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Kvalitativna i kvantitativna analiza humanih bioloških tragova minimalnih graničnih količina u forenzičkim analizama DNK

Qualitative and quantitative assesment of biological traces in forensic DNA analysis

Ćelije kože ostavljene na površini bilo kog predmeta nakon kontakta sa njim mogu biti izvor DNK materijala. Ova vrsta biološkog materijala, u odnosu na količinu DNK koju je moguće izolovati iz nje, obično je mnogo manje izdašna u odnosu na tragove koji se rutinski analiziraju (krv, semena tečnost, pljuvačka). Iako visoko osetljive tehnike analize omogućavaju dobijanje rezultata iz minimalnih količina DNK, još uvek postoje znatne poteškoće u radu sa ovakvim uzorcima, i to iz više razloga. Različit kvalitet i kvantitet izolovane DNK, te posebno izraženi stohastički efekti samo su neki od njih. Zbog toga je proces analize DNK materijala izolovan iz ovakvih uzoraka posebno kompleksan. Biološki uzorci ove vrste se vrlo često nalaze u izuzetno malim količinama (doslovno tragovima), što se postojećim rutinskim metodama analize DNK ne mogu pouzdano interpretirati. U cilju poboljšavanja rezultata analize DNK materijala dobijenog izolovanjem biološkog materijala zaostalog na površini dodirnutih predmeta, analizirano je više od 700 otisaka prstiju. Svaki korak procesa je testiran posebno u cilju dobijanja STR profila koji su bar 70% kompletni, te stoga mogu biti uneti u baze podataka. Kompletnost profila je određivana na osnovu broja dobijenih alela iz 15 testiranih lokusa (najviše moguće 30 alela). Testirani su sledeći koraci u proceduri dobijanja DNK profila: prikupljanje uzoraka, izolovanje DNK, umnožavanje STR lokusa kao i detekcija proizvoda umnožavanja kapilarnom elektroforezom. Ćelije su prikupljene korišćenjem mikrosfera, lepljivih traka, lepila ili pamučnih briseva natopljenih različitim rastvorima. DNK materijal je izolovan korišćenjem interno razvijenih metoda i/ili komercijalno dostupnih kompleta hemikalija za njenu izolaciju. Izolovana DNK je umnožavana Identifiler® kompletom, korišćenjem različitog broja PCR ciklusa...

Skin flakes left on an object after it has been touched or handled could be a source of DNA. These skin flakes tend to be deposited in considerably smaller amounts than from routinely tested cells of blood, semen or saliva. Although, highly sensitive DNA analysis procedures are able to provide results from trace amounts of DNA there are still some fundamental difficulties inherent to these samples, including variability in quality and quantity of extracted DNA and exaggerated stochastic effects, making it hard to reliably interpret DNA profiles of these samples. These types of samples could also carry skin flakes in trace, which currently applied methodology of testing frequently cannot interpret. In order to improve the results from touched DNA samples, over 700 fingerprints were tested. Each step of the workflow for genotyping was assessed with the goal to generate STR profiles that were at least 70% complete and therefore database eligible. The profiles were calculated from the number of obtained alleles with a maximum of 30 for the 15 amplified STR loci. The steps evaluated in the workflow included sample collection, DNA extraction, STR amplification and detection utilizing capillary electrophoresis. Cells were collected using microglobes, tapes, glues, or cotton swabs moistened with different solutions. DNA extraction was assessed with methods designed in the laboratory and commercially available extraction kits. Extracted DNA was amplified with Identifiler® kits using various number of PCR cycles. These comparisons led to the best method that generated a database eligible STR profiles from almost 70% of tested fingerprints. This method suggested collection of fingerprints by swabbing with cotton swab moistened in detergent solution, then extracting DNA using a commercially available extraction kit that uses enzyme activated at a high temperature, followed by amplification at higher PCR cycle number and analysis at longer injection time and higher voltage during capillary electrophoresis...

Model za ocenu ekološke ispravnosti kuća za individualno stanovanje na području Beograda

Model for environmental assessment of single family houses in Belgrade area

Osnovna svrha istraživanja sprovedenog u doktorskoj disertaciji jeste definisanje metodologije jedinstvenog procesa sveobuhvatnog ekološkog vrednovanja kuća za individualno stanovanje na području Beograda. U skladu sa tim, model za ocenu ekološke ispravnosti formiran je prvo kao opšti, a zatim su iz njega, zbog karakteristika sektora individualnog stanovanja na području Beograda, izvedena tri posebna modela, i to za ocenu: novoprojektovanih, obnovljenih i postojećih kuća za individualno stanovanje. Model je struktuiran po: kategorijama, podkategorijama i kriterijumima (najpreciznijim temama) ocene ekološke ispravnosti. Za određivanje kategorija, koje predstavljaju osnovna polja ocene ekološke ispravnosti, presudan značaj imali su ekološki uticaji kuća za individualno stanovanje na životnu sredinu, dok su se karakteristike područja Beograda, postojeća domaća i inostrana regulativa i druge smernice, kao i razvijeni inostrani modeli odrazili na definisanje sadržaja tih kategorija. Svi definisani kriterijumi u modelu podeljeni su na obavezne uslove i mere sa dobrovoljnim ispunjenjem kojima su dodeljeni ekološki poeni. Sabiranjem broja dodeljenih ekoloških poena, prvo za pripadajuće kriterijume, zatim podkategorije i konačno kategorije dobija se ukupni broj ekoloških poena dodeljen kući čija se ekološka ispravnost ispituje. Uspostavljen je minimalni prag ekološke ispravnosti ispod kojeg kuća koja se ocenjuje ne može biti okarakterisana kao ekološki ispravna, a iznad kojeg se, u zavisnosti od broja dodeljenih poena, ona svrstava u jedan od tri nivoa ekološke ispravnosti. Kući koja je postavljena u najoptimalniji položaj prema okruženju (sa minimalnim tokovima od prirode i ka njoj) biće dodeljen maksimalni broj ekoloških poena. Na taj način definisana je idealna kombinacija primenjenih mera, relevantnih za područje Beograda i za tip kuća za individualno stanovanje. Nakon što su definisane struktura modela i metodologija ocene, kreiran je elektronski alat u računarskom programu EXCEL, čime je stvorenom metodu data upotrebna vrednost. Rad alata testiran je na primerima postojećih kuća za individualno stanovanje na području Beograda, čime je utvrđeno da je model funkcionalan, a da su rezultati koji se preko njega dobijaju tačni. Struktura opšteg modela iz kojeg su ponikli posebni kreirana je segmentno, što je, na jednoj stani, omogućilo jednostavnije razdvajanje varijati, a na drugoj, još važnije, integrisanje životnog ciklusa kuće sa kriterijumima preko kojih se ocena ekološke ispravnosti sprovodi. Određivanje uticajnih faktora za formiranje strukture modela za ocenu ekološke ispravnosti, izvršeno u ovoj doktorskoj disertaciji na primeru kuća za individualno stanovanje, od značaja je za kreiranje modela za ocenu ekološke ispravnosti drugih tipova arhitektonskih objekata i na drugim predmetnim područjima, što definiše pravce budućih naučnih istraživanja u oblasti.

The aim of the research conducted in this doctoral dissertation was to define methodology for developing a system for comprehensive environmental assessment of individual houses in the area of Belgrade. According to this, model for environmental assessment was initially formed as general. Three separate models for assessments of new, renewed and existing houses, based on characteristics of individual housing sector in Belgrade, were derived later. Model structure is consisted of categories, subcategories and criteria (the most precise items) of environmental assessment. Environmental impacts of individual houses were found as the most relevant factor for determination of categories (basic fields of environmental assessment). Characteristics of the area of Belgrade, existing domestic and international legislative and other sources, as well as internationally developed models, on the other side, all influenced the content of categories. All defined criteria were divided on prerequisites and voluntary measures which carry a certain number of ecological points. By summing the numbers of ecological points, first by criteria, then subcategories and finally categories, the total number of achieved ecological points is obtained. Minimum threshold below which the house cannot be defined as environmentally correct is set; above this threshold, according to the total number of the obtained ecological points, the house will be classified among three set levels of environmental quality. Maximum number of ecological points will be given to the house set in optimal position towards environment (with minimal flows towards nature and from it). Therefore, the ideal combination of applied measures relevant for the area of Belgrade and for the type of individual houses is determined. Defined model structure and methodology for assessment opened the road to the creation of electronic tool in EXCEL computer program by which the practical value has been given to the model. The tool was tested on examples of existing individual houses in Belgrade; it is confirmed that the model is functional and that the obtained results are correct. The structure of general model, from which three separate were later derived, was created in segments; from one side, this enabled simplified separation of variants, and from other, more important, integration of life cycle with criteria of environmental assessment. Determination of factors influencing the structure of model for environmental assessment, conducted in this doctoral dissertation on the example of individual houses, is significant for the creation of models for environmental assessment of other types of buildings and in other areas, which defines the direction of future researches in the field.

Uloga HMGB1 proteina u modifikaciji i integraciji signalnih puteva apoptoze i autofagije u ćelijama jetre pacova sa indukovanim dijabetesom

The role of HMGB1 protein in the modification and integration of signaling pathways of apoptosis and autophagy in liver cells of diabetic rats.

Ćelijska smrt je jedan od uzroka patoloških promena u jetri tokom dijabetesa. Protein koji može imati važnu ulogu u ovim procesima, bilo kao marker nekroze ili aktivator signalnih puteva koji vode ćelijskoj smrti ili preživljavanju, je HMGB1. U vanćelijsku sredinu HMGB1 dospeva iz nekrotičnih, oštećenih ćelija i aktiviranih imunskih ćelija. U ovoj doktorskoj disertaciji ispitivana je uloga ovog proteina u određivanju profila ćelijske smrti u jetri pacova sa dijabetesom kroz njegov doprinos u modulaciji procesa apoptoze i autofagije. Rezultati ovog istraživanja pokazali su da je nivo HMGB1 proteina u serumu i jetri pacova sa dijabetesom u korelaciji sa nivoom oštećenja jetre i nekroze, što ga svrstava među potencijalne biološke markere za praćenje dijabetičnih komplikacija u jetri. Na aktivaciju procesa apoptoze i autofagije HMGB1 protein deluje dvojako, kroz aktivaciju nizvodnih signalnih puteva pokrenutih interakcijama vanćelijskog HMGB1 sa TLR4 i RAGE receptorima, i kroz uticaj citoplazmatskog HMGB1 na formiranje autofagozoma kroz interakcije sa Beklinom1. Pokazano je da nivo vanćelijskog HMGB1 proteina i njegov doprinos profilu ćelijske smrti u jetri zavisi od intenziteta prisutnog oksidativnog stresa. U uslovima oksidativnog stresa prisutnog u nelečenom dijabetesu, HMGB1 favorizuje puteve apoptoze i doprinosi ćelijskoj smrti i oštećenju jetre. Smanjenjem oksidativnog stresa i nivoa vanćelijskog HMGB1 melatoninom ili etil piruvatom, favorizuje se delovanje citoplazmatskog HMGB1 u pravcu protektivne autofagije i uklanjanja oštećenih unutarćelijskih struktura. Ovi podaci ukazuju na terapeutski značaj melatonina i etil piruvata u regulaciji aktivnosti HMGB1 u dijabetesu i na ulogu HMGB1 proteina u regulaciji i povezivanju apoptoze i autofagije.

Cell death underlies hepatic pathological changes in diabetes. HMGB1 is a protein with important roles in these changes, whether as a necrotic marker or a pathway activator determining cell death or survival. HMGB1 is released into the extracellular space by damaged or necrotic cells and activated immune cells. The role of HMGB1 in determining the profile of cell death in the liver of diabetic rats was studied by examining its role in the modulation of apoptosis and autophagy. The levels of HMGB1 protein in the serum and liver of diabetic rats correlate with the degree of necrosis and liver damage, thus rendering HMGB1 as a potential biological marker of diabetic complications. To activate apoptosis and autophagy, HMGB1 protein acts by stimulating downstream signaling pathways triggered by interactions of extracellular HMGB1 with TLR4 and RAGE receptors, and through the activity of cytoplasmic HMGB1 on the formation of autophagosomes via interaction with Beclin1, which has a central role in autophagy. Extracellular expression of HMGB1 and its contribution to the cell death profile in the liver depends on the level of oxidative stress. In oxidative stress accompanying untreated diabetes, HMGB1 promotes apoptosis and resulting liver damage. The reduction of oxidative stress by melatonin and HMGB1 release by ethyl pyruvate stimulates the cytoprotective role of cytosolic HMGB1 in autophagy activation These results points to the therapeutic significance of melatonin and ethyl pyruvate in the regulation of HMGB1 activity in diabetes, and the role of HMGB1 in the regulation and interplay between apoptosis and autophagy.

