood.
Aims and methods
The aim of our study was to investigate markers that participate in
processes of cell proliferation and apoptosis in patients with MPN. Therefore, the research
included the study of JAK2-V617F and FLT3-ITD mutations, molecular markers involved
in the process of proliferation. These mutations were detected by PCR-based methods.
Using qPCR method, the proportion of V617F mutant allele was determined. The method
of DNA sequencing has been used to analyze part of intron 14 of the JAK2 gene, containing
the SNP rs12343867. We further characterized its function in vitro and in vivo, and
analyzed its effect on transcription of JAK2 gene. In this segment of the study we used
methods of recombinant DNA, transient transfection of K562 cells, a functional CAT assay
and EMSA assay. Using RT-qPCR we studied resistance of MPN granulocytes to
apoptosis, monitoring the expression of markers of both internal (BCL2, BAX and
BCL2L12) and external (FASR) apoptotic pathway.
Results, discussion and conclusions Out of a total of 199 analyzed patients with MPN,
JAK2-V617F mutation was detected in 53% of patients with ET, 78% of patients with PV
and 38% of patients with MF. As to the proportion of V617F allele, we found that patients
with ET have low level of mutant allele (27%), patients with PV harbor higher mutant
allele proportion (44%), whereas in patients with MF V617F mutant allele is predominant
(81%). Studying the presence of FLT3-ITD mutation, we concluded that this mutation is
neither present in JAK2-V617F negative (15/33), nor in JAK2-V617F positive (18/33)
subjects. Our results indicate that in the early stages of MPN, FLT3-ITD mutation is not an
event that influences the proliferative capacity of the malignant clone.
Functional analysis of intron 14 of the JAK2 gene, harboring SNP rs12343867, has
demonstrated that this DNA region has a strong potential for transcriptional repression. For
the first time it was shown that some intronic element of the JAK2 gene is a transcriptional
regulatory element. It was found that protein complex that interacts with this regulatory
region contains transcription factor MEIS1/2, which is specifically bound only when
nucleotide C is present within the intron sequence. We also found that JAK2 gene
expression is under the strong influence of JAK2-V617F mutation, and that the haplotype
has no significant role in the regulation of JAK2 gene expression.
Investigation of apoptotic markers in patients with MPN has shown altered
expression of genes involved in both external and internal apoptotic pathway. It was also
noted that the JAK2-V617F mutation does not affect the level of expression of these
apoptotic markers. This result demonstrates that apoptosis is deregulated in patients with
MPN, being an important mechanism of accumulation of the malignant clone.
In summary, the results of this study contributed to better understanding of the
molecular basis of MPN and shed new light on the transcriptional regulation of JAK2 gene,
providing another aspect of complex JAK2 gene action in the etiology of these disorders. | true |
Ekološke karakteristike i morfološko-genetička diferencijacija populacija crnke (Umbra krameri Walbaum, 1792) sa područja Bačke, Mačve i Semberije | Ecological characteristics and morphological-genetic differentiation in populations of European mudminnoow (Umbra krameri Walbaum, 1792) from Bačka, Mačva and Semberija | Crnka (Umbra krameri Walbaum, 1792) je jedina autohtona i reliktna vrsta iz
roda Umbra prisutna u Evropi, endemična za basene Dunava i Dnjestra. U okviru
današnjeg diskontinuiranog areala, usled isušivanja i zagađivanja staništa, populacije
crnke značajno su smanjene poslednjih decenija XX veka. Iz navedenih razloga crnka se
nalazi na IUCN Crvenoj listi ugroženih vrsta i ima status ranjive vrste (VU). Na
nacionalnom nivou ima status kritično ugrožene vrste (CR) i nalazi se u neposrednoj
opasnosti od iščezavanja.
U ovoj disertaciji su uz distribuciju vrste u Srbiji i Bosni i Hercegovini, sa
novozabeleženom populacijom u Srbiji (lokalitet Lugomir), prikazani i rezultati analize
ekoloških karakteristika i morfološko-genetičke diferencijacije istraživanih populacija, a
dat je i status ugroženosti vrste na nacionalnom nivou sa ugrožavajućim faktorima i
merama zaštite i očuvanja.
Za potrebe rada uzorkovano je ukupno 76 jedinki iz tri populacije, dve iz Srbije
(23 jedinke sa lokaliteta Lugomir i 21 jedinka sa lokaliteta Bakreni Batar) i jedna iz
Bosne i Hercegovine (32 jedinke sa lokaliteta Gromiželj). Za svaki istraživani lokalitet
određeni su osnovni geografski parametri (geografska dužina, širina i nadmorska visina)
i fizičko-hemijske karakteristike staništa (širina vodenog basena, dubina vode i mulja,
temperatura vode i vazduha, pH vrednost, elektroprovodljivost, koncentracija kiseonika,
saturacija kiseonikom, koncentracije amonijaka, nitrita i fosfata). Sve jedinke u uzorku
su prvo fotografisane za potrebe analiza geometrijske morfometrije, a nakon toga su im
merene totalna (TL) i standardna (SL) dužina tela i težina tela za potrebe analize
strukture populacija. Takođe, jedinkama su uzete gonade za utvrđivanje pola, uzorci
digestivnog trakta za analizu ishrane, uzorci krljušti za određivanje starosti i uzorci
tkiva za analizu genetičke varijabilnosti. Na svim istraživanim lokalitetima utvrđen je
kvalitativni sastav biljaka i riba, a izvršeno je i uzorkovanje beskičmenjaka iz mulja i
submerzne vegetacije u cilju utvrđivanja njihovog kvalitativnog i kvantitativnog sastava
i analize ishrane crnke. Sva tri istraživana lokaliteta prema analiziranim fizičko-
Rezime
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vii
hemijskim parametrima i sastavom zajednice biljaka i riba predstavljaju tipična staništa
crnke.
Analizom
procentualne
zastupljenosti
glavnih
grupa
beskičmenjaka
ustanovljeno je da su na svim istraživanim lokalitetima prisutni predstavnici Mollusca,
Isopoda, Odonata, Coleoptera, Heteroptera i Chironomidae. Među najzastupljenijim
beskičmenjacima nalaze se predstavnici grupe Isopoda, koji su prisutni na sva tri
lokaliteta, zatim grupa Heteroptera i Megaloptera u Bakrenom Bataru, odnosno
Heteroptera i Odonata u kanalima na Lugomiru, kao i Odonata i Chironomidae na
lokalitetu Gromiželj.
Struktura populacija analizirana je na osnovu izmerene totalne dužine i težine
tela i utvrđenog pola i starosti uzorkovanih jedinki. Totalne dužine tela uzoraka sa
lokaliteta Lugomir i Bakreni Batar imaju približnu vrednost koja se kreće od 53,3 do
82,4 mm, odnosno 60,1 do 83,2 mm, sa izmerenim težinama od 1,5 do 5,9 g, odnosno
od 1,5 do 6,6 g. U uzorku sa lokaliteta Gromiželj totalna dužina tela iznosi od 24,3 do
102,9 mm a težina od 0,1 do 16,4 g. U svim uzorcima dominiraju jedinke mlađih
uzrasnih kategorija (1+ i 2+), s tim da su na Gromiželju detektovane i juvenilne jedinke
(0+), kao i jedinke starijih uzrasnih kategorija (3+ i 4+). U odnosu na polnu strukturu, u
uzorcima iz Lugomira i Bakrenog Batara dominiraju mužjaci, a iz Gromiželja ženke.
Analizom tempa dužinskog i težinskog rastenja ustanovljena je statistički
značajna razlika u totalnoj dužini tela samo kod uzrasne klase 1+ između jedinki sa
lokaliteta Gromiželj i Bakreni Batar. Za uzorak sa Gromiželja utvrđeni su i parametri
rasta prema modelu von Bertalanffy-ja (L∞ = 117,76, K = 0,449, t0 = 0,447). Dobijene
vrednosti faktora alometrije b variraju od 2,619 do 3,684, što ukazuje na to da ribe iz
sva tri uzorka imaju alometrijski rast. Opadajuće vrednosti Fultonovog faktora kondicije
u odnosu na dužinu tela registrovane su samo kod ženki i na nivou uzorka sa lokaliteta
Lugomir, dok u ostalim slučajevima Fultonov faktor kondicije raste sa porastom dužine.
Alometrijski faktor kondicije neznatno opada sa porastom dužine na nivou uzoraka sa
sva tri lokaliteta i odvojeno po polovima, izuzev kod mužjaka sa lokaliteta Lugomir,
kod kojih vrednost alometrijskog faktora kondicije raste sa porastom dužine.
Na osnovu želudačno-crevnog sadržaja uzoraka analizirana je ishrana crnke
(indeks vakuiteta, spektar ishrane, prosečan broj komada plena po stomaku predatora i
širina niše). U odnosu na vrste plena, predstavnici familije Chironomidae
Rezime
viii
najzastupljeniji su kod jedinki sa lokaliteta Lugomir, dok je kod jedinki iz Gromiželja i
Bakrenog Batara najčešći plen Asellus aquaticus (Isopoda).
Primenom metoda geometrijske morfometrije prvi put je kod crnke analizirana
varijabilnost spoljašnje morfologije, odnosno veličine i oblika tela. Korišćenjem
molekularno-genetičkih metoda u kojima su kao genetički markeri upotrebljene
ponavljajuće sekvence jedarne DNK – mikrosateliti i mitohondrijalna DNK (citohrom
b), utvrđena je i genetička raznovrsnost analiziranih populacija. Poređenjem rezultata
geometrijsko-morfometrijskih i molekularno genetičkih analiza uočava se visok stepen
njihove podudarnosti. Dunavska populacija sa lokaliteta Lugomir se diferencirala u
odnosu na savske populacije sa lokaliteta Bakreni Batar i Gromiželj, koje pokazuju
znatno veći stepen međusobne sličnosti i srodnosti. Na ovako jasnu diferencijaciju
upućuje vizuelno razdvajanje analiziranih populacija po prvoj kanonijskoj osi,
statističke značajnosti izračunatih Prokrustovih distanci, UPGMA fenogram, analize
citohroma b mtDNK i mikrosatelitskih lokusa ("neighbour-joining" neukorenjeno stablo
individua i "STRUCTURE" analiza). Uočeni fenetički i genetički odnosi analiziranih
populacija crnke najverovatnije su posledica istorijskih, geografskih i ekoloških faktora.
Sadašnje prisustvo vrste na tri istraživana lokaliteta u Srbiji i Bosni i
Hercegovini, koji se nalaze izvan postojećih granica areala navedenih u savremenoj
literaturi, dopunjava sliku o njenom rasprostranjenju u Evropi i sugeriše mogućnost
postojanja dodatnih populacija. Rezultati istraživanja ovog rada predstavljaju doprinos
poznavanju ekologije crnke, kao i morfološke i genetičke varijabilnosti njenih
populacija. U skladu sa nacionalnom legislativom, uz donošenje odgovarajućih planskih
dokumenata, saznanja do kojih se došlo predstavljaju osnovu za primenu aktivnih mera
zaštite ove, na globalnom nivou, ugrožene vrste. | European mudminnow (Umbra krameri Walbaum, 1792) from Bačka, Mačva and
Semberija
ABSTRACT
European mudminnow (Umbra krameri Walbaum, 1792) is the only
autochthonous and relic species of the genus Umbra present in Europe, and it is
endemic for the Danube and Dniester river basins. In scope of its present discontinuous
range, due to habitat desiccation and pollution, the populations of European
mudminnow significantly decreased during the last decades of the 20th century. For this
reason it is listed on the IUCN Red List of Threatened Species as Vulnerable (VU). At
the national level, it is listed as Critically Endangered (CR), with a serious threat to be
soon extinct.
The range of the species in Serbia and in Bosnia and Herzegovina is presented in
this dissertation, including a newly recorded population in Serbia (locality Lugomir),
along with the results of ecological and morphological-genetic differentiation of the
analysed populations, and the threat status at the national level with threat factors and
conservation measures.
A total of 76 individuals from three populations, two from Serbia (23 individuals
from Lugomir and 21 individuals from Bakreni Batar) and one from Bosnia and
Herzegovina (32 individuals from Gromiželj), were sampled. Basic geographical
parameters (longitude, latitude, altitude) were determined for each locality, along with
physical and chemical habitat parameters (width of the water basin, water and silt depth,
water and air temperature, pH, electroconductivity, oxygen concentration, oxygen
saturation, ammonia, nitrites, phosphates). All sampled individuals were photographed
for the purpose of geometric morphometrics analyses, and their total (TL) and standard
(SL) body length and body weight were measured for population stucture analyses.
Furthermore, gonads were taken for sex determination, digestive tract for diet analysis,
scales for age determination, and tissue samples for genetic variability analysis. Species
composition of aquatic plants and fishes was determined at all localities, and
invertebrates from mud and submersed vegetation were sampled with the aim to
determine their diversity and to analyse the diet of European mudminnow. For their
Abstract
x
physical-chemical parameters and aquatic plant and fish species composition all three
studied localities represent typical habitats of European mudminnow.
The analysis of invertebrate assemblages showed that representatives of
Mollusca, Isopoda, Odonata, Coleoptera, Heteroptera, and Chironomidae were present
at all localities. Among the most abundant invertebrates are Isopoda, which were
present at all three localities, followed by Heteroptera and Megaloptera in Bakreni
Batar, Heteroptera and Odonata in channels of Lugomir, and Odonata and
Chironomidae in Gromiželj.
Population structure was analysed according to measured total body lengths and
weights, and determined sex and age of specimens. Total body lengths in Lugomir and
Bakreni Batar are similar and range from 53.3 to 82.4 mm, and from 60.1 to 83.2 mm,
respectively, as well as weights, ranging from 1.5 to 5.9 g, and from 1.5 to 6.6 g,
respectively. In the sample from Gromiželj, the total body length ranges from 24.3 to
102.9 mm, and weight from 0.1 to 16.4 g. Younger age classes (1+ and 2+) dominate in
all samples, and in Gromiželj even juvenile individuals were detected (0+), as well as
individuals from older age classes (3+ and 4+). The sex ratio indicates that males
dominate in the samples from Lugomir and Bakreni Batar, and females from Gromiželj.
Length and weight growth analysis showed that there is a statistically significant
difference only in total body lengths of individuals aged 1+ from Gromiželj and Bakreni
Batar. The parameters of the von Bertalanffy's growth model for the sample from
Gromiželj were L∞ = 117.76, K = 0.449, and t0 = 0.447. The estimated values of the
allometric coefficient b vary from 2.619 to 3.684, indicating that individuals from all
three samples have an allometric growth. Fulton's condition factor decreases with
increasing body length only in females and in the overall sample from the locality
Lugomir, while in other cases it increases with increasing body length. The allometric
condition factor slightly decreases with increasing body length in all three overall
samples and between sexes, except in males from the locality Lugomir, in which it
increases with increasing body length.
The diet of European mudminnow was analysed according to its gastrointestinal
contents (vacuity index, food spectrum, average number of prey items per predator
stomach, and niche breadth). The representatives of the family Chironomidae were the
most frequent prey of specimens from the locality Lugomir, while Asellus aquaticus
(Isopoda) was the most frequent prey of specimens from Gromiželj and Bakreni Batar.
Abstract
�
xi
The morphological variability, namely body size and shape, was analysed for the
first time in European mudminnow by using the methods of geometric morphometrics.
By applying the molecular-genetic methods, in which repetitive sequences of nuclear
DNA were used – microsatellites and mitochondrial DNA (cytochrome b), the genetic
diversity of the analysed populations was determined. The comparison of the results of
geometric morphometrics and molecular genetic analyses indicates a high level of their
congruence. The Danube River population from the locality Lugomir is differentiated
from the Sava River populations from the localities Bakreni Batar and Gromiželj, which
show a significantly higher level of mutual similarity and relatedness. Such clear
differentiation is indicated by visual separation of the analysed populations across the
first canonical axe, as well as by statistical significance of the estimated Procrustes
distances, the UPGMA phenogram, mtDNA cytochrome b analysis, and microsatellite
loci analysis ("neighbour-joining" unrooted tree of individuals and "STRUCTURE"
analysis). Observed phenetic and genetic relationships of the analysed populations of
European mudminnow are most probably the consequence of historical, geographical,
and ecological factors.
The current presence of European mudminnow at three studied localities in
Serbia and in Bosnia and Herzegovina, which are positioned outside the range limits
cited in literature, supplement the knowledge on its distribution in Europe and suggest a
possibility for existence of other populations, which all represents a good basis for
species conservation and return to its extinct habitats. The results of this research
represent a contribution to the knowledge of ecology of this species and of its
morphological and genetic population variability. In keeping with the national
legislation, following the adoption of adequate documents and measures, the obtained
data offer a good basis for conservation and management of this globally threatened
species.
� | true |
Uticaj indukovane mutageneze na reproduktivno ponašanje Drosophila subobscura i uloga seksualne selekcije u odnosu na obim genetičkih opterećenja | The influence of induced mutagenesis on reproductive behavior of Drosophila subobscura and the role of sexual selection in relationto the level of mutational load | Prema teorijskim očekivanjima seksualna selekcija može smanjivati mutaciona
opterećenja u populacijama. Mužjaci koji su boljeg genetičkog kvaliteta bi trebalo da
budu uspešniji u parenju u poređenju sa mužjacima lošijeg genetičkog kvaliteta. Na taj
način ženke, prilikom izbora partnera za parenje, mogu da eliminišu one mužjake koji
nose štetne alele, i da smanje mogućnost prenošenja štetnih alela u sledeću generaciju.
Seksualna selekcija će redukovati mutaciona opterećenja i povećavati adaptivnu
vrednost populacije, ako je većina mutacija štetna i za adaptivnu vrednost i za osobine
koje utiču na uspešnost mužjaka u parenju.
Istraživanja poslednjih godina su dala rezultate koji idu u prilog ovoj teoriji, ali
i
oprečne
rezultate.
Ovakva
kontradiktornost
proizilazi
iz
kompleksnosti
problematike usled različitih reproduktivnih strategija vrsta, kao i različitih
reproduktivnih strategija mužjaka i ženki. Takođe, osobine koje utiču na komponente
adaptivne vrednosti, kao i na uspešnost u parenju, su veoma složene, pod
determinacijom velikog broja gena, i u čijoj osnovi leže kompleksne genske
interakcije.
Iako se kao osobine koje su cilj seksualne selekcije najčešće analiziraju
sekundarne polne karakteristike, i mnoge druge osobine određuju atraktivnost
mužjaka. To su i morfološke, fiziološke, ponašajne i druge osobine koje utiču na
uspešnost u parenju, a mogu se nazvati osobinama koje su zavisne od opšteg stanja
mužjaka (eng. condition-dependent traits).
U cilju provere hipoteze, da seksualna selekcija utiče na smanjenje mutacionih
opterećenja, u ovom radu na vrsti Drosophila subobscura, od istog genetičkog pula su
formirane dve eksperimentalne grupe koje su se razlikovale u nivou mutacionih
opterećenja. U jednoj grupi su mutacije indukovane gama jonizujućim zračenjem,
dozom od 30Gy. Efekat seksualne selekcije na redukciju mutacionih opterećenja je
praćen preko različitih komponenti reproduktivnog ponašanja mužjaka (odigravanje
udvaranja, latenca udvaranja, intenzitet udvaranja, odigravanje kopulacije, latenca
kopulacije i trajanje kopulacije), sa ciljem da se ispita da li su nosioci mutacija manje
uspešni u parenju. Dobijeni rezultati ukazuju da seksualna selekcija D. subobscura
može da utiče na smanjivanje mutacionih genetičkih opterećenja preko uspešnosti u
parenju mužjaka. Heterozigotni nosioci indukovanih mutacija su pokazali manju
uspešnost u parenju, i kasniji početak udvaranja. Kako je uspešnost u parenju složena
komponenta adaptivne vrednosti, determinisana velikim brojem gena, ona reflektuje
veliki broj osobina koje su važne za reproduktivni uspeh mužjaka.
Postavljena hipoteza je proverena i analizom dve komponente adaptivne
vrednosti: fekunditeta i preživljavanja od stadijuma jaja do adulta, u zavisnosti od
nivoa mutacionih opterećenja, kao i primenjenog sistema parenja (bez mogućnosti
izbora partnera i sa višestrukom mogućnošću izbora). Dobijeni rezultati ne mogu da
potvrde ulogu seksualne selekcije u eliminaciji mutacionih opterećenja za obe
komponente adaptivne vrednosti kod D. subobscura. Pozitivna uloga seksualne
selekcije u eliminaciji štetnih alela je potvrđena samo za fekunditet, ali samo kad su
indukovana mutaciona opterećenja. S obzirom da je za većinu eksperimentalnih
grupa utvrđeno postojanje seksualnog konflikta, odnosno manje vrednosti fekunditeta
kad ženke nisu imale mogućnost izbora, dobijeni rezultati ukazuju da potencijalni
negativni efekti seksualnog konflikta mogu biti prevaziđeni u uslovima povećanog
broja mutacija. Genetička struktura populacija, koja podrazumeva i veličinu
mutacionih opterećenja, može biti važan faktor od koga će i zavisiti delovanje
seksualne selekcije na adaptivnu vrednost. | According to theoretical predictions sexual selection can reduce mutational
load in populations. Males of good genetic quality should be more successful in
matings, compared to the males of low genetic quality. In this way females, through
the choice of mating partners, can eliminate males carriers of deleterious alleles, and
reduce transmission of deleterious alleles to the next generation. Sexual selection will
purge mutational load and increase fitness if most mutations were deleterious to both
nonsexual fitness and condition-dependent traits affecting mating success.
Studies in recent years have yielded results that support this theory, but
opposite results are obtained, as well. This contradiction arises from different
reproductive strategies of species, and different reproductive strategies of males and
females. Traits that affect fitness components, as well as mating success, are under
polygenic determination, with very complex gene interactions.
Although secondary sexual traits were usually analyzed as target of sexual
selection, many other traits can determine male attractiveness. Traits such as
morphological, physiological, behavior and other, affecting mating success, can be
termed as condition-dependent traits.
In order to test this hypothesis, that sexual selection can reduce mutational
load, two experimental groups were formed from the same population genetic pool of
Drosophila subobscura, which differed in the level of mutational load. Mutations were
induced in one group, using 30Gy ionizing radiation. The effect of sexual selection in
reduction of mutational load was measured through different components of male
mating behavior (courtship occurrence, courtship latency, courtship intensity, mating
occurrence, latency to mating and duration of mating). The aim was to evaluate if
mutation carriers were less successful in matings. Obtained results indicate that
sexual selection can reduce mutational load via male mating success. Potential
carriers of mutations were less succesfful in matings and courted later. As mating
success is the complex fitness component, determined by a large number of genes, it
reflects a large number of traits important to male reproductive success.
A hypothesis was tested through analysis of two fitness components : fecundity
and egg-to-adult viability, depending on the level of mutational load, as well as applied
mating system (no choice and multiple choice). The obtained results can not confirm
the role of sexual selection in reducing mutational load for both measured fitness
components. The positive role of sexual selection in reducing mutational load was
confirmed only for fecundity, and only when mutational load was induced.
Considering that sexual conflict was confirmed for the most of the experimental
groups, i.e. less fecundity values when females had no choice, the obtained results
suggest that potential negative effects of sexual conflict could be overcome in
conditions of increased number of mutation. Genetic structure of population,
includung the range of mutational load, could be important factor affecting the action
of sexual selection and overall fitness. | true |
Efekti kombinovane transkranijalne magnetne stimulacije i deprivacije spavanja u tretmanu major depresija | Effects of combined transcranial magnetic stimulation and sleep deprivation in the treatment of major depression | Cilj rada. Cilj ovog rada je procena efekata kombinovane primene repetitivne
transkranijalne magnetne stimulacije (rTMS) niske frekvencije (1 Hz) i parcijalne
deprivacije spavanja (PDS) kod osoba obolelih od major depresije, koje prethodno
pokazuju nezadovoljavajući odgovor na dva različita antidepresiva (ispunjavaju
kriterijume terapijske rezistencije). Uporedo sa dugoročnim praćenjem (do 6 meseci)
efekata ovog tretmana, analiziran je i genetički BDNF Val66Met polimorfizam, kao
mogući perimisivni faktor plasticiteta odgovoran za predikciju efikasnosti primenjenih
terapijskih strategija.
Ispitanici i metode. Ukupno 20 osoba obolelih od major depresije, koje su ispunjavale
kriterijume terapijske rezistencije tretirano je na navedeni način kombinacijom rTMS i
PDS, uporedo sa njihovom prethodnom medikamentoznom terapijom. Kombinovani
protokol rTMS i PDS sprovodio se tokom dve uzastopne nedelje, i za to vreme su
ispitanici svakog dana, izuzev pauze za vikend dobijali stimulaciju rTMS (frekvencija 1
Hz, intenzitet 110% praga izazivanja motornog odgovora) primenjivanu iznad desnog
dorzolateralnog prefrontalnog korteksa (DLPFK), u jutarnjim časovima. U svakoj
nedelji po jednom, opisanoj rTMS, prethodila je redukcija noćnog spavanja uz buđenje
u pola 2. Ispitanici su randomski podeljeni u grupu koja je primala rTMS+PDS i grupu
koja je primala placebo (sham) rTMS stimulaciju + PDS. Procene efekta vršene su
standardnim instrumentima kliničke procene (modifikovana Hamiltonova skala za
procenu depresivnosti - HDRS-24; Montgomery-Asberg skala za procenu depresivnosti
- MADRS, i skala opšteg kliničkog utiska), i to: pre početka primene protokola,
neposredno po završetku, jednu nedelju kasnije, tri i šest meseci kasnije (dugoročna
praćenja sprovedena su isključivo kod ispitanika koji su bili podvrgnuti aktivnom
tretmanu).
Rezultati. Primenjeni protokol stimulacije pokazao je antidepresivni efekat, kod osoba
obolelih od unipolarne depresije na nepromenjenoj dozi antidepresivnih lekova koji se
održavao tokom čitavog šestomesečnog perioda praćenja. Neposredno nakon 2 nedelje
protokola ispitanici koji su primali aktivni rTMS imali su prosečno poboljšanje od 39%
(mereno HDRS-om). Međutim, relevantno poboljšanje (arbitrarno definisano kao
smanjenje HDRS ≥ 50%) imalo je 36% ispitanika tretiranih rTMSom. Dodatnih 45%
ispitanika pokazalo je manje značajan, odnosno delimičan odgovor (definisan kao
smanjenje HDRS ≥ 25%). Međutim, analizom efekata, nakon tri meseca od
započinjanja stimulacije, značajno poboljšanje postojalo je kod 64% ispitanika, a čak i
nakon šest meseci praćenja, ovaj efekat se održavao kod 55%. Takođe, tzv. potpuna
remisija (povlačenje simptoma bolesti, definisano kao HDRS ≤ 10 poena) postignuto je
kod 5 od 11 ispitanika. Poštujući ograničenja procene koja se zasniva na malom uzorku,
ipak je zanimljivo navesti da su svi aktivnom rTMS stimulacijom tretirani pacijenti sa
Val66Val BDNF genotipom postigli kriterijum značajnog poboljšanja, a sa jednim
izuzetkom i kriterijum remisije. Suprotno ovome, samo jedan od njih sa Val66Met
BDNF genotipom, postigao je značajno poboljšanje, a kod jednog stimulacija u
potpunosti nije imala efikasnost, nakon čega je preveden na drugu terapiju.
Zaključak. Primena kombinovane terapije rTMS i PSD kod obolelih od terapijski
rezistentne unipolarne depresije, pokazuje visoku efikasnost, kako neposredno, tako i
nakon dugoročnog praćenja. Genetički BDNF polimorfizam Val66Met mogući je
prediktor nezadovoljavajućeg odgovora na primenu ove metode. | Objectives. The main aim of this study was to evaluate potential synergistic
antidepressant effect of two therapies: repetitive transcranial magnetic stimulation
(rTMS) applied at 1 Hz associated with partial sleep deprivation (PSD) in patients with
treatment resistant major depression.
Besides the long- term (during the next 6 months) evaluation of effecacy of this
treatment, potential predictive role of brain-derived neurotrophic factor (BDNF)
Val66Met genetic polymorphism in rTMS treatment response was analysed, as a factor
of neuronal plasticity, also involved in patogenesis of major depression
Subjects and methods Twenty patients with diagnoses of treatment resistant major
depression, at fixed doses of antidepressants, were treated with combination of rTMS
and PSD. rTMS was applied during two consecutive weeks (5 days/week) with
frequency of 1Hz, at 110% intensity of resting motor threshold, over the right dorso-
lateral prefrontal cortex (DLPFC), in the morrning hours.
Partial sleep deprivation was applied once during every week, as late partial sleep
deprivation (patients were woke up at 01.30 a.m.). Patients were randomly assigned to
receive rTMS+PSD, or sham+PSD.
Clinical evaluation was done using the following rating scales: the modified Hamilton
depression rating scale (HDRS-24), Montgomery-Asberg rating scale (MADRS ) and
Clinical global impression scale (CGI-S), at baseline, after 2 weeks of treatment and one
week after treatment, as well as long-term follow-up (only patients treated with active
rTMS).
Results. Study results suggest clinically relevant response, lasted up to 6 months in
patients with unipolar major depression at fixed doses of antidepressants.
After 2 weeks of treatment, patients treated with active rTMS had the average
improvement of 39% score reduction on HDRS. Total antidepressant response (defined
as HDRS score reduction ≥ 50%) had 36% of patients treated with active arm. Another
45% of patients showed only partial clinical response (HDRS score reduction ≥ 25%).
However, in 3 months follow-up period 64% of patients had significant improvement,
and was sustained in 55% of patients after 6 months. Total clinical remission (defined as
HDRS ≤ 10 ) was achieved in 5 of 11 patients treated with active rTMS.
Taking into account the obvious drawback of this study related to the small number of
patients in the active treatment arm, it`s still interesting to point out that all BDNF
Val66Val patients treated with active rTMS had significant improvement, but also
criteria of remission, with only one exception. In contrast, only one of BDNF Val66Met
patients had significant improvement and in another one with this genotype rTMS was
totally unsuccesfull, and patient was excluded from this study and treated with another
therapies.
Conclusions. This study suggest high antidepressant effecacy of combined rTMS and
PSD in patient with treatment resistant major depression. Antidepressant effect was
obvious immediately after treatment and was sustained during the long-term follow-up.
BDNF Val66Met genetic polymorphism is possible predictor of unsuccesfull treatment
response. | true |
Razvoj metaboličkog sindroma izazvanog kombinacijom stresa i ishrane obogaćene fruktozom - doprinos glukokortikoida u visceralnom masnom tkivu i hipotalamusu ženke pacova | The development of metabolic syndrome induced by the combination of stress and fructose enriched diet - the contribution of glucocorticoids in visceral adipose tissue and hypothalamus of female rats | Savremen način života karakterišu prekomeran unos fruktoze i svakodnevni
stres koji koincidiraju sa pojavom metaboličkog sindroma (MetS), vodećeg faktora
rizika za razvoj dijabetesa tipa 2 i bolesti srca. Narušen signalni put glukokortikoidnih
hormona (GH), regulatora metabolizma i odgovora na stres, može biti uključen u
patofiziologiju MetS-a.
Cilj ove doktorske disertacije bio je da se testira hipoteza da fruktoza u
kombinaciji sa hroničnim stresom dovodi do poremećaja u signalnom putu GH
odgovornih za razvoj karakteristika MetS-a kod ženki pacova. U tom cilju ispitani su
efekti devetonedeljnog izlaganja ishrani obogaćenoj fruktozom i nepredvidivom stresu
na prereceptorski metabolizam GH, ekspresiju i funkciju glukokortikoidnog receptora i
njime regulisanih gena uključenih u metabolizam masti u visceralnom masnom tkivu
(VMT). Pod istim uslovima ispitani su i leptinski signalni put u hipotalamusu i
inflamatorni status oba pomenuta tkiva.
Rezultati pokazuju da dugoročna ishrana obogaćena fruktozom stimuliše
adipogenezu i de novo lipogenezu u VMT-u, dok hronični nepredvidivi stres promoviše
lipolizu. Iako suprotni, ovi efekti su najverovatnije posledica aktivnosti GH. Nijedan od
tretmana nije uticao na leptinski signalni put niti na regulaciju unosa hrane u
hipotalamusu. Takođe, rezultati sugerišu da je inflamacija u VMT-u jedna od prvih
posledica konzumiranja fruktoze budući da se razvija pre nego što se mogu uočiti
gojaznost i inflamacija u hipotalamusu.
Sumarno, fruktoza svojim lipogenim uticajem uzrokuje metaboličke i
fiziološke promene koje doprinose razvoju metaboličkog sindroma, dok su posledice
hroničnog stresa izraženije u odnosu na efekte fruktoze i predominantno usmeravaju
metabolizam ka potrošnji energije, pri čemu u oba slučaja GH ostvaruju značajan
doprinos. | Hallmarks of modern lifestyle, fructose overconsumption and daily exposure to
unpredictable stress, coincide with the onset of metabolic syndrome (MetS), leading risk
factor for the development of type 2 diabetes and cardiovascular diseases.
Glucocorticoid hormones (GC), main regulators of metabolism and response to stress,
could be involved in the pathophysiology of MetS.
The aim of this doctoral dissertation was to test the hypothesis that the
combination of fructose diet and chronic unpredictable stress evokes disturbances in GC
signaling responsible for the development of MetS in female rats. We analyzed the
effects of 9 week exposure to fructose enriched diet and stress on GC prereceptor
metabolism, expression and function of glucocorticoid receptor and receptor regulated
genes involved in lipid metabolism in visceral adipose tissue (VAT). Also, we analyzed
leptin signaling in hypothalamus and inflammation in both tissues.
Results showed that long term fructose overconsumption stimulated
adipogenesis and de novo lipogenesis in VAT, while chronic stress promoted lipolysis.
Although opposing, these effects probably result from the GC activity. Leptin signaling
and appetite regulation in hypothalamus were not affected by these treatments. Also,
results suggest that VAT inflammation is one of the first consequences of fructose
overconsumption as it was developed before the onset of obesity and inflammation in
hypothalamus.
In summary, lipogenic effects of fructose elicit metabolic and physiologic
disturbances that contribute to the development of MetS, while chronic stress
predominantly directs metabolism to energy expenditure and exceeds the effects of
fructose. In both cases, GC play a significant role. | true |
Parametri antioksidacionog zaštitnog sistema i koncentracija teških metala u visceralnoj masi odabranih vrsta puževa i školjki Dunava, Tise i Velike Morave | Parameters of the antioxidant defence system and heavymetal concentrations in the visceral mass of selected snail and musselspecies from the Danube, Tisa and Velika Morava rivers | Slatkovodni mekušci su dobri model organizmi za proučavanje uticaja faktora sredine
u biomonitoring studijama zbog svojih karakteristika: slabe pokretljivosti, načina ishrane,
brojnosti, veličine tela i dr. Oni imaju sposobnost da u svom telu akumuliraju znatne količine
zagađujućih materija. Na osnovu analize sadržaja ovih materija u mekušcima moguće je
zaključivati o kvalitetu sredine.
Cilj ove doktorske disertacije bio je da se ispita uticaj različitih lokaliteta: reka Dunav,
Tisa i Velika Morava na promene u parametrima antioksidacionog zaštitnog sistema:
superoksid-dismutaze (SOD, EC 1.15.1.1), katalaze (CAT, EC 1.11.1.6), glutation-
peroksidaze (GSH-Px, EC 1.11.1.9), glutation-reduktaze (GR, EC 1.6.4.2), enzima faze II
biotransformacije glutation-S-transferaze (GST, EC 2.5.1.18), kao i koncentraciju glutationa
(GSH). Parametri su ispitivani u visceralnoj masi puža Viviparus acerosus i školjke
Corbicula fluminea. Interspecijske razlike su praćene analizom parametara antioksidacionog
zaštitnog sistema u visceralnoj masi puževa V. acerosus, Amphimelania holandrii i školjki C.
fluminea i Sinanodonta woodiana iz Velike Morave. Uticaj sezonskih faktora određivan je
tokom aprila i septembra u visceralnoj masi vrste V. acerosus iz Dunava, Tise i Velike
Morave. Analizirane su koncentracije 15 metala u visceralnoj masi sve četiri vrste
slatkovodnih mekušaca u aprilu.
Značajne razlike aktivnosti SOD u visceralnoj masi puža V. acerosus iz različitih reka
čine ovaj enzim pogodnim biomarkerom u ekofiziološkim studijama. Varijacije aktivnosti
GST u visceralnoj masi školjke C. fluminea iz različitih reka, ukazuju na to da se i ovaj enzim
može koristiti kao biomarker. Uporedni pregled parametara antioksidacionog zaštitnog
sistema četiri vrste slatkovodnih mekušaca izdvajaju A. holandrii kao potencijalnu
bioindikatorsku vrstu. Odsustvo jasnog sezonskog uticaja na GST u visceralnoj masi V.
acerosus izdvaja ovaj enzim kao potencijalno dobar biomarker.
Sposobnost bioakumulacije metala slatkovodnih mekušaca nije dovoljno istražena.
Prema podacima dobijenim u ovoj doktorskoj disertaciji V. acerosus predstavlja potencijalno
dobrog indikatora zagađenosti rečnih sistema metalima.
Biomarkeri imaju važno mesto u programima monitoringa i proceni biološkog efekta
različitih ksenobiotika. Biomarkeri otkrivaju stres prouzrokovan dejstvom zagađivača kao i
drugim promenama u životnoj sredini. Integracija biomarkera i hemijske analize neophodna
je kako bi se uspostavila veza između stresa i zagađenja. Dosadašnji literaturni podaci koji se
odnose na odgovor enzimskih i neenzimskih komponenti antioksidacionog zaštitnog sistema
kod slatkovodnih vrsta puževa su ograničeni što ukazuje na značaj podataka dobijenih u ovoj
studiji. | Freshwater molluscs are a good model organism for studying the effects of
environmental factors in biomonitoring studies due to their following characteristics: poor
mobility, diet, numbers, size. They have the ability to accumulate in their body significant
amounts of pollutants. By analysing the contents of these substances in molluscs we can draw
conclusions about the quality of the environment.
The aim of this PhD thesis was to investigate the effects of different localities, the
Danube, Tisa and Velika Morava Rivers, on changes in the parameters of the antioxidative
defence system: the enzymatic activities of superoxide dismutase (SOD; EC 1.15.1.1),
catalase (CAT; EC 1.11.1.6), glutathione-peroxidase (GSH-Px; EC 1.11.1.9), glutathione-
reductase (GR; EC 1.6.4.2), and phase II biotransformation enzyme glutathione-S-transferase
(GST; EC 2.5.1.18), and glutathione (GSH) concentration. The parameters were investigated
in the visceral mass of the snail Viviparus acerosus and mussel Corbicula fluminea.
Interspecies differences were followed by analysing the parameters of the antioxidative
defense system in the visceral mass of snails, V. acerosus, Amphimelania holandrii, and
mussels, C. fluminea and Sinanodonta woodiana, from the Velika Morava River. Seasonal
changes were determined during April and September in the visceral mass of species, V.
acerosus from the Danube, Tisa and Velika Morava. We analyzed the concentrations of 15
metals in the visceral mass of all four species of freshwater molluscs in April.
Significant differences in SOD activities in the visceral mass of the snail V. acerosus
from different rivers render this enzyme a suitable biomarker for ecophysiological studies.
Variations in GST activities in the visceral mass of the mussel C. fluminea from different
rivers can also be considered as a good biomarker. A comparative review of the parameters of
the antioxidative defence system in four species of freshwater molluscs distinguish A.
holandrii as a potential bioindicator species. The absence of a clear seasonal impact on GST
in the visceral mass of V. acerosus makes this enzyme a good biomarker.
The potential of freshwater molluscs to bioaccumulate metals studied in this
dissertation showed that V. acerosus is a good indicator of metal pollution in river systems.
Biomarkers have an important role in monitoring programs and assessments of the
biological effects of various xenobiotics. Biomarkers reveal the influence of stress caused by
pollution and other changes in the environment. The integration of biomarkers and chemical
analyses is necessary to establish a connection between stress and pollution. Previous
literature data related to the responses of enzymatic and nonenzymatic components of the
antioxidant defence system in freshwater snail species have been limited. This points to the
importance of the data obtained in this dissertation. | true |
Galektin-1 i funkcionalna svojstva ćelija trofoblasta čoveka in vitro | Galectin-1 and functional properties of human trophoblast cells in vitro. | U zoni interakcije majke i fetusa galektin-1 (gal-1) je eksprimiran na način koji
ukazuje na njegovu moguću ulogu u ćelijskoj adheziji, migraciji i invaziji trofoblasta,
procesima koji su ključni za implantaciju embriona i formiranje funkcionalne placente.
Međutim, uloga gal-1 u trofoblastu čoveka dosada nije bila ispitivana, što je predmet
istraživanja ovog doktorskog rada.
U tu svrhu korišćeni su odgovarajući funkcionalni in vitro testovi adhezije, migracije i
invazije na modelima invazivnog trofoblasta (izolovanom citotrofoblastu iz placenti prvog
trimestra normalne trudnoće, imortalizovanoj ekstravilusnoj ćelijskoj linija HTR-8/SVneo,
i, u manjoj meri, na horiokarcinomskoj ćelijskoj liniji JAr). Obzirom da gal-1 sintetišu i
ekstravilusne trofoblastne i decidualne stromalne ćelije in vivo, u ovom radu je ispitivan
mogući uticaj kako endogenog (upotrebom antitela prema gal-1 koja blokiraju i funkciju
gal-1), tako i egzogenog gal-1 (upotrebom dveju molekulskih formi rekombinantnog
humanog gal-1 dokazane biološke aktivnosti, od kojih jedna poseduje lektinsku aktivnost -
CS-gal-1, a druga ne - Ox-gal-1) na funkcionalna svojstva trofoblasta in vitro. Značaj
lektinskih interakcija za funkciju trofoblastnih ćelija ispitivan je funkcionalnim testovima u
prisustvu laktoze (inhibitornog šećera za lektinski tip interakcija galektina). Zbog činjenice
da se obrasci ekspresije, kao i funkcije galektina često znatno preklapaju, utvrđen je
galektinskog profila invazivnog trofoblasta na nivou RNK i proteina.
U ovom radu je po prvi put pokazan funkcionalni značaj nekog od galektina za
invazivnost trofoblasta čoveka in vitro, kvalifikujući gal-1 za važnog člana grupe molekula
koji učestvuju u trofoblastnoj invaziji. Pokazali smo da endogeni gal-1, kao i dodatni
egzogeni gal-1 (obe ispitivane molekulske forme) stimulišu invaziju trofoblasta, što
ukazuje na mogućnost da bi i u uslovima in vivo, gal-1, kako trofoblastnog, tako i
decidualnog porekla, mogao biti uključen u ovaj proces. Dobijeni rezultati su pokazali i da
se ovaj uticaj gal-1 na invazivnu sposobnost trofoblasta ostvaruje pre svega njegovom
lektinskom aktivnošću. Ovim radom je po prvi put utvrđen galektinski profil invazivnog
trofoblasta koji čine gal-1, gal-3, i do sada u trofoblastu čoveka neidentifikovani član
galektinske familije, gal-8.
Dobijeni rezultati predstavljaju napredak u razumevanju biološkog značaja gal-1 za
trofoblastnu funkciju in vitro. Pored toga, oni su i dobra osnova za dalja ispitivanja
mogućeg uticaja ovog lektina, kao i drugih članova galektinske familije koji su
eksprimirani u invazivnom trofoblastu čoveka na procese koji su ključni za uspešnost
trudnoće. | Galectin-1 (gal-1) is expressed at the fetomaternal interface in a manner suggestive of a
potential involvement in cell adhesion, migration and invasion of the human trophoblast.
These processes are considered essential for the embryo implantation and formation of the
functional placenta. However, no studies have examined the role of the gal-1 in the human
placenta. Hypothesis that gal-1 may be relevant for trophoblast invasion was addressed by
this thesis for the first time.
To this end appropriate functional tests of cell adhesion, migration and invasion were
performed
in
vitro,
using
invasive
trophoblast
models
(isolated
first
trimester
cytotrophoblast, immortalized extravillous HTR-8/SVneo cell line and choriocarcinoma
JAr cell line). Since gal-1 is synthesised by both extravillous cytotrophoblast and decidual
stromal cells in vivo, selected approaches addressed possible effect of both endogenous and
exogenous gal-1. Use of function blocking anti-gal-1 antibody provided an insight into
participation of endogenous gal-1, while supplementation by either molecular form of
biologically active recombinant human gal-1, CS-gal-1 (with lectin activity) or Ox-gal-1
(without lectin activity) indicated relevance of exogenous gal-1 for trophoblast invasion in
vitro. The importance of lectin-type interactions was investigated primarily using lactose
(an inhibitory sugar for galectins). Since the expression patterns and functions of galectins
often overlap, galectin profile of the invasive trophoblast at RNA and protein levels was
established here.
This is the first study to address the functional relevance of any galectin for human
trophoblast invasivness in vitro, qualifying gal-1 as an important member of the trophoblast
cell invasion machinery. It is documented here that endogenous gal-1, as well as additional
exogenous gal-1 (both forms, but particularly the form with lectin activity) participate in
trophoblast invasion, suggesting possible involvement of trophoblastic and decidual gal-1
in this process in vivo. Galectin profile of the invasive trophoblast was established here for
the first time, which comprised of gal-1, gal-3 and gal-8.
The obtained findings improve current understanding of the biological relevance of
the gal-1 for the trophoblast function in vitro. These results provide the basis for further
investigation of the mechanisms and effector molecules involved in gal-1 induced
processes. Potential relevance of other expressed members of the galectin family for the
invasive trophoblast remains to be elucidated. | true |
Značaj fitoplanktona kao indikatora eutrofikacije u akvatorijumu Bokokotorskog zaliva | Importance of phytoplankton as indicator of eutrophication in aquatorium of the Boka Kotorska Bay | Fitoplanktonski organizmi su primarna karika u lancima ishrane, odnosno primarni producenti organskih supstanci. Sa jedne strane se ističe njihova važnost u ishrani, a sa druge njihov povećan razvoj može da dovede do neželjenih posljedica za ekosistem. Cilj ovog istraživanja je bio da se prikaže kvalitativno i kvantitativno stanje fitoplanktona u Bokokotorskom zalivu, uključujući tu u posljednjem slučaju i biomasu fitoplanktona, a izraženu preko koncentracije hlorofila a. Preko kvalitativnog i kvantitativnog aspekta fitoplanktona može se stvoriti okvirna slika o stepenu eutrofikacije u zalivu i ukazati na moguće opasnosti i mjere koje treba preduzeti da bi se izbjegle negativne posljedice. Sve nabrojane biološke parametre nije moguće odvojiti od fizičko-hemijskih parametara sa kojima su u međusobnoj interakciji i koji direktno utiču na količinu i sastav fitoplanktona. Brojnost fitoplanktona je često dostizala vrijednosti 106 ćelija/l, što je karakteristično za eutrofno područje i nije bila ispod 105 ćelija/l tokom cijelog istraživanja, što opet ukazuje na povećanu eutrofikaciju. Fitoplanktonska grupa koja je dominirala tokom istraživanja su bile dijatomeje, koje su eurivalentna grupa organizama. Druga grupa organizama koja je bila prisutna su dinoflagelate, koje su interesantne s obzirom da mnoge od njih proizvode toksine koji mogu biti štetni za čovjeka. Među dijatomejama zabilježene su vrste koje su karakteristične za područja bogata nutrijentima, što govori o promjenama u zalivu. Alge koje su nađene, a koje preferiraju povećanu količinu nutrijentima su Chaetoceros affinis, Dactyliosolen fragilissimus, Leptocylindrus danicus, Navicula spp., Pseudonitzschia spp., Thalassionema nitzschioides i dr. Analizirani su indeksi diverziteta Shannon-ov indeks diverziteta (H’), recipročan Simpson-ov indeks(1/D) i Margalef-ov indeks (d) za područje Bokokotorskog zaliva i nađeno je da su indeksi bili manji kada je brojnost fitoplanktona bila veća i kada je bila prisutna dominacija određenih vrsta, kao npr. u julu mjesecu kada je dominirala vrsta Thalassionema nitzshioides. To ukazuje na činjenicu da je diverzitet manji kada je prisutna dominacija određenih vrsta. U odnosu na biomasu fitoplanktona izraženu koncentracijom hlorofila a, prema raznim kriterijumima (Håkanson et al., 1994; Ignatiades 2005), zalivsko područje je okarakterisano kao mezo-eutrofno područje. S obzirom da parametri koji pokazuju stepen eutrofikacije ukazuju da Bokokotorski zaliv počinje da dobija karakteristike eutrofnog, treba preduzeti mjere da bi se spriječilo dalje napredovanje prema još višem stepenu eutrofije. Bokokotorski zaliv je jedan osjetljiv ekosistem, a naročito Kotorski zaliv koji je zatvoreniji, sa slabijom dinamikom vodenih masa i pod većim pritiskom eutrofikacije. slabijom dinamikom vodenih masa i pod većim pritiskom eutrofik | Phytoplankton organisms are primary link in the food chain and primary producers of organic substances. On one side, it is emphasissed their importance in the diet and, on the other, their excessive growth can lead to undesirable consequences for the ecosystem. The aim of this study is to present qualitative and quantitative situation of phytoplankton in the Boka Kotorska Bay, including in the last case phytoplankton biomass too, which is expressed through the concentration of chlorophyll a. Through qualitative and quantitative aspects of phytoplankton can be create a general overview of the degree of eutrophication in the Bokakotorska Bay and to point out possible hazards and precautions to be taken to avoid negative consequences. All these biological parameters can not be separated from the physical-chemical parameters with which they are in interaction and which directly affect the quantity and qualitative composition of phytoplankton. The abundance of phytoplankton often reached values of 106 cells/l, which is typical for an eutrophic area and abundance of phytoplankton wasn’t below 105 cells/l throughout the study, which again points to increased eutrophication. Phytoplankton group that has dominated almost during all investigated period were diatoms, which are eurivalent group of organisms. Another group of organisms that were present were dinoflagellates, which are interesting because they produce toxins that can be harmful to humans. Among diatoms, it was found algae that are otherwise characteristic of nutrients-enriched areas, which indicate changes in the Bay. Noticed algae that prefer higher nutrients concentration were Chaetoceros affinis, Dactyliosolen fragilissimus, Leptocylindrus danicus, Navicula spp., Pseudo-nitzschia spp. and Thalassionema nitzschioides. The following diversity were analyzed: Shannon's diversity index (H '), the reciprocal of Simpson's index (1 / D) and Margalef's index (d) for the Boka Kotorska Bay. It was found that indices were lower when phytoplankton abundance was higher and when the dominance of certain species was noticed, such in current study it was in July, when dominated species Thalassionema nitzshioides. That suggests that diversity is lower when occurrs predominance of certain species. In relation to phytoplankton biomass expressed in chlorophyll a concentration, according to various criteria (Håkanson et al., 1994; Ignatiades 2005), Boka Kotorska Bay was identified as meso-eutrophic area. Considering that parameters which indicate the degree of eutrophication suggest that the Boka Kotorska Bay begins to take characteristic of eutrophic area, it should be taken measures to prevent further progression to an even higher degree of eutrophication. Boka Kotorska Bay is one particularly sensitive ecosystem, especially the Kotor Bay, which is closed and with the weaker dynamics of water masses and under the growing pressure of eutrophication. | false |
Metilovanje promotora i ekspresija estrogenskog receptora beta (ERß) u invazivnim karcinomima dojke | Estrogen receptor beta (ERß) promoter methylation and expression in invasive breast cancers | Invazivni karcinom dojke je najčešći kancer kod žena. Pored genetičkih i
epigenetički faktori imaju važnu ulogu u njegovoj inicijaciji i progresiji. Cilj ove studije bio
je ispitati da li metilacija promotora ON, gena za ERβ protein (ESR2) utiče na njegovu
ekspresiju (na nivou iRNK i proteina) i utvrditi povezanost metilacionog indeksa promotora
ON sa nivoima ERβ1-iRNK i ERβ1 proteina, kao i sa kliničkim i patohistološkim
parametrima. U ovoj studiji analiziran je 131 arhivski uzorak kancera dojke. Pomoću
posebno dizajniranog PCR testa iz dva koraka, amplifikovan je i kvantifikovan specifični
region promotora ON ESR2 gena. Za kvantifikaciju ERβ1-iRNK korišćena je metoda
kvantitativnog RT-PCR u realnom vremenu. Metoda „Western Blot“ je korišćena za
relativnu kvantifikaciju ERβ1 proteinske izoforme. Dobijeni podaci, metilacioni indeks
promotora ON, nivoi iRNK i ERβ1 proteina, analizirani su i korelisani sa
kliničkopatološkim parametrima neparametrijskim statističkim testovima. Nađeno je da je
metilacioni indeks promotora ON ERβ gena značajno veći u grupi pacijentkinja sa
pozitivnim, u odnosu na grupu sa negativnim statusom aksilarnih limfnih čvorova. Takođe,
pronađena je značajna pozitivna korelacija metilacionog indeksa promotora ON ERβ gena
sa nivoom estrogenskog receptora α. Na osnovu dobijenih rezultata pokazano je da
metilacioni indeks promotora ON, ERβ gena može biti pouzdaniji marker prognoze kancera
dojke nego ekspresija ERβ1-iRNK ili proteina ERβ1. | Breast cancer is the most common invasive cancer in women. Beside genetic,
epigenetic factors influence its initiation and progression. The purpose of this study was to
test whether methylation of the promoter ON of the gene for ERβ protein (ESR2)
influences its expression (on mRNA and protein level) and to correlate the methylation
index of the ON promoter, ERβ1-mRNA and ERβ1 protein levels with clinicopathological
parameters. In this study, 131 archival samples of breast cancer tissue were used. Custom
designed two step PCR method, was done for amplification and relative quantification of
the specific region of the ON promoter of the ESR2 gene. Measuring of ERβ1-mRNA was
done by quantitative real time RT-PCR, and relative quantification of ERβ1 protein isoform
was done by Western blot. Statistical analysis was performed and results for methylation
index of ON promoter, mRNA and ERβ1 protein levels were correlated with
clinicopathological parameters. The methylation index of ERβ-ON promoter was
significantly higher in a group of patients with positive axillary lymph node status, than in a
group with negative one. A significant positive correlation between methylation index of
ON promoter and ERα protein levels was obtained. It can be concluded that methylation
index of the ERβ-ON promoter could be more confident parameter for prediction and/or
prognosis of breast cancer, than measuring of ERβ1-mRNA or ERβ1 protein levels. | true |
Uloga polimorfizama i ekspresije gena za hemokine CX3C ligand 1 i CXC ligand 16 i njihove receptore u nastanku i progresiji multiple skleroze u Srbiji | Roles of polymorphisms and expression of genes coding for chemokines CX3C ligand 1 and CXC ligand 16 and their receptors in the development and progression of multiple sclerosis in Serbia | Multipla skleroza je hroniĉna inflamatorna, autoimunska, demijelinizaciona i neurodegenerativna bolest centralnog nervnog sistema (CNS-a). Hemokini i njihovi receptori predstavljaju znaĉajne medijatore inflamacije koji uĉestvuju u patogenezi odreĊenih hroniĉnih inflamatornih i autoimunskih bolesti meĊu kojima je i multipla skleroza. Ciljni hemokini u ovoj studiji, CX3C ligand 1 (CX3CL1) i CXC ligand 16 (CXCL16), specifiĉni su po tome što postoje u dve forme - kao transmembranski adhezivni molekuli i kao solubilni hemoatraktanti koji nastaju nakon proteolitiĉkog seĉenja vanćelijskih hemokinskih domena njihovih transmembranskih formi. U toku inflamatornog odgovora, na membrani endotelnih vaskularnih ćelija eksprimirani su CX3CL1 i CXCL16, a na membrani leukocita receptori za CX3CL1 (CX3CR1) i CXCL16 (CXCR6), te ovi hemokini i njihovi receptori posreduju u prodiranju leukocita iz krvi u tkivo zahvaćeno inflamacijom, podsticanjem hemotaksije i adhezije leukocita za aktivirani endotel krvnog suda. Ova studija obuhvata genetsko-epidemiološku analizu polimorfizama zamena pojedinaĉnih nukleotida u kodirajućim regionima gena, koje rezultuju zamenama aminokiselina. To su polimorfizmi V249I i T280M u genu za CX3CR1, i I123T i A181V u genu za CXCL16. U prethodnim studijama je pokazano da ovi genski polimorfizmi menjaju funkcionalna svojstva CX3CR1 i CXCL16, kao i da su asocirani sa patogenezom odreĊenih hroniĉnih inflamatornih bolesti. Uzimajući to u obzir, ova studija je imala za cilj da po prvi put ispita asocijaciju navedenih polimorfizama u genima za CX3CR1 i CXCL16 sa nastankom i progresijom multiple skleroze. Primenom alel-specifiĉne PCR metode i PIRA PCR-RFLP metode detektovani su genotipovi polimorfizama V249I i T280M u genu za CX3CR1, kod zdravih kontrola i pacijenata sa multiplom sklerozom. UtvrĊeno je da haplotip I249T280 u genu za CX3CR1 ima znaĉajno veću uĉestalost kod pacijenata sa relapsno-remitentnom (RR) formom, u odnosu na pacijente sa sekundarno-progresivnom (SP) formom multiple skleroze, što znaĉi da ovaj haplotip ima protektivni efekat na progresiju RR u SP formu bolesti... | Multiple sclerosis is a chronic inflammatory, autoimmune, demyelinating and neurodegenerative disease of the central nervous system (CNS). Chemokines and their receptors are important mediators of inflammation, which are involved in pathogenesis of certain chronic inflammatory and autoimmune diseases including multiple sclerosis. Chemokines of interest in this study, CX3C ligand 1 (CX3CL1) and CXC ligand 16 (CXCL16), are specific in that they can exist either as transmembrane adhesion molecules or soluble chemoattractants being generated by proteolytic cleavage of their transmembrane forms’ extracellular domains. During the inflammatory response, CX3CL1 and CXCL16 are expressed on the surface of vascular endothelium, while the leukocytes produce membrane receptors for CX3CL1 (CX3CR1) and CXCL16 (CXCR6). Therefore, these chemokines and their receptors mediate the infiltration of leukocytes from blood into the inflamed tissue areas, by stimulation of both chemotaxis and adhesion of leukocytes to the activated endothelium of blood vessels. This study is based on genetic epidemiological analysis of single nucleotide polymorphisms, which are located in the coding regions of genes and result in amino acids’ substitutions. These are V249I and T280M substitutions in the gene coding for CX3CR1, and I123T and A181V substitutions in the gene coding for CXCL16. In previous studies these polymorphisms have been associated with the functional properties of CX3CR1 and CXCL16 as well as the pathogenesis of certain chronic inflammatory diseases. Therefore, this study aimed to investigate the association of the polymorphisms in CX3CR1 and CXCL16 genes with the development and progression of multiple sclerosis. Using the allele-specific PCR and PIRA PCR-RFLP methods, genotypes of CX3CR1 V249I and T280M polymorphisms were detected in healthy controls and patients with multiple sclerosis. Following statistical analysis showed significantly higher frequency of CX3CR1 I249T280 haplotype in patients with relapsingremitting (RR) form, compared to patients with secondary-progressive (SP) form of multiple sclerosis, so this haplotype had a protective effect on progression of RR to SP form of the disease... | false |
Analiza interakcija laktobacila sa crevnom mukozom pacova | Analysis of interactions of lactobacilli with rat gut mucosa | Probiotici predstavljaju žive mikroorganizme koji, nakon unošenja u gastrointestinalni
(GI) trakt, ostvaruju pozitivne efekte na zdravlje domaćina. Reakcija domaćina na unos
probiotika u velikoj meri zavisi od sposobnosti bliske interakcije bakterija sa epitelnim
ćelijama GI mukoze. S druge strane, s obzirom na nepoznatu antigenu prirodu unetih
bakterija, kod imunokompromitovanog domaćina je prisutan rizik od imunske reakcije
crevnog tkiva što može da dovede do narušavanja integriteta mukozne barijere i hronične
inflamacije u GI traktu.
Cilj istraživanja bio je ispitivanje efekta soja Lactobacillus fermentum BGHI14,
alohtonog za GI trakt pacova, nakon peroralnog unosa u zdrave pacove i u pacove sa
kolitisom indukovanim TNBS-om (trinitrobenzensulfonat). Metodološki, u eksperimentima
su praćeni: telesna masa pacova i ekspresija iRNK za parametre inflamacije (IL-1β, TNFα,
IL-17F), pokazatelje ćelijskog stresa (HSP70) i parametre uključene u održavanje
integriteta mukoze (TJP1); zatim su rađene histološke i biohemijske analize i analiza
sastava crevne mikroflore. Rezultati istraživanja su pokazali imunostimulišuće delovanje
soja BGHI14 na nivou tkiva kolona nakon unosa u GI trakt zdravih pacova u prvih 16 dana
peroralnog tretmana pacova. Iako se, histološki, sa produžetkom tretmana pacova sojem
BGHI14 tokom narednih 12 dana, imunska reakcija u tkivu kolona povukla, nivoi iRNK za
proinflamatorne IL-1β i TNFα citokine ostali su povišeni što ukazuje na rizik od
nekontrolisane inflamacije u slučaju naknadnog oštećenja epitelne barijere kolona.
U istraživanju je pokazano da je nakon imunostimulacije tkiva kolona sojem BGHI14,
u slučaju indukcije kolitisa, ali uz nastavak tretmana sojem BGHI14 došlo do indukcije
transkripcije iRNK za HSP70 protein, kako u ranoj (48 h) tako i u kasnoj (7 dana) akutnoj
fazi kolitisa. Povećanje transkripcije Hsp70 gena, sudeći po histološkim i inflamatornim
pokazateljima, kao i promenama telesnih masa pacova tokom eksperimenta, nije bilo
praćeno pozitivnim delovanjem soja. S druge strane, prestanak tretmana sojem BGHI14
nakon indukcije kolitisa doveo je do manje izraženog pada telesnih masa životinja u
poređenju sa kontrolnim pacovima sa kolitisom, iako bez pratećeg smanjenja inflamatorne
reakcije u tkivu kolona, bar u kasnoj akutnoj fazi bolesti (sedam dana od indukcije). Na
osnovu opisanog može se zaključiti da su se u interakciji soja BGHI14 sa zdravim tkivom
kolona aktivirali mehanizmi adaptivne imunosti u vidu memorijskih T-limfocita koji su
indukovani nakon povrede u slučaju kontinuiranog prisustva istog bakterijskog antigena.
Nasuprot, terapijski tretman pacova sojem BGHI14 doveo je do pozitivnog efekta u kasnoj
akutnoj fazi kolitisa ocenjujući po promenama telesnih masa životinja kao i na osnovu
parametara oštećenja mukozne barijere kolona. Dobijeni rezultati ukazuju da bi primena
imunostimulišućih probiotika neposredno po nastanku oštećenja crevnog tkiva predstavljala
efikasan pristup u lečenju inflamatornih oboljenja crevnog trakta, iako su na ovu temu
potrebna dodatna istraživanja.
S obzirom na značaj koji interakcija bakterija sa crevnom mukozom ima u ostvarivanju
njihovog probiotičkog potencijala, u istraživanju su testirani mehanizmi kojima se može
poboljšati vezivanje laktobacila kao potencijalnih probiotika za GI mukozu pacova. U tom
smislu praćeni su efekti proteina sa mucin-vezujućim domenom (MbpL) poreklom iz soja
Lactococcus lactis subsp. lactis BGKP1-20 na sposobnost vezivanja bakterijskog domaćina
za mukozu ileuma i kolona u uslovima ex vivo i in vivo, kao i u in vitro esejima sa
izolovanim želudačnim mucinom i mucin-produkujućim HT29-MTX ćelijama kolona.
Osim homolognog domaćina, za funkcionalna ispitivanja korišćen je potencijalni
probiotički soj Lactobacillus salivarius BGHO1. Rezultati su pokazali da eksprimirani
MbpL protein nije bio dovoljan za vezivanje soja BGHO1 za crevnu mukozu, iako je
pozitivno uticao na vezivanje BGKP1-20 soja za svinjski želudačni mucin i HT20-MTX
ćelije u kulturi. Moguće je da je fenotipski efekat ekspresije MucBP proteina kod
laktokokalnog BGKP1-20 soja veći u odnosu na BGHO1 soj, s obzirom na prisustvo drugih
potencijalnih adhezivnih faktora koji se sintetišu u laktobacilima.
Koristeći istu metodologiju testirana je uloga koju u vezivanju za mukozne površine GI
trakta ostvaruje agregacioni faktor AggL poreklom iz soja BGKP1-20. Agregacioni faktor
eksprimiran na površini bakterijskih ćelija često se dovodi u vezu sa povećanim afinitetom
bakterijskog domaćina za GI mukozu. Naši rezultati su pokazali da, iako bi, povećanjem
površinske hidrofobnosti mogao doprineti vezivanju za površinu kolona kod laktokokalnih
sojeva sa niskom ukupnom hidrofobnošću površine, eksprimirani agregacioni faktor
generalno otežava vezivanje bakterija za površine izložene dinamičkim peristaltičkim
kontrakcijama kakva je u najvećoj meri površina ileuma. Na taj način bi sojevi koji
sintetišu faktor agregacije mogli olakšati uklanjanje patogena ali bez sposobnosti
ostvarivanja direktnog efekta na imunski sistem domaćina. | Probiotics are life microorganisms that, after administration in gastrointestinal (GI)
tract, excert positive effects on host’s health. Host reaction to probiotic administration
largely depands on ability of bacteria to closely interact with epithelial cells of GI mucosa.
On the other hand, considering the unknown antigenic nature of administered bacteria, in
immune-compromised host there is a risk of immune reaction of gut tissue which could
lead to disturbance of mucosal barrier integrity and chronic inflammation in GI tract.
The aim of the study was to examine the effects of the strain Lactobacillus fermentum
BGHI14, which is alochtonous to GI tract of rats, after peroral administration in healthy
rats and in rats with TNBS (trinitrobenzenesulfonate)-induced colitis. Methodologically,
following parameters were examined in experiments: body masses of rats and expression of
mRNA of inflammatory cytokines (IL-1β, TNFα, IL-17F), as well as markers of cellular
stress (HSP70) and barrier integrity maintenanace (TJP1); next histological and
biochemical analyses and microflora profiling were performed. Results of the study
indicated immune-stimulating effects of BGHI14 in colon tissue after administration in GI
tract of healthy rats in first 16 days of peroral treatment of rats. Although, histologically,
with prolongation of BGHI14 treatment of rats for next 12 days, immune reaction in colon
tissue had retreated, mRNA levels of proinflammatory IL-1β and TNFα cytokines remained
elevated which points to the risk of uncontrolled inflammation in the case of subsequent
colon epithelial barrier damage.
In research it was shown that after immunostimulation of colon tissue with BGHI14, in
the case of colitis induction, but with sequel of treatment with bacteria, transcription of
Hsp70 gene was induced, in early (48 h) as well as late (7 days) acute phase of colitis. The
increase of Hsp70 gene transcription, according to histological and inflammatory markers,
as well as body mass changes during the experiment, was not followed by positive action of
the strain. On the other hand, the cessation of treatment with BGHI14 after colitis induction
had led to less pronounced body mass decrease compared to control colitic rats, but without
concomitant reduction in inflammatory scores in colon tissue, at least in late acute phase of
disease (seven days from induction). Based on the above, it could be concluded that
mechanisms of adaptive immunity including memory T-cells might had been activated
during interaction of BGHI14 with healthy colon tissue and those systems were induced
after the injury in the case of continuous presence of same bacterial antigen. Contrary,
therapeutic treatment with BGHI14 had brought about the protection in late acute colitis
phase according to body mass changes as well as indicators of mucosal barrier disruption.
Obtained results implicate that use of immune-stimulating probiotics immediately after
colon tissue damage would represent effective approach in treatment of bowel
inflammatory diseases, though more detailed research is needed in this area.
Considering the importance of interaction of bacteria with gut mucosa in achieving its
probiotic potential, in current research mechanisms for improvement of potential probiotic
lactobacilli binding to rat’s GI mucosa were tested. In this respect the effects of mucin-
binding domain containing protein (MbpL) from strain Lactococcus lactis subsp. lactis
BGKP1-20 in ability of bacterial host to bind to ileal and colonic mucosa in vivo and ex
vivo, as well as in assays in vitro with isolated mucin and mucin-producing HT29-MTX
colonic cells were tested. Aside from using the homologous host, for functional tests we
have used potential probiotic strain Lactobacillus salivarius BGHO1. Results have
demonstrated that the expression of MbpL protein was not sufficient for binding of BGHO1
to gut mucosa, although it influenced positively the binding of BGKP1-20 to pig gastric
mucin and HT29-MTX cells in culture. It is possible that in lactococcal strain BGKP1-20,
phenotypic effects of MucBP protein expression are more pronounced compared to
BGHO1 strain, because of presence of other potential adhesion factors synthesized in
lactobacilli strains.
Simultanously, utillizing the same methodology, the role of aggregation factor AggL
from BGKP1-20 in binding to GI tract mucosal surfaces was tested. Expression of
aggregation factor is commonly correlated to enhanced affinity of bacterial host to GI
mucosa. Our results have demonstrated that, although, by increasing surface
hydrophobicity, in lactococcal strains it could contribute to binding to colonic surface, the
expression of aggregation factor generally aggravates binding of bacteria to surfaces
exposed to dynamic peristaltic contractions such as those present in ileum. That way strains
expressing aggregation factor could facilitate removal of pathogens but without directly
affecting the host’s immune system. | true |
Biološka aktivnost etarskih ulja i ekstrakata odabranih vrsta iz familije Apiaceae | Biological activities of essential oils and extracts of selected Apiaceae species | U ovom radu analizirana je biološka aktivnost etarskih ulja i ekstrakata sledećih
vrsta familije Apiaceae: Eryngium serbicum Pančić, Seseli pallasii Besser, Seseli
libanotis (L.) Koch subsp. libanotis, Seseli libanotis (L.) Koch subsp. intermedium
(Rupr.) P. W. Ball, Peucedanum officinale L., Peucedanum longifolium W. et K.,
Peucedanum aegopodioides (Boiss.) Vand., Peucedanum alsaticum L., Pastinaca sativa
L., Heracleum sphondylium L., Tordylium maximum L., Cachrys cristata DC. i
Opopanax hispidus (Friv.) Griseb. Biljni materijal je ekstrahovan metanolom, acetonom
i etil-acetatom, dok su vodeni ekstrakti liofilizirani. Etarska ulja navedenih vrsta i
odabrani ekstrakti su testirani sa aspekta antimikrobne i antioksidativne aktivnosti. Kao
pozitivne kontrole korišćeni su komercijalni antibiotici i antimikotici, a u testovima za
utvrđivanje antioksidativne aktivnosti BHA i vitamin C. Antimikrobna aktivnost
etarskih ulja i ekstrakata testirana je na: Gram-negativne bacterije (Escherichia coli,
Pseudomons
aeruginosa,
Salmonella
typhimurium,
Salmonella
enteritidis
i
Enterobacter cloacae) i Gram-pozitivne bacterije (Listeria monocytogenes, Bacillus
cereus, Micrococcus flavus i Staphylococcus aureus), kao i na mikromicete (Aspergillus
niger, A. ochraceus, A. versicolor, A. fumigatus, Penicillium funiculosum, P.
ochrochloron, Trichoderma viride i Candida albicans). Kompozicija etarskih ulja je
analizirana
korišćenjem
GC
i
GC/MS
metoda.
Metoda
mikrodilucije
na
mikrotitracionim pločama je korišćena za testiranje antimikrobne aktivnosti.
Određivane su minimalne inhibitorne i minimalne baktericidne/fungicidne koncentracije
(MIC i MBC/MFC). DPPH i ABTS analize, kao i ukupan sadržaj fenola i flavonoida, su
korišćene za određivanje antioksidativne aktivnosti.
Etarsko ulje Eryngium serbicum je bilo najefikasniji antimikrobni agens, a slede
ga ulja vrste roda Peucedanum, dok je etarsko ulje Seseli pallasii imalo najnižu
antimikrobnu aktivnost. Acetonski i etil-acetatni ekstrakti su imali bolji antimikrobni
potencijal od metanolnih i vodenih ekstrakata. Salmonella typhimurium je najosetljivija
bakterija, dok je Listeria monocytogenes, pokazala dobru otpornost na ispitivana ulja i
ekstrakte. Rezultati testiranih gljiva pokazuju da je Aspergillus ochraceus bila
najosetljivija, dok je Candida albicans bila najrezistentnija.
Vodeni ekstrakti su bili najefikasniji u neutralizaciji DPPH radikala, dok su
acetonski ekstrakti bili najefikasniji kod ABTS testa. Ekstrakti Peucedanum officinale
su pokazali najbolju antioksidativnu aktivnost. | In this work the biological activity of essential oils and extracts from following
Apiaceae species was analyzed: Eryngium serbicum Pančić, Seseli pallasii Besser,
Seseli libanotis (L.) Koch subsp. libanotis, Seseli libanotis (L.) Koch subsp.
intermedium (Rupr.) P. W. Ball, Peucedanum officinale L., Peucedanum longifolium W.
et K., Peucedanum aegopodioides (Boiss.) Vand., Peucedanum alsaticum L., Pastinaca
sativa L., Heracleum sphondylium L., Tordylium maximum L., Cachrys cristata DC.,
Opopanax hispidus (Friv.) Griseb. Herbal samples, were extracted with methanol,
acetone and ethil-acetate, while aqueous extracts were dried by freeze-dryer. Essential
oils of above species, and selected extracts were evaluated for their antimicrobial and
antioxidant activities. As a positive control commercial antibiotics and fungicides were
used, while BHA and Vitamin C were used in antioxidant test. The antimicrobial
activity of essential oils and extracts was investigated against: Gram (-) bacteria
(Escherichia coli, Pseudomons aeruginosa, Salmonella typhimurium, Salmonella
enteritidis, Enterobacter cloacae) and Gram (+) bacteria (Listeria monocytogenes,
Bacillus cereus, Micrococcus flavus, Staphylococcus aureus), and micromycetes
(Aspergillus niger, A. ochraceus, A. versicolor, A. fumigatus, Penicillium funiculosum,
P. ochrochloron, Trichoderma viride, Candida albicans). The volatile constituents were
analyzed using GC and GC/MS procedures. Microdilution method on microtitration
plates was used for antimicrobial assay. Minimum inhibitory and minimum
antibacterial/antifungal concentrations (MIC and MBC/MFC) were determined. DPPH
and ABTS assay and total phenol and flavonoid content were used for antioxidant
activity determination.
Essential oil of Eryngium serbicum was the most effective antimicrobial agents,
followed by Peucedanum species, while Seseli pallasii essential oil showed the lowest
antimicrobial activity. Acetone and ethil-acetate extracts showed better antimicrobial
potential than methanol and aquous extracts. Salmonella typhimurium were the most
susceptible bacteria, while Listeria monocytogenes, showed good resistance to all of oils
and extracts investigated. The results show that the tested micromycetes Aspergillus
ochraceus was the most sensitive, while Candida albicans was the most resistant.
Aquous extracts were the most effective DPPH radical scavangers, while
acetone extracts were the most effective at ABTS test. Exstracts of Peucedanum
officinale was with highest antioxidant activity. | true |
Strukturna i funkcionalna analiza 5' regulatornog regiona gena SMAD4 u karcinomu pankreasa čoveka | Structural and functional analysis of smad4 gene 5' regulatory region in human pancreatic cancer | Karcinom pankreasa spada u najinvazivnije i najprogresivnije humane maligne bolesti. Genetske promene karakteristične za ovu bolest su aktivacija onkogena KRAS i inaktivacija tumor supresor gena p16, p53 i SMAD4. Novija istraživanja ukazuju na povezanost aktivacije onkogena KRAS i inaktivacije tumor supresor gena SMAD4 u pankreasnoj karcinogenezi. Struktura promotora gena SMAD4 nedovoljno je proučena i malo se zna o mehanizmima regulacije transkripcije ovog gena. Ova studija je imala za cilj da istraži genetske i epigenetske promene u 5’ regulatornom regionu gena SMAD4 kao potencijalne mehanizme njegove inaktivacije u karcinomu pankreasa, kao i njihovu udruženost sa prisustvom mutacije u kodonu 12 gena KRAS u malignom tkivu. Strukturna analiza promotora gena SMAD4 je pokazala da su dva mononukleotidna ponovka, -462T(15) and -4T(12), polimorfna u karcinomu pankreasa i kolorektalnom kanceru, dok je funkcionalna analiza pokazala da broj timina u elementu -4T(12) može biti od značaja za regulaciju transkripcije. Haplotip 14/10 je bio najčešći haplotip u tkivu karcinoma pankreasa, prisutan u 88% slučajeva. Haplotipovi 10/12 i 9/12 su otkriveni u po 4% slučajeva u tkivu kolorektalnog kancera. U najvećem broju tkiva karcinoma pankreasa (98%) bila je prisutna ili mutacija u kodonu 12 gena KRAS ili haplotip 14/10 u promotoru gena SMAD4. Rezultati ovog istraživanja ukazuju da haplotipovi 14/10, 10/12 i 9/12 u promotoru gena SMAD4 predstavljaju genetičke markere koji mogu biti povezani sa specifičnom malignom bolešću, a njihova analiza potencijalno iskorišćena za dijagnostikovanje i praćenje bolesti, pojedinačno ili u kombinaciji sa analizom mutacije u kodonu 12 gena KRAS. | Pancreatic carcinoma is one of the most invasive and progressive human malignant neoplasms. This malignancy is characterized by activation of KRAS oncogene and inactivation of p16, p53 and SMAD4 tumor supressor genes. Recent results show association between KRAS activation and SMAD4 inactivation in pancreatic carcinogenesis. The structure of the SMAD4 gene promoter has not been clearly defined and little is known about the transcription regulatory mechanisms of this gene. This study was aimed at analyzing genetic and epigenetic changes in the 5’ regulatory region as potential mechanisms of SMAD4 gene inactivation in pancreatic carcinoma and their coexistance with KRAS codon 12 mutation in malignant tissues. Structural analysis of SMAD4 gene promoter has shown that two mononucleotide repeat elements, -462T(15) and -4T(12), are polymorphic in pancreatic carcinoma and colorectal cancer tissues, while functional analysis indicated that -4T(12) element may be important for transcriptional regulation. The haplotype 14/10 was the most common haplotype in pancreatic carcinoma tissue, present in 88% of cases. Haplotypes 10/12 and 9/12 were detected in 4% of colorectal cancer tissues each. In majority of pancreatic carcinoma cases (98%) either KRAS codon 12 mutation or SMAD4 gene promoter haplotype 14/10 was present. These findings suggest that haplotypes 14/10, 10/12 and 9/12 in the SMAD4 gene promoter represent genetic markers that might be associated with specific malignancy and potentially exploited for diagnostics and monitoring of disease, alone or in combination with KRAS codon 12 mutation testing. | false |
Granična stanja armiranobetonskih montažnih dvopojasnih nosača od betona visokih čvrstoća | Limit states of reinforced high strength concrete prefabricated two-chord structures | U ovoj disertaciji izvršena je teorijska i eksperimentalna analiza linijskog montažnog
armiranobetonskog nosača sa čeličnim zetegama. Da bi se potvrdile teorijske postavke
proračuna, unapredila tehnologija grañenja i omogućilo praćenje ponašanja ovih sistema u
svim naponsko-deformacijskim stanjima, izvršena su eksperimantalna istraživanja sa betonom
visoke čvrstoće na pritisak. Eksperimentalno su ispitivana i analizirana dva nosača (modela)
“A“ i “B“, raspona 20.00 m u prirodnoj veličini koji su izrañeni od betona visokih čvrstoća od
75 MPa i 78 MPa, respektivno.
U uvodnom delu je navedeno da ovakvi nosači u velikoj meri zadovoljavaju zahteve
savremene tehnologije grañenja u koje spadaju: znatna redukcija sopstvene težine, brza i laka
montaža i izbegavanje potreba za betoniranjem na licu mesta. Pored toga, ispunjeni su svi
zahtevi u pogledu stabilnosti, funkcionalnosti i trajnosti ovakvih nosača. Kratkotrajno statičko
opterećenje, nanošeno je u sedam faza, a osma faza je izvršena tako što je nosač izložen udaru
u sleme pod punim opterećenjem sedme faze slobodnim padom tega od 5.00 kN sa visine
6.00m.
U poglavlju 2 je prikazan pregled najznčajnijih svojstava betona visoke čvrstoće,
metode projektovanja betonske mešavine, pregled radnih dijagrama betona u pojedinim
zemljama i njihovo poreñenje sa odgovarajućim analizama.
U poglavlju 3 dat je pregled najznačajnijih objekata od betona visoke čvrstoće koji su
realizovani u svetu sa osnovnim karakteristikama i recepture za beton na nekim objektima.
Pregled primene dvopojasnih sistema, uglavnom u našoj zemlji, prikazan je u poglavlju
4. Ovi konstrukcijski sistemi su nastali i unapreñeni na Grañevinskom fakultetu u Beogradu,
što je rezultiralo njihovom velikom primenom u našoj zemlji. Dat je pregled realizovanih
objekata sa karakterističnim detaljima autora sa ovog fakulteta i nekih detalja koje je autor
disertacije realizovao, sam ili u saradnji sa njima.
Peto poglavlje daje opis eksperimentalnih istraživanja: pripremu, izradu probnih
mešavina, pravljenje modela u prirodnoj veličini i nanošenje opterećenja sa merenjima opštih
i lokalnih deformacija nakon svake faze opterećenja.
U šestom poglavlju daje se teorijska analiza ovih dvopojasnih sistema. Izložena je
analiza stanja napona i deformacija, zatim analiza graničnih stanja ovih sistema u obliku
integro-diferencijalnih jednačina. Ukratko je prikazan jedan metod dvojice kiniskeh autora,
koji predstavlja uprošćenje metoda koji su predložili naši autori Ivković, Perišić, Aćić i
Pakvor. Izvršena je teorijska analiza ponašanja nosača usled dejstva udara i razvijen je
poseban računarski program na bazi konačnih elemenata i uporeñeni mereni i teorijski
rezultati koji se dobro slažu.
U sedmom poglavlju prikazuje se metodologija odreñivanja faktora graničnog
opterećenja koje nosač dovodi u stanje granične ravnoteže za aplicirano i jednako podeljeno
opterećenje na gornjem pojasu. Primenjeni postupak odreñivanja faktora graničnog
opterećenja i mehanizma loma je zasnovan na dualitetu statičkih i kinematičkih veličina, i
primenom linearnog programiranja odreñene karakteristične statičke i kinematičke veličine i
formulisani zaključci. Za ova opterećenja je izvršena analiza ponašanja sistema prema teoriji I
i II reda za elastično ponašanje nosača.
U osmom poglavlju je izvršena završna uporedna analiza, tj. poreñenje teorijskih i
eksprimentalnih rezultata i konstatovano njihovo dobro slaganje. | In this PhD thesis is carried out theoretical and experimental analysis of linear,
prefabricated reinforced concrete structures with a lower steel chord. To confirm the theoretical
calculations, improve construction technology and to enable monitoring of the behavior of these
systems in all stress-strain states was carried out an experimental research with concrete of high
compressive strength. Two girders (models) “A” and “B” are investigated experimentally in life-
size, made of high strengths concrete with a compressive strength 75 MPa and 78 MPa.
In the introductory chapter states that these structures largely meet the requirements of
modern construction technology which include: significant reduction of its own weight, quick
and easy assemblage, avoiding the need for concreting on site, and in addition met all the
requirements in terms of stability, functionality and durability of these structural systems. The
short-term static load was applied in seven stages, and in the eights stage the ridge of the loaded
structure was exposed to the impact of the weight free fall 5.00 kN from a height 6.00 m.
In Chapter 2 is given an overview of the most important features of high-strength concrete,
methods of concrete admixture design, review of operating diagrams for concrete in certain
countries and their comparison with the corresponding analysis.
In Chapter 3 is given an overview of the most important buildings constructed of high
strength concrete that have been realized in the world with the basic characteristics and
admixtures of concrete on some of these buildings.
Through Chapter 4 provides an overview of application of two-chord structural systems,
mainly in our country. These systems are created and developed in Faculty of Civil Engineering,
University of Belgrade, which led to their wide application in our country. The review of the
executed projects and characteristic details designed by authors from this faculty and some of the
details that the author has realized, either alone or in collaboration with them, is presented.
The fifth chapter mostly describes experimental research, preparation, making of test
mixtures, creating a life-size models, applying loads with measurements of general and local
deformation after each load stage.
The sixth chapter gives a theoretical analysis of the two-chord structures. Provides a stress
and strain analysis, and then the limit state analysis of the system in the form of integro-
differential equations. One method, given by two Chinese authors, that represents simplification
of the method for analysis of these systems, proposed earlier by Ivkovic, Perišić, Aćić and
Pakvor, is shortly explained.
The seventh chapter presents a methodology for determining the factor of limit load that
leads the structural system into a state of limit equilibrium for the applied load and equally
distributed load all along upper chord. Procedure for determining the factor of limit load and
mechanism of collapse is based on the duality of characteristic static and kinematic quantities.
Appling Linear programming these quantities are determined successfully and corresponding
conclusion are formulated. For all mentioned loadings, an analysis for elastic behaviour of
investigated structures according to the theory of the first and second order is carried out.
A comparative analysis, i.e. comparison of theoretical and experimental results, is
performed in Chapter 8, and good agreement is found between these results. | true |
Genetičko-epidemiološka analiza i analiza ekspresije gena renin-angiotenzin sistema (RAS) u karotidnoj aterosklerozi kod čoveka | Genetic-epidemiological analysis and analysis of gene expression of the renin-angiotensin system (RAS) in carotid atherosclerosis in humans | Karotidna ateroskleroza (KA) je hronična bolest koja započinje aktivacijom i
nakupljanjem inflamatornih ćelija koje se pune lipidima u zidu arterijskog krvnog suda.
Aterosklerotski plak sadrži lipidno jezgro, fibroznu kapu, akumulira glatke mišićne
ćelije (GMĆ) i proteine ekstraćelijskog matriksa (EĆM).
Renin-angiotensin system (RAS) ima ključnu ulogu u aktivaciji inflamacije u zidu
krvnog suda. Glavni molekuli RAS su: angiotenzin-konvertujući enzim (ACE),
angiotenzin-konvertujući enzim-homolog (ACE2), angiotenzin II receptori tipa 1
(ATR1) i tipa 2 (ATR2), ACE2 homolog (TMEM 27) i regulatorna RNK (miR-155).
Angiotenzin II (Ang II) je glavni efektorni molekul RAS koji se sintetiše aktivnošću
ACE, a razgrađuje aktivnošću ACE2. Ang II ostvaruje svoje efekte vezujuću se za
ATR1 i ATR2. Efekti Ang II imaju važnu ulogu u regulaciji vaskularne homeostaze i
to su: vazokonstrikcija, migracija, proliferacija i hipertrofija GMĆ, povećana sinteza
EĆM i povećana produkcija matriks-metaloproteinaza (MMPs). Ekspresija ATR1
regulisana je sa miR-155. Balans između aktivacije i represije RAS može biti odlučujući
u patološkom remodelovanju zida krvnog suda i patogenezi KA. Zato je važno ispitati
funkcionalnu aktivaciju RAS sistema na lokalnom (tkivnom) nivou u karotidama i to na
nivou ekspresije gena koji kodiraju za komponente RAS, enzime i receptore. Ekspresija
gena je regulisana preko prisustva/odsustva različitih alelnih varijanti funkcionalnih
polimorfizama u genima za RAS.
Genetičko-epidemiološka studija gena RAS je urađena po pacijenti - kontrole
dizajnu, na tri gena i tri polimorfizma. Ova studija obuhvatila je 750 ispitanika oba pola
iz populacije Srbije, 505 pacijenata i 246 kontrola iste etničke pripadnosti. Urađena je
studija asocijacije polimorfizma I/D u genu za ACE, polimorfizma A1166C u genu za
ATR1 i polimorfizma A/G -1332 u genu za ATR2 sa nastankom KA, kliničkim
parametrima KA, fenotipom plaka (stabilni (SAP) i nestabilni (NAP)), kliničkim
događajima (prolazni ishemijski događaj (TIA) i cerebrovaskularni insult (CVI)) u
Srbiji. Isti polimorfizmi su asocirani i sa ekspresijom gena u tkivu plaku KA. Analiza
ekspresije gena RAS na nivou iRNK je urađena po istom dizajnu, na šest gena. To su
ACE, ACE2, TMEM 27, ATR1, ATR2 i miR-155. Ova studija obuhvatila je tkivo KA
od 61 pacijenta po tipu SAP (45) i po tipu NAP (16). Studija ekspresije na nivou
proteina na tkivu KA plakova obuhvatila je (8) pacijenata, po tipu SAP (3) i po tipu
NAP (5) za ATR1, ATR2 i ACE2. U genotipizaciji ispitivanih polimorfizama korišćena
je reakcija amplifikacije DNK in vitro PCR i restrikciona analiza sintetisanih
fragmenata (RFLP). Relativna kvantifikacija iRNK za ACE, ACE2, ATR1, ATR2,
kolektrin (TMEM27) i miR-155 urađena je Real-time PCR metodom. Analiza ekspresije
gena RAS i miR-155 urađena je u programu REST (REST Software, QIAGEN), kao i
metodom 2-ΔΔCt i 2-ΔCt u GraphPad programu. Relativna kvantifikacija proteina urađena
je Western blot metodom i densinometrijom. Statistička obrada podataka urađena je
korišćenjem programskog paketa STATISTICA 8.0.
Ispitani polimorfizmi: I/D u genu za ACE, A1166C u genu za ATR1 i A/G -
1332 u genu za ATR2 nisu bili nezavisni faktori rizika za nastanak KA u ispitanom
uzorku u populaciji Srbije. Polimorfizam I/D u genu za ACE bio je nazavistan faktor
rizika za formiranje NAP u grupi pacijenata muškog pola. Nosioci genotipa II imali su
2.52 puta veći korigovani rizik za formiranje NAP od nosilaca genotipova koji sadrže
alel D. Utvrđena je statistički značajna razlika u distribuciji alela i genotipova
polimorfizma A1166C u genu za ATR1 između pacijenata sa SAP i NAP. Polimorfizam
A1166C u genu za ATR1 je nezavistan faktor rizik za formiranje NAP. Nosioci alela C,
po dominantnom modelu nasleđivanja, imaju 1.94 puta veći korigovani rizik za
formiranje NAP od nosilaca genotipa AA. Polimorfizam A/G -1332 u genu za ATR2
nije asociran sa rizikom za formiranje NAP. Polimorfizam A/G -1332 u genu za ATR2
je značajan nezavistan faktor rizika za nastanak CVI u grupi pacijenata muškog pola.
Hemizigot G/- je asociran sa 2.67 većim korigovanim rizikom za nastanak CVI u
odnosu na hemizigot A/-. Polimorfizam I/D u genu za ACE je asociran sa nastankom
TIA u grupi pacijenata muškog pola. Genotip DD, po recesivnom modelu nasleđiovanja
asociran je sa 2.2 puta većim korigovanim rizikom za nastanak TIA nego genotipovi
koji sadrže alel I. U tkivu KA plaka iRNK za ACE, ACE2, TMEM 27, ATR1 i miR-155
se eksprimiraju, a iRNK za ATR2 se ne eksprimira. U ovoj studiji relativni odnos
ekspresije gena za ACE/ACE2 u KA plaku čoveka je bio statistički značajno smanjen u
poređenju sa kontrolnim tkivom arterije bez znakova aterosklerozei u muškom (3.4
puta) i u ženskom (2.7 puta) polu. Relativni nivo ekspresije iRNK za ACE u poređenju
sa nivoom iRNK za ACE2 bio je statistički značajno niži. Nivo ekspresije iRNK za
ACE i ATR1 se nisu statistički značajno razlikovale po genotipovima polimorfizama
I/D i A1166C. Nivo ekspresije miR-155 se statistički značajno razlikovao u odnosu na
genotipove polimorfizma 1166 A/C u genu za ATR1 u grupi pacijenata ženskog pola.
Genotip CC je po recesivnom modelu nasleđivanja statistički značajno asociran sa višim
nivoom ekspresije miR-155. Nivo ekspresije iRNK za ACE, ACE2, ATR1 i TMEM27
se nije statistički značajno razlikovao između SAP i NAP. Nivo ekspresije proteina
ATR1 bio je statistički značajno viši u NAP u odnosu na SAP, dok se nivoi proteina
ACE2 i ATR2 nisu statistički značajno razlikovali između SAP i NAP. Nivoi ekspresije
proteina ATR1 i ATR2 nisu se statistički značajno razlikovali po genotipovima
ispitivanih polimorfizama A1166C i A/G -1332.
Ispitani genetički markeri RAS u KA bi mogli biti od kliničkog značaja kao
faktori modulacije nastanka i toka bolesti, za koje se do sada nije znalo. U različitim
podgrupama pacijenata sa KA asocijacija genetičkih faktora sa kliničkim parametrima
može imati potencijalnu primenu u prevenciji i terapiji KA u bliskoj budućnosti. | The carotid atherosclerosis (CA) is chronic disease, which begins with activation
inflammatory cells and accumulation lipid in these cells within blood vessel wall.
Atherosclerotic plaque containing a lipid core, fibrous cap, accumulates smooth muscle
cells (GMC) and proteins of the extracellular matrix (ECM).
The renin-angiotensin system (RAS) has a key role in activation of inflammation within
blood vessel wall. The main components of the system are: angiotensin I-converting
enzyme (ACE), angiotensin converting enzyme homolog (ACE2), angiotensin II
receptor type 1 (ATR1), angiotensin II receptor type 2 (ATR2), ACE2 homolog
(TMEM27) and regulatory RNA (miR-155). Angiotensin II (Ang II) is the main effector
molecule of the RAS, which is synthesized by the activity of ACE and is cleaved by
ACE2. Ang II achieves its effects by binding to ATR1 and ATR2. These effects has an
important role in the regulation of vascular homeostasis and include the following:
vasoconstriction, vascular smooth muscle cells (SMC) migration, proliferation and
hypertrophy, increased synthesis of extracellular matrix (ECM) and enhanced
production of matrix metalloproteinases (MMPs). The expression of ATR1 is regulated
by the miR-155. The balance between activation and repression of RAS can be decisive
in pathological remodeling of the vessel wall and the pathogenesis CA. It is therefore
important to examine the functional activation of RAS at the local (tissue) level in
carotid vessel wall at level of expression genes encoding for components of the RAS,
enzymes and receptors. The gene expression of components of RAS is regulated
through the presence/absence of different allelic variants of functional polymorphisms
in these genes.
The genetic epidemiological study for RAS genes was performed by case-control design
and consisted of three genes and three polymorphisms within these genes. This study
included 750 subjects of both sexes from the Serbian population, 505 patients and 246
controls of the same ethnic background. It is done genetic association study the ACE
I/D, ATR1 A1166C and ATR2 A/G -1332 polymorphisms with occurrence of CA,
clinical parameters of CA, (phenotypes of plaques (stable (SP) and unstable (USP)),
clinical events of disease (transient ischemic attack (TIA) and cerebrovascular insult
(CVI)) in Serbia. The same polymorphisms are associated with the with the expression
of genes in the tissue of carotid atherosclerotic plaques. Analysis of gene expression at
the level RAS mRNA is performed according to the same design, for the all six genes.
These genes were ACE, ACE2, TMEM27, ATR1, ATR2 and miR-155. This study
involved carotid atherosclerotic tissue of 61 patients, 45 patients with stable carotid
plaque characteristics and 16 patients with USP plaques. The study expression at the
level of proteins in carotid atherosclerotic tissue included 8 patients, 3 SP and 5 USP
plaques for ATR1, ATR2 and ACE2. Genotyping of examined polymorphisms is done
by reaction of amplification DNA in vitro (PCR) and restriction fragment length
polymorphism analysis (RFLP). Relative quantification of mRNA for ACE, ACE2,
ATR1, ATR2, collectrin (TMEM27) and miR-155 was performed by real-time PCR.
Analysis of the gene expression of RAS and miR-155 was performed by REST softwere
(REST Software, QIAGEN) and by 2-ΔΔCt and 2-ΔCt method in GraphPad softewere.
Relative quantification of protein was done by Western blot and densitometry. The
statistical analysis was performed using the software package STATISTICA 8.0.
The investigated polymorphisms in the human genes of the RAS: the ACE I/D
polymorphism, the ATR1 A1166C and ATR2 A/G -1332 were not independent risk
factors for the occurrence of CA in examined sample in population of Serbia. The ACE
I/D gene polymorphism was an independent risk factor for the formation of USP
plaques in group of male patients. The carriers of II genotype had 2.52 fold higher
adjusted OR for formation of USP plaques compared to those carriers containing allele
D. There was a significant statistical difference in the distribution of genotypes and
alleles of the ATR1 A1166C gene polymorphism between patients with SP and USP
carotid plaques. The ATR1 A1166C gene polymorphism was an independent risk factor
for the formation of USP plaques. The carriers of allele C according to the dominant
model inheritance had a 1.94 fold higher adjusted OR for the formation of USP plaques
compared to AA genotype carriers. The ATR2 A/G -1332 gene polymorphism is not
associated with the risk of the formation of USP plaques. The ATR2 A/G -1332 gene
polymorphism was a significant independent risk factor for CVI in the group of male
patients. The hemizygote G/- was associated with 2.67 fold gigher adjusted OR for CVI
compared to those carriers of hemizygote A/-. The ACE I/D polymorphism is associated
with an increased risk for the occurrence of TIA in male group of patients. The
genotype DD, according to the recessive model of inheritance is associated with 2.2
fold higher adjusted OR for the occurrence of TIA compared to those genotypes which
containing allele I.
In the tissue of carotid atherosclerotic plaques the mRNA expression level of ACE,
ACE2, TMEM27, ATR1 and miR-155 is determined whilst ATR2 is not. In this study,
the relative ratio of mRNA expression of ACE and ACE2 (ACE/ACE2) in human CA
plaques was significantly reduced compared with control tissue arteries without any
signs of atherosclerosis, 3.4 fold in males and 2.7 fold in female patients. The relative
mRNA expression level of ACE compared to the level of ACE2 was significantly
decreased. The relative mRNA expression levels of ACE and ATR1 were not
significantly different between genotypes of the gene polymorphisms, I/D and A1166C.
The relative expression level of miR-155 was significantly different in regard to
genotypes of polymorphism A1166C in female group in patients. The genotype CC
according to the recessive model of inheritance is significantly associated with r level of
miR-155. The relative level of ACE, ACE2, ATR1 and TMEM27 mRNA expression
were not significantly different betwereen stable and unstable carotid plaques. The
ATR1 protein expression level was significantly higher within unstable carotid plaque
in comparison to stable plaques, whereas protein levels of ACE2 and ATR2 did not
significantly differ between these phenotypes. The expression levels of proteins ATR1
and ATR2 are not significantly different between the investigated polymorphisms,
A1166C and A/G -1332.
The examined genetic markers of RAS in CA could have clinical importance as factors
that modulate the origin and course of the disease, which hitherto unknown so far. The
association of genetic markers with clinical parameters in various subgroups of patients
with CA could have potential application in the prevention and treatment of disease in
the near future. | true |
Bioakumulacioni potencijal odabranih biljnih vrsta iz porodice Brassicaceae sa serpentinskih staništa u Srbiji | Bioaccumulation potential of selected plant species of the family Brassicaceae from serpentine habitats in Serbia | Ultramafiti (serpentiniti) predstavljaju grupu magmatskih ili metamorfnih stena, koje se karakterišu visokim koncentracijama Mg, Fe, Ni, Cr i Co, kao i niskim koncentracijama Ca, P i K sadrže manje od 45% silicijuma (SiO2). Flora i vegetacija koje naseljavaju serpentinitske supstrate obično su značajno oskudnije i siromašnije u odnosu na okolna staništa. Mnoge biljne vrste nisu sposobne da rastu na serpentinitski staništima, zbog njihovog ekstremno niskog sadržaja esencijalnih elemenata (nitrijenata), kao i nepovoljnog odnosa Ca/Mg u zemljištu. Na osnovu sposobnosti usvajanja teških metala, biljke koje rastu na ultramafitskim zemljištima mogu se podeliti u dve grupe: tzv. „normalne“ i metal hiperakumulatorske vrste. Dok je većina serpentinitskih biljaka sposobna da raste na ovakvim zemljištima, bez usvajanja velikih koncentracija teških metala, hiperakumulatorske vrste mogu akumulirati i do 1000 mg kg-1 Ni u suvoj masi svojih nadzemnih delova (najčešće u listovima). Do danas je identifikovano oko 360 vrsta koje su hiperakumulatori nikla, od kojih najveći broj pripada porodici Brassicaceae. Daleko najveći broj hiperakumulatora zabeležen je u okviru roda Alyssum L. Osim vrsta iz ovog roda, hiperakumulatorske vrste mogu pripadati i sledećim vrstama i/ili rodovima koje naseljavaju mediteranske delove Evrope i Male Azije: Leptoplax emarginata (Boiss.) O.E. Schulz, nekoliko vrsta iz roda Bornmuellera Hausskn., dve vrste iz roda Pseudosempervivum (Boiss.) Grossh. (Cochlearia L.), jedna vrsta iz roda Aethionema, kao i značajan broj vrsta iz roda Thlaspi L. Na području Srbije je do danas utvrđeno da jedino vrsta Alyssum murale sa planine Goč ima sposobnost hiperakumulacije nikla, dok za vrste iz nekih drugih rodova kao što su Isatis, Cardamine, Rorippa, Erysimum ne postoje informacije o hiperakumulativnim potencijalima. Prema tome, bilo je neophodno sakupiti više informacija o akumulativnim kapacitetima biljaka iz porodice Brassicaceae sa serpentinitskih staništa u Srbiji, ali i proceniti njihov potencijal za fitoremedijaciju, pa su na osnovu ovih činjenica definisani ciljevi ove disertacije: 1) utvrđivanje osnovnih karakteristika staništa ispitivanih taksona, uključujući podatke o tipu vegetacije i/ili zajednice, nadmorskoj visini, nagibu terena, geološkoj podlozi i klimi; 2) utvđivanje osnovnih fizičkih (tekstura, tj. mehanički sastav) i hemijskih (pH u H2O, pH u 1 N KCl, procenat organske materije, koncentracija P2O5 i K2O) svojstava serpentinitskih zemljišta na istraživanim lokalitetima; 3) utvrđivanje koncentracije makroelemenata (Ca, Mg, Fe) i teških metala (Ni, Mn, Zn, Cu, Cr, Pb, Co, Cd) u zemljištima koja se razvijaju iznad serpentinitske podloge na više lokaliteta u Srbiji; 4) ispitivanje koncentracije makroelemenata i teških metala u podzemnim i nadzemnim delovima biljaka iz porodice Brassicaceae koje naseljavaju serpentinitska staništa u Srbiji; 5) na osnovu dobijenih rezultata, proceniti kapacitet pojedinih biljnih vrsta iz porodice Brassicaceae za toleranciju ili bioakumulaciju određenih mikroelemenata (pre svega teških metala); 6) otkriti nove nikl i/ili cink akumulatorske ili hiperakumulatorske vrste iz porodice Brassicaceae... | Ultramafics ('ultrabasic' or 'serpentine') represent magmatic or metamorphic rocks which are characterized by high concentrations of Mg, Fe, Ni, Cr and Co and low concentrations of Ca, P, and K and contain less than 45% silica (SiO2). Serpentine substrates commonly support vegetation and flora that is sparser and more stunted than that of other nearby soils. Many plant species are incapable of growing on serpentine substrates because they are extremely poor in essential nutrients and have a low Ca/Mg ratio. On the basis of heavy metal uptake, plants which grow on ultramafic soils can be divided into ‘normal’ and metal hyperaccumulators. While most serpentine plants are able to grow on these soils without excessive uptake of elements, hyperaccumulators take up more than 1000 mg kg-1 Ni and accumulate it into their leaf dry matter. To date, about 360 hyperaccumulators of Ni from ultramafic soils have been identified. They are mostly found in Brassicaceae family in temperate areas (especially Mediterranean and Turkey). By far the greatest number of nickelaccumulating species within any genus is found in Alyssum L. Besides the Alyssum species, nickel hyperaccumulation was also discovered in species that belong to other genera of the Brassicaceae family of Europe and Turkey. So far only the data about Ni accumulation in A. murale from the central Serbia was published, but there has been no information about the hyperaccumulative potential of the species from the ultramafic areas of Serbia that belong to some other genera (Isatis, Cardamine, Aethionema, Rorippa, Erysimum) of the Brassicaceae family. Hence, it is necessary to obtain more information about metal accumulation capacities of the plants from the Brassicaceae family growing on serpentine soils to evaluate their potential for phytoremediation including metal extraction (phytoextraction). Therefore, the aims of this thesis were to: 1) determine the basic habitat characteristics of the selected taxa from the Brassicaceae family, including information on the type of vegetation and/or community, altitude, slope, geological and pedological substrate and climate; 2) identify the basic physical (texture) and chemical (pH in H2O, pH in 1N KCl, percent of organic matter, contents of P2O5 and K2O) features of serpentine soil of the study sites; 3) determine the concentrations of the macro (Ca, Mg, Fe) as well as trace elements (Ni, Mn, Zn, Cu, Cr, Pb, Co, Cd) in soils that are developed over serpentinite substrates at several locations in the northwestern, western, southwestern and central Serbia; 4) examine the concentrations of the macro and trace elements in underground and aboveground parts (stems and/or leaves) of plants of the family Brassicaceae that inhabit serpentinite habitats in Serbia; 5) based on the obtained results, the assessment of the capacity of some plant species of the family Brassicaceae for tolerance and/or bioaccumulation of certain trace elements (with special attention to nickel and zinc); 6) identificationn and discovery of new potential hyperaccumulator species in the family Brassicaceae from Serbia.... | false |
Morfološka i genetička karakterizacija Aphidius colemani kompleksa vrsta (Hymenoptera, Braconidae) | Morphological and genetic characterization of Aphidius colemani species complex (Hymenoptera, Braconidae) | Aphidius colemani kompleks vrsta je definisan kao grupa vrsta sa pantropsko-
suptropskim rasprostranjenjem koja se odlikuje malim brojem brazdi, karakterističnog
položaja, na anterolateralnom regionu petiolusa. Predstavlja jednu od 10 grupa vrsta
(kompleksa) sa nerešenim taksonomskim statusom u okviru roda Aphidius. Morfološka
i genetička karakterizacija Aphidius colemani kompleksa izvršena je primenom
morfometrije i analize sekvenci barkoding regiona gena za subjedinicu jedan citohrom
oksidaze (COI). Morfometrijskim analizama obuhvaćeno je 250 jedinki A. colemani
kompleksa sa prostora 23 države sa četiri kontinenta. Analizom varijabilnosti
morfoloških karaktera pokazano je postojanje značajnih razlika u pogledu njihove
informativnosti u pogledu razdavjanja različitih grupa ovog kompleksa. UtvrĎeno je
koji su morfološki karakteri dovoljno informativni za razlikovanje taksona unutar ovog
kompleksa parazitskih osa. Molekularne analize su obuhvatile 54 jedinke iz 8 država.
Analizom sekvenci COI gena i rekonstrukcijom filogenetskih odnosa utvrĎeno je
postojanje skrivenog taksona unutar A. colemani kompleksa. Kombinacijom
rmorfometrijskih i molekularnih metoda utvrĎeno je da u okviru Aphidius colemani
kompleksa vrsta postoje najmanje tri taksona: Aphidius colemani, Aphidius
transcaspicus i Aphidius cf. colemani 1 (novi takson). UtvrĎeno je da novotkirvena
vrsta naseljava Aziju (Iran) i Južnu Ameriku (Čile). Pokazano je da ne postoji jasna
veza izmeĎu genetičke diferencijacije parazitoida ovog kompleksa i njihovih afidnih
domaćina, kao ni izmeĎu genetičke diferencijacije i geografskog porekla. | Aphidius colemani species complex has been defined as a group of species with
pantropical – subtropical distribution which is characterized by low number of grooves
(costulae) with specific position on the anterolateral region of the petiole. This group is
one of the 10 groups of species (complexes) with unsolved taxonomical status among
the genus Aphidius. Morphological and genetic characterization of the Aphidius
colemani complex was done by using morphometrics and DNA sequence analysis of the
barcoding gene for Cytochrome oxidase I (COI). In total 250 specimens of A. colemani
complex from 23 deferent countries on four continents were encompassed with
morphometric analysis. By analyzing variability of morphological characters,
statistically significant differences were noted in the sense of their informativity in
separation of deferent groups of the complex. Molecular analysis was performed on 54
specimens from eight deferent countries. By analyzing sequences of COI gene and the
reconstruction of phylogenetic relations, one hidden taxon was detected among the A.
colemani complex. Combination morphometric and molecular methods has shown the
existence of at least three distinct taxa: Aphidius colemani, Aphidius transcaspicus and
Aphidius cf. colemani 1 (new taxon). This new species can be found in Asia (Iran) and
South America (Chile). It has been shown that there are no clear connections between
genetic differentiation of the parasitoids from this complex and their aphid hosts, as
well as between genetic differentiation and geographic distribution. | true |
Efekat kadmijuma na rast i biohemijske osobine larvi gubara Lymantria dispar L. | Cadmium effect on growth and biochemical traits of Gypsy moth larvae (Lymantria dispar L.) | Gubar (Lymantria dispar L.) spada u red najinvazivnijih polifagnih vrsta insekata, koja naseljava šume, voćnjake i urbana staništa. U ovom radu je kod larvi gubara trećeg dana četvrtog larvenog stupnja, ispitivan akutni i hroničan uticaj dve koncentracije kadmijuma (10 i 30 μgCd/g suve hrane), kao i oporavak od istih, na različite osobine larvi: komponente adaptivne vrednosti (mortalitet, trajanje prvog stupnja, trajanje razvića do ulaska u četvrti larveni stupanj, masa i relativna brzina rasta larvi), aktivnosti enzima srednjeg creva (proteaza, tripsin, leucin aminopeptidaza, α i β-glikozidaza, esteraza i glutation-S-transferaza), kao i procenat mase srednjeg creva u odnosu na masu larve. Kod svih ispitivanih enzima, osim kod GSTa, prisutan je trend pada aktivnosti na kadmijumu u odnosu na kontrolu. Hronična izloženost kadmijumu nije pokazala značajan uticaj na mortalitet prvog stupnja, kao i ukupan mortalitet, masa larvi se smanjuje pri dugotrajnim tretmanima, dok se u toku akutnog tretmana brzina rasta larvi povećava. Analize varijanse su pokazale da se kod proteolitičkih enzima aktivnost menja u zavisnosti od koncentracije metala. Između proteolitičkih enzima na svim tretmanima najverovatnije postoji neka vrsta zajedničke regulacije sinteze ili sekrecije u prisustvu kadmijuma. Regulacioni mehanizmi β-glikozidaze i GSTa se razlikuju u odnosu na ostale proučavane enzime. Zbog ograničenih energetskih resursa postoji veliki broj negativnih korelacija između mase i enzima. Znak korelacija između α i β-glikozidaza se menja na stresu u odnosu na kontrolu. Na različitim tretmanima kadmijumom plastičnost enzima se menja. Pošto se indeks fenotipske plastičnosti ne menja u toku delovanja kadmijuma, promena osobina adaptivne vrednosti ili njihova plastičnost, se ne mogu uzeti kao dobri indikatori prisustva kadmijuma. Zbog velike osetljivosti leucin aminopeptidaze i esteraze u toku akutnog stresa, postojanja korelacija sa velikim brojem digestivnih enzima, velike plastičnosti odgovora u stresnim uslovima, ova dva enzima bi se, uz dalja istraživanja, mogla uzeti kao potencijalni biomarkeri na prisustvo kadmijuma. | The Gypsy moth (Lymantria dispar L.) represents one of the most invasive polyphagous insect species, which inhabits forests, orchards and even urban habitats. We investigated the acute and chronic effects of two cadmium concentrations (10 and 30 μgCd/g dry food), as well as the recovery from chronic stress on various traits of 4th instar gypsy moth larvae: fitness components (mortality, duration of development of the first larval instar, duration of development from hatching to molting into the 4th instar, larval mass and relative growth rate), midgut enzyme activities (activity of protease, trypsin, leucine aminopeptidase, α and β-glucosidase, esterase and glutathione S-transferase), as well as the percentage of the midgut mass in relation to the larval mass. All examined enzymes, with the exception of the glutathione S-transferase, showed a decrease of activity in relation to control. Larval mass decreased during the long-term treatment, while the acute treatment resulted in the growth rate increase. The analysis of variance showed that the activity of proteolytic enzymes depended on metal concentration. Significant broad-sense heritability was discovered after short-term exposure to cadmium. Regulation mechanisms of β-glucosidase and glutathione S-transferase differ in relation to the rest of the examined enzymes. There are a large number of negative correlations between larval mass and enzymes due to the limited energy resources. The correlation sign between α and β- glucosidases changes during the stress conditions in relation to the control. During different cadmium treatments plasticity of enzymes is changed. Changes of life history traits or their plasticity cannot be good indicators of cadmium presence. Due to a great sensitivity of leucine aminopeptidase and esterase during the acute stress, correlated response to a large number of digestive enzymes, great plasticity in stressful conditions, these two enzymes, with further research, could be potential biomarkers for cadmium presence. | false |
Uticaj kadmijuma na sistem antioksidativne zaštite i varijabilnost komponenti adaptivne vrednosti gubara Lymantria dispar L. | Cadmium effects on antioxidative defense system and on variability of fitness-related traits in Lymantria dispar L. gipsy moth | Kadmijum je jedan od najpotentnijih polutanata koji dospeva u životnu
sredinu, iz prirodnih i/ili antropogenih izvora, i ulazi u žive sisteme preko lanaca
ishrane, ostvarujući svoj toksični efekat na svim nivoima biološke organizacije. U
ovom radu ispitivan je uticaj kadmijuma u koncentracijama od 10 (C1), 30 (C2) i
50 µg Cd/g (C3) suve hrane na komponente adaptivne vrednosti, heritabilnost u
širem smislu, genetičke i fenotipske korelacije, fenotipsku plastičnost, cenu
plastičnosti, i na obim i pravce delovanja selekcije, kao i uticaj 50 µg Cd/g suve
hrane na sistem antioksidativne zaštite kod gubara tokom larvenog razvića,
razvića lutke i adultnog perioda. Gubar, Lymantria dispar L., je polifagna,
invazivna vrsta koja zbog svog štetnog delovanja na šumske ekosisteme spada u
grupu organizama od posebnog interesa za istraživanja fizioloških i evoluciono-
genetičkih odgovora koji utiču na promenu brojnosti populacije ove vrste.
Kadmijum dovodi do produžetka razvića kod mlađih larvenih stupnjeva,
do smanjenja mase lutke kod oba pola i skraćivanja razvića lutke i života adultnih
ženki. Polovi se značajno razlikuju u trajanju pojedinih faza larvenog razvića,
naročito u C3 grupi. Za većinu ispitivanih osobina postoji značajan nivo
heritabilnosti u širem smislu. Pri tome, prisustvo kadmijuma u C3 grupi dovodi do
značajnog povećanja heritabilnosti trajanja razvića larvi četvrtog stupnja i kod
mužjaka za trajanje larvenog razvića do 5. stupnja u odnosu na kontrolu i u
odnosu na ženke.
Značajna varijabilnost fenotipske plastičnosti postoji kod svih mlađih
larvenih stupnjeva u svim grupama, i kod oba pola za pojedine osobine u okviru
različitih sredina ukazujući na potencijal evolucije adaptivnog plastičnog
odgovora u stresnim uslovima sredine. Indeksi fenotipske plastičnosti po
Čeplikovoj i Lijovoj metodi pokazuju značajne vrednosti kod većine osobina, što
upućuje na moguć pravac odgovora familija za određene osobine na prisustvo
kadmijuma. Tokom razvića uočene su i značajno veće vrednosti heritabilnosti
plastičnosti u odnosu na heritabilnost svojstva za pojedine osobine što ukazuje da
je u heterogenoj sredini, koja je karakteristična za polifagne insekte, verovatnija
evolucija generalista.
Selekciona analiza je pokazala da postoji značajan potencijal populacija da
odgovore plastično na stresne uslove životne sredine. Selekcija favorizuje u C3
grupi mužjake koji imaju kraće larveno razviće, a u kontrolnoj, C1 i C2 grupi
ženke koje imaju duže razviće larve i lutke. Krupnije ženke su u selektivnoj
prednosti u stresnoj sredini, a mužjaci u optimalnim uslovima.
Većina fenotipskih i genetičkih korelacija između ispitivanih osobina je
bila pozitivna, pri čemu se broj korelisanih osobina smanjivao sa povećanjem
koncentracije kadmijuma u ishrani. Većina genetičkih korelacija za određene
osobine u okviru različitih sredina je bila takođe značajna i pozitivna, što je
karakteristično za generaliste.
Aktivnost superoksid dismutaze, katalaze, askorbat peroksidaze, glutation
S transferaze, glutation reduktaze i količina ukupnog glutationa i slobodnih SH
grupa tokom razvića gubara su pokazale varijabilnost u aktivnosti i količini
zavisno od stupnja razvića, ukazujući na postojanje oksidativnog stresa nastalog
prisustvom kadmijuma. | Cadmium is one of the most potent pollutants which enters the
environment through natural and/or anthropological sources and into the living
organisms though the food chain thus realizing its toxic effect in all levels of
biological organization. This work examines cadmium effects at 10 (C1), 30 (C2)
and 50 µg Cd/g (C3) concentrations of dry food on the fitness-related traits,
broad-sense heritability, genetic and phenotypic correlations, phenotypic
plasticity, cost of plasticity and on direction of selection force. Also, this work
examines the effects of 50 µg Cd/g of dry food on antioxidative defense system in
gypsy moth during larval development, pupal development and the adult period.
Lymantria dispar L. gypsy moth is a polyphagous, invasive species which, due to
its detrimental influence on forest ecosystems, is considered an organism of
particular interest for researches of physiological and evolutionary-genetic
responses which can influence population dynamics of this species.
Cadmium prolongs the development duration in early larval instars, it also
reduces larval mass in both genders and larval development and life expectancy in
adult females. Duration of certain larval instars is significantly different between
genders, especially in C3 group. Most of the examined traits showed a significant
level of broad-sense heritability. Thus, the presence of cadmium in C3 group
significantly increases the heritability of the fourth instar duration and the
heritability of development duration of male larvae from hutching until the 5th
instar in comparison with control and female larvae, respectively.
Significant variability of phenotypic plasticity is present in all of the early
instars regardless of the group, as well as in both genders for certain traits
depending on different environments which points to the potential of the
evolution of adaptive plastic response in stressful environments. An index of
phenotypic plasticity according to Cheplik and Li’s method shows significant
values for most of the traits pointing to to the possible direction of the families’
response for certain traits in the presence of cadmium. Significantly higher values
of heritability for plasticity compared to heritability of the traits indicate that in
heterogeneous environment, which is common for polyphagous insects, evolution
of generalist strategy is expected.
Selection analysis has proved that population has significant potential for
plastic response to stressful environment. When it comes to C3 group, the
selection favours male individuals characterized by shorter larval development,
while the female individuals which have longer larval and pupal development are
favoured in control group, C1 and C2 group. Larger females have selective
advantage in stressful environments, while large male individuals have the
advantage in optimal conditions.
Most of the phenotypic and genetic correlations among the analyzed traits
were positive ones, whereas the number of correlated traits tends to decrease
along with the increase of Cadmium in diets. Most of the genetic correlations
between traits within different environments were also significant and positive,
which is characteristic of generalists.
The ativities of superoxide dismutase, catalase, ascorbat peroxidase,
glutathione-S-transferase, glutathione reductase, total glutathione amount and the
amount of free SH groups during gypsy moth development showed variability in
the activity and the amount depending on the instar pointing to the existence of
the oxidative stress incurred by the presence of cadmium. | true |
Spektralne i fraktalne karakteristike električne aktivnosti mozga pacova pod akutnim uticajem etarskog ulja kamforovog drveta | Spectral and fractal characteristics of rat brain electrical activity after acute camphor tree essential oil administration | Odlučili smo se za istraživanje pojave epileptičnih napada kod pacova, izazvanih etarskim uljem kamforovog
drveta ili njegovim glavnim konstituentom 1,8-sineolom. Eksperimenti su izvedeni na odraslim pacovima
muškog pola Wistar soja. Operativni zahvati i akutna registrovanja elektrofizioloških signala su obavljeni
pod Nembutal anestezijom. Registrovani su lokalni potencijali polja kore velikog i malog mozga u uslovima
pre i 1 h posle intraperitonealne (i.p.) administracije etarskog ulja kamforovog drveta ili 1,8-sineola.
Pacovima je intraperitonealno ubrizgavano etarsko ulje kamforovog drveta (150-750 μl/kg t.m., u 1 ml
fiziološkog rastvora i.p.) ili 1,8-sineol (300-500 μl/kg t.m., u 1 ml fiziološkog rastvora, i.p.). Digitalizovani
elektrokortikalni signali (frekvence semplovanja: 256 Hz) su analizirani uz pomoć brze Furijeove
transformacije, a potom je izračunavana vrednost fraktalne dimenzije (FD) i Hurstovog eksponenta (H).
Etarsko ulje kamforovog drveta (≥450 μl/kg t.m.) ili 1,8-sineol (≥300 μl/kg t.m.) indukuju epileptičnu
aktivnost sa pojavom pojedinačnih i grupnih šiljaka velike amplitude u elektrokortikogramu sa povremenim
kloničnim konvulzijama ekstremiteta, 3-15 minuta nakon administracije supstanci. Na velikom mozgu,
nakon administracije etarskog ulja kamforovog drveta, kao i 1,8-sineola, u epohama sa epileptičnom
aktivnošću, bio je prisutan veliki porast ukupne snage kao i povećanje relativne snage u delta (0,1-4,0 Hz)
frekventnom opsegu, odnosno smanjenje relativne snage u teta (4,1-8,0 Hz), alfa (8,1-15,0 Hz), beta (15,1-
32,0 Hz) i gama (32,1-128,0 Hz) frekventnim opsezima. Potvrđeno je dominantno učešće delta (0,1-4,0 Hz)
frekventnog opsega a vrednost relativne snage se kretala od 47 do 87 %. Na malom mozgu, u epohama sa
epileptičnom aktivnošću, relativne snage frekventnih opsega su se različito ponašale u odnosu na veliki
mozak i različito nakon administracije 1,8-sineola u odnosu na etarsko ulje kamforovog drveta. Razlike
između velikog i malog mozga, kao i razlike u delovanju etarskog ulja kamforovog drveta i 1,8-sineola, bile
su izraženije u parametrima fraktalne analize. Na velikom mozgu pacova, nakon administracije etarskog ulja
kamforovog drveta, u epohama sa epileptičnom aktivnošću, postoji povećanje vrednosti FD u odnosu na
kontrolu, a na malom mozgu smanjenje. Suprotno, nakon administracije 1,8-sineola postoji smanjenje
vrednosti FD na velikom, odnosno povećanje na malom mozgu. Postojala je i dozna zavisnost odgovora
nakon administracije etarskog ulja kamforovog drveta, odnosno 1,8-sineola. Na velikom mozgu, povećanje
doze etarskog ulja kamforovog drveta, odnosno 1,8-sineola, u epohama sa epileptičnom aktivnošću, praćeno
je povećanjem relativne snage delta (0,1-4,0 Hz) frekventnog opsega, odnosno smanjenjem relativne snage
teta (4,1-8,0 Hz), alfa (8,1-15,0 Hz), beta (15,1-32,0 Hz) i gama (32,1-128,0 Hz) frekventnih opsega.
Suprotno, na malom mozgu, povećanje doze etarskog ulja kamforovog drveta u epohama sa epileptičnom
aktivnošću, praćeno je smanjenjem relativne snage delta (0,1-4,0 Hz), odnosno povećanjem relativne snage
teta (4,1-8,0 Hz), alfa (8,1-15,0 Hz) i beta (15,1-32,0 Hz) frekventnih opsega. Na malom mozgu, povećanje
doze 1,8-sineola u epohama sa epileptičnom aktivnošću, praćeno je povećanjem relativne snage teta (4,1-8,0
Hz), odnosno smanjenjem relativne snage gama (32,1-128,0 Hz) frekventnog opsega. I na velikom i na
malom mozgu pacova, promene vrednosti FD negativno su korelisane sa povećanjem doze kako etarskog ulja
kamforovog drveta tako i 1,8-sineola. Neophodno je dalje ispitati doznu zavisnost svakog konstituenta
etarskog ulja kamforovog drveta na aktivnost mozga kako bi se rasvetlila sigurna upotreba etarskih ulja
biljaka koje sadrže ove terpenoide. | We investigated the rat brain activity in acute seizures evoked by camphor tree essential oil or its main
constituent 1,8-cineole. The experiments were performed on adult male Wistar rats. The surgery and acute
electrophysiological recordings were performed under pentobarbital sodium anesthesia. Local field potentials
of cerebral and cerebellar parietal cortex were recorded before and 1 h after intraperitoneal (i.p.) camphor
essential oil or 1,8-cineole administration. The rats were intraperitoneally injected with camphor essential oil
(150-750 μl/kg b.w., in 1 ml of saline, i.p.) or 1,8-cineole (300-500 μl/kg b.w., in 1 ml of saline, i.p.). The
digitized electrocortical signals (sampling frequency: 256 Hz) were analyzed by the Fast Fourier Transform,
and were calculated Fractal dimension (FD) and Hurst exponent (H) also. Camphor essential oil (≥450 μl/kg
b.w.) and 1,8-cineole (≥300 μl/kg b.w.) induced seizure-like activity with single and multiple spiking of high
amplitudes in the parietal electrocorticogram and occasional clonic limb convulsions, 3-15 min after their
administration. On the cerebrum, after camphor essential oil and 1,8-cineole administration, in epochs with
epileptic activity, there was high increase in total power and an increase in relative power in delta (0,1-4,0
Hz) frequency range, as well as a decrease in relative power in theta (4,1-8,0 Hz), alpha (8,1-15,0 Hz), beta
(15,1-32,0 Hz) and gamma (32,1-128,0 Hz) frequency ranges. It was confirmed the dominant presence of the
delta (0,1- 4,0 Hz) frequency range and the values of the relative power ranged from 47 to 87 %. On the
cerebellum, in epochs with epileptic activity, the relative power of the frequency ranges were behaving
differently than cerebrum, as well as after administration of 1,8-cineole compared to the camphor essential
oil. The differences between the cerebrum and cerebellum, as well as differences in the activities of the
camphor essential oil and 1,8-cineole, were more pronounced in the parameters of fractal analysis. On the
cerebrum, after camphor essential oil aministration, in epochs with epileptic activity, there was an increase in
the FD value compared to the control, and the decrease on the cerebellum. In contrast, after 1,8-cineole
administration there was a decrease in FD value on the cerebrum, and increase on the cerebellum. There was
a dose dependence of response after the camphor essential oil and 1,8-cineole administration. On the
cerebrum, increasing the dose of the camphor essential oil and 1,8-cineole, in epochs with epileptic activity,
correlated with an increase in relative power in delta (0,1 to 4,0 Hz) frequency range and a decrease in
relative power in theta (4,1-8,0 Hz), alpha (8,1-15,0 Hz), beta (15,1-32,0 Hz) and gamma (32,1-128,0 Hz)
frequency ranges. In contrast, on the cerebellum, increasing the dose of the camphor essential oil in epochs
with epileptic activity, correlated with a decrease in relative power in delta (0,1 to 4,0 Hz) and increase in
relative power in theta (4,1 to 8,0 Hz), alpha (8,1 to 15,0 Hz) and beta (15,1 to 32,0 Hz) frequency ranges. On
the cerebellum, increasing the dose of 1,8-cineole, in epochs with epileptic activity, correlated with an
increase in relative power in theta (4,1-8,0 Hz) and a decrease in relative power in gamma (32,1-128,0 Hz)
frequency range. On the cerebrum and cerebellum, FD changes were negatively correlated with campor
essential oil and 1,8-cineole doses increasing. It is necessary to investigate further the dose dependent
effectiveness of each terpenoid constituent of camhor essential oil on the brain activity in order to elucidate
the relative safety of the usage of essential oils with these terpenoids. | true |
Konzervirana svojstva proteina virusa hepatitisa C genotipa 1b kao prognostičkih markera odgovora na kombinovanu terapiju pegilovanim interferonom i ribavirinom | Conserved properties of hepatitis C genotype 1b proteinsas prognostic markers of response to pegylated interferon and ribavirin combination therapy | Uvod: Infekcija virusom hepatitisa C (HCV) predstavlja značajan globalni
zdravstveni problem koji često vodi ka hroničnoj bolesti jetre i cirozi. Prema podacima
Svetske zdravstvene organizacije registrovano je preko 170 miliona ljudi inficiranih
virusom HCV. Sadašnja standardna terapija hroničnog HCV-a kod pacijenata
inficiranih genotipom 1b, koji predstavlja najčešći genotip kako u SAD, zapadnoj
Evropi i Srbiji, se sastoji od pegilovanog interferona u kombinaciji sa ribavirinom.
Međutim, kombinovana terapija je praćena brojnim neželjenim efektima i dovodi do
stabilnog virusološkog odgovora samo kod 50% pacijenata inficiranih genotipom 1.
Stoga bi jednostavan i pouzdan test, koji bi pre početka terapije mogao da predvidi
virusološki odgovor, bio od velike koristi u kliničkoj praksi. Metode: Identifikacija
konzervirane informacije sadržane u proteinima HCV koja korelira sa odgovorom na
standardnu kombinovanu terapiju rađena je bioinformatičkom analizom. Uzorci plazme
48 pacijenata sa hroničnom infekcijom HCV, genotipa 1b su klasifikovani u odnosu na
odgovor na kombinovanu terapiju. Za određivanje primarne strukture proteina HCV
primenjene su klasične metode molekularne biologije: reverzna transkripcija i lančana
reakcija polimeraze (Rt-PCR), apsolutna kvantifikacija sa PCR-om u realnom vremenu i
automatsko sekvenciranje. Rezultati: Na osnovu rezultata analize svih proteina virusa
HCV utvrđeno je da je informacioni sadržaj proteina p7 u korelaciji sa odgovorom na
kombinovanu terapiju. Rezultati dobijeni analizom proteinskih sekvenci, 48 pacijenata
sa teritorije Srbije su u saglasnosti sa predloženim bioinformatičkim kriterijumom.
Posebna pažnja je posvećena optimizaciji eksperimentalnih protokola i formiranju
homogenenih grupa u odnosu na osobenosti virusa (tip i podtip) i odgovora na
antivirusnu terapiju pacijenata. Zaključak: Na osnovu rezultata ove studije predložen je
bioinformatički kriterijum koji omogućava procenu odgovora hroničnih HCV bolesnika
inficiranih genotipom 1b na kombinovanu terapiju. | Background: Hepatitis C virus (HCV) infection is a major and rising global
health problem, affecting about 170 million people worldwide, according to WHO data,
and often leading to chronic liver disease and cirrhosis. The current standard therapy for
chronic HCV infection with pegylated interferon combined with ribavirin in patients
with the genotype 1 infection, the most frequent genotype in the USA, Western Europe
and Serbia, accompanied by numerous side effects, leads to a successful outcome in
only about 50% of individuals. Therefore, simple and accurate prediction of hepatitis C
treatment response is of great benefit to patients and clinicians. Methods: Identification
of the conserved information of the HCV proteins that correlate with the combination
therapy outcome was performed by bioinformatics analysis. Plasma samples of 48
chronic HCV patients from Serbia were classified according to the outcome of therapy.
To determine primary structure of HCV proteins classical methods of molecular
biology: reverse transcription and polymerase chain reaction (Rt-PCR), the absolute
quantification-Real Time PCR and DNA sequencing were applied. Results: Among the
HCV proteins that we have analyzed the informational property of the p7 of HCV
genotype 1b was best related to the therapy outcome. Findings obtained from analyzing
sequences 48 patients collected from Serbia were in perfect agreement with proposed
bioinformatics criterion. Special attention was paid to optimization experimental
protocols and forming homogeneous groups of patients regarding HCV genotype (type
and subtype) and therapy response. Conclusions: On the basis of the results in the
present study, a simple bioinformatics criterion that could be useful in assessment of the
response of HCV-infected patients to the combination therapy has been proposed. | true |
Mehanizam zaštitnog dejstva arilpiperazinskih liganada za dopaminske D2 receptore u azot monoksidom i 6-hidroksidopaminom izazvanoj smrti SH-SY5Y ćelija humanog neuroblastoma | Protective mechanism of arylpiperazine dopaminergic D2ligands on nitric oxide and 6-hydroxydopamine induced SH-SY5Y human neuroblastoma cell death | U ovom radu je ispitivan uticaj 20 novosintetisanih arilpiperazinskih
dopaminergičkih liganada na vijabilnost humanih SH-SY5Y neuroblastomskih ćelija
tretiranih donorom azot monoksida (engl. nitric oxide, NO) natrijum nitroprusidom
(engl. sodium nitroprusside, SNP) i uzročnikom oksidativnog stresa dopaminergičkim
neurotoksinom 6-hidroksidopaminom (6-OHDA). Supstanca koje je pružala najjaču
zaštitu od donora NO je bio N-{4-[2-(4-fenil-piperazin-1-il)-etil]-fenil}-pikolinamid
(arilpiperazin 6a), dok je
N-{3-[2-(4-fenil-piperazin-1-il)-etil]-fenil}-pikolinamid
(arilpiperazin 6b) najefikasnije štitio humanu neuroblastomsku ćelijsku liniju SH-SY5Y
od 6-OHDA. Arilpiperazin 6a je delimično sprečavao povećanje sadržaja superoksid
anjon radikala, smanjenje potencijala membrane mitohondrija i unutarćelijskog sadržaja
adenozin-trifosfata (ATP), aktivaciju kaspaza i sledstvenu fragmentaciju DNK koje je
izazivao NO. Uočeno smanjenje unutarćelijske koncentracije superoksida nije bilo
posledica direktne interakcije ispitivanog arilpiperazina sa O2
-∙, niti je supstanca 6a
uticala na akumulaciju NO unutar ćelije. Arilpiperazin 6a je sprečavao inhibiciju
protektivnog Akt, kao i aktivaciju proapoptotskih ERK, JNK i AMPK signalnih puteva
izazvane SNP-om, ukazujući na bitnu ulogu ovih molekula u njegovom zaštitnom
delovanju.
Potencijalni
značaj
arilpiperazina
6a
u
sprečavanju
neurodegenerativnih/neurozapaljenskih procesa posebno naglašava činjenica da je štitio
neuronima slične SH-SY5Y ćelije i od citotoksičnog efekta NO-a poreklom od
stimulisanih makrofaga. Slično, arilpiperazin 6b je sprečavao povećanje unutarćelijskog
sadržaja superoksid anjon radikala (O2
-∙), smanjenje potencijala membrane mitohondrija
i posledične apoptotske događaje – aktivaciju kaspaza i fragmentaciju DNK – koje je
izazivao 6-OHDA. Stabilizacija potencijala mitohondrijalne membrane pod dejstvom
arilpiperazina 6b se vremenski poklapala sa smanjenjem unutarćelijskog sadržaja O2
-∙,
što ukazuje da je supstanca 6b inhibirala oslobađanje superoksida iz oštećenih
mitohondrija stabilizacijom potencijala njihove membrane. Arilpiperazin 6b nije
pokazao značajnu sposobnost direktnog uklanjanja superoksid anjon radikala. Zaštitni
mehanizam supstance 6b nije uključivao interakciju sa JNK i ERK proapoptotskim
putevima, koje je aktivirao 6-OHDA. Međutim, arilpiperazin 6b je izazvao dodatnu
prolaznu aktivaciju preživljavajućeg Akt signalnog puta u ćelijama tretiranim
6-OHDA-om, pa se ne može isključiti doprinos Akt-a antiapoptotskom efektu
arilpiperazina 6b. Pored apoptoze, 6-OHDA je izazivao i citotoksičnu autofagiju u
SH-SY5Y ćelijama smanjujući aktivnost ključnog inhibitora autofagije u ćeliji − mTOR
proteina. Arilpiperazin 6b je delimično sprečavao inhibiciju aktivnosti mTOR-a i
posledičnu autofagiju zabeležene u prisustvu 6-OHDA. Ovaj efekat arilpiperazina 6b je
bio nezavisan od aktivnosti pozitivnog regulatora autofagije AMPK. Iako su oba
arilpiperazina pokazivala parcijalnu agonističku aktivnost na D2 receptorima njihov
neuroprotektivni efekat je bio nezavisan od vezivanja za dopaminske receptore, jer ga
visokoafinitetni blokatori D1/D2 receptora butaklamol (u slučaju 6a) ili selektivni
blokator D2 receptora sulpirid (u slučaju 6b) nisu poništavali. Ovi rezultati ukazuju da
arilpiperazini predstavljaju dobre kandidate za razvoj potencijalnih neuroprotektivnih
lekova. | We investigated the protective ability of 20 novel arylpiperazine-based
dopaminergic ligands against nitric oxide (NO) and dopaminergic neurotoxin
6-hydroxydopamine
(6-OHDA)-mediated
neurotoxicity.
The
most
potent
neuroprotective compound against NO-induced toxicity was N-{4-[2-(4-phenyl-
piperazin-1-yl)-ethyl]-phenyl}-picolinamide (arylpiperazine 6a), while N-{3-[2-(4-
phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolinamide
(arylpiperazine
6b)
most
effectively protected SH-SY5Y human neuron-like cells from 6-OHDA-generated
oxidative injury. Arylpiperazine 6a diminished the proapoptotic action of NO donor
sodium nitroprusside (SNP) by decreasing superoxide anion content, mitochondrial
membrane depolarization, decline in intracellular adenozine-triphosphate (ATP)
content, caspase activation and subsequent phosphatydilserine externalization/DNA
fragmentation. The observed decrease of intracellular superoxide concentration was not
mediated by direct O2
-∙ scavenging. Arylpiperazine 6a did not interfere with NO
accumulation within the cell. The protective effect of arylpiperazine 6a in NO-induced
stress was associated with activation of anti-apoptotic (Akt) and the inhibition of
proapoptotic (JNK, ERK, AMPK) signaling pathways. A potential therapeutic value of
the arylpiperazine 6a in neurodegenerative/neuroinflammatory diseases prevention was
additionally supported by the ability of this arylpiperazine to protect SH-SY5Y neuron-
like cells from macrophage-derived NO. Similarly, arylpiperazine 6b prevented
6-OHDA-induced increase in superoxide anion content, mitochondrial membrane
depolarization and following apoptotic related events – caspase activation and DNA
fragmentation. The stabilization of 6-OHDA-disrupted mitochondrial membrane
potential by arylpiperazine 6b correlated with the decrease in intracellular superoxide
anion (O2
-∙) content, suggesting that decline in O2
-∙ concentration resulted from
mitohondrial membrane stabilization. Arylpiperazine 6b did not posses significant O2
-∙
scavenging ability. The mechanism underlying the protection provided by the
compound 6b was independent of JNK- and ERK-proapoptotic pathways, as
6-OHDA-provoked activation of these proapototic pathways was not modified by
arylpiperazine 6b. However, arylpiperazine 6b caused additional transient activation of
pro-survival Akt signaling pathway in 6-OHDA-treated cells, which probably
contributed to its anti-apoptotic effect. In addition to apoptosis, 6-OHDA induced
deleterious autophagy in SH-SY5Y cells by silencing the activity of mTOR, a key
autophagy inhibitor in the cell. Arylpiperazine 6b partialy prevented 6-OHDA-triggered
decrease of mTOR activity and autophagy in AMPK-independent manner. Although
both compounds showed partial agonist activity on hD2 receptors, their neuroprotective
action was independent of dopamine receptor binding, as it was not affected by the
high-affinity D1/D2 receptor blocker butaclamol (6a) or selective D2 receptor blocker
sulpiride (6b). These results support further study of arylpiperazines as potential
neuroprotective drugs. | true |
Korelacija između proteinske i genske ekspresije HER-2 i topoizomeraze 2a kod infiltrativnog duktalnog karcinoma dojke | Correlation between protein and gene expression of HER-2 and topoisomerase 2a in infiltrative ductal breast carcinoma | Uvod: Karcinom dojke predstavlja jako heterogeno oboljenje kao i jedno od
najzastupljenijih malignih bolesti u opštoj ženskoj populaciji. Na osnovu prisustva i
nivoa ekspresije ER-R, PR-R i HER-2 receptora ostvarena je podela na četiri
molekularna tipa karcinoma dojke: luminalni A (ER-R+ i / ili PR-R+, HER-2-), luminalni
B (ER-R+ i / ili PR-R+, HER-2+), HER-2 pozitivni (ER-R- i / ili PR-R-, HER-2+) i
trostruko negativni (ER-R- i / ili PR-R-, HER-2-) tip karcinoma dojke. U oko 20 do 30%
karcinoma dojke javlja se amplifikacija HER-2 i TOP2A gena. Amplifikacija HER-2 i
TOP2A gena je povezana sa lošijim ishodom bolesti, agresivnijim tokom i dobrim
odgovorom pacijentkinja na terapiju Herceptinom i antraciklinima. Koamplifikacija
HER-2 i TOP2A gena je prisutna u 12 do 38% karcinoma dojke. Na osnovu rezultata
nekoliko studija postavljena je hipoteza po kojoj se amplifikacija TOP2A gena javlja
isključivo sa amplifikacijom HER-2 gena, mada postoje i drugačija, suprotna mišljenja.
Cilj: Cilj naše studije bio je da se odrede genski statusi HER-2 i TOP2A kao i njihova
korelacija sa apoptotskim i proliferativnim indeksom, nivoom ekspresije ER-R, PR-R i
HER-2 receptora, TOP2A, BRCA1, Bcl-2 i p53 proteina, osnovnim kliničko-patološkim
parametrima kod tumora dojke 60 pacijentkinja podeljenih u četiri molekularna tipa.
Materijal i metode: Šezdeset tkivnih uzoraka karcinoma dojke ispitivano je
imunohistohemijskom metodom na prisustvo i stepen ekspresije ER-R, PR-R, HER-2,
p53, Bcl-2, TOP2A, BRCA1, ssDNA, Ki67, kao i FISH metodom na stepen genske
modifikacije HER-2 i TOP2A. Stepen modifikacije HER-2 i TOP2A gena analiziran je
primenom HER-2 FISH pharmDxTM kit i TOP2A FISH pharmDxTM kit
(DakoCytomation).
Rezultati:
Kliničko-patološki podaci - Starost ispitanica se kretala u rasponu izmeñu 38 i 87
godina (64.79 ± 21). Veličina tumora se kretala u opsegu izmeñu 0.4 i 5.5 cm, dok je
histološki gradus G1 bio zastupljen kod 6 (10%) karcinoma, histološki gradus G2 kod
25 (41.7%) i histološki gradus G3 kod 17 (28.3%) karcinoma dojke. Kod 26 (43.4%)
ispitanica infiltracija regionalnih limfnih čvorova tumorskim ćelijama je bila odsutna,
dok je kod 22 (36.6%) ispitanice zabeležena pozitivna infiltracija regionalnih limfnih
čvorova. Metastaze su prisutne kod sedam ispitanica dok je kod 39 ispitanica
registrovan uredan nalaz bez prisustva mestastaza.
HER-2 i TOP2A genske aberacije kod ispitanica sa četiri različita molekularna tipa
karcinoma dojke - U luminalnom A tipu karcinoma dojke amplifikacija HER-2 gena je
zastupljena samo u jednom tumoru (1/24, 4.2%), dok je visok nivo amplifikacije
TOP2A gena zabeležen u tri karcinoma (3/24, 12.5%), nizak nivo amplifikacije u pet
(5/24, 20.8%), dok je delecija TOP2A gena zastupljena u karcinomu jedne ispitanice
(1/24, 4.2%).
Nizak nivo amplifikacije HER-2 gena zabeležen je u pet (5/26, 19.3%) dok je
visok nivo genske amplifikacije zabeležen u šest karcinoma (6/26, 23%) luminalnog B
tipa. Genske modifikacije TOP2A su bile učestalije u odnosu na HER-2 u tumorima
ispitanica luminalnog B tipa. Nizak nivo amplifikacije TOP2A karakterisao je tumore
šest ispitanica (6/26, 23.1%), visok nivo amplifikacije zabeležen je u karcinomima pet
ispitanica (5/26, 19.2%), dok je TOP2A genska delecija zastupljena u četiri tumora
(4/26, 15.4%).
Nizak nivo amplifikacije TOP2A gena zastupljen je samo u jednom tumoru (1/4,
25%) trostruko negativnog tipa. Visok nivo amplifikacije HER-2 gena zabeležen je kod
četiri tumora (4/6, 66.7%), a nizak nivo amplifikacije u samo jednom tumoru (1/6,
16.7%) HER-2 pozitivnog tipa. Genske modifikacije TOP2A su zabeležene u tumorima
kod četiri ispitanice, od toga je kod tri zabeležen nizak nivo amplifikacije (3/6, 50%), a
kod jedne visok nivo amplifikacije TOP2A gena (1/6, 16.7%).
Korelacija izmeñu genskog statusa HER-2 i TOP2A sa ekspresijom drugih bioloških
markera - Statistički značajna negativna korelacija karakterisala je odnos izmeñu HER-
2 genskog statusa i ekspresije ER-R, PR-R i Bcl-2 proteina, dok je statistički značajna
pozitivna korelacija karakterisala odnos stepena ekspresije HER-2 receptora i TOP2A
genskog statusa. U tumorima trostruko negativnog tipa takoñe je zabeležena statistički
značajna negativna korelacija izmeñu ekspresije TOP2A, BRCA1 proteina i HER-2
genskog statusa, dok je pozitivna korelacija karakterisala odnos izmeñu p53 proteina i
HER-2 genskog statusa. U tumorima ispitanica luminalnog A tipa, TOP2A genski status
negativno je korelirao sa stepenom ekspresije Bcl-2 proteina.