Efekat mutacije gena VHL na nivo ekspresije proteina mTOR signalnog puta u svetloćelijskom karcinomu bubrega

The effect of VHL gene mutation on the expression levels of mTOR signaling pathway proteins in clear cell renal cell carcinoma

Sporadični svetloćelijski karcinomi bubrega asocirani su sa mutacijama tumor supresorskog gena VHL kod 60-80 % pacijenata. U odsustvu funkcionalnog proteina VHL dolazi do akumulacije faktora indukovanih hipoksijom koji regulišu ekspresiju gena uključenih u procese angiogeneze, eritropoeze, ćelijskog metabolizma i preživljavanja. Pored specifičnog genetičkog profila i povišenog nivoa HIF-2α svetloćelijske karcinome bubrega karakteriše glikolitički metabolički put i povišena aktivnost mTOR signalnog puta. Nepovoljan energetski status i hipoksija blokiraju aktivnost mTOR signalnog puta preko proteina AMPK i REDD1. Ova studija imala je za cilj da utvrdi mogću asocijaciju mutacionog statusa gena VHL sa patohistološkim parametrima, nivoom HIF-2α iRNK i nivoom proteina HIF-2α, mTOR, TSC1, TSC2, eIF4E-BP1, p70S6K1, AMPK, REDD1 i PDK3 u svetloćelijskom karcinomu bubrega. Studija je obuhvatala 47 pacijenata kojima je urađena totalna nefrektomija zbog prisustva svetloćelijskog karcinoma bubrega. Genomska DNK izolovana iz uzoraka venske krvi i somatska DNK izolovana iz uzoraka tumorskog i neizmenjenog tkiva bubrega upotrebljene su za određivanje mutacionog statusa gena VHL i gubitka heterozigotnosti 3p lokusa sekvenciranjem, metodom amplifikacije višestruko ligiranih proba i fragmentnom analizom mikrosatelitnih markera. Metilacioni status promotora gena VHL ispitan je metil specifičnom PCR reakcijom nakon bisulfitne modifikacije DNK. Kvantitativni RT-PCR primenjen je za relativnu kvantifikaciju HIF-2α iRNK u tumorskom i neizmenjenog tkivu bubrega. Kvantitafikacija ekspresije proteina HIF-2α, mTOR, TSC1, eIF4E-BP1, p70S6K1, AMPK, REDD1 i PDK3 utvrđena je Western blot metodom. Semikvantitativna imunohistohemijska analiza korišćena je za utvrđivanje nivoa ekpresije proteina TSC2. Alteracija u genu VHL i/ili 3p regionu detektovana je kod 37/47 (78.7 %) analiziranih uzoraka tumorskog tkiva. Monoalelna inaktivacija gena VHL (prisustvo intragenske mutacije ili gubitak heterozigotnosti 3p lokusa) detektovana je kod 10/47 (21.3 %) pacijenata. Bialelna inaktivacija gena VHL utvrđena je kod 27/47 (57.4 %) pacijenata, odnosno 27/31 (87.1 %) nosioca intragenske mutacije. Uzorci tumorskog tkiva sa wtVHL genom imali su značajno veći gradus u odnosu na tumore sa monoalelnom i bialelnom inaktivacijom gena VHL. U našoj studiji, pokazana je asocijacija nivoa ekspresije proteina TSC1, REDD1 i PDK3 sa mutacionim statusom gena VHL. Ovo je prva studija rađena na uzorcima svetloćelijskog karcinoma bubrega u kojoj je ispitivana ekspresija i pokazana asocijacija nivoa proteina PDK3 sa mutacionim statusom gena VHL. Rezultati ove studije pokazali su naučnu opravdanost, jer pružaju informacije koje se odnose na nivo ekspresije komponenata mTOR signalnog puta u zavisnosti od mutacionog statusa gena VHL, što doprinosi potpunijem razumevanju molekularnih mehanizama koji su uključeni u proces onkogeneze bubrežnog tkiva.

Sporadic clear cell renal carcinoma is associated with mutations of the VHL gene in 60-80 % patients. The absence of a functional VHL protein leads to accumulation of hypoxia-inducible factors which regulate expression of genes involved in angiogenesis, erythropoiesis, cell metabolism and survival. Specific genetic background, overexpression of HIF-2α transcription factor as well as glycolytic metabolism and upregulated activity of mTOR pathway are the main characteristics of clear cell renal carcinoma. The lack of energy and hypoxia inhibit mTOR signaling through activity of AMPK and REDD1 proteins. The aim of present study was to determine association of VHL gene mutational status with pathohistological parameters, expression level of mRNA HIF-2α and expression levels of mTOR, TSC1, TSC2, eIF4E-BP1, p70S6K1, AMPK, REDD1 and PDK3 proteins in clear cell renal carcinoma. The study included 47 patients who underwent radical nephrectomy due to unilateral kidney tumor. DNA was extracted from peripheral venous blood, tumorous and corresponding healthy renal tissue. The VHL gene mutational status and loss of heterozigosity of 3p loci were detected using DNA sequencing, MLPA and analysis of microsatellite markers. The VHL gene promoter methylation analysis was examined by MS-PCR after bisulphite modification of DNA. Quantitative real time PCR was performed for relative quantification of HIF-2 alpha mRNA. Expression levels of HIF- 2α, mTOR, TSC1, eIF4E-BP1, p70S6K1, AMPK, REDD1 and PDK3 proteins was determined using Western blot method. Immunohistochemistry was performed for semi- quantitative analysis of TSC2 protein. Alteration of VHL gene and/or changes of 3p loci were detected in 37/47 (78.7 %) analysed tumorous samples. Monoallelic inactivation of VHL gene (intragenic mutation or loss of heterozygosity of 3p loci) occurred in 10/47 (21.3 %) tumorous samples. Biallelic inactivation of VHL gene were found in 27/47 (57.4 %) tumorous samples or 27/31 (87.1 %) samples with mutation in VHL gene. Tumors with wt VHL gene have shown significantly higher Furhman’s nuclear grade in comparison to tumors with monoallelic and biallelic inactivation of VHL gene. This study has shown association of VHL gene mutational status with expression levels of TSC1, REDD1, and PDK3 proteins. To our knowledge, this was the first study performed on clear cell renal cell carcinoma that has examined the expression level of PDK3 protein and its association with VHL gene mutational status. Scientific rationale for our study was results that provide information about the expression levels of mTOR signaling pathway components according to mutational status of VHL gene and contribute to better insights into molecular mechanisms involved in oncogenesis of renal tissue.

Model klasifikacije elemenata arhitektonskih objekata u tehničkoj dokumentaciji - formiranje i primena

A model for classifying building elements in the design documentation - establishment and application

Tema istraživanja je formiranje modela klasifikacije elemenata arhitektonskih objekata u tehničkoj dokumentaciji. Priroda procesa projektovanja i izgradnje objekata zahteva stvaranje i sakupljanje informacija širokog spektra u svakoj fazi životnog ciklusa arhitektonskog objekta. Specifičnosti ovog procesa zahtevaju jasne formate u kojima će informacije konzistentno da se prenose iz faze u fazu. Pravci razvoja građenja i tendencije visokorazvijenih sistema u svetu ukazuju da je potrebno obratiti više pažnje procesima koji prethode samoj gradnji, u koje sigurno spada i podrobno planiranje i usklađivanje formi izrade tehničke dokumentacije, što vodi najvažnijim ciljevima u izgradnji, a to su unapređenje kvaliteta, skraćenje vremena i kontrolisani troškovi građenja. Nivo obrađenosti tehničke dokumentacije, koja je neophodni element procesa ugovaranja za izgradnju objekta, potrebno je da odgovara kako domaćim, tako i svetskim standardima i da omogući lakše upravljanje procesom izgradnje i bržu i precizniju procenu troškova građenja u ranim fazama investicionog ciklusa. Analizom stanja u domaćoj i svetskoj praksi iskristalisao se i oblikovao predmet istraživanja: nedostatak jasno formulisane, naučno zasnovane nomenklature i klasifikacije, koja bi omogućila standardizaciju u izradi tehničke dokumentacije, a samim tim i unapredila odnose i procese u svim fazama životnog ciklusa arhitektonskih objekata.Formatizovana i standardizovana tehnička dokumentacija postaje zajednička nit za prenos podataka, opisa, praćenje i evaluaciju kroz sve faze projekta. Realizovano istraživanje ima za rezultat definisanje konkretnih kriterijuma, čijom primenom je formiran standardizovan model klasifikacije, baziran na elementima zgrade prema mestu i funkciji na objektu, neophodan je u cilju omogućavanja jasne komunikacije između učesnika u izgradnji u pogledu obima poslova i troškova u svakoj fazi životnog ciklusa zgrade. Ovako formiran model klasifikacije elemenata u tehničkoj dokumentaciji predstavlja konstantu u izuzetno dinamičnom procesu projektovanja i građenja arhitektonskog objekta. Istovremeno, model nudi fleksibilnost u daljem razvoju tehnologije i primene materijala, s obzirom da, postavljajući arhitektonski objekat u logičnu, centralnu poziciju u procesima investicione izgradnje, nije zavisan od ekonomskih i tehnoloških promena koje su neminovne.

The research is devoted to the creation of a model for classifying building elements and related sitework in the design documentation. The nature of the design and construction process requires creation and collection of a wide range of information at each stage of the of the building life cycle. Specifics of this process requires a clear format which will provide transmission of the information consistently among different phases. Directions of construction development and tendencies of the world’s highly developed systems indicate that it is necessary to pay more attention to the process preceding the construction, which certainly includes the detailed planning and coordination of design and building documentation, leading to the most important goals, such as improving quality, reducing time and controling construction costs. The processing of the design documentation is a necessary element in the contracting process for building construction and it is required in order to comply with both national and international standards and provide an easier management of the building process, as well as faster and more accurate construction cost estimates in the early stages of the investment cycle. Analysis of the state of local and global practice crystallized and formed the subject of research: the lack of clearly formulated, science-based nomenclature and classification, which would allow standardization in the development of the design documentation, and therefore improve the relations and processes in all stages of the building life cycle. Structured and standardized design documentation becomes the common link for the transmision of data, descriptions, monitoring and evaluation informations throughout all phases of the project. The research has resulted in the definition of specific criteria according to which a standardized model of classification was formed, based on the elements of the building according to the place and function of the building. The purpose of the model is to provide clear communication between participants in the building process in terms of workload and costs in each stage of the building life cycle. A model of classification based on building elements becomes a design documentation constant base in the highly dynamic process of designing and constructing. At the same time, the model provides flexibility in the further development of building technology and the application of the materials. The model is not dependent on the economic and technological changes that are inevitable, since, it sets the building into a logical, central position in the construction process.

Molecular characterization and phylogenetic relationships among European Aphidius Nees (Hymenoptera, Braconidae, Aphidiinae)

Молекуларна карактеризација и филогенетски односи европских врста рода Aphidius Nees (Hymenoptera, Braconidae, Aphidiinae)

U rod Aphidius klasifikovane su mnoge vrste koje su ekonomski značajne kao biološki agenti za kontrolu štetnih vrsta biljnih vašiju u staklenicima i na otvorenom polju. Obzirom da biologija, ekologija i taksonomski status mnogih vrsta nisu dovoljno istraženi, klasifikacija vrsta unutar roda se kontinuirano revidira. Delimične sekvence gena mitohondrijske DNK citohrom oksidaze subjedinica I (mtCOI) i jedarnog gena za faktor elongacije 1-α a (EF1-α) su markeri korišćeni u istraživanju taksonomskog statusa i filogenetiskih odnosa 33 vrste roda Aphidius u asocijaciji sa raličitim vrstama vaši i biljaka domaćina sakupljenih sa šireg arela u Evropi. Filogenetske analize su potvrdile da je rod Aphidius parafiletički. Na filogenetskom stablu konstruisanom maximum likelihood metodom jasno je izdvajanje sledeća 22 taksona kao zasebnih vrsta: A. erysimi, A. sonchi, A. linosiphonis, A. hieraciorum, A. arvensis, A. balcanicus, A. phalangomyzi, A. banksae, A. uzbekistanicus, A. sussi, A. silvaticus, A. avenae, A. rosae, A. ericaphidis, A. eadyi, A. viaticus, A. schimitscheki, A. ribis, A. setiger, A. asteris, A. matricariae i A. urticae Pored ovih „dobrih“ vrsta, izdovjilo se još pet klada: i) A. salicis i A. aquilus; ii) A. funebris, A. tanacetarius, A. absinthii; iii) A. ervi i A. microlophii; iv) A. chaetosiphonis i A. hortensis; v) A. rubi i A. rhopalosiphi. Vrste u ovim kladama se ne mogu jasno identifikovati na osnovu mitohondrijskog i jedarnog markera. Neuspeh u identifikaciji vrsta primenom ovih markera može se pripisati adaptivnoj divergenciji usled širenja kruga domaćina ili staništa i specijaciji, ili mitohondrijalnoj introgresiji prilikom hibridizacije primeraka srodnih vrsta. Za pojedine vrste sugeriše se revizija taksonomskog statusa, primenjujući savremeni integrativni pristup. Molekularna identifikacija mtCOI otkrila je u okviru kompleksa vrsta A. urticae tri kriptične vrste A. urticae s. str., A. rubi i A. silvaticus. Takođe, DNK barkoding metod je potvrdio prvi put u Evropi prisustvo vrste A. ericaphidis.

The genus Aphidius includes many species of economic importance that are used as biocontrol agents against numerous pest aphids in greenhouses and under open field conditions. However, classification within this genus is constantly revisited, in view of the fact that the biology, ecology and taxonomic status of many species are still understudied. Partial sequences of the cytochrome oxidase subunit I mitochondrial gene (mtCOI) and Elongation factor 1-α nuclear gene (EF1-α) were used to explore the taxonomic status and phylogenetic relationships of 33 European species from the genus Aphidius in different aphid /plant host associations over a wide area of distribution. Phylogenetic analyses clarified that Aphidius is in fact a paraphyletic group. Topology of the maximum likelihood tree showed separation of 22 taxa as independent species: A. erysimi, A. sonchi, A. linosiphonis, A. hieraciorum, A. arvensis, A. balcanicus, A. phalangomyzi, A. banksae, A. uzbekistanicus, A. sussi, A. silvaticus, A. avenae, A. rosae, A. ericaphidis, A. eadyi, A. viaticus, A. schimitscheki, A. ribis, A. setiger, A. asteris, A. matricariae and A. urticae. Besides “good” species, five more clades were distinguished: i) A. salicis and A. aquilus; ii) A. funebris, A. tanacetarius, A. absinthii; iii) A. ervi and A. microlophii; iv) A. chaetosiphonis and A. hortensis; v) A. rubi and A. rhopalosiphi. Taxa within five clades could not be clearly discriminated as separate species based on either mtCOI or EF1-α. Failure of the two markers to delimit these taxa could be attributed either to adaptive divergence due to host and/or habitat range expansion and speciation or to mitochondrial introgression via hybridization of sibling species. In any event, it is suggested that their taxonomic status be re-visited using an integrative approach. Molecular characterization revealed cryptic taxa associated with different hosts within the A. urticae group. Re-descriptions of A. urticae s. str., A. rubi and A. silvaticus are given. Also, mtDNA barcoding identified the presence of A. ericaphidis for the first time in Europe.