Preživljavanje i tip terapije - Tipovi primenjenih terapija kod ispitanica obuhvaćenih
ovom studijom sastojali su se od endokrine, hemoterapije, terapije zračenjem i terapije
primenom antitela Herceptina. Kod većine pacijentkinja iz sva četiri molekularna tipa
karcinoma dojke primenjena je kombinacija hemoterapijskih agenasa (AC, CMF),
hormonska terapija (tamoksifen, nolvadeks) i terapija zračenjem. Studija je obuhvatila
praćenje toka bolesti ispitanica u toku četvorogodišnjeg perioda (januar 2009. do
februara 2013.). U toku ovog vremenskog perioda kod dve ispitanice je nastupila smrt
kao direktna posledica ove bolesti dok kod njih 39 nije bilo znakova bolesti. Prosečan
period opšteg preživljavanja kao i preživljavanje bez pojave recidiva iznosi 34.8 meseci.
Prosečno najduži period opšteg preživljavanja registovan je kod ispitanica sa HER-2
pozitivnim tipom karcinoma dojke i iznosio je 44.8 ± 10.2 meseca. Najkraći period
opšteg preživljavanja karakterisao je ispitanice sa trostruko negativnim tipom
karcinoma dojke (iznosio je 19.5 ± 18 meseci).
Zaključak: Rezultati ove studije su pokazali da tumore luminalnog A i B tipa generalno
karakteriše mala veličina, odsustvo infiltracije regionalnih limfnih nodusa tumorskim
ćelijama i dugačak period opšteg preživljavanja u odnosu na tumore trostruko
negativnog tipa. Luminalne tipove tumora je takoñe najčešće odlikovao nizak nivo
ekspresije proteina p53, visok nivo očuvane ekspresije Bcl-2 proteina kao i niske
vrednosti indeksa proliferacije. Karcinome HER-2 pozitivnog tipa odlikovala je potpuno
redukovana ekspresije Bcl-2 proteina, nizak indeks apoptoze, kao i dominantno
prisustvo HER-2 i TOP2A genskih aberacija. Tumore luminalnog tipa karakteriše dobra
prognoza ishoda bolesti kod ispitanica, mala verovatnoća za pojavu metastaza, kao i
dobar odgovor na hormonsku terapiju (nolvadeks, tamoksifen) u poreñenju sa
ispitanicama čiji tumori imaju odlike trostruko negativnog i HER-2 pozitivnog tipa.
Takoñe, karcinomi dojke ispitanica sa luminalnim A i B tipom odlikuje visok stepen
zastupljenosti genskih aberacija TOP2A u odnosu na HER-2 genski status što opravdava
njegovu ulogu samostalnog prediktivnog i prognostičkog markera u karcinomu dojke.
Na osnovu sumiranja svih rezultata iznetih u ovoj disertaciji može se zaključiti
da luminalne tipove karcinoma dojke sa jedne strane, i trostruko negative i HER-2
pozitivne tipove karcinoma sa druge strane, karakteriše odreñeni obrazac ekspresije
ispitivanih bioloških markera što je značajno za izbor terapije, predviñanje odgovora na
terapiju i ishoda same bolesti kod ispitanica sa karcinomom dojke. | Introduction: Breast cancer is a heterogeneous disease and the most common
malignancy in women with increasing incidence in recent years. According to
immunohistochemical expression of estrogen receptor (ER-R), progesterone receptor
(PR-R) and HER-2 four different molecular subtypes of breast cancer were identified:
luminal A (ER-R+ and / or PR-R+, HER-2-), luminal B (ER-R+ and / or PR-R+, HER-
2+), HER-2 positive (ER-R- and / or PR-R-, HER-2+) and triple negative (ER-R- and / or
PR-R-, HER-2-) breast cancer subtypes. HER-2 and TOP2A genes are amplified in 20–
30 % of breast cancers. Amplification of HER-2 and TOP2A genes are associated with a
poor prognosis and predictive response to treatment by Herceptin and anthracycline.
Co-amplification of HER-2 and TOP2A has been seen in approximately 12–38 % of
patients with breast cancers. A few retrospective studies have reported that TOP2A gene
amplification occurs almost exclusively in conjunction with HER-2 gene amplification.
Aim: The aim of our study is to investigate HER-2 and TOP2A gene aberrations and
their correlation with apoptotic and proliferative indexes, Bcl-2, p53, ER-R, PR-R,
HER-2, TOP2A, BRCA1, clinicopathological parameters and overall survival in four
molecular subtypes of breast cancer.
Material and methods: Sixty paraffin-embedded breast cancer tissue samples were
studied immunohistochemically for the expression of ER-R, PR-R, HER-2, p53, Bcl-2,
ssDNA, Ki67, BRCA1, TOP2A and FISH for HER-2 and TOP2A gene aberrations.
HER-2 and TOP2A gene copy number were analyzed by HER-2 FISH pharmDxTM kit
and TOP2A FISH pharmDxTM kit (DakoCytomation), respectively.
Results:
Clinicopathological parameters - The ages of patients ranged from 38 to 87 years
(64.79 ± 21). The tumor size ranged between 0.4 and 5.5 cm, and the histological grades
were G1, G2 and G3 in 6 (10 %), 25 (41.7 %), and 17 (28.3 %) patients, respectively. In
26 patients (43.4 %) lymph node status was negative; while in 22 patients (36.6 %) was
detected positive lymph node status. Distant metastasis characterized 7 (15.3 %)
patients and in 39 (84.7 %) patients we did not find a distant metastasis.
HER-2 and TOP2A gene aberrations in four molecular subtypes of breast
cancer - Among 24 patients of luminal A subtype, only in one case (1/24, 4.2 %) a low
level of HER-2 gene amplification was detected, while 3 cases (3/24, 12.5 %) were
highly amplified for TOP2A, 5 cases (5/24, 20.8 %) had low level of TOP2A gene
amplification and in one case (1/24, 4.2 %) TOP2A gene deletion was detected. We
characterized 5 cases with low (5/26, 19.3 %) and 6 cases with high (6/26, 23 %) level
of HER-2 gene amplification in total 26 patients of luminal B subtype of breast cancer.
In the same subtype, a low level of TOP2A gene amplification was detected in 6 cases
(6/26, 23.1 %), high level in 5 cases (5/26, 19.2 %) and in 4 cases (4/26, 15.4 %) were
detected TOP2A gene deletion. In patients with triple negative subtypes of breast
cancer, only one case (1/4, 25 %) had a low level of TOP2A gene amplification whereas
all other cases did not have HER-2 or TOP2A gene aberrations. In HER-2 positive
subtype of breast cancer, we characterized 4 cases (4/6, 66.7 %) with high amplified
HER-2 gene, and one case (1/6, 16.7 %) with a low level of HER-2 gene amplification.
TOP2A gene had a low level of amplification in 3 cases (3/6, 50 %) and in one case
(1/6, 16.7 %) a high level of TOP2A gene amplification was registered.
Correlation between HER-2 and TOP2A gene aberrations with expression of other
biological markers - HER-2 gene aberrations correlated negatively with ER-R, PR-R
and Bcl-2 protein, while statistically significant positive correlation was detected
between HER-2 gene aberrations and HER-2 receptor expression and TOP2A gene
aberrations. HER-2 gene aberrations correlated positively with p53 protein expression in
triple negative breast cancer subtype. Statistically significant negative correlation was
detected between the expression of TOP2A and BRCA1 protein in comparison with the
HER-2 gene status in triple negative breast cancer subtype. In luminal A molecular
breast cancer subtype, TOP2A gene aberration negatively correlated with Bcl-2 protein
expression.
Survival analysis and therapy - Treatment was nonrandomized and consistent with
endocrine therapy, chemotherapy, radiotherapy, Herceptin and their different
combinations. In the most patients from all four molecular subtypes of breast cancer a
combination of chemotherapy agents (AC, CMF), hormonal therapy (tamoxifen,
nolvadex) and radiotherapy was applied. Follow-up of the patients extended from
January 2009. to February 2013. Two patients died of breast carcinoma and 39 patients
did not have symptoms of remission. The median of disease free survival (DFS) and
overall survival (OS) were 34.8 months. We found that DFS and OS were significantly
longer (44.8 ± 10.2 months) in patients with HER-2 positive molecular subtype. The
shortened OS characterized patients with triple negative breast cancer subtype (19.5 ±
18 months).
Conclusion: The results of this study showed that luminal A and B subtypes of breast
cancer are generally characterized by small tumor size, negative lymph node status, as
well as a longer period of overall survival compared to the triple negative breast cancer
subtype. Breast cancers from luminal A and B subtypes are characterized with low
levels of p53 protein expression and proliferative index, but high level of Bcl-2 protein
expression. In HER-2 positive breast cancer subtype tumor has negative expression for
Bcl-2 protein, low level of apoptotic index, and high level of HER-2 and TOP2A gene
aberrations. Luminal A and B breast cancers have a more favourable prognosis, small
occurrence of metastases and good response to hormonal therapy (nolvadex, tamoxifen),
compared to the triple negative and HER-2 positive tumors. Also, patients in luminal A
and B breast cancer subtypes are characterized by a high proportion of cases with
amplification and deletion of TOP2A gene in comparison to HER-2 gene status,
justifying TOP2A significance as an individual prognostic marker regarding
anthracycline therapy.
According to presented results we concluded that the luminal A and B breast
cancer subtypes on one side and triple negative and HER-2 positive breast cancer
molecular subtypes on the other side share similar specific model of expression of
studied biological markers that are important for the choice of therapy, prediction
response to therapy and outcome of the disease in patients with breast cancer. | true |
Detekcija mutacija u p53 genu kod bolesnica sa primarnim operabilnim karcinomom dojke | Detection of p53 gene mutation in operable breast cancer patients | Uvod: Zbog svoje uloge u očuvanju genomskog integriteta, produkt normalnog p53 gena se često naziva i "čuvar genoma". Pokazano je da u ćeljama gde je p53 izmenjen dolazi do aberantne replikacije DNK, stoje praćeno genomskom nestabilnošću. Gubitak funkcije p53 povezan je ne samo sa nemogućnošću popravke DNK, već i sa apoptozom. Podaci ukazuju da p53 ima kritičnu ulogu u aktiviranju apotoze pri odgovoru na različite vidove hemioterapije u karcinomu dojke. Cilj ovog rada bio je da se utvrdi da li p53 mutacije utiču na odgovor na hemioterapiju i da li p53 mutacije mogu dati informaciju o prognozi bolesnica sa karcinomom dojke. Bolesnice i metode. U rad je bilo uključeno 100 bolesnica, od toga 70 sa metastazama u regionalnim limfnim čvorovima i 30 bolesnica bez metastaza u regionalnim limfnim čvorovima. Sve osim jedne bolesnice sa metastazama u limfnim čvorovima su primile adjuvantnu hemioterapiju. 65/70 bolesnica je primilo CMF (ciklofosfamid, metotreksat, 5-fluorouracil) po modifikovanom intravenoznom protokolu, i 5/70 je primilo FAC (5-fluorouracil, adriablastin, ciklofosfamid). Bolesnice su praćene u intervalu 6-127 meseci, medijana 48 meseci. DNK je izolovana fenolskom ekstrakcijom iz uzoraka rutinski prikupljanih za odredjivanje receptora za steroidne hormone. Specifični DNK regioni p53 gena koji odgovaraju eksonima 5-8, umnoženi su lančanom reakcijom polimeraze (PCR). Detekcija mutacija izvršena je SSCP (konformacioni polimorfizam jednolančane DNK) elektroforezom. Gelovi su bojeni srebrom. Amplifikacija c-myc gena odredjena je diferenijalnim PCR-om. Receptori za steroidne hormone odredjivani su biohemijskom saturacionom metodom sa aktivnim ugljem. Hi-kvadrat i Fisher-ov egzaktni test, kao i Kaplan-Meir-ov i Log-Rank test su korišćeni za statističku obradu podataka. Rezultati: p53 mutacije detektovane su kod 21/100 bolesnica. Ukupno je pronadjeno 25 mutacija (po dve mutacije detektovane su kod 4 bolesnice), i njihova raspodela po ispitivanim eksonima nije bila jednaka: 10 u eksonu 5, 6 u eksonu 6, 8 u eksonu 7 i samo jedna mutacija u eksonu 8. Učestalost p53 mutacija bila je znatno viša kod bolesnica sa metastazama u limfnim čvorovima u odnosu na bolesnice bez metastaza u limfnim čvorovima. p53 mutacije bile su predominantno locirane kod bolesnica sa invazivnim duktalnim tipom karcinoma dojke. S obzirom da je kod bolesnica bez metastaza u regionalnim limfnim čvorovima pronadjena samo jedna mutacija, korelacija izmedju detektovanih mutacija i klničkog toka bolesti analizirana je samo kod bolesnica sa metastazama u limfnim čvorovima. Od ovih bolesnica, 32/70 je imalo ponovno javljanje bolesti (relaps). U prvih 24 meseci praćenja, bolesnice sa p53 mutacijama su imale značajno kraći period do ponovnog javljanja bolesti (DFI), u odnosu na bolesnice bez p53 mutacija. Takodje, u grupi bolesnica sa relapsom pokazan je značajno kraći period do progresije bolesti (PFI) kod bolesnica sa p53 mutacijama u odnosu na bolesnice bez mutacija. U odnosu na ukupno preživljavanje (OS), značajno kraće preživljavanje zabeleženo je kod bolesnica sa p53 i/ili c-myc alteracijama u odnosu na bolesnice bez detektovanih genskih alteracija. Zaključak: Pojava ranog relapsa kod bolesnica sa p53 mutacijama koje su primile hemioterapiju, ukazuje na prediktivni značaj p53. Rezultati dobijeni za OS, ističu značaj istovremenog odredjivanja alteracija više gena u nastojanju da se definiše molekularni profil svakog tumora, što za krajnji cilj ima individualni terapijski pristup. | Background: the main function of normal p53 gene product is to preserve genome integrity by acting as the "guardian of the genome". It has been shown that in p5 3-defective cells DNA aberrant replication is followed by genome instability. Loss of p53 function seems to be connected not only with abrogation of DNA reapair, but also with abrogation of apoptosis. The evidence indicates that p53 play a critical role in implementing apoptosis in response to treatment with different chemotherapeutics in breast cancer. The aim of this study was to evaluate whether tumor's p53 mutations affect response to chemotherapy, and whether p53 mutations provide prognostic information for breast cancer patients. Patients and methods: 100 breast cancer patients, among 70 lymph node-positive and 30 lymph node-negative were included. All except one node-positive patients underwent chemotherapy as adjuvant treatment. 65/70 patients received CMF (cyclophosphamide, methotrexate, 5-fluorouracil) modified intravenous protocol, and 5/70 FAC (5-flurouracil, adriablastine, cyclophosphamide) protocol. The patients were followed-up from 6 to 127, median 48 months. DNA was isolated by phenol extraction from samples routinely collected for steroid receptor determinations. Specific DNA regions corresponding to exons 5-8 were amplified by polimerase chain reaction (PCR). Detection of mutations was done by single stranded conformation polymorphism - SSCP - electrophoresis. Gels were silver stained. C-myc amplification was detected by differential PCR. Steroid receptors were measured by five- point dextrane coated charcoal (DCC) assay. Chi-square and Fisher exact test, as well as Kaplan-Meir and Log-Rank tests were used for statistical evaluation. Results: p53 mutations were detected in 21/100 patients. Totally 25 mutations were found (in 4 patients double mutations were detected). They were not equally spread within examined exons - 10 in exon 5, 6 in exon 6, 8 in exon 7 and only one mutation in exon 8. The incidence of p53 mutations was significantly higher in lymph node-positive than in lyph node-negative patients. Further, p53 mutations were predominantly located in invasive ductal histological type. Since the node-negative patients only one mutation was found, the correlation between detected mutations and the course of disease was examined in lymph node-positive group of patients. Among them, 32/70 patients developed relapse of disease. In the first 24 months of follow-up, patients with p53 mutations had significantly shorter disease free interval (DFI), than the patients without p53 mutations. Also, significantly shorter progression free interval (PFI) in relapsed patients with p53 mutations than in relapsed patients without p53 mutation was found. Concerning overall survival (OS), significantly shorter OS was found in the patients with p53 and/or c-myc gene alterations than in the patients without gene alterations. Conclusion: In conclusion, occurrence of early relapse in chemotherapy treated patients with p53 mutations, supports data about p53 predictive value. The results obtained for OS indicate importance of simultaneous screening for multiple gene alterations in attempt to define specific tumor molecular profile, finally resulting with individual therapeutic approach. | false |
Analiza fosforilacije glukokortikoidnog receptora u limfocitima zdravih ljudi i pacijenata sa akutnom epizodom depresivnog poremećaja | Analysis of lymphocyte glucocorticoid receptor phosphorylation in healty subjets and patients with current episode of depressive disorder | Depresivni poremećaj (DP) je multifaktorijalno oboljenje, široko rasprostranjeno u svetu, i rasvetljavanje mehanizama koji su u osnovi ovog poremećaja predstavlja jedan od najvećih izazova biomedicinskih istraţivanja današnjice. Narušena funkcija hipotalamo-hipofizno-adrenalne (HHA) ose, kao i signalizacija preko glukokortikoidnog receptora (GR), čest su nalaz kod pacijenata sa DP-om. Takođe, određeni stepen disfunkcije HHA ose i samog GR-a uočavaju se i kod zdravih ljudi i pretpostavlja se da to predstavlja jedan od faktora rizika za razvoj kliničke depresije. S druge strane, uspešno lečenje pacijenata sa depresijom, praćeno je normalizacijom aktivnosti HHA ose i funkcije GR-a. GR je ligand-zavisan transkripcioni faktor, koji nakon vezivanja glukokortikoida (krajnjih produkata aktivnosti HHA ose) prelazi iz citoplazme u jedro gde reguliše ekspresiju velikog broja gena. Vaţan način regulacije funkcije GR-a postiţe se njegovom fosforilacijom koja je posredovana aktivnošću kinaza, koje se aktiviraju u odgovoru na različite ćelijske signale. Fosforilacija GR-a na serinu 211 (S211) stimuliše ulazak receptora u jedro i generalno se smatra biomarkerom njegove povećane transkripcione aktivnosti, dok je za fosforilaciju GR-a na serinu 226 (S226), koja je posredovana c-Jun N-terminalnim kinazama (eng. c-Jun N-terminal kinases, JNK), pokazano da inhibira njegovu transkripcionu aktivnost i stimuliše izlazak GR-a iz jedra u citoplazmu. Među genima stimulisanim glukokortikoidima su GILZ (eng. glucocorticoid-induced leucine zipper, GILZ), vaţan posrednik anti-inflamatornih efekata GR-a, i FKBP5 (eng. FK506 binding protein 5, FKBP5), koji ima vaţnu ulogu u ograničavanju glukokortikoidne signalizacije u ćeliji, sprečavajući translokaciju GR-a iz citoplazme u jedro... | Major depressive disorder (MDD) is a multifactorial illness that a ffects people across the globe and u derstanding mechanisms underlying this disorder represents a major challenge of biomedical research today. One of the most consistent findings among patients with MDD is dysfunctional hypothalamic – pituitary – adrenal (HPA) axis, caused by altered function of the glucocorticoid receptor (GR). In healthy population, individuals with impaired HPA axis and GR function are also observed, and these features are believed to represent increased risk factors for depression . On the o ther hand, successful antidepressant treatments ameliorate many of the neuroendocrine disturbances observed in depression . The GR is ligand - activated transcriptional factor that apon glucocorticoid binding (glucocorticoids are end products of HPA axis acti vity) translocate from the cytoplasm to the nucleus where it modulates the expression of wide variety of genes. Phoshor y lation of the GR, mediated by activity of diverse cellular kinases, is imprtant way of regulating GR function. The GR phosphorylation at S211 promotes GR translocation to the nucleus and enhances its transcriptional activity , while GR phosphorylation at S226, mediated by c - Jun N - terminal kinases (JNK), inhibits its transcriptional activity and promotes GR nuclear export. Some of m yriad of genes whose expression is stimu late d by the GR are glucocorticoid - induced leucine zipper (GILZ), an important mediator of glucocorticoid anti - imflamatory actions, and FK506 binding protein 5 (FKBP5), which is a part of an intracellular ultra - short negative feedback loop for GR activity... | false |
Citogenetička, palinološka i filogeografska istraživanja roda Ramonda (Gesneriaceae) na Balkanskom poluostrvu | Cytogenetics, palynology and phylogeography of genus Ramonda (Gesneriaceae) in the Balkan peninsula | Familija Gesneriaceae obuhvata biljne vrste pretežno rasprostranjene u tropskim
u subtropskim oblastima Starog i Novog sveta. Samo mali broj vrsta naseljava umerene
oblasti, a među njima je i jedinih 5 vrsta ove familije u Evropi: Ramonda myconi,
endemit Pirinejskog poluostrva, kao i R. nathaliae, R. serbica, Haberlea rhodopensis i
Jankaea heldreichii, četiri endemične vrste Balkanskog poluostrva. Svih pet vrsta su
relikti Tercijera, vremena kada je klima u Evropi bila toplija i vlažnija. Za vreme
Ledenog doba, one su našle skrovište u klisurama i kanjonima gde ih najčešće nalazimo
i danas. Ovi „živi fosili“ danas nepovoljne uticaje spoljašnje sredine u velikoj meri
preživljavaju zahvaljujući poikilohidričnosti, pa se još nazivaju i „biljke koje
vaskrsavaju“.
Danas se dve balkanske vrste roda Ramonda odlikuju disjunktnim arealima.
Ramonda nathaliae zastupljena je u Makedoniji, S Grčkoj, na obroncima planine Šare
na Kosovu i u JI Srbiji. Najveći deo areala vrste R. serbica nalazi se u Albaniji, a
prisutna je i na SZ Grčke, u Z Makedoniji, JZ i SI Crnoj Gori, JZ, JI i SI Srbiji, kao i na
SZ Bugarske. Jedina zona u kojoj se areali dve vrste susreću nalazi se u JI Srbiji, gde na
dva lokaliteta, Oblik i Radovanski Kamen, obrazuju simpatrijske populacije. Obe vrste
prevashodno naseljavaju krečnjačku podlogu, s tim što se R. nathaliae može naći i na
serpentinitu, škriljcima i granitu. Ova vrsta, takođe, bolje podnosi otvorenija staništa, a
njene populacije se mogu naći i na većem rasponu nadmorskih visina, zbog čega se
smatra otpornijom i tolerantijom.
Ciljevi ovog rada su višestruki: da se utvrde morfološke osobine polena i semena
sve tri vrste roda Ramonda, jer detalji njihove građe do sada nisu istraživani; da se
procene vijabilnost njihovog polena i klijavost semena; da se utvrdi broj hromozoma,
kao i veličina genoma kod tri vrste; da se proveri da li postoje hibridne jedinke u
simpatrijskim populacijama; da se pomoću molekularnih markera utvrdi stepen
sličnosti i filogeografske veze između vrsta R. nathaliae i R. serbica. U tu svrhu, polen
i seme analizirani su pomoću svetlosnog i scanning elektronskog mikroskopa, broj
hromozoma određen je standardnim kariološkim tehnikama, veličina genoma
procenjena je pomoću protočne citometrije, a genetički diverzitet primenom
molekularne AFLP tehnike.
Ramonda myconi i R. nathaliae su diploidi sa 2n = 48, dok je R. serbica
heksaploid sa 2n = 144 hromozoma. U jednoj populaciji ustanovljeno je i postojanje
nekoliko individua sa većim brojem hromozoma (2n = 8x = 192 i 2n = 10x = ~230).
Prosečna veličina genoma kod R. myconi je 2C = 2,55 pg, R. nathaliae 2C = 2,34 pg i
R. serbica 2C = 7,85 pg, s tim što retki dekaploidi ove vrste imaju 2C = 11,65 pg.
Monoploidne veličine genoma R. myconi (1Cx = 1,27 pg) i R. serbica (1Cx = 1,31 pg)
su slične, dok R. nathaliae ima manju vrednost ovog parametra (1Cx = 1,17 pg).
Polenova zrna sve tri vrste su mala, 3–kolporatna, uglavnom sferoidnog oblika.
Kod diploidnih vrsta su slične veličine (R. myconi E = 12,57 µm; R. nathaliae E =
15,19 µm), dok su kod heksaploidne R. serbica veća (E = 19,80 µm) u skladu sa većom
veličinom genoma. Egzina je mikroretikularno-perforatna, kod R. nathaliae više
perforatna, kod R. serbica sa krupnim lumenima unutar kojih je veliki broj perforacija,
dok je kod R. myconi intermedijarna između dve balkanske rođake.
Semena su sitna, izduženog oblika, kod R. myconi u proseku duga 814,47 µm i
široka 217,77 µm, kod R. nathaliae duga 639,23 µm, a široka 228,09 µm i kod R.
serbica prosečne dužine 714,36 µm i širine 254,84 µm. Na površini semena uočljive su
trouglaste strukture najupadljivije kod R. myconi (visine 46 – 87 µm), nešto manje kod
R. serbica (32 – 44 µm), a najmanje kod R. nathaliae (21 – 30 µm), čija je moguća
uloga bolje pričvršćivanje za podlogu. Masa semena je umereno korelisana sa
veličinom genoma i brojem hromozoma. Veličine polena i semena ne predstavljaju
dobre karaktere za razlikovanje tri vrste roda Ramonda. Razlike koje se mogu koristiti
u tu svrhu su karakteristike površine egzine polena, kao i površinske strukture
semenjače.
Zahvaljujući opsežnim istraživanjima u simpatrijskim populacijama, otkriveni su
prirodni hibridi nastali ukraštanjem R. nathaliae i R. serbica. Hibridne individue su
najčešće tetraploidi sa 2n = 96 hromozoma i prosečnom veličinom genoma od 2C =
5,14 pg. Pronađen je i mali broj individua čija veličina genoma odgovara penta (2C =
6,19 pg) i oktoploidima (2C = 10,04 pg). Polen hibridnih individua je veoma heterogen
po broju kolpi (3-, 9- i 12-kolporatna zrna) i veličini polenovih zrna, a semena su
izuzetno sitna, 2–3 puta manja od semena roditeljskih vrsta i veoma male klijavosti
(1%). Ovi rezultati pokazuju da se u simpatrijskim populacijama dešavaju složeni
procesi ukrštanja dve reliktne vrste, introgesije i dupliciranja genoma.
U ekološkom pogledu, veoma interesantna je populacija R. nathaliae sa
serpentinita čije se individue odlikuju manjim prečnikom rozete, najmanjim polenovim
zrnima, tanke egzine i slabe vijabilnosti, najmanjim semenima, pa čak i najmanjom
veličinom genoma među ispitivanim populacijama ove vrste.
AFLP analiza genetičke strukture populacija dve vrste potvrdila je jasnu
taksonomsku razdvojenost R. nathaliae i R. serbica. Kao endemične vrste kod kojih
nema samooprašivanja, ali je prisutno vegetativno razmnožavanje, one se odlikuju
malim genetičkim diverzitetom, tako da su vrednosti očekivane heterozigotnosti (H)
kod R. nathaliae od 0,062 do 0,128, a kod R. serbica od 0,087 do 0,140. Varijabilnost
unutar populacija (R. nathaliae 72%, R. serbica 86%) veća je od varijabilnosti između
populacija (R. nathaliae 28%, R. serbica 14%). Genetičke distance i indeksi fiksacije
su veći između populacija R. nathaliae, na osnovu čega se može zaključiti da je ona
starija vrsta čije su populacije duže vreme međusobno izolovane.
Današnji genetički diverzitet populacija ovih reliktnih i endemičnih vrsta sa
Balkanskog poluostrva oslikava kako uticaj Ledenog doba tako i recentnih uslova na
njihovim staništima. Obe vrste su svakako imale više refugijalnih staništa u kojima su
preživele nepovoljne klimatske prilike tokom glacijacije. Iz njih su, nakon otopljavanja
ponovo počele da šire svoj areal, a novoobrazovane populacije odlikuju se manjim
genetičkim diverzitetom usled efekta osnivača i uskog grla. Kako su takve populacije
podložnije nepovoljnim uticajima spoljašnje sredine, neophodno je sprovesti njihovu
adekvatnu zaštitu. | Family Gesneriaceae comprises plant species mostly distributed in the tropics
and subtropics of both the Old and New Worlds. Only a small number of species
inhabits temperate regions. Among them, there are the only 5 species of this family in
Europe: Ramonda myconi, endemic of the Iberian Peninusla, as well as R. nathaliae, R.
serbica, Haberlea rhodopensis i Jankaea heldreichii, four endemic species of the
Balkan Peninsula. All five species are Tertiary relicts, remnants from the times when
the climate was much warmer and more humid. During the Ice Age, they have found
shelter in canyons and gorges where can be often found today as well. These „living
fossils“ are poikilohydric, „resurrection plants“ what helps them survive the
inhospitable environmental conditions.
Balkan species of the genus Ramonda are today characterized by disjunct
distribution. Ramonda nathaliae is restricted to Macedonia, N Greece, slopes of Mt.
Šara in Kosovo and few localities in SE Serbia. The largest part of the R. serbica range
is situated in Albania, but is also present in NW Greece, W Macedonia, SW and NE
Montenegro, SW, SE and NE Serbia and NE Bulgaria. The ranges of two species co-
occur only in two localities in SE Serbia, Oblik and Radovanski Kamen, establishing
sympatric populations. Both species prefer limestone rocks, but R. nathaliae can also
be found on serpentine, schist and granite. This species is often found in more open
habitats and in higher altitudes why it is considered as more resistant and tolerant than
R. serbica.
The aims of this study are: to investigate morphology of both pollen and seeds of
three Ramonda species; to estimate pollen viability and germination capacity of their
seeds; to determine chromosome numbers and genome size of three species; to explore
if there are hybrids in sympatric populations; to look into genetic diversity and
phylogeography of R. nathaliae and R. serbica. Pollen grains and seeds were examined
by light and scanning electron microscopy, chromosome number by standard
karyological techniques, genome size was estimated by flow cytometry and genetic
diversity by molecular AFLP method.
Ramonda myconi and R. nathaliae are diploids with 2n = 48, while R. serbica is
a hexaploid with 2n = 144 chromosomes. Few individuals with larger chromosome
numbers were detected in one population (2n = 8x = 192 and 2n = 10x = ~230).
Average genome size in R. myconi is 2C = 2.55 pg, R. nathaliae 2C = 2.34 pg and R.
serbica 2C = 7.85 pg, while rare decaploids of this last species have 2C = 11.65 pg.
Monoploid genome sizes of R. myconi (1Cx = 1.27 pg) and R. serbica (1Cx = 1.31 pg)
are similar, while this parameter has a lower value in R. nathaliae (1Cx = 1.17 pg).
Pollen grains of three Ramonda species are small, 3-colporate, mostly spheroidal
in shape. They are of similar size in diploid species (R. myconi E = 12.57 µm; R.
nathaliae E = 15.19 µm), while R. serbica, as a hexaploid with larger genome size, has
larger pollen grains as well (E = 19,80 µm). Exine ornamentation is microreticulate-
perforate, in R. nathaliae more perforate, in R. serbica with larger lumens with more
perforations, while R. myconi exine ornamentation characteristics are intermediary
between two Balkan relatives.
Seeds are small, elongated, 814.47 µm long and 217.77 µm wide in R. myconi,
639.23 µm long and 228.09 µm wide in R. nathaliae and with 714.36 µm average
length and 254.84 µm width in R. serbica. There are auriculate structures on the seed
surface, most pronounced in R. myconi (46 – 87 µm high), little bit smaller in R.
serbica (32 – 44 µm) and least high in R. nathaliae (21 – 30 µm). Their possible role is
better surface adhesion. Seed mass is moderately correlated to genome size and
chromosome number. While size of pollen grains and seeds are not good characters for
differentiation of three Ramonda species, differences in fine pollen exine and seed
surface ornamentations can be used for this purpose.
Natural hybrids between R. nathaliae and R. serbica are found in the scope of
extensive research performed in sympatric populations. Hybrid individuals are
tetraploid with 2n = 96 chromosomes and average genome size of 2C = 5.14 pg. Small
number of individuals could be penta (2C = 6.19 pg) or octoploids (2C = 10.04 pg) on
the basis of their 2C-values. Pollen from hybrids is very heterogeneous in number of
colpi (3-, 9- and 12-colporate) and the size of the grains. Seeds are very small, 2-3
times smaller than those from parental species and germinate weekly (c. 1%). These
results reveal complex processes of hybridisation between two relict species,
introgression and genome duplications.
Population of R. nathaliae from serpentine substrate is ecologically very
interesting. Plants have smaller rosettes, smallest pollen grains with thin exine and
lower viability, smallest seeds and even smallest genome size among investigated
populations of this species.
AFLP analysis of genetic population structure confirmed clear taxonomic
differentiation of R. nathaliae and R. serbica. As endemics without self-fertilisation,
but with vegetative reproduction present, they are characterized by small genetic
diversity. Expected heterozygosity (H) ranges from 0.062 to 0.128 in R. nathaliae
populations and from 0.087 to 0.140 in R. serbica. Variability within populations (R.
nathaliae 72%, R. serbica 86%) is higher than among populations (R. nathaliae 28%,
R. serbica 14%). Genetic distances and fixation indices are higher among R. nathaliae
populations suggesting that this is an older species whose populations are separated for
a longer period of time.
Today’s genetic population diversity of these relic and endemic species from the
Balkan Peninsula reflects the influences of the Ice Age, but of more recent conditions
in their habitats as well. Both species had several refugia where they survived harsh
conditions during glaciations. They began spreading from there after climate warming,
so newly established populations have lower genetic diversity because of the founder
and bottleneck effects. They can be more sensitive to environmental stress and
therefore must be adequately protected. | true |
Strukturne i funkcionalne promene paraštitastih žlezda i bubrega nekon primene steroida, izoflavona i kalcijuma u animalnom modelu andropauze | Structural and functional changes of parathyroid glands and kidneys after treatment with steroids, isoflavones and calcium in an animal model of the andropause | Postepeno smanjenje koncentracije testosterona u cirkulaciji tokom procesa starenja,
koje je u čvrstoj vezi sa povećanom učestalošću nastanka kardiovaskularnih oboljenja,
benignih i malignih oboljenja prostate i osteoporoze, označava se kao andropauza.
Terapije steroidnim hormonima, kao i konvencionalna terapija Vit D i kalcijumom, koje
se primenjuju u prevenciji i tretmanu menopauzalnih i andropauzalnih simptoma kod
oba pola, ispoljavaju i neželjena dejstava i povećavaju rizik od nastanka
kardiovaskulanih oboljenja, hiperfosfaturije, hipertrofije prostate benignog karaktera,
kancera dojke i prostate. Zbog brojnih negativnih efekata primenjivanih terapija, u
poslednje vreme sve je veći broj istraživanja koja teže pronalaženju rešenja sa ciljem
prevazilaženja navedenih problema. Veliku pažnju privlače izoflavoni soje, genistein i
daidzein, koji su predmet istraživanja brojnih studija, prvenstveno zbog svog
blagotvornog efekta na simptome starenja kod oba pola.
Cilj ovog istraživanja bio je rasvetljavanje dejstava steroida, izoflavona i kalcijuma na
ključne regulatore homeostaze Ca2+ i Pi u animalnom modelu andropauze, i utvrđivanje
njihovog potencijalnog efekta u očuvanju metabolizma minerala koji je narušen tokom
procesa starenja.
Mužjaci pacova Wistar soja, starosti 15 meseci, su orhidektomisani (Orx) i lažno
operisani (SO) u ketaminskoj anestaziji (15 mg/kg b.w.). Dve nedelje nakon operacije
Orx životinje su podeljene u eksperimentalne grupe (n=8), kojima su subkutano
aplikovani testosteron-propionat (5mg/kg t.m.; TP), estradiol-dipropionat (0.625 mg/kg
t.m.; EDP), vitamin D (50 µg/kg t.m.; Vit D), genistein (30 mg/kg t.m.; G), daidzein (30
mg/kg t.m.; D) i intramuskularno kalcijum (28.55 mg/kg t.m.; Ca), svakog dana tokom
3 nedelje. Svaka tretirana grupa je imala svoje kontrolne SO i Orx grupe za poređenje,
kojima je aplikovan odgovarajući volumen adekvatnog rastvarača. Tokom izvođenja
eksperimenta, životinjama su bile slobodno dostupne voda i laboratorijska hrana, koja
nije sadržala sastojke soje ni leguminoza, sa kazeinom kao izvorom proteina. Životinje
su dekapitovane 24h nakon poslednjeg tretmana, paraštitaste žlezde i levi bubrezi su
izolovani, izmereni i pripremljeni za tehnike svetlosne i elektronske mikroskopije, dok
su desni bubrezi zamzrnuti u tečnom azotu i čuvani na -80˚C do korišćenja i izolacije
RNK. Analiza tkiva je izvršena korišćenjem svetlosne mikroskopije, tehnike
imunohistohemijskog bojenja i metoda stereološke analize, te transmisione elektronske
mikroskopije, a ekspresija gena je određena reakcijom lančanog umnožavanja u
realnom vremenu. Biohemijskim metodama određene su koncentracije PTH, Ca2+, Pi i
kreatinina u serumu, kao i koncentracije Ca2+, Pi i kreatinina u urinu. Dobijeni podaci su
statistički obrađeni.
Volumen paraštitastih žlezda značajno je povećan kod Orx pacova u odnosu na SO
grupu. Kod Orx životinja tretiranih TP-om, EDP-om ili Vit D volumen paraštitastih
žlezda je značajno smanjen u poređenju sa Orx pacovima. Volumenska gustina glavnih
ćelija je značajno smanjena, dok je volumenska gustina intersticijuma značajno
povećana nakon tretmana EDP-om u odnosu na Orx grupu životinja. Tretman
genisteinom je uzrokovao značajno povećanje volumena paraštitastih žlezda, dok je
tretman daidzeinom značajno smanjio volumen paraštitastih žlezda, u poređenju sa
istom vrednošću kod Orx pacova. Tretmani genisteinom ili daidzeinom su statistički
značajno smanjili volumensku gustinu glavnih ćelija, u poređenju sa Orx grupom
životinja. Nakon tretmana genisteinom i daidzeinom, volumenska gustina intersticijuma
je značajno povećana u odnosu na Orx grupu životinja. Kod životinja tretiranih
kalcijumom, volumen paraštitastih žlezda je značajno smanjen, dok je volumenska
gustina intersticijuma značajno povećana, u poređenju sa Orx pacovima. Kod Orx
životinja nivo PTH u serumu je značajno povećan u odnosu na SO grupu, dok je nivo
PTH nakon tretmana TP-om, EDP-om, Vit D, izoflavonima ili kalcijumom značajno
smanjen u poređenju sa Orx životinjama. Intezitet NaPi 2a signala je redukovan kod
Orx životinja, u odnosu na SO grupu. Tretmani TP-om, Vit D, genisteinom ili
daidzeinom su značajno povećali nivo ekspresije iRNK za NaPi 2a i jačinu
imunofluorescentnog signala za NaPi 2a na apikalnoj membrani ćelija proksimalnih
tubula, u poređenju sa Orx grupom. Tretman EDP-om je značajno smanjio nivo iRNK
za NaPi 2a, kao i prisustvo kotransportera na apikalnoj membrani ćelija proksimalnih
tubula, u odnosu na Orx pacove. Jačina imunofluorescentnog signala za PTH1R je
povećana nakon Orx, u poređenju sa SO grupom. Nakon tretmana EDP-om signal za
PTH1R je neznato promenjen u odnosu na Orx životinje, dok su tretmani TP-om, Vit D,
genisteinom, daidzeinom ili kalcijumom značajno smanjili jačinu imunofluorescentnog
signala za PTH1R, u odnosu na Orx životinje. Ekspresija FGFR i Klotho receptora nije
značajno promenjena kod Orx grupe u poređenju sa SO pacovima. Tretman EDP-om je
značajno povećao nivo ekspresije FGFR i Klotho receptora u odnosu na Orx kontrolnu
grupu. Tretmani genisteinom ili daidzeinom su značajno smanjili ekspresiju FGFR, dok
je ekspresija Klotho receptora povećana nakon genisteina, odnosno smanjena nakon
daidzeinskog tretmana, u poređenju sa Orx životinjama. Koncentracije Ca2+ i Pi u
serumu su značajno smanjenjene nakon Orx, dok su koncentracije Ca2+ i Pi u urinu
povećane, u poređenju sa SO kontrolnom grupom životinja. Nakon tretmana TP-om,
EDP-om, Vit D, genisteinom, daidzeinom ili kalcijumom koncentracije Ca2+ u serumu
su povećane u odnosu na isti parametar kod Orx pacova. Koncentracija Pi u serumu je
povećana nakon tretmana TP-om, genisteinom, daidzeinom i kalcijumom, u poređenju
sa Orx kontrolom. Aplikovanje TP, EDP, Vit D, genisteina, daidzeina ili kalcijuma je
uzrokovalo snižavanje koncentracije Ca2+ u urinu, u odnosu na Orx životinje. Tretmani
Vit D, genisteinom, daidzeinom ili kacijumom su snizili koncentraciju Pi u urinu, dok je
tretman EDP-om povećao koncentraciju Pi u urinu, u poređenju sa Orx grupom.
Dobijeni rezultati pokazuju da tretmani andropauzalnih mužjaka pacova steroidima,
izoflavonima i kalcijumom ispoljavaju izvesne razlike u jačini istosmernih efekata na
ključne regulatore homeostaze Ca2+ i Pi, kod orhidektomisanih mužjaka pacova
srednjeg životnog doba. Naime, tretmani izoflavonima ispoljavaju jači stimulatoran
efekat na ekspresiju NaPi 2a kotransportera, intenzivnije snižavaju stepen zastupljenosti
PTH1R, prisustvo i ekspresija FGFR u tubulima bubrega su izraženije redukovani, a
promene u smanjenju koncentracija Ca2+ i Pi u urinu, odnosno povećanju koncentracija
Ca2+ i Pi u serumu su više izražene, u poređenju sa blažim efektima tretmana steroidnim
hormonima i kalcijumom. Navedene promene mogu ponovno uspostaviti homeostazu
narušenu procesom starenja. | Andropause is defined as a gradual decline of serum testosterone concentration during
ageing, which is related to an increased incidence of cardiovascular diseases, benign and
malignant prostate diseases and osteoporosis. Using steroid hormones, as well as
vitamin D and calcium in the treatment of osteoporosis and other ageing symptoms, in
both genders, have some undesirable side effects, such as increased risk of
cardiovascular diseases, hyperphosphaturia and prostate cancer.Taking into account the
potentially harmful aspects of the hormone replacement therapy, an increasing emphasis
is placed on the alternative, plant-originated therapeutics for osteoporosis.
Accumulating evidence suggests that soy isoflavones may represent a promising
alternative remedy for aging symptoms in both genders.
This study aimed to examine the effects of steroids, isoflavones and calcium on the
structural and functional changes in parathyroid glands and specific functional proteins
in the kidney tubules, responsible for Ca and Pi regulation, in an animal model of the
andropause, and to determine their potential impact in the preservation of mineral
metabolism impaired with ageing.
Fifthteen-month-old Wistar rats were orchidectomised (Orx) or sham (SO) operated
under ketamine anesthesia. After 2 weeks of recovery, Orx animals were divided into
experimental groups and treated subcutaneously with testosterone-propionate (5 mg/kg
b.w.; TP), estradiol-dipropionate (0.625 mg/kg b.w.; EDP), vitamine D (50 µg/kg b.w.;
Vit D), genistein (30 mg/kg b.w.; G), daidzein (30 mg/kg b.w.; D) or calcium
intramuscularly (28.55 mg/kg b.w.; Ca) every day, for 3 weeks. Every treated group had
a coresponding SO and Orx control groups treated with the proper amount of vehicle,
following the same regime. Animals were fed a soy-free diet with corn oil as the fat
source. The rats were decapitated 24h after the last injection, parathyroid glands and left
kidneys were excised, their weight was measured and they was processed for light and
electron microscopic examinations. Techniques of histochemical staining and
immunohistochemical labeling, transmission electron microscopy, and stereological
analyses were performed. Gene expression levels were determined with Real-time PCR.
Concentrations of PTH, Ca2+, Pi and creatinin levels in serum and concentrations of
Ca2+, Pi and creatinin in urine were determined biochemically. The obtained data were
statistically processed.
The volume of parathyroid glands in Orx rats was increased compared to the SO group.
After treatments with TP, EDP or Vit D the parathyroid glands volumes were decreased,
when compared to Orx animals. The volume density of chief cells after treatment with
EDP was decresed, while the volume density of interstitium was increased, comparing
to Orx rats. Treatment with genistein led to an increase of parathyroid gland volume,
while treatment with daidzein decreased the same parameter, with regards to Orx group.
The volume densities of chief cells were decreased after treatments with genistein or
daidzein, while the volume densities of interstitium were increased, in comparison with
Orx animals. Administration of calcium to Orx rats provoked the reduction of
parathyroid glands volume, while the volume density of interstitium after same
treatment was increased, comparing to the Orx rats. The lack of steroid hormones,
provoked by Orx, led to the increment of serum PTH, when compared to the SO control
group, while treatments with TP, EDP, Vit D, genistein, dadizein or calcium decreased
the mentioned parameter, comparing to the Orx group. NaPi 2a expression in Orx
animals was reduced in regards to its abundance in SO animals, although it was
increased in TP, Vit D, genistein or daidzein groups, compared to the Orx rats. The
treatment with EDP attenuated NaPi 2a expression, in comparison with Orx group. In
Orx rats, the staining for PTH1R was stronger when compared to SO group, while the
treatments with TP, Vit D, genistein, daidzein or calcium induced reduction of the
PTH1R immunofluorescence, compared to Orx animals. The intensity of the PTH1R
signal after EDP treatment was slightly reduced in regards to its abundance in Orx
group. FGFR and Klotho receptor expression weren’t significantly changed in Orx
animals, when compared to the SO controls. After treatment with EDP, the expression
of FGFR and Klotho receptors was increased, when compared to the Orx rats.
Administration of genistein or daidzein caused the decrease in FGFR expression level,
compared to Orx animals. Genistein treatment of Orx rats induced enhancement of
Klotho receptor expression level, while daidzein treatment decrease it, in comparison
with Orx rats. In Orx animals, Ca2+ and Pi serum concentrations were decreased, while
urine Ca2+ and Pi content was increased, in comparison with the SO control. After
treatments with TP, EDP, Vit D, genistein, daidzein or calcium serum
Ca2+concentrations were increased, when compared to Orx animals. Treatments with
TP, genistein, daidzein or calcium induced the increment of Pi serum concentrations,
with regards to Orx rats. Aplication of TP, EDP, Vit D, genistein, daidzein or calcium to
Orx rats led to decrease Ca2+ urine concentrations, comparing to Orx animals. Urine Pi
concentrations after treatment with Vit D, genistein, daidzein or calcium were
decreased, while EDP treatment induced enhancement of Pi urine concentration, with
regards to Orx rats.
Our results showed that the treatments with steroids, isoflavones and calcium of
andropausal male rats demonstrate some differences in the level of the same direction
effects, demarcating widespread, commonly used therapies and alternative approaches
in the regulation of Ca2+ and Pi homoestasis. Application of isoflavones led to the
stronger increment in abundance and the expression level of NaPi 2a cotransporter,
intensively decreased the presence of PTH1R, abundance and expression of FGFR in
the kidney tubules was noticeable reduced, and decreased the concentrations of Ca2+
and Pi in urine, as well as more pronouncely increased the concentrations of Ca2+ and Pi
in the serum, when compared to the milder effects of treatments with steroids and
calcium. The listed changes may reestablish mineral homeostasis, disturbed by the
aging process. | true |
Modulacija apoptotskih signalnih puteva u ćelijama mozga odraslih pacova nakon hroničnog tretmana deksametazonom | Modulation of apoptotic signaling pathways in brain cells of adult male rats after chronic dexamethasone treatment | Deksametazon, jak sintetski glukokortikoid, se dugi niz godina koristi kao lek u
tretmanu različitih bolesti poput psorijaze, adrenalne insuficijencije, bakterijalnog
meningitisa, moždanih trauma, šloga, alergija, spazma bronhija, reumatidnog artritisa,
itd; ali i kao pomoćni lek u hemo- i radioterapiji. Brojne studije ukazuju da
deksametazon može regulisati sinaptičku plastičnost, povećati vijabilnost ćelija i
njihovu proliferaciju u in vivo i in vitro uslovima. Međutim, uprkos širokoj primeni u
terapijske svrhe primećeno je da deksametazon ispoljava i niz negativnih efekata u
mozgu, na primer apoptozu u granularnom sloju dentatnog girusa hipokampusa kod
mladih i starih pacova, kao i smanjenje kognitivnih funkcija i motornog razvoja. Usled
neusklađenosti rezultata mnogih studija, proučavanje sistemskih efekata, kao i efekata
deksametazona u mozgu predstavljaju interesantno polje istraživanja prvenstveno jer se
efekti niskih doza ovog sintetskog glukokortikoida ne mogu ispitivati centralno usled
blokiranja njegovog ulaska dejstvom MDR p-glikoproteina i/ili nekim drugim
mehanizmom. Stoga, za potrebe eksperimenta, odrasli mužjaci pacova Wistar soja su
podeljeni u dve grupe– kontrolne jedinke i životinje tretirane deksametazonom
(100 g/kg/dan) tokom 7 uzastopnih dana. 28 h nakon završetka hroničnog tretmana
životinje su žrtvovane. Sistemski efekat deksametazona je praćen promenom
biometrijskih parametara (telesna masa, masa timusa i nadbubrežnih žlezdi), kao i
koncentracije kortikosterona u serumu i moždanom tkivu. Kako bi se utvrdilo da li male
doze deksametazona dovode do apoptoze u hipofizi, hipotalamusu, hipokampusu i
prečeonoj kori korišćen je DNK fragmentacioni esej, dok se metodama histološkog
bojenja hipokampusa i prečeone kore (fluoro-jade B i krezil ljubičastim) istraživao obim
neuralne smrti i morfološke promene. Western blot i RT-PCR analizama su ispitivane
promene u ekspresiji proteina i iRNK markera procesa smrti odnosno preživljavanja
ćelija.
Rezultati ove studije su pokazali da hroničan tretman malim dozama deksametazona
uzrokuje hipoaktivnost hipotalamo-hipofizno-adrenalne ose, koja se ogleda u smanjenju
telesne mase, mase timusa i nadbubrežnih žlezdi, kao i kortikosterona u serumu.
Smanjena koncentracija kortikosterona u hipokampusu i prečeonoj kori ukazuje na
pojavu hipokortikoidnog stanja, praćenog nepromenjenom aktivacijom GR-a i
mogućom povećanom aktivacijom MR-a, što generalno štiti ćelije od apoptoze.
Primenjeni DNK fragmentacioni esej nije pokazao prisustvo apoptoze u svim
ispitivanim moždanim strukturama. Histološko bojenje hipokampusa i prečeone kore je
potvrdilo odsustvo neuralne smrti i morfoloških promena. Analizirani markeri apoptoze
odnosno preživljavanja u mozgu ukazuju da je u osnovi kasnih efekata tretmana malim
dozama deksametazona aktivacija AKT kinaze i MR-a, koji su odgovorni za dalju
regulaciju ekspresije ispitivanih nishodnih apoptotskih molekula. Naime, u
hipotalamusu, hipokampusu i prečeonoj kori je uočeno pomeranje ravnoteže između
pro- i anti-apoptotskih članova Bcl-2 familije ka anti-apoptotskim, a u hipofizi ta
promena nije detektovana. Dobijeni rezultati ukazuju na tkivno-specifičnu regulaciju
ekspresije molekula smrti odnosno preživljavanja. U hipotalamusu i hipokampusu, u
ranijoj vremenskoj tački je uočena pojava apoptoze, dok se kasnije aktiviraju
anti-apoptotski mehanizmi, što oslikava osetljivost ovih struktura na hroničan tretman
deksametazonom i fluktuacije koncentracije glukokortikoida.
Na osnovu dobijenih rezultata može se zaključiti da hroničan tretman malim dozama
deksametazona ima kapacitet da oblikuje odgovor hipotalamo-hipofizno-nadbubrežne
ose. U mozgu zdravih odraslih Wistar pacova, deksametazon dovodi do nastanka
hipokortikoidnog stanja, aktiviranja anti-apoptotske kinaze AKT i MR-a, povećanja
ekspresije Bcl-2 molekula i promena u ekspresiji ispitivanih pro-apoptotskih molekula.
Ovi podaci ukazuju da bi upravo ovi ispitivani molekuli mogli biti ključni za
apoptotsku-rezistenciju i deksametazonom-posredovanu ćelijsku protekciju, koje su
uočene u mozgu pacijenata tokom i nakon hemo- i radioterapije. | For many years, dexamethasone, a potent synthetic glucocorticoid, has been used as a
medication in the treatment of psoriasis, adrenal insufficiency, bacterial meningitis,
brain trauma, stroke, allergies, bronchial spasm, rheumatoid arthritis, etc., and as a co-
medicament in chemo- and radiotherapy. Numerous studies suggest that dexamethasone
is able to regulate synaptic plasticity, enhance cell viability and proliferation in vivo and
in vitro. However, dexamethasone exerts a number of adverse reactions in the brain,
such as apoptosis in the hippocampal granular layer of dentate gyrus in young and old
rats, as well as reduced cognitive and motor development. The dexamethasone-induced
systemic effects and dexamethasone-provoked effects in the brain are an interesting
field of research, mainly because the effects of low-dose dexamethasone treatment
could not be tested directly in brain tissue due to the central blocking action of MDR
p-glycoprotein and/or some other mechanism. Therefore, for the purposes of the
experiment, adult male Wistar rats were divided into two groups – controls and animals
treated with dexamethasone (100 g/kg/day) per 7 days. 28 h upon chronic treatment,
rats from both groups were sacrificed. Late systemic effects of dexamethasone were
monitored by alterations of biometric parameters (body weight, thymus and adrenal
glands mass) and level of corticosterone in serum and brain tissue. Further, using DNA
fragmentation assay, present study aimed to determine whether low dose
dexamethasone treatment is able to cause apoptosis in the pituitary gland,
hypothalamus, hippocampus and prefrontal cortex, while histological staining methods
(fluoro-jade B and cresyl violet staining) were applied to investigated the extent of
neuronal death and morphological changes in hippocampus and prefrontal cortex.
Changes in protein and mRNA expression of cell death and cell survival markers were
examined by Western blot and RT-PCR analyzes.
The results obtained in this thesis revealed that chronic low dose dexamethasone
treatment caused hypoactivity of hypothalamus-pituitary-adrenal axis, reflected in the
reduction of body weight, thymus and adrenal glands masses, as well as levels of
corticosterone in serum. Decreased concentration of corticosterone in hippocampus and
prefrontal cortex of dexamethsaone-treated animals indicates the occurrence of
hypocorticoid state, accompanied by unaltered activation of GR and possible increased
activation of MR, which in general protects cells from apoptosis. DNA fragmentation
assay showed unaltered profile of apoptosis in all investigated brain structures of both
experimental groups. Histological staining of hippocampus and prefrontal cortex
confirmed the absence of neuronal death and morphological changes. The analyzed
markers of apoptosis and survival in the brain suggest that late effects of low dose
dexamethasone treatment are based on the activation of AKT kinase and MR.
Activation of these anti-apoptotic proteins is responsible for the further regulation of
investigated downstream apoptotic molecules. In the hypothalamus, hippocampus and
prefrontal cortex the shift balance between Bcl-2 family members towards anti-
apoptotic Bcl-2 was observed, while in the pituitary gland this change is not detected.
The obtained outcome indicates the occurrence of tissue-specific regulation of cell death
and/or survival molecules. Furthermore, in the earlier time point, apoptosis was
observed in the hypothalamus and hippocampus; however the anti-apoptotic
mechanisms were activated later, indicating the sensitivity of these structures to chronic
low dose DEX treatment and fluctuations in the concentration of glucocorticoids.
In summary, chronic low dose dexamethasone treatment displayed the capacity to
significantly shape the response of hypothalamic-pituitary-adrenal axis. In the brain of
healthy adult Wistar rats, dexamethasone created the hypocorticoid conditions, triggered
the anti-apoptotic kinase AKT and MR activation, increased the expression of Bcl-2
molecule and altered the expression of investigated pro-apoptotic molecules. These data
point that investigated molecules might be crucial for apoptosis-resistance and
dexamethasone-mediated cell protection observed during and after chemo- and
radiotherapy of patients with brain tumours. | true |
Uticaj imunoglobulina sa specifičnošću značajnom za antifosfolipidni sindrom na ekspresiju karakterističnih proteina i invazivnost ekstravilusnog trofoblasta čoveka in vitro | The effect of immunoglobulins with specificity significant for antiphospholipid syndrome on expression of characteristic proteins and invasiveness of human extravillous trophoblast in vitro | Antifosfolipidni sindrom (APS) je autoimunsko oboljenje koje klinički
karakteriše ponavljana pojava tromboze i/ili poremećaji u trudnoći, a serološki prisustvo
antifosfolipidnih antitela (aPL). Prisustvo aPL predstavlja najčešći stečeni faktor rizika
za gubitak trudnoće i komplikacije u trudnoći. Smatra se da je glavni antigen u APS β2-
glikoprotein I (β2GPI), evolutivno konzerviran protein, koji je po sekvenci i
konformaciji sličan velikom broju proteina mikrobskog porekla. Do nedavno se
smatralo da su tromboza i inflamacija u placenti najznačajniji mehanizmi kojima aPL
ostvaruju patološki uticaj na tok i ishod trudnoće, ali novija saznanja ukazuju da aPL
antitela direktno utiču na proces placentacije.
U ovom radu je ispitivan uticaj imunoglobulina sa specifičnošću značajnom za
antifosfolipidni sindrom na ekstravilusni trofoblast čoveka in vitro. Studija je obuhvatila
ispitivanje efekata poliklonskih aPL koja su prečišćena iz seruma pacijenata sa APS i
monoklonskog antitela 26 (MAb 26) prema tetanus toksoidu, koje poseduje i
specifičnost prema β2GPI. Time je omogućeno bolje razumevanje uloge heterogene
populacije aPL koja je prisutna u serumu pacijenata, kao i antitela jasno definisane
specifičnosti prema β2GPI u procesima koji dovode do defektne placentacije.
Ispitivanjem in vitro uticaja imunoglobulina sa specifičnošću značajnom za APS
na invazivnost ekstravilusnog trofoblasta čoveka, testom ćelijske invazije u Matrigel,
pokazano je da aPL+ IgG i MAb 26 smanjuju invazivnost trofoblasta. Uočeni efekat
nije bio posledica smanjene vijabilnosti i proliferacije ćelija, niti povećane apoptoze.
Oba ispitivana antitela su uticala na ekspresiju karakterističnih proteina koji imaju ulogu
u ćelijskoj invazivnosti. Efekat aPL+ IgG na trofoblastnu invaziju je posredovan
inhibitornim delovanjem na matriksnu metaloproteinazu (MMP)-9, integrinske
subjedinice α1, α5 i β1 i sekretovani galektin-1, dok MAb 26 svoje efekte ostvaruje
inhibitornim delovanjem na MMP-2 i -9, α1 subjedinicu integrina i galektin-1.
Vezivanje aPL+ IgG i MAb 26 za ekstravilusnu trofoblastnu ćelijsku liniju
HTR-8/SVneo, kao i vezivanje MAb 26 za citotrofoblast (CT) izolovan iz placente
prvog trimestra, je potvrđeno imunocitohemijskom analizom. Ispitivana antitela se
vezuju za ćelije trofoblasta i u odsustvu egzogenog β2GPI i drugih faktora prisutnih u
fetalnom telećem serumu, ukazujući na ekspresiju endogenog β2GPI ili još uvek
nedefinisanog receptora koji bi mogao da direktno vezuje antitela. Iz literature je
poznata internalizacija aPL u pojedine ćelijske tipove (endotelne ćelije, makrofage,
sinciciotrofoblast), što predstavlja nov koncept na osnovu koga bi aPL mogla ostvariti
patološki efekat. Analiza HTR-8/SVneo ćelija nakon inkubacije sa aPL+ IgG i MAb 26
je pokazala lokalizaciju antitela unutar ćelija. Broj ćelija kod kojih se uočava
intracelularna lokalizacija imunoglobulina, kao i intenzitet bojenja, u direktnoj je
korelaciji sa dužinom trajanja inkubacije. Internalizacija MAb 26 u HTR-8/SVneo ćelije
je aktivan proces koji zavisi od temperature na kojoj se inkubacija odvija, što ukazuje na
endocitozu kao mogući mehanizam internalizacije antitela. Sa druge strane, odsustvo
internalizacije imunoglobulina koji su korišćeni kao kontrola (aPL- IgG i mIgG)
ukazuje da proces internalizacije verovatno nije posredovan Fc receptorom. Nije uočena
zavisnost od tipa ekstravilusnih trofoblastnih ćelija za internalizaciju MAb 26.
Ispitivanje lokalizacije MAb 26 u odnosu na odeljke unutar ćelije, primenom
odgovarajućih markera, je potvrdilo unutarćelijsko prisustvo MAb 26. Nakon 2 h
najveća blizina u detektovanim signalima kod dvojnog bojenja je uočena za MAb 26 i
kaveolin-1, kao i klatrin, što ukazuje na lokalizaciju MAb 26 neposredno uz ćelijsku
membranu.
Dobijeni rezultati predstavljaju napredak u razumevanju mehanizama i molekula
uključenih u patološke promene placente pod delovanjem aPL. Pored toga, biološki
efekat izazvan korišćenim anti-β2GPI antitelom, obzirom na način njegovog nastanka
posredstvom molekulske mimikrije, otvara nove pravce u istraživanju autoimunosti. | Antiphospholipid antibody syndrome (APS) is an autoimmune multisystemic
disorder clinically characterized by recurrent thrombosis and pregnancy morbidity, and
presence of antiphospholipid antibodies (aPL) in sera of affected individuals. Presence
of aPL is the most common acquired risk factor for pregnancy loss and pregnancy-
related
complications.
Main
antigen
in
APS
is
β2-glycoprotein
I
(β2GPI), an evolutionarily conserved protein, which shows similarity in sequence and
conformation with a number of microbial proteins. Until recently, thrombosis and
inflammation were thought to be the main aPL related mechanisms leading to
complications in pregnancy. More recent findings, however, indicate that aPL can exert
direct effects of on placentation.
This research was conducted to investigate the effects of immunoglobulins with
specificity significant for APS on human extravillous trophoblast in vitro. The study
included polyclonal aPL derived from APS patients (aPL+ IgG) and monoclonal
antibody 26 (MAb 26) raised to tetanus toxoid with additional β2GPI specificity,
originating from conformational similarity between tetanus toxoid and β2GPI.
Effects of immunoglobulins with specificity important for APS on human
extravillous trophoblast invasion in vitro were investigated using Matrigel invasion
assay. The test showed that both aPL+ IgG and MAb 26 induce a decrease in
trophoblast invasiveness, which did not result from a change in cell viability,
proliferation, or apoptosis. Both antibodies, however, did affect the expression of
characteristic proteins that are known to act as mediators of cell invasion. The aPL+ IgG
induced decrease in trophoblast invasiveness was accompanied by reduction in matrix
metalloproteinase (MMP)-9, integrin subunits α1, α5 and β1, and in levels of secreted
galectin-1. Similarly, the inhibitory effect of MAb 26 on trophoblast invasion was
accompanied by a decrease in MMP-2 and -9, integrin subunit α1, and galectin-1
expression.
Immunocytochemistry revealed binding of both aPL+ IgG and MAb 26 to
extravillous trophoblast cells. The observed binding was persistent in the absence of
exogenous β2GPI and other factors derived from fetal calf serum used for cell culture.
That implies expression of endogenous β2GPI, or putative receptor(s) that can bind aPL
directly. Internalization of aPL in to various cell types (endothelial cells, macrofages,
syncitiotrophoblast) has been reported, and represents a novel mechanism of aPL
induced effects. Analysis of both types of trophoblast cells following incubation with
aPL+ IgG and MAb 26 revealed intracellular localization of the antibodies in some of
the cells. Number of cells with intracellular localization of the antibodies, as well as the
staining intensity, positively correlated with the duration of incubation. MAb 26
internalization into HTR-8/SVneo cell was active and temperature dependent process
indicating involvement of endocytosis. The absence of significant internalization of
immunoglobulins used as control (aPL- IgG or mIgG) implies that the process was not
Fc receptor mediated. No cell type specific difference was observed in the
internalization of MAb 26. Further analysis of MAb 26 cell localization, using confocal
microscopy and specific markers of cell compartments, confirmed the intracellular
presence of MAb 26. After 2 h of exposure the highest proximity of the signal to the
MAb 26 was determined for caveolin-1 and clathrin coated vesicles, indicating that
MAb 26 localizes near inner side of cell membrane.
The original results presented in this study contribute to the understanding of the
mechanisms and molecules that may be involved in defective placentation induced by
aPL. In addition, the effectiveness of anti-β2GPI antibody used, likely derived through
molecular mimicry, opens new avenues in research of autoimmunity. | true |
Aktivnost komponenti adenozinskog signalnog sistema astrocita u modelu moždane povrede in vivo i in vitro | Activity of the astrocytes' adenosine signaling system components in model of traumatic brain injury in vivo and in vitro | Glavno obeležje traumatske povrede mozga (TPM) je reaktivna astroglioza
koja, između ostalog, uzrokuje i promene u signalizaciji purinima. Posebno važan
aspekt purinske signalizacije u patološkim procesima centralnog nervnog sistema
predstavlja
dinamika
promena
vanćelijskih
koncentracija
neuroprotektora
adenozina. Stoga je cilj ove doktorske teze bio ispitivanje ekspresije i funkcije
komponenti adenozinskog signalnog sistema astrocita u in vivo i in vitro modelu
moždane povrede, sa posebnim osvrtom na ulogu ekvilibrišućih nukleozidnih
transportera (ENT).
U in vivo studiji, izvedenoj na modelu ubodne lezije kore prednjeg mozga
pacova, je pokazano da povreda dovodi do dinamičnih promena u ekspresiji ENT,
ektonukleotidaza i adenozinskog A1 receptora. Sem toga, povreda uzrokuje i
ćelijsku re-distribuciju ENT1/2 i ushodnu regulaciju transportera na reaktivnim
astrocitima, što je posebno izraženo sedmog dana nakon ozlede.
Uloga astrocita u orkestraciji adenozinskog signalnog sistema nakon
povrede je detaljnije ispitana in vitro, nakon skarifikacije astrocitnog jednosloja.
Rezultati su pokazali da skarifikacija povećava ekspresiju ENT1 i ENT2 tek u
kasnijim vremenima. Bifazna promena u ekspresiji ekto-5`-nukleotidaze (e-5NT) je
iskazana prvobitnim smanjenjem i potom povećanjem ekspresije u kasnijim
vremenima nakon skarifikacije. Pored toga, skarifikacija astrocitnog jednosloja
vodi promenama u koncentracijama adenozina i njegovih metabolita u ćelijskom
medijumu. Naime, porast koncentracija adenozina u ranim vremenima nakon
povrede, bio je praćen padom u kasnijim vremenima. Blokiranje ENT
dipiridamolom (DPM) je dovelo do promena u koncentracijama adenozina nakon
skarifikacije, ukazavši na ulogu ENT1/2 u kontroli vanćelijskih koncentracija ovog
nukleozida.
Kako bi se nagovestili mogući putevi regulacije ENT, kultura astrocita je
podvrgnuta tretmanima adenozinom, ATP, DPM i blokatorom A1 receptora
DPCPX. Rezultati su pokazali da ATP ushodno reguliše oba transportera i e-5NT,
dok adenozin povećava ekspresiju ENT2 a smanjuje ekspresiju ENT1. Takođe,
drugačiji efekat na ekspresiju ENT1/2 su imali i DPM i DPCPX, potvrdivši da su
ova dva transportera regulisana drugačijim putevima.
Na kraju smo želeli da uporedimo efekte mehaničke povrede i CoCl2
izazvane hipoksije nakon četvoročasovnog na aktivnost komponenti adenozinskog
sistema. Za razliku od skarifikacije, CoCl2 vodi ranom povećanju ekspresije ENT2,
e-5NT i HIF-1α, kao i daleko većem povećanju vanćelijskih koncentracija
adenozina.
Dobijeni rezultati predstavljaju pregled eskpresije i aktivnosti najvažnijih
komponenti adenozinskog sistema astrocita nakon povrede, dajući dobru osnovu
za dalja ispitivanja uloge astrocita u kontroli kruženja adenozina u fiziološkim i
patološkim stanjima, kao i potencijalne kandidate za buduće terapije TPM. | Reactive astrogliosis is a hallmark of traumatic brain injury (TBI), which,
among the others alterations, causes changes in purinergic signaling. Due to its
neuroprotective features, fluctuations of adenosine extracellular concentration are
particularly important aspect of purinergic signaling in brain pathology. Hence,
herein given thesis aimed to investigate expression and function of astrocytes’
adenosine signaling system components after brain injury in vitro and in vivo, with
special regard to the role of equilibrative nucleoside transporters (ENT).
In vivo study, performed on a model of cortical stub injury of rat forebrain,
showed that injury caused dynamic changes in expression of ENTs,
ectonucleotidases, and adenosine A1 receptor. Moreover, injury induced cell
redistribution of ENT1/2 and upregulation of transporters on reactive astrocytes,
which is especially pronounced seven day after the impact.
The role of astrocytes in orchestration of adenosine signaling system after
the injury was examined in more details in vitro, after scratch wound injury of
astrocytic monolayer. Results have shown that scarification induced upregulation
of ENT1 and ENT2 in later time points. Biphasic alteration in expression of e-5NT
was shown in early downregulation followed by upregulation of the enzyme in
later time points after the induction of scratch wound. Beside, scarification of
astrocytic monolayer caused changes in concentration of adenosine and its
metabolites in extracellular medium. The rise of adenosine concentration was
noted early after the injury, which was followed by drop of the concentration in
later time points examined. Blocking of ENT with dipyridamole (DPM) resulted in
changes of observed adenosine concentration after the scarification, pointing out
that ENT1/2 have significant role in controlling extracellular concentration of this
nucleoside.
In order to identify possible regulation pathways for ENT, astrocytes where
treated with adenosine, ATP, DPM and DPCPX, antagonist of A1 receptor. Results
have shown that ATP upregulates both transporters as well as e-5NT, while
adenosine enhances expression of ENT2 and diminishes expression of ENT1.
Moreover, DPM and DPCPX have shown different effect on ENT1/2 expression,
confirming that regulation of these two transporters depends on different
pathways.
Toward the end, we wanted to compare effects of mechanical injury to those
caused by chemically induced hypoxia after 4-hour CoCl2 treatment, on activity of
the components of adenosine system. Unlike the sratch wound injury, CoCl2 causes
early upregulation of ENT2, e-5NT and HIF-1α, as well as much greater increase in
extracellular adenosine concentration.
Herein given results represent an overview of activity and expression the
most important components of adenosine signaling system after the injury,
therefore giving good foundation for future research of astrocyte role in control of
adenosine in both physiological and pathological conditions, as well as for
investigation of potential candidates for future TBI therapies. | true |
Izolacija i karakterizacija bioaktivnih sekundarnih metabolita iz odabranih sojeva roda Streptomyces | Isolation and characterization of bioactive secondary metabolites from selected Streptomyces strains | UNIVERZITET U BEOGRADU
BIOLOŠKI FAKULTET
Nada N. Stanković
IZOLACIJA I KARAKTERIZACIJA
BIOAKTIVNIH SEKUNDARNIH
METABOLITA IZ ODABRANIH SOJEVA
RODA Streptomyces
Doktorska disertacija
Beograd, 2012
UNIVERSITY OF BELGRADE
FACULTY OF BIOLOGY
Nada N. Stanković
ISOLATION AND CHARACTERIZATION
OF BIOACTIVE SECONDARY
METABOLITES FROM SELECTED
Streptomyces STRAINS
Doctoral Dissertation
Belgrade, 2012
MENTORI:
dr Branka Vasiljević, naučni savetnik
Institut za molekularnu genetiku i genetičko inženjerstvo,
Univerzitet u Beogradu
dr Đorđe Fira, vanredni profesor
Biološki fakultet, Univerzitet u Beogradu
ČLANOVI KOMISIJE:
dr Branka Vasiljević, naučni savetnik
Institut za molekularnu genetiku i genetičko inženjerstvo,
Univerzitet u Beogradu
dr Đorđe Fira, vanredni profesor
Biološki fakultet, Univerzitet u Beogradu
dr Jasmina Nikodinović-Runić, viši naučni saradnik
Institut za molekularnu genetiku i genetičko inženjerstvo,
Univerzitet u Beogradu
DATUM ODBRANE:
Ova teza urađena je u Laboratoriji za molekularnu genetiku i ekologiju
mikroorganizama u perodu od 2006. – 2012. godine.