Promena urbanističko-arhitektonske paradigme na primeru beogradskih stambenih naselja

Change of urban and architectural paradigm exemplified by Belgrade housing estates: the period of Yugoslav late socialism (1965-1991)

Ovaj rad istražuje dinamiku i pravce promene urbanističko-arhitektonske paradigme na primeru stambenih naselja projektovanih i/ili izgrađenih u Beogradu između 1965. i 1991. godine. Vremenski okvir rada obuhvata tri krupna istorijska procesa koji su oblikovali teoriju i praksu socijalističkog samoupravljanja u Jugoslaviji: privrednu reformu iz 1965. godine, donošenje Ustava SFRJ 1974. godine i demontiranje socijalističkog društveno- simboličkog poretka tokom kasnih 80-ih godina. Promena paradigme se u radu razmatra u kontekstu ovih procesa, to jest, u pogledu marginalne pozicije koju je participacija krajnjih korisnika zauzimala u okviru promene paradigme. Ukoliko je samoupravljanje teorijski postavljeno kao vrhunski oblik participacije i ukoliko je inostrani razvoj paradigmi prisutnih u beogradskom urbanizmu i arhitekturi perioda obuhvatio koncepte poput otvorene forme, participativnog projektovanja i samogradnje, onda je cilj ovog rada utvrđivanje pozicije koju je promena paradigme zauzimala u širem okviru socijalističke proizvodnje prostora, a u svetlu nedostatka participacije krajnjih korisnika. Oslanjajući se na tezu francuskog neomarksiste Anri Lefevra (Henri Lefebvre) o proizvodnji prostora kao društvenoj praksi, te na savremene teorije nepotpunosti društva, rad polazi od pretpostavke da je samoupravljanje, bez obzira na marginalnost participacije, bilo deo urbanizma i arhitekture beogradskih stambenih naselja, to jest, da su se kasnomoderna, postmoderna i tradicionalna paradigma perioda permanentno odnosile prema samoupravljanju i antagonizmima koji su određivali njegovu teoriju i praksu. U pogledu isključivanja krajnjih korisnika iz urbanističkog i arhitektonskog projektovanja rad polazi od pretpostavke da je ovaj postupak građen uporednim delovanjem više aktera, uključujući one u čije ime su malobrojni primeri otvorene forme, participativnog projektovanja i samogradnje i nastupali. Istraživanjem institucionalnih kanala kojima je decenijama regulisana proizvodnja prostora beogradskih stambenih naselja, te preklapanjem rezultata ovog istraživanja sa utvrđenim oblicima delovanja urbanista i arhitekata i krajnjih korisnika, rad zaključuje da se promena urbanističko-arhitektonske paradigme odvijala u nekoliko instanci otuđenja koje su činile uzaludnim svaki pokušaj doslednijeg praćenja smernica zvaničnih diskursa socijalističkog samoupravljanja. Intervenišući u pogledu zatečenih društvenih podela urbanizam i arhitektura perioda rezultirali su odnosom razlike prema stanju i dinamici kasnomoderne i tradicionalne paradigme na prostoru Zapada, ali ne snažnijim isticanjem participacije krajnjih korisnika, već upravo u senci odsustva i ignorisanja istog. Konačno, rad zaključuje da i pored oštrog suprotstavljanja između delova discipline koji su cinično podrivali i pravoverno pratili emancipatorske smernice zvaničnih diskursa, ove dve opcije su u kontekstu kasnog socijalizma igrale istovetnu ulogu anticipatora novog društvenog poretka postsocijalizma, što se prepoznaje i kao zajedničko tradicionalnom urbanizmu i postmodernoj arhitekturi perioda.

This dissertation investigates dynamics and directions of the change of urban and architectural paradigm exemplified by the housing estates designed and/or built in Belgrade between 1965 and 1991. The dissertation’s time frame envelops three major historical processes shaping the theory and practice of socialist self-management in Yugoslavia: the economic reform of 1965, the enactment of the 1974 SFRY Constitution and the deconstruction of socialist socio-symbolic order during the late 1980s. The paradigm change is discussed in the context of the processes, that is, in the light of the marginal position occupied within it by participation of end users. If self- management was conceived theoretically as the ultimate form of participation and if the foreign development of paradigms present in Belgrade urbanism and architecture of the period involved concepts such as open form, participatory design and self-built, than the goal of this dissertation is to establish the position paradigm change occupied within the wider framework of socialist production of space, in the light of a lack of user participation. Supported by the thesis of the French neo-Marxist philosopher Henri Lefebvre, regarding the production of space as a social practice, and by contemporary theories of society’s lack of closure, the dissertation’s hypothesis is that self-management, regardless of the marginality of participation, was indeed part of the urbanism and architecture of Belgrade housing estates, or that late modern, postmodern, and traditional paradigms of the period had constantly addressed self-management and the antagonisms which had determined its theory and practice. Regarding the exclusion of end users from urban and architectural design, the dissertation’s hypothesis is that this procedure was shaped by the parallel strivings of several actors, including those in whose name rare examples of open form, participatory design and self-built operated. By investigating the institutional channels which had for decades regulated the production of space of Belgrade housing estates, and by overlapping the results of this investigation with the determined forms of action of planners and architects and end users, the dissertation concludes that the change of urban and architectural paradigm took place on several levels of alienation which made futile every attempt of consistent following of the guidelines of official discourses of socialist self-management. Acting in regards to encountered social divisions, urbanism and architecture of the period resulted in difference towards the conditions and dynamics of the late modern and traditional paradigms of the West, but not by emphasising participation in a stronger manner, but by the absence and ignorance of it. Finally, the dissertation concludes that regardless of harsh confrontation between parts of the discipline which cynically undermined and rightly followed the emancipatory guidelines of the official discourses, these two options played the same role of the harbinger of new social order of post-socialist capitalism, which is also recognized as common to traditional urbanism and postmodern architecture of the period.

Funkcionalnost i obrazac signalnih puteva Lajdigovih ćelija odraslih pacova nakon primene anaboličkih androgenih steroida

Functionality and pattern of signaling pathways of Leydig cells in adult rats after administration of anabolic androgenic steroids

Lajdigove ćelije intersticijuma testisa su primarno mesto sinteze androgenih hormona, a dominantno testosterona (T). Ovaj hormon je zajedno sa svojim metabolitom dehidrotestosteronom (DHT) neophodan, kako za produženje vrste obezbeđivanjem pravilnog razvoja i funkcionisanja muškog reproduktivnog sistema, tako i za opšte zdravlje individue. U cilju tretiranja brojnih kliničkih poremećaja, kao i u svrhu kontracepcije, sintestisani su derivati androgena čija se primena u klinici zasniva na dejstvu koje ostvaruju posredstvom androgenih i/ili anaboličkih efekata, te su zajedničkim imenom nazvani anabolički androgeni steroidi (AAS). Nažalost, AAS se često zloupotrebljavaju, ne samo od strane profesionalnih i rekreativnih sportista, nego i velike populacije adolescenata, iako je dobro poznato da njihova upotreba u neterapeutske svrhe može izazvati niz neželjenih zdravstvenih posledica. Stoga su AAS svrstani u grupu farmakoloških preparata čija je upotreba strogo regulisana i nisu dostupni bez lekarskog recepta. Uprkos široko rasprostranjenoj kliničkoj upotrebi i zloupotrebi AAS, kao i velikom interesovanju naučne zajednice za ovu problematiku, nisu u potpunosti razjašnjeni molekulski događaji koji su posledica njihove kratkoročne i dugoročne primene. S obzirom na značaj T za reprodukciju i produženje vrste, ali i zdravlje i kvalitet života indivudue, kao i široku primenu i zloupotrebu T i njegovih derivata, neophodno je okarakterisati precizne molekulske događaje nastale kao posledica poremećene homeostaze T. Ovo je važno zbog toga što, prema trenutno dostupnoj literaturi, ne postoji dovoljan broj podataka o funkcionalnosti i obrascima signalnih puteva Lajdigovih ćelija, čija je osnovna uloga sinteza i sekrecija T. Stoga je glavni cilj ovog istraživanja bio da ispita funkcionalnost i obrasce signalnih puteva važnih za održavanje steroidogene funkcije Lajdigovih ćelija, narušene primenom egzogenih agonista i/ili antagonista T u in vivo ili in vitro uslovima. U tu svrhu primenjen je derivat T, testosteron-enantat (TE), koji se najčešće upotrebljava u kliničkoj praksi, ali se i u najvećoj meri zloupotrebljava i najprodavaniji je na tzv. “crnom” tj. ilegalnom tržištu. Ovakav model daje mehanistički pristup, ali ima i translacioni aspekt, s obzirom na to da su upotrebljavane doze/koncentracije T koje se koriste u kliničkoj praksi ili se zloupotrebljavaju. Rezultati su pokazali da in vivo aplikacija TE inhibira steroidogenu funkciju Lajdigovih ćelija odraslih pacova, kao i relativnu ekspresiju gena za komponente cAMP-PRKA signalizacije, kao glavnog regulatora steroidogeneze ovih ćelija. Suprotno, primena TE je stimulisala ekspresiju enzima koji učestvuje u sintezi T - hidroksisteroid dehidrogenaze 3 beta (HSD3B), zatim androgenog receptora (AR), kao i elemenata JAK-STAT, uključenih u regulaciju ekspresije HSD3B. Sistemska in vivo blokada AR je potpuno ili delimično prevenirala TE-poremećenu homeostazu intersticijumske tečnosti testisa (TIF), funkcionalnost Lajdigovih ćelija, kao i poremećen obrazac ekspresije komponenti steroidogene mašinerije i cAMP-PRKA signalizacije. In vitro blokada AR ciproteron-acetatom u kombinaciji sa T omogućila je razdvajanje tzv. direktih autokrinih efekata T od onih posredovanih jukstakrinim, parakrinim, endokrinim i nervnim faktorima. Svi ovi rezultati potvrđuju uključenost T i AR u kompleksnim mehanizmima regulacije koji su važni u odgovoru Lajdigovih ćelija neophodnom za očuvanje i sprečavanje potpunog gubitka steroidogene funkcije, ali istovremeno predstavljaju prikaz molekulskih događaja koji su posledica primene AAS. Stoga, rezultati imaju fundamentalan značaj sa aspekta rasvetljavanja mehanizama delovanja T u regulaciji funkcije Lajdigovih ćelija tokom poremećene homeostaze organizma. Pored toga, rezultati su prmenljivi u kliničkoj i forenzičkoj praksi, s obzirom na to da je u in vivo modelu korišćen derivat T, TE, u sličnom maniru i u dozi koja se najčešće upotrebljava u kliničkoj praksi, ali se u najvećoj meri i zloupotrebljava.

Leydig cells of testis interstitium represent the major site for synthesis of androgenic hormone, primarily testosterone (T). This hormone, together with its metabolite dihydrotestosterone (DHT), is required, not only for the continuation of the species by ensuring proper development and functioning of male reproductive system, but for overall health of an individual as well. For the purpose of treatment of multiple clinical disorders, as well as for contraception purposes, androgen derivatives have been synthesized, the clinical application of which is based on the influence they have through anabolic and/or androgenic effects, thus having a common name anabolic androgenic steroids (AASs). Unfortunately, AASs are often abused, not only by professional and recreational athletes, but by a large population of adolescents as well, although it is well known that non- therapeutic use thereof may cause a series of adverse health effects. Therefore, AASs are classified into a group of pharmacological preparations, the use of which is strictly regulated and which are not available without a medical prescription. Despite widespread clinical use and abuse of AASs, as well as the great interest shown by the scientific community in this field, molecular events resulting from their short-term and long-term use have not been fully clarified. Given the importance of T for reproduction and continuation of the species, but also for health and quality of life of an individual, and the widespread use and abuse of T and its derivatives, it is necessary to characterize precise molecular events resulting from disturbed T homeostasis. This is important because, according to the currently available literature, there is insufficient data on the functionality and patterns of signaling pathways of Leydig cells, the basic role of which is the synthesis and secretion of T. Therefore, the main aim of this study is to examine the functionality and patterns of signaling pathways relevant for maintaining the steroidogenic function of Leydig cells, impaired by the use of exogenous T agonists and/or antagonists under in vivo or in vitro conditions. To this end, a T derivative known as testosterone-enanthate (TE) is applied, which is most commonly used in clinical practice, but is largely abused and best-selling product in the so- called "black" or illegal market. Such model provides a mechanistic approach, but has also a translational aspect, since the applied T doses/concentrations are used in clinical practice or are abused. The results have shown that in vivo application of TE inhibits the steroidogenic function of Leydig cells in adult rats, as well as gene expression for cAMP-PRKA signaling components, being the main regulator of steroidogenesis of such cells. On the contrary, the application of TE has stimulated the expression of an enzyme involved in the synthesis of T - 3-beta-hydroxysteroid dehydrogenase (HSD3B), androgen receptor (AR), and JAK-STAT elements, engaged in HSD3B expression regulation. Systemic in vivo blockade of AR has completely or partially prevented TE- disturbed homeostasis of testicular interstitial fluid, functionality of Leydig cells, as well as disturbed expression pattern of steroidogenic machinery components and cAMP-PRKA signaling. In vitro blockade of AR by cyproterone acetate in combination with T has enabled the separation of the so-called direct autocrine effects of T from those mediated by juxtacrine, paracrine, endocrine and nerve factors. All these results confirm the involvement of T and AR in complex regulatory mechanisms relevant for the response by Leydig cells required for the preservation and prevention of complete loss of steroidogenic function, but at the same time represent an overview of molecular events arising from the application of AASs. Hence, the results have fundamental significance from the aspect of clarifying the mechanism of T effect in regulating the function of Leydig cells during disrupted organism homeostasis. In addition, the results are applicable both in clinical and forensic practice, since in vivo model uses T derivative - TE in a similar manner and with a dose most commonly used in clinical practice, but largely abused, too.