U toku duge izrade ove teze skoro da nema kolega i zaposlenih u IMGGI koji nisu na
neki način doprineli da eksperimntalni rad i laboratorijski život idu glatko i budu lakši.
Ja im se na tome iskreno zahvaljujem.
Zahvaljujem se dr Đorđu Firi što se (više puta) prihvatio mentorstva nad mojom tezom.
Takođe mu se zahvaljujem na kritičkom čitanju i stručnoj oceni teze.
Mojim dragim kolegama iz LMGEM, onima koji su ovde i onima koji su otišli negde
dalje, posebno se zahvaljujem. Mojim „najstarijim“ koleginicama Tanji, Sandri, Ivani M.
za dugogodišnje druženje i deljenje svega kroz šta smo u ovih 14 godina prošli, Lidiji,
koleginici iz MBT lab dana, veselom triju Tanja/Lidija/Sanja, Saši, Vanjici, kao i Miloju i
Ivani pionirima PKS problematike u ovoj laboratoriji, svima od srca hvala.
„Šefici“, dr Branki Vasiljević zahvaljujem se na kolegijalnom i ljudskom odnosu koji ima
prema članovima svoje laboratorije, na razumevanju, strpljenju kao i otvorenosti za
nove predloge i ideje. Zahvaljujem joj se i na uloženom trudu u oceni ove teze.
Jasmini se zahvaljujem jednostavno na svemu. Bez njenog entuzijazma, ideja, energije i
truda koji je uložila, ova teza ne bi u ovom obliku ugledala svetlost dana.
Mojim najdražima Vladi, Urošu i Irini posvećujem ovu tezu. Zbog njih je sve vredno
truda.
Oktobar 2012.
Izolacija i karakterizacija bioaktivnih sekundarnih metabolita iz odabranih sojeva
roda Streptomyces
REZIME:
Aktinomicete su Gram-pozitivne zemljišne bakterije poznate kao proizvođači
bioaktivnih sekundarnih metabolita. U cilju pronalaženja novih bioaktivnih jedinjenja
pretraživana je kolekcija aktinomiceta Laboratorije za molekularnu gentiku i ekologiju
mikroorganizama Instituta za molekularnu gentiku i genetičko inženjerstvo Univerziteta
u Beogradu po više kriterijuma – bioaktivnosti, prisustvu gena za poliketid sintaze
(PKS), pigmentaciji i profilu apsorpcije ukupnih ekstrakata kultura u ultraljubičastom i
vidljivom (UV/Vis) delu spektra. Na osnovu ovoga izdvojena su tri izolata NP10, JS520
i Streptomyces durmitorensis, od ranije poznati proizvođač didehidroroflamikoina
(DDHR). Izolati NP10 i JS520 identifikovani su kao pripadnici roda Streptomyces.
Streptomyces sp. NP10 akumulirao je masne kiseline račvastog niza, pre svega
izopalmitinsku kiselinu, i ciklične dipeptide sastavljene od prolina u kombinaciji sa
valinom, izoleucinom ili alaninom koji su imali stimulativno dejstvo na trofoblastne
ćelije u kulturi u niskim koncentracijama (1 ng/ml i 1 µg/ml) i citotoksično dejstvo u
visokim (1 mg/ml) koncentracijama. Pokazano je da Streptomyces sp. JS520 ima
sposobnost proizvodnje velike količine pigmenta undecilprodigiozina (UP) sa
antibakterijskim, antimikotičkim, antioksidativnim i UV protektivnim osobinama koji je
imao citotoksično dejstvo na trofoblastne ćelije u kulturi u visokim koncentracijama
(1 ng/ml). Citotoksičnost polienskog makrolidnog antibiotika DDHR iz S. durmitorensis
je potvrđena, a pokazan je i njegov antimikotički efekat. PKS klaster zadužen za sintezu
DDHR je subkloniran u kozmidnu biblioteku i delimično sekvenciran. Optimizacijom
uslova gajenja količina proizvedenog UP povećana je 2,12 puta, a DDHR 1,1 puta. | Actinomycetes are Gram-positive soil bacteria known as producers of secondary
bioactive metabolites. In the search for new bioactive compounds collection of
actinomycetes from the Laboratory for Microbial Molecular Genetics and Ecology
(Institute of Molecular Genetic and Genetic Engineering, University of Belgrade) was
screened according to several criteria - bioactivity, the presence of the genes for
polyketide synthase (PKS), pigmentation, and crude culture extracts absorption profile
in ultraviolet and visible (UV/Vis) spectra. Based on these we have isolated three strains
NP10,
JS520,
and
Streptomyces
durmitotensis,
previously
renewed
for
didehydroroflamicoin (DDHR) production. Isolates NP10 and JS520 were identified to
belong to the genus Streptomyces. Streptomyces sp. NP10 accumulated branched chain
fatty acids, predominately isopalmitic, and cyclic dipeptides consisting of proline in
combination with valine, isoleucine or alanine, that have a stimulating effect on the
trophoblast cell line in culture at low concentrations (1 ng/ml and 1 mg/ml) and
cytotoxic effect at high concentrations (1 mg/ml). Streptomyces sp. JS520 was
determined to be exceptionally good producer of pigment undecylprodigiosine (UP)
with antibacterial, antifungal, antioxidant and UV protective activities showing
cytotoxic effects on trophoblast cell line in culture at high concentrations (1 ng/ml).
Cytotoxicity of polyene macrolide antibiotic DDHR from S. durmitotensis was
confirmed and it’s antifungal effects were shown. PKS cluster responsible for DDHR
synthesis was subcloned in the cosmid library and partially sequenced. Optimization of
culture conditions resulted in 2.12 times increased production of UP, and 1.1 times
increased production of DDHR. | true |
Uticaj ishrane obogaćene ugljenim hidratima na morfološke i ultrastrukturne odlike pankreasa pacova u uslovima eksperimentalno indukovanog hipotireoidizma | The effect of carbohydrate rich diet on rat pancreas morphological and ultrastructural characteristics in experimentally induced hypothyroidism | Pankreas je žlezda sa egzokrinom i endokrinom funkcijom. Proizvodi njegove
egzokrine komponente od vitalnog su značaja za proces varenja hrane. Endokrine ćelije
pankreasa grupisane u Langerhansova ostrvca proizvode insulin i glukagon, hormone
koji regulišu metabolizam pre svega ugljenih hidrata i neophodni su za održavanje
optimalne koncentracije glukoze u krvi. Sekretna aktivnost pankreasa, naročito njegove
endokrine komponente, pod kontrolom je kompleksnih endokrinih, parakrinih i
neuralnih mehanizama.
Hormoni tireoidne žlezde ključni su regulatori bazalnog metabolizma, rasta i
razvića. Potrebni su za normalno funkcionisanje gotovo svih organa i organskih sistema,
a njihov značaj u metabolizmu ugljenih hidrata poznat je već decenijama. Funkcije
endokrinog pankreasa i tireoidne žlezde, kao i efekti koje ostvaruju njihovi hormoni,
uzajamno su povezani i međusobno zavisni. U kliničkoj praksi relativno je česta
koegzistencija disfunkcije tireoidne žlezde i razvoj insulinske rezistencije, koja je
predvorje nastanka dijabetesa tipa 2.
Ishrana bogata prostim šećerima, uz smanjen nivo fizičke aktivnosti
karakteristika je savremenog načina života, naročito u industrijski razvijenijim
zemljama sveta. Pri kontinuiranom unosu ugljenih hidrata pankreas radi pod povećanim
opterećenjem, intenzivno proizvodeći insulin, što dugoročno gledano može dovesti do
patofizioloških procesa na nivou unutarćelijskih organela i eventualno do gubitka β-
ćelija, odnosno do dijabetesa tipa 2.
Osnovni cilj ove teze bio je da se detaljnom analizom stekne uvid u morfo-
funkcionalne karakteristike pankreasa pacova pod uticajem ishrane obogaćene
saharozom, u uslovima sistemskog hipotireoidizma indukovanog metimazolom.
U tronedeljnom eksperimentu korišćeni su mužjaci albino pacova Wistar soja,
držani pod standardnim laboratorijskim uslovima. Životinje su u odnosu na ponuđeni
rastvor u vodi za piće podeljene u četiri grupe. Kontrolna grupa životinja pila je čistu
česmensku vodu, hipotireoidizam je indukovan 0.02% rastvorom metimazola, dok je
pankreas funkcionalno opterećen unosom 10% rastvora saharoze. Pacovi poslednje
grupe pili su kombinovani rastvor metimazola i saharoze, već pomenutih koncentracija.
Pankreas je na uobičajen način pripremljen za morfološku i stereološku analizu
na nivou svetlosne i elektronske mikroskopije. Pored rutinskih metoda bojenja
hematoksilinom i eozinom, toluidin plavim i AZAN-om, korišćene su metode
imunohistohemije za detekciju insulina, glukagona, somatostatina, pankreasnog
polipeptida, PDX1, α-SMA i Ki67 proteina, kao i specifično bojenje propidijum
jodidom.
Dobijeni rezultati pokazali su sledeće:
- Tronedeljni tretman metimazolom doveo je do uspostavljanja umerenog
sistemskog hipotireoidizma, nezavisno od režima ishrane. Nivoi tireoidnih hormona u
cirkulaciji bili su smanjeni, a životinje su zaostajale u rastu.
- Kod hipotireoidnih životinja, bez obzira na način ishrane, ukupan energetski
unos bio je smanjen usled sniženja stope bazalnog metabolizma i smanjenja energetskih
potreba organizma. Energetski unos eutireoidnih životinja kojima je ponuđen rastvor
saharoze bio je znatno iznad kontrolnih vrednosti i to prevashodno na račun ugljenih
hidrata, ali se to nije negativno odrazilo na homeostazu glukoze.
- Manja apsolutna masa pankreasa životinja svih eksperimentalnih grupa
posledica je proporcionalnog smanjivanja egzokrine i endokrine komponente pankreasa,
koje se kod obe grupe hipotireoidnih životinja dešavalo paralelno sa manjim prirastom
životinja. Smanjenje relativne mase pankreasa eutireoidnih životinja prehranjivanih
saharozom delom je bilo posledica blagog povećanja telesne mase, a moguće delom i
promena na nivou ćelijske degranulacije/egzocitoze.
- Rezultati morfološke analize pokazali su da je sintetska i sekretna aktivnost
egzokrinog pankreasa eutireoidnih životinja koje su unosile rastvor saharoze bila
povišena u odnosu na životinje iz kontrolne grupe. Mehanički efekat zapremine
želudačnog i crevnog sadržaja imao je ključnu regulatornu ulogu u ovim procesima.
- Egzokrini pankreas hipotireoidnih normalno hranjenih životinja bio je u stanju
niže aktivnosti, zbog opšteg sniženja metaboličkih procesa i manjeg unosa hrane. U
acinusnim ćelijama aktivirani su mehanizmi zimofagije kao zaštite od unutraćelijske
aktivacije digestivnih enzima i autodigestije.
- Acinusne ćelije egzokrinog pankreasa hipotireoidnih životinja koje su unosile
rastvor saharoze bile su izložene većem izazovu nego ćelije normalno hranjenih
životinja, kojem su uglavnom uspešno izlaze u susret, uz blage morfo-funkcionalne
adaptacije. Pojedinačne ćelije trpele su stres endoplazminog retikuluma i, moguće,
podlegle ireverzibilnim oštećenjima.
- Primenjeni tretman nije doveo do promene zastupljenosti i distribucije
pojedinih tipova endokrinih ćelija u Langerhansovim ostrvcima.
- Kod normalno hranjenih hipotireoidnih životinja postojao je zastoj u
sintezi/sekreciji insulina, manifestovan akumulacijom sitnijih granula u β-ćelijama, koji
je donekle kompenzovan sintetskom aktivnošću ćelija koje ko-eksprimiraju insulin i
glukagon.
- β-ćelije životinja koje su unosile rastvor saharoze, bez obzira na tireoidni
status, većinom su bile funkcionalne insulin-produkujuće ćelije. Kod eutireoidnih
životinja nalazile su se u stanju pune sintetske i sekretne aktivnosti, ali bez hipertrofije i
hiperplazije.
- U grupi eutireoidnih saharozom prehranjivanih pacova detektovane su i
ekstrainsularne β-ćelije u acinusima, koje trenutno nisu bile funkcionalne u sintezi i
sekreciji insulina, i koje predstavljaju rezervne insulinske ćelije. Najverovatniji put
nastanka ovih ćelija je transdiferencijacijom acinusnih ćelija.
- Unos rastvora saharoze finu strukturu β-ćelija hipotireoidnih pacova udaljava
od grupe tretirane samo metimazolom i približava je kontroli, što se naročito
manifestuje ultrastrukturno-stereološkim odlikama granula.
- Negativan efekat metimazola na sintezu i sekreciju insulina, uz istovremeno
povećanje zahteva za ovim hormonom usled unosa saharoze, aktivirao je mehanizme
koji produžavaju poluživot insulina. To je dovelo do blagog rasta nivoa ovog hormona u
krvi i stvaranja uslova za razvoj insulinske rezistencije. | The pancreas is a gland with both exocrine and endocrine function. The products
of its exocrine component are vital to the food digestion. Its endocrine function is based
on secretory action of several types of endocrine cells that cluster together to form islets
of Langerhans. Some of them produce insulin and glucagon, hormones that regulate
primarily carbohydrate metabolism and are essential for maintaining optimal blood
glucose concentrations. Secretory action of the pancreas, especially its endocrine
component, is controlled by a complex endocrine, paracrine and neural mechanisms.
Thyroid hormones are key regulators of the basal metabolism, growth and
development. They are necessary for normal function of almost all organs and organ
systems; their importance in the metabolism of carbohydrates has been known for
decades. The functions of the endocrine pancreas and thyroid gland, and effects of their
hormones are mutually connected and interdependent. In clinical practice the
coexistence of thyroid dysfunction and the development of insulin resistance, which is a
good prognostic factor for type 2 diabetes occurrence, is relatively frequent.
A diet rich in simple sugars, along with reduced physical activity is a feature of
modern lifestyle in developed countries of the world. Continuous intake of
carbohydrates represents a form of stress in which pancreas intensively produces
insulin, which in the long term can lead to pathophysiological processes at the level of
intracellular organelles, and eventually to loss of β-cells, i.e. to type 2 diabetes.
The aim of this work was to gain an insight into the detailed morpho-functional
characteristics of the rat pancreas under the influence of sucrose rich diet, in terms of
systemic hypothyroidism induced by methimazole.
The obtained results show that:
- Moderate systemic hypothyroidism was established after three-week
methimazole treatment, regardless of the diet. Levels of thyroid hormones in the
circulation were reduced, and the animals fell behind in growth.
- In hypothyroid animals, regardless of the diet, total energy intake was reduced
due to lower basal metabolism rate and reduced energy requirements. In sucrose fed
euthyroid animals energy input was significantly above control values, primarily at the
expense of carbohydrates, but negative impact on glucose homeostasis was not evident.
- Reduced absolute mass of the pancreas in all experimental animals resulted
from the proportional reduction of exocrine and endocrine components of the organ, and
in both groups of hypothyroid animals occurred in parallel with a smaller body mass
gain. Pancreas relative mass reduction in the sucrose fed euthyroid animals was in part
the consequence of a slight increase in body mass, and partially as we assume the result
of the increased cell degranulation/exocytosis.
- The results of morphological analysis showed that exocrine pancreas of the
euthyroid animals with sucrose diet was more active in synthesis and secretion than of
control animals. The mechanic effect of the gastric and intestinal contents had a key
regulatory role in these processes.
- Exocrine pancreas of normally fed hypothyroid animals was less active, due to
a general reduction of metabolic processes and lower food intake. In acinar cells the
zymophagy is initiated as a protection mechanism from digestive enzyme's activation
and autodigestion.
- Acinar cells in sucrose fed hypothyroid animals were exposed to a greater load
than the cells of normally fed animals. This challenge has been successfully overcome,
with slight morpho-functional adaptations. In individual cells rER stress could lead to
possibly irreversible damage.
- The applied treatment did not induce changes in the number and distribution of
any cell type in the islets of Langerhans.
- In normally fed hypothyroid animals there is a delay in the insulin
synthesis/secretion, manifested by accumulation of smaller granules in β-cells, which
was somewhat compensated by synthetic activity of insulin and glucagon co-expressing
cells.
- Most of the β-cells in sucrose consuming animals regardless of thyroid status
were functional insulin-producing cells. In euthyroid animals they were active regarding
synthesis and secretion, and no hypertrophy or hyperplasia was detected.
- In the sucrose fed euthyroid group of animals extra-insular acinar β-cells were
detected. At that moment they were not functional in the synthesis and secretion of
insulin and could be considered the back-up cells. Most likely these cells originated by
acinar cell transdifferentiation.
- Sucrose intake in hypothyroid rats distinguishes β-cells fine structure away
from those treated only with methimazole and approaches them to the control, which is
especially manifested by granule's ultrastructural and stereological features.
- The methimazole negative effect on the synthesis and secretion of insulin, with
increased requirements for this hormone due to the intake of sucrose, activate the
mechanisms that prolong the half-life of insulin. This led to a slightly increased insulin
circulation level and created conditions for the development of insulin resistance. | true |
Analiza p53 gena i HPV infekcija u karcinomima grlića materice i jajnika | Analysis of p53 gene and HPV infection in cervical and ovarian carcinomas | Uvod: Ginekološki maligniteti predstavljaju veoma raznoliku grupu kancera, među kojima su najučestaliji karcinomi grlića materice a najsmrtonosniji karcinomi jajnika. Podaci o povezanost p53 mutacija (TP53 po HUGO nomenklaturi), polimorfizma kodona 72 i infekcije humanim papiloma virusima (HPV) sa kliničko-histopatološkim karakteristikama, nastankom ovih maligniteta, kao i odgovorom na antikancersku terapiju su kontradiktorni. Malo se zna o međusobnoj povezanosti TP53 mutacija, polimorfizma kodona 72 i HPV infekcije. Zato postoji potreba za ispitivanjem pomenutih potencijalnih biomarkera ovih maligniteta. Cilj: Ispitivanje povezanosti TP53 mutacija, polimorfnih varijanti kodona 72 i HPV infekcije sa demografskim karakteristikama, kliničko-histopatološkim karakteristikama karcinoma grlića materice i karcinoma jajnika i karakteristikama bolesnica sa ovim karcinomima, kao i ispitivanje međusobne povezanosti pomenutih potencijalnih biomarkera i njihovog uticaja na antikancersku terapiju. Materijal i metode: U radu su analizirana 53 uzorka karcinoma grlića materice i 54 uzorka karcinoma jajnika. Kontrolnu grupu činilo je 95 uzoraka briseva grlića materice žena sa urednim ginekološkim i normalnim Papa nalazom, kao i odsustvom prethodne istorije postojanja prekancerskih i kancerskih lezija ginekološke regije. DNK je izolovana metodom isoljavanja. Egzoni 4-8 TP53 gena su amplifikovani lančanom reakcijom polimeraze (PCR). Preliminarni skrining mutacija vršen je metodom konformacionog polimorfizma jednolančane DNK (SSCP), a automatskim sekvenciranjem DNK je potvrđivano prisustvo i utvrđivan tip mutacija. Polimorfizam kodona 72 TP53 gena je ispitivan analizom polimorfizma dužine restrikcionih fragmenata (RFLP). Prisustvo HPV infekcije je detektovano putem amplifikacije dela L1 virusnog gena. HPV16 i HPV18 tipovi u karcinomima grlića materice su detektovani amplifikacijom dela E7, odnosno E1 virusnih gena, dok je genotipizacija HPV u karcinomima jajnika vršena sekvenciranjem DNK. Za statističku obradu podataka korišćeni su Fišerov egzaktni, χ2 , odds ratio i Log-Rank test... | Introduction: Gynecological malignancies present a various group of cancers; among them the most frequent are carcinomas of cervix and the most aggressive are ovarian carcinomas. Conflicting data about correlation of TP53 mutations, codon 72 polymorphism and HPV infection with clinicopathological characteristics, the origin of these malignancies and with the response to anti-cancer therapy, as well as correlation between mentioned biomarkers, are pointing out the necessity of analysis of these biomarkers. Goal: Examination of correlation of TP53 mutations, codon 72 polymorphic variants and HPV infection with demographic features, clinicopathological characteristics of ovarian and cervical carcinomas, patient’s characteristics, as well as examination of interconnection of these biomarkers and their possible predictive values to anti-cancer therapy. Material and methods: 53 samples of cervical carcinomas and 54 samples of ovarian carcinomas were analyzed. Control group was consisted of 95 cervical smears of gynecological healthy women with normal Papa test results and without previous history of pre- and cancer lesion of gynecological region. DNA was extracted by salting-out procedure. Exons 4-8 of TP53 gene were amplified by polymerase chain reaction (PCR). Preliminary screening of mutations was done by single strand conformation polymorphism (SSCP) and automatic DNA sequencing was employed to confirm the presence and establish the type of mutation. Codon 72 polymorphism was assessed by restriction fragment-length polymorphism (RFLP). Presence of HPV infection was detected through amplification of one part of L1 viral gene. Types HPV16 and HPV18 in cervical carcinomas were detected by amplification of one part of E7 or E1 viral gene, while HPV genotyping in ovarian carcinomas were performed by DNA sequencing. Fisher exact, χ2, odds ratio and Log-Rank tests were employed for statistical analysis... | false |
Genetička transformacija kičice (Centaurium erythraea Rafn.) AtCKX1 i AtCKX2 genima | Genetic transformation of Centaury (Centaurium erythraea Rafn.) using AtCKX1 and AtCKX2 genes | Biljna vrsta Centaurium erythraea Rafn., u našem narodu poznata kao kičica, od davnina se
upotrebljava kao lek za sniženje temperature i nivoa šećera u krvi, lečenje anemije, žutice i
gihta, za jačanje apetita i povećanje stomačne sekrecije. Zbog poznatih lekovitih svojstava,
kičica se nalazi u mnogim farmakopejama. Kao početni materijal za uspostavljanje kulture
korenova in vitro korišćeni su vrhovi adventivnih korenova prosečne dužine ~10 mm koji
su izolovani od sterilno dobijenih klijanaca kičice i dalje su gajeni na čvrstoj MS hranljivoj
podlozi bez regulatora rastenja. Proces organogeneze u kulturi korenova kičice je spontan.
Posle sedam dana gajenja u kulturi na bazalnom delu početnog eksplantata uočavaju se
prve promene. Koren zadebljava i tkivo počinje da puca celom dužinom bazalnog dela.
Tokom četvoronedeljnog pasaža, duž razraslog dela eksplantata, formira se veliki broj
adventivnih pupoljaka. U cilju dobijanja transgenih biljaka sa sniženim nivoom endogenih
citokinina, za transformaciju su korišćena dva bakterijska soja Agrobacterium tumefaciens
GV3101 koji nose pBinHTX plazmid sa dva različita gena, AtCKX1 odnosno AtCKX2, koji
kodiraju enzim citokinin oksidazu/dehidrogenazu (CKX). Efikasna integracija AtCKX1
gena (30% ) i AtCKX2 gena (28.2% ) određena je PCR analizom genomske DNK. U cilju
što boljeg određivanja nivoa ekspresije AtCKX gena kičice prvo je bilo neophodno urađena
je optimizacija PCR protokola sa prajmerima za qPCR. Ovaj postupak sastojao se u
pronalaženju optimalne temperature vezivanja prajmera uz istovremenu primenu različitih
aditiva odnosno pojačivača PCR reakcije. Dodavanje 5% DMSO-a u qPCR reakcije
značajno je poboljšalo specifičnost amplifikacije AtCKX1 i AtCKX2 gena kičice. qRT-PCR
analizom potvrđena je ekspresija AtCKX1 i AtCKX2 gena kod 50% odnosno 64%
analiziranih linija. U ovom radu uspešno je pokazano da ekspresija AtCKX gena u
transgenim biljkama kičice značajno utiče na metabolizam citokinina. Ključni faktor u
kontroli nivoa citokinina u biljnim tkivima predstavlja CKX enzim, jedini do sada poznati
enzim koji učestvuje u katabolizmu citokinina. Specifična aktivnost CKX enzima
određivana je u izdancima i korenovima svih transgenih AtCKX linija. Analiza količine i
sadržaja ukupnih kao i pojedinačnih grupa endogenih citokinina netransfromisanih biljaka
kičice omogućila je, po prvi put, određivanje citokiniskog profila kičice in vitro. Takođe je
pokazano da se promene u metabolizmu citokinina prilično nepredvidivo odražavaju na
količinu auksina tj. količinu ukupne endogene IAA u samom biljnom tkivu. Uspešno je
pokazano da ekpresija AtCKX gena takođe utiče i na neke morfološke parametre
transformisanih biljaka kičice. Promenjena citokininska homeostaza utiče na smanjenje
morfogenetskog potencijala transformisanih biljaka kičice, ali ne utiče na značajno
odstupanje prirasta mase izdanaka u odnosu na kontrolu. Rezultati analize fotosintetičkih
pigmenata kičice ukazuju na to da sniženje citokinina može biti jedan od preduslova, ali ne
i sam signal za početak senescencije. Dominantne farmakološki aktivne supstance kičice su
sekoiridoidi i ksantoni. Sadržaj sekoiridoidnih komponenti biljaka in vitro ne razlikuje se
mnogo u odnosu na biljke iz prirode dok se količina ksantonskih jedinjenja značajno
povećava kod biljaka gajenih in vitro. Većina transgenih AtCKX linija kičice produkuje
manje sekoiridoida u odnosu na biljke in vitro i biljke iz prirode. Povećana akumulacija
ksantona određena je u skoro svim AtCKX linijama kičice u poređenju sa biljkama iz
prirode. Imajući u vidu da su ksantoni sve više u upotrebi zbog svojih farmakoloških
svojstava, transgene AtCKX linije kičice mogle bi biti koristan izvor biljnog materijala u
proizvodnji novih lekova. | Plant species Centaurium erythraea Rafn., commonly known as common centaury, has
been used for centuries to cure febrile conditions, regulate blood sugar, treat anemia,
jaundice and gout and to increase appetite and stimulate digestion. Because of its numerous
therapeutic properties centaurii herba is officially recognized as a drug in a number of
pharmacopoeias. Root tips of ≈10 mm average length, isolated from seedlings obtained
under sterile conditions, were used as initial explants in this work. The in vitro culture of
non-transformed centaury roots was established on solid half-strength MS nutrient medium
without growth regulators. Organogenesis is a spontaneous process in centaury root culture.
The first changes were noticeable on the basal part of the initial explants, after seven days
of growth on MS medium. The root explants thickened and began to burst over the entire
length of the basal part. During four weeks of subculture, a large number of adventitious
buds were observed on initial explants. In order to obtain transgenic centaury plants with
lowered endogenous cytokinin levels, in vitro cultivated root explants were transformed
with Agrobacterium tumefaciens GV3101 bearing a pBinHTX plasmid with either AtCKX1
or AtCKX2 genes encoding isoforms of cytokinin oxidase/dehydrogenase (CKX). PCR
analysis of genomic DNA confirmed the effecient integration of both AtCKX1 (30%) and
AtCKX2 (28.2%) genes. In order to investigate the expression of AtCKX genes in transgenic
centaury plants, an optimization of PCR protocol with qPCR primers was necessary.
Optimization of PCR protocol implicated finding the optimal annealing temperature and
simultaneous application of different PCR additives and enhancers. Addition of 5% DMSO
significantly improved the specificity of qPCR amplification of AtCKX1 and AtCKX2
genes. qRT-PCR analysis indicated that AtCKX1 and AtCKX2 genes were expressed in 50%
and 64% of the analyzed transgenic lines, respectively. In this work we clearly demonstrate
a significant effect of AtCKX genes on cytokinin metabolism in transgenic centaury plants.
Since CKX is the only known enzyme involved in cytokinin catabolism, it is considered a
key factor in controlling the cytokinin levels in plant tissues. CKX enzyme activity was
determined in shoots and in roots of all AtCKX transgenic lines. The analysis of the content
of total cytokinins and individual groups of endogenous cytokinins in non-transformed
centaury plants enabled, for the first time, a determination of cytokinin profile of this
species in vitro. In this work we also showed that changes in the cytokinin metabolism have
unpredictable effects on the endogenous IAA levels in plant tissues. The expression of
AtCKX genes also affected some morphological traits of the transgenic centaury plants. We
observed that a decrease of endogenous cytokinins caused a reduction of morphogenetic
potential of transgenic centaury plants but did not significantly affect the biomass
production in comparison to control plants. Analysis of photosynthetic pigments of
centaury indicated that a decrease of endogenous cytokinins might be a prerequisite rather
than a signal for the onset of senescence. The main active pharmacological compounds of
the centaury are secoiridoids and xanthones. The content of the secoiridoids found in plants
grown in vitro was similar to that in plants collected from nature, whereas the quantity of
xanthones was significantly elevated in plants grown in vitro, in comparison to plants from
nature. Most of the obtained AtCKX transgenic centaury lines produced less secoiridoids
than plants from natural habitat or plants grown in vitro. On the contrary, it was determined
that the majority of the AtCKX transgenic centaury lines accumulated more xanthones than
plants grown in nature. As xanthones are increasingly being used for their pharmacological
properties, AtCKX transgenic centaury plants could be used as a useful source of plant
material for the production of novel drugs. | true |
Populaciona dinamika, ishrana i ekotoksikologija velikog kormorana Phalacrocorax carbo (Linnaeus, 1758) na Carskoj bari | Population dynamics, diet and ecotoxicology of the GreatCormorant Phalacrocorax carbo (Linnaeus, 1758) in the Carska bara marshland | kormorana i naseljava sve kontinente izuzev Južne Amerike i Antarktika.
Politipska je vrsta u okviru koje se razlikuje pet podvrsta, a Evropu naseljavaju dve: P.
carbo carbo – naseljava obalu severnog Atlantika, i P. carbo sinensis – naseljava
centralnu i južnu Evropu. Brojnost ove vrste konstantno raste od 70-ih godina prošlog
veka. Veliki kormoran se u potpunosti hrani ribom. Rast broja kormorana širom Evrope
doveo je do nezadovoljstva uzgajivača ribe i ribara, što je pokrenulo mnogobrojna
istraživanja ove vrste u mnogim evropskim zemljama.
U prošlosti, veliki kormoran se u Srbiji gnezdio u plavnim oblastima duž velikih
reka (Obedska bara, Kovilj, Carska bara, Apatin...), ali je 60-ih i 70-ih godina prošlog
veka, usled isušivanja močvara i meliorativnih radova, gotovo nestao kao gnezdarica.
Sredinom 80-ih godina ponovo počinju da se formiraju kolonije velikog kormorana na
području Vojvodine uz velike reke, i od tada je njegova brojnost u stalnom porastu.
Početkom ovog veka počinju da se formiraju kolonije i južno od Save i Dunava.
Istraživanja u okviru ove disertacije sprovedena su na području Specijalnog
rezervata prirode „Carska bara“, u periodu od 2007. do 2010. godine. Brojnost gnezdeće
populacije praćena je prebrojavanjem (cenzusom) aktivnih gnezda, dok je van tog
perioda brojnost praćena prebrojavanjem ptica na zajedničkom noćilištu. Gnezdeća
populacija je bila najbrojnija 2007. godine, kada je sastojala od 410 do 440 parova, dok
je najmanja brojnost zabeležena 2010. godine, kada se gnezdilo 250 do 270 parova. Van
perioda gnežđenja najveća brojnost velikih kormorana zabeležena je tokom novembra
2008. i 2009. godine (po 1950 jedinki).
Ispitivanje ishrane velikih kormorana na području SRP „Carska bara“ vršeno je
analizom gvalica i povraćenog želudačnog sadržaja. Ukupno je prikupljeno 587 gvalica,
na osnovu kojih su identifikovani ostaci 2433 jedinke riba i 529 povraćenih riba.
Registrovano je učešće 21 vrste riba u ishrani kormorana na ovom području, od čega 12
V
iz porodice Cyprinidae. Najznačajnije vrste riba u ishrani bile su babuška (Carassius
gibelio) i šaran (Cyprinus carpio), kako u periodu gnežđenja tako i van njega. Prosečna
dužina plena iznosila je 17,53 cm, a kretala se u opsegu od 6,30 cm do 43,50 cm, dok je
prosečna težina iznosila 94,77 g i kretala se u opsegu od 1,45 g do 625 g. Vrednost
dnevnog unosa hrane (masa riba po gvalici) tokom zimskog perioda od 2007. do 2010.
godine iznosila je 412 ± 215 g, a tokom sezone gnežđenja 405 ± 181 g.
Procenjuje se da kormorani pričinjavaju značajne štete akvakulturi, ne samo
direktnim konzumiranjem, već i povređivanjem gajene ribe pri neuspešnim napadima,
što izaziva stres i promenu njihovog ponašanja. S obzirom da se na istraživanom
području nalazi i najveći ribnjak u Srbiji (ribnjak Ečka), navedeni fenomen je takođe
analiziran u okviru ovih istraživanja. Površina tela zahvaćena povredama (površinskim i
subepitelijalnim) iznosila je prosečno oko 11% ukupne površine tela. Najčešće uočene
histopatološke promene na mestima povreda bile su: broj i položaj pigmentnih ćelija u
dermisu i enormno zadebljanje epidermalnog sloja (u više od 80% uzoraka), zatim
poremećena struktura sloja epidermisa, povećan broj alarmnih ćelija i izmenjena
morfologija pokrovnih ćelija (u više od 75% uzoraka), kao i mioliza (u oko 50%
uzoraka).
Pored navedenih istraživanja, analizirane su i koncentracije 20 teških metala i
mikroelemenata (Al, As, B, Ba, Cd, Co, Cr, Cu, Fe, Hg, Li, Mg, Mn, Mo, Ni, Pb, Si, Se,
Sr i Zn) u različitim tkivima kormorana (mišić, jetra, crevo, pero i kost), kao i u
različitim tkivima (mišić, jetra, škrge, gonade i bubreg) njegovog dominantnog plena na
ovom području - babuški i šaranu. Analiza je vršena metodom induktivno spregnute
plazme – optičko emisione spektrometrije (ICP-OES). Analiza glavnih komponenti
ukazuje da se jetra kormorana izdvaja po povećanim koncentracijama Hg, Fe, Cu i Mo,
perje po visokim koncentracijama Al i Si, a kosti po visokim koncentracijama Sr i Mg.
Razlike između tri starosne grupe kormorana zabeležene su samo u pogledu
koncentracije elemenata u njihovim perima, dok nije bilo značajnijeg razdvajanja
uzrasnih kategorija na osnovu koncentracije elemenata u preostala četiri tkiva. U
poređenju sa plenom, kormorani se razlikuju po značajno višim koncentracijama Fe, Hg
i Cu u mišićima. Toksična koncentracija žive iznad 4 μg/g suve mase registrovana je u
jetri juvenilnih i jetri i perima subadultnih kormorana. Koncentracija žive bila je
VI
značajno korelisana među svim analiziranim tkivima kormorana. Kormorani su imali i
značajno više koncentracije Hg i Fe u mišićima i jetri u odnosu na svoj dominantni plen.
Rezultati dobijeni ovim istraživanjem doprineće boljem razumevanju navika u
ishrani velikog kormorana kao i monitoringu njegovih populacija u Srbiji, a u skladu sa
Evropskim planom za upravljanje populacijama kormorana (European Cormorant
Management Plan, Rezolucija Evropskog parlamenta P6 TA(2008)0583). | The Great Cormorant Phalacrocorax carbo (L.) is the most widely distributed
species of cormorants, as it inhabits all continents with the exception of South America
and Antarctica. It is a polytypic species with five subspecies. Europe is inhabited by two
subspecies: nominate P. carbo carbo – which inhabits the North Atlantic coasts, and P.
carbo sinensis – which inhabits Central and Southern Europe. The abundance of Great
Cormorant populations is constantly increasing since 1970s. The Great Cormorant is a
fish eating bird. The increase in the number of cormorants has resulted in dissatisfaction
of fish farmers and fishermen, which in turn instigated numerous studies aimed at
assessing their actual impact on fish in many European countries.
In the past, the Great Cormorant nested in floodplain areas along the large rivers
in Serbia (Obedska Bara, Kovilj, Carska Bara, Apatin, etc.). However, in the 1960s and
1970s, due to drying of wetlands and melioration, it almost disappeared as a nesting
species from this region. In the mid-1980s, Great Cormorants again began to form
colonies in the province of Vojvodina along large rivers and, since then, the number of
Great Cormorants in Serbia is constantly increasing. At the beginning of this century,
Great Cormorants started to form colonies south of the Sava and the Danube rivers.
Research activities within this dissertation were conducted in the Special Nature
Reserve "Carska Bara" during 2007-2010. The abundance of the breeding population
was monitored by counting active nests (census). Outside of this period, the number was
determined by counting birds at overnight roosting locations. The breeding population
was the largest in 2007, when it numbered 410 to 440 pairs, and the lowest in 2010,
when 250 to 270 pairs were nesting. Outside of the nesting period, the highest number
of Great Cormorants was observed during November 2008 and 2009 (1950 specimens
each).
Examination of the diet of Great Cormorants in the SRP "Carska Bara" was
performed by analysis of pellets and regurgitated stomach contents. A total of 587
VIII
pellets were collected, in which remains of 2433 fish specimens were identified, as well
as 529 regurgitated fish. Twenty-one fish species was registered in the diet of
cormorants in the research area, with 12 species from the family Cyprinidae. The most
important fish species in the Great Cormorants’ diet are the Prussian carp (Carassius
gibelio) and the common carp (Cyprinus carpio), both during the nesting and the
wintering period. The average length of the prey was 17.53 cm, ranging from 6.30 cm to
43.50 cm, and the average weight was 94.77 g, ranging from 1.45 g to 625 g. The
amount of food intake (weight of fish in pellets) during the winter season from 2007 to
2010 was 412 ± 215 g, while during the breeding season it was 405 ± 181 g.
It is estimated that cormorants cause considerable damage to fish in aquaculture,
not only by direct consumption of fish, but also by injuring them during unsuccessful
attacks, which causes stress and change of their behaviour. Considering that the largest
fishpond in Serbia (fishpond Ečka) is located in the research area, this phenomenon was
also included in the research. The body area affected by injuries (surface and
subepithelial) represented about 11% on average of the total body area. The most
commonly observed histopathological changes in injured areas were the number and
position of pigmented cells in the dermis and the enormously thickened epidermal layer
(in more than 80% of samples), disturbed epidermal structure, increased number of
alarm cells, and altered morphology of overlaying cells (in more than 75% of samples),
and miolysis (in about 50% of the samples).
The research also included the analysis of 20 heavy metals and trace elements
(Al, As, B, Ba, Cd, Co, Cr, Cu, Fe, Hg, Li, Mg, Mn, Mo, Ni, Pb, Si, Se, Sr, and Zn) in
different tissues (muscle, liver, intestine, feather, and bone) of Great Cormorants and in
different tissues (muscle, liver, gills, kidney and gonad) of their main prey (Prussian
carp and common carp) within the research area. The analysis was performed by
inductively-coupled plasma optical spectrometry (ICP-OES). A principal components
analysis (PCA) indicated that the cormorant liver tissue was differentiated from the
other four tissues by higher concentrations of Hg, Fe, Cu, and Mo, feathers by higher
concentrations of Al and Si, and bones by higher concentrations of Sr and Mg.
Differentiation among the three age classes of cormorants was observed only with
regard to elemental concentrations in their feathers: subadult cormorants were
differentiated by higher Hg and Zn concentrations, while the adults were differentiated
IX
by higher concentrations of Fe. In comparison to their prey, cormorants were
differentiated by higher concentrations of Fe, Hg, and Cu in the muscle. Toxic mercury
concentrations above 4 μg/g dry weight were determined in the liver of juvenile
cormorants, as well as in the liver and feathers of subadults. Hg concentrations were
significantly correlated among all studied tissues of cormorants. Cormorants had
significantly higher concentrations of Fe and Hg in muscle and liver than the two
studied fish species.
The results obtained in this study will contribute to a better understanding of the
dietary habits of the Great Cormorant, as well as to the monitoring of its populations in
Serbia, in keeping with the European Cormorant Management Plan, European
Parliament resolution P6 TA(2008)0583). | true |
Diverzitet mikromiceta na objektima kulturne baštine i testiranje fungicida primenljivih u konzervaciji | Micromycetes diversity on cultural heritage objects and testing the fungicides applicable in conservation | Cilj istraživanja doktorske disertacije je bio proučavanje biodeterioracije objekata
kulturne baštine u Srbiji. Na osnovu predloga stručnih timova u institucijama za zaštitu
objekata kulturne baštine izvršen je odabir istraživanih objekata i predmeta. Spomeni-
ci kulture obuhvaćeni u istraživanju bili su: crkva Svete Trojice manastira Sopoćani i
crkva Svete Bogorodice manastira Gradac kod Novog Pazara, crkva Svetog Nikole u
Paležu, Ajfelova prevodnica kod Bečeja, spomenik Neznanom junaku na Avali, Bran-
kov most u Beogradu i srednjovekovni kameni stećci sa lokaliteta Mramorje i Rasti-
šte kod Perućca. Opservacijom in situ opisani su simptomi biodeterioracije: pojava bi-
opatine, izmenjena tekstura kamene površine, fenomen „biopitting“ i drugi. Sa mesta
sa vidljivim simptomima biopatine izvršeno je uzorkovanje fragmenata biofilma adhe-
zivnom trakom sa kamenog supstrata. Direktnim posmatranjem fototrofne komponen-
te biofilma, svetlosnim mikroskopom, identifikovane su prisutne cijanobakterije i alge,
uzročnici različite obojenosti supstrata. Crvena biopatina na fasadi crkve svete Bogoro-
dice je posledica prisusutva kolonija Gloeocapsa sanguinea (C.Agardh) Kützing dok je
Trentepohlia aurea (Linnaeus) C.F. P. Martius izazvala pojavu narandžaste biopatine na
istom objektu. Crna katranasta biopatina na Ajfelovoj prevodnici je posledica prisusutva
kolonija Nostoc sp., dok je Haematococcus pluvialis Flotow uzročnik narandžaste bio-
vi
patine na spomeniku Neznanom junaku. Cijanobakterija Oscillatoria sp. i zelena alga
Cylindrocistis brebissonii (Ralfs) de Bary su uzročnici zelene biopatine na istom objek-
tu. U okviru fototrofne komponente biofilma na površini kamenog supstrata 5 istraži-
vanih spomenika kulturne baštine u Srbiji identifikovano je 17 taksona cijanobakterija i
13 taksona algi, što sugeriše nizak diverzitet fototrofa. Najčešće identifikovana vrsta na
kamenom supstratu objekata materijalne kulturne baštine je cijanobakterija Gloeocap-
sa violacea (Corda) Rabenhorst a najčešće identifikovana alga je Desmococcus oliva-
ceus (Persoon ex Acharius) J.R.Laundon. Mikromicete prisutne na površini spomenika
kulture i umetničkih predmeta uzorkovane su metodom sterilnog brisa, izolovane su
monosporične kulture i identifikovane do nivoa roda ili vrste. U okviru fungalne ko
mponente biofilma na površini kulturnoistorijskih spomenika u Srbiji identifikovano je
28 taksona. Zastupljenost identifikovanih mikromiceta je različita na ispitivanim obje
ktima. Cladosporium cladosporioides (Fresen.) G.A. de Vries i vrste rodova Alterna-
ria i Fusarium bile su prisutne na svim ispitivanim objektima. Epicoccum purpurascens
Ehrenb je izolovan, sa visokom učestalošću, na četiri objekta. Najveći broj vrsta mi-
kromiceta (13) zabeležen je na kamenu peščaru Ajfelove prevodnice. Mikološka ana-
liza zidnih slika u srpskim sakralnim objektima pokazala je njihovu kolonizaciju vrsta-
ma rodova Alternaria, Aspergillus, Cladosporium, Epicoccum, Emericella, Nigrospora
i Penicillium. Izolacija i identifikacija mikromiceta sa umetničkih predmeta sačinjenih
od različitih tipova materijala, pokazala je različit afinitet mikromiceta da kolonizuju
ove supstrate. Analizirani su umetnički predmeti iz zbirki Muzeja istorije Jugoslavije,
Muzeja grada Beograda, Galerije Slika „Sava Šumanović“ i zbirke Muzeja savremene
umetnosti. Najveći diverzitet mikromiceta zabeležen je na umetničkim predmetima od
drveta, na kome su zabeležene vrste rodova Absidia, Alternaria, Aspergillus, Chaetomi-
um, Cladosporium, Epicoccum, Neurospora, Penicillium, Rhizopus, Syncephalastrum,
Trichoderma i Ulocladium. Umetničke slike su do sada retko proučavane sa stanovi-
šta biodeterioracije i fungalne kontaminacije. Tokom ovog istraživanja pokazano je da
i bojeni slojevi slika i slikarska platna mogu biti kolonizovani različitim vrstama mi-
vii
kromiceta. Sa slikarskih boja izolovane su vrste rodova: Alternaria, Aspergillus, Cla-
dosporium, Drechslera, Fusarium, Neurospora, Paecylomyces, Penicillium, Rhizopus,
Trichoderma i Ulocladium, dok su sa slikarskih platna izolovane vrste rodova: Alter-
naria, Aspergillus, Chaetomium, Cladosporium, Penicillium, Trichoderma, Ulocladium
i Wardomyces. Najmanji diverzitet zabeležen je na fotografskom papiru (vrste rodova
Fusarim, Humicola, Paecylomyces, Rhizopus, Trichoderma i Ulocladium) i na tkanina-
ma (Aspergillus i Penicillium vrste). Mikološka analiza vazduha zatvorenih prostorija
različite namene u institucijama kulture, izvršena je metodom pasivne sedimentacije, a
procena brojnosti gljiva je određena metodom po Omelijanskom. Kvantitativna anali-
za vazduha, poređenjem sa standardima Svetske zdravstvene organizacije, pokazala je
umerenu kontaminaciju vazduha zatvorenih prostorija ispitivanih institucija kulture. Do-
minirale su vrste rodova Cladosporium, Penicillium, Trichoderma i Aspergillus. U cilju
pronalaženja neagresivnih, efikasnih i ekološki bezbednih agenasa, koji se mogu kori-
stiti u procesu prevencije i sanacije u okviru konzervatorskih postupaka, testirana je an-
tifungalna aktivnost etarskih ulja odabranih aromatičnih biljka (Rosmarinus officinalis
L., Lavandula angustifolia Miller, Origanum vulgare L. i Helichrysum italicum (Roth)
G. Don i benzalkonijum hlorida, osnove nekih komercijalnih biocida. Test mikromicete
su izolati sa objekata kulturne baštine Aspergillus fumigatus Fresen, Aspergillus niger
Tiegh, Aspergillus ochraceus G. Wilh, Aspergillus versicolor (Vuill.) Tirab., Bipolaris
spicifera (Bainier) Subram, Emericella nidulans (Eidam) Vuill., Epicoccum purpuras-
cens Ehrenb, Penicillium spp. i Trichoderma viride Pers. Hemijski sastav etarskih ulja
određen je kombinacijom metoda gasne hromatografije i gasne hromatografije sa spek-
trometrijom masa. Sastav etarskog ulja R. officinalis pokazuje dominaciju komponente
1.8cineol (44,28 %.). Dominantne komponente etarskog ulja L. angustifolia su linalol
(37,61%) i linalol acetat (34,86 %), dok je karvakrol (64,06%) dominirao u etarskom
ulju O. vulgare. Najzastupljenija komponenta etarskog ulja H. italicum je γkurkumen
(22,45 %). Kombinacijom različitih metoda za ispitivanje antifungalne aktivnosti (me-
toda mikroatmosfere, mikrodiluciona metoda, makrodiluciona metoda i metoda neutra-
viii
lizacije biocida) pokazano je da svi testirani agensi poseduju antifungalnu aktivnost ra-
zličitog stepena. Etarsko ulje O. vulgare i benzalkonijum hlorid pokazali su snažnu anti-
fungalnu aktivnost, etarska ulja R. officinalis i L. angustifolia umerenu, dok je najslabije
dejstvo zasbeleženo za H. italicum. Dobijeni rezultati sugerišu mogućnost implementa-
cije etarskih ulja kao potencijalno novih fungicida u sistem konzervacije i sanacije obje-
kata kulturne baštine. | The aim of presented PhD thesis was research of biodeterioration of cultural he-
ritage objects in Serbia. The objects and artefacts for this study were chosen accor-
ding to the recomendations of the experts in the field of cultural heritage conservation.
Cultural heritage monuments chosen for this study were The Church of The Holly Tri-
nity of the Monastery Sopoćani (Novi Pazar), The Holly Virgin Church of the Mo-
nastery Gradac (Novi Pazar), Saint Nicholas church (Palež), Eiffel’s Lock (Bečej),
Monument of the Unknown Hero (Avala), bridge „Brankov most“ (Belgrade) and me-
dieval tombstones on two localities Mramorje and Rastište (Perućac). During in situ
observations of examined stone monuments clear signs of biodeterioration were ob-
served, such as presence of biopatinas, changed texture of stone surface, biopitting
and other. Biofilm fragments were sampled using adhesive tape from the stone sur-
faces with visible biopatinas and examined using light microscopy in order to identi-
fy cyanobacteria and algae as phototrophic components of biofilm. It was shown that
causative agent of red biopatina on the facade of Holly virgin church was Gloeocapsa
sanguinea (C.Agardh) Kützing colonies, while Trentepohlia aurea (Linnaeus) C.F. P.
caused the orange biopatina on the same monument. Black gelationous patina on Ei-
fel’s Lock was caused by Nostoc sp. colonies. Haematococcus pluvialis Flotow cau-
x
sed the orange biopatina on the Monument of the Unknown Hero. On the same mo-
nument the green biopatina caused by Oscillatoria sp. and Cylindrocistis brebissonii
(Ralfs) de Bary was documented. The total of 17 taxa of cyanobacteria and 13 taxa of
algae from all five examined monuments suggested low diversity of phototrophs. Cya-
nobacteria Gloeocapsa violacea (Corda) Rabenhorst and green algae Desmococcus
olivaceus (Persoon ex Acharius) J.R.Laundon were the phototrophs most freuqently
found during this research. Fungi were sampled using sterile cotton swabs from the
surface of the monuments, and cultivated in laboratory in order to obtain pure cul-
tures and identified to the species or genus level. The total of 28 fungal taxa were
documented on the surface of the stone monuments. Cladosporium cladosporioides
(Fresen.) G.A. de Vries along with species belonging to genera Alternaria and Fu-
sarium were present on all examined surfaces. Epicoccum purpurascens Ehrenb was
isolated with high frequency. The biggest fungal diversity was documented on sand-
stone substrata of the Eifel’s Lock (13). Fungi isolated from wall pantings in Serbian
monasteries and churches belonged to genera Alternaria, Aspergillus, Cladosporium,
Epicoccum, Emericella, Nigrospora and Penicillium Isolation and identification of
fungi from museum artefacts suggested different susceptility of different substrata to
be colonized by fungi. Examined artefacts belonged to the collections of the Museum
of Yugoslav History, Belgrade City Museum, Galery „Sava Šumanović” and Museum
of Contemporary Art. The wooden substrata were the most susceptible for fungal co-
lonization and fungi belonging to genera Absidia, Alternaria, Aspergillus, Chaetomi-
um, Cladosporium, Epicoccum, Neurospora, Penicillium, Rhizopus, Syncephalastrum,
Trichoderma and Ulocladium were isolated from wooden artefacts. Canvas paintings,
which in general have seldomly been investigated for biodeterioration, can be also
colonized by fungi. This research showed that fungi belonging to genera Alternaria,
Aspergillus, Cladosporium, Drechslera, Fusarium, Neurospora, Paecylomyces, Peni-
cillium, Rhizopus, Trichoderma and Ulocladium colonize pictorial surface, while Al-
ternaria, Aspergillus, Chaetomium, Cladosporium, Penicillium, Trichoderma, Ulocla-
xi
dium and Wardomyces colonize the canvases. Photographic papers and textiles were
less susceptible for fungal colonization. From the surfaces of examined photographs
fungi Fusarim sp, Humicola sp, Paecylomyces sp, Rhizopus sp, Trichoderma sp. and
Ulocladium sp. were isolated. Examined textiles were colonized by Aspergillus and
Penicillium species. Sampling of airmycota in indoor environment in Serbian cultural
institutions was carried out using passive sedimentation method and the fungal pre-
velance in the air was estimated using Omelyansky’s method. The obtained results
were compared with World Health Organization’s recommendations and low risk for
human health was proved. Cladosporium spp., Penicillium spp, Trichoderma spp. and
Aspergillus spp. were the most dominant airborne fungi in examined indoor environ-
ments. In order to find efficaceous and non agressive and safe chemical agents which
can be used in cultural heritage conservation, the antifungal actrivity of selected aro-
matic plants (Rosmarinus officinalis L., Lavandula angustifolia Miller, Origanum vul-
gare L. and Helichrysum italicum (Roth) G. Don) along with biocide benzalconium
chloride were tested against fungi isolated from cultural heritage objects (Aspergillus
fumigatus Fresen, Aspergillus niger Tiegh, Aspergillus ochraceus G. Wilh, Aspergillus
versicolor (Vuill.) Tirab., Bipolaris spicifera (Bainier) Subram, Emericella nidulans
(Eidam) Vuill., Epicoccum purpurascens Ehrenb, Penicillium spp. and Trichoder-
ma viride Pers.). The chemical composition of essential oils were carried out using
gas chromatography and gass chromatography mass spectrometry. The main compo-
nent of R. officinalis essential oil was 1.8cineole(44,28 %.), the main components
of L. angustifolia oil were linalool (37,61%) and linalool acetate (34,86 %). Carva-
crol (64,06%) was the main component of O. vulgare oil, while γcurcumene were
the main component of H. italicum oil. Using different methods for testing antifungal
activity (microatmosphere method, microdilution method, macrodilution method and
biocide neutralization method) all tested agents showed different antifungal potential.
O. vulgare oil and biocide were the strongest inhibitors fungi, while oils of R. offici-
nalis and L. angustifolia showed moderate antifungal activity. H. italicum oil was the
xii
weakest inhibitor of fungal growth. However, presented research could be the first
step in implementation of essential oils as potentialy novel fungicides in cultural heri-
tage conservation. | true |
Uticaj ledenih doba na filogeografske obrasce šumskih ptica: Phylloscopus collybita, Prunella modularis i Certhia familiaris | Impact of ice ages on phylogeographic patterns of forest bird species: Phylloscopus collybita, Prunella modularis and Certhia familiaris | izolaciju populacija u refugijumima za vreme glacijalnih maksimuma, i nakon toga
rekolonizaciju šireg dela Evropskog kontinenta za vreme povlačenja lednika. Da bi se
ova paradigma testirala, analizirani su filogenetski obrasci unutar tri šumske vrste ptica
(Phylloscopus collybita, Prunella modularis
i Certhia familiaris) koje su
rasprostranjene širom Evroazije. Obrasci geografske varijacije jednog mitohondrijalnog
i jednog nuklearnog gena su analizirani kod običnog zviždka (P. collybita). Kod
običnog popića (P. modularis) u istraživanjima je korišćen jedan mitohondrijalni gen i
10 nuklearnih gena, dok kod kratkokljunog puzića (C. familiaris) tri mitohondrijalna i
tri nuklearna gena. Analiza mitohondrijalnog gena kod običnog zviždka je pokazala
postojanje šest filogenetskih linija koje odgovaraju postojećim podvrstama. Kod
običnog popića, analize mitohondrijalnog gena, kao i analize nuklearnih gena i
razgraničavanja podvrsta su ukazale na postojanje tri odvojene evolutivne linije: jedna
je rasprostranjena na prostoru Pirinejskog poluostrva, druga na području Kavkaza i treća
na području Apeninskog, Balkanskog poluostrva i ostatka Evrope. Kratkokljuni puzić se
sastoji od dve filogenetske linije koje su se razdvojile u srednjem Pleistocenu: jedna
paleoendemična koja ima alopatričko rasprostranjenje ograničeno na ostrvo Korzika i
region Kavkaza i drugu, mlađu i široko rasprostranjenu, koja je prisutna u većem delu
Evroazije i severnog dela Kine. Rezultati dobijeni u ovom istraživanju naglašavaju
značaj glacijacija u oblikovanju evolucione istorije sve tri istraživane vrste ptica na
prostoru Evroazije. | Hewitt’s paradigm for effects of Pleistocene glaciations on European populations
assumes their isolation in peninsular refugia during glacial maxima, followed by
recolonization of broader Europe during interstadials. To test this paradigm, we
analyzed the phylogenetic relationships within three forest bird species (Phylloscopus
collybita, Prunella modularis and Certhia familiaris) which ranges includes parts of
Eurasia. For Common Chiffchaff (P. collybita) the patterns of intraspecific geographic
variation in one mitochondrial and one nuclear gene were analyzed, for Dunnock (P.
modularis) one mitochondrial and 10 nuclear genes and for Eurasian Treecreeper (C.
familiaris) three mitochondrial and three nuclear genes. Common Chiffchaff subspecies
were differentiated in their mtDNA, and formed six distinct mtDNA lineages.
Regarding Dunnock, mtDNA gene tree, multilocus species tree and species delimitation
analyses indicate the presence of three distinct lineages: one in the Iberian refugium,
one in the Caucasus refugium, and one comprising the Italian and Balkan refugia and
broader Europe. Eurasian Treecreeper comprises of two lineages that diverged during
the mid-Pleistocene: one palaeoendemic lineage has an allopatric range nowadays
restricted to the Corsica Island and the Caucasus region whereas the second one, more
recent and widespread, is distributed over most of Eurasia and in northern China.
Results obtained in this study highlight the importance of glaciations in shaping the
evolutionary history of these three forest bird species in Eurasia. | true |
Razvoj tehnoloških postupaka u cilju prevencije nastanka tabanskih lezija brojlerskih pilića | Development of technological processes TI in order to prevent the occurrence of footpad lesions in broiler chickens | U okviru doktorske disertacije prikazani su rezultati ispitivanja razliĉitih tehnoloških postupaka u cilju prevencije nastanka oštećenja tabanskih jastuĉića brojlerskih pilića. Istraţivanja su sprovedena u 4 ogleda i to u jednom u kome su vršena ispitivanja fiziĉko – hemijskih i higroskopnih osobina materijala koji se mogu koristiti kao prostirka, kao i tri biološka ogleda koji su imali za cilj da ispitaju uticaj razliĉitih faktora na oštećenja tabanskih jatuĉića kod brojlerskih pilića. Cilj bioloških ogleda je bio da odgovore na pitanje da li se razliĉitim tretmanima prostirke, primenom razliĉitih preparata i ishranom moţe uticati na prevenciju nastanka tabanskih lezija kod brojlerskih pilića. Pored toga, cilj ovih ogleda je bio da se utvrdi kako primenjene tehnologije utiĉu na proizvodne rezultate brojlera. Od fiziĉkih karakteristika materijala koji se mogu koristiti kao prostirka u tovu brojlera ispitivane su sposobnost vezivanja i otpuštanja vode, a od hemijskih sadrţaj suve materije, sadrţaj sirovih vlakana kao i pojedine frakcije (NDF, ADF, hemiceluoza, celuloza i lignin). Biološki ogledi su sprovedeni u objektima eksprimentalne farme (mikro ogled) i u proizvodnim uslovima (makro ogled). U biološkim ogledima kao prostirka je korišćena pšeniĉna slama, a tretmani kojima je pokušano poboljšanje njenih karakteristika su bili seckanje, kao i dodatak mikrobiološko-enzimskog preparata (Micropan Complex®) i lignina. Uticaj ishrane na oštećenja tabanskih jastuĉića ispitivan je u biološkom ogledu, ishranom brojlerskih pilića smešama sa smanjenom energijom i dodavanjem enzima koji poboljšava varenje hrane. Kao rezultat uticaja pomenutih tretmana ispitivani su kvalitet prostirke (vlaga, pH i emisija amonijaka), proizvodnih parametri u tovu brojlera (telesna masa, utošak hrane, konverzija, mortalitet i proizvodni indeks), ponašanje brojlera, kao i nastanak i stepen oštećenja tabanskih jastuĉića. Ocena oštećenja tabanskih jastuĉića je vršena makroskopski (primenom skale od 0-3, a u skladu sa zahvaćenom površinom) i mikroskopski, histološkom analizom koţe tabanskih jastuĉića. Rezultati ispitivanja fiziĉko-hemijskih karakteristika razliĉitih materijala pokazuju da pšeniĉna slama upija velike koliĉine vode, a usled niskog sadrţaja NDF slabo optušta vodu te shodno tome predstavlja loš izbor za prostirku u pogledu nastanka oštećenja tabanskih jastuĉića. MeĊutim, rezultati su pokazali da seckanje slame, kao tehnološki postupak, moţe uticati na nastanak i stepen oštećenja tabanskih jastiĉića, ali da dovodi i do povećanja telesnih masa brojlera kao i promena u ponašanju pilića. Dodatak mikrobiološko-enzimskog preparata dovodi do smanjenja pojave i stepena oštećenja tabanskih jastuĉića brojlera gajenih i na seckanoj i na nesckanoj slami. Rezultati takoĊe pokazuju da se, u proizvodnim uslovima, primenom lignina u prostirci od neseckane slame moţe smanjiti oštećenje tabanskih jastuĉića. Upotreba smeša koje sadrţe smanjene koliĉine energije uz primenu enzima dovodi do prevencije u pogledu nastanka tabanskih lezija, bez znaĉajnog uticaja na proizvodne rezultate. Histološka analiza tabanskih jastuĉića je pokazala da sistem ocenjivanja koji se zasniva na zahvaćenoj površini tabanskih jastuĉića u saglasnosti sa stepenom i ozbiljnošću oštećenja koţe tabanskih jastuĉića i da moţe predstavljati standardi model za ocenjivanje na liniji klanja. Na osnovu rezultata ove disertacije se moţe zakljuĉiti da primenjeni tehnološki postupci mogu znaĉajno uticati na prevenciju oštećenja tabanskih jastuĉića, bez negativnog uticaja na proizvodne rezultate u tovu brojlera. | Within this dissertation, the results of various technological processes are shown in order to prevent the occurrence of damage to the foot pads of broiler chickens. The study was conducted in four trials, one in which we investigated the physical - chemical and hygroscopic properties of materials that can be used as litter, as well as three biological trials which were aimed to examine the influence of various factors on the damage to the foot pads in broiler chickens. The objective of the biological experiments was to answer the question whether the various treatments of litters, usage of different preparations and nutrition can prevent occurrence of foot lesions in broiler chickens. In addition, the aim of this experiment was to determine how the applied technology affects performance of broiler chickens. Among physical properties of materials that can be used as litter in fatting broilers, absorption and release of water were investigate, and among chemical properties there were content of dry matter, crude fiber content as well as individual fractions (NDF, ADF, hemicelluloses, cellulose and lignin). Biological experiments were conducted in the facilities of experimental farm (micro experiment) and in production conditions (macro experiment). In biological experiments wheat straw was used as litter and treatments which attempted to improve its characteristics were chopping and addition of microbial-enzyme preparation (Micropan Complex®) and lignin. Feeding influence on damage to the foot pad was tested in a biological experiment, by feeding broilers with reduced energy diet and by adding enzymes that improve digestion. As a result of the impact of the above mentioned treatments there were tested the litter quality (moisture, pH and ammonia emissions), production parameters in fattening broilers (body weight, feed consumption, conversion, mortality and production index), the behavior of broilers, as well as the onset and degree of damage to the foot pads. Assessment of the damage to the foot pads was done macroscopically (on a scale from 0-3, and according to the affected area) and microscopically, by histological analysis of skin of foot pads. Test results of physic-chemical characteristics of different materials indicate that wheat straw absorbs large amounts of water, due to the low content of NDF it releases water slowly and consequently represents a bad choice for a litter in terms of damage to the foot pads. However, the results showed that chopping straw as a technological procedure may influence the onset and degree of damage to the foot pads, but it also leads to an increase in body weight of broilers as well as changes in their behavior. Addition of microbial-enzyme preparation leads to a decrease in the occurrence and level of damage to the foot pads in broilers grown on both chopped and unchopped straw. The results have also shown that, under production conditions, using lignin in the unchopped straw litter it can reduce the damage to the foot pads. The use of mixtures containing a reduced amount of energy with the use of the enzyme leads to prevention in terms of occurrence of foot lesions, without significant impact on performance. Histological analysis of foot pads have showed that the grading system that is based on the affected area of foot pads in accordance with the extent and severity of damage of the skin of foot pads and that it can represent a model for the assessment of standards on the slaughter line. Based on the results of this dissertation it can be concluded that the applied technological procedures can significantly influence the prevention ofdamage to the foot pads, with no negative impact on production results in fattening broilers. | false |
Fluktuirajuća asimetrija kao indikator sredinskog stresa u populacijama zidnog guštera (Podarcis muralis Laurenti 1768) u urbanim i ruralnim sredinama | Fluctuating asymmetry as an indicator of environmental stress in populations of the common wall lizard (Podarcis muralis Laurenti 1768) in urban and rural environments | Uticaj sredinskog stresa na stabilnost razvića merističkih karakteristika
(femoralne pore, subdigitalne lamele, supracilijarne granule) i oblika dorzalne strane
glave ispitivan je poređenjem nivoa fluktuirajuće asimetrije (FA) navedenih
karakteristika između populacija zidnog guštera (Podarcis muralis) koje naseljavaju
urbana i ruralna staništa. Takođe, ispitano je i da li postoje razlike u nivou varijanse
simetrične komponente oblika glave, alometriji simetrične komponente oblika i u nivou
odstupanja od grupne alometrije između populacija koje naseljavaju ova dva tipa
sredina. Pored toga, analizirano je i da li sredinski stres ima uticaja na performansu,
kondicioni indeks i prevalencu i intenzitet krvnih parazita kao i da li jedinke koje su
zaražene krvnim parazitima i jedinke sa niskim kondicionim indeksom pokazuju viši
nivo asimetrije oblika glave. Osnovna hipoteza je bila da će populacije koje naseljavaju
suboptimalne uslove životne sredine pokazivati viši nivo FA merističkih karakteristika,
viši nivo FA oblika glave, viši nivo varijanse simetrične komponente oblika. Pored toga
hipoteza je bila i da će postojati razlike u alometriji simetrične komponente oblika
glave, nivou morfološke integracije između dva modula dorzalne strane glave između
dva tipa populacija kao i veće odstupanje od grupne alometrije u urbanim
populacijama. Takođe, pretpostavka je bila i da će urbane populacije pokazati veću
prevalencu i intenzitet krvnih parazita, niži kondicioni indeks ali i da će inficirane
jedinke i jedinke sa nižim kondicionim indeksom imati viši nivo FA oblika glave.
U poređenju sa ruralnim populacijama, urbane su pokazale statistički značajno
viši nivo FA za sve analizirane merističke karakteristike što ukazuje na to da je
stabilnost razvića ovih karakteristika u ovoj sredini narušena. Takođe, uočeno je
postojanje razlika u razvojnoj stabilnosti između analiziranih karakteristika što ukazuje
na to da razvojna stabilnost može biti specifična za morfološke karakteristike. Nivo
fluktuirajuće asimetrije je bio niži kod karakteristika koje su od većeg funkcionalnog
značaja, što ukazuje da je kontrola razvića ovih karakteristika bila veća. Pored toga,
uočeno je i da postoji značajna korelacija između FA femoralnih pora i subdigitalnih
lamela što ukazuje na to da su ove karakteristike integrisane. To je, najverovatnije,
posledica deljenja razvojnih puteva jer se obe karakteristike nalaze na istom delu tela.
Takođe je pokazano da je razviće glave narušeno u urbanim populacijama na šta
ukazuje značajno viši nivo FA oblika glave. Pored toga, pokazano je i da je nivo
varijanse simetrične komponente oblika veći u urbanim populacijama što ukazuje na to
da je kanalisanost razvića takođe narušena. Uočena je i značajna korelacija između
varijanse oblika unutar i između jedinki što pokazuje da su zajednički ili slični
molekularni mehanizmi odgovorni za kanalisanost razvića i razvojnu stabilnost.
Značajne razlike između nivoa morfološke integracije nisu nađene između urbanih i
ruralnih sredina. Dalje, uočena je značajna razlika u veličini glave između ova dva tipa
populacija gde su urbane jedinke imale manju veličinu glave. Ovaj rezultat može biti
posledica sporijeg rasta glave, ranijeg prestanka rasta glave ili rezultat alokacije
energije na neke druge procese. Dalje, nađeno je da između dva tipa populacija postoje
značajne razlike u alometriji simetrične komponente oblika glave što ukazuje na to da
su i mehanizmi koji regulišu alometriju oblika narušeni. Takođe, odstupanje od grupne
alometrije je bilo veće u urbanim sredinama što znači da veličina i njene posledice
objašnjavaju manji deo varijanse u urbanim nego u ruralnim sredinama.
Analiza performanse je pokazala da značajne razlike između dva tipa populacija
ne postoje. Nivo asimetrije ekstremiteta je imao značajnog uticaja na performansu pa su
jedinke sa izraženijom asimetrijom zadnjih ekstremiteta bivale sporije. Značajne razlike
u FA ekstremiteta između tipova sredina nisu nađene.
Urbane populacije su takođe okarakterisane i manjim vrednostima kondicionog
indeksa. Imajući u vidu značajnu korelaciju između kondicionog indeksa,
reproduktivne vrednosti i ostalih komponenti adaptivne vrednosti realno je očekivanje
da je adaptivna vrednost u urbanim sredinama smanjena. Pored toga, uočena je i
značajna korelacija između nivoa FA oblika glave i kondicionog indeksa, gde su
jedinke sa manjim kondicionim indeksom pokazivale viši nivo FA. Ovaj rezultat može
biti posledica neuhranjenosti, ali i slučajnost, jer su u urbanim sredinama jedinke
pokazivale viši nivo FA oblika glave, ali i niži kondicioni indeks. Između dva tipa
populacija nisu uočene razlike u prevalenci krvnih parazita, dok je intenzitet bio
značajno veći u urbanoj sredini, najverovatnije kao posledica slabijeg imunog sistema.
Dalje, uočeno je i da jedinke inficirane krvnim parazitima pokazuju viši nivo FA oblika
glave u obe analizirane sredine. Uočena korelacija može biti rezultat direktnog uticaja
parazita tako da jedinke alociraju značajne količine energije na borbu protiv parazita te
nemaju dovoljno energije koju bi uložili u stabilnost razvića. | Effects of environmental disturbance on developmental stability of meristic
traits (femoral pores, subdigital lamelae, supraciliar granules) and dorsal head shape
were evaluated by comparing fluctuating asymmetry (FA) levels between populations
of the common wall lizard (Podarcis muralis) inhabiting urban and rural areas.
Moreover, possible differences in degree of head shape variation, head shape allometry
and in deviation from within-group allometric lines were tested between these two
population types. Also, it was analysed whether environmental disturbance affects
locomotor performance, condition index, and prevalence and intensity of blood
parasites but also if infected animals and animals with low body condition show higher
degree of head shape asymmetry.
The main hypothesis were that individuals inhabiting suboptimal environments
will show higher FA degree of meristic traits and head shape as well as higher degree
of head shape variance. Also, it was hypothesised that, due to stressfull conditions,
there will be differences in symmetric head shape allometry, differences in the degree
of morphological integration between two head modules and differences in the degree
of deviation from within-group allometric lines between the two types. Moreover,
higher prevalence and intensity of blood parasites and lower body condition were
expected in urban populations, as well as higher head shape asymmetry in infected
individuals and in individuals with low body condition.
Compared to rural populations, the urban ones showed significantly higher
degree of FA for all analysed meristic traits, which suggests that developmental
stability of these traits is affected in urban environments. Differences in developmental
stability were found between the three traits meaning that developmental stability may
be trait specific. Degree of fluctuating asymmetry was lower in functionaly significant
traits, suggesting that it may be under stricter developmental control. Significant
correlation was found between signed FA of femoral pores and subdigital lamelae,
possibly a sign of morphological integration as these traits develop in the same body
part and it is likely that they share same developmental pathways.
Apart from meristic traits, head development was also shown to be affected as
indicated by higher degree of head shape FA in urban populations. Moreover, it was
shown that urban populations show higher degree of head shape variance suggesting
that canalisation is also affected. Significant correlation between the variation within-
and between individuals was found indicating that these two phenomenons, FA and
developmental stability, may depend on the same or partialy overlaping molecular
mechanisms. No significant differences in the degree of morphological integration
between the two types were found. Mean head size was shown to differ between the
two types, with urban lizards having smaller mean head size. This may be a result of
slower head growth in urban animals, earlier cessation of growth, or due to allocation
of energy to some other processes. Moreover, significant differences between the two
population types in head shape allometry were observed, meaning that mechanisms
regulating head shape allometry may also be affected. Higher deviation from the group
allometric line was also detected in urban populations meaning that size and its
consequences explain less variation in urban than in rural environments.
The analysis of locomotor performance showed that there are no significant
differences between urban and rural animals. Hindlimb asymmetry significantly
affected running performance with individuals with more asymmetric hindlimbs
running slower. Degree of hindlimb FA was similar in both environments.
Urban populations were also characterised by lower body condition. Having in
mind significant correlation between condition index, reproductive value and other
components of fitness, it is not unlikely that lower body condition has fitness costs.
Significant correlation was observed between the degree of head shape FA and
condition index, where head shape FA tended to increase with a reducing body
condition. This result may be a consequence of malnutrition but also a result of
statistical coincidence, as urban lizards showed both high head shape FA and low body
condition. There were no differences in blood parasite prevalence between the two
types, while intensity was significantly higher in urban lizards, most likely as a result of
reduced immunocompetence. Also, infected animals showed higher head shape FA in
both population types. The observed pattern may be due to effect of parasites, since
infected animals need to allocate significant amounts of energy to immune sistem so
that they lack energy needed to invest in stabile development. | true |
Struktura topološki zatvorene ORI sekvence dihidrofolatreduktaznog lokusa u interakciji sa inicijacionim proteinom ORC | Structure of topologically closed ORI sequence from dihydrofolate reductase locus in interaction with initiation protein ORC | Replikacija je jedan od osnovnih procesa u životnom ciklusu ćelije. Cilj
replikacije je udvajanje genetičkog materijala. Da bi genetički materijal bio pravilno
umnožen i kasnije razdvojen na ćerke ćelije, razvijen je veliki broj mehanizama
regulacije replikacije. Jedan od načina regulacije je pravilan izbor i aktivacija mesta
inicijacije replikacije.
Broj i kompleksnost mesta inicijacije replikacije varira među vrstama. Kod
evolutivno nižih eukariota su jednostavnija, dok su kod viših eukariota
kompleksnija. Način prepoznavanja ovih mesta od strane inicijacionih proteina nije
u potpunosti razjašnjen.
Ori sekvenca, locirana nizvodno od gena DHFR (dihidrofolat reduktaza)
hrčka, sadrži tri definisana ori mesta inicijacije replikacije: ori β, ori β' i ori γ. Ori β
sekvenca DHFR je jak replikator i pokazuje aktivnost na ektopičnim mestima.
Premda je ori sekvenca DHFR jedna od najbolje okarakterisanih ori sekvenci kod
metazoa i u okviru nje definisani funkcionalno značajni regioni (AT bogati region,
DNR region, BEND, RIP60 region, IR 4 bp), njihova funkcija nije u potpunosti
razjašnjena.
Protein ORC (eng. Origin recognition complex) je heteroheksamer odgovoran
za prepoznavanje mesta inicijacije replikacije. Pretpostavka je da nekoliko
elemenata ima ulogu u prepoznavanju ori sekvence od strane ORC kompleksa kod
metazoa.
Funkcija ljudskog proteina Orc4 (HsOrc4) do sad nije u potpunosti
razjašnjena. Našim prethodnim istraživanjima, pokazali smo da HsOrc4
prepoznaje nekanonske strukture i ima sposobnost za utiče na formiranje TAT
tripleksa i homoadeninskih struktura.
Dosadašnja istraživanja su vršena na linearnim fragmentima, pa je u ovom
radu funkcija proteina HsOrc4 ispitivana na cirkularnoj, topološki zatvorenoj
DNK, odnosno plazmidu. Za analizu je odabrana sekvenca ori β DHFR, uklonirana
u plazmid u kojem zauzima strukturu najsličniju in vivo topologiji.
U ovom radu je analizirana struktura funkcionalno značajnih regiona ori β
DHFR sekvence: AT bogatog regiona, DNR, BEND, RIP60. Ispitivano je vezivanje
proteina HsOrc4 za superspiralizovanu formu plazmida i eventualna uloga u
remodelovanju topologije plazmida i strukture odabranih funkcionalnih regiona
ori β DHFR sekvence.
Analize su vršene nukleaznim i topoizomeraznim esejom. Nukleaza MB
(eng. Mung Bean) prepoznaje i iseca jednolančane DNK regione, pa je korišćena za
detektovanje nekanonskih struktura unutar plazmida. Topoizomeraza I relaksira
plazmid i na taj način „zaključava“ promene topologije izazvane interakcijom sa
proteinom HsOrc4.
Ovim istraživanjem je pokazano da topološki zatvoreni AT bogati region ori
β DHFR formira nekanonsku strukturu. U interakciji sa proteinom HsOrc4, ova
struktura nestaje. Moguće je da protein direktnom interakcijom modifikuje ovu
strukturu ili da je promena strukture posledica izmene topologije celokupnog
plazmida. Ostali ispitivani regioni ne zauzimaju nekanonske strukture na
neutralnom pH. Na kiselom pH 5, DNR region zauzima alternativnu strukturu,
dok AT region zauzima istu strukturu. Opstanak nekanonske strukture u okviru
AT bogatog regiona na različitim pH vrednostima unutar topološki zatvorene
sekvence ori β DHFR, za razliku od DNR regiona, sugeriše da strukture nisu
podjednako osetljive na promene pH.
Pokazano
je
da
se
protein
HsOrc4
preferencijalno
vezuje
za
superspiralizovanu formu plazmida i modifikuje topologiju plazmida. Ove
promena topologije su reverzibilne, jer nestaju po uklanjanju proteina i da bi sve
bile „zapamćene“, potrebno je prisustvo Topoizomeraze I. Protein HsOrc4 se
intenzivnije vezuje za plazmid na pH 5 u odnosu na pH 7,5, formirajući komplekse
velikih dimenzija.
Na osnovu rezultata ovog istraživanja možemo zaključiti da protein HsOrc4
uvodi topološke promene u plazmidu, koje se ispoljavaju upravo na nivou
funkcionalno značajnih elemenata ori sekvence. | Replication is one of the fundamental processes in the life cycle of the cell.
The aim of replication is a duplication of genetic material. To make genetic
material properly duplicated and subsequently divided into daughter cells, a large
number of mechanisms of regulation of replication have been developed. One way
of regulation is the proper selection and activation of replication initiation.
The number and complexity of the origins of replication (ori) varies among
species. They are simpler at evolution lower eukaryotes, while in higher
eukaryotes are more complex. The way of recognizing these places by initiation
proteins is not fully understood.
Ori sequence, located downstream of the DHFR gene (dihydrofolate
reductase) hamster, contains three defined origins of replication: ori β, ori β' and ori
γ. Ori β DHFR sequence is strong replicator and shows activity at ectopic places.
Although the sequence of ori DHFR is one of the best characterized ori sequences in
metazoans and within defined functionally important regions (AT rich region,
DNR region, BEND, RIP60 region, IR 4 bp), their function is still not completely
understood.
ORC (Origin recognition complex) is heterohexamer protein responsible for
identifying origins of replication. It is assumed that several elements play a role in
recognizing ori sequences of the ORC complex in metazoans.
The function of the human protein Orc4 (HsOrc4) has not been fully
elucidated. Our previous studies showed that HsOrc4 recognize non-canonical
structures and has the ability to influence at formation of TAT triplexes and
homoadenine structures.
Our previous studies are done on linear fragments, and in this work we
examined the function of protein HsOrc4 the circular, topologically closed DNA,
(plasmid). For the analysis of the ori β DHFR, this sequence was subcloned into the
plasmid which forms the structure similar to topology in vivo.
In this work we analyzed the structure of functionally important regions of
ori β sequence DHFR: AT rich region, DNR, BEND, RIP60. Also, we investigated
binding properties of the protein HsOrc4 to supercoiled form of plasmid, and its
possible role in the remodeling of plasmid topology and structure of selected
regions of functional β DHFR ori sequences.
We performed assays with topoisomerase and nuclease. MB nuclease
(Mung Bean) recognizes and cut single stranded DNA regions, and it is used to
detect non-canonical structures within the plasmid. Topoisomerase I relax plasmid
and thus "lock" topology changes caused by interaction with protein HsOrc4.
This research has shown that topologically closed AT rich region of ori β
DHFR sequence form noncanonical structure. In the interaction with the protein
HsOrc4, this structure disappears. It is possible that protein modifies this structure
in direct interaction or the structural change is a consequence of changes in the the
topology of the entire plasmid. Other regions studied do not form non-canonical
structures at neutral pH. At acidic pH 5, DNR region occupies an alternative
structure, while the AT region occupies the same structure. The survival of non-
canonical structures in the AT rich regions at different pH values in the sequence
of topological closed DHFR ori β, in contrast to the DNR region, suggest that the
structures are not equally sensitive to pH changes.
It is shown that the protein preferentially binds to HsOrc4 supercoiled form
of plasmids and modify the topology of plasmids. These topology changes are
reversible, since they disappear as protein is removed and for its "remembering"
requires the presence of Topoisomerase I. Protein HsOrc4 intensely bind to the
plasmid at pH 5 than at pH 7.5, forming large complexes.
Based on the results of this study, we conclude that protein HsOrc4
introduces topological changes in plasmid that are expressed at the level of
functionally important elements of ori sequences. | true |
Ontogenetska fenotipska plastičnost i materinski efekti tokom ranih stupnjeva razvića Iris pumila L. (Iridaceae) | Ontogenetic phenotypic plasticity and maternal effects during early stages of development in Iris pumila L.(Iridaceae) | Prikazana su proučavanja veličine i obrazaca fenotipske plastičnosti tokom ranih faza procesa razvića biljaka kao odgovor na promenu intenziteta svetlosti, kao i doprinos materinskih faktora ekspresiji juvenilnog fenotipa. Za ovo istraživanje izabrane su dve prirodne populacije I. pumila koje su izložene različitim svetlosnim uslovima u Deliblatskoj Peščari. Eksperimentalni dizajn obuhvatao je tri svetlosna intenziteta. Istražene su tri faze juvenilnog perioda I.pumila pri čemu je na svakom klijancu izvršeno merenje većeg broja osobina. Na osnovu procena i poredjenja različitih aspekata variranja ispitivanih osobina dobijeni su sledeći zaključci: 1. Veličina i pravac fenotipskog odgovora bili su specifični za osobinu, kao i za deo primenjenog svetlosnog gradijenta. Dobijeni rezultati pokazuju da je plastičnost specifične lisne površine (SLA) najizraženiji odgovor na nizak intenzitet svetlosti; 2. Heritabilnost i genetičke korelacije kod vrste I.pumila variraju između različitih svetlosnih sredina;3. Plastičnost osobina, merena preko indeksa plastičnosti, bila je veća kod biljaka iz populacije Šuma; 4. Genotipovi I.pumila, ali i populacije, značajno su se razlikovali u ontogenetskim normama reakcije. Razlike razvojnih putanja izmedju populacija bile su manje u poređenju sa razlikama između svetlosnih tretmana; 5. Korelacije izmedju osobina značajno su se menjale tokom razvića; 6. Koeficijent materinskog efekta (m2) i koeficijent materinske varijacije (CVM) bili su specifični za osobinu i svetlosni tretman. Zavisnost ispoljavanja materinskih vanjedarnih uticaja od potomačkih sredinskih faktora pokazuje da selektivna vrednost materinskih genotipova zavisi od sredine u kojoj se odvija razviće potomaka; 7. U ranim fazama razvića I.pumila postoji značajna ontogenetska varijabilnost ispoljavanja materinskog efekta. Sa starošću klijanaca smanjuje se stepen ispoljavanja materinskih efekata. Materinske korelacije se značajno menjaju tokom ontogenije pri čemu su ove promene i sredinski zavisne. | Study was about the size and patterns of phenotypic plasticity during the early stages of plant development in response to different light intensities, and the contribution of maternal factors expression of juvenile phenotype. For this study two natural populations of I.pumila were chosen. They were exposed to different light conditions in Deliblato sands. The experimental design consisted of three light intensities. The three phases of the juvenile period I.pumila where investigated. On each seedling a number of measurements were taken. Based on the evaluation and comparison of different aspects of the variation of plant traits the following conclusions were obtained: 1. The size and direction of phenotypic responses were specific for the trait, as well as to the part of the light gradient. The results show that the plasticity of specific leaf area (SLA) is most pronounced in response to low light intensity; 2. Heritability and genetic correlation in I. pumila vary significantly between different light environments; 3. Phenotypic plasticity, measured by the index of plasticity, was higher in plants from “Šuma” population; 4. Genotypes, as well as populations, of I.pumila, have significantly different ontogenetic reaction norms. The differences between the populations developmental pathways were lower in comparison with the differences between the light treatment; 5. Trait correlations significantly changed during development; 6. The coefficient of maternal effects (m2) and the coefficient of maternal variation (CV) were trait-specific and treatment-specific. The selective value of maternal genotype depends on the environment in which offspring development occurs; 7. In the early stages of development of I. pumila there was no significant ontogenetic variation of maternal effects. The degree of maternal effects decreased with seedlings age. Maternal correlations significantly change during ontogeny. These changes were environmentally dependent, too. | false |
Uspostavljanje rezistentnih tumorskih ćelijskih linija kao modela za testiranje novih hemioterapeutika : molekularna karakterizacija rezistencije nastale dugotrajnim izlaganjem paklitakselu | The establishing of resistant cancer cell lines as a model for testing of new chemotherapeutics: molecular characterization of resistance developed after continuous treatment with paclitaxel | Glavni uzrok neuspeha hemioterapije u lečenju kancera je pojava višestruke
(engl. „multi-drug“) rezistencije (MDR). Efikasnost paklitaksela (PTX) je često
ograničena pojavom rezistencije. Cilj ove doktorske disertacije je bio ispitivanje
molekularnih i fenotipskih promena u toku razvoja MDR-a indukovanih PTX-om kod
ćelijskih linija humanog karcinoma debelog creva (DLD1) i glioblastoma (U87).
Takođe je testirana upotrebljivost dobijenih MDR modela u evaluaciji četiri anti-kancer
agensa.
Kontinuirani tretman PTX-om doveo je do razvoja MDR-a kod obe ispitivane
ćelijske linije koje su postale rezistentne na strukturno i funkcionalno različite
hemioterapeutike. Nakon potvrde prisustva ukrštene rezistencije kod novo-
uspostavljenih ćelijskih linija DLD1-TxR i U87-TxR, analizirana je ekspresija
membranskih trasportera uključenih u razvoj MDR-a na nivou iRNK. Ćelije su imale
povišen nivo ekspresije mdr1 gena i smanjen nivo ekspresije mrp1 gena. Prekomerna
ekspresija P-glikoproteina (P-gp), koji kodira mdr1 gen, je uočena kod obe MDR
ćelijske linije. Analiza na protočnom citofluorimetru je pokazala da je akumulacija P-
gp supstrata (rodamina 123 i doksorubicina) kod DLD1-TxR i U87-TxR ćelija značajno
manja u poređenju sa odgovarajućim parentalnim ćelijama, DLD1 i U87. Značajno
smanjenje ekspresije gst-π gena i koncentracije glutationa (GSH) je uočeno kod U87-
TxR ćelija. Sekrecija vaskularnog endotelijalnog faktora rasta (VEGF) je inhibirana u
jednokratnom tretmanu kod ćelijskih linija karcinoma debelog creva i u kontinuiranom
tretmanu kod ćelijske linije glioblastoma. Analiza ćelijskog ciklusa je pokazala da je
jednokratni tretman PTX-om kod ćelijskih linija humanog karcinoma debelog creva
praćen porastom subG0 faze, odnosno povećanjem procenta mrtvih ćelija, dok je kod
ćelija glioblastoma došlo do umiranja tokom interfaze (G1, S ili G2). MDR tumorske
ćelijske linije su stekle nove strukturne i numerićke hromozomske aberacije. Sticanje
MDR fenotipa kod U87-TxR ćelija je praćeno smanjenem jednog nivoa ploidije.
Takođe je uočen gubitak hromozomskog regiona 6q kod obe rezistentne ćelijske linije,
kao i inaktivacija p53 tumor spresor gena kod U87-TxR ćelija i PTEN tumor supresor
gena kod DLD1-TxR ćelija.
Dalje je analizirana anti-kancer aktivnost i potencijal za reverziju MDR-a Akt
inhibitora (GSK690693), Ras inhibitora (Tipifarnib) i dva inhibitora P-gp-a (jatrofanski
diterpenoidi Eufodendrofana H-Euph H i Eufodendrofane S -Euph S). Njihova
efikasnost varira u zavisnosti od razlika u tipu ćelija, postojanja MDR fenotipa ili
promena u tumor supresorima. Tipifarnib, Euph H i S su značajno povećali senzitivnost
MDR ćelijskih linija na PTX.
Kontinuirani tretman PTX-om dovodi do prekomerne ekspresije P-gp-a i
sticanja MDR fenotoipa kod ćelijskih linija humanog karcinoma debelog creva i
glioblastoma. MDR ćelije su stekle nove molekularne i citogenetske karakteristike, dok
su suprimirani neki mehanizmi MDR-a i progresije tumora kao što su GSH
detoksifikacioni sistem i sekrecija VEGF-a. Ovi rezultati ukazuju da tretman PTX-om
može imati veliki značaj u terapiji karcinoma debelog creva i glioblastoma, čak i u
prisustvu MDR-a. Uprkos ograničenjima o kojima se govori u literaturi u vezi
upotrebljivosti MDR in vitro modela, ispitivanja promena izazvanih veštačkim
razvojem MDR-a su i dalje neophodna i aktuelana. Zbog toga, ove MDR tumorske
ćelijske linije predstavljaju značajan eksperimentalni model za testiranje novih anti-
kancer agenasa. | Multi-drug resistance (MDR) is a major obstacle to successful cancer treatment.
The efficacy of paclitaxel (PTX) is often limited by appearance of drug resistance. The aim of
this study was to explore molecular and phenotypic alterations during development of MDR
induced by PTX in human colon carcinoma (DLD1) and glioblastoma (U87) cell lines. We
also tested the usefulness of developed MDR models in the evaluation of four anti-cancer
agents.
Continuous treatment with PTX led to the development of MDR in both tested
cancer cell lines that became resistant to structurally and functionally unrelated
chemotherapeutics. After confirmation of the cross-resistance in newly established DLD1-TxR
and U87-TxR, we analyzed the mRNA expression of membrane transporters involved in
MDR. The cells had increased levels of mdr1 gene expression, while mrp1 was decreased.
Over-expression of P-glycoprotein (P-gp), coded by mdr1, was observed in both MDR cancer
cell lines. Flow cytometry analyzes showed that the accumulation of P-gp substrates
(rhodamine 123 and doxorubicin) in DLD1-TxR and U87-TxR was significantly lower
compared to DLD1 and U87, respectively. The significant depletion of gst-π gene expression
and glutathione (GSH) concentration was observed in U87-TxR. Vascular Endothelial Growth
Factor (VEGF) secretion was inhibited by single PTX treatment of colon cancer and in
continuous treatment of glioblastoma cell lines. The analysis of cell cycle kinetics revealed
extensive cell death in colon cancer cells that were accumulated in subG0 phase after PTX
treatment, while glioblastoma cells died through interphase (G1, S or G2). The MDR cancer
cell lines acquired novel structural or numerical chromosomal aberrations. Polyploidy
reduction was observed after development of MDR in U87-TxR. Losses of 6q in both resistant
cancer cell lines and inactivation of p53 in U87-TxR and PTEN in DLD1-TxR were also
revealed.
We evaluated the anti-cancer activities and MDR reversal potential of the Akt
inhibitor (GSK690693), the Ras inhibitor (Tipifarnib) and two P-gp inhibitors (jatrophane
diterpenoids Euphodendrophane H-Euph H and Euphodendrophane S -Euph S). Their effects
vary due to the cell-type differences, existence of MDR phenotype or tumor suppressors’
alterations. Tipifarnib, Euph H and S, significantly sensitized MDR cancer cells to PTX.
In conclusion, continuous PTX treatment caused the over-expression of P-gp and
acquisition of MDR in colon cancer and glioblastoma cell lines. MDR cancer cells obtained
new molecular and cytogenetic characteristics, while some mechanisms of MDR and tumor
progression such as GSH detoxification system and VEGF secretion were suppressed. The
results implicate the PTX treatment as an important clinical tool for colon carcinoma and
glioblastoma treatment even in the presence of MDR. Despite limitations discussed in
literature due to the usefulness of MDR in vitro models, further examinations of changes
provoked by artificially developed MDR are still emerging. Therefore, our MDR cancer cell
lines present valuable tool for pre-clinical evaluation of new anti-cancer agents. | true |
Efekat grelina na regulaciju antioksidativnih enzima i inducibilne azot-monoksid-sintaze u jetri pacova | The effect of ghrelin on the regulation of antioxidantenzymes and inducible nitric oxide synthase in rat liver | U poslednjih nekoliko godina, objavljeni naučni podaci sve više ukazuju na
antioksidantne i antiinflamatorne efekte grelina. U ovoj doktorskoj disertaciji su izučavani
efekti grelina na ekspresiju enzima jetre: superoksid dismutaze (engl. Superoxide Dismutase,
SOD), katalaze (engl. Catalase, CAT), glutation peroksidaze (engl. Glutathione peroxidase, GPx)
i glutation reduktaze (engl. Glutathione reductase, GR), kao i ekspresiju njihovog uzvodnog
regulatora, nuklearnog faktora-kB (engl. Nuclear factor- kappa B, NFκB). Takođe su izučavani
efekti grelina na regulaciju enzima inducibilne azot-monoksid-sintaze (engl. Inducible nitric
oxide synthase, iNOS; NOS2) u jetri pacova. Jedan od ciljeva istraživanja u okviru ove
doktorske disertacije je bio i utvrđivanje uloge kinaze regulisane ekstraćelijskim signalima
(engl. Extracellular regulated protein kinase, ERK1/2) i protein kinaze B (engl. Protein kinase B,
Akt) u grelinom regulisonoj aktivnosti i ekspresiji antioksidatnivnih enzima i iNOS u jetri
pacova.
Kontrolni, normalno hranjeni mužjaci pacova, Wistar soja (mase 200-250g) kao i
mužjaci pacova koji su bili na dijeti bogatoj mastima (engl. High fat diet, HF), tretirani su
grelinom (0,3nmol/5μl) intracerebroventrikularnim (ICV) injeciranjem, u bočne cerebralne
komore svaka 24 h, u trajanju od 5 dana. Dva sata nakon poslednjeg tretmana, životinje su
žrtvovane i izolovane su jetre. Za utvrđivanje nivoa ekspresije proteina: iNOS,
antioksidativnih enzima, fosforilacije Akt na Ser473, ERK 1/2 i NFκB, subjedinica p50 i p65,
primenjena je Western blot metoda. Dobijeni rezultati pokazuju da dolazi do povećanja
ekspresije proteina za enzime antioksidativne zaštite: Cu/Zn-SOD (p<0,05), Mn-SOD
(p<0,05), CAT (p<0,01), GPx, (p<0,001), kao i GR (p<0,01) u grupi normalno hranjenih
životinja tretiranih grelinom u poređenju sa kontrolnim životinjama. Grelin značajno
smanjuje ekspresiju iNOS (p<0,001) kao i fosforilaciju NFκB subjedinica p65 (p<0,001) i
p50 (p<0,05) u grupi normalno hranjenih životinja u poređenju sa kontrolnom grupom
pacova. Nivo fosforilacije Akt na Ser473 značajno je veći u grelinom tretiranoj grupi nego kod
kontrolnih životinja (p<0,01).
Tretman gojaznih pacova grelinom doveo je do značajnog povećanje ekspresije
enzima antioksidativne zaštite: Cu/Zn-SOD (p<0,05), Mn-SOD (p<0,05), CAT (p<0,05),
GPx (p<0,05) kao i ekspresije transkripcionog faktora NFκB p65 (p<0,001), dok je značajno
smanjio fosforilaciju ERK1/2 (<0,05) i Akt (p<0,01) u poređenju sa ne-tretiranim gojaznim
pacovima.
Dobijeni rezultati pokazuju da grelin reguliše ekspresiju antioksidativnih enzima i
iNOS kod kontrolnih, normalno hranjenih pacova preko Akt/ERK1/2 kinaza. Takođe,
grelin smanjuje posledice indukovanog oksidativnog stresa kod gojaznih pacova povećanjem
ekspresije i aktivnosti enzima antioksidativne zaštite.
U zaključku, rezultati ove doktorske disertacije pokazuju da u jetri normalno
hranjenih pacova, aktivacija Akt i ERK1/2 je uključena u regulaciju ekspresije
antioksidativnih enzima i iNOS posredovane grelinom. | In recent years, there have been prominent findings suggesting that ghrelin has
antioxidant and anti-inflammatory effects. In this study we investigated the effects of ghrelin
on protein expression of liver antioxidant enzymes superoxide dismutases (SODs), catalase
(CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) as well as protein
expression of their upstream regulator, nuclear factor kappa B (NFκB). We have also studied
the effects of ghrelin on liver inducible nitric oxide synthase (iNOS) protein expression.
Furthermore, we aimed to investigate whether extracellular regulated protein kinase (ERK
1/2) and protein kinase B (Akt) are involved in ghrelin regulated liver antioxidant enzymes
and iNOS protein expression. Male Wistar rats (200-250g) were treated with ghrelin
(0,3nmol/5µl) injected into the lateral cerebral ventricle every 24 h for 5 day, 2 h after the
last treatment the animals were sacrificed and liver excised. For protein expression of
antioxidant enzymes, iNOS, phosphorylation of Akt at Ser473, ERK1/2 and nuclear factor B
(NFκB) subunits 50 and 65, Western blot method was used. Results show significantly higher
protein expression of Cu/Zn SOD (p<0,05), Mn-SOD (p<0,05), CAT (p<0,01), GPx,
(p<0,001), and GR (p<0,01) in the liver isolated from ghrelin treated animals compared with
control animals. On the contrary, ghrelin significantly (p<0,001) reduced protein expression
of iNOS. In addition, phosphorylation of NFκB subunits p65 and p50 were significantly
(p<0,05) decreased by ghrelin when compared with controls. Phosphorylation of ERK1/2
and phosphorylation of Akt at Ser473 were significantly higher in ghrelin treated than in
control animals (p<0,05 for ERK1/2; p<0,01 for Akt). In conclusion, results from this study
show that activation of Akt and ERK1/2 are involved in ghrelin-mediated regulation of
protein expression of antioxidant enzymes and iNOS in the rat liver.
Title of doctoral dissertation: “The effect of ghrelin on the regulation of antioxidant
enzymes and inducible nitric oxide synthase in rat liver” | true |
Ispitivanje uloge komponenti ESL1 antigena Trichinella spiralis u oblikovanju imunskog odgovora Dark agouti pacova | The role of Trichinella spiralis ESL1 antigens in shaping of immune response in Dark agouti rats | Tokom hronične faze infekcije helmint, Trichinella spiralis, komunicira sa
imunskim sistemom domaćina posredstvom ekskretorno-sekretornih produkata (ESL1)
mišićnih larvi i kreira sredinu u kojoj dominiraju Th2 i regulatorni tip imunskog
odgovora. Međutim, mehanizmi koji leže u osnovi ovakve polarizacije imunskog
odgovora još uvek nisu poznati. Ova studija je posvećena istraživanju molekula ESL1
antigena uključenih u interakciju sa receptorima za molekulske obrasce i signalnih
puteva koji tom interakcijom bivaju pokrenuti u dendritskim ćelijama, a koji dovode do
sledstvene
polarizacije
T
ćelijskog
odgovora.
ESL1
antigeni,
primenjeni
intraperitonealno, statistički značajno povećavaju produkciju IL-4 i IL-10, a ne utiču na
nivo produkcije TGF-β i IFN-γ in vivo. Promena ugljenohidratne strukture ESL1
antigena (perjodatom tretirani ESL1, pESL1) snažno smanjuje produkciju IL-4 i IL-10,
što ukazuje na važnost glikana za pokretanje Th2 i anti-inflamatornog imunskog
odgovora. In vitro eksperimenti sa ESL1 i njegovim komponentama: 7C2C5Ag (antigen
koji sadrži 45, 49 i 53-kDa glikoproteine) i rTsp53 (rekombinantni p53) su pokazali da
svi ispitivani antigeni dovode do nepotpunog sazrevanja dendritskih ćelija, koje je
praćeno smanjenom produkcijom IL-12p70 i povećanom produkcijom IL-10 i
kapacitetom da promovišu Th2 i regulatorni imunski odgovor. Modifikacija glikana
ESL1 nije uticala na fenotip dendritiskih ćelija, ali je dovela do smanjenja produkcije
citokina IL-12p70 i IL-10 u tretiranim dendritskim ćelijama, što je rezultiralo sniženom
produkcijom IL-10 i TGF-β od strane T ćelija. Prisustvo intaktnih glikana je bitno za
indukciju Th2 i regulatornog odgovora in vitro. Ispitivanjem interakcije receptora
sličnih Toll proteinu (engl. Toll-like receptors, TLR) i ESL1 antigena, pokazano je da
ESL1 antigeni angažuju TLR2 i TLR4, dok se 7C2C5Ag vezuje samo za TLR2 i da su
te interakcije zavisne od prisustva intaktnih ugljenohidratnih struktura. ESL1, pESL1,
rTsp53 i 7C2C5Ag dovode do aktivacije MAP kinaza ERK1/2 i p38 u signalnom putu
koji biva pokrenut interakcijom dendritiskim ćelijama sa ovim T. spiralis antigenima,
dok pESL1 dovodi do slabe aktivacije obe ispitivane MAP kinaze. Ova studija je po
prvi put pokazala da komponente ESL1 antigena (7C2C5Ag and rTsp53) imaju
imunomodulatorne karakteristike i da su glikani ESL1 antigena važni za polarizaciju
imunskog odgovora. | The helminth Trichinella spiralis communicates with the host immune system
through excretory-secretory products (ESL1). ESL1 antigens drive immune response
towards Th2 and regulatory type. However, there is no information about mechanisms
by which ESL1 influences such polarization. Here, we investigated the impact of ESL1
and its components on immune response, Pattern Recognition Receptors involved in the
interaction with these antigens and subsequently provoked signaling events in dendritic
cells . ESL1 antigens significantly increased the production of IL-4 and IL-10, while
they did not affect the levels of TGF-β and IFN-γ in vivo. Changes in the structure of
ESL1 glycans greatly reduced the production of IL-4 indicating their importance for
Th2 immune response. In vitro experiments with ESL1 and ESL1 components:
7C2C5Ag (containing 45, 49, and 53-kDa glycoproteins) and rTsp53 (recombinant p53)
resulted in the same semimature dendritic cell phenotype accompanied by reduced
production of IL-12p70 and elevated production of IL-10 and the capacity to promote
Th2 and regulatory responses. Modification of ESL1 glycans (periodate treated ESL1,
pESL1) did not change dendritic cell phenotype but had an impact on cytokine
production of dendritic cells (reduced IL-12p70 and IL-10). Dendritic cells treated with
pESL1 provoked low production of IL-10 and TGF-β in T cells, indicating the
importance of intact glycans for Th2 and regulatory response induction. Considering
investigation of Toll-like receptors (TLR) involved in the interaction with ESL1
antigens, we have found that TLR2 is engaged by ESL1 and 7C2C5Ag, while TLR4
interacts only with ESL1. These interactions are impaired with changes in carbohydrate
structure of ESL1. ESL1, 7C2C5Ag and rTsp53 transiently activated ERK1/2 MAP
kinase, weakly activated p38 kinase and these activations were glycan dependent. This
study indicates for the first time that some components of ESL1 (7C2C5Ag and rTsp53)
possess immunomodulatory properties and that glycans on ESL1 are important for
immune response polarization. | true |
Nanoplazmonski metamaterijali za novu generaciju hemijskih, biohemijskih i bioloških senzora | Nanoplasmonic metamaterials for a new generation of chemical, biochemical and biological sensors | Potreba za osetljivim senzorima hemijskih, biohemijskih i bioloških agensa u današnjem svetu sve je veća, od industrije, preko biomedicine, do očuvanja okoline i protivterorističke odbrane. MeĎu najosetljivijim i najsvestranijim napravama ove vrste su elektromagnetni senzori, naročito oni koji svoju funkciju zasnivaju napovršinskimplazmonima-polaritonima (surface plasmons polaritons, SPP) na meĎupovršini metal-dielektrik. Sa dolaskom nanotehnologija otvorila se mogućnost da se proizvedu metal-dielektrični nanokompoziti, čime je omogućen novi stepen slobode u dobijanju SPP senzora i stvorena nova grana elektromagnetne optike koja je nazvana plazmonika. Posebno bitan proboj predstavljaju elektromagnetni metamaterijali koji se mogu definisati kao veštačke strukture sa elektromagnetnim, odnosno optičkim osobinama, kakve se ne pojavljuju uobičajeno u prirodi. Moţda najpoznatiji primer sutzv. „levoruki“ metamaterijali, strukture čiji efektivni indeks prelamanja u odreĎenom opsegu talasnih duţina dostiţe negativne vrednosti. Ova disertacija okrenuta je razmatranju upotrebljivosti plazmonskih metamaterijala za visoko osetljive senzore hemijskih, biohemijskih i bioloških agensa. Osnovna ideja je da se kontrolisanim strukturiranjem metal-dielektričnih nanokompozita obezbedi projektovanje novih elektromagnetnih modova. Oni će se zatim iskoristiti za prevazilaţenje principskih ograničenja konvencionalnih plazmonskih senzora i povećanje njihove osetljivosti i selektivnosti. Koncept metamaterijala s jedne strane daje mogućnost inţenjeringa frekventne disperzije i time formiranja optičkih modova neostvarivih u prirodnim materijalima, a s druge dozvoljava modifikovanje prostorne disperzije, odnosno kontrolisano menjanje optičkog prostora (tzv. transformaciona optika). Jedna od osnovnih posledica ovoga jeste mogućnost lokalizovanja optičkog polja na duboko podtalasne domene, što dovodi do drastičnogpovećanja osetljivosti hemijskih, biohemijskih i bioloških senzora. Druga posledica je mogućnost formiranja plazmonskih nanokompozita koji uvoĎenjem novih modova omogućuju selektivno očitavanje prisustva ciljanih agensa. Najzad, u takvim v strukturama konstrukcijski se obezbeĎuje efikasna spregasa snopom za očitavanje, što je jedan od velikih problema kod konvencionalnih SPP senzora... | The need for sensitive sensors of chemical, biochemical and biological analytes in the contemporary world is ever increasing, from industry, to biomedicine, to environmental protection and homeland defense. Among the most sensitive and most versatile devices of this kind are electromagnetic sensors, especially those with their function based on surface plasmons polaritons (SPP) at a metal-dielectric interface. The advent of nanotechnologies ensured a possibility to fabricate metal-dielectric nanocomposites, thus ensuring a novel degree of freedom in the fabrication of SPP and creating a new branch of electromagnetic optics, the plasmonics. An especially important breakthrough are electromagnetic metamaterials that can be defined as artificial structures with electromagnetic or optic properties which do not occur usually in nature. Maybe the best known example are the so-called "left-handed" metamaterials, the structures whose effective refractive index in a given wavelength range reaches negative values. This dissertation is dedicated to a consideration of the applicability of plasmonic metamaterials for highlysensitive sensors of chemical, biochemical and biological analytes. The basic idea is to ensure the design of new electromagnetic modes through controlled structuring of metal-dielectric nanocomposites. These modes are further used to overcome the fundamental limitations of conventional plasmonic sensors and to increase their sensitivity and selectivity. The concept of metamaterial from one side offers an opportunity to engineer the frequency dispersion and thus to design optical modes unattainable in natural materials, while from the other side it allows the modification of the spatial dispersion, i.e. controlled changing of the optical space (the so-called transformation optics). One of the main consequences is the possibility to concentrate the optical field into deep subwavelength domains, which leads to a drastic increase of the sensitivity of chemical, biochemical and biological sensors. Another consequence is the possibility to form plasmonic nanocomposites which by introducing new modes ensure selective readout of vii the targeted analytes. Finally, the geometry of such structures ensures efficient coupling with a readout beam, which is a large problem with the conventional SPP sensors... | false |
Genetički markeri oksidativnog stresa kod bolesnika sa manifestnim dijabetesom tipa 2 i aterosklerozom | Genetic markers of oxidative stress in patients with manifested diabetes type 2 and atherosclerosis | Ateroskleroza je kompleksno i multifaktorsko oboljenje koje obuhvata veliki
broj tipova ćelija kao i brojne fiziološke procese. Veruje se da oksidativni stres igra
važnu ulogu u inicijaciji i progresiji ateroskleroze. Kardiovaskularne bolesti (CVD)
predstavljaju vodeći uzrok smrtnosti u Srbiji i pronalaženje markera oksidativnog stresa,
uključujući i genske varijante, doprinelo bi smanjenju broja obolelih.
Studija asocijacije, koja je obuhvatala 140 obolelih od diabetes mellitus tip 2 sa
kardiovaskularnim komplikacijama (DM+A), 60 obolelih od neke od kliničkih
manifestacija ateroskleroze (A) i 100 zdravih kontrola (K) je sprovedena da bi se
utvrdila značajnost polimorfizama gena koji su povezani sa oksidativnim stresom,
metabolizmom lipida i detoksifikacijom kao markera oksidativnog stresa i faktora rizika
za nastanak oboljenja. Za studiju su odabrani polimorfizmi gena čiji produkti imaju
antioksidativnu ulogu u plazmi (pon1 Q192R, L55M i C(-107)T), antioksidativnu ulogu
i ulogu u metabolizmu lipida u plazmi i ćeliji (apoE 112/158 polimorfizam) i
antioksidativnu i detoksifikujuću ulogu u ćeliji (GSTM1*0, GSTT1*0 i GSTP1
Ile105Val). Genomska DNK je izolovana ili iz ćelija periferne krvi ili iz epitelnih ćelija
bukalne sluzokože. Genotipizacija je rañena restrikcionom digestijom PCR produkata i
analizom
dužine
restrikcionih
fragmenata
(PCR-RFLP)
na
agaroznom
ili
poliakrilamidnom gelu i real-time PCR metodom. Hi-kvadrat i Fišerov egzaktni test su
upotrebljeni za utvrñivanje razlika u distribuciji učestalosti ispitivanih genotipova i
alela, a logističkom regresionom analizom utvrñivan je rizik za oboljevanje.
Hipertenzija, hiperlipidemija i pozitivna porodična istorija za KVB su faktori
rizika za kardiovaskularne komplikacije u čijoj osnovi je ateroskleroza kod obe grupe
pacijenata, a pozitivna porodična istorija za KVB je faktor rizika za aterosklerozu kod
obolelih od T2DM. Učestalosti alela i genotipova Q192 polimorfizma pon1 gena
pokazale su značajnu razliku izmeñu pacijenata sa T2DM i kontrola (p<0.0001) i
izmeñu pacijenata sa T2DM i grupe bez T2DM (sa aterosklerozom) (p<0.0001).
Rezultati logističke regresione analize su pokazali značajno povećanje rizika za razvoj
kliničkih manifestacija ateroskleroze kod dijabetičara koji nose R alel i genotipove QR i
RR u poreñenju sa kontrolama (za R alel:OR=2,22, p<0,0001; za QR genotip: OR=3,36,
p<0,0001; za RR genotip: OR=4,29, p=0,0012) i u poreñenju sa grupom A (za R alel:
OR=2,28, p<0,001; za QR genotip: OR=3,7, p<0,0001; za RR genotip: OR=4,27,
p=0,006). Uočena je i značajna razlika u učestalostima GSTM1 nultog genotipa i GSTT1
nultog genotipa izmeñu obolelih bez T2DM i kontrolne grupe, a logistička regresiona
analiza je pokazala 2 puta veći rizik za oboljenje kod osoba sa GSTM1 nultim
genotipom (OR= 2.0, p<0.03) i više od 2 puta povećan rizik kod pacijenata sa GSTT1
nultim genotipom (OR=2.26, p<0.03). Učestalost kombinovane delecije GSTM1 i
GSTT1 se značajno razlikovala meñu grupama. Dijabetičari, nosioci dvostrukog nultog
genotipa su imali približno 4,5 puta uvećan rizik za razvoj ateroskleroze (OR=4,4,
p=0,05) a rizik od ateroskleroze bio je čak 15 puta veći kod nosilaca nultih genotipova
bez T2DM (OR=15,05, p<0,0001) u poreñenju sa kontrolnom grupom. Takoñe, uočili
smo i značajno povećanje rizika za aterosklerozu kod nosilaca dvostruke delecije kod
pacijenata bez T2DM u poreñenju sa dijabetičarima (za DM+A grupu: OR=0,29,
p=0,001). Nije nañena statistički značajna razlika u učestalosti alela i genotipova
poreñenjem grupa za L55M i C(-107)T polimorfizme pon1 gena i 112/158
polimorfizam apoE gena. Nije uočena povezanost sa rizikom od bolesti ni za jedan od
alela i genotipova ova tri polimorfizma.
Možemo zaključiti da su polimorfizam Q192R pon1 gena kao i kombinovana
delecija GSTM1 i GSTT1 gena genetički markeri oksidativnog stresa kod obolelih od
diabetes mellitus tip 2 sa kliničkim manifestacijama ateroskleroze, a da su GSTM1 nulti
genotip i GSTT1 nulti genotip, pojedinačno ili udruženo, faktori podložnosti za razvoj
ateroskleroze kod pacijenata bez T2DM. | Atherosclerosis is a complex and multifactorial disorder that involves many cell
types and organs as well as many physiological processes. It is believed that the
oxidative stress plays a critical role in the initiation and progression of atherosclerosis.
Cardiovascular diseases (CVD) are the leading cause of death in Serbia and discovering
oxidative stress markers, including gene variants, would contribute to reducing the
number of patients.
A association study, comprised 140 patients with diabetes mellitus type 2 and
cardiovascular complications (DM+A), 60 patients with clinical manifestations of
atherosclerosis (A) and 100 healthy individuals (K), has been undertaken in order to
estimate the relevance of polymorphisms in genes related to oxidative stress, lipid
metabolism, and detoxification as oxidative stress markers and disease risk factors.
Polymorphisms in genes whose products exert an antioxidative role in the plasma (pon1
Q192R, L55M i C(-107)T), in both plasma and cells (apoE 112/158 polymorphisms)
and antioxidative and detoxication role in cells (GSTM1*0, GSTT1*0 i GSTP1
Ile105Val) have been selected for the study. Genomic DNA was isolated from
peripheral blood cells or from buccal epithelial cells. The genotyping was performed
using restriction digestion of PCR products and by analysis of restriction fragment
length (PCR-RFLP) on agarose or polyacrilamide gel electrophoresis and the real-time
PCR method. Chi-square and Fisher exact test were used for determination of
differences in the analyzed genotype and allele distribution frequencies, and logistic
regression analysis was used for disease risk assessment.
Hypertension, hyperlipidemia and positive family history of CVD are risk
factors for atherosclerosis-based cardiovascular complications in both patient groups
and positive family history of CVD is a risk factor for atherosclerosis in T2DM patients.
Allele and genotype frequencies for Q192R polymorphism of pon1 gene showed
significant difference between patients with T2DM and controls (p<0.0001) and
between patients with T2DM and the group without T2DM (with atherosclerosis)
(p<0.0001). The results of logistic regression analysis showed a significant increase of
risk for atherosclerosis development in diabetics that carry R allele and genotypes QR
and RR compared with controls (for R allele :OR=2,22, p<0,0001; for QR genotype:
OR=3,36, p<0,0001; for RR genotype: OR=4,29, p=0,0012) and compared with group
A (for R allele : OR=2,28, p<0,001; for QR genotype: OR=3,7, p<0,0001; for RR
genotype: OR=4,27, p=0,006). A significant difference in GSTM1 null genotype and
GSTT1 null genotype frequencies between patients with atherosclerosis without T2DM
and control group were observed and logistic regression analysis showed a 2 fold
increase in the risk for disease in patients with the GSTM1 null genotype (OR= 2.0,
p<0.03) and more than 2 fold increase of the risk in patients with GSTT1 null genotype
(OR=2.26, p<0.03). GSTM1 and GSTT1 combined deletion frequencies were
significantly different between groups. In the group of diabetics, carriers of double null
genotypes had an approximately 4.5 fold increase of the risk for atherosclerosis in
patients with T2DM (OR=4.4, p=0.05) and 15 fold increase in risk in patients without
T2DM (OR=15.05, p<0.0001) compared with control group. Also, we observed a
significant increase of the risk for atherosclerosis in double null carriers in patients
without T2DM compared with diabetics (for DM+A group: OR=0.29, p=0.001). No
statistically significant difference was found after comparing allele and genotype
frequencies between groups for the L55M and C(-107)T polymorphisms in pon1 gene
and 112/158 polymorphism in apoE gene. No association with disease risk could be
observed for any of the alleles or genotypes of these three polymorphisms.
In conclusion, Q192R polymorphism of pon1 gene and combined deletion of GSTM1
and GSTT1 genes are genetic markers of oxidative stress in patients with diabetes
mellitus type 2 with clinical manifestations of atherosclerosis and that GSTM1 null and
GSTT1 null genotypes, alone or in combination are susceptibility factors for the
development of atherosclerosis in patients without T2DM. | true |
Morfofunkcionalne osobine reproduktivnog sistema nezrelih i acikličnih ženki pacova nakon tretmana fitoestrogenima iz soje | Morphofunctional features of reproductive system of immature and middle-aged female rats after treatments with soy phytoestrogens | Fitoestrogeni iz soje, pre svega genistein i daidzein, su veoma zastupljeni u životu
savremenog čoveka, kako kroz ishranu, tako i kroz pomoćna lekovita sredstva. Zbog svog
selektivno modulatorskog, tj. tkivno specifičnog estrogenskog delovanja, fitoestrogeni
poslednjih godina postaju atraktivno polje intenzivnih studija. Period gašenja reproduktivne
funkicije kod žena, tj. menopauza, vrlo često je praćen tegobama i poremećajima koje su
posledica smanjenja koncentracije endogenog estradiola, odnosno funkcije ovarijuma.
Stoga je jedna od mogućih terapijskih aplikacija genisteina i daidzeina upravo sprečavanje i
ublažavanje ovih tegoba, a kao alternativa supstitucionoj hormonskoj terapiji. Međutim,
njihovo delovanje na organe ženskog reproduktivnog sistema, naročito u periodu
menopauze, još uvek nije u potpunosti istraženo.
Cilj ove studije obuhvata rasvetljavanje dejstava fitoestrogena na reproduktivni
sistem nezrelih i acikličnih ženki pacova. Od posebnog interesa je jasno definisanje
potencijala fitoestrogena u poboljšanju funkcije reproduktivnog sistema u animalnom
modelu menopauze, kao i poređenje efekata njihove aplikacije sa onima koje ima aplikacija
terapijske doze estradiol dipropionata, koji se koristi kao lek izbora u prevenciji i terapiji
menopauzalnih simptoma.
Za ispitivanje estrogenske aktivnosti fitoestrogena (genisteina i daidzena) korišćen je
uterotrofni esej po protokolu Organizacije za ekonomsku saradnju i razvoj (OECD).
Ženkama pacova starosti 19 dana aplikovani su fitoestrogeni (genistein i daidzein), tokom
tri dana u tri doze (visokoj, srednjoj i niskoj). U okviru protokola predviđene su i kontrolne
grupe - pozitivne kontrolne grupe, koje su primale 17α-etinil estradiol (u dve doze),
negativne kontrolne grupe koje su primale rastvarače, kao i intaktna kontrolna grupa.
Nakon uterotrofnog eseja, utvrđeno je koja doza fitoestrogena (genisteina i
daidzeina) ispoljava minimalan estrogenski efekat. Ta doza je, tokom četiri nedelje,
aplikovana acikličnim ženkama pacova starosti 12 meseci, koje predstavljaju
eksperimentalni model menopauze. Na ovaj način utvrđeno je da li ispitivani fitoestrogeni
(genistein i daidzein) imaju efekat na strukturu i funkciju organa reproduktivnog sistema,
hipofize, ovarijuma, uterusa i vagine. Dodatno su efekti fitoestrogena poređeni sa efektima
koje u organima reproduktivnog sistema izaziva terapijska doza estradiol dipropionata.
Promene u strukturi i funkciji organa reproduktivnog sistema analizirane su
histološkim,
imunofluorescentnim,
savremenim
stereološkim,
morfometrijskim
i
molekularno biološkim metodama. Imunofluorescentno obeležavanje specifičnih antigena,
omogućilo je vizuelizaciju gonadotropnih i laktotropnih ćelija hipofize, i izračunavanje
relativnog intenziteta fluorescentnog signala. Istim metodama, detektovano je i prisustvo
laktoferina, kao i steroidnih receptora u uterusu nezrelih i acikličnih ženki pacova.
Metodama savremene stereologije, uz pomoć newCast stereološkog softverskog sistema,
određeni su volumeni organa, volumenske gustine osnovnih gradivnih faza organa, kao i
broj i volumen pojedinačnih granuloza luteinskih ćelija u žutim telima. Izračunavanje visine
endometrijalnog epitela, broja endometrijalnih žlezda, broja ovarijalnih folikula u različitim
stadijumima folikulogeneze i atrezije kao i broja, kako pojedinačnih klasa, tako i ukupnog
broja žutih tela, omogućila je primena morfometrijskih metoda. Molekularno biološka
metoda, reakcija lančanog umnožavanja u realnom vremenu “Real-time” PCR, korišćena je
za određivanje relativne ekspresije gena za estrogenski receptor alfa (ERα), estrogenski
receptor beta (ERβ) i progesteronski receptor (PR) u uterusu acikličnih ženki pacova.
Dobijeni rezultati su statistički obrađeni.
Uterotrofnim esejem pokazano je da genistein u dozama od 35 i 50 mg/kg telesne
mase na dan ispoljava estrogensko delovanje, koje se ogleda u gravimetričkom porastu
uterusa, te možemo reći da deluje kao agonista estradiola, dok daidzein ne izaziva ove
efekte. Evaluacijom visoke, srednje i niske doze ustanovljeno je da genistein aplikovan u
visokoj i srednjoj dozi, ima ulogu estrogenskog agoniste u pogledu svih ispitivanih
karakteristika u uterusu, hipofizi i vagini nezrelih ženki pacova. Međutim, iste doze su u
ovarijumima uslovile antagonističko delovanje. Zahvaljući uključivanju dodatnih analiza,
osetljivost uterotrofne probe je povećana i ustanovljeno je da i daidzein poseduje
estrogensko delovanje u svim ispitivanim dozama, koje se ogleda u povećanju broja i
volumenske gustine endometrijalnih žlezda i stimulaciji gonadotropnih i laktotropnih ćelija
hipofize. Minimalne efektivne doze, tj. doze koje izazivaju estrogenski efekat u organima
ženskog reproduktivnog sistema, su doze od 35 mg/kg telesne mase na dan genisteina i
daidzeina.
U animalnom modelu menopauze, tretman estradiolom je izazvao inhibiciju
gonadotropnih ćelija, koja se ogleda, ne samo u smanjenju intenziteta fluorescentnog
signala, već i u smanjenju veličine ćelija. Istovremeno, estradiol je delovao stimulatorno na
laktotropne ćelije, povećanjem njihove zastupljenosti, kao i povećanjem relativnog
intenziteta fluorescentnog signala immunoobeleženog prolaktina. Nakon tretmana
genisteinom i daidzeinom, nasuprot estradioloskom tretmanu, izostaju promene u
relativnom intenzitetu fluorescentnog signala unutar gonadotropnih i laktotropnih ćelija.
Međutim, fitoestrogeni su izazvali promene u morfologiji ćelija. Gonadotropne ćelije su
veće, dok su laktotropne ćelije nešto manje u poređenju sa kontrolnom grupom. Međutim,
fitoestrogeni su u navedenim ćelijskim populacijema uslovili promenu oblika, dok se u
citoplazmi uočavaju neobojeni delovi koji joj daju izgled vakuoliziranosti.
Hroničan tretman terapijskim dozama estradiola, u ovarijumima acikličnih ženki,
generalno je pokazao negativan uticaj. U grupi malih folikula izazvao je značajno povećanje
atretičnih, primordijalnih, primarnih i preantralnih folikula. Slična slika je uočena i u grupi
antralnih folikula. Tretman estradiolom, uslovio je povećanje broja i volumena žutih tela,
koje za posledicu ima povećanje volumena ovarijuma. Međutim, hronična aplikacija kako
genisteina, tako i daidzeina, uslovila je višestruko povećanje broja folikula inicijalne faze
folikulogeneze (primordijalnih i primarnih). Sa druge strane, genistein i daidzein nisu uticali
na promenu apsolutnog broja atretičnih primordijalnih folikula, dok su značajno smanjili
atreziju primarnih folikula. Tretmani fitoestrogenima nisu uticali na promenu sume
preantralnih i antralnih folikula, dok su izazvali smanjenje apsolutnog broja atretičnih
antralnih folikula. Tretman genisteinom, nije uticao ni na promenu pojedinačnih klasa žutih
tela, kao ni na promenu njihovog ukupnog broja. Za razliku od genisteina, daidzein je
povećao brojnost zrelih žutih tela, što je uzrokovalo povećanje njihovog ukupnog broja,
usled čega je došlo i do povećanja apsolutnog volumena ovarijuma. Međutim, usled
očuvanja zdravih folikula, oba tretmana su uzrokovala povećanje volumena folikularnog
parenhima.
Tretman estradiolom izazvao je povećanje apsolutne i relativne mase uterusa, koje
je posledica povećanja volumensih gustina endometrijuma, endometrijalnog epitela i
endometrijalnih žlezda. Ekspresija ERα je smanjena, PR povećana, dok promene u eksperiji
ERβ izostaju. Nakon tretmana genisteinom, uočena je povećana masa uterusa, kao i
povećana volumeska gustina endometrijuma. Genistein je smanjio ekspresiju ERα, i
povećao ekspresiju PR i ERβ. Za razliku od genisteina, daidzein nije izmenio masu uterusa,
njegove morfometrijske i stereološke karakteristike, kao ni ekspresiju ERα i PR, dok je
uslovio povećanje ERβ.
Aplikacija estradiola izazvala je hipertrofiju vaginalnog epitela, praćenu
deskvamacijom ćelija. Nakon tretmana fitoestrogenima, promena opšte histološke slike
vaginalnog epitela nije zapažena u poređenju sa kontrolnim presecima. Međutim detaljnom
analizom uočava se odsustvo piknotičnih jedara, dok se mogu zapaziti ćelije u mitozi.
Genistein i daidzein, u poređenju sa estradiolom, ispoljavaju niz pozitivnih efekata
na reproduktivni sistem acikličnih ženki, a posebno na nivou ovarijuma. Imajući u vidu
da menopauza, između ostalog, nastaje usled smanjene funkcije ovarijuma, rezultati ove
studije imaju veliki značaj i primenu, s obzirom na to da očuvanje funkcije ovarijuma
značajno utiče na kvalitet života žena. Stoga se može reći da genistein i daidzein mogu
biti uspešna alternativa supstitucionoj hormonskoj terapiji. | Soy phytoestrogens, primarily genistein and daidzein, are widely used through diet
and as dietary supplements. Since they may act as “natural” selective modulators of
estrogen receptor, scientific interest for phytoestrogens is constantly increasing. Cessation
of a women’s reproductive function, i.e. menopause, is often accompanied with disorders
that are consequence of reduced ovarian function and low estrogen concentration.
Therefore, one of the possible therapeutic application of genistein and daidzein is
prevention and alleviation of menopausal symptoms, as an alternative to hormone
replacement therapy.
The aim of this study is to examine the effects of phytoestrogens on the
reproductive system of immature and middle-aged female rats. Of particular interest is to
define the potential of phytoestrogens for improvement of reproductive system function in
an animal model of menopause, and to compare these effects with the effects of
therapeutic doses of estradiol dipropionate, commonly used in prevention and treatment of
menopausal symptoms.
Uterotrophic assay was used in order to test estrogenic activity of genistein and
daidzein, according to the protocol of the Organization for Economic Cooperation and
Development (OECD). Immature female rats received phytoestrogens (genistein and
daidzein), in three doses (high, medium and low) for three consecutive days, from 19th till
21st day of life. According to the protocol, control groups were formed - the positive
control groups that received 17α - ethynyl estradiol (in two doses), negative control groups
that received solvents, and the intact control group.
After uterotrophyc assay, minimal dose of genistein and daidzein that exerts
estrogenic effect was determined. This dose was administered to 12 months old, middle-
aged female rats, for four weeks. After this period it was analyzed whether genistein and
daidzein exerted beneficial effects on the structure and function of the pituitary, ovary,
uterus and vagina. These effects were then compared with the effects of the therapeutic
dose of estradiol dipropionate.
Changes in the structure and function of the organs of reproductive system of
immature and middle-aged female rats were analyzed by histological, immunofluorescence,
design-based
stereological,
morphometric
and
molecular
biological
methods.
Immunofluorescent labeling of specific antigens allowed the visualization of the pituitary
lactotropic and gonadotropic cells, and the calculation of the relative intensity of
fluorescent signal, i.e. concentration of antigen within the cell. In the same manner, the
presence of lactoferrin and steroid receptors in the uterus of immature and middle-aged
female rats was analysed. Unbiased, design-based stereological methods were used for
determination of volume of reproductive organs, volume density of their basic
compartments, as well as volume, number and volume density of granulosa lutein cells of
the corpora lutea. The endometrial epithelium height, endometrial gland number, number of
ovarian follicles at different stages of folliculogenesis and atresia, as well as the number of
individual classes and the total number of corpora lutea were performed using morphometric
methods. "Real-time" PCR was used to determine the relative gene expression of estrogen
receptor alpha (ERα), estrogen receptor beta (ERβ) and progesterone receptor (PR) in the
uterus of middle-aged female rats. All results were statistically analyzed.
Uterotrophic assay showed that genistein, at doses of 35 and 50 mg/kg b.w.,
increased gravimetric uterine growth and acted as an estrogen agonist, while daidzein, in
any dose used, did not cause these effects. It has been shown that genistein, in medium and
high dose, acted as an estrogen agonist in terms of all tested parameters in uterus, pituitary
gland, and vagina. However, the same doses in the ovaries caused antagonistic effects. By
including additional analyses, the sensitivity of uterotrophic assay was increased. This
resulted in an observation that daidzein also exerts estrogenic activity in all tested doses.
The estrogenic activity of daidzein was reflected in the increase of endometrial gland
number and volume density, and stimulation of pituitary gonadotropic and lactotropic cells.
The minimal effective dose, i.e. the dose that causes the estrogenic effect in the organs of
female reproductive system, is the dose of 35 mg/kg b.w. per day of genistein and daidzein.
Chronic estradiol treatment of middle-aged female rats caused an inhibition of
gonadotropic cells, which was reflected not only in the reduction of fluorescent signal
intensity, but also in reduction of the cell size. At the same time, estradiol stimulated
lactotrops, by increasing their density and the relative intensity of the immunolabeled
prolactin in cells.
In contrast to estradiol treatment, genistein and daidzein did not cause changes in
the relative intensity of the fluorescent signal within the gonadotropic or lactotropic cells.
However, changes in cell morphology were observed after treatment with both
phytoestrogens. Gonadotropic cells were larger in size, while lactotropic cells were smaller
comparing to the controls. All types of examined pituitary cells were changed in shape with
unevenly stained cytoplasm, that is, immunolabeled parts of cytoplasm were separated by
unstained regions, which gave cells the appearance of vacuolization.
Chronic treatment with therapeutic doses of estradiol, in the ovaries of acyclic
females, generally had a negative effect. In the group of small follicles, estradiol caused a
significant increase of atretic, primordial, primary and preantral follicles. Similar effect was
observed in the group of antral follicles. Estradiol treatment caused an increase of volume
and number of corpora lutea, which resulted in the ovary volume increase. In contrast to
estradiol, chronic application of genistein and daidzein caused a significant increase in the
number of follicles in initial stage of folliculogenesis (primordial and primary follicles),
without affecting total number of atretic primordial follicles, but significantly reducing the
number of primary atretic follicles. Treatments with phytoestrogens did not affect total
number of preantral and antral follicles, while they caused a decrease of atretic antral
follicle number. Genistein treatment did not change the number of individual classes of
corpora lutea, or their total number. Unlike genistein, daidzein increased the number of
mature corpora lutea, which resulted in an increase of their total number, and ovary volume.
However, due to the preservation of healthy follicles, both treatments caused an increase of
follicular parenchyma volume.
Estradiol treatment increased the absolute and the relative uterine weights. This is
result of increased volume density of endometrium, endometrial epithelium and
endometrial glands. Estradiol caused decrease of ERα mRNA and increase of PR mRNA
expression, while ERβ mRNA expression was not changed. Genistein caused an increase
of uterine weight and endometrial volume density, decrease of ERα mRNA expression, and
increase of PR and ERβ mRNA expression. In cotrast to genistein treatment, daidzein did
not change uterine weight, or morphometric and stereological characteristics of uterus.
Expression of ERα and PR mRNA was not affected, while expression of ERβ mRNA was
increased.
In vagina, application of estradiol caused hypertrophy of the epithelium, followed
by desquamation of epithelial cells. After treatment with phytoestrogens, changes in general
histological appearance of the vaginal epithelium were not observed. However, detailed
light microscopic analysis revealed less numerous pyknotic nuclei, and more numerous
mitotic cells.
Comparing to estradiol, genistein and daidzein, exhibited numerous beneficial
effects on the reproductive system of middle-aged females, especially in the ovary. Bearing
in mind that menopause, among other things, is caused by reduced ovarian function, the
results of this study are significant and applicable, given that the preservation of ovarian
function can significantly improve the quality of life of women. Therefore, it can be
concluded that genistein and daidzein could be a successful alternative to estrogen
replacement hormone therapy. | true |
Uloga inhibicije protein-kinaze aktivirane adenozin-monofosfatom u indukciji apoptoze i autofagije u tumorskim ćelijskim linijama | The role of adenosine monophosphate-activated protein kinase inhibition in apoptosis and autophagy induction in tumor cell lines | U ovoj doktorskoj disertaciji ispitivan je uticaj inhibicije intracelularnog
energetskog senzora protein kinaze aktivirane adenozin-monofosfatom (AMPK) na
indukciju apoptoze i autofagije u tumorskim ćelija. Farmakološki inhibitor AMPK
dorzomorfin indukovao je G2/M blokadu ćelijskog ciklusa, praćen apoptozom koju
karakteriše aktivacija kaspaza, eksternalizacija fosfatidilserina i fragmentacija DNK u U251
humanim i C6 pacovskim ćelijama glioma, dok na vijabilitet primarnih pacovskih astrocita
i ćelija mišjeg melanoma B16 nije imao uticaja. Mehanizam indukcije apoptoze
dorzomorfinom bio je posredovan stimulacijom produkcije reaktivnih vrsta kiseonika i
inhibicijom ekspresije antiapoptotskog Bcl-2 proteina. Dorzomorfin je inhibirao
fosforilaciju i enzimatsku aktivnost AMPK, što je za posledicu imalo smanjenje
fosforilacije njenog supstrata acetil-CoA karboksilaze. Aktivatori AMPK metformin i
AICAR delimično su neutralisali blokadu ćelijskog ciklusa, oksidativni stres i apoptozu
indukovanu dorzomorfinom. Mala interferirajuća RNK (siRNK) koja sprečava ekspresiju
humanog AMPK enzima je poput dorzomorfina zaustavila proliferaciju ćelija u G2/M fazi
ćelijskog ciklusa, ali nije izazvala oksidativni stres i apoptozu u U251 ćelijama. Dakle,
inhibicija AMPK je neophodna, ali ne i dovoljna za indukciju apotpoze dorzomorfinom u
ćelijama glioma.
U ovoj studiji takođe je pokazano da dorzomorfin indukuje autofagiju u ćelijama
kancera. Indukcija autofagije u U251 ćelijama detektovana je fluorescentnim bojenjem
unutarćelijskih kiselih vezikula akridin oranžom, indukcijom beklina-1, degradacijom p62
proteina i konverzijom LC3-I u formu asociranu sa autofagozomima LC3-II u odsustvu i
prisustvu proteolitičkih inhibitora. Prisustvo autofagozomima sličnih vezikula potvrđeno je
transmisionom elektronskom mikroskopijom. Inhibicija AMPK i Raptora indukovana
dorzomorfinom u U251 ćelijama bila je paradoksalno asocirana sa smanjenjem fosforilacije
AMPK/Raptorom inhibiranog glavnog represora autofagije mTOR i njegovog supstrata
p70S6K. Fosforilacija mTOR aktivatora Akt i PI3K-aktivirane Src kinaze bile su takođe
inhibirane u ćelijama tretiranim dorzomorfinom. Poništavanje ekspresije AMPK sa siRNA
nije redukovalo aktivnost Akt/mTOR/p70S6K signalnog puta, a AMPK aktivatori
metformin i AICAR nisu uspeli da blokiraju autofagiju indukovanu dorzomorfinom.
Inhibitori autofagije bafilomicin i hlorokin značajno su povećali citotoksičnost
dorzomorfina prema U251 ćelijama, što je pokazano povećanjem oslobađanja laktat
dehidrogenaze, fragmentacije DNK i aktivacije kaspaze-3. Sličan efekat dorzomorfin je
imao i prema ćelijama pacovskog glioma C6, mišjeg fibrosarkoma L929 i mišjeg
melanoma B16. Pošto je ranije pokazano da dorzomorfin suprimira AMPK-zavisnu
autofagiju u različitim ćelijskim tipovima, rezultati ove studije sugerišu da efekat
dorzomorfina na autofagiju zavisi od doze, konteksta i/ili vrste ćelija. Imajući u vidu da
dorzomorfin indukuje autofagiju u ćelijama kancera nezavisno od inhibicije AMPK,
inhibicijom Akt/mTOR signalnog puta, rezultati ove studije ukazuju na neophodnost opreza
pri interpretaciji rezultata eksperimenata u kojima se dorzomorfin koristi kao inhibitor
AMPK, ali takođe sugerišu da bi dorzomorfin, sam ili u kombinaciji sa inhibitorima
autofagije, mogao biti potencijalni kandidat za terapiju tumora. | In this doctoral dissertation the effect of intracellular energy sensor AMP-activated
protein kinase (AMPK) inhibition on induction of apoptosis and autophagy in tumor cells
was investigated. Pharmacological AMPK inhibitor dorsomorphin caused G2/M cell cycle
block, accompanied by apoptotic cell death characterized by caspase activation,
phosphatidylserine exposure and DNA fragmentation in U251 human and C6 rat glioma
cells, while it had no effect on viability of primary rat astrocytes and B16 mouse melanoma
cells. The mechanisms underlying the pro-apoptotic action of dorsomorphin involved
induction of oxidative stress and down-regulation of antiapoptotic molecule Bcl-2.
Dorsomorphin diminished AMPK phosphorylation and enzymatic activity, resulting in
reduced phosphorylation of its target acetyl CoA carboxylase. AMPK activators metformin
and AICAR partly prevented the cell cycle block, oxidative stress and apoptosis induced by
dorsomorphin. The small interfering RNA (siRNA) targeting of human AMPK mimicked
dorsomorphin-induced G2/M cell cycle arrest, but failed to induce oxidative stress and
apoptosis in U251 glioma cells. Therefore, AMPK inhibition is required, but not sufficient
for dorsomorphin-mediated apoptotic death of glioma cells.
In this study, it was also reported that dorsomorphin can induce autophagy in cancer
cells. The induction of autophagy in U251 human glioma cell line was demonstrated by
acridine orange staining of intracellular acidic vesicles, Beclin 1 induction, p62 decrease
and conversion of LC3-I to autophagosome-associated LC3-II in the absence and presence
of proteolysis inhibitors. The presence of autophagosome like vesicles was confirmed by
transmission electron microscopy. Dorsomorphin-mediated inhibition of AMPK and Raptor
in U251 cells was associated with paradoxical decrease in phosphorylation of
AMPK/Raptor-repressed mTOR, a major negative regulator of autophagy, and its
downstream target p70S6K. The phosphorylation of mTOR activator Akt and PI3K-
activating kinase Src was also impaired in dorsomorphin-treated cells. The siRNA-
mediated AMPK silencing did not reduce the activity of the Akt/mTOR/p70S6K pathway
and AMPK activators metformin and AICAR failed to block dorsomorphin-induced
autophagy. Autophagy inhibitors bafilomycin and chloroquine significantly increased the
cytotoxicity of dorsomorphin towards U251 cells, as confirmed by the increase in lactate
dehydrogenase release, DNA fragmentation and caspase-3 activation. Similar effects of
dorsomorphin were also observed in C6 rat glioma, L929 mouse fibrosarcoma and B16
mouse melanoma cell lines. Since dorsomorphin has previously been reported to suppress
AMPK dependent autophagy in different cell types, results in this study suggest that the
effects of dorsomorphin on autophagy might be dose-, cell type- and/or context-dependent.
By demonstrating the ability of dorsomorphin to induce autophagic response in cancer cells
via AMPK inhibition-independent downregulation of Akt/mTOR pathway, results warrant
caution when using dorsomorphin to inhibit AMPK-dependent cellular responses, but also
suggest that dorsomorphin, alone or in combination with autophagy inhibitors, could be
potential candidate for anticancer therapy. | true |
Procena preostalog veka strukturalnih elemenata sa inicijalnim oštećenjima pod dejstvom spektra termomehaničkih opterećenja | The assessment of residual life of structural elements with an initial damage under thermomechanical loading spectrum | Predmet istraživanja u doktorskoj disertaciji je procena preostalog
veka struktura sa inicijalnim oštećenjima pod dejstvom spektra termo-
mehaničkih opterećenja. Prvo su proučavane strukture sa inicijalnim
oštećenjima samo pod dejstvom termičkog opterećenja, i za zadate granične
uslove određivani su parametri mehanike loma. Za pouzdanu proceduru
određivanja parametara mehanike loma gde se prevashodno misli na faktor
inteziteta napona korišćene su numeričke metode i teoretske postavke
linearno elastične mehanike loma.
Strukture koje su proučavane se uglavnom odnose na delove vazduhoplova
koji su od vitalnog značaja za njihov ispravan i pouzdan rad. Analizirani su
delovi polja oplate sa geometrijskim diskontinuetima i inicijalnim
oštećenjima pod dejstvom termičkog opterećenja. Takođe su analizirani delovi
pogonskih grupa kod vazduhoplova. Posebno je proučavan deo lopatica kod
mlaznih motora kojima je na samom kraju nanet sloj termičke zaštite zbog
visokih radnih temperatura. Detaljna analiza strukture u graničnom sloju
između osnovnog sloja lopatice i termičke zaštite, kada dolazi do pojave
naprslina može imati značaja u samoj fazi projektovanja kao i u toku
eksploatacije pogonskih grupa kod vazduhoplova. Još jednom delu strukture
pogonskih grupa kod vazduhoplova posvećena je posebna pažnja u analizi sa
aspekta mehanike loma u okviru doktorske disertacije. Delovi veze diska rotora
i korena lopatice turbine predstavljaju najopterećenije delove strukture.
U daljim istraživanjima u okviru doktorske disertacije, detaljnjo je
analiziran strukturalni element sa površinskom naprslinom izložen
mehaničkim i termičkim opterećenjima. Za raličitu geometriju površinske
naprsline i različite nivoe mehaničog opterećenja i temperaturnih
gradijenata, izračunate su vrednosti faktora inteziteta napona. Određivanje
funkcionalne zavisnost faktora inteziteta napona u odnosu na položaj vrha
naprsline za različite geometrije oblika naprsline, predstavlja značajna
saznanja o ponašanju takvih struktura sa aspekta mehanike loma.
Takođe, kod vazdzhoplovnih konstrukcija susrećemo se sa delovima
struktura koji su spojeni zavarivanjem. Kao posledica procesa zavarivanja
javljaju se zaostali naponi koji mogu nepovoljno uticati na ponašanje strukture
pod dejstvom dinamičkog opterećenja i stoga je u okviru doktorske disertacije
posvećena posebna pažnja određivanju zaostalih napona primenom metode
konačnih elemenata. Precizno određena raspodela zaostalih napona kao i njihov
intezitet mogu značajno smanjiti preostali vek strukture sa aspekata zamora
materijala. | The subject of doctoral dissertation is research into the residual life of structures
with initial damages under the influence of the spectrum of thermo-mechanical loads.
First, the structures with initial damages under the influence of thermal load only were
studied, and for given boundary conditions the parameters of fracture mechanics were
determined. For a reliable procedure of determining fracture mechanics parameters,
which primarily refers to the stress intensity factor, numerical methods and theoretical
approaches of linear elastic fracture mechanics were used.
The structures that were studied are mainly related to aircraft parts that are vital
for their proper and safe operation. The parts of the plate with geometric discontinuities
and initial damages under the influence of thermal load were analyzed. The components
of power engine of aircraft were also analyzed. In particular, a part of the blades in jet
engines was researched, at the end of which the layer of thermal protection was applied
due to high operating temperatures. The detailed analysis of structures in the boundary
layer between the basic blade and thermal protection when cracks appear can be very
important in the design phase and during the operation of aircraft power engines. In this
doctoral dissertation special attention was devoted to one more part of the power engine
structure of aircraft, in the analysis in terms of fracture mechanics. The parts of the
connection between the turbine rotor and the turbine blade root are the most overload
parts of the structure
In the further research in this doctoral dissertation, structural element with a
surface crack subjected to mechanical and thermal loads was analyzed. For various
geometry of surface crack and different levels of mechanical loads and temperature
gradients, the values of stress intensity factor were calculated. Determination of the
functional dependence of the stress intensity factors in relation to the position of crack
tip for different types of crack geometry represents significant knowledge about the
behavior of such structures in terms of fracture mechanics.