Prinos i kvalitet biomase lucerke (Medicago sativa L.) u zavisnosti od sistema gajenja

Yield and quality of alfalfa biomass (Medicago sativa L.) as affected by production management

Značaj lucerke u srpskoj poljoprivredi, kao i njena velika tržišna vrednost nameće stalnu potrebu za unapređenjem proizvodnje ove krmne biljke koja zauzima velike površine, kako u svetu tako i u našoj zemlji. Sveobuhvatni cilj doktorske disertacije je da se oceni uticaj različitih sistema gajenja (režima kosidbe) na prinos i hranljivu vrednost lucerke, doprinese rešavanju praktičnih pitanja u proizvodnji lucerke (redefiniše optimalna količina semena za setvu) i odredi fenološka faza u momentu kosidbe u kojoj se postiže balans prinosa i kvaliteta suve materije lucerke. Trogodišnje istraživanje (2010-2012) je izvedeno na oglednim poljima Odeljenja za krmno bilje Instituta za ratarstvo i povrtarstvo u Novom Sadu, na dva lokaliteta (Rimski Šančevi i Čenej). Poljski ogled je postavljen po slučajnom blok sistemu u tri ponavljanja na oba lokaliteta. U radu su analizirane četiri sorte lucerke (Banat VS, Nijagara, NS Medijana ZMS V i NS Alfa); dve setvene norme od 8 i 16 kg/ha i tri sistema kosidbe u zavisnosti od fenološke faze razvoja biljaka: I – rani (10% cvetalih biljaka), II – srednje rani (50% cvetalih biljaka), i kasni (početak zametanja mahuna). Tretmani su aranžirani po sistemu podeljenih parcela (split-plot metod), a kao osnovna parcela (veličina 72 m2) korišćena je setvena norma, dok su sve ostale kombinacije sorti i režima kosidbe tretirane kao podparcele (veličina 6 m2). Dimenzije podparcele su 5 m dužina i 1,2 m širina, sa međurednim razmakom od 20 cm. Razmak između blokova je 1 m. Među ispitivanim sortama nije bilo značajne razlike u visini prinosa i komponentama prinosa suve materije lucerke, kao ni u pogledu njihovog kvaliteta. Različite količine semena za setvu lucerke, od 8 i 16 kg/ha, nisu uticale na prinos suve materije kao ni na komponente prinosa (visina biljaka, udeo lista u prinosu, broj izdanaka i broj biljaka). U umereno-kontinentalnim klimatskim uslovima, kakvi vladaju u većem delu u Republike Srbije, nema opravdanja povećanja setvene norme iznad 16 kg/ha jer se setvom većih količina semena ne obezbeđuje dugoročna korist u proizvodnji lucerke. Sistemi gajenja, odnosno kosidba lucerke u različitim fazama starosti useva (početak cvetanja, puno cvetanje i faza zelenih mahuna) imala je značajan efekat na prinos, komponente prinosa i hranljivu vrednost ispitivanih sorti lucerke. Najveći prinos zelene krme ostvaren je pri ranoj kosidbi u fazi 10% cvetalih biljaka, dok je prinos suve materije bio identičan pri košenju u fazi 10% i 50% cvetalih biljaka i iznosio je 15,9 t ha-1. Sistem kosidbe značajno je uslovljavao visinu biljaka, pri čemu su najviše biljke (81,5 cm) izmerene pri košenju u fazi zelenih mahuna. Najveći udeo lista od 500,7 g/kg suve materije dobijen je kada se lucerka kosila na početku cvetanja. Učestalost kosidbe je značajno uticala na broj izdanaka po jedinici površine, koji se značajno smanjio pri kosidbi u kratkim vremenskim intervalima, odnosno u fazi početka cvetanja biljaka. Fenološka faza razvoja u momentu kosidbe nije uticala na broj biljaka po jedinici površine. Gustina lucerišta bila je slična pri košenju u fazi 10% i 50% cvetalih biljaka (240,7 i 246,3 bilj./m2). Rezultati analize o hranljivoj vrednosti lucerke ukazuju na značajan uticaj uslova sredine i faze razvoja u momentu kosidbe za sadržaj sirovih proteina, dok je sadržaj vlakana bio uslovljen samo fazom u momentu košenja (sistemom kosidbe). Bolji kvalitet suve materije lucerke ostvaren je u vlažnoj godini (2010) u poređenju sa sušnom godinom (2011). Sadržaj sirovih proteina u suvoj materiji lucerke se smanjivao zbog smanjenja udela lista, dok se sadržaj NDF-a, ADF-a i ADL-a povećavao od faze početka cvetanja do faze zelenih mahuna. Kosidbom lucerke u četvorootkosnom i petootkosnom sistemu dobijeni su slični prinosi suve materije (15,9 t ha-1), dok se primenom trootkosnog sistema kosidbe ne iskorišćava genetički potencijal NS sorti, dobija se loš kvalitet i svarljivost (veći udeo vlakana), i dolazi do značajnog smanjenja prinosa suve materije lucerke (13,1 t ha-1). Kosidba lucerke u početku cvetanja biljaka (pet otkosa u godini) daje bolji kvalitet suve materije, odnosno veći sadržaj proteina i manji sadržaj vlakana (NDF, ADF i ADL).

The importance of alfalfa in Serbian agriculture, as well as its high market value, imposes a constant need for the improvement of this forage crop production that occupies large areas both in the world and in our country. The overall objective was to evaluate the effect of different growing systems (cutting regimes) on yield and nutritional value of alfalfa, contribute to solving practical issues in alfalfa production (redefine the optimal amount of seed for sowing) and specify the phenological stage in the moment of cutting when the balance of yield and dry matter quality of alfalfa is achieved. Three-year study (2009-2012) was carried out on experimental fields of Forage Crops Department of the Institute of Field and Vegetable Crops in Novi Sad, at two sites (Rimski Šančevi and Čenej). Field trial was set up in a randomized block design with three replications at both sites. Four cultivars of alfalfa were studied in this dissertation (Banat VS, Nijagara, NS Medijana ZMS V and NS Alfa); two sowing rates from 8 and 16 kg/ha and three cutting systems depending on crop phenological stage of development: I – early (10% of flowering plants), II – medium early (50% of flowering plants), and late (beginning of pod setting). Treatments were arranged by split-plot method, and the basic plot (size 72 m2) was used as the sowing rate, while all the other combinations of cultivars and cutting regimes were treated as sub-plots (6 m2). Sub-plot dimensions were 5 m in length and 1.2 m in width, with interrow distance of 20 cm. Inter-block distance was 1 m. There were no significant differences in yield and yield components of alfalfa dry matter, or in their quality, among the tested cultivars. Different amounts of seed for alfalfa sowing, from 8 and 16 kg/ha, did not affect the dry matter yield, or the yield components (crop height, leaf ratio in yield, number of shoots, and number of plants). In moderate-continental climatic conditions, which are present in the majority of the Republic of Serbia, there is no justification for increasing sowing rate above 16 kg/ha, because higher sowing rates do not provide long-term benefit in alfalfa production. Growing systems or cutting alfalfa in different stages of crop maturity (beginning of flowering, full flowering and green pods stage) had significant effect on the yield, yield components and nutritional value of tested alfalfa cultivars. The highest yield of green forage was achieved in early cutting at the stage of 10% flowering plants, while the dry matter yield was identical in cutting at the stage of 10% and 50% flowering plants and amounted to 15.9 t ha-1. Cutting system significantly conditioned plant height, when the highest plants (81.5 cm) were measured in cutting at the stage of green pods. The highest leaf ratio from 500.7 g/kg of dry matter was obtained when alfalfa was cut at the beginning of flowering. Frequency of cutting significantly affected the number of shoots per area unit, which notably decreased during cutting in short time intervals, i.e. at the beginning of flowering stage. Phenological development stage at the moment of cutting did not affect the number of plants per area unit. Alfalfa field density was similar to cutting at the stage of 10% and 50% flowering plants (240.7 and 246.3 plants/m2). Results of alfalfa nutritional value analysis indicated the significant effect of environmental conditions and development stage at the moment of cutting on crude protein content, while fibre content was conditioned only by the stage at the moment of cutting (cutting system). Alfalfa dry matter had better quality in wet year (2010) than in dry year (2011). Content of crude proteins in alfalfa dry matter decreased due to the reduction of leaf ratio, while the contents of NDF, ADF and ADL increased from the beginning of flowering stage to the stage of green pods. Similar yields of dry matter were achieved by cutting alfalfa with four and five cuts (15.9 t ha-1), while three cuts system did not use the genetic potential of NS cultivars, lead to bad quality and digestibility (higher fibre ratio) and to significant yield decrease of alfalfa dry matter (13.1 t ha-1). Cutting alfalfa at the beginning of flowering (five cuts a year) gives better quality of dry matter, i.e. higher protein content and lower fibre content (NDF, ADF i ADL).

Karakterizacija strukture i funkcije HMGB1 proteina u jetri pacova tokom eksperimentalno izazvanog dijabetesa tipa 1

Structural and functional characterization of HMGB1 protein in rat liver during experimentally-induced diabetes type 1

Oksidativni stres i hronična inflamacija smatraju se glavnim uzrocima pojave dijabetičnih komplikacija, među kojima su i oštećenja jetre. Važnu ulogu medijatora ovih procesa može imati endogeni protein HMGB1, koji u vanćelijsku sredinu dospeva iz nekrotičnih, oštećenih i aktiviranih ćelija. Kako je u dijabetesu ponašanje HMGB1 proteina slabo izučavano, u ovoj doktorskoj disertaciji ispitivan je doprinos HMGB1 oštećenjima jetre pacova sa streptozotocinom-izazvanim DT1. Pokazano je da nivo oštećenja jetre tokom dijabetesa koreliše sa prisustvom vanćelijskog HMGB1. Ovaj protein, u dijabetičnoj jetri, biva strukturno modifikovan acetilacijom, fosforilacijom i O-GlcNAc glikozilacijom što koreliše sa njegovim izlaskom iz jedra ćelija u citoplazmu i povećanjem njegovog prisustva u jetri i serumu. Rezultati u vezi sa snižavanjem nivoa vanćelijskog HMGB1 tretmanom dijabetičnih pacova melatoninom ili etil piruvatom, ukazuju da HMGB1 doprinosi oštećenju jetre u dijabetesu održavanjem stanja hronične inflamacije, stišavanjem antioksidativne odbrane i stišavanjem regeneracije. Vanćelijski HMGB1 kroz interakcije sa TLR4 receptorom aktivira MAPK/NF-κB p65 i JAK1/STAT3 signalne puteve, doprinoseći povećanju produkcije proinflamacijskih citokina TNF-α i IL-6 i akutno-faznog proteina haptoglobina. Podsticanjem NF-κB p65 inflamacijskog puta, HMGB1 deluje negativno na citoprotektivni odgovor u dijabetičnoj jetri tako što onemogućava aktivnost Nrf2 proteina, odgovornog za stišavanje inflamacije i produkciju antioksidativnih enzima. Na stišavanje regenerativnog potencijala jetre, aktivirana HMGB1/TLR4 osa utiče preko uvećanja prisustva negativnih regulatora ćelijskog ciklusa - proteina p53 i p21, i smanjenjem nivoa ciklina D1. Dobijeni rezultati ukazuju na složenost delovanja HMGB1 proteina u dijabetesu i na značaj sprečavanja oslobađanja HMGB1 ili blokade HMGB1/TLR4 ose u cilju odlaganja nastanka oštećenja jetre.

Oxidative stress and chronic inflammation are considered to be the main causes of diabetic complications, one of which is liver damage. An important mediator of these processes may be the endogenous HMGB1 protein, when released into the extracellular environment from the necrotic, damaged or activated cells. As the HMGB1 role in diabetes was insufficiently studied, in this doctoral dissertation the contribution of HMGB1 to liver damage of streptozotocin-induced diabetic rats was investigated. It has been shown that the level of liver damage in diabetes correlates with the presence of extracellular HMGB1. In diabetic liver, this protein is structurally modified by acetylation, phosphorylation, and O- GlcNAc glycosylation, which correlates with its translocation from the nucleus to the cytoplasm and an increase in its presence in the liver and serum. Reduction of the level of extracellular HMGB1 by melatonin or ethyl pyruvate treatment of diabetic rats, shows that HMGB1 contributes to diabetic liver damage by maintaining a chronic inflammation, by lowering antioxidant defense and by reducing regeneration. Extracellular HMGB1 activates MAPK/NF-κB p65 and JAK1/STAT3 signaling pathways through interactions with the TLR4 receptor, thus contributing increased production of proinflammatory cytokines TNF- α and IL-6 and the acute-phase protein, haptoglobin. By stimulating the NF-κB p65 inflammatory pathway, HMGB1 acts negatively on the cytoprotective response of the diabetic liver, by disabling Nrf2 protein activity, which is responsible for reduction of inflammation and antioxidant enzymes production. Activated HMGB1/TLR4 axis reduces regenerative potential of the liver by increasing the presence of negative cell cycle regulators - proteins p53 and p21, and also by decreasing the level of cyclin D1. The obtained results indicate the complexity of HMGB1 protein action in diabetes and underlines the importance of preventing the release of HMGB1 or blockage of HMGB1/TLR4 axis in order to delay the occurrence of liver damage.