Also, the aircraft constructions encounter with parts of structures that are
connected by welding. As the result of welding processes, the residual stresses that can
adversely affect the behavior of structure under dynamic loading occur, and therefore, in
this doctoral dissertation special attention is devoted to the determination of residual
stresses using finite element method. Precisely determined distribution of residual
stresses, as well as their intensity can significantly reduce the residual life of structures
from the aspects of fatigue life of material. | true |
Fitohemijska i molekularno-genetička karakterizacija populacija kičice (Centaurium erythraea Rafn) sa Balkanskog poluostrva | Phytochemical and molecular characterization of centaury (Centaurium erythraea Rafn) populations from the Balkan Peninsula | Kičica (Centaurium erythraea Rafn) je farmakološki atraktivna
biljna vrsta koja pokazuje čitav spektar bioloških aktivnosti, a čiji je
biodiverzitet
nedovoljno
istražen.
Osnovni
cilj
predstavljenog
istraživanja jeste analiza genetičkog i fitohemijskog diverziteta
kičice na teritoriji Balkanskog poluostrva. U tom smislu, formirana je
kolekcija semena kičice poreklom iz prirodnih populacija, koja
oslikava diverzitet vrste na ovom području. Istraživanja su uključila 43
populacije ove vrste, od kojih je 41 poreklom sa Balkanskog poluostrva. Za
procenu genetičkog diverziteta optimizovane su tehnike RAPD (Random
Amplified Polymorphic DNA) i TRAP (Target Region Amplification
Polymorphism) molekularnih marker sistema. DNK upotrebljena u ovim
analizama je izolovana iz biljaka gajenih u uslovima in vitro, kako bi se
obezbedila
autentičnost
DNK
kičice
i
eliminisala
mogućnost
kontaminanacije stranom DNK. Za potrebe dobijanja što većeg broja
informativnih jedinica, upotrebljeno je 16 desetomernih prajmera u
slučaju RAPD markera, pri čemu je dobijeno ukupno 408 različitih PCR
produkata, dok su za TRAP markere upotrebljene kombinacije tri
specifična i tri arbitrarna prajmera i dobijeno je 767 fragmenata DNK.
Specifični prajmeri korišćeni u TRAP analizama, konstruisani su na
osnovu sekvenci RNK kičice sa visokim stepenom homologije sa DNK
sekvencama za sledeće enzime: geraniol 10-hidroksilaza, citohrom P450
reduktaza i sekologanin sintaza, objavljenih u elektronskim bazama
podataka. Primenom molekularnih markera dobijen je visok procenat
polimorfizma, a informativnost odabranih prajmera ili kombinacija
prajmera je potvrđena pomoću više parametara. Ustanovljena je vrlo
niska unutarpopulaciona genetička varijabilnost kičice, što može da
se objasni velikim udelom samooplodnje i geitonogamije u nastanku
Rezime/Summary
semena kod prirodnih populacija, kao i snažnim genetičkim driftom. S
obzirom da se kičica javlja u malim i fragmentisanim populacijama,
između kojih verovatno ne postoji protok gena, očekivana je i visoka
genetička diferenciranost analiziranih populacija, što je potvrđeno i
metodama molekularnih markera. Osim toga, molekularnim markerima
vrlo uspešno su razdvojene diploidne od tetraploidnih populacija
kičice. Tetraploidne populacije se prilično jasno diferenciraju u
posebne grupe koje su povezane sa njihovim geografskim poreklom,
naročito kada su primenjeni RAPD markeri. Značajna korelacija
pokazana je između genetičkih sličnosti populacija kičice sa
Balkanskog poluostrva, dobijenih pomoću dva tipa molekularnih markera
i geografskih distanci lokaliteta sa kojih ove populacije potiču.
Određivanje
i
kvantifikacija
osnovnih
jedinjenja
sekundarnog
metabolizma kičice, sekoiridoidnih glikozida, dalo je značajan
doprinos proceni varijabilnosti analiziranih populacija kičice.
Sadržaj svercijamarina, dominantnog sekoiridoidnog glikozida u
nadzemnim delovima biljaka gajenih u uslovima staklare, pokazao se kao
najznačajniji faktor međupopulacione varijabilnosti kičice. Uz to,
pokazana je značajna korelacija između sadržaja sekoiridoidnih
glikozida i genetičke sličnosti populacija, korišćenjem oba tipa
molekularnih markera, s tim što je snažnija korelacija primećena u
slučaju TRAP markera. Predstavljena procena genetičkog i fitohemijskog
diverziteta
kičice
daju
osnovu
za
eventualnu
zaštitu
ukupnog
biodiverziteta ove vrste na Balkanskom poluostrvu, kao i za odabir
genotipova za zasnivanje i unapređenje njene plantažne proizvodnje. | Фитoхeмиjскa и мoлeкулaрнo-гeнeтичкa кaрaктeризaциja
пoпулaциja кичицe (Centaurium erythraea Rafn)
са Балканског полуострва
Резиме
Кичица (Centaurium erythraea Rafn) је фармаколошки атрактивна
биљна врста која показује читав спектар биолошких активности, а чији је
биодиверзитет
недовољно
истражен.
Основни
циљ
представљеног
истраживања јесте анализа генетичког и фитохемијског диверзитета
кичице нa тeритoриjи Бaлкaнскoг пoлуoстрвa. У том смислу, формирана је
колекција семена кичице пореклом из природних популација, која
осликава диверзитет врсте на овом подручју. Истраживања су укључила 43
популације ове врсте, од којих је 41 пореклом са Балканског полуострва. За
процену генетичког диверзитета оптимизоване су технике RAPD (Random
Amplified Polymorphic DNA) и TRAP (Target Region Amplification
Polymorphism) молекуларних маркер система. ДНК употребљена у овим
анализама је изолована из биљака гајених у условима in vitro, како би се
обезбедила
аутентичност
ДНК
кичице
и
елиминисала
могућност
контаминанације страном ДНК. За потребе добијања што већег броја
информативних јединица, употребљено је 16 десетомерних прајмера у
случају RAPD маркера, при чему је добијено укупно 408 различитих PCR
продуката, док су за TRAP маркере употребљене комбинације три
специфична и три арбитрарна прајмера и добијено је 767 фрагмената ДНК.
Специфични прајмери коришћени у TRAP анализама, конструисани су на
основу секвенци РНК кичице са високим степеном хомологије са ДНК
секвенцама за следеће ензиме: гераниол 10-хидроксилаза, цитохром P450
редуктаза и секологанин синтаза, објављених у електронским базама
података. Применом молекуларних маркера добијен је висок проценат
полиморфизма, а информативност одабраних прајмера или комбинација
прајмера је потврђена помоћу више параметара. Установљена је врло
ниска унутарпопулациона генетичка варијабилност кичице, што може да
се објасни великим уделом самооплодње и геитоногамије у настанку
Резиме/Summary
семена код природних популација, као и снажним генетичким дрифтом. С
обзиром да се кичица јавља у малим и фрагментисаним популацијама,
између којих вероватно не постоји проток гена, очекивана је и висока
генетичка диференцираност анализираних популација, што је потврђено и
методама молекуларних маркера. Осим тога, молекуларним маркерима
врло успешно су раздвојене диплоидне од тетраплоидних популација
кичице. Тетраплоидне популације се прилично јасно диференцирају у
посебне групе које су повезане са њиховим географским пореклом,
нарочито када су примењени RAPD маркери. Значајна корелација
показана је између генетичких сличности популација кичице са
Балканског полуострва, добијених помоћу два типа молекуларних маркера
и географских дистанци локалитета са којих ове популације потичу.
Одређивање
и
квантификација
основних
једињења
секундарног
метаболизма кичице, секоиридоидних гликозида, дало је значајан
допринос процени варијабилности анализираних популација кичице.
Садржај сверцијамарина, доминантног секоиридоидног гликозида у
надземним деловима биљака гајених у условима стакларе, показао се као
најзначајнији фактор међупопулационе варијабилности кичице. Уз то,
показана је значајна корелација између садржаја секоиридоидних
гликозида и генетичке сличности популација, коришћењем оба типа
молекуларних маркера, с тим што је снажнија корелација примећена у
случају TRAP маркера. Представљена процена генетичког и фитохемијског
диверзитета
кичице
дају
основу
за
евентуалну
заштиту
укупног
биодиверзитета ове врсте на Балканском полуострву, као и за одабир
генотипова за заснивање и унапређење њене плантажне производње.
Кључне речи: Centaurium erythraea, Балканско полуострво,
фрагментација станишта, популације, молекуларни маркери, RAPD, TRAP,
генетичка варијабилност, секоиридоидни гликозиди
Научна област: Биологија
Ужа научна област: Физиологија биљака
УДК број: 582.923.1-11(497)(043.3)
Резиме/Summary
Phytochemical and molecular characterization of centaury
(Centaurium erythraea Rafn) populations
from the Balkan Peninsula
Summary
Centaury
(Centaurium
erythraea
Rafn)
is
a
medicinal
and
pharmacologically attractive plant species which has a wide range of biological
activity. The main objective of presented study was to estimate genetic and
phytochemical diversity of centaury within the Balkan Peninsula. With that
purpose, seed collection of natural centaury populations was established. The
study included 43 populations of C. erythraea, among which 41 originated from
the Balkan Peninsula. Molecular marker techniques, such as RAPD (Random
Amplification Polymorphic DNA) and TRAP (Target Region Amplification
Polymorphism) were optimized to investigate the genetic diversity among these
populations. DNA used in these analyses originated from in vitro grown plants
to ensure the authenticity of centaury DNA by eliminating the possibility of its
contamination by extraneous DNA. To acquire sufficient informational
characters, 16 decameric primers were applied in RAPD assays, which produced
408 different PCR products, while TRAP assays included the combinations of 3
specific and 3 arbitrary primers, resulting in 767 fragments of DNA. Specific
primers used for TRAP analyses were constructed according to the RNA
sequences, showing high homology with DNA sequences published in electronic
data bases for following enzymes: geraniol 10-hydroxylase, cytochrome P450
reductase and secologanin synthase. Molecular markers provided a high
percentage of polymorphism. The informativeness of single primers (RAPD) of
primer combinations (TRAP) was confirmed by various parameters. Low
within-population genetic variability of natural populations of centaury could be
explained by the high levels of selfing or geitonogamy in the process of
fertilization and by strong genetic drift. Due to the fact that centaury grows in
small, fragmented populations and that gene flow between them probably does
not exist, a high genetic differentiation of analyzed populations was expected
and this was confirmed by molecular markers. Moreover, molecular markers
Резиме/Summary
efficiently differentiated diploid populations from tetraploids. Tetraploid
populations were clearly clustered into groups related to their geographic origin,
especially when RAPD markers were analyzed. Genetic similarity data between
the Balkan Peninsula populations, obtained with either RAPD or TRAP markers,
significantly correlated with the geographic distances between localities of their
origin. Identification and quantification of secondary metabolite compounds,
secoiridoid glycosides, highly contributed to the estimation of diversity of
centaury populations in the analyzed region. A dominant secoiridoid glycoside
in aerial parts of plants grown under greenhouse conditions was swertiamarin,
which pointed to the interpopulation variability of centaury. Furthermore, a
significant correlation between the secoiridoid glycosides content and genetic
similarity of populations has been obtained, though a higher correlation was
recorded for TRAP markers. The genetic and phytochemical diversity estimation
of centaury from the Balkan Peninsula provides a basis for future biodiversity
conservation efforts and also for high-productive genotype selection and field
production improvement. | true |
Evolucija ekstremiteta velikih mrmoljaka (Triturus cristatus complex, Salamandridae, Caudata) | Limb evolution in crested newts (Triturus cristatus complex, Salamandridae, Caudata) | Integracija, modularnost i razvojna ograničenja predstavljaju važne
komponente fenotipske varijabilnosti. U ovoj tezi analiziran je uticaj ovih
komponenti na morfološku evoluciju ekstremiteta pet vrsta velikih krestatih
mrmoljaka.
Veliki krestasti mrmoljci predstavljaju važne model organizme za
istraživanja u oblasti morfološke integracije i razvojnih ograničenja, posebno ako se
analiziraju ekstremiteti. To je grupa blisko srodnih vrsta sa poznatim filogenetskim
odnosima, i izraženom vezom izmedju morfološke forme/tipa (oblika tela i
dužine ekstremiteta) i preference prema akvatičnoj/terestričnoj sredini. Akvatične
vrste se odlikuju izduženim telom i kratkim nogama, dok su vrste sa više
terestričnim životnim stilom snažnije i robusnije građe sa dugim nogama. Takođe,
kao grupa sa složenim životnim ciklusom, gde larveni, juvenilni i adultni stupanj
imaju različite morfologije, ekološke sredine i preference, načine ishrane i
lokomocije, veliki krestasti mrmoljci imaju posebno mesto u izučavanju
ontogenetskih putanja tokom razvića. Ontogenetska promena niše i tranzicija
između akvatične i terestrične sredine je povezana sa procesom metamorfoze. Iako
ekstremiteti mrmoljaka ne prolaze kroz drastične promene strukture tokom tog
procesa, funkcionalni zahtevi za efikasnom lokomocijom u te dve sredine su bitno
različiti. Pretpostavka je da razlike između vrsta u pogledu stepena korišćenja
akvatične tj. terestrične lokomocije, može biti faktor koji doprinosi razlikama u
dužinama skeletnih elemenata i različitim obrascima korelacija između elemenata
ekstremiteta, oslikavajući adaptacije na različite uslove sredine i način života (više
ili manje akvatičan-terestričan).
Analiza morfološke integracije i modularnosti je utvrdila postojanje dva
važna aspekta variranja elemenata ekstremiteta velikih krestastih mrmoljaka.
Odgovarajući ontogenetski stupnjevi su sličniji između vrsta nego sukcesivni
ontogenetski stupnjevi u okviru jedne iste vrste, sa najvećim nivoom integracije u
juvenilnom stupnju. Visoki nivo integracije prisutan u juvenilnom stupnju
predstavlja funkciju brzog, koordinisanog rasta skeleta ekstremiteta tokom perioda
u okviru koga sistemski hormoni imaju odlučujuću ulogu, dok je niska ukupna
integracija na adultnom stupnju je najverovatnije rezultat mnogih lokalnih i
globalnih-sistemskih faktora koji menjaju jedan drugome efekat u vremenu i
prostoru, pri čemu bifazični životni ciklus sa različitim načinima lokomocije, imaju
odlučujuću ulogu u ontogenetskim promenama morfološke integracije i
modularnosti. Razlike u obrascima integracije između više akvatičnih i više
terestričnih vrsta mrmoljaka su predstavljene nižim stepenom korelacija između
homologih elemenata ekstremiteta, i povećanjem korelacija između elemenata
unutar ekstremiteta kod terestričnih vrsta velikih mrmoljaka (kao T. karelinii), dok
više akvatične vrste (kao T. dobrogicus) imaju suprotan obrazac variranja sa većim
korelacijama između homologih elemenata ekstremiteta nego između elemenata
unutar ekstremiteta.
Ova studija je pokazala važan uticaj razvojnih ograničenja u kreiranju
morfološke varijabilnosti ekstremiteta velikih krestastih mrmoljaka. Uporedna
analiza osifikacionih sekvenci ekstremiteta utvrdila je postojanje nekoliko
sinapomorfnih heterohroničnih promena kod velikih krestastih mrmoljaka, koje
uključuju kašnjenje u sekvenci drugog prsta i ubrzanje metakarpalnog elementa III
i metatarzalnog elementa V. Ove heterohronične promene nisu adaptivne ali su
važne za razmatranje morfološke evolucije ekstremiteta kod velikih krestastih
mrmoljaka jer uključuju promenu preaksijalne ka postaksijalnoj dominaciji u
razvojnoj sekvenci, koja nije poznata kod repatih vodozemaca. Analiza
alometrijskih obrazaca variranja ekstremiteta velikih mrmoljaka je pokazala dva
važna rezultata: osobenosti obrazaca rasta kod vrste T. dobrogicus, kao i zajednički
obrazac rasta nakon završene metamorfoze kod svih vrsta velikih mrmoljaka. Ovi
rezultati ukazuju na verovatni uticaj i ulogu kako prirodne selekcije, tako i
unutrašnjih-razvojnih mehanizama koji regulišu variranje dužine ekstremiteta
tokom diverzifikacije velikih mrmoljaka.
Obrasci variranja u basipodijumu su slični medju analiziranim vrstama sa
većim variranjem u broju karpalnih nego tarzalnim elemenata. Značajan podatak
ove studije je da je u basipodijumu vrste T. dobrogicus došlo do gubitka jednog od
karpalnih elemenata (centrale 1). Specifičnost ekstremiteta dunavskog mrmoljka se
dopunjuje i posebnim planom organizacije karpusa i tarzusa koji odlikuje nizak
nivo osifikacije praćen rastresitim pakovanjem skeletnih lemenata. Sve ove odlike
ukazuju na postojanje heterohronične promene (pedomorfoze) kada je u pitanju
skelet ekstremiteta.
Rezultati ove studije su pokazali značajan uticaj morfološke integracije,
modularnosti i razvojnih ograničenja u kreiranju fenotipske varijabilnosti u
ekstremitetima velikih krestastih mrmoljaka. | Integration, modularity and developmental constraints are three related
components of phenotypic variability. Their impacts on morphological evolution
on limbs in five species of crested newts were analyzed in this thesis.
Crested newts are valuable model organisms in studies of morphological
integration and developmental constraints, especially when concern limbs. They
are closely related group of species with known phylogenetic relations, and each
characterized by a specific body-to-limb conformation associated with their
respective ecologies. Aquatic species of newts have small, elongate bodies and
small limbs while more terrestrial species are with large, stocky bodies and robust
limbs. Also, as group with complex life cycle, where larval, juvenile and adult
stage have different morphologies, ecologies and different diet and locomotor
preferences, crested newts have special place in studies of changes in ontogenetic
trajectories. Ontogenetic niche change and transition between aquatic and
terrestrial environment is connected with process of metamorphosis. Although
limbs in newts do not undergo drastic changes in structure during that process,
functional demands for high efficient locomotion in both environments are quit
different. It is assumed that the differences between species in degree of aquatic
and terrestrial locomotion may be the factor which can attribute to changes in
length of skeletal elements and different patterns of correlations between limb
elements, reflecting adaptations on different environments and life styles of
analyzed species of crested newts.
Analysis of morphological integration and modularity has found different
aspects of variation patterns in limbs of crested newts. Equivalent ontogenetic
stages of different species of crested newts show higher concordance in the
correlation pattern than successive ontogenetic stages within species, with highest
level of integration in juvenile stage. High integration in juveniles is function of the
rapid, coordinated growth of the limb skeleton during this time period, in which
systematic hormones are thought to play a major role. While, low integration in
adult stage are probably due to many locally and globally-systematic factors which
change effect one to another in time and space, although biphasic life cycle with
different locomotion modes have decisive role in ontogenetic changes of
morphological integration and modularity. Differences in patterns of integrations
between aquatic and more terrestrial species of newts were observed, with lower
correlation between homologous limb elements and higher correlation within-limb
elements in more terrestrial species, the reverse pattern occurs in more aquatic
species.
This study showed important role of developmental constraints in creating
phenotypic variability in limbs of crested newts. Comparative analyses revealed
some synapomorphic heterochronic shifts specific to crested newts, including
delay of the ossification in the second finger and accelerations in metacarpal III and
metatarsal V. These shifts involve a change from pre-axial to post-axial dominance
in a developmental sequence uncommon to caudate salamanders. No adaptive
explanation of these shifts is apparent. Allometric analyses showed two important
results: specific growth patterns in T.dobrogicus and similar growth pattern after
metamorphosis in all species of crested newts. These results are reflecting impact
of natural selection as well as internal developmental mechanisms which regulated
variation in length of limbs during morphological diversification of crested newts.
All studied species shared the same basipodial pattern, within which the
carpus exhibited much more variation than the tarsus. Important point in this
study is lost of one carpal element (centrale 1). Specific pattern observed in T.
dobrogicus is followed with low ossification and loose package of carpale elements.
It is suggested that the uniqueness of T. dobrogicus originates from heterochronic
process.
Results of this study showed important influence of morphological
integration, modularity and developmental constraints in creating phenotypic
variability in limbs of crested newts. | true |
Molekularna i asocijativna analiza svojstava klasa pšenice (Triticum aestivum L.) | Molecular and association analysis of spike-related traits in wheat (Triticum aestivum L.) | Uvod: Hlebna pšenica (Triticum aestivum L.) je jedna od najvažnijih žitarica
široko zastupljena u proizvodnji u svetu, odnosno po ukupnoj proizvodnji zrna nalazi se
na trećem mestu iza kukuruza i pirinča. Zahvaljujući značaju ove biljne vrste posebno u
ljudskoj ishrani, priporitet u selekciji je usmeren na poboljšanje postojećih i stvaranje
novih sorti sa povećanim genetičkim potencijalom za prinos, kako bi se zadovoljile i
obezbedile kvantitativne potrebe za ovom žitaricom. Razvoj savremenih metoda i
primena najnovijih tehnologija iz oblasti biotehnologije, sa posebnim akcentom na
korišćenje molekularnih markera, otvorila je mogućnost implementacije molekularnog
oplemenjivanja u klasične oplemenjivačke programe. Na ovaj način se putem marker-
asistirane selekcije ostvaruje efikasnije, preciznije i ekonomičnije stvaranje prinosnijih
sorti. Najvažnije agronomske osobine nalaze se pod kontrolom većeg broja gena i
lokusa za kvantitativna svojstva (QTL), te su i osobine klasa koje predstavljaju
najvažnije komponente prinosa, svojstva koja se nalaze pod većim uticajem faktora
spoljašnje sredine. Danas se za otkrivanje i precizno definisanje hromozomskih regiona
odgovornih za ekspresiju ovih važnih osobina primenjuje jedan od najperspektivnijih
pristupa tzv. asocijativno mapiranje koje visokom rezolucijom mapiranja olakšava
primenu marker-asistirane selekcije.
Cilj istraživanja je bio da se utvrde osnovni parametri genetičke varijabilnosti
primenom mikrosatelita, da se proceni genetička struktura u ispitivanoj populaciji na
osnovu molekularnih podataka, da se analiziraju svojstva klasa kao najznačajnijih
komponenti prinosa pšenice kao i adaptivnih svojstava tokom pet vegetacionih sezona.
Takođe, postavljen je i cilj da se ispita neravnoteža vezanosti između hromozoma, i da
se analiziraju veze između markera i svojstava u svim ispitivanim godinama primenom
najsavremenijeg pristupa - asocijativnog mapiranja. Krajnji cilj bio je da se identifikuju
značajni i stabilni lokusi odgovorni za ekspresiju pojedinačnih karakteristika klasa i
svojstava adaptabilnosti i da se utvrde poželjni aleli u okviru lokusa koji su imali
najveći uticaj na fenotipsku varijabilnost ispitivanih osobina. Na ovaj način bi bilo
moguće odabrati genotipove koji su nosioci poželjnih alela za pojedine karakteristike
klasa, koji bi mogli da posluže kao roditelji u narednim ukrštanjima, a u cilju
poboljšanja postojećih genotipova ili stvaranja novih sorti pšenice sa željenim
osobinama klasa.
Materijal i metode: Molekularnom analizom je obuhvaćeno 283 genotipa
pšenice odabranih na osnovu višegodišnje fenotipske ocene velikog broja agronomski
važnih svojstava koja su ukazala na prisustvo značajnog stepena varijabilnosti. Ovaj
materijal je reprezentativni deo genetičke kolekcije pšenice koji pripada Odeljenju za
strna žita Instituta za ratarstvo i povrtarstvo u Novom Sadu. Genotipovi su nastali u
različitim oplemenjivačkim centrima i poreklom su iz 24 zemlje sa svih pet kontinenata.
Od 41 mikrosatelitskog markera, 30 je pokazalo efikasnost i značajan stepen
polimorfnosti čime su iskorišćeni za analizu svih genotipova primenom fragmentne
analize na genetičkom sekvenatoru Genetic Analyzer 3130. Fenotipskom ocenom je
obuhvaćeno ukupno 10 osobina (8 svojstava klasa i 2 adaptivne osobine) koje su
merene i beležene tokom pet vegetacionih sezona (1994/95., 1995/96., 1996/97.,
1997/98., 1998/99.). Obrada statističkih podataka je vršena u sledećim programima:
PowerMarker, Excel Mikrosatellite Kit, GenAlEx, Structure, Tassel i Statistica.
Rezultati: Fenotipskom evaluacijom ispitivanih osobina utvrđena je značajna
varijabilnost u ispitivanom materijalu. U 30 mikrosatelitskih lokusa svih genotipova
zabeleženo je ukupno 349 alelnih varijanti sa prosečnim brojem od 11,5 alela po lokusu
i prosečnom PIC vrednošću od 0,68. Procenjena struktura populacije u programu
STRUCTURE je pokazala distribuciju genotipova unutar šest subpopulacija na osnovu
vrednosti verovatnoće, dok je ΔK metod grupisao genotipove unutar tri postojeća
klastera. Slična distribucija genotipova dobijena je i primenom analize glavnih
koordinata (PCoA). Asocijativna analiza je pokazala da je 8 osobina (izuzev mase klasa
i mase zrna po klasu) pokazalo stabilne veze sa dvanaest markera u tri i više ispitivanih
godina. Detektovano je prisustvo trideset i tri stabilna QTL za svojstva klasa i adaptivna
svojstva, sa prosečnim uticajem na fenotipsku varijabilnost koja se kretala od 4,7% do
40,7% u zavisnosti od datog svojstva. Ovi značajni QTL locirani su na sledećim
hromozomima: 1B, 2A, 2B, 2D, 5B, 6A, s tim da su dominirali markeri sa druge
hromozomske grupe. Izdvojeni su i poželjni aleli u stabilnim QTL, gde se izdvajaju
QTL za broj klasića po klasu i broj zrna po klasu, kao najvažnije komponente klasa.
Tako su genotipovi koji su nosioci poželjnih alela za pojedina svojstva izabrani putem
marker-asistirane selekcije, kao roditelji za naredna ukrštanja u toku oplemenjivačkog
procesa.
Zaključak: 1. Polimorfni mikrosatelitski markeri su se pokazali informativni i
značajni u utvrđivanju genetičke varijabilnosti u oplemenjivačkom materijalu pšenice.
2. Procenjen je i odgovarajući stepen prisustva genetičke strukture u populaciji kao
preduslov asocijativne analize.3. Potvrđena je i značajna fenotipska varijabilnost
ispitivanih
osobina
klasa
i
adaptivnih
osobina.
4.
Utvrđeno
prisustvo
interhromozomskog LD je posledica rekombinacija i/ili epistatičkih interakcija nastalih
tokom selekcije. 5. Primenom i komparacijom različitih modela asocijativne analize
(GLM i MLM) pri utvrđivanju značajnih i stabilnih veza marker-svojstvo smanjena je
mogućnost detekcije lažno-pozitivnih veza. 6. Većina otkrivenih QTL je detektovana u
prethodnim istraživanjima na različitim mapirajućim populacijama, a sada je njihov
značaj potvrđen i na relevatnom oplemenjivačkom materijalu i u našim agro-ekološkim
uslovima. Pored ranije poznatih, detektovano je i nekoliko novih QTL. 7. Izdvojeno je
nekoliko genotipova koji su za pojedine osobine imali po nekoliko poželjnih alela u
okviru dva i više lokusa.
Ova disertacija predstavlja doprinos na polju molekularnog oplemenjivanja koje
bi zajedno sa klasičnim metodama oplemenjivanja omogućilo stvaranje genotipova sa
poboljšanim karakteristikama klasa čime bi se indirektnim putem postiglo povećanje
genetičkog potencijala za prinos. | Introduction: Bread wheat (Triticum aestivum L.) is one of the most important
cereals worldwide and in terms of total grain production wheat is positioned on third
place after maize and rice. Due to a great importance for human diet, the priority in
wheat breeding is focused on improving of existing and creating new varieties with
increased genetic yield potential, in order to meet quantitative demands for this grain.
The development of modern methods and the use of new technologies in the field of
biotechnology, with particular emphasis on the use of molecular markers, opened the
possibility of implementing molecular breeding in classical breeding programs. In this
way, the creation of high yielding varieties is more efficient, accurate and cost-effective.
The most important agronomic traits are controlled by a large number of genes and
quantitative trait loci (QTL). As the most important yield components, spike-related
traits are under strong influence of numerous environmental factors. In the last few
years, association mapping has been considered as one of the most promising methods
for the exploration of precise detection chromosome regions responsible for the
expression of these important traits. This high resolution perspective approach
facilitates the application of marker-assisted selection into breeding process.
The aim of this study was to determine the basic parameters of genetic
variability by using microsatellites, to assess the genetic structure in the population
based on molecular data, to analyze the spike characteristics as the most important yield
components and the adaptability traits during five growing seasons. Moreover, linkage
disequilibrium, and the associations between SSR markers and traits investigated were
examined by use of association mapping. Furthermost, the aim was to identify
significant and stable loci responsible for expression of spike-related and adaptability
characteristics, to identify desirable alleles within loci that have had the greatest impact
on the phenotypic variability. Genotypes which are carriers of favorable alleles for each
spike characteristics could serve as parents in future crosses, in order to improve the
existing genotypes in terms of specific spike characteristics.
Materials and Methods: Molecular analysis included 283 wheat genotypes
were selected upon results of multi-year phenotypic assessment of a large number of
agronomical important traits which indicated the presence of a significant degree of
variability. This material is a representative part of the genetic collection of wheat
which belongs to the Small Grains Department of Institute of Field and Vegetable Crops
in Novi Sad. Genotypes were created in different breeding centers, originating from 24
countries spread on all five continents. Of the 41 microsatellite markers, 30 showed
efficiency and a significant degree of polymorphism so they were used for analysis of
all genotypes by using fragment analysis on Genetic Analyzer 3130. A total of 10
properties (8 spike characteristics and 2 adaptability traits) were evaluated, measured
and recorded during five growing seasons (1994/95, 1995/96, 1996/97, 1997/98,
1998/99.). Statistical data processing was performed by the following programs:
PowerMarker, Excel Mikrosatellite Kit, Genalex, Structure, Tassel and Statistica.
Results: The phenotypic evaluation of the studied traits showed significant
variability in the breeding material. A set of 30 microsatellite markers positioned near
important agronomic loci were used to evaluate genetic diversity, resulting in a total of
349 allelic variants in all genotypes with an average number of 11.5 alleles per locus
and an average PIC value of 0.68. The population structure estimated by model-based
clustering distributed the genotypes into six subpopulations according to log probability
of data, whereas Evanno`s ΔK method grouped genotypes within three existing clusters.
Moreover, internal genetic structure using principal coordinates (PCoA) analyze
separated genotypes in a similar distribution as obtained by program Structure.
Association analysis showed that 8 traits investigated (except spike weight and grain
weight per spike) had a stable associations with 12 markers in three or more years. A
total number of 33 stable QTLs were detected for all traits investigated, with an average
effect on the phenotypic variation ranged from 4.7% to 40.7% depending on certain
traits. A significant QTLs were located on following chromosomes: 1B, 2A, 2B, 2D,
5B, 6A with a major concentration of the markers on chromosome group 2. Likewise,
desirable alleles were determined in stable QTLs, with emphasis on QTLs for number of
spikelets per spike and number of grains per spike, as the most important yield
components. Thus genotypes that possess favorable alleles responsible for certain traits
could be selected through marker-assisted selection, as parents for the next crossing
during the breeding process.
Conclusion: 1. The polymorphic microsatellites proved to be informative and
relevant markers in determination of the genetic diversity in wheat breeding materials.
2. The evaluated collection of wheat genotypes shows genetic diversity, and population
structure is an important tool and prerequisite for association analysis. 3. In addition to
molecular and phenotypic evaluation of the traits investigated have shown and
confirmed significant level of variability in breeding material. 4. The presence of inter-
chromosomal LD is a consequence of recombination and/or epistatic interactions
occurred during the selection. 5. The different statistical models (GLM and MLM)
evaluated increase the accuracy and power of the associations. 6. The new chromosome
regions identified as responsible for spike-related traits are useful for wheat breeding
programs in our agro-ecological conditions. 7. The genotypes which are carriers of
favorable alleles for particular traits are highlighted and could be useful for further
breeding program.
This thesis is a contribution to the field of molecular breeding, and implemented
with the conventional breeding methods could create genotypes with improved spike
characteristics and consequently increase of wheat yield potential. | true |
In vitro ispitivanje antitumorske aktivnosti ekstrakata endemične biljne vrste Helichrysum zivojinii Černjavski et Soška | In vitro investigation of antitumor activities of extracts of endemic plant species Helichrysum zivojini Černjavski et Soška U | Bioaktivni sastojci biljnih vrsta nalaze se u centru pažnje istraživanja u savremenoj
onkologiji zbog svoje moguće uloge u hemioprevenciji, odnosno inhibiciji različitih koraka
procesa maligne transformacije. Antikancerski potencijal biljnih jedinjenja zasniva se na
mogućnosti redukcije slobodnih radikala, regulacije karcinogen-aktivirajućih i karcinogen-
detoksifikujućih enzima, kao i mogućnosti inhibicije inflamatornih citokina, zatim na
mogućnosti da dovedu do promena u regulaciji faktora rasta i ciljnih molekula signalnih
puteva koji kontrolišu ćelijski rast, proliferaciju i apoptozu, kao i angiogenezu, invaziju i
metastazu malignih ćelija.
Osnovni cilj istraživanja je bio da se ispita citotoksičnost, odnosno odrede intenzitet i
mehanizmi citotoksičnog dejstva pet ekstrakata izolovanih u vidu frakcija iz endemične
biljne vrste Helichrysum zivojinii Černjavski et Soška prema specifičnom malignom
ćelijskom tipu, kao i da se odredi selektivnost u antitumorskom dejstvu prema nizu
humanih malignih ćelija, kako u odnosu na poreklo - tip tumora, tako i u odnosu na zdrave
mononuklearne ćelije periferne krvi (PBMC). Kako bi se doprinelo boljoj proceni
antitumorskog potencijala ekstrakata, ispitan je i uticaj ekstrakata na invazivnost humanih
metastatskih malignih ćelija i na angiogenezu endotelijalnih ćelija. Hemijska
karakterizacija biljnih ekstrakata je imala za cilj da omogući razumevanje veze između
kvalitativnog sastava i intenziteta citotoksične aktivnosti ekstrakata.
Citotoksična aktivnost pet ekstrakata endemične biljne vrste Helichrysum zivojinii
Černjavski et Soška je ispitana na sledećim humanim malignim ćelijskim linijama: HeLa
(adenokarcinom cerviksa), Fem-x (melanom), K562 (mijeloidna leukemija), MDA-MB-361
(adenokarcinom dojke), MDA-MB-231 (adenokarcinom dojke), kao i prema EA.hy926
ćelijama (transformisane humane endotelijalne ćelije umbilikalne vene). Obzirom da su pri
primeni antitumorskih agenasa u direktnom kontaktu sa njima i zdrave imunokompetentne
ćelije koje su uključene u imunsku kontrolu razvoja tumora, njihova vijabilnost je od
izuzetnog značaja za opštu kontrolu tumora. Zato je ispitano citotoksično dejstvo biljnih
ekstrakata na normalne PBMC. Ispitivani ekstrakti (heksanski (1), dihlormetanski (2), etil-
acetatni (3), n-butanolski (4) i metanolski ekstrakt (5)), su ispoljili selektivnu dozno-
zavisnu citotoksičnu aktivnost prema ciljnim malignim ćelijskim linijama, kao i prema
zdravim imunokompetentnim PBMC koje su bile stimulisane da proliferišu, dok je njihova
citotoksična aktivnost bila manje izražena prema nestimulisanim PBMC. Svi ispitivani
ekstrakti su pokazali znatno viši intenzitet citotoksične aktivnosti prema HeLa, Fem-x i
K562 ćelijama u odnosu na intenzitet aktivnosti prema normalnim PBMC, kako prema
nestimulisanim PBMC tako i prema PBMC koje su mitogenom bile stimulisane da
proliferišu. Biljni ekstrakti su pokazali slabiji intenzitet citotoksične aktivnosti prema
nestimulisanim PBMC u odnosu na PBMC koje su bile stimulisane fitohemaglutininom da
proliferišu. Veoma je važno da se istakne da su heksanski (1) i dihlormetanski ekstrakt (2)
ispoljili visoku selektivnost u antitumorskom dejstvu prema ciljnim HeLa i K562 malignim
ćelijama u odnosu na normalne PBMC.
Morfološka analiza tipa ćelijske smrti HeLa ćelija, kao i analiza distribucije HeLa ćelija
u određenim fazama ćelijskog ciklusa nakon dejstva ekstrakata, pokazale su da je svaki od
pet testiranih ekstrakata pri višim koncentracijama indukovao apoptozu u ciljnim HeLa
ćelijama. Primenom specifičnih inhibitora kaspaza je utvrđeno da su ispitivani ekstrakti
uzrokovali apoptozu HeLa ćelija posredstvom unutrašnjeg puta aktivacije i spoljašnjeg puta
aktivacije.
Analiza uticaja ekstrakata na migraciju i invazivnost humanih MDA-MB-231 ćelija
adenokarcinoma dojke je pokazala da je heksanski ekstrakt (1) primenjen pri niskoj
netoksičnoj koncentraciji ispoljio najizraženije supresivno dejstvo na migraciju, kao i na
invazivni potencijal ciljnih ćelija. Dihlormetanski ekstrakt (2) pri niskoj netoksičnoj
koncentraciji je ostvario značajan inhibitorni efekat na invazivni potencijal MDA-MB-231
ćelija.
Svaki od pet testiranih ekstrakata biljke Helichrysum zivojinii primenjen pri niskoj
netoksičnoj koncentraciji pokazao je svojstvo da inhibira migraciju humanih
transformisanih endotelijalnih ćelija umbilikalne vene EA.hy926, odnosno ostvarili su
supresivno dejstvo na inicijalni korak u procesu angiogeneze. Pored značajnog inhibitornog
dejstva na migraciju ciljnih EA.hy926 ćelija, heksanski ekstrakt (1) je ispoljio i izraženu
antiangiogenetsku aktivnost. Ostali testirani ekstrakti su ispoljili blag antiangiogenetski
efekat in vitro.
In vitro ispitivanje antitumorske aktivnosti pet ekstrakata endemične biljne vrste
Helichrysum zivojinii Černjavski et Soška je ukazalo na značajan antitumorski potencijal
heksanskog (1) i dihlormetanskog (2) ekstrakta, obzirom da su pomenuti ekstrakti ispoljili
izraženo citotoksično dejstvo prema ciljnim malignim ćelijskim linijama, zatim visoku
selektivnost u antitumorskom dejstvu prema specifičnim malignim ćelijskim tipovima u
odnosu na normalne PBMC, kao i antiinvazivna i antiangiogenetska svojstva. | Bioactive constituents of plants are in the center of attention of modern cancer research
due to their prospective role in cancer chemoprevention based on the suppression of
different stages in malignant transformation. The anticancer potential of plant compounds
could be attributed to their ability to scavenge free radicals, regulate carcinogen-activating
and –detoxifying enzymes and inhibit inflammatory cytokines, than to induce changes in
the regulation of growth factors and target molecules in oncogenic signal transduction
pathways implicated in cell growth, proliferation, apoptosis, as well as in angiogenesis,
invasion and metastasis of cancer cells.
The aim of this research was to investigate the cytotoxicity, more exactly to determine
the intensity and mechanisms of the cytotoxic actions of the five extracts isolated as
fractions from the endemic plant species Helichrysum zivojinii Černjavski et Soška against
specific malignant cell type, as well as to determine the selectivity in their antitumor
actions against malignant cell lines of different tumor origin and against healthy peripheral
blood mononuclear cells (PBMC). To fully evaluate the antitumor potential of extracts, the
examination of their effects on the invasiveness of human metastatic malignant cells as well
as on the angiogenesis of endothelial cells was done. Chemical characterization of plant
extracts was performed in order to understand the relation between qualitative composition
and intensity of cytotoxic action of the investigated extracts.
The cytotoxic activities of the five extracts of the endemic plant species Helichrysum
zivojinii Černjavski et Soška were tested against selected human malignant cell lines: HeLa
(cervix adenocarcinoma), Fem-x (melanoma), K562 (myelogenous leukemia), MDA-MB-
361 (breast adenocarcinoma), MDA-MB-231 (breast adenocarcinoma), as well as against
transformed human umbilical vein endothelial EA.hy926 cells. Considering the possible
effects of applied antitumor drugs on normal healthy immunocompetent cells, which are
implicated in the immune control of tumor development, their viability is significant for
tumor control. For that reason, the cytotoxic actions of the plant extracts were evaluated
against normal PBMC. The investigated extracts (hexane extract (1), dichloromethane
extract (2), ethyl-acetate extract (3), n-butanol extract (4) and methanol extract (5)) exerted
selective dose-dependent cytotoxic actions against target malignant cell lines and against
healthy immunocompetent PBMC stimulated to proliferate, while their cytotoxic actions
were not as pronounced against unstimulated PBMC. All of these extracts exhibited
considerably higher intensities of cytotoxic action against HeLa, Fem-x and K562 cells
when compared to intensities of action against PBMC, both resting and mitogen-stimulated.
The plant extracts showed lower intensity of cytotoxic activity against resting PBMC in
comparison to PBMC stimulated to proliferate by the mitogen phytohemagglutinin. It is
very important to stress that hexane extract (1) and dichloromethane extract (2) exhibited
highly selective antitumor actions against target HeLa and K562 malignant cells in
comparison to their actions against normal PBMC.
Morphological analysis of the mode of HeLa cell death in addition to analysis of
changes in the cell cycle phase distribution of target HeLa cells treated with the extracts,
showed that each of the five investigated extracts applied at a higher concentration induced
apoptotic cell death. The apoptotic mechanisms induced by the tested extracts were
determined using specific caspase inhibitors; it was demonstrated that extracts induced
apoptosis in target HeLa cells through the activation of both intrinsic and extrinsic
signaling pathways.
Examination of effects of the extracts on the migration and invasiveness of human breast
adenocarcinoma MDA-MB-231 cells showed that hexane extract (1) applied at a low non-
toxic concentration exhibited the most prominent inhibitory effect on the migration as well
as on the invasive potential of target cells. Dichloromethane extract (2) applied at a low
non-toxic concentration showed the notable inhibition of MDA-MB-231 cells invasion.
Each of the five tested Helichrysum zivojinii extracts applied at a non-toxic
concentrations was found to inhibit migration of human transformed umbilical vein
endothelial EA.hy926 cells and therefore exerted suppressive action on this initial step in
angiogenesis. The prominent inhibitory effect of the hexane extract (1) on EA.hy926 cells
migration was associated with a notable antiangiogenic action of this extract. The other four
tested extracts demonstrated mild antiangiogenic activities in vitro.
In vitro investigation of antitumor activities of the five extracts of the endemic plant
species Helichrysum zivojinii Černjavski et Soška points to significant antitumor potential
of the hexane extract (1) and dichloromethane extract (2) which can be attributed to their
pronounced cytotoxic actions against target malignant cell lines, high selectivity in their
antitumor actions against specific malignant cell types in comparison to normal PBMC, as
well as to exerted antiinvasive and antiangiogenic properties. | true |
Gen za veliku subjedinicu šaperonina (groEL gen) kao dodatni molekularni marker za diferencijaciju fitoplazmi srodnih 'Candidatus Phytoplasma asteris' | Molecular chaperonin large subunit gene (gro EL gene) as an additional marker for differntiation of 'Canidatus Phytoplasma asteris' - related strains | Na teritoriji Republike Srbije su u periodu od 2009. do 2011. godine sakupljeni uzorci biljaka sa simptomima karakterističnim za prisustvo fitoplazmi. Upotrebom PCR metode pomoću univerzalnih prajmera P1/P7 i R16F2n/R16R2 u njima su detektovane fitoplazme. Upotrebom restrikcionih enzima Tru1I, HhaI, RsaI i Tsp509I na R16F2n/R16R2 amplikone, ove fitoplazme su na osnovu dobijenih restrikcionih profila identifikovane kao Aster yellows fitoplazme (ribozomalna grupa 16SrI), podgrupe 16SrI-B, I-C i I-P, odnosno kao Stolbur fitoplazme (ribozomalna grupa 16SrXII). Upotrebom iste PCR-RFLP metode kod 19 sojeva Aster yellows fitoplazmi iz kolekcije i tri soja poreklom iz šargarepe iz Srbije, potvrĎena je pripadnost podgrupama 16SrI-A, I-B, I-C i I-F preuzeta iz literature. TakoĎe je utvrĎena pripadnost odreĎenoj podgrupi kod četiri soja iz kolekcije, koji se prvi put koriste u analizi i o kojima nema literaturnih podataka. Upotrebom novodizajniranih prajmera AYgroesF/AYampR i AYgroelF/AYgroelR umnoţen je groEL gen kod sva 34 testirana soja Aster yellows fitoplazmi. Analizom restrikcionih profila dobijenih pomoću Tru1I i AluI restrikcionih enzima na AYgroelF/R amplikone, utvrĎeno je postojanje šest, odnosno osam različitih profila na osnovu kojih su svi testirani sojevi svrstani u devet groELI podgrupa (groELI-I do groELI-IX). Na osnovu groEL gena podgrupe 16SrI-A i I-C su dalje diferencirane u dve groELI podgrupe, 16SrI-B u tri, dok podgrupe 16SrI-M i I-L nisu pokazale nikakve meĎusobne razlike u odnosu na jedan deo podgrupe 16SrI-B. Podgrupe 16SrI-F i I-P se razlikuju od ostalih podgrupa i na nivou groEL gena kao i na nivou 16S rDNK. TakoĎe je utvrĎeno da sedam novodetektovanih sojeva Aster yellows fitoplazmi iz Srbije pripada podgrupama groELI-III, I-VII i I-IX. Analizom tuf gena, dela rp operona i secY gena kod 22 odabrana od 34 testirana soja soja Aster yellows fitoplazmi moguće je testirane sojeve klasifikovati u šest tufI i sedam rpI i secYI podgrupa na osnovu MboI, Tsp509I i Tru1I restrikcionih profila za tuf gen, HhaI, AluI i Tsp509I za rp gen i Tsp509I i Tru1I za secY gen. Podgrupa 16SrI-A je dalje diferencirana u dve tufI podgrupe, odnosno 16SrI-A i I-B u po dve rpI i secYI podgrupe, dok podgrupe 16SrI-B, I-M i I-L nisu pokazale nikakve meĎusobne razlike. Podgrupe 16SrI-C, I-F i I-P se razlikuju od ostalih podgrupa i na nivou sva tri testirana gena kao i na nivou 16S rDNK | The plant samples with symptoms typical for phytoplasma infection were collected in Serbia from 2009 till 2011. In those samples phytoplasmas were detected by PCR technique with universal primers P1/P7 and R16F2n/R16R2 and identified according to their Tru1I, HhaI, RsaI and Tsp509I restriction profiles of digested R16F2n/R16R2 amplicons as Aster yellows phytoplasma (ribosomal group 16SrI), subgroups 16SrI-B, I-C and I-P or as Stolbur phytoplasma (ribosomal group 16SrXII). Applying the same PCR-RFLP method on 19 strains of Aster yellows phytoplasmas from collection and three strains from carrot from Serbia, it was confirmed that this strains belong to subgroups 16SrI-A, I-B, I-C and I-F as stated in literature. Also the subgroups were determined for four strains from collection that are used in analyses for the first time and that have no literature data. Using newly designed primers AYgroesF/AYampR and AYgroelF/AYgroelR, the groEL gene was successfully amplified in all 34 strains of Aster yellows phytoplasma tested. RFLP analyses of AYgroelF/AYgroelR amplicons with Tru1I i AluI restriction enzymes revealed existence of six and eight different restriction profiles, respectively, according to which all tested strains were classified in nine groELI subgroups (groELI-I till groELI-IX). On the basis of groEL gene, subgroups 16SrI-A and I-C were further differentiated into two groELI subgroups, 16SrI-B into three, while subgroups 16SrI-M and I-L showed no difference to some strains belonging to subgroup 16SrI-B. Subgroups 16SrI-F and I-P could be differentiated from other subgroups on the basis of groEL gene as on the basis of 16S rDNA. The seven newly detected Aster yellows strains from Serbia were affiliated to subgroups groELI-III, I-VII and I-IX. RFLP analyses with MboI, Tsp509I and Tru1I restriction enzymes of tuf gene, HhaI, AluI and Tsp509I of rp gene and Tsp509I and Tru1I of secY gene, classified 22 selected Aster yellows strains into six tufI subgroups and seven rpI and secYI subgroups. Subgroup 16SrI-A was further differentiated into two tufI subgroups and subgroups 16SrI-A and I-B were further differentiated into two rpI and secYI subgroups each. On the other hand subgroups 16SrI-B, I-M and I-L showed no mutual differences, while subgroups 16SrI-C, I-F and I-P could be differentiated from other subgroups on the basis of all three genes tested as on the basis of 16S rDNA. RFLP analysis of tuf gen with HpaII restriction enzyme showed that all 116 tested Stolbur phytoplasma strains from Serbia belong to tuf type II variant. For further analyses, 39 Stolbur strains were selected out of 116 detected and two strains from grapevine from Croatia were also included in analyses as tuf type I reference strains to gain a larger picture of variability of groEL gene in Stolbur phytoplasmas. | false |
Modulacija funkcije humanih dendritskih ćelija kombinovanom primenom agonista endozomnih Toll-sličnih receptora, Dektin-1 receptora i proinflamatornih citokina | Modulation of the function of human monocyte derived dentritic cells by combined use of the endosomal Toll-like receptors, Dectin-1receptor and proinflamatory cytokines | Dendritske ćelije (DĆ), najpotentnije antigen-prezentujuće ćelije
(APĆ), integrišu signale koje primaju sa različitih receptora u jedinstveni odgovor.
Ključni značaj u ostvarivanju funkcija DĆ imaju receptori za prepoznavanje
konzervisanih struktura, tzv. molekularnih obrazaca patogena (engl. Pattern
Recognition Receptor, PRR). Aktivacija pojedinačnih PRR, posebno Toll-sličnih
receptora (engl. Toll-like receptor, TLR) ili lektinskih receptora C-tipa poput dektina-1,
dovodi do sazrevanja DĆ, dok je za razvoj efikasnog imunskog odgovora neophodna
kooperacija više receptora.
Dektin-1 receptor je, pored TLR, jedini receptor urođenog imuniteta čija
aktivacija samostalno indukuje signalnu kaskadu koja dovodi do sazrevanja MoDĆ sa
sposobnošću indukcije Th1 i Th17 odgovora. Imajući navedno u vidu, sledeći cilj ovog
istraživanja je bio da se ispita efekat kombinovane primene Poly (I:C) i kurdlana,
agonista dektin-1 receptora, na funkcionalne i fenotipske karakteristike MoDĆ.
Za modulaciju sazrevanja i funkcija DĆ, pored stimulacije PRR, značajnu ulogu
imaju i citokini prirodnog imuniteta koji se produkuju prilikom infekcije. Najnovija
istraživanja ukazuju na ulogu TNF-α, jednog od najznačajnijih proinflamatornih
citokina, u sazrevanju DĆ u ranim fazama infekcije kao i razvoju antigen-specifičnog
odgovora. Naredni cilj našeg istraživanja bio je ispitivanje dozno- i vremenski-
zavisnog efekta kombinovane primene TNF-α i Poly (I:C) na funkcionalne i fenotipske
karakteristike MoDĆ.
Poslednji deo istraživanja se odnosio na ispitivanje uticaja signala stečenog
imuniteta koje DĆ dobijaju tokom interakcije sa T-limfocitima, uključujući signalizaciju
preko CD40 receptora i receptora za IFN-γ na njihove karakteristike.
Metode. Nezrele MoDĆ, dobijene kultivacijom humanih monocita, su
stimulisane samim Poly (I:C) ili njegovom kombinacijom sa loksoribinom, kurdlanom
ili TNF-α tokom 48h. Da bi ispitali uticaj signala stečenog imuniteta, MoDĆ stimulisane
samim Poly (I:C) ili u kombinaciji sa TNF-α dalje su kultivisane u prisustvu ćelija J558
transfektovanim ligandom za CD40, solubilnog CD40L ili IFN-γ. U cilju ispitivanja
uticaja kinetike aktivacije na kapacitet MoDĆ da polarizuju imunski odgovor, MoDĆ su
stimulisane kombinacijama različitih koncentracija Poly (I:C) i TNF-α tokom 24h i 48h.
Protočnom citofluorimetrijom su analizirane fenotipske karakteristike MoDĆ.
Alostimulatorna sposobnost MoDĆ je određena testom mešane leukocitne reakcije.
Produkcija citokina je određena ELISA metodom i testom za detekciju citokina pomoću
imunofluorescentnih kuglica.
Rezultati.
Stimulacija MoDĆ optimalnom koncentracijom Poly (I:C) dovela je do povećanja
ekspresije HLA-DR, CD86, CD40, CD54, CD83 i CCR7 molekula, povećanja produkcije IL-
12, umerene produkcije IL-23 i niske produkcije IL-10. Ovako stimulisane MoDĆ dovode
do povećane produkcije IFN-γ i umerene produkcije IL-17 tokom kokultivacije sa CD4+
T limfocitima.
U preliminarnim eksperimentima nezrele MoDĆ su stimulisane
različitim koncentracijama Poly (I:C) (5 μg/ml, 10 μg/ml, 25 μg/ml i 50 μg/ml),
loksoribina (34 μg/ml i 85 μg/ml) i kurdlana (10 μg/ml , 50 μg/ml, 100 μg/ml i 200
μg/ml). Na osnovu fenotipskih i funkcionalnih karakteristika MoDĆ procenjeno je da je
optimalna koncentracija za aktivaciju MoDĆ za Poly (I:C) 25 μg/ml, za loksoribin 85
μg/ml i za kurdlan 100 μg/ml, dok je koncentracija Poly (I:C) od 10 μg/ml i loksoribina
od 34 μg/ml suboptimalna. Optimalne i suboptimalne koncentracije agonista su dalje
korišćene za stimulaciju nezrelih MoDĆ u ovom istraživanju.
MoDĆ kultivisane u prisustvu suboptimalnih koncentracija agonista TLR3 i
TLR7 povećano su eksprimirale HLA-DR, CD86, CD83, CD54 i CD40 molekule i
produkovale su veće nivoe IL-27, IL-23 i IL-10, u poređenju sa ćelijama tretiranim
pojedinačnim agonistima. Ovako pripremljene MoDĆ su dovele do povećanja
produkcije IFN-γ i IL-17 u kokulturi sa alogenim CD4+ T limfocitima. Tretman MoDĆ
suboptimalnom koncentracijom Poly (I:C) i optimalnom koncentracijom loksoribina u
poređenju sa efektima pojedinačnih agonista, doveo je do povećanja ekspresija CD86,
smanjenja HLA-DR, povećanja produkcije IL-12 i IL-23 i smanjenja produkcije IL-10 i IL-
27. MoDĆ diferencirane na ovaj način u kokulturi sa CD4+ T ćelijama su indukovale
povećanje produkcije IFN-γ i smanjenje produkcije IL-4 i IL-10. Stimulacija MoDĆ
optimalnim koncentracijama oba TLR agonista dovela je do smanjenja ekspresije HLA-
DR, CD83 i CD40 molekula, kao i povećanja produkcije IL-12, IL-27 i IL-10, dok je nivo
IL-23 bio značajno smanjen, u poređenju sa efektima pojedinačnih TLR agonista. MoDĆ
stimulisane na ovaj način su u kokulturi sa CD4+ T limfocitima dovele do povećanja
produkcije IFN-γ i smanjenje produkcije IL-4, IL-10 i IL-17.
Stimulacija MoDĆ kurdlanom dovela je do povećanja produkcije IL-23 koji je
tokom kokultivacije sa CD4+ T limfocitima usmerio odgovor T limfocita u Th17 pravcu
uz manje izražen Th1 odgovor. MoDĆ kultivisane u prisustvu Poly (I:C) i kurdlana su
ispoljile fenotip zrelih ćelija i stimulisale su proliferaciju alogenih CD4+ T limfocita.
Kostimulacija TLR3 i dektina-1 na MoDĆ je dodatno povećala produkciju IL-12, IL-23 i
IL-10 u poređenju sa efektima angažovanja pojedinačnih receptora. Ovi rezultati su u
korelaciji sa indukcijom snažnijeg Th1 i Th17 imunskog odgovora, procenjivanog na
osnovu produkcije IFN-γ od strane naivnih (CD45RA+) CD4+ T limfocita, odnosno IL-17
od strane memorijskih (CD45RO+) CD4+ T limfocita.
MoDĆ stimulisane optimalnom koncentracijom Poly (I:C) i TNF-α ispoljile su
povećane nivoe CD80, CD86 i CD54 molekula i smanjile produkciju IL-12, što je uticalo
na smanjenje proliferacije T limfocita i produkcije IFN-γ u kokulturi sa alogenim CD4+
T ćelijama, u poređenju sa efektom MoDĆ tretiranih samim Poly (I:C). Dozno-zavisni
efekti Poly (I:C) i TNF-α na funkciju MoDĆ su izraženiji nakon 24h kultivacije u
poređenju sa 48h, što je procenjeno na osnovu smanjenja produkcije IL-12 i IL-23 od
strane MoDĆ i smanjenog nivoa IFN-γ i IL-17 u kokulturi sa CD4+ T ćelijama. Uticaj
interakcije CD40 i CD40L je najizraženiji u slučaju produkcije IL-12 od strane MoDĆ
tretiranih najvećom koncentracijom Poly (I:C) i TNF-α. Smanjenje produkcije IL-12 od
strane MoDĆ stimulisanih ovom kombinacijom je dovelo do smanjenja produkcije IFN-
γ u kokulturi sa CD4+ T ćelijama. Povezivanje CD40 molekula na MoDĆ stimulisanim
najvećom koncentracijom Poly (I:C) i TNF-α je dovelo do smanjenja produkcije IL-23 i
povećanja IL-10. Stoga su ovako pripremljene MoDĆ u kokulturi sa CD4+ T ćelijama
dovele do smanjenja produkcije IL-17, a povećanja IL-10.
Povezivanje CD40 molekula ili tretiranje sa IFN-γ nezrelih i MoDĆ
diferenciranih u prisustvu Poly (I:C) značajno je povećalo njihov alostimulatorni
potencijal. Povezivanje CD40 molekula na nezrelim MoDĆ dovelo je do povećanja
produkcije IL-12 i IL-23, što je praćeno povećanjem produkcije IFN-γ tokom
kokultivacije ovako stimulisanih ćelija i CD4+ T limfocita. Međutim, povezivanje CD40
molekula na MoDĆ pretretiranim sa Poly (I:C) dovelo je do povećanja produkcije IL-12,
IL-23 i IL-10. Ovako stimulisane ćelije u kokulturi sa CD4+ T ćelijama dovele su do
povećanja produkcije IL-17, a smanjenja produkcije IFN-γ i IL-10. Nezrele MoDĆ
stimulisane sa IFN-γ produkovale su veći nivo IL-12, a u kokulturi sa CD4+ T limfocitima
su dovele do smanjenja produkcije IL-5 i IL-17. Suprotno tome, tretman sa IFN-γ je
doveo do smanjenja produkcije IL-12, a povećanja produkcije IL-10 od strane MoDĆ
prethodno stimulisanih sa Poly (I:C). MoDĆ pripremljene na ovaj način su tokom
kokultivacije sa T limfocitima dovele do smanjenja IFN-γ a povećanja nivoa IL-10.
Zaključak. | Dendritic cells (DCs) are the most potent antigen presenting
cells (APCs) which receive and integrate multiple signals to initiate and direct a
response appropriate to extracellular milleu. These APCs perform these functions
mostly due to the expression of a wide variety of pattern recognition receptors (PRRs).
PRRs discriminate self-tissues from infectious non-self tissues through molecular
pattern (MPs) recognition. Although triggering of a single pattern recognition
receptor (PRR), especially Toll-like receptors (TLRs) or C-type lectins, results in
phenotypic changes in DCs, for functional maturation cooperativity between multiple
PRRs is needed in order to achieve an effective immune response.
Recent studies have shown that the ligation of Dectin-1, C-type lectin receptor,
on MoDCs elicits their maturation. Dectin-1, a DC-associated C-type lectin, is the first
of many PRRs which mediate their own signaling and induces the maturation of DCs
capable of eliciting the generation of different T helper (Th) effectors. The next aim of
this work was to study the response of MoDCs to the combined effect of Poly (I:C) and
curdlan, selective Dectin-1 agonists.
Tumor necrosis factor (TNF)-α is important for early DC maturation and as a
bridge between initiation of the inflammatory cascade and generation of the antigen-
specific response. To gain insight into this scientific problem we investigated the
kinetics of maturation and the length of exposure of MoDCs to a pathogen (mimicked
by Poly (I:C) in our study) in an inflamed tissue (mimicked by TNF-α) on phenotypic
and functional characteristics of MoDCs.
Finally, little is known about how subsequent interaction of MoDCs with T cell-
derived stimuli, such as CD40 or interferon-γ (IFN-γ), modulates MoDC functions.
Therefore, this problem was the last objective of this study.
Methods. Immature MoDCs (iMoDCs), generated from human monocytes, were
treated with Poly (I:C) alone or in combination with loxoribine, curdlan or TNF-α for
48h. To investigate the influence of T-cell derived stimuli, MoDCs cultivated for 24h
with Poly (I:C) alone or in combination with TNF-α were incubated either with CD40
ligand (L)-transfected J558 cells, soluble CD40L or IFN-γ for additional 24h. To examine
the influence of kinetics of activation on the Th polarizing capability of MoDCs, we
stimulated MoDCs with different doses of Poly (I:C) in combination with TNF-α for 24h
and 48h. Phenotypic characteristics of MoDCs were determined by flow cytometry.
Allostimulatory capability of MoDCs was tested using a mixed leukocyte reaction
assay. Cytokine production was measured by ELISA and FlowCytomix.
Results.
Optimal concentration of Poly (I:C) stimulated the maturation of MoDCs as
judged by the up-regulation of HLA-DR, CD86, CD40, CD54, CD83 and CCR7 expression.
Poly (I:C)-treated MoDCs were potent producers of interleukin (IL)-12, moderate
producers of IL-23 and weak producers of IL-10, which was followed by high
production of IFN-γ and moderate production of IL-17 by allogeneic CD4+ T cells.
In preliminary experiments iMoDCs were treated with Poly (I:C) (5
μg/ml, 10 μg/ml, 25 μg/ml and 50 μg/ml), loxoribine (34 μg/ml and 85 μg/ml) or
curdlan (10 μg/ml , 50 μg/ml, 100 μg/ml and 200 μg/ml). Based on phenotypic
characteristics and functional capabilities of MoDCs, the concentrations of Poly (I:C)
(25 μg/ml), loxoribine (85 μg/ml) and curdlan (100 μg/ml) were found to be optimal
for activation of MoDCs, while the concentrations of Poly (I:C) (10 μg/ml) and
loxoribine (34 μg/ml) were found to be suboptimal. For stimulation of iMoDCs we used
these concentrations of the agonists.
The combined treatment of MoDCs with suboptimal concentrations of TLR3 and
TLR7 agonists resulted in slight potentiation of HLA-DR, CD86, CD83, CD54 and CD40
molecules and stimulation of IL-27, IL-23 and IL-10 secretion, compared to effects of
single agonists. This was followed by up-regulated secretion of IFN-γ and IL-17 in the
co-culture with allogeneic CD4+ T cells. When the suboptimal concentration of Poly
(I:C) was combined with the optimal concentration of loxoribine, MoDCs down-
regulated HLA-DR and up-regulated CD86 expression, enhanced the production of IL-
12 and IL-23 and down-regulated the levels of IL-10 and IL-27, compared to the effects
of single agonists. MoDCs pretreated in this way stimulated the production of IFN-γ
and lowered the levels of IL-4 and IL-10 by CD4+ T cells. The treatment of MoDCs with
optimal concentrations of both TLR agonists was followed by down-regulation of HLA-
DR, CD83 and CD40 expression and augmented the production of IL-12, IL-27 and IL-10,
whereas the level of IL-23 was significantly lower, compared to relevant controls.
These MoDCs promoted the production of IFN-γ and inhibited the production of IL-4,
IL-10 and IL-17 in co-culture, compared to the effect of corresponding controls.
The combination of Poly (I:C) and curdlan induced phenotypic maturation of
MoDCs with the capability to stimulate alloreactive response. Such treated MoDCs up-
regulated the production IL-12, IL-23 and IL-10, compared to the effect of Poly (I:C),
alone. The opposite effect was observed for IFN-γ production. When combined, these
agonists primed MoDCs to further increase the production of IFN-γ by CD4+ T cells in
co-culture, especially those of naïve (CD45RA+) phenotype, and IL-17 by memory
(CD45RO+) CD4+ T cells.
MoDCs stimulated with optimal concentration of Poly (I:C) and TNF-α up-
regulated the expression of CD80, CD86 and CD54 molecules and lowered the
production of IL-12 which was followed with inhibition of cellular proliferation and
IFN-γ production in co-culture with allogeneic CD4+ T cells, compared to the effect of
Poly (I:C)-treated MoDCs. Dose- and time- dependent effect of Poly (I:C) and TNF-α on
the functions of MoDCs were more pronounced after 24h than 48h of stimulation, as
judged by down-regulation of IL-12 and IL-23 production by this cells and reduced
levels of IFN-γ and IL-17 in co-cultures with CD4+ T cells. The influence of CD40:CD40L
interaction was most prominent in the case of production of IL-12 when MoDCs were
pretreated with the highest dose of Poly (I:C) and TNF-α. This effect was reflected on
reduced production of IFN-γ by allogeneic CD4+ T cells co-cultured with MoDCs treated
with this combination. The addition of CD40L lessen the levels of IL-23 and increased
the production of IL-10 in cultures of MoDCs stimulated with the highest dose of Poly
(I:C) and TNF-α which was reflected on significant reduction of the levels of IL-17 and
up-regulation of IL-10 produced by allogeneic CD4+ T cells.
Ligation of CD40 or treatment with IFN-γ of iMoDCs or Poly (I:C)-treated MoDCs
significantly up-regulated their allostimulatory activity. Ligation of CD40 on iMoDCs
up-regulated the production of IL-12 and IL-23 which was accompanied by increased
secretion of IFN-γ in co-culture. Stimulation of CD40 on Poly (I:C)-treated MoDCs
significantly enhanced the production of IL-12, IL-23 and IL-10. However, such treated
MoDCs decreased the production of IFN-γ and IL-10 and up-regulated the secretion of
IL-17. iMoDCs treated with IFN-γ up-regulated IL-12 production, but lowered the
production of IL-5 and IL-17 by CD4+ T cells. Treatment of Poly (I:C)-activated MoDCs
with IFN-γ down-regulated the production of IL-12 and up-regulated IL-10 by these
cells and increased/decreased the levels of IL-10/ IFN-γ, respectively, in co-culture
with CD4+ T cells.
Conclusions. | true |
Prognostički značaj aberantne metilacije CpG ostrvaca u folikularnim i difuznim B-krupnoćelijskim limfomima čoveka | Prognostic significance of aberrant CpG islands methylation in human follicular and diffuse large B-cell lymphoma | Difuzni B-krupnoćelijski limfom (DBKL) i folikularni limfom (FL),
predstavljaju najučestalije entitete nehočkinovih limfoma i zajedno čine oko 50 % svih
B-ćelijskih neoplazija. To su klinički i biološki heterogena oboljenja, sa visoko
varijabilnim odgovorom na terapiju i uprkos značajnom napretku u terapiji, u velikom
broju slučajeva ostaju neizlečiva. Aktuelni terapijski pristupi su praćeni brojnim
neželjenim efektima i dovode do dugotrajne remisije i preživljavanja kod svega 50 %
bolesnika. Najznačajniji klinički prediktor ishoda bolesti do danas je internacionalni
prognostički indeks (IPI za DBKL, odnosno, FLIPI za FL), koji obuhvata odgovarajuće
kliničke prognostičke parametre. Uprkos tome, oboleli koji pripadaju istim IPI/FLIPI
kategorijama ispoljavaju značajne razlike u odgovoru na terapiju i preživljavanju, što
ukazuje na postojanje značajne biološke heterogenosti u okviru svake IPI/FLIPI
kategorije. Zbog toga je neophodno pronalaženje novih prognostičkih molekularnih
parametara, koji bi omogućili precizniju podelu obolelih u grupe različitog stepena
rizika i izbor odgovarajuće terapije. Primenom PCR metode specifične za metilaciju
(MSP), ispitivan je metilacioni status p16, p15, MGMT i DAPK gena kod obolelih od
DBKL i FL, kao bi se utvrdio njihov potencijalni prognostički značaj. Kod obolelih od
DBKL, metilacija nijednog gena nije pokazala prognostički značaj, mada je uočena
tendencija ka akumulaciji metilacije p15 gena kod ispitanika sa povoljnijim kliničko-
patološkim karakteristikama. Kod obolelih od FL metilacija p16, p15 i MGMT gena bi
mogla da ima određeni prognostički značaj, ukoliko se kombinuje sa FLIPI, gradusom
tumora, odnosno starosnim dobom obolelihi. Istovremena metilacija MGMT i DAPK
gena bi mogla da ukaže na grupu obolelih potencijalno hemorezistentnih na primenjenu
hemoterapiju i sklonu recidivima bolesti. | Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) are the
most comon subgroups of non-Hodgkin’s lymphoma and comprise approximately 50 %
of all cases. They represents clinically and biologically heterogeneous diseases,
characterized with highly variable response to the treatment, and remain incurable
despite the significant advances in therapy. Actual therapy regimens are agressive, and
long-term remission and survival are ashieved in only 50 % of patients. The strongest
clinical predictor of outcome to date in DLBCL and FL patients is the International
Prognostic Index (IPI for DLBCL and FLIPI for FL) which includes several clinico-
pathological prognostic parameters. However, patients with identical IPI/FLIPI still
exhibit marked variability in survival, suggesting the presence of significant biological
heterogenity within the same risk category. So, it is important to investigate additional
molecular markers in order to further stratify patients into diferent risk groups, choose
appropriate treatement strategy and improve prognosis. In this study, methylation-
specific polymerase chain reaction (MSP) was used to investigate the role and potential
prognostic significance of the methylation status of p16, p15, MGMT and DAPK genes
in patients with DLBCL and FL. No one of four examined genes showed prognostic
significance in patients with DLBCL, though we observed a tendency toward
accumulation of p15 methylation with favorable clinico-pathological parameters.
However, in patients with FL, our results suggest that promoter methylation of p16, p15
and MGMT genes could have some prognostic value when used in combination with the
FLIPI, tumor grade and patients age, respectively. Concurrent methylation of MGMT
and DAPK genes could be the marker of tumor chemoresistance and disease recurrence. | true |
Kliničko genetska analiza pacijenata sa familijarnom amiotrofičkom lateralnom sklerozom u populaciji Srbije | Clinical and genetic analysis of patients with familiar amyotrophic lateral sclerosis in Serbian population | Amiotrofična lateralna skleroza (ALS) nastaje usled primarnog oštećenja
motornih neurona na nivou kore velikog mozga, jedara moždanog stabla i prednjih
rogova kičmene moždine. Oko 90% ALS pacijenata čini tzv. sporadičnu ALS (SALS),
dok 5-10% pacijenta ima genetsku ili familijarnu osnovu bolesti (FALS). Do sada je
opisano više od 20 gena povezanih sa ALS. Nisu poznate epidemiološke i kliničke
karakteristike pojedinih formi FALS u Srbiji. U svetu nedostaju podaci o zahvaćenosti
centralnog nervnog sistema (CNS) u sklopu FALS.
Ciljevi ovog istraživanja su bili: molekularno-genetsko ispitivanje pacijenata sa ALS na
prostoru Srbije; određivanje tipičnih kliničkih znakova u subpopulaciji pacijenata sa
FALS; ispitivanje korelacija kliničkih i genetskih karakteristika, uključujući poređenje
nalaza neuropsihološkog testiranja i morfoloških specifičnosti mozga i kičmene
moždine analiziranih pomoću savremenih metoda magnetne rezonancije (MR).
Metod: U ovo ispitivanje uključen je 241 pacijent iz hospitalnog registra Klinike za
neurologiju Kliničkog centra Srbije. Pacijenti su testirani na sledeće mutacije: SOD1
(svih 5 egzona), TDP 43 (egzon 6), FUS (egzoni 14 i 15), c9orf72 i ANG. Genetska
grupa od 37 ispitanika poređena je klinički sa 32 SALS pacijenta bez verifikovane
mutacije. Neuropsihološko testiranje je sprovedeno kod 22 SOD1 pozitivna ispitanika, 5
SOD1 negativnih ispitanika pozitivnih na druge ALS gene i 82 zdrave kontrole.