Efekti hronične primene losartana i tempola u eksperimentalnom modelu fokalno segmentne glomeruloskleroze kod spontano hipertenzivnih pacova

Effects of chronic losartan and tempol treatments on experimental focal segmental glomerulosclerosis in spontaneously hypertensive rats

Dosadašnja istraživanja pokazala su da renin angiotenzin sistem (RAS) ima važnu ulogu u patogenezi i progresiji fokalno segmentne glomeruloskleroze (FSGS). Oksidativni stres je prisutan u hroničnoj bubrežnoj slabosti i doprinosi progresiji bolesti. Povezanost oksidativnog stresa i RAS u procesima progresije FSGS još je nedovoljno razjašnjena. Stoga su istraživanja u okviru ove disertacije bila usmerena ka ispitivanju efekata hronične primene tempola (sakupljač slobodnih radikala, SOD mimetik), losartana (blokator receptora za angiotenzin II tipa 1, AT1R), kao i njihove kombinacije, na usporavanje progresije FSGS izazvane adrijamicinom (ADR) kod spontano hipertenzivnih (SH) pacova. Životinje su inicijalno deljene u dve eksperimentalne grupe, kontrolnu (SHC) i grupu koja je primila ADR 2 mg/kg i.v. dva puta u intervalu od 21 dan. Nakon druge injekcije ADR, životinje su dobijale vodu (SHADR), losartan (SHADR+L), tempol (SHADR+T) i kombinovani tretman (SHADR+T+L) gavažom. Na kraju šeste nedelje tretmana vršena su hemodinamska merenja i uzorkovanje krvi, urina i bubrega. Na osnovu biohemijskih parametara vršena je procena lipidnog statusa i bubrežne funkcije. Urađena je histopatološka analiza bubrega. Imunihistohemijskom metodom ispitivani su proteini citosketeta, nestin i vimentin, čije izmenjene ekspresije su pokazatelj oštećenja bubrežnog tkiva. Matriksna metaloproteinaza-1 (MMP-1), koja učestvuje u degradaciji komponenti vanćelijskog mariksa i očuvanju integriteta glomerula, određivana je ELISA metodom. Ispitivani su parametri oksidativnog stresa: lipidna peroksidacija i nivo karbonilovanih proteina (PCOs); aktivnosti enzima antioksidativnog sistema: superoksid dismutaze (SOD), katalaze (CAT), glutation peroksidaze (GPx) i glutation reduktaze (GR); kao i antioksidativni kapacitet. Primenom imunohistohemijske, Western blot i ELISA metode u bubrezima su određivane ekspresije Nox2 i Nox4 izoforme katalitičke subjedinice NADPH oksidaze, glavni izvor reaktivnih kiseoničnih vrsta u ćeliji, uključene u proces inflamacije. Ekspresija proteina tri izoforme azot-monoksid sintaze, inducibilna (iNOS), endotelna (eNOS) i neuronska (nNOS), određivane su Western blot i imunohistohemijskom metodom u tkivu bubrega. Određivani su ukupni metaboliti NO-a (nitriti, nitrati) u urinu i bubrezima. Hronični pojedinačni tretmani losartanom i tempolom smanjuju strukturna oštećena bubrega izazvana ADR (fokalno segmentnu glomerulosklerozu, atrofiju i dilataciju tubula, intersticijsku inflamaciju i fibrozu), ali u kombinaciji sinergistički ne deluju jer ne menjaju ukupni zbir morfoloških promena u ovom eksperimentalnom dizajnu. Pojedinačni tretmani losartanom i tempolom smanjuju ekspresije nestina i vimentina u bubrežnim telašcima i ne detektuju se u tubulointersticijumu. Nakon kombinovanog tretmana ekspresija nestina je detektovana u podocitima i miofibroblastina intersticijuma, a vimentina u bubrežnim telašcima, tubulocitima proksimalnih tubula i okolnim fibroblastima i miofibroblastima intersticijuma, što ukazuje na prisutna oštećenja u tim delovima bubrega. Pojedinačni tretmani losartanom i tempolom poboljšavaju bubrežnu funkciju jer smanjuju proteinuriju i albuminuriju, ali tempol u kombinaciji sa losartanom dodatno ne sprečava gubitak proteina urinom i smanjuje brzinu glomerulske filtracije. Losartan, tempol i kombinovani tretman smanjuju koncentraciju MMP-1 u bubrezima. Losartan normalizuje vrednosti triglicerida, ukupnog holesterola i LDL holesterola, a tempol povećava nivo HDL holesterola u plazmi, međutim u kombinaciji ne utiču na poboljšanje lipidnog statusa. Losartan, tempol i kombinovani tretman poboljšavaju oksidativni status, odnosno povećavaju antioksidativni kapacitet, a smanjuju lipidnu peroksidaciju u plazmi i karbonilaciju proteina u bubrezima. Takođe, povećavaju aktivnosti enzima antioksidativnog sistema u eritrocitima. U bubrezima losartan povećava aktivnosti SOD, GPx i GR, dok tempol u kombinaciji sa losartanom povećava aktivnost SOD. Losartan povećava ekspresiju Nox2 izoforme u bubrezima i sprečava periglomerularnu i peritubularnu inflamaciju intersticijuma, ukazujući na anti- inflamatornu ulogu NADPH oksidaze. Pojedinačni tretmani losartanom i tempolom povećavaju ekspresiju sve tri izoforme NOS, a kombinovani tretman povećava ekspresiju iNOS u bubrezima, dok su ekspresije konstitutivnih izoformi NOS snižene. U bubrezima losartan, za razliku od tempola, povećava koncentraciju metabolita NO, dok kombinovani tretman povećava produkciju metabolita NO povećavanjem koncentracije nitrita. Svi ispitivani tretmani smanjuju ukupnu produkciju metabolita NO putem urina kod tretiranih pacova. Losartan snižava sistolni i srednji arterijski pritisak, a povećava protok i smanjuje vaskularni otpor u bubrežnoj arteriji. Tempol povećava frekvencu srčanih kontrakcija i smanjuje minutni volumen, dok kombinovani tretman povećava protok i smanjuje vaskularni otpor u karotidnoj arteriji ne menjajući krvni pritisak. Rezultati ove disertacije pokazuju da u ranoj fazi adrijamicinske nefropatije kod SH pacova hronična blokada AT1R losartanom u kombinaciji sa tretmanom tempolom ne sprečava strukturna oštećenja, niti poboljšava funkciju bubrega, što može ukazati na to da u datim eksperimentalnim uslovima izostaje njihov udružen povoljan efekat na usporavanje progresije FSGS.

Previous studies have shown that renin angiotensin system (RAS) plays an important role in the pathogenesis and progression of focal segmental glomerulosclerosis (FSGS). Oxidative stress is involved in the development and progression of chronic kidney disease. However, the association of oxidative stress and RAS in the progression of FSGS has not been completely elucidated. In this study we investigated the effects of chronic tempol (free radical scavenger, SOD mimetic), losartan (selective angiotensin II type 1 receptor (AT1R) blocker), and their combined treatment in slowing down the progression of FSGS, in spontaneously hypertensive (SH) rats with adriamycin (ADR) nephropathy. Animals were initially divided into two experimental groups: control (SHC) and group that received ADR 2 mg/kg i.v. twice in an interval of 21 days. After the second injection of ADR, the animals were given tap water (SHADR), losartan (SHADR+L), tempol (SHADR+T) or combined treatment (SHADR+T+L) by gavage. Hemodynamic measurements were performed, blood, urine, and kidney samples were taken for biochemical and histopathological analysis. Immunohistochemical method was used for protein expression and localization of the nestin and vimentin, proteins of cytoskeleton network, and change in their protein expression is a marker of cell injury. Matrix metalloproteinase-1 (MMP-1), which participates in the degradation of the extracellular matrix components and preserving the integrity of the glomerulus, was determined by the ELISA method. The parameters of oxidative stress: lipid peroxidation, protein carbonyl content (PCOs); antioxidant defense: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and antioxidant capacity were analysed. Immunohistochemical, Western blot and ELISA method were used for analysis of the Nox2 and Nox4 protein expression, isoforms of the catalytic subunit of NADPH oxidase, the main source of reactive oxygen species in the cell, involved in the inflammation process. Protein expression of three isoforms of azote monoxide synthase, inducible (iNOS), endothelial (eNOS), and neuronal (nNOS) were determined by Western blot and immunohistochemical method in the kidney. Total metabolites of NO (nitrites, nitrates) were measured in the kidney and urine. Chronic losartan and tempol treatments decrease structural damage in the kidney induced by ADR (focal segmental glomerulosclerosis, atrophy and tubal dilatation, interstitial inflammation and fibrosis), but in combination they do not show synergistic effect because there is no decrease in total sum of morphological changes in the kidney. Individual losartan and tempol treatments decrease the expression of nestin and vimentin in glomeruli and Bowman’s capsule, with no detection in tubulointerstitium. After combined treatment nestin expression was detected in the podocytes and interstitial myofibroblasts, and vimentin expression in the epithelial cells of proximal tubules and interstitial fibroblasts and myofibroblasts, indicating the injury in these area of the kidney. Losartan and tempol individually improve renal function by reducing proteinuria and albuminuria, but tempol in combination with losartan does not further decrease proteinuria and reduces the rate of glomerular filtration. All treatments reduce the concentration of MMP-1 in the kidney. Losartan normalizes the triglyceride, total cholesterol and LDL cholesterol levels, and tempol increases HDL cholesterol levels in plasma, but in combination losartan and tempol do not improve the lipid status. All treatments improve the oxidative status, they increase the antioxidant capacity, and reduce lipid peroxidation in plasma and protein carbonyl content in the kidney. They also increase the activity of the antioxidant enzymes in erythrocytes. Losartan increases SOD, GPx and GR activities in the kidney, while tempol in combination with losartan increases SOD activity. Losartan increases Nox2 expression and prevents periglomerular and peritubular interstitial inflammation of the kidney, indicating the anti-inflammatory role of NADPH oxidase. Losartan and tempol treatments increase three NOS isoforms expression, while combined treatment increases the iNOS expression, while expressions of NOS constitutive isoforms have been reduced in the kidney. Losartan, unlike tempol, increases the concentration of NO metabolites in the kidney, while combined treatment increases the production of NO metabolites by increasing the concentration of nitrite. All treatments decrease the total production of NO metabolites by urine in treated rats. Losartan decrease systolic and mean arterial pressure and increases blood flow and decreases vascular resistance in the renal artery. Tempol increases the frequency of heart contractions and decreases the minute volume, while combined treatment increases the blood flow and reduces vascular resistance in the carotid artery without altering blood pressure. In conclusion, results from this study show that in the early phase of adriamycin nephropathy in SH rats, the chronic AT1R blockade by losartan in combination with tempol treatment neither prevent structural damage nor improve kidney function, which may indicate that in these experimental design their combined treatment does not contribute to further slowing down of FSGS progression.

Ekspresionizam u arhitekturi XX veka u Srbiji

Expressionism in XX century architecture in Serbia.

Kada se u nauci pominje reč „ekspresionizam”, u većini slučajeva se misli na stilski pravac, koji se javio u arhitekturi i umetnostima početkom XX veka i koji je trajao do 1930. godine. Sa druge strane, postoji i šire tumačenje pojma, po kome ekspresionizam nije samo stilski pravac, već jedna od ključnih stvaralačkih tendencija, tj. temeljni činilac u svim umetnostima tokom istorije. Oslanjajući se na šire tumačenje, u radu je razmatrana mogućnost postojanja ekspresionizma u arhitektonskoj kulturi u Srbiji tokom XX veka i pored uvreženog mišljenja, da on nije imao značajnijeg uticaja na arhitekturu u našoj sredini. Kritičkom analizom teorijskih stavova u literaturi, razmotrene su sličnosti i razlike između osnovnih estetskih kategorija „ekspresije” i „ekspresivnosti”. Takođe, je konstatovana njihova relacija sa fenomenom ekspresionizma, a sve sa ciljem jasnijeg tumačenja i evidentiranja njegove pojave u arhitekturi u Srbiji. Studijom najznačajnijih ekspresionističkih ostvarenja u arhitekturi u svetu, su ekstrahovani ekspresionistički principi i obeležja, čije je postojanje istraženo na referentnim arhitektonskim primerima iz Srbije. Komparativnom analizom odabranih arhitektonskih ostvarenja, koji su nastali tokom XX veka u Srbiji i koji su u literaturi označeni kao ekspresionistički, u istraživanju je izveden zaključak, da ekspresionizam predstavlja temeljni princip i jednu od ključnih stvaralačkih tendencija u arhitekturi XX veka u Srbiji. Takođe, ekspresionizam se ne javlja u istom periodu kad i u drugim delovima Evrope (1905-1930), već je prelomni trenutak za njegovu pojavu početak šezdesetih godina XX veka, kada se javljaju prvi projekti i realizacije, kod kojih je ekspresionistički izraz u celosti sproveden, što je suprotno onome što je do sada važilo u nauci.

When the word „expressionism” is mentioned in science, it usually refers to style movement which appeared in architecture and the arts at the beginning of the XX century and lasted until 1930. However, there is a wider interpretation of the term, which defines expressionism not only as a style movement but as one of the key creative tendencies, i.e. the essential factor in all arts throughout history. By relying on the wider interpretation, this paper discussed potential presence of expressionism in architectural culture in Serbia during the XX century, even though the prevailing opinion claimed that expressionism did not have a more significant influence on the architecture in Serbia. By critically analyzing theoretical positions in literature, we examined the similarities and differences between basic aesthetic categories: „expression” and „expressiveness”. We also confirmed their relation to the phenomenon of expressionism, with an aim of providing a clearer interpretation of it, as well as establishing its presence in Serbian architecture. By studying most prominent expressionist achievements in world architecture, we extracted expressionistic principles and characteristics whose presence we examined on leading architectural examples in Serbia. By comparative analysis of selected architectural objects, made in XX century in Serbia and marked in reference books as expressionist, our paper concludes that expressionism represents the key principle and one of the essential creative tendencies in XX century architecture in Serbia. Moreover, expressionism in Serbia did not emerge at the same time as in other parts of Europe (1905-1930), the turning point leading to its appearance was the beginning of the sixties in the XX century, when the first projects and realizations appeared, implementing complete expressionist articulation, which opposed the existing viewpoints in science.

Efekat elektromagnetnog polja (50Hz, 0,25mT) na komponente adaptivne vrednosti i morfometriju krila kod Drosophila subobscura Collin, 1936 (Insecta, Diptera)

Effect of electromagnetic field (50 Hz, 0.25 mT) on fitness components and wing morphometrics of Drosophila subobscura Collin, 1936 (Insecta, Diptera)

Od svog nastanka život na Zemlji se odvijao u prisustvu prirodnih elektromagnetnih polja (EMP) koja su relativno jednostavna i potiču od malobrojnih emitera elektromagnetne energije. Živa bića su se tokom svoje evolucije prilagodila ovom konstantnom sredinskom faktoru, ali su danas pored prirodnih, u sve većoj meri izložena EMP poreklom od izvora koje je stvorio čovek. Intenzitet i frekvencija ovih polja su daleko veći u odnosu na polja prirodnog porekla, a tu spadaju najvećim delom EMP mrežne frekvencije (u okviru dela spektra ekstremno niske frekvencije (ENF) od 50 ili 60 Hz), polja koji potiču od uređaja masovnih (RTV predajnici, 100-500 MHz) i mobilnih komunikacija (800-900 MHz i 1800-2200 MHz). EMP ENF imaju veliki biofizički značaj jer su sveprisutna u životnoj sredini usled prenosa i upotrebe električne energije. Elektromagnetna polja antropogenog porekla predmet su izučavanja različitih bioloških disciplina, ali pre svega sa aspekta zdravlja ljudi. Izlaganje organizama ELM ENF, čak i tokom kraćeg vremenskog perioda, ima za posledicu narušavanje ravnoteže bioloških sistema, kao i poremećaje na različitim nivoima organizacije, ali još uvek ne postoji opšte prihvaćena teorija o mehanizmima dejstva EMP ENF u biološkim sistemima. Pored toga, sredinske promene, usled prisustva EMP ENF, mogu imati uticaj na ekološke i evolucione procese koji utiču na stepen i obim promena u genetičkoj strukturi i evoluciji populacije. Stoga je od suštinskog značaja dublje razumevanje mehanizama koji omogućavaju organizmima da se manje ili više uspešno prilagođavaju sredinskim izazovima, kao i mogućnost da se predvide i prate odgovori populacija na promene u životnoj sredini koja ih okružuje. Tokom poslednje tri decenije izvedene su brojne studije na Drosophila sa ciljem ispitivanja bioloških efekata EMP ENF. Eksperimentalno izlaganje individualnih genotipova Drosophila EMP ENF tokom različitih faza životnog ciklusa, rezultuje različitim odgovorima koji su često oprečni, jer efekat delovanja EMP ENF na rast i metabolizam organizama, bilo inhibitorni, bilo stimulativni, zavisi od kompleksnih veza između frekvencije, jačine polja, dužine izlaganja, i stanja i tipa ćelija ili organizama. Postoje brojni podaci o uticaju EMP ENF na razviće, fekunditet, vijabilitet i druge komponente adaptivne vrednosti, praćeni na jedinkama Drosophila u laboratorijskim uslovima. Međutim EMP ENF, kao sveprisutni abiotički sredinski faktor, može imati efekat na genetičku stukturu populacija, utičući i na nivo genetičke varijabilnosti populacija. Takva genetička varijabilnost može biti glavni osnov za delovanje drugih evolucionih mehanizama na nivou populacije. Cilj ovog rada je ispitivanje efekata EMP ENF, kao kompleksnog sredinskog faktora, na populaciono-genetičkom nivou. Eksperiment evaluira mrežu fenotipskih odgovora na EMP ENF koristeći genetičku varijabilnost linija u okviru populacije vrste D. subobscura. Analizirani fenotipovi opisuju finu saradnju između genotipa i sredine potencirajući značajnost njihove interakcije za opstanak populacija u uslovima stresa kojima su jedinke/populacije izložene tokom većeg broja generacija. Jedinke Drosophila subobscura izlagane su u embrionalnom i ranom postembrionalnom razviću EMP ENF od 50 Hz i 0,25 mT, tokom tri sukcesivne generacije. Rezultati ispitivanja komponenti adaptivne vrednosti su pokazali da jedinke već tokom prve generacije izlaganja EMP ENF odgovaraju na prisustvo ovog sredinskog faktora, i to dužim embrionalnim razvićem (jaje-adult), slabijim preživljavanjem, skraćivanjem dužine života, i trendom povećanja fekunditeta u stresnim uslovima, u odnosu na jedinke iz kontrolne grupe koje su čitavo razviće provele izvan dejstva EMP. Nakon treće generacije, jedinke koje su izlagane navedenom EMP takođe imaju slabije preživljavanje i kraći životni vek, ali njihovo razviće traje kraće, a u pogledu fekunditeta uočava se trend smanjivanja u poređenju sa jedinkama iste generacije iz kontrolnih uslova. Prosečna vrednost komponenti adaptivne vrednosti opada tokom generacija, što je posebno izraženo u značajnom smanjenju preživljavanja kroz generacije kod jedinki izloženih EMP od 50 Hz i 0,25 mT. Ovi rezultati takođe pokazuju da uticaj EMP ENF tokom niza sukcesivnih generacija ima kumulativno dejstvo. Fenotipske korelacije analiziranih komponenti adaptivne vrednosti pokazuje da EMP ENF kao sredinski faktor utiče na kovariranje analiziranih osobina, menjajući smer i smanjujući stepen povezanosti osobina. Izlagane jedinke iz druge i treće generacije pokazuju varijabilnost fenotipske plastičnosti u dužini razvića, što može može biti važno za adaptaciju na veštačka EMP ENF. Jedinke posmatranih IF linija, koji se međusobno mogu razlikovati u osetljivosti i fiziološkim mehanizmima odgovora na sredinske uslove, variranjem dužine razvića delimično kompenzuju sredinski stres nastao usled uticaja EMP od 50 Hz i 0,25 mT. Uticaj EMP ENF na jedinke D. subobscura izlagane tokom embrionalnog i ranog post-embrionalnog razvića ogleda se i kroz promenu veličine, ali ne i oblika krila. Jedinke koje su bile izlagane veštačkom EMP ENF u prvoj generaciji imaju veća krila u odnosu na jedinke iz kontrolne grupe, i to kod oba pola, za razliku od jedinki iz treće generacije kojih je došlo do smanjenja veličine krila. Promena veličine krila izlaganih jedinki tokom tri generacije je pokazatelj uticaja EMP ENF na procese razvića tokom embrionalne i larvalne faze. Rezultati pokazuju da se pod delovanjem EMP ENF na puteve i mehanizme tokom razvića menja i konačni fenotip organizama. Rezultati ovog rada su pokazali da EMP ENF predstavlja faktor spoljašnje sredine koji ima uticaj na rast, razviće i adaptivnu vrednost jedinki D. subobscura, pa na populacionom nivou ovaj faktor može predstavljati selekcioni pritisak za komponente adaptivne vrednosti, fenotip i genetičku varijabilnost populacije. Analizirani efekti EMP od 50 Hz i 0,25 mT na populacionom nivou se ogledaju u smanjivanju vrednosti pojedinih komponenti adaptivne vrednosti populacije D. subobscura, a samim tim i na sposobnost populacije da adekvatno odgovori na dodatne i/ili buduće promene u životnoj sredini. Na osnovu svega iznetog, može se zaključiti da EMP ENF može da se klasifikuje kao stresogeni faktor antropogenog porekla.

Since its beginning, the life on Earth has been evolved in the presence of natural electromagnetic fields (EMFs) which are relatively simple and and with few EM energy emitters. During their evolution living organisms have well adapted to this constant environmental factor, but in nowadays, they are being increasingly exposed to artificial EMF’s. The intensity and frequency of these fields are far higher than in the fields of natural origin, and includes mostly EMP power frequency (within the spectrum of extremely low frequency (ELF) of 50 or 60 Hz), fields originating from the unit of mass (radio and television transmitters, 100-500 MHz) and mobile communications (800-900 MHz and 1800-2200 MHz). The ELF EMFs have great biophysical significance since they are part of ubiquitous electric power transmission and consumption. The artificial EMF’s have been a subject of various biological studies, focused mainly on the effects to human health. Exposing organisms to ELF EMFs, even for a short period, results in a disturbance of equilibrium of biological systems, as well as in disorder/disturbance on different levels of the organization. Nevertheless, there is no generally accepted theory about the ELF EMF mechanisms and their effects on biological systems. Furthermore, environmental changes caused by ELF EMF can have an effect on ecological and evolutionary processes that affect the level and extent of genetic structure and evolutionary changes within a population. Therefore, it is of utmost importance to not only thoroughly understand the mechanisms that enable organisms to adapt to environmental challenges, but to anticipate and observe populations’ responses to environmental changes. In the last three daecades, there have been numerous biological studies on ELF EMF effects on Drosophila. The results of exposing Drosophila individuals to ELF EMF during different life stages are often contradictory because the ELF EMF effects on growth and metabolism can be either inhibitory or stimulative depending on the complex relations between the frequency, field intensity, exopusre duration, and condiotion and type of cells or organisms. There are many data on ENF EMP effects on development, fecundity, viability, and other fitness components observed on Drosophila in laboratory conditions. Nevertheless, the ENF EMP, being one of the ubiqutous abiotic environmental factors, can affect populations’ genetic structures and levels of populations’ genetic variability. This kind of the genetic variability can serve as a basis for other evolutionary mechanisms within a population. The goal of this research is to analyse the effects of ENF EMP, complex environmental factor, on a populational-genetic level. The experiment evaluates network of phenotypic responses to ELF EMF by using the genetic variability of IF lines whitin population of D. subobscura. Analized phenotypes describe delicate cooperation between genotype and the environment, emphasizing the importance of their interaction for population survival in the conditions of stress, to which the individuals/populations are exposed during the generations. Individuals of D. subobscura were exposed to ELF EMF of 50 Hz and 0,25 mT in embryonal and early post-embryonal developmental stages during three successive generations. The results of fitness components analysis showed that individuals responded to exposure with ELF EMF already in the first generation. The response was reflected in longer embrionic development (egg-to-adult), lower survival, shortening of longevity, and by the trend of fecundity increase in stressful conditions, when compared to individuals of the control group whose development was completed outside of the EMP. In the third generation, individuals exposed to EMF also had lower survival and shorter life span, but their development was faster, while fecundity showed trend of decrease, when compared to control individuals of the corresponding generation. Average values of fitness components decreased through generations, which can be observed in particular by lowered survival through generations of individuals exposed to 50 Hz and 0.25 mT EMF. The results also show that the effect of EMF ELF is cummulative during generations. Phenotypic correlations of fitness components indicate that EMF ELF as an environmental factor affects covariation of the analized traits by changing the direction and by lowering the degree of trait interconnection. Exposed individuals from second and third generation show variation of phenotypic plasticity of developmental time, which can be important for adaptation to artificial EMF ELF. By varying the developmental time, individuals of the analized IF lines, which can differ in sensitivity and physiological mechanisms of response to environmental factors, partially compensate environmental stress caused by EMF ELF. The effect of ELF EMF on individuals of D. subobscura exposed during embryonic and early post-embryonic development, is reflected through the change of wing size as well, but not through the wing shape. Individuals of both sexes exposed to an artificial EMF ELF in first generation had larger wings compared to control group individuals. Contrary to that, in exposed individuals of the third generation there has been a decrease of wing size . Change of wing size of the exposed individuals during three generations is the indicator of the EMF ELF effect on developmental processes during embrionic and larval phase. The results also show that the effect of EMF ELF on developmental mechanisms changes final phenotype of individuals as well. The results showed that EMF ELF represents environmental factor which influence growth, development and fitness of D. subobscura individuals. Therefore, on a population level this factor can represent selection pressure for fitness components, phenotype and population genetic variability. Analized effects of 50 Hz and 0,25 mT EMF on population level are reflected in the decrease of particular fitness components of D. subobscura population, and by that in the ability of population to adequately respond to added and/or future changes in the environment. Based on all the evidence presented here, the conclusion is that ELF EMF can be clasified as a stressongenic factor of the anthropogenic origin.

Interakcija inicijacionog proteina ORC sa replikatorom regiona lamin B2

Interaction of initiation protein Orc with lamin B2 replication region

Inicijacija replikacije DNK kod eukariota započinje vezivanjem proteinskog kompleksa ORC za diskretna mesta u genomu nazvana ori-sekvence ili ori. Specifične sekvence odgovome za prepoznavanje i vezivanje kompleksa ORC, kao i mehanizam kojim ORC selektuje orisekvence još uvek nisu poznati. Jedna od malobrojnih zajedničkih karakteristika mesta uključenih u vezivanje inicijacionog proteina jeste visok sadržaj AT bp i kratkih nizova (dA)-(dT) što ukazuje na mogućnost da ori-sekvence obrazuju sinonimne stmkture koje odreduju specifičnost vezivanja inicijacionog proteina. Radi testiranja ove pretpostavke, analizirana je struktura i oblik humane ori-sekvence lamin B2 i detaljno ispitana DNK vezivna specifičnost proteina HsOrc4. Ovaj protein predstavlja jednu od subjedinica kompleksa ORC i pokazuje istu DNK-vezivnu aktivnost kao i kompleks ORC. Utvrđeno je da ori-sekvenca, pod uslovima neutralnog pH, niske ili umerene jonske jačine i u prisustvu jona Mg2+ zauzima altemativnu formu koju karakteriše prisustvo denaturisanog regiona, okceta, i povećana elektroforetska pokretljivost u nativnoj PAGE. Stabilno prisustvo denaturisanog regiona u okviru fragmenta DNK omogućeno je formiranjem Hoogsteen-ovih vodoničnih veza izmedu centralnih pirimidina okceta i adenina jednog od kratkih nizova (dA)-(dT). Kao rezultat ove interakcije nastaje dvolančana petlja koja u osnovi sadrži intramolekulski tripleks. Struktura intramolekulskog tripleksa formira se u regionu koji interaguje sa inicijacionim proteinom i može predstavljati jedan od elemenata odgovornih za prepoznavanje i vezivanje ovog proteina. U eksperimentima direktnog vezivanja proteina HsOrc4 utvrdeno je da on ne prepoznaje kratke nizove (dA)-(dT) i (dA), dok se za (dT) slabo vezuje. Najveći afmitet protein je pokazivao za trolančane strukture tipa TAT. Kompeticija jedno-, dvo- i trolančanih molekula DNK vezivanju HsOrc4 za ori-sekvencu pokazala je da protein razlikuje trolančanu strukturu od jedno- i dvolančane i specifično se vezuje za nju. Sudeći po efikasnosti kompeticije, trolančana DNK bila je veoma slična prirodnim vezivnim mestima proteina HsOrc4. Svojstvo ori-sekvence da formira intramolekulski tripleks i specifično vezivanje proteina HsOrc4 za tripleks sugeriše da trolančana struktura može predstavljati deo mehanizma kojim inicijacioni protein prepoznaje i selektuje mesta inicijacije replikacije.

In complex eukaryotes DNA replication is initiated by binding of origin recognition complexes (ORCs) to specific genomic sites called origins of replication. Consensus sequence required for this event and the mechanism by which ORC is localized to origins of replication remain poorly understood. General features of genomic regions involved in initiator protein binding are AT-richness and frequent occurrence of short (dA)-(dT) runs. Such distribution of A and T residues opens a possibility that origins of replication build mutually equivalent unorthodox structures which are recognised by initiation protein. In order to test this hypothesis, a study of structure and shape of the human lamin B2 origin was performed. DNA binding activity of protein HsOrc4, one of ORCs subunits that exhibited similar DNA binding properties as the whole complex, was also tested. It was shown that, at neutral pH, low or moderate ionic strength and in presence of Mg" ions, lamin B2 ongm adopted alternative helical form, characterized by a single unpaired region and faster migration in native polyacrylamide geis. It was proposed that these properties reflected the ability of origin DNA to form double stranded loop with intramolecular triplex in its base. Triplex was kept together by Hoogsteen hydrogen bonding between Central pyrimidines of unpaired region and complementary double stranded sequence. Since intramolecular noncanonical structure formed in origin sequences protected by ORC in vivo and in vitro, it could represent the element responsible for site-specific ORC binding. In order to test this notion, binding specificity of HsOrc4 was tested in direct and competition DNA binding experiments. The protein did not recognize (dA) or (dA)-(dT), but it exhibited very low affmity for (dT) and a very high affmity for TAT triplex. Consistent with that, triple stranded DNA competed very well with origin DNA for HsOrc4 binding, whereas single or double stranded DNA exhibited much less significant competitive effect. As judged by its competitive efficiency, triple stranded DNA was very similar to naturally ocuring DNA binding sites of HsOrc4. In conclusion, formation o f intramolecular triplex within origin DNA and its specific recognition by HsOrc4 suggest that triple stranded structure might play a role in selection of eukaryotic origins of replication.