Primenjivani su testovi za kognitivni skrining, procenu opšteg intelektualnog nivoa,
pažnje, pamćenja, vizuospacijalnih i konstrukcionih, egzekutivnih i jezičkih funkcija.
Od 69 pacijenta sa ALS, MR pregledom mozga i vratnog dela kičmene moždine na
aparatu od 1,5T je obuhvaćena grupa od 23 genetski determinisanih pacijenata i 11 iz
grupe SALS. Kontrolna grupa bila je sastavljena od 34 uparena zdrava ispitanika.
Analizirana je debljina korteksa, mikrostruktura puteva bele mase, funkcionalna
povezanost delova mozga u stanju mirovanja, kao i analiza strukture kičmene moždine.
Rezultati: Ukupno 37 (15,4%) od 241 pacijenta imalo je jednu ili više mutacija u
testiranim ALS genima. U genetskoj grupi 27 pacijenta je imalo mutaciju u SOD1 genu
(72,9%), šest pacijenata je imalo mutaciju u genu za c9orf72 (16,2 %), dva pacijenta
mutaciju u ANG genu (5.4%), jedan pacijent je imao mutaciju u TDP43 genu (2,7%), a
jedan kombinovanu mutaciju c9orf72, FUS i ANG (2,7%). Svi SOD1 pozitivni pacijenti
imali su spinalni početak bolesti i u većini slučajeva (85,2%) prvi simptomi su bili u
donjim udovima. Bulbarni početak je bio češći kod SOD1 negativnih FALS pacijenata.
Senzitivni poremećaji su zabeleženi kod 14 (51,9%) SOD1 pozitivnih i dva (20,0%)
SOD1 negativna pacijenta (p>0,05). Poremećaji sfinktera registrovani su samo u SOD1
pozitivnoj grupi (p<0.01). Opšti kognitivni skrining je pokazao lošije rezultate kod ALS
u poređenju sa zdravima (p<0.01). SOD1 pozitivni pacijenti su postigli znatno niži
rezultat na egzekutivnom testu nego zdrave kontrole (p <0.01). Sa druge strane, SOD1
negativni pacijenti su pokazali lošiji rezultat u: opštem intelektualnom nivou, verbalnoj i
vizuospatijalnoj memoriji, egzekutivnoj funkciji (p<0.01), pažnji, kao i jezičkim
funkcijama (p<0.05) u poređenju sa zdravim kontrolama. Nisu utvrđene značajne
razlike između pacijenata sa SOD1 ALS, SALS pacijenata i zdravih kontrola u debljini
korteksa. U poređenju sa zdravim kontrolama, SALS pacijenti imali su značajno
smanjenje frakcione anizotropije i levog i desnog kortikospinalnog puta (p=0,001 i
p=0,02) i povećanu radijalnu difuzivnost levog kortikospinalnog puta (p=0,01).
Pacijenti sa SOD1 ALS imali su promene koje su bile između kontrola i sALS
pacijenata (p=0,06). Posmatrajući senzorimotornu mrežu, SALS pacijenti su pokazali
povećanu funkcionalnu povezanost desnog precentralnog korteksa u poređenju sa
kontrolama, što pokazuju i voxel-wise analiza i analiza regiona od interesa (ROI)
(Grafikon 2). Uz to, ROI analiza je pokazala povećanu funkcionalnu povezanost i levog
precentralnog korteksa pacijenata sa SALS u odnosu na kontrole (p=0,05). SOD1 ALS
pacijenti imali su značajno smanjenje srednje vrednosti površine poprečnog preseka
cervikalne kičmene moždine od nivoa C2 do nivoa C7 u odnosu na SALS pacijente.
Zaključak: U populaciji pacijenata sa ALS na prostoru Srbije, oko 15% je imalo
genetsku osnovu bolesti, pri čemu su mutacije u SOD1 genu činile više od dve trećine.
Pacijenti sa SOD1 ALS su često imali spinalni početak bolesti, senzorne i sfinkterijalne
smetnje, egzekutivnu disfunkciju, te oštećenje kortikospinalnog puta i atrofiju kičmene
moždine. SOD1 negativni FALS pacijenti su obično imali bulbarni početak bolesti i širu
afekciju kognitivnih funkcija. | Amyotrophic lateral sclerosis (ALS) is caused by damage of motor
neurons in bran cortex, brainstem and the spinal cord. About 90% of ALS patients have
sporadic disease form (SALS), while 5-10% of patients have a genetic or familial
disease (FALS). So far, more than 20 genes have been associated with ALS. The
epidemiological and clinical characteristics of certain forms of FALS in Serbia are not
known. Furthermore, there is a lack of data on central nervous system (CNS)
involvement in FALS worldwide, both in terms of neuroimaging and cognitive testing.
The objectives of this research were: molecular-genetic testing of ALS patients in
Serbia; determination of typical clinical signs in subpopulation of patients with FALS;
correlations of clinical and genetic characteristics, including neuropsychological testing
and morphological specificities of the brain and spinal cord analyzed using modern
magnetic resonance imaging (MRI) methods.
Method: This study included 241 patients from the Neurology Clinic, Clinical Center of
Serbia. Patients were tested for the following mutations: SOD1 (all 5 exons), TDP 43
(exon 6), FUS (exon 14 and 15), c9orf72 and ANG. A genetic group of 37 subjects was
compared clinically with 32 SALS patients without a verified mutation.
Neuropsychological testing was performed in 22 SOD1 positive subjects, 5 SOD1
negative subjects positive for other ALS genes and 82 healthy controls (HCs). Cognitive
screening, as well as tests of general intellectual level, attention, memory, visuospacial,
executive and linguistic functions were applied. Of the 69 patients with ALS, MRI
examination of the brain and cervical spinal cord on the 1.5T equipment were conducted
on 23 genetically determined patients and 11 SALS subjects. The control group was
composed of 34 subjects macthed for gender and age. Cortical thickness analysis, white
matter microstructural assessment, functional MRI at resting state, and the analysis of
the spinal cord were made.
Results: A total of 37 (15.4%) of 241 patients had one or more mutations in tested ALS
gene. In the genetic group, 27 patients had a mutation in the SOD1 gene (72.9%), six
patients had a mutation in the c9orf72 gene (16.2%), two in the ANG gene (5.4%), one
in the TDP43 gene (2.7%), and one had combined mutations of c9orf72, FUS and ANG
(2.7%). All SOD1 positive patients had the spinal onset of the disease, and in most
cases (85.2%), the first symptoms were in the lower limbs. The bulbar start was more
common in SOD1 negative FALS patients. Sensory disorders were observed in 14
(51.9%) SOD1 positive and two (20.0%) SOD1 negative patients (p>0.05). Sphincter
disorders were registered only in the SOD1 positive group (p<0.01). General cognitive
screening showed poorer results in ALS compared to HCs (p<0.01). SOD1 positive
patients achieved a significantly lower result on an executive tests than HCs (p<0.01).
On the other hand, SOD1 negative patients showed worse results in: general intellectual
level, verbal and visuospatial memory, executive functions (p <0.01), attention and
linguistic functions (p<0.05) compared to HCs. Significant differences were not found
between patients with SOD1 ALS, SALS patients and HCs regarding cortical thickness.
Compared to HCs, SALS patients had a significant reduction in fractional anisotropy in
both left and right corticospinal pathways (p=0.001 and p=0.02) and increased radial
diffusivity of the left corticospinal pathway (p=0.01). Patients with SOD1 ALS were
between controls and SALS patients (p=0.06). Regarding the sensorimotor network,
SALS patients showed an increased functional correlation of the right prefrontal cortex
compared to controls, which is shown both by voxel-wise analysis and analysis of the
region of interest (ROI). In addition, ROI analysis showed increased functional
correlation of left prefrontal cortex of patients with SALS compared to controls
(p=0.05). SOD1 ALS patients compared to SALS patients had a significant reduction in
mean surface area of the cervical spinal cord cross-section from level C2 to level C7.
Conclusion: In the population of ALS patients in Serbia, about 15% had a genetic basis
of the disease, with mutations in the SOD1 gene comprising more than two thirds of
FALS cases. Patients with SOD1 ALS often had spinal disease onset, sensory and
sphincteric disturbances, executive dysfunction, as well as corticospinal damage and the
spinal cord atrophy. SOD1 negative FALS patients usually had bulbar onset and a
broader cognitive dysfunction. | true |
Uticaj prolaktina na ćelije trofoblasta na čoveka in vitro | The effect of prolactin on human trophoblast in vitro | Prolaktin (PRL) je polipeptidni hormon koji utiče na rast i diferencijaciju različitih
ćelija, a uključen je i u brojne fiziološke procese. Sintetiše ga i luči hipofiza, ali i različita
druga tkiva i ćelije. Prolaktin je jedan od glavnih proteina koji sintetiše i sekretuje
decidualizovani endometrijum. Produkcija decidualnog prolaktina se povećava u trudnoći
nakon implantacije, i dostiže maksimum u tkivu decidue 20 do 25 nedelje gestacije.
Delovanje PRL se ostvaruje vezivanjem za PRL receptore (PRLR) na ćelijskoj
membrani. Poznato je da se za prolaktinski receptor vezuje najmanje tri liganda - prolaktin,
placentni laktogen i hormon rasta. Do sada je za placentu poznato da je receptor za
prolaktin ispoljen u decidui, trofoblastu horiona, epitelu amniona i sinciciotrofoblastu na
kraju trudnoće. Prolaktin u različitim ćelijama i tkivima utiče na ekspresiju adhezionih i
proteolitičkih molekula koji su značajni za degradaciju ekstraćelijskog matriksa i ćelijsku
migraciju. Međutim, njegova uloga u trofoblastu nije dovoljno poznata.
Ovim radom je po prvi put ispitivan uticaj prolaktina na svojstva invazivnog
ekstravilusnog trofoblasta prvog trimestra trudnoće. Detektovana je ekspresija PRLR, kao i
profil prisutnih formi receptora na izolovanim citotrofoblastnim ćelijama i HTR-8/SVneo
trofoblastnoj imortalizovanoj ćelijskoj liniji. Pokazali smo da PRL stimuliše adheziju,
migraciju i invaziju trofoblasta in vitro. Kao potencijalne efektore ispitivali smo integrine,
gal-1 i matriksne metaloproteinaze-2 i -9. Dobijeni rezultati ukazuju da PRL stimuliše
ekspresiju subjedinica integrina α1, α5, kao i gal-1. Ispitivali smo i efekat PRL na
vijabilnost, proliferaciju i apoptozu HTR-8/SVneo ćelija. PRL je blago stimulisao
vijabilnost i uticao na povećanje broja adherentnih ćelija, dok apoptoza nije bila značajno
promenjena.
Dobijeni rezultati predstavljaju napredak u razumevanju fiziološke uloge PRL
značajne za funkciju humanog trofoblasta. Takođe, ovaj rad pruža celovitiji uvid u proces
diferencijacije ranog trofoblasta i predstavlja dobru osnovu za dalja ispitivanja biološke
uloge prolaktina, kao i drugih članova ove familije proteina u humanom trofoblastu. | Prolactin (PRL) is a polypeptide hormone which has impact on the growth and
differentiation of various cell types, and is known to participate in numerous physiological
processes. It is synthesized and secreted by the pituitary gland and many other tissues and
cell types. Prolactin is also one of the major proteins synthesized and secreted by
decidualized endometrium. The production of decidual prolactin increases after
implantation, and peaks in decidual tissue at 20 to 25th week of gestation.
The action of PRL is exerted through binding to its receptors (PRLR) at the surface
of target cells. It is known that prolactin receptor has at least three ligands – prolactin,
placental lactogen and growth hormone. Regarding localization of prolactin receptor in
placenta, so far it has been found in decidua, chorionic trophoblast, amniotic epithelium and
synciciotrophoblast at term. Prolactin affects the expression of adhesion and proteolytic
molecules which are important for degradation of extracellular matrix and cell migration.
However, its role in trophoblast is not well defined.
In this study the effect of prolactin on the function of first trimester of pregnancy
extravillous trophoblast was studied for the first time. The expression of PRLR, as well as
the profile of different isoforms was examined in both cytotrophoblast and HTR-8/SVneo
trophoblast derived cell line. It is shown here that PRL stimulates trophoblast cell adhesion,
migration and invasion in vitro. Potential effectors were sought among integrins, gal-1 and
matrix metalloproteinases-2 and -9. The results showed that PRL stimulated the expression
of integrin subunits α1, α5, as well as gal-1. When investigating the effect of PRL on cell
viability, proliferation and apoptosis of HTR-8/SVneo cells, PRL was found to slightly
stimulate cell viability and adherent cell number, while apoptosis was not altered.
The results of this study represent a step further in the understanding of the
physiological role of PRL in human trophoblast. Moreover, this work gives a better insight
in to the process of early trophoblast differentiation and represents a good basis for further
studies of the biological role of prolactin, as well as other members of this family of
proteins in the human trophoblast. | true |
Identifikacija psihijatrijskih i psiholoških prediktora efikasnosti medicinske nutritivne terapije gojaznosti | Identification of psychiatric and psychological predictors of medical nutrition therapy for obesty outcome | Gojaznost je hronično oboljenje sa značajnim nepovoljnim efektima na zdravlje, a broj gojaznih osoba u svetu dostigao je epidemijske razmere. Medicinska nutritivna terapija predstavlja terapiju izbora kod lečenja gojaznosti, ali je njena uspešnost ograničena. Praćena je značajnim procentom odustajanja kao i ponovnim dobijanjem na težini. Cilj ove studije je bio da se proceni da li psihijatrijski i specifični psihološki faktori mogu da utiču na uspešnost odnosno neuspešnost terapije. Metode: Studijom su obuhvaćene ukupno 84 žene predgojazne i gojazne I stepena starosne dobi 20 do 40 godina, koje su bile motivisane za medicinsku nutritivnu terapiju. Sve ispitanice su prošle individualno savetovanje i dobile dnevni nutritivno balansiran plan ishrane koji je podrazumevao kalorijsku restikciju od 300-500kcal u odnosu na procenjene dnevne energetske potrebe. Na početku studije primenjeni su upitnici: Lista simptoma od 90 pitanja (90-item Symptom Check-list) i Baratova skala impulsivnosti (Barratt Impulsiveness scale - BIS 11), Skala opštih stavova i verovanja (The General Attitude and Belief Scale - GABS 55), Upitnik frustracione netolerancije (The Frustration Discomfort Scale - FDS) kao i za ovu studiju dizajniran socio-demografski upitnik. Nakon 6 meseci primene kalorijski restriktivnog načina ishrane ponovljena su antropometrijska merenja. Ispitanice su podeljene u dve grupe: 40 ispitanica (48 %) koje su izgubile ≥ 10% od početne telesne mase (Uspešne) i druga od 44 ispitanice (52 %) koje su izgubile < 10% od početne telesne mase (Neuspešne). Rezultati. Nije bilo značajne razlike između grupa ispitanica u odnosu na sociodemografske karakteristike (godine života, stepen obrazovanja, zaposlenost, bračni status, navika pušenja, uzimanje alkohola), antropometrijske parametre na početku studije kao i u skoru opšte psihopatologije i dimenzija simptoma... | Obesity is a chronic disease and health threatening condition. The numbers of obese people in the world have taken epidemic proportions. Medical nutritional therapy is the first choice in the treatment of obesity but it is also accompanied with a great percentage of attrition and a significant weight regain. The aim of our study was to evaluate if psychiatric and specific psychological factors could be the predictors of successful weight loss. Method. Study sample consisted of eighty four consecutive overweight/obese women, 20 to 40 years old, who were willing to adhere to medical nutrition therapy. All participants received a personalized nutritional counseling and daily balanced diet treatment with treatment with 300-500 caloric restriction from estimated daily energy requirement. At the beginning of the study 90-item Symptom Check-list (SCL-90), 30-item Barratt Impulsiveness scale (BIS-11), The General Attitude and Belief Scale (GABS 55), The Frustration Discomfort Scale - FDS as well as the questionnaire with socio-demographic data designed for this study were administrated. At the end of six months of caloric restriction conventional diet therapy and control weight measurement, patients were divided into two groups: 40 participants (48 %) who lost ≥10% of their initial body weight (Successful) and 44 participants (52 %) who lost <10% of their initial weight (Unsuccessful). Results. There were no significant differences between both groups in demographic data (age, level of education, employment, marital status, smoking, alcohol consumption), baseline anthropometric parameters and general psychopathology as well as symptom dimensions. Successful group had higher levels of pshisical activity... | false |
Uticaj starenja i dugotrajne dijetalne restrikcije na metabolizam holesterola u prednjem mozgu pacova | Effects of aging and long-term dietary restrictions on cholesterol metabolism in the rat forebrain | Starenje centralnog nervnog sistema (CNS) praćeno je brojnim promenama koje
dovode do narušavanja nervnih funkcija, pre svega kontrole motorike i kognitivnih
sposobnosti. Smatra se da su kognitivni poremećaji tokom starenja posledica smanjene
sinaptičke plastičnosti, naročito u korteksu i hipokampusu, regionima mozga izuzetno
važnim za procese učenja i pamćenja. Najvažniji faktor sredine kojim se može uticati na
starenje uopšte, a samim tim i na starenje mozga je režim redukovane ishrane, bez
pothranjenosti. Pored toga što se već nekoliko decenija zna da redukovana ishrana
produžava životni vek mnogih vrsta, uključujući i sisare, pokazano je da ona odlaže i
ublažava starosno zavisne promene u nervnom sistemu.
Holesterol je najzastupljeniji lipid u ćelijama sisara. Učestvujući u izgradnji
fosfolipidnog
dvosloja
bioloških
mambrana,
on
reguliše
njihovu
fluidnost,
propustljivost i rigidnost, a samim tim i funkcionalna svojstva membranskih proteina,
poput jonskih kanala i transmiterskih receptora. Metabolizam holesterola predstavlja
važan aspekt sinaptičke plastičnosti, jer je pokazano da je holesterol neophodan za
biogenezu i transport sinaptičkih vezikula, kao i za prvilnu funkcionalnu organizaciju
lipidnih ostvaca. Od svih organa u telu, mozak sadrži najviše holesterola. Održavanje
homeostaze holesterola neophodno je za normalno i neometano funkcionisanje CNS.
Cilj ovog istraživanja bio je da se vidi kako starenje i dugotrajna dijetalna
restrikcija (DR) utuču na metabolizam holesterola u mozgu. Mužjaci pacova soja Wistar
stari 3 meseca (m) bili su podeljeni u dve grupe. Prva grupa, označena kao ad libitum
(AL), dobijala je svakog dana 100% dnevnog unosa hrane, dok je druga grupa
(označena kao DR) stavljena na režim redukovane ishrane koji je podrazumevao
dobijanje 100% dnevnog unosa hrane svakog drugog dana. Životinje su žrtvovane
nakon dostizanja starosti od 12 i 24m. Životinje stare 3m su predstavljale kontrolnu
starosnu grupu. Metabolizam holesterola određivan je na nivou proteina koji su
uključeni u sintezu holesterola (HMGCR), njegov transport (ApoE), katabolizam
(CYP46) i regulaciju (LXRα) metodom Western blota i imunohistohemijski.
Zastupljenost
samog
holesterola,
njegovih
prekursora
(lanosterol,
latosterol,
dezmosterol) i metabolita (24S-hidroksiholesterol, 27-hidroksiholesterol, holestanol)
određena je gasnom hromatografijom-masenom spektrometrijom (GC-MS). U
primenjenim eksperimentalnim uslovima istom metodom analizirana je i zastupljenost
biljnih sterola u svetlu sve veće upotrebe hrane obogaćene biljnim analozima holesterola
u ljudskoj ishrani. Pored toga, praćena je i veza između sadržaja holesterola i
neurotrofinske signalizacije.
Praćenje promena u metabolizam holesterola pokazalo je da u serumu tokom
starenja dolazi do povećanja količine holesterola, bez uticaja režima ishrane. Dugotrajna
DR delovala je suprotno starosno-zavisnom povećanju količine holesterolskih
prekursora i holestanola, održavajući njihov nivo na nivou 3m starih životinja.
Zastupljenost 24S-hidroksiholesterola nije bila promenjena u analiziranim uslovima,
dok dijeta nije imala uticaja na povećanje količine 27-hidroksiholesterola tokom
starenja. Tokom starenja je takođe došlo do povećanja količine ispitivanih biljnih sterola
(sa izuzetkom brasikasterola), dok je DR režim održavao njihov nivo na nivou 3m
starih životinja.
Rezultati su pokazali da je tokom starenja došlo do smanjivanja količine
proteina HMGCR u korteksu pacova, bez obzira na režim ishrane, kao i do smanjenja
nivoa proteina LXRα, koje je bilo pojačano dijetalnom restrikcijom. Količina proteina
ApoE bila je povećana tokom starenja. Dijeta u grupi životinja starih 12m delovala je
suprotno starenju, smanjujući količinu ovog proteina u odnosu na starosnu kontrolu.
Nivo proteina CYP46 bio je povećan u grupi životinja starih 24m koje su bile
podvrgnute dijeti. U hipokampusu pacova tokom starenja došlo je do smanjenja količine
proteina HMGCR, CYP46 i LXRα, bez obzira na primenjeni režim ishrane. Ni starenje
ni DR nisu uticali na ćelijsku lokalizaciju ekspresije CYP46. Količina proteina ApoE u
grupi najstarijih pacova koji su bili na režmu DR bila je povećana i u odnosu na
starosnu kontrolu, i u odnosu na 3m stare životinje.
Tokom starenja u korteksu je došlo do povećanja količine holesterola, dok u
hipokampusu nisu detektovane pormene. Sa izuzetkom lanosterola u grupi 12mAL
holesterolski prekursori su bili manje zastupljeni tokom starenja u obe ispitivane
strukture. Dijetalna restrikcija u grupi 12mAL održavala je nivo lanosterola na nivou
kontrolnih 3m starih životinja. U grupi 24m starih životinja, pod uticajem dijete došlo je
do povećanja količine dezmosterola u hipokampusu u odnosu na starosnu kontrolu,
mada je ta količina i dalje bila manja od one izmerene kod 3m starih životinja. Količina
holestanola bila povećana tokom starenja bez obzira na režim ishrane. Količina 24S-
hidroksisterola nije se menjala ni tokom starenja, ni pod delovanjem dijete, ukazujući da
ispitivani eksperimentalni uslovi nisu uticali na katabolizam holesterola u mozgu
pacova.
Zastupljenost biljnih sterola u obe moždane strukture bila je povećana tokom
starenja, dok je redukovana ishrana delovala suprotno starenju, smanjujući njihovu
količinu u odnosu na odgovarajuće starosne kontrole. Jedino se količina brasikasterola u
korteksu nije menjala u analiziranim eksperimentalnim uslovima.
U korteksu pacova tokom starenja došlo je do povećanja količine proteina
BDNF, a do smanjenja nivoa njegovog aktiviranog receptora pTrkB. Dijetalna
restrikcija je u grupi životinja starih 12m dodatno povećala količinu BDNF, a u
najstarijoj grupi delovala suprotno starenju, održavajući količinu ovog trofina na
kontrolnom nivou. U 12m starim životinjama dijeta je održavala nivo aktiviranog
receptora na nivou izmerenom kod životinja starih 3m. Ukupan receptor u korteksu nije
se menjao u analiziranim uslovima. Promene u trofinskoj signalizaciji u hipokampusu
tokom starenja i pod uticajem dugotrajne dijete detektovane su samo kroz povećanje
samog proteina BDNF u grupi 12m starih životinja na režimu redukovane ishrane u
odnosu na 3 meseca stare životinje.
Rezultati dobijeni u okviru ove disertacije pokazuju da se metabolizam holesterola
u korteksu i hipokampusu pacova menja tokom starenja i pod delovanjem dugotrajne
dijetalne restrikcije. Najveće promene uočene su na nivou prekursora holesterola,
ukazujući na smanjenu stopu sinteze holesterola, dok eliminacija holesterola iz mozga
određena njegovim katabolitom, 24S-OHC, nije bila promenjena pod datim
eksperimentalnim uslovima. Uticaj dugotrajne dijetalne restrikcije na metabolizam
holesterola bio je izraženiji u hipokampusu, nego u korteksu, i bio je sličan uticaju dijete
ispoljenom u serumu pacova. Veza između holesterola i neurotrofinske signalizacije bila
je izraženija u korteksu nego u hipokampusu. Rezultati ove studije pokazali su kapacitet
dugotrajne dijetalne restrikcije da utiče na metabolizam holesterola u mozgu smanjujući
njegovu sintezu, pri čemu količina holesterola ostaje nepromenjena, ukazujući na
mogućnost korišćenja redukovane ishrane u regulaciji metabolizma holesterola kako u
periferiji, tako i u mozgu. | Ageing of the central nervous system is associated with a number of changes
that disturb nerve function, especially motor control and cognitive abilities. It is
believed that cognitive disorders in aging are consequence of reduced synaptic
plasticity, especially in the cortex and hippocampus, brain regions extremely important
for learning and memory processes. Dominant factor that can influence aging in general,
and therefore the aging of the brain is reduced diet regime, without malnutrition. In
addition to being known for decades that reduced caloric intake prolongs life span of
many species, including mammals, it is shown that it delays and alleviates age-
dependent changes.
Cholesterol is the most abundant lipid in mammalian cells. By participating in
the construction of phospholipid bilayer of biological membranes, it regulates their
fluidity, permeability and rigidity, and consequently the functional properties of
membrane proteins, such as ion channels and transmmitters receptors. Cholesterol
metabolism represents one aspect of synaptic plasticity, as has been shown that
cholesterol is essential for biogenesis of synaptic vesicles, and vesicle transport, as well
as for proper functional organization of lipid rafts. The brain contains the most
cholesterol of all the organs in the body. Maintenance of cholesterol homeostasis is
essential for proper functioning of the central nervous system.
The aim of this study was to analyze the influence of aging and long-term
dietary restriction on cholesterol metabolism in the rat serum, cortex and hippocampus.
The experiments were performed on 3-, 12-, and 24-month-old male Wistar rats fed ad
libitum (AL), or exposed to long term DR (100% every other day-EOD) starting from
the age of 3. At the indicated time points, the rats were killed by decapitation. The
expression of proteins involved in cholesterol synthesis (HMGCR), transport (ApoE),
catabolism (CYP46) and regulation (LXRα) was determined using Western blot and
immunohistochemistry. The levels of cholesterol, its precursors (lanosterol, lathosterol,
desmosterol)
and
metabolites
(24S-hydroxycholesterol,
27-hydroxycholesterol,
cholestanol) were determined by gas chromatography/mass spectrometry (GC/MS). The
same method was used to analyze the amount of phytosterols, given the increasing use
of foods enriched with plant analogues of cholesterol in human nutrition. Additionally,
the link between cholesterol content and neurotrophin signaling in the rat cortex and
hippocampus was analyzed at protein level.
In rat serum cholesterol levels were increased during ageing, regardless of diet
regime. Long-term dietary restriction counteracted age-dependent increase in the
amount of cholesterol precursors, maintaining their level at the one measured in 3
month old animals. The same influence of dietary restrictions was detected on the age-
dependent increase in the cholestanol levels. The amount of 24S-hydroxycholesterol
remained stable under experimental conditions, while dietary restriction had no effect
on elevated 27-hydroxycholesterol levels during ageing. Ageing has led to increased
phytosterols levels, while reduced dietary intake maintained their content on control
levels measured in 3-month-old animals.
During aging, there was a reduction in HMGCR protein in the rat cortex,
regardless of the diet, and reduction in the levels of LXRα protein, which has been
reinforced by dietary restriction. The amount of ApoE protein was increased during
ageing. Dietary restriction in the group of 12-month-old animals acted contrary to aging,
reducing the amount of this protein in relation to age control. CYP46 protein levels
were increased in the group of 24-month-old animals exposed to diet. In the rat
hippocampus aging has caused decreased levels of HMGCR, CYP46 and LXRα
proteins, regardless of the diet. The amount of ApoE protein in the oldest group of rats
that were exposed to dietary restriction was increased in comparison to age-control, and
compared to 3 month old animals.
Aging induced slight but significant increase of cholesterol content in the rat
cortex, while there were no changes in the amount of cholesterol in the hippocampus.
With the exception of lanosterol at 12- month- old AL- fed animals, cholesterol
precursors were less common in aging in both brain regions examined. Dietary
restriction in the group 12mAL animals prevented the increase of lanosterol amount,
keeping it at the level detected in 3-month-old animals. In the hippocampus of 24-
month old animals, reduced diet intake increased desmosterol amount in relation to age
control, although the amount was still less than that measured in 3-month-old controls.
Cholestanol content was increased during ageing, regardless of diet regime. The levels
of 24S-hydroxycholesterol remained stable under experimental conditions, indicating
that experimental conditions had no influence on brain cholesterol catabolism.
Amount of plant sterols in both brain structures was increased during aging,
while the reduced diet intake acted contrary to the aging, decreasing their levels in
relation to age-appropriate controls. Only the amount of brassicasterol in the cortex did
not change in the analyzed experimental conditions.
In the rat cortex ageing influenced increased BDNF protein levels, and
decreased levels of activated receptor pTrkB. Dietary restriction in the group of 12-
month-old animals further increased the amount of BDNF, while in the oldest group
acted contrary to aging, keeping the amount of this trephine on the control level. At 12-
month-old animals dietary restriction maintained levels of activated receptors at the one
measured in animals aged 3 months. The total receptor in the cortex did not change in
the analyzed conditions. Changes in trephine signaling in the hippocampus during aging
and under the influence of long-term dietary restriction were detected only through the
increase in BDNF protein in the group 12 months old animals exposed to reduced
feeding compared to 3 month old animals.
The results obtained in this thesis showed that the cholesterol metabolism in the
rat cortex and hippocampus have changed during ageing and under the influence of
long-term dietary restriction. The greatest changes were observed at the level of
cholesterol precursors, indicating a reduced rate of cholesterol synthesis, while the
elimination of cholesterol from the brain determined by its derivate, 24S-
hydroxycholesterol, was not altered under the experimental conditions. Effect of long-
term dietary restriction on cholesterol metabolism was more pronounced in the
hippocampus than in the cortex, and was similar to the impact of reduced dietary intake
manifested in the rat serum. The link between cholesterol and neurotrophine signaling
was more evident in the cortex than in hippocampus. The results of this study showed
the capacity of long-term dietary restriction to influence cholesterol metabolism in the
brain by reducing its synthesis, whereas the amount of cholesterol remained unchanged,
and point to long-term DR as a possible alternative to statin use for its ability to
preserve cholesterol homeostasis in the brain. | true |
Funkcionalne i imunofenotipske karakteristike NK ćelija regionalnih limfnih čvorova obolelih od melanoma i njihova in vitro aktivacija IL-2 i IL-15 | Functional and immunophenotypic characteristics of NK cells from regional lymph nodes of melanoma patients and their in vitro activation with IL-2 and IL-15 | Melanom, maligni tumor melanocita, iako ima brojna imunogenična
svojstva, predstavlja tumor kože sa najvišom incidencom smrtnosti. Za
anititumorsku imunost u melanomu posebno su značajne ćelije prirodne ubice (NK
ćelije), efektorske ćelije sistema urođene imunosti koje mogu neposredno da
prepoznaju maligno transformisane ćelije i da ih liziraju citolitičkim enzimima
(perforin i granzimi) kao i da produkcijom citokina i hemokina regulišu adaptivnu i
urođenu amtitumorsku imunost. NK ćelije su prisutne u mnogim tkivima i
organima. Kod melanoma su uglavnom proučavane NK ćelije u perifernj krvi i u
tumorskom tkivu, dok NK ćelije regionalnih limfnih čvorova (LČ) obolelih nisu do
sada ispitivane.
NK ćelije su CD3-CD56+ i obuhvataju dve funkcionalno različite subpopulacije,
imunoregulatornu, CD3-CD56sjajno+ i citotoksičnu, CD3-CD56potmulo+. Funkcija NK
ćelija je regulisna balansom signala koji potiču od aktivacionih i inhibitornih NK-
ćelijskih receptora. Aktivacioni receptori prepoznaju stresogene ligande na
tumorskim ćelijama, dok inhibitorni ubilački receptori slični imunoglobulinima
(engl. Immunoglobulin-like Killer Receptors, KIR) vezivanjem za MHC molekule
klase I inhibiraju NK ćelijsku funkciju i pored toga omogućavaju toleranciju NK
ćelija na ćelije sopstvenog organizma.
NK ćelije su prisutne u T ćelijskoj zoni LČ, gde se odvija njihovo sazrevanje i
diferencijacija. U fiziološkim uslovima u LČ perifernom cirkulacijom dospeva
uglavnom nezrela (CD3-CD56sjajno+) NK ćelijska subpopulacija koja čini veći deo
NK ćelijske populacije u mirujućim limfnim čvorovima. Pod uticajem endogenih
citokina u LČ ova subpopulacija sazreva u citotoksičnu CD3-CD56potmulo+
perforin+CD16+KIR+ subpopulaciju koja zatim napušta LČ. U patološkim stanjima
dolazi do povećane zastupljenosti CD16+KIR+ NK ćelija u reaktivnim LČ što može
biti posledica povećane produkcije citokina urođene imunosti od strane dendritičnih
ćelija, kao i Th1 citokina od strane stimulisanih T ćelija antigenima u LČ, kao i
selektivne migracije CD16+KIR+ NK ćelija u LČ.
Među citokinima γc familije, interleukini IL-2 i IL-15 su posebno značajni za
diferencijaciju i sazrevanje NK ćelija, a takođe su pokazali i najizraženiji
stimulativni efekat na citotoksičnu aktivnost NK ćelija.
Ciljevi rada: S obzirom na to da regionalni LČ predstavljaju prvu barijeru u
limfogenom širenju tumora kao i na značaj uticaja mikrosredine na funkciju i
fenotip NK ćelija, cilj ovog rada bio je da se uporede funkcionalne i
imunofenotipske karakteristike CD3-CD56+ NK ćelija u tumor-infiltrisanim
(infiltirsanim) i neinfiltrisanim regionalnim LČ bolesnika sa melanomom.
S obzirom na to da je melanom imunogeničan tumor koji je slabo osetljiv na
zračnu i na hemioterapiju, cilj ovog rada bio je da se ispita sposobnost IL-2,
citokina koji se već više od dve decenije primenjuje u lečenju metastatskog
melanoma i citokina novije generacije IL-15, koji je neophodan za razvoj i
sazrevanje NK ćelija, da indukuju ciototoksičnu aktivnost NK ćelija ispitivanih
regionalnih LČ.
Materijal i metode: Uzorci tkiva regionalnih LČ 45 bolesnika sa melanomom
kože u kliničkim stadijumima bolesti II-IV uzeti su neposredno nakon hirurške
intervencije. Iz uzetih uzoraka tkiva LČ izolovane su mononukleane ćelije (MNĆ).
Citotoksična aktivnost NK ćelija određivana je u odnosu na NK-osetljivu K562
tumorsku ćelijsku liniju radioaktivnim 51Cr testom iz sveže izolovanih MNĆ kao i
nakon 72 h i 7 dana in vitro tretmana medijumom za ćelijsku kulturu, medijumom
sa IL-2 (200 IU/ml) i medijumom sa IL-15 (25 ng/ml) na 37ºC u vlažnoj atmosferi
sa 5% CO2. Metodom protočne citometrije određivani su: produkcija IFNγ u CD3-
CD56+ NK ćelijama, procenat CD3-CD56+ NK ćelija, CD3-CD56potmulo+ i CD3-
CD56sjajno+ subpopulacija, kao i ekspresija aktivacionih (NKG2D, CD16),
inhibitornih KIR (CD158a, CD158b) receptora kao i aktivacionih antigena (CD25,
CD69, HLA-DR) na CD3-CD56+ NK ćelijama i njihovim subpopulacijama. RT-
PCR metodom nakon 72 h in vitro tretmana ovim citokinima je praćena
transkripcija gena za perforin, a Western blot metodom ekspresija perforina,
pSTAT1, pSTAT5 i Bcl-2 molekula. Za poređenja ispitivanih parametra
infiltrisanih u odnosu na neinfiltrisane LČ korišćen je statistički Mann Whitney
test, a značajnost promena nakon tretmana citokinima je utvrđivana Wilcoxon
testom sume rangova. U statističkoj analizi korelacione povezanosti korišćen je
Spearman test.
Rezultati: U neinfiltrisanim i i infiltrisanim regionalnim LČ bolesnika sa
melanomom dobijena je slična niska citotoksična funkcija NK ćelija. Produkcija
IFNγ kao drugog merila funkcionalnosti NK ćelija, snižena u ukupnoj populaciji
CD3-CD56+ NK ćelija samo u tumor-infiltrisanim LČ što može biti posledica
prisustva imunosupresivnih citokina i drugih biološki aktivnih molekula koji kod
ovih bolesnika mogu biti kako lokalnog, tako i sistemskog porekla. U infiltrisanim
LČ nađena je veća zastupljenost CD3-CD56+ NK ćelija usled povećanja
zastupljenosti CD3-CD56potmulo+ subpopulacije. Ovaj rezutat zajedno sa povećanjem
zastupljenosti CD16+ i CD158b+ CD3-CD56+ NK ćelija u infiltrisanim LČ ukazuje
na zreliji fenotip NK ćelija usled ulaska tumora u LČ. Nađena povećana ekspresija
CD69 antigena na CD3-CD56+ NK ćelijama u infiltisanim LČ ukazuje na aktivaciju
ove populacije sa invazijom tumora u LČ, pri čemu ekspresija NKG2D
citotoksičnog receptora ostaje nepromenjena. Ove razlike u imunofenotipskim
parametrima usled infiltrisanosti LČ tumorskim ćelijama su više ispoljene kada je
zahvaćen veći broj LČ kao i kada je stepen invazije primarnog tumora u kožu veći.
Nakon in vitro tretmana citokinima pokazano je da za razliku od tretmana
interleukinom IL-15, tretman interleukinom IL-2 dovodi do porasta ukupne
populacije CD3-CD56+ NK ćelija uvećanjem CD3-CD56sjajno+ subpopulacije u
neinfiltrisanim i u infiltrisanim regionalnim LČ bolesnika sa melanomom. In vitro
tretmani interleukinima IL-2 i IL-15 višestruko povećavaju nisku citotoksičnost NK
ćelija, kao i transkripciju i sintezu perforina u obe grupe regionalnih LČ. Ova dva
citokina na CD3-CD56+ NK ćelijama obe grupe regionalnih LČ povećavaju
ekspresiju ranog aktivacionog antigena CD69, citotoksičnog NKG2D receptora, a
povećanjem ekspresije CD16 i CD158b inhibitornog KIR-a doprinose njihovom
zrelijem fenotipu. Uočeni efekti ispitivanih citokina praćeni su povećanjem nivoa
fosforilisanog STAT5 signalnog molekula.
Zaključak: U ovom radu opisani su funkcija i fenotip do sada neispitivane
populacije NK ćelija regionalnih LČ bolesnika sa melanomom. Dobijeni rezultati
ukazuju na to da metastiziranje melanoma u regionalne LČ nije dovelo do promene
citotoksične funkcije prisutnih NK ćelija, već do povećane zastupljenosti CD3-
CD56+ NK ćelija zrelijeg CD16+CD158b+ fenotipa. In vitro tretmanima sa IL-2,
citokinom koji se primenjuje u terapiji ovog tumora i sa IL-15, citokinom čija se
terapijska primena još uvek ispituje, postignuto je višestruko povećanje
citotoksične aktivnosti NK ćelija regionalnih LČ bolesnika sa melanomom koje
može da ukaže na potencijalni značaj ove populacije NK ćelija u novijim
terapijskim pristupima kao što je adoptivni tarnsfer NK ćelija kod bolesnika sa
melanomom. | Introduction: Melanoma, malignant neoplasm of melanocytes, despite of its
immunogenicity is skin cancer with the highest mortality rate. In antitumor
immunity im melanoma, natural killer (NK) cells as innate immune system effector
cells play an important role as they are able to recognize malignantly transformed
cells, lyse them by cytolitic enzymes (perforin and gramzymes) and also regulate
adaptive and innate antitumor immunity by production of cytokines and
chemokines. NK cells are present in many tissues and organs. In melanoma NK
cells have been studied mostly in peripheral blood and in tumor tissue, while NK
cells in regional lymph nodes (LN)s have not been investigated.
NK cells are CD3-CD56+ and comprise two functionally distinct subsets,
immunoregulatory, CD3-CD56bright+ and cytotoxic, CD3-CD56dim+. NK cell
function is regulated by the balance of signals mediated by activating and
inhibitory NK cell receptors. Activating receptors recognize stress induced ligands
on tumor cells, while the inhibitory immunoglobulin-like killer receptors (KIR) by
binding to MHC class I molecules inhibit NK cell function and enable self
tolerance.
NK cells are located mostly in T cell zone of LNs, where NK cell
differentiation and maturation may occur. In physiological conditions, by
peripheral circulation mostly immature CD3-CD56bright+ NK cell subset migrates to
LNs and represents the majority of NK cell population in resting LNs. Endogenous
cytokines in LN mediate maturation of this subset into cytotoxic CD3-CD56dim+
perforin+CD16+KIR+ subset that subsequently leaves LN. In pathological
conditions, increase in CD16+KIR+ NK cell population in reactive LNs may be the
consequence of increased production of innate immunity cytokines by dendritic
cells, as well as Th1 cytokine production by antigen stimulated T cells in LNs, or
selective migration of CD16+KIR+ NK cells into LNs.
Among γc cytokines, IL-2 and IL-15 are most important for NK cell
differentiation and maturation, and are also the most potent stimulators of NK cell
cytotoxic activity.
Objectives: As regional LNs represent first barrier to lymphogenic tumor
invasion and considering the importance of microenvironment in shaping NK cell
function and phenotype, the aim of this study was to compare functional and
immunophenotypic characteristics of CD3-CD56+ NK cells from tumor-infiltrated
(infiltrated) and not infiltrated regional LNs of melanoma patients.
Since melanoma is an immunogenic tumor insensitive to irradiation and
chemotherapy, the aim of this study was to investigate the ability of IL-2, cytokine
that has been used more than two decades in metastatic melanoma treatment and
IL-15, cytokine of novel generation which is essential for NK cell differentiation
and maturation, to induce cytotoxic activity of NK cells from investigated regional
LNs.
Material and methods: Tissue samples of regional LNs were obtained from 45
melanoma patients in clinical stages II-IV immediately after surgical intervention.
Mononuclear cells (MNC) were isolated from obtained LN samples. NK cell
cytotoxic activity was evaluated against NK-cell sensitive K562 tumor cell line
using radioactive 51Cr test on freshly isolated MNC as well as after 72 h and 7 day
in vitro treatments in cell culture medium alone, medium supplemented with IL-2
(200 IU/ml) and with IL-15 (25 ng/ml) on 37ºC in humid atmosphere with 5% CO2.
Flow cytometry method was used for evaluation of: IFNγ production in CD3-
CD56+ NK cells, percentage of CD3-CD56+ NK cells, CD3-CD56dim+ and CD3-
CD56bright+ NK cell subsets, as well as the expression of activating receptors
(NKG2D, CD16), inhibitory KIRs (CD158a, CD158b) and activation antigens
(CD25, CD69, HLA-DR) on CD3-CD56+ NK cells and their two subsets. After 72 h
in vitro cytokine treatments perforin transcription was estimated by RT-PCR, while
perforin, pSTAT1, pSTAT5 and Bcl-2 expression was estimated by Western blot.
The investigated parameters between infiltrated and not infiltrated regional LNs
were compared by statistical Mann Whitney test, while the significance of
differences after cytokine in vitro treatments was evaluated by Wilcoxon rank sum
test. For correlation analysis Spearman's correlation coefficient was evaluated.
Results: In both not infiltrated and tumor- infiltrated regional LNs of melanoma
patients were shown similar low levels of NK cell cytotoxic activity. Production of
IFNγ, as another aspect of NK cell function, in entire CD3-CD56+ NK cell
population was lower only in tumor-infiltrated LNs which could be influenced by
immunosupresive cytokines and other biologically active molecules that might be
of local or systemic origin. Tumor-infiltrated LNs also show higher percentage of
CD3-CD56+ NK cells due to the increased presence of CD3-CD56dim+
subpopulation. This finding, together with increased percentage of CD16+ and
CD158b+ CD3-CD56+ NK cells in tumor-infiltrated LNs, indicates more mature NK
cell phenotype in LNs invaded by tumor cells. Furthermore, CD3-CD56+ NK cells
in infiltrated LNs show increased expression of CD69 antigen which may indicate
NK cell activation alongside with tumor invasion into LNs, while the expression of
NKG2D cytototoxic receptor remained similar. The obtained differences in
immunophenotypic parameters caused by tumor infiltration of LN are more
apparent when more regional LNs are involved as well as if the degree of primary
tumor invasion into skin was higher.
After cytokine in vitro treatments it was shown that unlike IL-15, IL-2
treatment increased the percentage of CD3-CD56+ NK cell population by increasing
CD3-CD56bright+ subset in both not infiltrated and tumor-infiltrated regional LNs of
melanoma patients. Both IL-2 and IL-15 in vitro treatments increased low NK cell
cytotoxicity, as well as perforin transcription and synthesis in both groups of
regional LNs. These two cytokines on CD3-CD56+ NK cells in both regional LN
groups increased the expression of early activating antigen CD69, cytotoxic
NKG2D receptor, and also by increasing the expression of CD16 receptor and
CD158b inhibitory KIR contributed to more mature NK cell phenotype. The effects
of investigated cytokines are followed by increased level of phosphorylated STAT5
signaling molecule.
Conclusion: In this study were analyzed the function and immunophenotype of
NK cell population from regional LNs of malanoma patients which have not been
investigated before. The obtained results indicate that metastatic invasion of
melanoma into regional LNs did not alter cytotoxic function of resident NK cells,
while it increased the percentage of CD3-CD56+ NK cells of more mature
CD16+CD158b+ phenotype. In vitro treatments with IL-2, cytokine that has been
used in melanoma treatment and IL-15, cytokine that is still under investigation for
its therapeutic application, strongly induced the increase in NK cell cytotoxic
activity which may indicate the therapeutic potential of this NK cell population in
novel approaches such as adoptive NK cell transfer in melanoma patients. | true |
Predeono-ekološka analiza diverziteta i interakcija sistema žitne vaši - parazitoidi (Homoptera; Hymenoptera) | Landscape scale study of diversity and interaction in cereal aphids - parasitoids system (Homoptera; Hymenoptera) | Intenzifikacija poljoprivrede predstavlja jedan od globalnih procesa sa izrazito
negativnim efektima na biodiverzitet. Održavanje visokog nivoa biodiverziteta
neophodno je za obezbeđivanje ekosistemskih usluga, od kojih su neke, poput biološke
kontrole štetočina, od velike važnosti u agroekosistemima. Rezultati sve većeg broja
studija ukazuju na to da elementi i karakteristike kompozicije i konfiguracije
poljoprivrednih predela imaju ključnu ulogu u očuvanju, odnosno, narušavanju
agrobiodiverziteta. Predeono-ekološki pristup u agroekologiji proučava uticaj različitih
obrazaca i procesa u savremenoj poljoprivrednoj praksi na mehanizme degradacije
biodiverziteta i gubitak funkcionalnosti njegovih komponenti. Posebno je značajno
upoznati obrazce i trendove u trofičkim interakcijama koje povezuju intenzivno
korišćene agroekosisteme i okolna poluprirodna i prirodna staništa. U ovom
istraživanju, analizirani su efekti predeonih karakteristika na diverzitet i interakcije
model sistema žitne vaši – parazitoidi na području Pančevačkog rita. Istraživanje je bilo
podeljeno u tri dela kako bi se ispitali efekti tri različita aspekta predeone heterogenosti.
Cilj prvog dela istraživanja bio je određivanje efekta predeone kompleksnosti izražene
preko procenta prirodnih i poluprirodnih staništa (heterogenost tipova staništa) na model
sistem. Odabrane su dve jasno diferencirane kategorije predela: 1) kompleksni predeli
(>50% poluprirodnih staništa) i 2) jednostavni predeli (<30% poluprirodnih staništa)
kako bi se testirala razlika između ovih kategorija. Ukupno je analizirano 50 predeonih
sektora u toku četiri godine (2008-2011). Drugi deo istraživanje bio je koncipiran da se
odrede efekti predeone kompleksnosti izražene preko veličine pojedinačnih polja
(heterogenost konfiguracije poljoprivrednih površina) na model sistem. Ponovo,
određene su dve jasno diferencirane kategorije predela: 1) predeli sa pretežno malim
poljima (<3 ha) i 2) predeli sa velikim poljima (>20 ha). Analizirano je 24 predeona
sektora u toku dve godine (2008-2009). Treći deo bio je fokusiran na određivanje
efekata različitog tipa ivice polja na model sistem. Kontrastirana su dva tipa ivice polja:
1) ivica sa poluprirodnom vegetacijom i 2) ivica bez poluprirodne vegetacije.
Analizirano je 12 predeonih sektora u toku dve godine (2008-2009). Dodatno,
analizirana je promena brojnosti biljnih vašiju tokom sezona sa namerom da se odredi
nivo parazitiranosti koji odgovara efektu uspešne biološke kontrole. U toku celokupnog
istraživanja zabeležene su 4 vrste biljnih vašiju, 7 vrsta parazitoida i 9 vrsta
hiperparazitoida. Rezultati prvog dela istraživanja ukazuju na to da kompleksni predeli
koji sadrže veći procenat prirodnih i poluprirodnih staništa podržavaju veću brojnost
biljnih vašiju kao i da imaju veći procenat parazitiranosti, diverzitet parazitoida i
diverzitet hiperparazitoida. Uočena povećana brojnost biljnih vašiju u kompleksnim
predelima je u suprotnosti sa početnim očekivanjima, ali je potvrđeno da kompleksni
predeli podržavaju veću uspešnost parazitoida. Rezultati drugog dela istraživanja
pokazuju da ne postoji jasno izražen efekat predeone kompleksnosti izražene preko
veličine pojedinačnih polja na proučavani model sistem. Suprotno očekivanjima, nije
uočena razlika u brojnosti biljnih vašiju i procentu parazitiranosti između predela sa
malim i predela sa velikim poljima. Razlika je uočena jedino u promeni brojnosti biljnih
vašiju i procentu parazitiranosti između faza generalno i između faza u zavisnosti od
predela što ukazuje da je efekat biološke kontrole izraženiji u jednostavnim predelima
sa velikim poljima, a da povećavanju brojnosti biljnih vašiju pogoduju kompleksni
predeli sa malim poljima. Rezultati trećeg dela pokazuju da je brojnost biljnih vašiju
veća na ivici polja, posebno na ivici sa poluprirodnom vegetacijom, a da tip ivice polja
nema nikakav efekat na parazitiranost. Takođe, diverzitet parazitoida nije bio značajno
različit u odnosu na tip i udaljenost od ivice polja. Procenjeni nivoi parazitiranosti koji
odgovaraju stagnaciji brojnosti biljnih vašiju između faza ukazuju na to da se efekat
uspešne biološke kontrole pojavljuje u intervalu 19-27% parazitiranosti. Primećeno je
da postoji varijabilnost u stepenu ispoljavanja efekata predela na model sistem na nivou
godina, ali da se određeni generalni trendovi mogu uočiti. | Agricultural intensification and associated farming practices are among the most
significant human impacts on the global environment. Increase in agricultural land-use
area and the intensification of crop management are causing numerous environmental
problems, including loss of biodiversity and degradation of some key ecosystem
services, such as biological control. Numerous studies have emphasized the importance
of landscape scale effects in these processes. To determine how agricultural
intensification affects agrobiodiversity and accompanied ecological service (biological
control), we examined effects of key landscape features on aphid–parasitoid complex in
winter wheat agroecosystems in Pančevački rit region. Study was organized in three
parts. Aim of the first part was to determine effect of landscape compositional
heterogeneity defined through proportion of crop vs. non-crop land in landscape sectors.
Two broadly contrasting classes were selected: complex landscapes, characterized by
more than 50% of non-crop land cover (forests, fallows, pastures, hedgerows and
shrubs), and simple landscapes, with much less than 30% of non-crop land. Total of 50
landscape sectors were sampled in course of four years (2008-2011). In the second part
of the study, effects of configurational landscape heterogeneity of crop habitats were
tested through contrasting landscape sectors dominated by small fields (field size
averaging less than 3 ha) and landscape sectors dominated by large fields (field size
averaging more than 20 ha). Total of 24 sectors were sampled in two years (2008-2009).
In the third part, aim was to analyze edge effects of field margin types on aphid–
parasitoid interactions. Two markedly different margin types were selected: one
bordering on irrigation canal with permanent semi-natural shrub vegetation, and the
other close to service road without semi-natural vegetation. Total of 12 fields were
sampled in two years (2008-2009). Additionally, changes in aphid population growth
and parasitism rates between phases were monitored to determine parasitism threshold
value for effective biological control. In total, 4 aphid species, 7 parasitoid species and 9
hyperparasitoid species were found. Results show that complex landscapes with higher
proportion of non-crop land support larger aphid populations and higher parasitism
rates, as well as higher diversity of both parasitoids and hyperparasitoids. Second part of
the study revealed non-significant landscape effects on overall aphid abundances and
parasitism rates. Contrary to initial expectation, no differences in analyzed parameters
between small- and large-field landscapes were detected. Only detected effect was
differential change during season in aphid population growth (stronger in small-field
landscapes) and in parasitism rate (higher in large-field landscapes). Results of the third
part show that larger aphid population are found near field margin, especially near
margin bordering on permanent semi-natural shrub vegetation. Contrary to initial
expectations, field margin type had no effect on parasitoids. Parasitism threshold value
for effective biological control was estimated to be in 19-27% range. Values above this
range regularly resulted in drop of aphid population densities. It was observed that there
is temporal variability in the strength of landscape effects on aphid–parasitoids
complex, but that general trends and conclusion how different landscape features affect
model system can be proposed. | true |
Varijabilnost genoma humanog virusa Parvovirus B19 (Parvoviridae, Erythrovirus) | Genome variability of Human Parvovirus B19 (Parvoviridae, Erythrovirus) | Uvod: Infekcija parvoB19 virusom koja se prenosi respiratornim i vertikalnim
putem, ali i preko zaražene krvi i krvnih derivata, može predstavljati ozbiljan
zdravstveni problem za rizične grupe pacijenata. Ovaj, po dimenzijama mali DNK
virus bez omotača je vrlo otporan na standardne tehnike inaktivacije virusa u
proizvodnji derivata krvi, a pouzdanu dijagnostiku otežava varijabilnost genomske
sekvence virusa. Određivanje karakteristika nukleotidnih sekvenci izolata B19
virusa od značaja je za razvoj dijagnostičkih metoda i daje uvid u puteve nastanka i
održavanja genomske varijabilnosti B19 virusa. Cilj ovog rada bio je da se odredi
učestalost B19 virusa kod dobrovoljnih davalaca krvi i kod pacijenata sa sumnjom
na B19 infekciju i da se na osnovu molekularno genetskih karakteristika dobijenih
genomskih sekvenci, a u poređenju sa genomskim sekvencama dostupnim u
bazama podataka, odredi evolutivna dinamika varijabilnosti parvoB19 virusa i
filogenetski i filogeografski odnosi ispitivanih B19 izolata.
Metode: Uzorci plazme dobrovoljnih davalaca i plazme i krvi pacijenata sa
sumnjom na B19 infekciju analizirani su na prisustvo B19 DNK primenom lančane
reakcije polimeraze (PCR), kako metodama iz literature tako i razvijenom
internom metodom. Za uzorke sa detektovanim B19 genomom, direktnim
nukleotidnim sekvenciranjem određena je genomska sekvenca. Bioinformatičkom
analizom u odnosu na genomske sekvence B19 virusa dostupne u bazama
podataka određen je genotip izolovanih virusnih genoma, evolutivni pritisci na
osnovu odnosa nesinonimnih i sinonimnih zamena (dN/dS) za nukleotidne
sekvence koje kodiraju virusne proteine, kao i genetičke distance i filogenetski
odnosi B19 izolata dobijenih u Srbiji u odnosu na izolate iz drugih geografskih
regija.
Rezultati: Za 5 izolata iz Srbije, utvrđena je sekvenca B19 genoma u regiji
kompletne cDNK. Iz baze podataka izdvojeni su i analizirani izolati sa poznatom
sekvencom u regiji kompletne ili skoro kompletne cDNK, kojih je do 2012. godine
bilo ukupno 146, od toga 117 genotipa 1. Analizom odnosa dN/dS za virusne
proteine na osnovu većeg broja nukleotidnih sekvenci u odnosu na dosadašnje
literaturne podatke procenjen je uticaj pozitivne i negativne selekcije na
varijabilnost virusnog genoma. Određene su genetičke distance i filogenetski
odnosi u okviru populacije izolata iz Srbije, i u odnosu na druge izolate čije su
genomske sekvence dostupne u nukleotidnim bazama podataka.
Zaključak: Na osnovu rezultata ove studije potvrđeno je prisustvo B19 genotipa 1 u
humanoj populaciji Srbije i određena genomska sekvenca izolata. Utvrđeno je da
su rekombinacije kod B19 virusa redak događaj, što ukazuje na to da su manje
važan mehanizam u evoluciji B19 virusa. Analizom dobijenih genomskih sekvenci i
sekvenci iz baza podataka potvrđeno je dominantno delovanje negativne selekcije
(p=0.05) na varijabilnost kodirajućih sekvenci i to izraženije za glavne virusne
proteine (VP1, VP2 i NS1), i slabije izraženo za male virusne proteine (7.5 kDa, 11
kDa, protein X). Zamena F554L (NS1 gen: C2276T) u sekvenci NS1 proteina
fiksirana je u populaciji izolata iz Srbije a na kodonu 554 utvrđeno je delovanje
pozitivne selekcije (p=0.095). Na osnovu filogenetske analize cDNK dobijenih
izolata i 117 izolata iz različitih delova sveta klasterizacija B19 virusa u okviru
genotipa 1 je nezavisna od geografskog porekla izolata. Do sada je u grupi DNK
humanih virusa, samo za hepatitis B virus (HBV), genotip D, filogeografska analiza
kopletnih genoma pokazala jasnu klasterizaciju na osnovu geografskog porekla.
Visok nivo genetičke distance između dva izolata B19 iz Srbije uzeta od istog
pacijenta u razmaku od godinu dana, ukazuje na postojanje koinfekcije sa najmanje
dve kvazivrste B19 virusa od kojih jedna kvazivrsta seleksionisana kao
dominantna u toku pokazane perzistentne infekcije. | Background: Parvovirus B19 infection, being transmitted via the respiratory
route, vertically from mother to fetus as well as through blood-derived products
administered parenterally, can represent a serious health problem for risk
patients groups. This small non-enveloped DNA virus is extremely resistant to
standard viral inactivation techniques, while a reliable diagnosis is challenged by
the virus genome sequence variability. Characterization of B19 isolates nucleotide
sequences is valuable for development of diagnostic methods, and it gives an
insight into the genesis and maintenance of the B19 virus genome variability.
Aim of this thesis was to determine the frequency of B19 viremia in voluntary
blood donors and in patients with the B19 infection symptoms, investigate the
level and evolutionary dynamics of the B19 genome variability as well as to
examine phylogenetic and phylogeographic relations of the B19 genomes obtained
in Serbia with the isolates from the other parts of the world on the bases of the
almost complete B19 cDNA sequences.
Methods: Plasma samples of the voluntary blood donors, and plasma and blood
samples of the B19 infection suspected patients were analysed for the B19 DNA
presence by the polymerase chain reaction (PCR), using the methods described in
the literature as well as internally developed method. For the B19 genome positive
samples genome sequences were determined by direct sequencing. Bioinformatic
analysis was performed by using the generated data as well as the database
information in order to determine a genotype of the isolated genomes,
evolutionary pressures (based on dN/dS ratio) for virus protein coding sequences,
and genetic distances and phylogenetic relations of the B19 isolates from Serbia
compared to the other geographic regions.
Results: For 5 isolates from Serbia, the B19 genome sequence was determined for
the complete cDNA. Database isolates with the known, complete or almost complete
cDNA, available until the end of 2012 (146 isolates in total, out of which 117 were
genotype 1) were chosen and used for the analysis. Positive and negative selection
influence for the virus protein coding sequences was determined by the dN/dS ratio
analysis by using larger data set than described in the literature so far. Genetic
distances and phylogenetic relations were determined within Serbian population,
as well as in comparison to the other populations with the known B19 genome
sequence available in the nucleotide database.
Conclusions: Based on the results of this study, B19 genotype 1 presence was
confirmed in the population of Serbia, and the genome sequence of viral isolates
was determined. It was shown that the recombination events for the B19 virus
were rare and therefore less significant mechanism in the B19 virus evolution.
Analysis of the isolates from Serbia and database sequences confirmed the
dominant effect of the negative selection (p=0.05) on coding sequences, with the
effect more pronounced for major virus proteins (VP1, VP2 and NS1), and less
pronounced for the small virus proteins (7.5 kDa, 11 kDa, protein X), respectively.
F554L substitution (NS1 gene: C2276T) in the NS1 protein sequence is fixated in
Serbian B19 population, and for the codon 554, the positive selection (p=0.095) is
confirmed. Based on the phylogenetic analysis of cDNA of the obtained isolates
and 117 isolates from different parts of the world, clusterization within the B19
genotype 1 is geographically independent. Up to now, in the group of the human
DNA viruses, the pronounced geographical clusterization is demonstrated for the
genotype D3 of the hepatitis B virus (HBV), by the phylogeographic analysis of
the complete genome sequences. High value of the genetic distance demonstrated
in comparison of two samples taken from the same patient one year apart
suggests co-infection with at least two quasispecies of the B19 virus, one of which
was selected as dominant during the demonstrated persistent infection. | true |
Hromozomska, mikrosatelitska i mitohondrijalna DNK varijabilnost populacija Drosophila subobscura u klisurama i kanjonima Srbije | Chromosomal, microsatellite and mitochondrial DNA variability of Drosophila subobscura population from gorges and canyons in Serbia | Drosophila subobscura je vrsta sa širokim Palearktičkim rasprostranjenjem. Naseljava gotovo ceo evropski kontinent, severnu Afriku, Malu Aziju i neka atlanska ostrva. Genetička struktura prirodnih populacija ove vrste je opisana upotrebom nekoliko genetičkih markera. Najtemeljnije je izučena varijabilnost hromozomskog inverzionog polimorfizma, po kome populacije D. subobscura pokazuju prostornu i vremensku struktuiranost. Klinalni obrasci ovog tipa varijabilnosti upućuju na značaj selektivnih procesa u njenom oblikovanju, dok razlike između geografski bliskih područja između kojih postoji geografska barijera upućuju na značaj istorijskih procesa. Naime, uočena varijabilnost oslikava procese rekolonizacije Evrope nakon završetka poslednje glacijacije iz primarnog pribežišta lociranog u regionu koji obuhvata Balkan, Malu Aziju i Kavkaz, ali i sekundarnog u regionu Pirinejskog poluostrva. Mikrosatelitska varijabilnost izučavana je u nekoliko evropskih populacija D. subobscura, uglavnom iz zapadnog dela Evrope. Pokazano je da su populacije slabo struktuirane po ovom genetičkom markeru. Populacije u južnom delu areala odlikuju se nešto većom genetičkom raznovrsnošću. Donekle je slična i slika varijabilnosti prirodnih populacija po mitohondrijalnoj DNK (mtDNK), u slučaju koje je zabeležena homogenost distribucije haplotipova između populacija. U velikoj većini do sada analiziranih populacija zabeleženo je prisustvo dva dominantna haplotipa i veliki broj endemičnih haplotipova niske učestalosti. Pokazano je da nekoliko faktora oblikuje varijabilnost mtDNK D.subobscura. Među njima su najznačajniji istorijski procesi, genetički drift, i prirodna selekcija koja najverovatnije deluje preko cito-nukleusne koadaptacije između alelskih kombinacija u okviru inverzija i mtDNK varijanti... | Drosophila subobscura shows a wide Palearctic distribution. It inhabits most of Europe, Northern Africa, Asia Minor and some Atlantic islands. The genetic structure in natural populations of this species has been extensively studied with respect to several genetic markers. The variability of chromosomal inversion polymorphism has been most thoroughly studied showing spatial and temporal structure in natural populations. The clinal pattern of chromosomal variability suggests the action of natural selection on inversion polymorphism, while abrupt changes in gene arrangement frequencies between closely related populations separated by geographical barrier suggest the importance of historical processes on the genetic structure. After the decay of the ice caps, Drosophila subobscura, most probably, recolonized Europe mainly from the Balkan Peninsula, Asia Minor and Caucasus, but also, to a lesser degree, from the Iberian Peninsula. Several populations of D. subobscura, mostly from the Western Europe, have been studied with respect to microsatellite variability. The analysis showed absence of structuring of genetic variation, with slightly higher genetic variability in Southern populations. There is a widespread genetic homogeneity of mitochondrial DNA (mtDNA) variability throughout the species range with high prevalence of two almost equally frequent haplotypes, and a number of population specific, rare haplotypes. Several factors account for the observed distribution in mtDNA variability: historical processes, genetic drift and natural selection, most probably acting through cytonuclear coadaptation of alleles inside inversions and mtDNA haplotypes... | false |
Antioksidativno, antimikrobno i alelopatsko dejstvo tri endemične vrste roda Nepeta (Lamiaceae) | Antioxidative, antimicrobial and allelopathic effects of three endemic Nepeta species (Lamiaceae) | Nepeta rtanjensis Diklić i Milojević, N. sibirica L. i N. nervosa Royle ex
Bentham (fam. Lamiaceae) su endemične vrste roda Nepeta, koje su nedovoljno
fitohemijski okarakterisane i slabo ispitane u pogledu biološke aktivnosti. Radi se o
vrstama koje se odlikuju različitim kvalitativnim i kvantitativnim sadržajem
nepetalaktona, zbog čega predstavljaju idealne objekte u istraživanjima biološke
aktivnosti različitih izomera ovog monoterpenskog jedinjenja. S obzirom da se radi o
retkim vrstama, a u slučaju N. rtanjensis i o krajnje ugroženoj vrsti, biljni materijal za
potrebe eksperimenata je dobijen mikropropagacijom. Istraživanje je obuhvatilo
fitohemijske analize sekundarnih metabolita tri vrste roda Nepeta gajenih in vitro, kao i
analizu njihovih bioaktivnih svojstava kao što su antimikrobno, antioksidativno i
alelopatsko dejstvo.
Fitohemijska karakterizacija tri vrste roda Nepeta gajenih in vitro, omogućila je
identifikaciju i kvantifikaciju glavnih grupa sekundarnih metabolita (terpenoida i
fenolnih jedinjenja) koji se u ovim uslovima produkuju. Metode koje su korišćene u
fitohemijskim analizama su: GC-MS, GC-FID, PTR-MS, Headspace GC-MS, HPLC-
UV, HPLC-MS, UHPLC/DAD/HESI-MS/MS. Dominantno isparljivo jedinjenje iz
grupe terpenoida je monoterpenski lakton - nepetalakton, pri čemu je kod vrste
N. rtanjensis većinski prisutan tran,cis- izomer a kod N. sibirica cis,trans- izomer
nepetalaktona. Kod vrste N. nervosa nepetalakton je detektovan samo u tragovima. Kod
ispitivanih vrsta, ruzmarinska kiselina se javlja kao dominantno fenolno jedinjenje, dok
su ostale fenolne kiseline (hlorogena, neohlorogena i kafeinska kiselina) prisutne u
znatno nižoj koncentraciji.
Metanolni ekstrakti N. rtanjensis, N. sibirica i naročito N. nervosa, pokazuju
značajnu antioksidativnu aktivnost u ABTS i DPPH testovima, kao i visoki kapacitet za
redukciju gvožĎa u FRAP testu. Značajna antioksidativna aktivnost se pripisuje
fenolnim jedinjenjima, na prvom mestu ruzmarinskoj kiselini.
Nepetalakton ne utiče značajno na antioksidativni potencijal metanolnih ekstrakata, ali
postoji mogućnost antagonističkog delovanja izmeĎu fenolnih kiselina i nepetalaktona.
Metanolni ekstrakti tri vrste roda Nepeta pokazuju značajnu antimikrobnu aktivnost
protiv osam vrsta bakterija i osam vrsta gljiva, a aktivnost potiče od monoterpenskih
laktona i fenolnih jedinjenja. Najveću antimikrobnu aktivnost pokazuje metanolni
ekstrakt N. rtanjensis, sledi N. sibirica, i na kraju N. nervosa. Stereohemija
nepetalaktona bitno odreĎuje njegovu antimirobnu aktivnost, pri čemu je trans,cis-
izomer aktivniji od cis,trans-izomera.
PotvrĎeno je da vrste N. rtanjensis i N. sibirica poseduju jedinjenja koja mogu
uticati na rastenje i razviće drugih biljnih vrsta u njihovom neposrednom okruženju, i na
taj način aktivno učestvovati u alelopatskim interakcijama. Alelopatsko dejstvo
N. rtanjensis i N. sibirica potiče od dominantnog bioaktivnog jedinjenja ovih vrsta-
nepetalaktona. Stereohemija nepetalaktona značajno odreĎuje njegov alelopatski
potencijal, pri čemu tran,cis- izomer pokazuje veću aktivnost od cis,trans- izomera.
Alelopatsko dejstvo nepetalaktona se ogleda kroz usporenu dinamiku klijanja semena
test vrste Lepidium sativum L., ali i kroz efekat na biohemijske procese koji su posledica
poremećenog antioksidativnog sistema biljaka. Alelopatsko dejstvo nepetalaktona, a
naročito njegovog trans,cis- izomera, dovodi do promena u antioksidativnom sistemu
klijanaca kresa narušavanjem normalnih modela ekspresije i aktivnosti antioksidativnih
enzima (POD, CAT, SOD, PPO). Primećena je inhibicija aktivnosti, kao i izmenjen
profil izoformi POD, CAT, Fe-SOD i CuZn-SOD kod klijanaca kresa. Trans,cis-
nepetalakton dovodi do povećanog sadržaja ukupnih fenola u prvim fazama rastenja i
razvića klijanaca L. sativum, što je praćeno ranijom indukcijom ekspresije i povećanom
aktivnošću PPO. Nepetalakton tokom alelopatskih interakcija indukuje odreĎene
modifikacije metabolizma sinapata kod klijanaca kresa, koje se generalno mogu opisati
usporenim prevoĎenjem sinapoil- holina do sinapoil- glukoze, i potom do sinapoil-
malata.
Alelopatska svojstva N. rtanjensis i N. sibirica ukazuju na mogućnost uspešne
primene ovih vrsta i njihovih bioaktivnih komponenti u poljoprivrednoj praksi, kao
bioherbicida, a u cilju suzbijanja i kontrole korovskih vrsta. U ovom radu potvrĎeno je
alelopatsko dejstvo N. rtanjensis i N. sibirica u slučaju poljoprivrednih kultura kao što
su L. sativum, Lactuca sativa L. sorta „Majska kraljica“, Lotus corniculatus L. sorta
„Bokor“, Brassica napus L., i korovskih vrsta poput Stellaria media (L.) Vill., Rumex
crispus L., i Arabidopsis thaliana (L.) Heynh. ekotip Kolumbija. | Nepeta rtanjnsis Diklić and Milojević, N. sibirica L. and N. nervosa Royle ex
Bentham (fam. Lamiaceae) are endemic species of genus Nepeta that are insufficiently
phytochemically characterized and poorly tested in a sense of their biological activity.
These three species are characterized by different qualitative and quantitative
nepetalactone content, what makes them ideal objects in investigating the biological
activity of different stereoisomers of this compound. Considering that these species are
rare, and extremely endangered in case of N. rtanjensis, plant material for the
experiments was obtained by micropropagation. The research included phytochemical
characterization of in vitro grown three Nepeta species , as well as analysis of their
bioactivities such as antimicrobial, antioxidative activity and allelopathic potential.
Phytochemical characterization of three Nepeta species grown in vitro, involving
GC-MS, GC-FID, PTR-MS, Headspace GC-MS, HPLC-UV, HPLC-MS, and
UHPLC/DAD/HESI-MS/MS analyses, enabled identification and quantification of the
main secondary metabolites (terpenes and phenolics). The dominant volatile compound
from terpenes group is monoterpene lacton - nepetalactone, whereby its trans,cis-
isomer is mostly present in N. rtanjensis and its cis,trans- isomer in N. sibirica. In
N. nervosa, nepetalactone has been detected only in traces. In all investigated species,
rosmarinic acid is a dominant phenolic compound, while the other phenolic acids
(chlorogenic, neochlorogenic and caffeic acids) are present in significantly lower
concentrations.
The results of ABTS and DPPH assays showed that methanol extracts of
N. rtanjensis, N. sibirica and especially N. nervosa, possess considerable antioxidant
activities, and the FRAP assay revealed high ferric reducing capacity for all the samples
tested.. Significant antioxidant activity is attributed to phenolic acids, in the first place
to rosmarinic acid.