Ekspresija antioksidativnih enzima i transkripcionog faktora Nrf2 kod pacijentkinja sa benigno, premaligno i maligno transformisanim endometrijumom

Expression of antioxidant enzymes and transcription factor Nrf2 in patients with benign, premalignant and malignant endometrial transformation .

Tokom čitavog životnog ciklusa aerobni organizmi su izloženi brojnim endogenim i egzogenim faktorima koji indukuju povećanje produkcije ROS-a. Prisutni u fiziološkim koncentracijama, reaktivni molekuli ROS-a imaju značajnu ulogu u važnim ćelijskim procesima kao što su regulacija signalnih kaskada i genske ekspresije. U visokim koncentracijama ROS mogu oksidovati ćelijske proteine, lipide i DNK i time dovesti do promena strukture i funkcije, oštećenja, pa i smrti ćelije. Kada koncentracija slobodnih radikala premaši fiziološki nivo, smatra se da se ćelija nalazi u stanju oksidativnog stresa. Kako bi sprečili nastanak i umanjili posledice ove vrste stresa, živi organizmi su razvili moćan antioksidativni sistem zaštite (AOS). Ovaj sistem uključuje seriju mehanizama kojima se nivo slobodnih radikala održava u uskom opsegu između fiziološke i toksične koncentracije. Najvažnije enzimske komponente AOS su: superoksid dismutaze (SOD), katalaza (CAT), glutation peroksidaza (GPx) i glutation reduktaza (GR). Ovi enzimi uklanjaju višak ROS-a i učestvuju u održavanju nivoa redukovanog glutationa, čime obezbeđuju očuvanje osnovnih životnih funkcija i sprečavaju nastanak oštećenja i bolesti ćelije. Zbog tako važne uloge AOS, regulacija funkcionalne ekspresije njegovih komponenti je naročito značajna za fiziološke i patološke procese u aerobnim organizmima. Jedan od najvažnijih regulatornih molekula u tom sistemu je Nrf2 (NF-E2 related factor 2). Nrf2 je transkripcioni faktor koji indukuje ekspresiju mnogih citoprotektivnih proteina uključujući i antioksidativne enzime, zbog čega ima značajnu ulogu u regulaciji oksidativnog stresa. Savremena istraživanja na različitim model-sistemima ukazuju na povezanost oksidativnog stresa i kancerogeneze. Razvoj kancera je višestepeni proces koji se razvija kroz tri faze: inicijaciju, promociju i progresiju, a oksidativni stres je povezan sa svakim od ova tri stadijuma transformacije ćelije. Osim toga i sam antioksidativni profil je u ćelijama kancera izmenjen u odnosu na zdrave, normalne ćelije, pa ovi molekuli mogu biti i značajni biomarkeri u dijagnostici, proceni rizika i stepena kancerogeneze. Kancer endometrijuma je jedno od tri najčešća oboljenja reproduktivnih organa žene. Pokazano je da benigne i premaligne promene na uterusu prethode samoj malignoj transformaciji tkiva, pa se ova stanja mogu smatrati fazama kancerogeneze. Uprkos brojnim istraživanjima, molekulski mehanizmi uključeni u višestepeni proces nastanka endometrijalnog kancera još uvek nisu potpuno razjašnjeni. Zato je cilj ove disertacije bio da se ispitaju promene ekspresije četiri najvažnija antioksidativna enzima u krvi i tkivu pacijentkinja sa benignim, premalignim i malignim transformacijama endometrijuma, kao i mehanizam njihove regulacije transkripcionim faktorom Nrf2. U istraživanju su korišćeni tkivo endometrijuma i venska krv pacijentkinja sa dijagnozama: polypus endometrii, uterus myomatosus, hyperplasia simplex endometrii, hyperplasia complex endometrii i adenocarcinoma endometrii. Polypus endometrii i uterus myomatosus su smatrani benignim, a hyperplasia simplex i hyperplasia complex endometrii premalignim transformacijama uterusa. Nakon adekvatne pripreme, u uzorcima krvi i endometrijuma svih pet grupa pacijentkinja prvo je određivana koncentracija ukupnih proteina, a zatim urađena SDS PAGE elektroforeza, transfer na nitroceluloznu membranu i hemiluminiscentna detekcija proteinskih molekula za CuZnSOD, CAT, GPx, GR i Nfr2, kao i za aktin u odnosu na koga su količine ispitivanih proteina normalizovane. Osim toga, u uzorcima endometrijuma svih pet grupa pacijentkinja je izolovana RNK i nakon reverzne transkripcije RNK u cDNK, TaqMan Real-Time PCR metodom, u odnosu na POLR2 kao endogenu kontrolu, određene su količine mRNK za CuZnSOD, CAT, GPx, GR i Nfr2. Rezultati ovih eksperimenata su pokazali sledeće: 1. U odnosu na ispitanice sa polipom i miomom, u endometrijumu pacijentkinja sa hiperplazijom simpleks i hiperplazijom kompleks zabeleženo je smanjenje, a u tkivu adenokarcinoma povećanje nivoa Nrf2 proteina. Uočene promene regulisane su na nivou transkripcije, osim u tkivu adenokarcinoma u kome je porast nivoa Nrf2 proteina regulisan nekim posttranskripcionim mehanizmom. 2. Nivo CuZnSOD mRNK i količina CuZnSOD proteina u endometrijumu pacijentkinja sa hiperplazijom simpleks i hiperplazijom kompleks značajno opadaju u odnosu na pacijentkinje sa polipom i miomom. U tkivu adenokarcinoma, međutim, vrednosti ovih parametara su značajno povećane u odnosu na obe benigne i obe premaligne transformacije. Zabeležene promene CuZnSOD u različitim fazama transformacije su na nivou transkripcije i translacije određene promenama nivoa transkripcionog faktora Nrf2. 3. U odnosu na kontrolne grupe pacijentkinja sa polipom i miomom, nivo CAT mRNK i CAT proteina je značajno smanjen u tkivu pacijentkinja sa premalignim i malignim lezijama uterusa. Takođe je pokazano da Nrf2 ne utiče direktno na transkripciju gena za CAT, ali delujući na druge mehanizme uključene u translacione i posttranslacione procese u ekspresiji CAT verovatno može uticati na nivo ovog enzima u endometrijumu. 4. Tokom kancerogeneze, u premaligno transformisanom endometrijumu dolazi do povećanja, a u adenokarcinomu do smanjenja nivoa GPx proteina. Nivo GPx je pozitivno korelisan sa količinom transkripcionog faktora NRF2, a mehanizam regulacije ekspresije ovog enzima se menja sa stepenom transformacije tkiva. 5. U toku maligne transformacije endometrijuma zabeleženo je povećanje ekspresije glutation reduktaze. Porast GR proteina u toku kancerogeneze rezultat je ne samo intenzivirane transkripcije pod uticajem Nrf2, nego i postojanja dodatnog, posttranskripcionog mehanizma kojim se postiže povećanje nivoa GR proteina u transformisanim ćelijama uterusa. 6. Što se tiče ekspresije AOE u krvi ispitivanih pacijentkinja, uočeno je da u odnosu na kontrolnu grupu, kod ispitanica sa benignim promenama na uterusu (polip i miom) dolazi do smanjenja nivoa Nrf2 i GR, porasta nivoa GPx, dok relativne količine CuZnSOD i CAT proteina nisu značajno promenjene. Nešto izraženije promene su uočene u krvi pacijentkinja sa dijagnozama hyperplasia simplex, hyperplasia complex i adenocarcinoma endometrii, kod kojih je zabeležen pad nivoa Nrf2, CuZnSOD i GR proteina, porast nivoa GPx i nepromenjena ekspresija CAT. Takođe, pokazano je da je ekspresija sva četiri ispitivana AOE u krvi pozitivno korelisana sa nivoom transkripcionog faktora Nrf2. Rezultati ove doktorske disertacije ukazuju na postojanje i značaj specifičnosti ekspresije antioksidativnih enzima i transkripcionog faktora Nrf2 kod ginekoloških pacijentkinja sa dijagnozama: polypus endometrii, uterus myomatosus, hyperplasia simplex endometrii, hyperplasia complex endometrii i adenocarcinoma endometrii. Uočene promene potvrđuju važnu ulogu ispitivanih antioksidativnih enzima u kompleksnim molekulskim interakcijama koje se nalaze u osnovi transformacije ćelija endometrijuma. Očigledno je da ovi AO molekuli ne samo da utiču na nastanak lezija u tkivu uterusa, nego predstavljaju i važan faktor za progresiju benignih promena u premaligni i maligni fenotip. Na osnovu svega izloženog može se zaključiti da rezultati ove doktorske disertacije značajno doprinose razumevanju molekulskih osnova kancerogeneze endometrijuma. Bolje poznavanje mehanizama maligne transformacije uterusa i primena ovih znanja u kliničkoj praksi mogli bi doprineti kvalitetu prevencije, dijagnostike i terapije ginekoloških oboljenja, čime bi se poboljšali tok i prognoza bolesti, a time povećala i stopa preživljavanja ovih pacijentkinja.

During the life cycle aerobic organisms are exposed to a number of endogenous and exogenous sources of ROS. Present in low to moderate concentrations these reactive molecules play an important role in many physiological processes such as regulation of signaling cascades and gene expression. In high concentrations, ROS can oxidize cellular proteins, lipids and DNA, causing changes in structure and function, damage and even cell death. When the concentration of free radicals exceeds the physiological level, a cell is said to be in a state of oxidative stress. To prevent the onset and reduce the consequences of oxidative stress, living organisms have developed powerful antioxidant system (AOS). This system includes a set of mechanisms to maintain the level of free radicals in the narrow range between physiological and toxic concentrations. The most important enzyme components in AOS are: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR). These enzymes are involved in the removal of ROS and participate in maintaining level of reduced glutathione, thus ensuring the preservation of physiological functions and inhibit the development of cell damage and disease. Because of such an important role of AOS, the regulation of the functional expression of its components is of particular importance for physiological and pathological processes in aerobic organisms. One of the most important regulatory molecules in this system is Nrf2 (NF-E2 related factor 2). Nrf2 is a transcription factor that induces expression of many cytoprotective proteins including antioxidant enzymes, and therefore play an important role in the regulation of oxidative stress. Previous studies have clearly demonstrated a link between oxidative stress and carcinogenesis. Cancer is a multistage process that develops over three stages: initiation, promotion and progression and oxidative stress is associated with each of them. Moreover, antioxidant profile is altered in cancer cells compared with healthy, normal tissue, so these molecules may be important biomarkers in the assessment of risk and the degree of carcinogenesis. Endometrial cancer is one of the three most common diseases of female reproductive organs. It has been shown that benign and premalignant changes precede the malignant transformation of the uterus, which is why these conditions may be considered as stages of carcinogenesis. Despite numerous studies, the molecular processes involved in multi-stage development of endometrial cancer are not yet fully known. Therefore, the aim of this dissertation was to examine the changes in expression of the four most important antioxidant enzymes in the blood and tissues of patients with benign, premalignant and malignant endometrial transformations, as well as the mechanism of their regulation by transcription factor Nrf2. In this research we used endometrial tissue and venous blood of patients diagnosed with: polypus endometrii, uterus myomatosus, hyperplasia simplex endometrii, hyperplasia complex endometrii and adenocarcinoma endometrii. Polypus endometrii and uterus myomatosus were considered as benign, while hyperplasia simplex and hyperplasia complex endometrii were considered as premalignant transformation of the uterus. After adequate sample preparation, we initially determined the total protein concentration in blood and endometrial tissue of all five groups of patients. Then we performed SDS PAGE electrophoresis, transfer to nitrocellulose membrane and chemiluminescent detection of CuZnSOD, CAT, GPx, GR and Nfr2 protein molecules. The protein levels were normalized with respect to β-actin. Furthermore, in endometrial samples of all five groups of patients, RNA was extracted and underwent reverse transcription into cDNA, followed by the TaqMan Real-Time PCR method by which the amounts of CuZnSOD, CAT, GPx, GR and Nfr2 mRNA were determined in respect to POLR2 as endogenous control. The results of these experiments showed the following: 1. In comparison to patients with polyps and myomas, in endometrium of women with hyperplasia simplex and hyperplasia complex decreased levels of Nrf2 were recorded, while in adenocarcinoma tissue Nrf2 protein level was increased. The observed changes were regulated at the transcriptional level, except in adenocarcinoma tissue in which increase in Nrf2 level was regulated by some post-transcriptional mechanism. 2. CuZnSOD mRNA level and the amount of CuZnSOD protein in the endometrium of patients with hyperplasia simplex and hyperplasia complex were decreased compared to patients with polyps and myomas. In adenocarcinoma tissue, however, the values of these parameters were significantly increased compared to both benign and both premalignant transformations. Observed transcriptional and translational CuZnSOD variability in different stages of endometrial transformation were the consequences of changes in the level of transcription factor Nrf2. 3. Compared to the control groups with polyps and myomas, levels of CAT mRNA and CAT protein were significantly decreased in the tissues of patients with premalignant and malignant lesions of the uterus. It is also shown that Nrf2 had no direct effect on the CAT gene transcription, but acting on other translational and posttranslational processes can probably influence the level of this enzyme in the endometrium. 4. During carcinogenesis, increase of GPx protein level was detected in premalignant endometrium, while decline in the the amount of this enzyme was measured in adenocarcinoma tissue. GPx level was positively correlated with the amount of transcription factor Nrf2, but mechanism that regulates the expression of this enzyme varied with the degree of tissue transformation. 5. In the course of malignant endometrial transformation, increased expression of glutathione reductase was observed. Consequent increased level of GR protein was the result not only of the intensified transcription under the influence of Nrf2, but also of an additional, posttranscriptional mechanism activated in transformed cells of the uterus. 6. As regards AOE expression in the blood of examined patients, it was observed that in comparison to the control group, in groups of women with benign endometrial changes a decline in Nrf2 and GR levels were measured, GPx level was increased, while the relative amounts of CuZnSOD and CAT protein did not change significantly. Somewhat more pronounced changes were observed in the blood of patients diagnosed with hyperplasia simplex, hyperplasia complex and adenocarcinoma, characterized by declined protein levels of Nrf2, CuZnSOD and GR, increased level of GPx and unaltered expression of CAT. It was also shown that the blood expression levels of all investigated AOE correlated positively with the amount of Nrf2. The results of this dissertation indicate the existence and importance of the specific expression pattern of antioxidant enzymes and transcription factor Nrf2 in gynecological patients diagnosed with: polypus endometrii, uterus myomatosus, hyperplasia simplex endometrii, hyperplasia complex endometrii and adenocarcinoma endometrii. The observed changes confirm the important role of the examined enzymes in complex molecular interactions that underlie the transformation of endometrial cells. It is obvious that these AO molecules not only influence the development of lesions in the uterine tissue, but they are also an important factor for the progression of benign changes in premalignant and malignant phenotype. It can be concluded that the results of this dissertation contribute significantly to a better understanding of the molecular processes involved in carcinogenesis. Implementation of this knowledge in clinical practice could contribute to the quality of prevention, diagnosis and treatment of gynecological patients, may lead to to improvement in the disease course and prognosis and, consequently, may result in increased survival rate of cancer patients.