Nepetalactone doesn’t contribute notably to the antioxidant potential of the methanol
extracts, but there is a possibility of antagonistic action between phenolic acids and
nepetalactone. The methanol extracts of three Nepeta species show significant
antimicrobial activity against eight bacterial and eight fungal species, and the activity
originates from monoterpenic lactones and phenolic acids. The highest antimicrobial
activity was recorded for N. rtanjensis methanol extract, while extracts of N. sibirica
and N. nervosa were less efficient. Nepetalactone stereochemistry substantially
determines its antimicrobial activity, whereby the trans,cis- isomer is more active than
the cis,trans- one.
It has been confirmed that N. rtanjensis and N. sibirica possess compounds that
can influence growth and development of other plant species in their proximate
surrounding, thus actively participating in allelopathic interactions. Allelopathic effect
of N. rtanjensis and N. sibirica originates from the dominant bioactive compound of
these species - nepetalactone. Stereochemistry of nepetalactone considerably determines
its allelopathic potential, with trans,cis- isomer being more active than the cis,trans-
nepetalactone. Allelopathic effect of nepetalactone is reflected through slugged dynamic
of seed germination in the test species Lepidium sativum L., but also through its effect
on biochemical processes that are the consequence of plant’s disturbed antioxidative
system. Allelopathic effect of nepetalactone, especially of its trans,cis- isomer, leads to
the changes in antioxidative system of cress seedlings by violating normal models of
antioxidative enzymes expression and activity (POD, CAT, SOD, PPO). In cress
seedlings, the inhibition of activities and changed profiles of POD, CAT, Fe-SOD and
CuZn-SOD isoforms were observed. Trans,cis- nepetalactone action leads to the
increased content of total phenols in the first phases of growth and development of
L. sativum seedlings, which is followed by earlier induction of expression and increased
PPO activity. During allelopathic interactions, nepetalactone induces certain
modifications of sinapate metabolism in cress seedlings, which in general can be
described by decreased conversion of sinapoylholin to sinapoylglucose, and further to
sinapoylmalate.
Allelopathic features of N. rtanjensis and N. sibirica indicate the possibility of
successful application of these species and their bioactive components in agricultural
practice, as bioherbicides, toward weed control. In this thesis the allelopathic effect of
N. rtanjensis and N. sibirica was confirmed in case of crops L. sativum, Lactuca sativa
L. cv. „Majska kraljica“, Lotus corniculatus L. cv. „Bokor“, and Brassica napus L., and
weeds Stellaria media (L.) Vill., Rumex crispus L., and Arabidopsis thaliana (L.)
Heynh. ecotype Colombia. | true |
Analiza antitumorskog delovanja agenasa modifikovanih azot-monoksidom, Sakvinavir-NO i GIT-27NO na kanceru kolona in vitro i in vivo | In vitro and in vivo evaluation of antitumor activity of NO-modified compounds, Saquinavir-NO and GIT-27NO in colon cancer | Iz poznate uzročno-posledične veze infekcije i inflamacije, s jedne strane i
karcinogeneze i progresije tumora, s druge strane, proistekla je ideja o upotrebi anti-
inflamatornih i anti-infektivnih agenasa u terapiji kancera. Međutim, problem
gastrotoksičnosti anti-inflamatornih, kao i generalne toksičnosti anti-viralnih lekova uz
činjenicu da je reč o agensima niske biološke iskoristljivosti, ozbiljne su barijere u
razmatranju njihove primene u terapiji bolesti za koje inicijalno nisu namenjeni. Kako
bi se prevazišla farmakološka ograničenja pomenutih jedinjenja, a imajući u vidu
poznato svojstvo azot-monoksida (NO) da neutrališe toksičnost supstanci na različitim
nivoima, anti-inflamatornom agensu VGX-1027 i anti-retroviralnom agensu sakvinaviru
(Saq) adekvatnom hemijskom intervencijom je dodata grupa dizajnirana da donira NO.
Prednost novodobijenih jedinjenja GIT-27NO i Saq-NO nad poznatim nesteroidnim
anti-inflamatornim agensima modifikovanim kovalentnim vezivanjem NO (NO-
NSAID) je odsustvo molekula “nosača”, koji je inače odgovoran za genotoksičnost NO-
NSAID. Strukturna promena ova dva jedinjenja doprinela je njihovom snažnom
antitumorskom svojstvu, što je potvđeno brojnim in vitro i in vivo studijama, uz
umanjenu toksičnost u slučaju GIT-27NO ili potpunom gubitku toksičnosti Saq-NO
tretmana.U ovoj studiji je po prvi put ispitano antitumorsko delovanje GIT-27NO i Saq-
NO in vitro i in vivo na modelu kancera kolona, jednog od najtežih formi maligniteta.
Kako su oba agensa NO-derivati, definisana je uloga NO u njihovom antitumorskom
delovanju. Na molekularnom nivou, determinisani su glavni unutarćelijski događaji
pokrenuti u odgovoru na tretman pomenutim jedinjenjima. Na kraju, imajući u vidu
značaj mikrosredine za rast i progresiju tumora, ispitana je sposobnost GIT-27NO i Saq-
NO da povrate osetljivost ćelija kancera kolona na antitumorski imunski odgovor
posredovan TRAIL molekulom.
GIT-27NO i Saq-NO smanjuju vijabilitet ćelijskih linija kancera kolona CT26CL25
poreklom od miša, i HCT116 poreklom od čoveka, in vitro i in vivo, ali je značaj
otpuštanja NO za antitumorsko delovanje ovih supstanci potpuno različit. Dok je pad
vijabiliteta ćelija tretiranih sa GIT-27NO posledica visoke unutarćelijske koncentracije
NO i konsekventno generisanog oksidativnog i nitrozativnog stresa, antitumorsko
delovanje
Saq-NO
nije
posredovano
oslobađanjem
kvantitativno
značajnih
koncentracija ovog slobodnog radikala. Kod ćelija kancera kolona, GIT-27NO indukuje
akumulaciju tumor supresora p53, promenu ravnoteže pro- i antiapoptotskih molekula i
depolarizaciju mitohondrijskih membrana što ishoduje pokretanjem ćelijske smrti
apoptozom zavisnom od kaspaza. Nasuprot njemu, tretman ćelija kancera kolona sa
Saq-NO rezultovao je trajnom inhibicijom proliferacije i smanjenjem metastatskog
potencijala in vitro i in vivo. U osnovi uočenog antitumorskog delovanja Saq-NO nalazi
se snažna inhibicija fosforilacije ribozomalnog S6 proteina koja korelira sa prolaznom
aktivacijom Akt, akumulacijom tumor supresora p53 i smanjenjem ekspresije ciklina
D3. Za razliku od GIT-27NO, Saq-NO je pokazao sposobnost da vrati osetljivost ćelija
kancera kolona na antitumorski imunski odgovor posredovan TRAIL molekulom,
ukidajući rezistenciju na membranskom i unutarćelijskom nivou kroz povećanje
membranske ekspresije DR5 receptora i smanjenje nivoa XIAP.
Opšte uzev, sa rezultatima ove doktorske disertacije zaokružena su pretklinička
ispitivanja GIT-27NO i Saq-NO i trasiran je put ka kliničkim ispitivanjima pomenutih
agenasa na pacijentima obolelim od kancera kolona. | Concept of using anti-inflammatory and anti-infectious agents in cancer therapy is
based on a known relationship between inflammation and infection on one side and
carcinogenesis and tumor progression on the other side. However, the problem of
gastrotoxicity of anti-inflammatory and general toxicity of antiviral drugs, along with
their low bioavailability, marks a strong barrier when considering them in therapies for
which they were not initially intended for. In order to overcome the pharmacological
limits of these compounds, nitric-oxide (NO) with its documented feature to neutralize
toxicity of drugs on various levels was added to anti-inflammatory agent VGX-1027
and antiretroviral agent saquinavir (Saq). The advantage of new compounds, GIT-27NO
and Saq-NO, over known non-steroid anti-inflammatory agents modified by covalent
binding NO moiety (NO-NSAID) is the absence of “carrier” molecule, which is
responsible for genotoxicity of NO-NSAID. Structural change in these compounds
resulted in potentiation of antitumor action, confirmed by numerous in vitro and in vivo
studies, along with reduced toxicity of GIT-27NO or complete loss of toxicity of Saq-
NO treatment.
In this study, antitumor potential of GIT-27NO and Saq-NO was tested in vitro and in
vivo for the first time in colon cancer model, one of the most severe forms of
malignancy. As both agents are NO-derivates, the role of NO in their antitumor action
was defined. On molecular level, main intercellular events triggered by the treatment
were determined. Finally, considering the importance of microenvironment for tumor
growth and progression, the ability of GIT-27NO and Saq-NO to re-establish colon
cancer cell sensitivity to TRAIL-mediated antitumor immune response was tested.
GIT-27NO and Saq-NO reduced the viability of mouse CT26CL25 and human HCT116
colon cancer cell lines, both in vitro and in vivo. The importance of NO release for
antitumor action was quite different. While cell viability reduction under GIT-27NO
treatment was due to accumulation of high intracellular concentration of NO and
consecutively generated oxidative and nitrosative stress, antitumor action of Saq-NO
was not mediated by a release of quantitatively relevant amount of this free radical.
GIT-27NO induced the accumulation of p53 tumor suppressor, changed pro- and
antiapoptotic molecule ratio and triggered mitochondrial membrane depolarization
which resulted in cell death via caspase-dependent apoptosis.
Opposite to GIT-27NO, Saq-NO treatment of colon cancer cells resulted in permanent
inhibition of cell proliferation and reduction of metastatic potential, demonstrated in
vitro and in vivo. Strong inhibition of ribosomal S6 protein phosphorylation that
correlates with transient Akt activation, accumulation of p53 tumor suppressor and a
reduction in cyclin D3 expression were found to be responsible for Saq-NO antitumor
action. Unlike GIT-27NO, Saq-NO demonstrated the ability to reestablish colon cancer
cell sensitivity to TRAIL-mediated antitumor immune response, canceling the resistance
on both membrane and intracellular level by restoring the presence of DR5 receptor and
reducing the expression of XIAP.
To conclude, this doctoral dissertation summarizes preclinical research of GIT-27NO
and Saq-NO and sets the road for further clinical research on colon cancer patients. | true |
Uloga transkripcionog faktora DREB2A i malih RNK miR398a/b i miR408 u odgovoru graška (Pisum sativum L.) na dehidrataciju | The role of DREB2A transcription factor and small RNAsmiR398a/b and miR408 in dehidratation response of pea (Pisum sativum L.) | Jedan od glavnih faktora abiotičkog stresa kod biljaka je dehidratacija. U ovom radu
ispitivan je uticaj dehidratacije na ekspresiju gena za transkripcioni faktor DREB2A i malih
RNK miR398 i miR408 graška, u cilju sagledavanja značaja ovih molekula u odgovoru graška
na dehidrataciju i rehidrataciju.
Parcijalna cDNK (PsDREB2A) koja kodira za DREB2A transkripcioni faktor graška je
dobijena primenom metode PCR sa prajmerima dizajniranim na osnovu sekvenci DREB2A gena
iz model leguminoze Medicago truncatula. Dobijena sekvenca (495 bp) deponovana je u
GeneBank (HM229349). PoreĎenje nukletidnih sekvenci pokazalo je da je dobijena sekvenca
najsličnija sa sekvencom za DREB2A iz M. truncatula, kao i sa EREBP (ethylene responsive
element binding protein) iz iste vrste. Analiza izvedene aminokiselinske sekvence pokazala je
da PsDREB2A ima strukturu karakterističnu za DREB2A protein - prisustvo konzervisanog
AP2 domena (17-55 ak), kao i da ima najveću homologiju sa DREB2A i sa ERBP proteinom iz
M. truncatula. Na osnovu kompjuterske predikcije posttranslacionih modifikacija proteina
utvrĎeno je prisustvo jednog potencijalnog mesta za sumoilaciju proteina, i to na poziciji
Lys157 (MKQE), kao i prisustvo nekoliko potencijalnih mesta za fosforilaciju, najverovatnije
protein kinazom A.
U cilju ispitivanja ekspresije gena za DREB2A transkripcioni factor graška vršene su
analize iRNK korena i nadzemnog dela graška u normalnom fiziološkom stanju, kao i u
uslovima dehidratacije u trajanju 7 i 10 dana, i rehidratacije. Primenjena je metoda Real-time
PCR uz upotrebu proba specifičnih za PsDREB2A. Pokazano je da se profil ekspresije razlikuje
u korenu i nadzemnom delu biljaka. Kod korena maksimalni nivo ekspresije PsDREB2A
utvrĎen je nakon 10 dana tretmana, dok je kod nadzemnog dela biljaka maksimum dostignut
nakon 7 dana dehidratacije.
TakoĎe je ispitivana eskpresija konzervisanih mikro RNK- miR398a/b i miR408 tokom
dehidratacije i rehidratacije biljaka. Northern blot analiza, uz upotrebu odgovarajućih LNA
proba, pokazala je da su ispitivane mikro RNK najviše zastupljene kod kontrolne grupe biljaka.
Tokom stresa, odnosno dehidratacije, dolazi do progresivnog smanjenja nivoa odgovarajućih
miRNK, kako u korenovima, tako i u nadzemnim delovima dehidratisanih biljaka.
Osim toga, Metodom Real-time PCR su detektovane promene u ekspresiji potencijalnih
target gena ispitivanih mikro RNK – CSD, COX5b i P1B-ATP-aza. Od navedenih gena jedino kod
COX5b gena nije utvrĎena promena u nivou zastupljnosti iRNK tokom dehidratacije i
rehidratacije biljaka. Primećeno je da je eskpresija CSD gena u inverznoj korelaciji sa
ekspresijom odgovarajuće miRNK, tako da je najniži nivo ekspresije zabeležen kod kontrolne
grupe biljaka, dok se pod tretmanima nivo ekspresije CSD povećava. Ekspresija P1B-ATP-aze je
promenjena dejstvom dehidratacije jedino kod nadzemnog dela biljaka, a najveća promena je
zabeležena kod rehidratisanih biljaka.U slučaju korenova ekspresija ovog gena je nepromenjena. | Adverse environmental stresses, such as drought, low temperature and soil salinity have a
strong influence on agricultural production and sustainability. A major limitation to yield and
quality in many crop species is water availability throughout or at critical times during the
growing season. We investigated the effect of dehydration on expression of DREB2A
transcription factor in pea, as well as on the expression of conserved miRNAs-miR398a/b and
miR408.
Partial cDNA (PsDREB2A), which encodes a DREB2A transcription factor in pea, was
obtained by PCR with primers designed according to DREB2A gene from the model legume
Medicago truncatula. The resulting sequence (495 bp) has been deposited in Gene Bank
(HM229349). Comparison of nucleotide sequences showed that the obtained sequence is most
similar to the sequence of DREB2A from M. truncatula, and with EREBP (ethylene responsive
element binding protein) from the same species. Analysis of deduced amino acid sequence
showed that PsDREB2A has a structure characteristic of the DREB2A protein - presence of AP2
domains (17-55 a.a.), and has the highest homology with DREB2A and ERBP protein from M.
truncatula. Computer prediction of post-translational modification of protein revealed the
presence of a potential site for protein sumoylation, at the position of Lys157 (MKQE), and the
presence of several potential sites for phosphorylation.
To study the expression of PsDREB2A we performed the analysis of mRNA level in pea
root and shoot in a normal physiological state, as well as during dehydration for 7 and 10 days,
and rehydration. Real-time PCR using a probe specific for PsDREB2A has been applied. It has
been shown that the expression profile was different in roots and aerial parts of the plants. In the
roots the maximum level of expression PsDREB2A was determined after 10 days of treatment,
while in the above-ground parts of plants the maximum was rached after 7 days of dehydration.
We also studied the expression of conserved micro RNAs- miR398a/b and miR408
during dehydration and rehydration of plants. Northern blot analysis, using the appropriate LNA
probes, showed that RNAs were most abundant in the control group of plants. During
dehydration stress, there was a progressive reduction of the level of investigated miRNAs, both
in roots and in the above-ground parts of plants..
In addition, using Real-time PCR changes in the expression level of potential target genes
of selected miRNAs - CSD, COX5b and P1B-ATPase have been investigated. The level of
COX5b gene has not been changed dehydration and rehydration of plants. It is noted that the
CSD expression level correlated inversely with the expression of the corresponding miRNA, so
that the lowest level of expression was noted in the control group of plants, while under
treatment the level of expression of CSD has been increased. Expression of P1B-ATP-ase was
changed during dehydration only in above-ground parts of plants, and the biggest change was
observed in rehydrated plants. In the roots the expression of this gene was unaffected. | true |
Karakterizacija procesa programirane ćelijske smrti i senescencije u listovima Nicotiana tabacum L. in vitro | Characterisation of programmed cell death and senescence in leaves of Nicotiana tabacum L. in vitro | Senescencija
lista
predstavlja
kompleksnu,
genetički
kontrolisanu
poslednju fazu ontogeneze lista koja rezultira koordinisanom razgradnjom
makromolekula i mobilizacijom njegovih komponenti u druge delove biljke.
Na ćelijskom nivou, ona obuhvata promene strukture, metabolizma i genske
ekspresije koje dovode do smrti, čiji se tip označava kao programirana
ćelijska smrt (PCD). Kao eksperimentalni model u ovom radu korišćene su
in vitro gajene biljke duvana (Nicotiana tabacum L.) koje obezbeđuju set listova u
kojima
postoji
gradacija
senescencije.
Istraživanje
je
obuhvatilo
citohistološku i imunohistohemijsku analizu dinamike strukturnih
promena mezofila listova, kao i ispitivanje metaboličkih, biohemijskih i
molekularnih procesa tokom senescencije lista. Utvrđen je redosled
degenerativnih promena u strukturi ćelija tokom fiziološkog starenja
listova. U senescentnim listovima detektovani su tipični simptomi
programirane ćelijske smrti, koja po svojim morfološkim odlikama
odgovara autofagnoj ćelijskoj smrti, i pokazano da internukleozomalna
fragmentacija DNK prethodi ultrastrukturnim promenama kao što su
degradacija hloroplasta i kondenzacija hromatina. Sadržaj hlorofila i
citosolnih proteina identifikovani su kao pouzdani biomarkeri stepena
senescencije listova. Hemijskom analizom endogenih regulatora rastenja
identifikovano je i kvantifikovano više od 20 različitih citokinina i
pokazano da je napredovanje senescencije u korelaciji sa opadanjem sadržaja
ukupnih citokinina, kao i sa smanjenjem nivoa citokinina trans-zeatinskog
tipa. Takođe je uočena jasna korelacija između smanjenja nivoa endogene IAA,
odnosno ABA i napredovanja senescencije. Imunohistohemijskom analizom
mezofila listova biljaka tretiranih citokininima pokazano je da visoke
doze citokinina BA ubrzavaju senescenciju, čime indukuju programiranu
smrt
ćelija.
Analiza
aktivnosti
pojedinih
enzimskih
komponenti
antioksidativnog sistema pokazala je značajne promene u antioksidativnom
statusu listova tokom fiziološkog starenja. Napredovanje senescencije je u
jasnoj korelaciji sa smanjenjem aktivnosti CAT i povećanjem ukupne
aktivnosti POD. Primarnu antioksidativnu zaštitu tokom starenja
obezbeđuju SOD, a u proces odbrane od štetnog dejstva ROS u inicijalnoj
fazi senescencije uključuju se POD. Molekularna analiza senescencije
pokazala je aktivaciju gena koji kodiraju enzime uključene u konverziju i
remobilizaciju produkata razlaganja u senescentnim listovima. qRT-PCR
metodom pokazana je indukcija ekspresije gena koji kodiraju cistein
proteaze odgovorne za remobilizaciju aminokiselina, kao i cistein
proteazu iz grupe metakaspaza uključenu u kontrolu ćelijske smrti.
Indukcija ekspresije Gln1-3 i GDH2, SAG-gena uključenih u mobilizaciju
azota u senescentnim listovima, naporedo sa padom ekspresije GS2 ukazala je
da se asimilacija amonijuma tokom senescencije postepeno premešta iz
hloroplasta u citosol mezofilnih ćelija. | Leaf senescence constitutes the final stage of leaf development that results in the
coordinated degradation of macromolcules and the subsequent nutrient
relocation to other plant parts. Leaf senescence involves an integrated action at
the cellular, tissue, organ and organism levels under the control of a highly
regulated genetic program. During senescence, leaf cells undergo orderly changes
in cell structure, metabolism and gene expression leading to cell death,
recognized as a form of programmed cell death (PCD). In vitro grown Nicotiana
tabacum L. has been chosen as a model plant system, since a single plant provides
a large set of leaves in which there is a gradation of senescence. The dynamics of
ultrastructural changes in leaf mesophyll cells, as well as metabolic, biochemical
and molecular changes during leaf senescence were investigated. We determined
the time sequence of senescence-related events in mesophyll cells during
physiological ageing of leaves. Typical symptoms of autophagic PCD were
detected in senescing leaves using cytological analysis and TUNEL assay. We
showed that PCD-specific nuclear DNA fragmentation occurs during natural
tobacco leaf senescence but that it starts early in leaf development, thus
preceeding ultrastructural changes such as chloroplast degradation and
chromatin condensation. Chlorophyll and soluble protein content were identified
as reliable biomarkers for estimating the relative age of a particular leaf along the
main plant axis. Using HPLC/MS more than 20 different cytokinins were
identified and quantified in tobacco leaves. The correlation of senescence with
decline in total cytokinin content and the levels of trans-Z was found. The
progression of leaf senescence correlated with a gradual decrease in endogenous
levels of both IAA and ABA. Immunohistological analysis showed that high levels
of cytokinin benzyladenine accelerate senescence thus inducing PCD. The
activities of different antioxidative enzymes were analyzed during physiological
ageing of leaves. Progression of senescence is correlated with decrease in CAT
activity and a concomitant increase in POD activity. Primary antioxidative
protection in the course of ageing is provided by SOD, whereas POD are
implicated in defense against detrimental ROS action in an initial phase of
senescence. Molecular analysis of leaf senescence has revealed activation of genes
encoding enzymes involved in conversion and remobilization of degradation
products in senescing leaves. Quantitative RT-PCR showed that the expression of
cysteine proteases responsible for amino acid remobilization, typically associated
with leaf senescence and not found in other forms of PCD, was induced in
senescent leaves. Induced expression of SAGs involved in nitrogen mobilization
in senescing leaves, Gln1-3 и GDH2, contiguous with decrease in GS2 expression
indicated that, during senescence, ammonia assimilation is progressively shifted
from the chloroplasts to the cytosol of leaf mesophyll cells. | true |
Fiziloška i fitohemijska karakterizacija biljaka vrste Rindera umbellata (Waldst. & Kit.) Bunge gajenih in vitro | Physiological and phytochemical characterization of plant species Rindera umbellata (Waldst. & Kit.) Bunge cultivated in vitro | Rindera umbellata je retka i krajnje ugrožena biljna vrsta u Srbiji. Da bi
se uspostavili protokoli za ex situ zaštitu i obezbedio materijal za
molekularne i fitohemijske analize uspostavljena je in vitro kultura ove slabo
ispitane vrste. Optimizacija uslova gajenja zasnivala se na variranju vrste i
koncentracije ugljenih hidrata (saharoza, glukoza i fruktoza) i fitohormona
(giberelna i jasmonska kiselina) u podlozi za gajenje. Takođe je ispitivan uticaj
navedenih regulatora rastenja na produkciju farmaceutski važnih sekundarnih
metabolita.
In vitro rastenje i razviće biljaka vrste R. umbellata je pod uticajem kako
vrste, tako i koncentracije primenjenog šećera. Utvrđeno je da saharoza
predstavlja najbolji izvor ugljenika za in vitro rast, ožiljavanje i aklimatizaciju
biljaka, a da su koncentracije od 0,3 i 1 M svih primenjenih šećera
inhibitorne. Giberelna kiselina stimuliše rast i izduživanje stabla ovih
rozetastih biljaka, ali ne utiče značajno na proces ožiljavanja. Jasmonska
kiselina je stimulisala in vitro rast i ožiljavanje R. umbellata, naročito pri
nižim koncentracijama i na kratkotrajnim tretmanima. Najviša primenjena
koncentracija ovog fitohormona (200 µM) inhibirala je sve posmatrane procese.
Uočena su značajna variranja u kvantitativnom i kvalitativnom
sadržaju sekundarnih metabolita zavisno od načina gajenja, fenofaze i
testiranog biljnog organa. Biljke vrste R. umbellata u prirodnim uslovima
sintetišu nezasićene i zasićene pirolizidinske alkaloide. Biljke gajene in
vitro
sintetišu
veoma
niske
koncentracije
isključivo
zasićenih
pirolizidinskih alkaloida.
Fenolna jedinjenja detektovana su u listovima i korenu ovih biljaka. Od
fenolnih kiselina biljke iz prirode sadrže više ruzmarinske kiseline u
nadzemnim delovima, dok je sadržaj litospermične kiseline B u korenovima
višestruko veći od količine ruzmarinske kiseline. Sličan odnos sadržaja ovih
jedinjenja zapažen je i u biljkama koje su gajene u in vitro kulturi. Iako su
najviše primenjene koncentracije saharoze bile inhibitorne za rastenje, biljke
pri ovim uslovima sintetišu najviše fenolnih jedinjenja, flavonoida i
ruzmarinske kiseline. Giberelna kiselina ne utiče na produkciju analiziranih
fenolnih jedinjenja u izdancima, dok u korenovima sadržaj ukupnih fenolnih
jedinjenja raste, a flavonoida, ruzmarinske kiseline i litospermične kiseline
B opada srazmerno povećanju koncentracije giberelne kiseline u podlozi.
Sadržaj ukupnih fenola, flavonoida, litospermične kiseline B i
ruzmarinske kiseline meren u izdancima neposredno nakon tretmana jasmonskom
kiselinom zavisi od koncentracije datog hormona u podlozi i od dužine trajanja
tretmana. Nakon oporavka na podlozi bez hormona nije uočena statistički
značajna razlika u produkciji fenolnih kiselina između kontrolne grupe i
tretiranih izdanaka. Kod većine biljaka aplikacija jasmonske kiseline
stimuliše biosintezu velikog broja sekundarnih metabolita, uključujući i
fenolna jedinjenja. Kod vrste R. umbellata efekat jasmonske kiseline je
kratkotrajan
i
vremenom
se
gubi.
Sadržaj
ruzmarinske
kiseline
i
litospermične kiseline B određuje nivo antioksidativne aktivnosti vrste R.
umbellata. Biljke sakupljene u prirodi produkuju manje sekundarnih metabolita i
pokazuju manju antioksidativnu aktivnost od biljaka gajenih in vitro.
U uzorcima R. umbellata potvrđena je ekspresija tri specifična gena iz
fenilalaninske „grane“ biosintetskog puta RA (PAL, 4CL, i CAH), gena za
NADPH:citohrom P450 reduktazu (CPR) i gena za citohrom P450 zavisnu
monooksigenazu (CYP98A). U ispitivanim uzorcima nije detektovana ekspresija
gena za TAT, HPPR i RAS, tj. enzima druge „grane“ koji kontrolišu put
konverzije L-tirozina. | Rindera umbellata is a rare and critically endangered plant species in Serbia. In
order to optimize protocols for ex situ conservation and provide the materials for
molecular and phytochemical analysis, a suitable method for in vitro propagation of this
plant species was established. Optimization of the growing conditions was based on
varying the type and concentration of carbohydrates (sucrose, glucose and fructose) and
phytohormones (gibberellic acid and jasmonic acid) in the substrate. Furthermore, the
effect of selected growth regulators on the production of pharmaceutically important
secondary metabolites was investigated.
In vitro growth and development of R. umbellata plants were significantly
affected by both the type and the concentration of applied sugars. It was found that
sucrose is the best carbon source for in vitro growth, rooting and acclimatization of the
plants, and that the concentrations of 0.3 and 1 M of all applied sugars had an inhibitory
effect. Gibberellic acid stimulated growth and stem elongation, but did not significantly
affect the process of rooting. Jasmonic acid stimulated in vitro growth and rooting of R.
umbellata, especially at lower concentrations, and after short-term treatments.
Significant variations in quantitative and qualitative contents of secondary
metabolites are noticed, which depended on the method of cultivation, growth stages
and plant organs tested. Plants of R. umbellata grown under natural conditions
synthesized both unsaturated and saturated pyrrolizidine alkaloids. In vitro grown plants
synthesized a very low concentration of saturated alkaloids.
Phenol compounds were detected in the leaves and roots of the plants. The
content of lithospermic acid B in the roots was several times greater than the amount of
rosmarinic acid. A similar relation between the contents of rosmarinic acid and
lithospermic acid B was observed in plants that were grown in vitro. Gibberellic acid
did not affect the production of phenolic compounds in the shoots. However, total
phenol content in roots increased, while the concentration of flavonoids, rosmarinic acid
and lithospermic acid B decreased in relation to increasing concentrations of gibberellic
acid.
Total phenolics, flavonoids, lithospermic acid B, and rosmarinic acid content
was measured in shoots immediately after the treatment and depended on both, the
concentration of the phytohormones in the medium and the treatment duration. After
growth recovery on medium without phytohormones there was no statistically
significant difference in the production of phenolic acids between the control group and
the treated plants. Although the application of jasmonic acid stimulates the biosynthesis
of a large number of secondary metabolites, including phenolic compounds, in R.
umbellata jasmonic acid effect was transient.
Wild grown plants produced fewer amounts of secondary metabolites and
exhibited a lower antioxidant activity then plants grown in vitro. The endogenous level
of rosmarinic acid and lithospermic acid B determines the antioxidant activity of R.
umbellata.
In R. umbellata samples it was confirmed the expression of three genes from
phenylalanine "branch" of rosmarinic acid biosynthetic pathway (PAL, 4CL and CAH),
the gene for NADPH: cytochrome P450 reductase (CPR) and the gene for cytochrome
P450-dependent monooxygenase (CYP98A). However, in all the samples tested it was
not confirmed gene expression of enzymes from the second "branch", that control the
time of conversion of L-tyrosine, namely TAT, HPPR and RAS. | true |
Distribucija različitih polutanata u tkivima pet komercijalnih vrsta riba iz Dunava kod Beograda | Distribution of different pollutants in tissues of five commercial fish species from the Danube river near Belgrade | Akvatični ekosistemi su danas ugroženi na globalnom nivou, stoga je u
cilju
održivog
upravljanja
vodenim
resursima
neophodno
uspostaviti
kontinuirani monitoring njihovog kvaliteta. Položaj riba na vrhu lanca
ishrane i upotreba u ishrani ljudi čini ih važnim za procenu kontaminacije
akvatičnih ekosistema toksičnim supstancama.
U okviru ove disertacije vršena je procena distribucije različitih
polutanata na tri lokacije na Dunavu: Batajnica, Veliko ratno ostrvo i Grocka,
u periodu od novembra 2012. godine do marta 2014. godine. Na osnovu analize
relativne zastupljenosti (abundancije) i masenog udela prisutnih vrsta riba u
ukupnom ulovu, kao i bioloških i ekoloških karakteristika samih vrsta, na
ispitivanim lokalitetima najpre jeodređenokoje vrste bi mogle biti optimalne
indikatorske vrste, tako da su u daljim istraživanjima korišćeni štuka,
deverika, krupatica,somi smuđ.
Na uzorcima soma,primenom masene spektrometrije sa induktivno
spregnutom plazmom (ICP-MS),ispitana je distribucija 11 elemenata u tkivima,
kao i njihove koncentracijeu različitim segmentima mišića, škrga, jetre i
crevakako bi se utvrdio potencijal korišćenja ovih tkiva u monitoringu
populacija riba. Potencijalna korelacija između koncentracije metala i
elemenata u tragovima u mišiću sa koncentracijama u krljuštima i analnom
peraju ispitana je na jedinkama štuke, deverike, krupatice, soma i smuđa, u cilju
razvoja neinvazivnih metoda monitoringa kvaliteta ribljeg mesa. Takođe,
ispitano je i prisustvo akumulacije organohlornih zagađivača (polihlorovanih
bifenila i pesticida) kod jedinki deverike i smuđa primenom gasne
hromatografije sa detektorom elektronskog zahvata(GC-ECD). Histopatološke
promene praćene su na škrgama i jetri deverike i smuđa, kako bi se utvrdio
potencijal korišćenja histoloških analiza u razvoju metoda monitoringa
populacija riba.
IV
Rezultati su ukazali na visok nivo diferencijalne akumulacije
elemenata među analiziranim tkivima u somu. Najviši sveukupni nivo
akumulacije uočen je u kičmenom pršljenu, bubrezima i jetri, dok su minimalne
vrednosti uočene u žučnoj kesi, mišiću, mozgu i ribljem mehuru. Akumulacija
elemenata u mišiću i koži,kao i u škržnim filamentima i luku, značajno se
razlikovala za većinu elemenata, dok između dva ispitivana dela jetre,
proksimalnog i distalnog, nije bilo razlika u koncentracijama analiziranih
elemenata. U proksimalnom i medijalnom segmentu creva uočen je isti
akumulacioni nivo, dok su u oba segmenta zabeležene značajno više
koncentracije Co i Zn i niže koncentracije Mn u odnosu na distalni segment.
Nije uočena značajna korelacija koncentracija elemenata između mišića i
krljušti kod ispitivanih vrsta, pa se može zaključiti da metoda korišćenja
krljušti
za
procenu
kontaminacije
mišića
datim
elementimanije
zadovoljavajuća, mada se može koristiti kao metoda ranog upozorenja na
kontaminaciju tkiva. Koncentracije organohlornih zagađivača bile su ispod
propisanih maksimalno dovozvoljenih vrednosti. Na akumulaciju organohlornih
jedinjenja utiču kako položaj ribe u lancu ishrane tako i sadržaj lipida,
međutim u ovom istraživanju nisu uočene značajne razlike u koncentracijama
PCB i pesticida između deverike i smuđa. Histopatološkom analizom utvrđene
su više prosečne vrednosti nivoa histopatoloških promena u škrgama
deverike, što je i očekivano imajući u vidu da su škrge riba u stalnom kontaktu
sa supstancama koje se nalaze u vodi koja ih okružuje, pa predstavljaju
relevantne indikatore izloženosti riba zagađivačima u vodi. Ukupni
histopatološki indeks jetre nije se značajno razlikovao među vrstama. | Aquatic ecosystems are today globally endangered;thus, in order to ensure
sustainable water resources management, it is necessary to establish continuous
monitoring of their quality. Considering that fish are situated at the top of the food chain
and used for human nutrition, it makes them important for the assessment of
contamination of aquatic ecosystems with toxic substances.
In scope of this dissertation,the estimation of the distribution of different pollutants
was carried out at three localities on the Danube: Batajnica, Veliko Ratno Ostrvo, and
Grocka, in the period from November 2012 to March 2014. Based on the analysis of
abundance and mass fraction of individual species in the total catch, as well as their
biological and ecological characteristics, the optimal indicator species for this study
were determined, and accordingly the following species were used northern pike,
freshwater bream, white bream, wels catfish, and pike-perch.
The distribution of 11 elements wasassessed in the wels catfish tissue samples by
inductively coupled plasma mass spectrometry(ICP-MS), as well as their concentrations
in different segments of muscles, gills, liver, and intestine, in order to determine the
potential for the use of these tissues in monitoring fish populations. The potential
relationship of metal and trace element concentrations in the muscle and their
concentrations in scales and anal fins was assessed in northern pike, freshwater bream,
white bream, wels catfish and pike-perch in order to develop non-invasive methods for
monitoring the quality of fish meat. Using the gas chromatography with electron capture
detector(GC-ECD), concentrations of organochlorine pollutants (polychlorinated
biphenyls and pesticides) in freshwater bream and pike-perch were determined.
Histopathological changes were assessed in gills and liver of freshwater bream and
pike-perch in order to determine the potential for using histological analyses in the
development of methods for monitoring fish populations.
VI
The results indicated a high level of differential accumulation of elements
among the analyzed tissues in wels catfish. The highest overall accumulation level was
observed in vertebra, kidneys, and liver, while minimal values were observed in
gallbladder, muscle, brain, and swim bladder. The accumulation of elements in the
muscle and skin, as well as in the gill filaments and gill arch, differed significantly
among most elements, while between the two examined parts of the liver, proximal and
distal, there were no differences in concentrations of the analyzed elements. The same
accumulation level was observed in the proximal and medial segments of the intestine,
while both segments had significantly higher concentrations of Co and Zn and lower
Mn concentration in comparison with the distal segment. There were no significant
correlations between muscle and scales in element concentrations in all investigated
species, which indicates that the method of using scales is inadequate for assessing
muscle contamination with these elements, but that it can be used as an early warning
method for assessing overall tissue contamination. Concentrations of organochlorine
contaminants were below the prescribed maximum allowed levels. While the
accumulation of organochlorine compounds is affected by both the position of the fish
in the food chain and the lipid content, no differences in concentrations of
polychlorinated biphenyls and pesticides between the freshwater bream and pike-perch
were observed in this study. Histopathological analysis revealed higher average
histological scores in the gills of freshwater bream, which was expected, given that the
gills are in constant contact with the substances found in the surrounding water, which
makes them good indicators of fish exposure to pollutants in water. The total
histopathological index of the liver did not differ significantly between species. | true |
Identifikacija genskih lokusa (QTL) uključenih u kontrolu odgovora kukuruza (Zea mays L.) na stres suše | Identification of quantitative trait loci (QTLs) for drought tolerance response in maize (Zea mays L.) | Suša je jedan od najvažnijih ograničavajućih faktora u proizvodnji kukuruza, pa
je poboljšanje tolerantnosti na sušu od veoma velikog značaja u programima
oplemenjivanja. Stvaranje genotipova tolerantnih na sušu je veliki izazov zbog variranja
u pojavljivanju, intenzitetu i trajanju stresa suše, zbog genetičke kompleksnosti ovog
stresa i izraženih interakcija genotip-sredina. Identifikacija kvantitativnih lokusa
vezanih za tolerantnost na sušu je važno sredstvo za indirektnu selekciju pomoću
molekularnih markera. Istovremena primena konvencionalnog sa molekularnim
oplemenjivanjem, marker-asistiranom selekcijom i genetičkim inžinjeringom može
doprineti bržem načinu za poboljšanje tolerantnosti na sušu kod gajenih biljaka.
Identifikacija lokusa za kvantitativna svojstva (QTL) za tolerantnost na sušu kod
kukuruza je urađena na genetičkom materijalu dobijenom ukrštanjem linija DTP79
(izvor tolerantnosti na sušu) i B73 (linija osetljiva na sušu). Za konstruisanje
molekularne mape neophodne za identifikaciju QTL-ova, korišćena je F2 generacija
(John Innes Centre (JIC), Norwich, United Kingdom). Podaci o vrednostima svojstava
potencijalno uključenih u odgovor kukuruza na stres suše su dobijeni analizom F3
familija u poljskim ogledima. Molekularna mapa i dobijene vrednosti za ispitivana
svojstva su korišćene za detekciju QTL-ova. Na osnovu informacija o QTL-ovima je
vršeno utvrđivanje postojanja uzročne povezanosti između dva ili više svojstava.
Fenotipske korelacije su izračunate primenom Pearson-ovog koeficijenta i bile
su visoko značajne za izvestan broj ispitivanih osobina. Identifikacija QTL-ova je
urađena primenom ANOVA metode i metoda statističkog programa WinQTL
karotgrafera, v 2.5. Prikazani su samo rezultati dobijeni primenom CIM (WinQTL
karotgrafer, v 2.5) i ANOVA metode. Za ispitivanih 26 osobina primenom CIM metode
je detektovano 147 QTL-ova, od kojih je 93 detektovano i primenom ANOVA metode.
Ukupna fenotipska varijabilnost objašnjena identifikovanim QTL-ovima za sva
analizirana svojstva je bila u ospegu od 11.85% (RSV) do 91.16% (ŠL3). Detektovani
su hromozomski regioni u kojima se preklapaju QTL-ovi za pojedine osobine, koje su
istovremeno pokazale i značajnu međusobnu fenotipsku korelaciju, što ukazuje na
moguće postojanje vezanih gena ili plejotropnih efekata koji utiču na ove osobine. Neki
od detektovanih QTL-ova su identifikovani i u radovima drugih autora, na istoj poziciji
u genomu kukuruza, što znači da su oni zajednički različitom genetičkom materijalu.
Ovi QTL-ovi mogu biti od značaja u ekspresiji ispitivanih svojstava, ukazujući na
mogućnost njihovog uspešnog korišćenja u marker - asistiranoj selekciji. | Drought is one of the most important limiting factors in maize production.
Improving drought tolerance is therefore of outmost importance for breeding. Achieving
this goal is a big challenge due to the variability in timing, intensity and duration of
drought, genetic complexity of drought tolerance and large genotype by environment
interactions. Identification of quantitative loci for drought tolerance is very imporatant
for indirect selection using molecular markers. Conventional breeding together with
molecular breeding, marker-assisted selection and genetic engineering could contribute
to drought tolerance improvement of cultivated plants.
Quantitative trait loci (QTLs) for drought tolerance were identified in a maize
population derived from a cross between two lines - DTP79 (drought tolerant) and B73
(drought sensitive). Map construction (John Innes Centre (JIC), Norwich, United
Kingdom) was done using F2 generation and the coresponding F3 progenies were
evaluated for the traits potentialy related to drought tolerance in field trials. Phenotypic
and marker data were used for QTL detection. Possible causal relationships between
two or more traits were determined according to information about QTLs.
Phenotypic correlations calculated using Pearson’s coefficients were highly
significant for some of the analyzed traits. ANOVA and methods of WinQTL
Cartographer 2.5 were employed to identify QTLs. Results of ANOVA and CIM option
in WinQTL cartographer, v 2.5 are presented. A total of 147 QTLs were detected using
CIM for the analyzed traits. Out of this number, 93 were also detected using ANOVA.
Percent of phenotypic variability determined for the identified QTLs for all of the traits
ranged from 11.85% (RWC) up to 91.16% (LW3). Mapping analysis identified genomic
regions associated with two or more traits in a manner that was consistent with
phenotypic correlation between traits, supporting the possible existence of linked genes
or pleiotrophy that influence these traits. Some of the QTLs mapped herein, were also
identified in the works of other authors, at the same position in the maize genome,
indicating that they could represent regions that are common to various populations.
These QTLs may be important in the expression of the traits, indicating the possibility
of their successful use in marker-assisted selection. | true |
Mikrobiom šljive (Prunus domestica L.) i potencijal odabranih izolata za biokontrolu patogena šljive | Plum microbiome (Prunus domestica L.) and potential of selected isolated for biocontrol of plum phytopathogens | Šljiva (Prunus domestica L.) ima izuzetan značaj kao voćarska kultura u Srbiji i
tradicionalno se prerađuje u rakiju šljivovicu. S obzirom da je godišnji prinos šljive
ugrožen različitim bolestima, naročito izazivačima bakterioznog raka i mrke truleži
neophodno je naći efikasne agense za njihovo suzbijanje, a naročito alternativu upotrebi
hemijskih pesticida. U ovom radu je prvi put analiziran mikrobiom filosfere četiri sorte
šljive (Čačanska lepotica, Čačanska rodna, Požegača i Ranka), obuhvatajući ranu (maj) i
kasnu fenološku fazu (juli) razvića ploda. Indeksi alfa i beta diverziteta su pokazali da
su zajednice bile heterogene i da je njihov sastav zavisio od sezone uzorkovanja i sorti
šljive. U ukupnoj bakterijskoj zajednici je metagenomskim pristupom utvrđeno da su
predstavnici razdela Proteobacteria bili najdominantniji, a najzastupljenije vrste su bile
iz rodova: Methylobacterium, Sphingomonas i Hymenobacter. Tradicionalnim
kultivabilnim pristupom i analizom sekvenci gena za 16S rRNK identifikovane su
Pseudomonas syringae i Pseudomonas graminis kao najdominantnije vrste. Analiza
mikobioma je ukazala na dominatnu zastupljenost predstavnika razdela Ascomycota.
Vrste iz rodova Aureobasidium i Cryptococcus su bile najučestalije u maju, dok su u
julu predstavnici različitih rodova dominirali na sortama šljive (Cryptococcus,
Metschnikowia, Fusarium i Hanseniaspora). U okviru kolekcije kultivabilnih gljiva,
identifikovano je 11 izolata Monilinia laxa na osnovu filogenetske analize sekvenci ITS
regiona. Odabrani izolati M. laxa su prouzrokovali tipične simptome na ubranim
plodovima sorti Požegača i Čačanska rodna, a testiranjem potencijalnih bakterijskih
patogena su izdvojeni izolati P. syringae ČL2/2 i P7/16_2, koji su izazivali nekroze na
ubranim listovima šljive svih ispitivanih sorti.
Analizom antagonističkog potencijala kolekcije bakterija i kvasaca poreklom sa
sve četiri sorte je izdvojen izolat Bacillus thuringiensis R3/3 sa snažnom
antibakterijskom
aktivnošću
protiv
autohtonih
i
referentnih
fitopatogena.
Karakterizacijom B. thuringiensis R3/3 je utvrđeno da ima genetički potencijal da
produkuje kurstakin i N-acil homoserin laktonazu, a u in vitro uslovima je pokazana
sposobnost
za
“quorum
quenching”,
produkciju
hidrolitičkih
egzoenzima,
preživljavanje uslova suše i redukciju rasta fitopatogena u ko-kulturi.
U okviru ove studije prvi put je pokazana antifungalna aktivnost autohtonog
izolata Pseudomonas synxantha P4/16_1 na M. laxa. In vitro je utvrđen potencijal za
redukciju rasta M. laxa u opsegu 80 – 88% kao i za proizvodnju antifungalnih
isparljivih organskih jedinjenja i fenazin-1-karboksilne kiseline. Sirovi benzenov
ekstrakt P. synxantha P4/16_1 je in vitro inhibirao rast micelije M. laxa u opsegu od 57
do 63%, dok je ex situ zabeleženo 100% redukcije rasta M. laxa na plodu sorte Ranka.
Svetlosnom i skenirajućom elektronskom mikroskopijom su zabeležene in vitro
morfološke promene na hifama izolata M. laxa izazvane punom kulturom i benzenovim
ekstraktom P. synxantha P4/16_1. HPLC-MS analiza benzenovog ekstrakta je
detektovala prisustvo derivata fenazina i strukturnih izomera hidroksibenzoeve kiseline.
Pored antifungalne aktivnosti, za P. synxantha P4/16_1 je utvrđeno da ima potencijal za
preživljavanja suše, intenzivnu produkciju siderofora kao i da ispoljava in vitro
antibakterijsku aktivnost u formi benzenovog ekstrakta na fitopatogenim sojevima.
Filosfera šljive se pokazala kao dobra osnova za izolaciju dva biokontrolna
kandidata sa potencijalom za suzbijanje značajnih bolesti šljive. Bacillus thuringiensis
R3/3 je pokazao potencijal za dalje istraživanje u biokontroli bakterioza šljive, dok je P.
synxantha P4/16_1 obećavajući kandidat za ispitivanja potencijalne upotrebe u
biološkoj kontroli izazivača mrke truleži. | Plum (Prunus domestica L.) has a great importance as a fruit crop in Serbia and
it is traditionally processed into slivovitz brandy. Considering that the annual plum yield
is significantly affected by various pathogens, especially those causing bacterial canker
and brown rot, it is necessary to find effective agents for their control, and in particular,
an alternative to the chemical pesticides. In this research, microbiome of four plum
cultivars phyllosphere (Čačanska lepotica, Čačanska rodna, Požegača i Ranka) was
analyzed for the first time, including the early (May) and late phenological stages (July)
of fruit development. Alpha and beta diversity indices had shown that the communities
were heterogenous and that their composition depended on sampling season and plum
cultivars. The metagenomic approach revealed that the representatives of the phylum
Proteobacteria were the most dominant in the total bacterial community, and that the
most represented species were from the genera: Methylobacterium, Sphingomonas and
Hymenobacter. Traditional cultivable approach and 16S rRNA sequence analyses
identified Pseudomonas syringae i Pseudomonas graminis as two most dominant
species. Mycobiome analysis revealed the dominance of the phylum Ascomycota
representatives. Aureobasidium and Cryptococcus species were the most abundant in
May, while in July representatives of different genera dominated the plum cultivars
(Cryptococcus, Metschnikowia, Fusarium and Hanseniaspora). From the collection of
cultivable fungi, eleven Monilinia laxa isolates were identified based on phylogenetic
analysis of the ITS region sequences. The selected M. laxa isolates caused typical
symptoms on the harvested fruits of the Požegača and Čačanska rodna cultivars, and the
testing of potential bacterial pathogens revealed P. syringae ČL2/2 and P7/16_2
isolates, which caused necrosis on the harvested plum leaves of all tested cultivars.
Antagonistic potential analysis of bacterial and yeast isolates from all four
cultivars singled out Bacillus thuringiensis R3/3 with strong antibacterial activity
against native and reference phytopathogens. Characterization of B. thuringiensis R3/3
indicated that isolate had genetic potential to produce kurstakin and N-acyl homoserine
lactonase, and has shown the in vitro ability of quorum quenching, production of
hydrolytic exoenzymes, surviving drought conditions and reducing phytopathogen
growth in co-culture.
This study demonstrated, for the first time, the antifungal activity of the
indigenous isolate Pseudomonas synxantha P4/16_1 against M. laxa. The in vitro
potential of M. laxa growth reduction in the range of 80-88%, as well as the production
of antifungal volatile organic compounds and phenazine-1-carboxylic acid has been
determined. The crude benzene extract of P. synxantha P4/16_1 in vitro inhibited M.
laxa mycelium growth in the range of 57 to 63%, while ex situ was observed 100%
reduction of M. laxa growth on Ranka cultivar fruit. Light and Scanning electron
microscopy recorded in vitro morphological changes on hyphae of M. laxa isolates
caused by full culture and benzene extract of P. synxantha P4/16_1. The HPLC-MS
analysis of benzene extract detected the presence of phenazine derivatives and structural
isomers of hydroxybenzoic acid. Besides antifungal activity, for P. synxantha P4/16_1
has been shown to have the potential for survival of drought, intensive production of
siderophores, and that exhibit in vitro antibacterial activity in the form of benzene
extract on phytopathogenic strains.
The plum phyllosphere has shown to be a good basis for isolating two biocontrol
candidates with the potential to suppress significant plum diseases. Bacillus
thuringiensis R3/3 has shown potential to be further investigated in the biocontrol of
plum bacteriosis, while P. synxantha P4/16_1 is a promising candidate for trials of
potential use in the biological control of brown rot pathogen. | true |
Praćenje uticaja pastrmskog ribnjaka na nivo aktivnosti antioksidativnih enzima kod larve Ephemera danica (Insecta, Ephemeroptera) | Monitoring the effect of trout farms on the antioxidative enzyme activity level in Ephemera danica larvae (Insecta, Ephemeroptera) | U monitoringu površinskih voda neophodno je pored praćenja abiotičkih faktora uključiti i biotičku komponentu tj. analizirati sastav i strukturu zajednica vodenih organizama. Da bi se dobila kompletnija slika o stanju vodenog ekosistema koriste se organizmi bioindikatori. Larva Ephemera danica ima specifične morfološke i funkcionalne karakteristike te je pogodan model organizam za studije biomonitoringa. Cilj doktorske disertacije je praćenje uticaja ispusnih voda pastrmskog ribnjaka na nivo aktivnosti enzima antioksidativne zaštite kod larvi E. danica, ispitivanje stepena bioakumulacije teških metala u telu larvi, vodi i sedimentu reke Skrapež, utvrđivanje faunističkog sastava makrozoobentosa istraživane tekućice. Hidrobiološka istraživanja reke Skrapež sprovedena su tokom 2015. (proleće, leto i jesen) i 2016. godine (zima) na jednom lokalitetu iznad i tri lokaliteta nizvodno od pastrmskog ribnjaka. Dvofaktorska analiza varijanse je pokazala da aktivnost SOD karakteriše veća sezonska varijabilnost, aktivnost GPx karakteriše značajnija longitudinalna varijabilnost i u svim sezonama dolazi do statistički značajnog porasta aktivnosti GPx na drugom lokalitetu, što ukazuje da organsko zagađenje poreklom sa ribnjaka indukuje oksidativni stres kod vrste E. danica. Koncentracija ukupnog glutationa (GSH) dostiže maksimum u jesen na drugom lokalitetu, što je negativan uticaj pastrmskog ribnjaka. Analiza CIA je pokazala postojanje statistički značajne kostrukture između PCA korelacione matrice koncentracija teških metala u sedimentu i u telu E. danica. Indeks saprobnosti ukazuje na uticaj ribnjaka kroz pogoršanje kvaliteta vode nizvodno od njega. Njegov uticaj je i u smanjenju procenta sekača nizvodno od ribnjaka, a povećanju sakupljača, aktivnih i pasivnih sakupljača filtratora. | biotic component, i.e., to analyses the composition and structure of communities of aquatic organisms. Bioindicator organisms are used to get a more complete picture of the state of an aquatic ecosystem. The larva of Ephemera danica has specific characteristics that make it a suitable model organism for biomonitoring studies. The purpose of the doctoral dissertation was to monitor the influence of outlet water of a trout farm on the level of activity of antioxidative defense enzymes in larvae of E. danica; test the degree of bioaccumulation of heavy metals in the body of larvae, water, and sediment, and establish faunistic composition of the macrozoobenthos. Hydrobiological investigation of the Skrapež River was conducted during 2015 (spring, summer, and fall) and 2016 (winter) at one locality upstream from the trout farm and three localities downstream from it. Two-factor analysis of variance showed that SOD activity is characterized by high seasonal variability, GPx activity is characterized by considerable longitudinal variability, and a statistically significant increase of GPx activity occurs in all seasons at the second locality, which indicates that organic pollution originating from the fish farm induces oxidative stress in the species E. danica. The concentration of total glutathione (GSH) attains a maximum in the fall at the second locality, which is a negative effect of the trout farm. Use of CIA revealed the existence of a statistically significant contracture between PCA of the correlation matrix of heavy metal concentrations in the sediment and in the body of E. danica. The saprobity index indicates influence of the fish farm in the form of a worsening of water quality downstream from it. Influence of the fish farm manifested in a decrease in the choppers at localities downstream from it, and an increase in the gatherers (active and passive gatherer-filterers). | false |
Karakterizacija površinskih molekula bakterijskih ćelija odgovornih za potencijalnu probiotičku aktivnost prirodnih izolata laktobacila | Characterization of surface molecules from bacterial cells involved in the potential probiotic activity of natural lactobacilli isolates | Probiotički potencijal bakterija u velikoj meri zavisi od površinskih karakteristika
bakterijske ćelije. Stoga su u ovom radu analizirane površinske komponente ćelija
odgovorne za agregacione sposobnosti i produkciju egzopolisaharida (EPS) prirodnih
izolata laktobacila. Sojevi laktobacila korišćeni u ovom radu su izolovani iz autohtonih
sireva proizvedenih u domaćinstvima prema tradicionalnoj tehnologiji. Odabrani laktobacili
koji su ispoljavali autoagregaciju (BGAR75, BGGR2-68, BGGR2-82, BGDP9-85,
BGDP1-84, BGNJ1-3, BGNJ1-61, BGNJ1-70), kao i dva odabrana soja koja ne agregiraju
(BGAR76 i BGGR2-20), su klasifikovani na osnovu poređenja nukleotidnih sekvenci gena
za 16S rRNK sa NCBI bazom podataka i svrstani u grupu Lactobacillus casei. Soj
BGDU4-71 je determinisan sekvenciranjem 16S rDNK kao Lactobacillus delbrueckii
subsp. bulgaricus, a soj BGCG11, proizvođač egzopolisaharida (EPS-CG11), je
determinisan AFLP metodom kao Lactobacillus paraplantarum. U cilju karakterizacije
faktora uključenih u proces agregacije u ovom radu su analizirani brzina i tip
autoagregacije. Brzina autoagregacije (utvrđivana spektrofotometrijski) kao i oblik agregata
su varirali kod sojeva. Najbrže su agregirali i formirali najkrupnije agregate sojevi BGSJ2-
8, BGDP1-84 i BGNJ1-6. Karakterizacija prirode faktora koji utiču na sposobnost
autoagregacije izabranih sojeva laktobacila je rađena intenzivnim pranjem sojeva koji
agregiraju u bidestilovanoj vodi i u PBS rastvoru. Uočeno je da se autoagregacija gubila
intenzivnim pranjem u bidestilovanoj vodi kod svih sojeva, osim kod BGDP1-84, na
osnovu čega je zaključeno da je prisustvo jona neophodno za formiranje agregata. Osim
toga, pokazano je da su neki od faktora autoagregacije proteinske prirode s obzirom da se
ova karakteristika gubila nakon tretmana proteinazom K. Lb. paracasei subsp. paracasei
BGSJ2-8 je formirao koagregate sa Listeria innocua ATCC33090, Escherichia coli
ATCC25922 ili sa Salmonella enterica ser. Typhimurium TR251, dok njegov derivat
BGSJ2-81 koji je izgubio sposobnost autoagregacije nije koagregirao. Spektrofotometrijsko
praćenje ovih interakcija je pokazalo da je nakon tretmana proteinazom K koagregacija bila
narušena, što ukazuje da su u proces koagregacije, kao i autoagregacije, uključeni faktori
proteinske prirode. Analiza površinskih karakteristika sojeva koji autoagregiraju metodom
adhezije za heksadekan, pokazala je da svi sojevi koji imaju sposobnost autoagregacije
ispoljavaju visoku hidrofobnost ćelijske površine, dok je u sojevima (kao i derivatu BGSJ2-
81) koji ne agregiraju ćelijska površina hidrofilna. Drugi deo rada je obuhvatao analizu
probiotičkog potencijala soja Lb. paraplantarum BGCG11, producenta egzopolisaharida
EPS-CG11 u in vitro modelima. U radu su takođe korišćeni i Muc derivati soja BGCG11
(NB1, NB4, NB16), dobijeni čišćenjem plazmida tretmanom novobiocinom. Pomenuti
derivati produkuju znatno manje EPS-a različitog sastava od EPS-CG11. Analiza
preživljavanja simuliranog prolaska kroz gastrointestinalni trakt (GIT) kao i adhezija za tri
ćelijske linije (Caco-2, HT29, HT29-MTX) je rađena brojanjem kolonija pre i nakon
tretmana/interakcije i određivanjem procenta preživljavanja/adhezije u odnosu na početni
broj bakterija. BGCG11 i Muc derivati (NB1, NB4, NB16) su preživljavali (1-2%)
simulirani prolazak kroz (GIT) ukoliko su bili resuspendovani u 10% obranom mleku. EPS-
CG11 je izolovan i prečišćen iz supernatanta kulture BGCG11 gajene u minimalnom
medijumu sa glukozom kao jedinim izvorom šećera. HPLC metodom sa MALLS
detektorom je praćena promena molarne mase i količine EPS-CG11 polimera nakon
simulirane digestije kroz želudačni i crevni sok. Rezultati su pokazali da su masa i količina
EPS-CG11 ostale nepromenjene, što znači da enzimi GIT ne utiču na stabilnost prečišćenog
EPS-CG11. Tokom testiranja sposobnosti adhezije za ćelijske linije soj BGCG11, kao i
kontrolni probiotički soj Lb. rhamnosus GG, su pokazali sličan procenat adhezije, dok su se
Mucderivati NB1, NB4 i NB16 statistički značajnije vezivali u većem procentu za sve tri
ćelijske linije. Svi testirani sojevi laktobacila su pokazali veoma nizak procenat adhezije za
HT29-MTX ćelijsku liniju, što je najverovatnije posledica prisustva mukusne barijere
(HT29-MTX ćelijska linija, za razliku od Caco-2 i HT29, produkuje mucine). Probiotički
potencijal soja BGCG11 je takođe praćen in vitro analizom indukcije imunog odgovora
limfocita periferne krvi dobrovoljnih davalaca u prisustvu mrtvih ćelija BGCG11,
Mucderivata i prečišćenog EPS-CG11. Imuni odgovor limfocita je praćen preko
proliferacije limfocita (korišćenjem kita za proliferaciju) i produkcije citokina (IFNγ,
TNFα, IL-12, IL-10, IL-1β i IL-17) metodom protočne citometrije. Zapaženo je da su
limfociti periferne krvi proliferisali u prisustvu laktobacila (BGCG11 i Mucderivata NB1,
NB4 i NB16) dok EPS-CG11 nije uticao na proliferaciju limfocita, što znači da su neki
drugi molekuli, a ne EPS zaslužni za proliferaciju limfocita. Kad se gleda generalno tip
imunog odgovora koji izaziva u prisustvo mrtvih laktobacila, može se reći da je prisustvo
soja BGCG11 i EPS-CG11 (u koncentraciji od 100 g/ml) indukovalo anti-inflamatorni i
pro-Th17 odgovor, dok su Muc derivati NB1, NB4 i NB16 indukovali inflamatorni
odgovor limfocita. Dalje je rađena detaljnija analiza uticaja potencijalnih patogenih
mikroorganizama, samih ili u koinkubaciji sa BGCG11, derivatom NB1 i EPS-CG11 na
HT29-MTX ćelijsku liniju. Analiziran je citotoksični efekat na HT29-MTX ćelije
(merenjem nivoa aktivnosti laktat dehidrogenaze) i nivo produkcije IL-8 (metodom
ELISA). Rezultati su poređeni sa efektom koji izaziva Lb. rhamnosus GG i njegov izolovan
i prečišćen EPS-GG u istim uslovima testiranja. Od patogenih bakterija samo je Listeria
monocytogenes LMG13305 izazivala značajniju lizu ćelija, a taj nivo citotoksičnog efekta u
HT29-MTX ćelijama je bio smanjen nakon koinkubacije L. monocytogenes LMG13305 sa
EPS-CG11. Nivo produkcije IL-8 u HT29-MTX ćelijama nakon koinkubacije patogenih
sojeva sa laktobacilima (BGCG11, Mucderivat NB1 i Lb. rhamnosus GG) ili sa EPS
molekulina (EPS-CG11 ili EPS-GG) je bio različit u odnosu na inkubaciju samo sa
patogenim sojevima. BGCG11 i NB1 značajno povećavaju indukciju IL-8 u prisustvu
Salmonella enterica ser. Thyphymurium LMG15660, Shigella sonnei LMG10473 i
Yersinia enterocolitica LMG7899. Samo je BGCG11 značajno povećao IL-8 u koinkubaciji
sa L. monocytogenes LMG13305. EPS-CG11 je snizio nivo ovog citokina u koinkubaciji sa
L. monocytogenes LMG13305, a oba tipa EPS molekula (EPS-CG11 i EPS-GG) su
značajno smanjili nivo produkcije IL-8 u koinkubaciji sa Clostridium difficile LMG21717.
Može se zaključiti da različiti molekuli sa površine kako samih patogena, tako i laktobacila
modifikuju nespecifični imuni odgovor HT29-MTX ćelijske linije, dok prisustvo
prečišćenih EPS molekula uglavnom stišava taj odgovor, a važno je reći da se razlikuje
efekat EPS-CG11 i EPS-GG polimera, kao i Lb. rhamnosus GG u odnosu na Lb.
paraplantarum BGCG11. Na kraju, u ovom radu je lokalizovan i okarakterisan potencijalni
operon EPS-CG11. Tehnikama molekularne genetike (kloniranjem, sekvenciranjem, DNK-
DNK hibridizacijom) iz plazmidne DNK soja BGCG11 dobijeno je 26463 bp sekvence
velikog plazmida pCG1, koji nedostaje Muc derivatima. Na ovom plazmidu je lokalizovan
potencijalni operon EPS-CG11: region od oko 15 kb sa 15 otvorenih okvira čitanja (ORF –
“open reading frame“). Na osnovu homologije aminokiselinske sekvence ORF-ovi su
identifikovani kao: primarna glikoziltransferaza (ORF 1), glikoziltransferaze (ORF 2, 3, 4,
5 i ORF 8), polisaharid polimeraze (ORF 6 i 7), regulatori dužine lanca polisaharida (ORF
9, 10 i 11) i regulatori biosinteze EPS-a (ORF 10 i 11). Nizvodno od pomenutih ORF-ova
je transpozaza, dok su ORF 13, 14, 15 i 16 pokazali homologiju na aminokiselinskom
nivou sa proteinima koji sintetišu dTDP-ramnozu (geni rfbACBD) koja ulazi u sastav EPS-
CG11. Na ovaj način je dobijena nova i originalna struktura genskog operona EPS-CG11,
koji je po prvi put okarakterisan u vrsti Lactobacillus paraplantarum, koji je specifičan po
svojoj plazmidnoj lokalizaciji. | The probiotic potential of bacteria depends on the surface characteristics of bacterial
cells. Keeping this in mind, in this work the surface components of the cells responsible for
the aggregation ability and the production of exopolysaccharides (EPS) were analyzed from
the natural isolates of lactobacilli. Lactobacilli strains used in this work were isolated from
autochthonous cheeses produced in households according to the traditional technology.
Selected lactobacilli showing the autoaggregation ability (BGAR75, BGGR2-68, BGGR2-
82, BGDP9-85, BGDP1-84, BGNJ1-3, BGNJ1-61, BGNJ1-70) as well as two selected
strains which do not form aggregates (BGAR76 and BGGR2-20) according to the
comparison of their 16S rRNA gene sequence to the NCBI database were classified in the
group Lactobacillus casei. The strain BGDU4-71 using the 16S rRNA gene sequence was
determined as Lactobacillus delbrueckii subsp. bulgaricus and the strain BGCG11, the
producer of the exopolysaccharide (EPS-CG11), was determined by AFLP methodology as
Lactobacillus paraplantarum. In order to characterize factors involved in the aggregation
process the kinetics and the type of autoaggregation were analyzed. The kinetics
(spectrofotometrically determined) as well as the shape of the aggregates was variable
among the strains. Strains with fastest and the largest aggregates were BGSJ2-8, BGDP1-
84 and BGNJ1-6. The characterization of the nature of factors involved in autoaggregation
of selected strains of lactobacilli was performed by exhaustive washing of the strains in
distilled water and in PBS solution. It was noticed that the autoaggregation ability was lost
after exhaustive washing in distilled water in all tested strains except BGDP1-84, which led
to the conclusion that the presence of some ions was necessary for the formation of the
aggregates. Besides, it was shown that some of the factors promoting autoaggregation were
of proteinaceous nature, since the ability was lost after the proteinase K treatment. Lb.
paracasei subsp. paracasei BGSJ2-8 was able to form coaggregates with Listeria innocua
ATCC33090, Escherichia coli ATCC25922 or with Salmonella enterica ser. Typhimurium
TR251, while its derivative BGSJ2-81 that was not able to autoaggregate, did not show
coaggregation. Spectrofotometrical measurements of the interaction showed that the
coaggregation was disturbed after proteinase K treatment, which indicated that
coaggregation, as well as the autoaggregation, involve some factors with proteinaceous
nature. The analysis of the surface characteristics of strains showing autoaggregation by
hexadecane adhesion method showed that all strains with the autoaggregation ability were
highly hydrophobic, while the ones that do not aggregate (as well as for the derivative
BGSJ2-81) have hydrophilic cell surface . The other part of the work was including the
analysis of the probiotic potential of the strain Lb. paraplantarum BGCG11, which
produces exopolysaccharide EPS-CG11, in in vitro model systems. In this part of the work,
the Muc derivatives from the strain BGCG11 (NB1, NB4, NB16), obtained by novobiocin
plasmid curing, were included in analyses. The derivatives produce significantly lower
amount of EPS that differs from EPS-CG11 in composition. The analysis of survival in
simulated transit trough gastrointestinal tract (GIT), as well as the adhesion to three cell
lines (Caco-2, HT29, HT29-MTX) were performed by counting of bacterial colonies before
and after the treatment/interaction and the percentage of survival/adhesion was calculated
referring to the number of bacteria before the incubation with cell lines. BGCG11 and
Muc derivatives (NB1, NB4 and NB16), resuspended in 10% skimmed milk, survived (1-
2%) the simulated GIT transit. The EPS-CG11 was isolated and purified from the
supernatant of the liquid BGCG11 culture, cultivated in minimal media with glucose as the
only sugar source. HPLC method with MALLS detector was used to monitor the change in
molar mass and the amount of EPS-CG11 polymer after simulated transit trough gastric and
intestinal digestion. Obtained results showed that the molar mass and the amount of EPS-
CG11 remained unchanged, indicating that the enzymes in GIT did not affect the stability
of the purified EPS-CG11. Testing of the adhesion ability of BGCG11, as well as the
control probiotic strain Lb. rhamnosus GG, showed similar percentage of adhesion for
these two lactobacilli, while Mucderivatives NB1, NB4 and NB16 expressed statistically
significant higher percentage of adhesion to all three cell lines. All tested strains showed
very low percentage of adhesion to HT29-MTX cell line, what is the most likely due to the
presence of the mucus barrier (HT29-MTX cell line, produces mucins, while Caco-2 cells
do not). The probiotic potential of the strain BGCG11 was also monitored by in vitro
analysis of the induction of the immune response in the presence of UV-inactivated bacteria
(BGCG11, Mucderivatives) and purified EPS-CG11, in human peripheral blood
lymphocytes (PBLs) isolated from healthy volunteers. The immune response was
monitored by the proliferation of PBLs (by using kit for the proliferation measurement) and
the cytokine production (IFNγ, TNFα, IL-12, IL-10, IL-1β and IL-17) by flow cytometry. It
was noticed that PBLs proliferated in the presence of lactobacilli (BGCG11 and
Mucderivatives NB1, NB4 and NB16), while EPS-CG11 did not affect to the
proliferation of PBLs, which led to the conclusion that some other molecules, rather than
EPS were involved in inducing of the proliferation. Generally analyzing the type of the
immune response, the presence of BGCG11 and the EPS-CG11 (in concentration of 100
g/ml) induced anti-inflammatory and a pro-Th17 response, while Muc derivatives NB1,
NB4 and NB16 induced inflammatory response in PBLs. Furthermore, the detailed analysis
of the influence of potential pathogenic microorganisms, alone or in coincubation with
BGCG11, derivative NB1 and EPS-CG11, on HT29-MTX cell line was performed. The
cytotoxic effect in HT29-MTX cell line (performed by measurement of the lactate
dehydrogenase activity) and the analysis of the production IL-8 (by ELISA method) was
performed. Obtained results were compared to the effects of Lb. rhamnosus GG and its
isolated and purified EPS-GG in the same experimental conditions. From all tested
pathogens, only Listeria monocytogenes LMG13305 induced significant cell lyses in
HT29-MTX cells, which was reduced after coincubation of L. monocytogenes LMG13305
with EPS-CG11. The level of the IL-8 production in HT29-MTX cells after coincubation of
pathogens with lactobacilli (BGCG11, Mucderivative NB1 and Lb. rhamnosus GG) or
with EPS molecules (EPS-CG11 or EPS-GG) was different in comparison to levels
obtained after incubation only with pathogenic strains. BGCG11 and NB1 induced
significantly higher level of IL-8 in the presence of Salmonella enterica ser.
Thyphymurium LMG15660, Shigella sonnei LMG10473 and Yersinia enterocolitica
LMG7899. In coincubation with L. monocytogenes LMG13305, only BGCG11 increased
the level of IL-8 production. EPS-CG11 decreased the level of this cytokine in coincubation
with L. monocytogenes LMG13305, while both type of EPS molecules (EPS-CG11 and
EPS-GG) significantly decreased the IL-8 production in coincubation with Clostridium
difficile LMG21717. The conclusion is that different molecules from the cell surface of
pathogens, and lactobacilli modify nonspecific immune response of HT29-MTX cell line,
while the presence of purified EPS molecules showed silencing of this response. Hence, it
is important to see whether the effects of EPS-CG11 and EPS-GG are different, since the
difference was seen when Lb. rhamnosus GG and Lb. paraplantarum BGCG11 strains
were. At the end of this work, the potential EPS-CG11 operon was localized and
characterized. Using techniques of molecular genetics (cloning, sequencing, DNA-DNA
hybridization) 26463 bp of the sequence of large plasmid pCG1, lacking in Muc
derivatives, were obtained. In this plasmid the potential operon EPS-CG11 was localized:
the region of 15 kb with 15 open reading frames (ORFs). Based on the amino acid sequence
homology these ORFs were identified as: the priming glycosyltransferase (ORF 1),
glycosyltransferases (ORF 2, 3, 4, 5 and ORF 8), polysaccharide polymerases (ORF 6 and
7), chain length determinators (ORF 9, 10 and 11) and the EPS biosynthesis regulators
(ORF 10 and 11). Downstream from these ORFs was the transposase, while ORF 13, 14, 15
and 16 showed homology at the amino acid level with proteins involved in synthesis of
dTDP-rhamnose (rfbACBD genes) that is a part of the EPS-CG11 composition. In this way,
the new and original structure of the EPS-CG11 gene operon was for the first time
characterized in Lactobacillus paraplantarum species. Moreover, this operon is also
specific by its plasmid localization. | true |