Optimizacija i karakterizacija elektrolita na bazi jonskih tečnosti pogodnih za litijum jonske baterije

Optimization and characterization of ionic liquid based electrolytes for Li-ion batteries

U ovoj doktorskoj disertaciji ispitivani su elektroliti na bazi jonskih tečnosti pogodni za primenu u litijum jonskim baterijama. Fizičko-hemijska svojstva binarnih smeša jonskih tečnosti sa dicijanamidnim i bis(trifluorometilsulfonil)imidnim anjonima i molekulskih rastvarača ispitana su u celom opsegu molskih udela i na različitim temperaturama. Na osnovu izmerenih gustina, viskoznosti i električne provodljivosti izračunati su različiti fizičko hemijski parametri i diskutavne interakcije između komponenata smeša. Ispitana je termička i elektrohemijska stabilnost odabranih elektrolita. Dodatkom litijumove soli u odabrane binarne smeše dobijeni su ternarni sistemi koji su okarakterisani u zavisnoti od koncentracije litijumove soli. Odabrani elektroliti upotrebljeni su za ispitivanje performansi litijum jonske ćelije sa anatas TiO2 nanotubularnim elektrodama. Cikličnom voltametrijom i galvanostatskim cikliranjem su ispitane performanse ćelije u toku 150 ciklusa punjenja i pražnjenja. Na osnovu ciklovoltametrijskih merenja izračunati su koeficijenti difuzije i energija aktivacije za difuziju.

In this doctoral dissertation, Ion liquid-based electrolytes were tested for use in lithium-ion batteries. The physicochemical properties of binary mixtures of ionic liquids with dicyanamide and bis (trifluoromethylsulfonyl) imide anions and molecular solvents were examined throughout the range of molar proportions and at different temperatures. Based on the measured densities, viscosity and electrical conductivity, various physical chemical parameters and discrete interactions between the components of the mixture are calculated. Thermal and electrochemical stability of selected electrolytes was examined. By addition of lithium salt to the selected binary mixtures, ternary systems were characterized which were characterized by the concentration of lithium salt. The selected electrolytes were used to test the performance of the lithium-ion cell with anatomic TiO2 nanotubular electrodes. Cyclic voltammetry and galvanostatic cycling tested the cell's performance during the 150 charge and discharge cycles. Based on cyclotoltametric measurements, the diffusion coefficients and activation energies for diffusion were calculated.

Molekularna i morfološka karakterizacija vrsta iz kompleksa Praon dorsale- yomenae (Hymenoptera, Braconidae)

Molecular and morphological characterization in Praon dorsale- yomenae species complex (Hymenoptera, Braconidae)

Molekularna i morfološka varijabilnost unutar kompleksa vrsta Praon dorsale- yomenae analizirana je primenom tri nezavisne metode: “tradicionalne” morfometrije, geometrijske morfometrije i analize citohrom oksidaza I sekvenci mitohondrijalne DNK i 28S D2 gena nuklearne DNK. Za morfometrijske analize korišćena je 421 jedinka (270 ţenki i 151 muţjak) sakupljena sa preko 30 razliĉitih afidnih domaćina širom Palearktika. Molekularne analize su obuhvatile 47 jedinki u asocijaciji sa razliĉitim domaćinima. Primenom diskriminantne kanonijske analize utvrĊeno je koji su karakteri dovoljno informativni za morfološku diferencijaciju analiziranih taksona. CVA analizom su prikazane razlike u obliku krila izmeĊu vrsta. Varijabilnost oblika krila meĊu biotipovima analizirana je primenom PCA. Pokazano je da postoji znaĉajan efekat domaćina na varijabilnost oblika krila meĊu biotipovima vrsta P. dorsale i P. longicorne, dok je meĊu biotipovima vrste P. yomenae utvrĊen znaĉajan efekat geografske distribucije. Analize oblika krila ukazale su na to da se vrste „dorsale- yomenae“ kompleksa mogu morfološki razlikovati na osnovu duţine radijalnog i povratnog nerva. Geometrijska morfometrija primenjena u cilju vizualizacije suptilnih razlika u obliku krila pokazala je veću osetljivost u razdvajanju analiziranih taksona „dorsale-yomenae“ grupe u odnosu na „tradicionalnu” morfometriju. Ispitali smo validnost prethodno korišćenih morfoloških karaktera u identifikaciji „dorsale- yomenae“ grupe. TakoĊe smo ukazali na nove karaktere koji se mogu koristiti u kljuĉevima za identifikaciju. UtvrĊeni su razliĉiti obrasci obojenosti krilne nervature unutar kompleksa vrsta „dorsale-yomenae“. Na osnovu rezultata molekularnih analiza potvrĊen je status vrsta P. longicorne, P. dorsale, P. yomenae i P. volucre. UtvrĊeni su novi domaćini/asocijacije za vrstu P. dorsale. Otkrivena je nova vrsta za nauku koja parazitira vaš Staticobium limonii na Limonium angustifolium. Vrsta P. megourae nije dobila molekularnu potvrdu pa su za rasvetljavanja statusa ovog taksona potrebna dodatna istraţivanja.

Molecular and morphological variability of Praon dorsale-yomenae species complex was analyzed using the three independent methods: “traditional” morphometrics, geometric morphometrics and DNA sequence analysis of two gene regions (COI mtDNA and 28S D2). For morphometric analysis we used 421 specimens (270 females and 151 males) reared from about 30 different species of aphid hosts throughout Palaearctic. Molecular analysis included 47 specimens in association with different aphid hosts. Discriminant Canonical Analysis was performed in order to determine which characters were informative for morphological diferentiation of analyzed taxa. To explore differences in wing shape between species we performed Canonical Variate Analysis. Variation in wing shape among biotypes was investigated using Principal Component Analysis. We found that great variablity in wing shape of P. dorsale and P. longicorne biotypes was influenced by the host, while great effect of geographical distribution was noticed between P. yomenae biotypes. Analysis of wing shape demonstrated that „dorsale-yomenae“ species can be morphologically discriminated on the basis of length of forewing radial and recurrent vein. Geometric morphometrics applied to visualize subtle shape differences had more sensitivity than „traditional“ morphometrics in distiguishing taxa within „dorsale-yomenae“ group. We tested the validity of morphological characteristics previously used for identification in „dorsale-yomenae“ group, also indicating a new characters that could be used in identification keys. Different colour patterns of wing venation in „dorsale-yomenae“ species complex were found. On the basis of molecular results P. longicorne, P. dorsale, P. yomenae and P. volucre was confirmed as separate species. We presented a new tritrophic association for P. dorsale. A new parasitoid species from Staticobium limonii infesting Limonium angustifolium was described. P. megourae was not confirmed as separate species using molecular markers, thus the clarification of its status requires further research.

Ispitivanje probiotičkih i imunomodularnih karakteristika prirodnih izolata enterokoka u modelima in vitro i in vivo

Examination of probiotic and immunomodulatory characteristics of natural isolates of enterococci on in vitro and in vivo models

Enterokoke pripadaju grupi mleĉnokiselinskih bakterija koje su široko rasprostranjene u prirodi. Predominantno se nalaze u gastrointestinalnom traktu životinja, od insekata do ĉoveka i preko fecesa se oslobaĊaju u spoljašnju sredinu, nakon ĉega naseljavaju razliĉita staništa. Imaju kontroverzni status zbog uloge u bolniĉkim infekcijama i širenja gena odgovornih za rezistenciju na antibiotike. S druge strane, neki sojevi se koriste kao probiotici za životinje i ĉoveka, kao i starter kulture u mlekarskoj industriji. Da bi se enterokoke koristile kao probiotici neophodno je da budu bezbedne za korišćenje, da nemaju hemolitiĉku i želatinoznu aktivnost i da nisu otporne na antibiotike. Enterokoke sintetišu antimikrobne proteine, bakteriocine, i na taj naĉin regulišu brojnost drugih bakterija i modulišu odgovor domaćina na infekciju. One mogu da sintetišu i biomolekule koji interaguju sa ćelijama domaćina i na taj naĉin mogu da menjaju razliĉite signalne puteve unutar ćelija domaćina. Biomolekuli sintetisani od strane probiotiĉkih enterokoka nazivaju se postbiotici. Primena postbiotika predstavlja bezbednu alternativu korišćenja enterokoka u biomedicini. Ova doktorska disertacija imala ja za cilj da ispita probiotiĉka i imunomodulatorna svojstva prirodnih sojeva enterokoka izolovanih iz fermentisanih mleĉnih proizvoda sa podruĉja Zapadnog Balkana. Definisana su tri cilja: 1. da se pronaĊu sojevi bezbedni za korišćenje u biomedicini; 2. da se ispitaju probiotiĉke karakteristike tih sojeva u modelima in vitro; i 3. da se ispita imunomodulatorni efekat odabranog soja u modelima in vitro i in vivo. U cilju ispitivanja bezbedne upotrebe enterokoka kao probiotika testirano je ukupno 75 sojeva iz fermentisanih mleĉnih proizvoda koje pripadaju vrstama: Enterococcus durans (50 sojeva), Enterococcus faecium (15 sojeva), Enterococcus faecalis (6 sojeva), Enterococcus italicus (3 soja) i Enterococcus hirae (1 soj). Hemolitiĉku aktivnost pokazalo je 18,7% (14/75), dok je želatinaznu aktivnost imalo 6,7% (5/75) testiranih sojeva. Na osnovu rezultata testa mikrodilucije pokazana je visoka uĉestalost rezistencije na ciprofloksacin 48,2% (27/56), dok su u manjoj meri testirani sojevi rezistentni na gentamicin 10,7% (6/56). Na osnovu rezultata sposobnosti formiranja biofilma u primenjenim uslovima, devet sojeva nema sposobnost formiranja biofilma, 11 sojeva ima mogućnost formiranja slabog biofilma, BGGO9-28 ima sposobnost formiranja jakog biofilma, dok sojevi BGTRK4-42 i BGZLM1-5 pokazuju veoma jaku sposobnost formiranja. Na osnovu prisustva gena koji kodiraju razliĉite faktore virulencije u genomu ispitivanih enterokoka, uoĉava se visoka zastupljenost gena koji kodiraju adhezine. Uoĉeno je da 30,4% sojeva poseduje tri gena koji kodiraju razliĉite faktore virulencije, po 21,7% sojeva sadrži dva, odnosno ĉetiri gena, 17,4% ima pet gena i po 4,3% ispitivanih enterokoka sadrži 6 ili 7 gena koji kodiraju faktore virulencije, dok prisustvo, odnosno odsustvo gena u okviru fsr operona varira od analiziranog soja. Gen koji kodira želatinazu, gelE gen, prisutan je kod 5 sojeva, dok je prisustvo gena za serinsku proteazu potvrĊeno kod 12 sojeva. Nije pronaĊeno prisustvo gena koji kodiraju hijaluronidazu i citolizin aktivirajući protein. Analizom preživljavanja simuliranih uslova gastrointestinalnog trakta pokazano je da testirane enterokoke preživljavaju izmeĊu 94,4% - 98,8% ± 1,3% želudaĉni sok, 87,1% - 97,9% ± 2,9% dvanaestopalaĉni sok i 82,7% - 92,9% ± 3,0% crevni sok. Da bi ostvarile svoj probiotiĉki efekat, enterokoke moraju da se vežu za mucin, proteine ekstraćelijskog matriksa i ćelije domaćina. Procenat vezivanja enterokoka za mucin iznosio je 71,4% ± 3,1%, za kolagen 74,1% ± 4,1% i fibronektin 73,9% ± 2,7%. Analizom vezivanja za HT29-MTX ćelijsku liniju pokazano je da se enterokoke vezuju u proseku 86,7% ± 5,4%. Sojevi En. faecium BGZLM1-5 i BGPAS1-3 pokazali su antimikrobni efekat na Listeria monocytogenes ATCC19111. L. monocytogenes je patogena bakterija koja se prenosi hranom, narušava crevnu epitelnu barijeru i dovodi do razvoja listerioze, posebno kod imunokompromitovanih pacijenata. Dalje je analiziran potencijal soja En. faecium BGPAS1- 3, u modulaciji odgovora diferenciranih Caco-2 intestinalnih epitelnih ćelija na infekciju L. monocytogenes ATCC19111. Pokazano je da soj BGPAS1-3 sintetiše antilisterijski molekul proteinske prirode, koji je vezan za ćelijski zid i delimiĉno otporan na visoku temperaturu. Pored toga, soj BGPAS1-3 ubijen izlaganjem visokoj temperaturi može da spreĉi narušavanje ĉvrstih veza izmeĊu diferenciranih Caco-2 ćelija inficiranih sa L. monocytogenes ATCC19111, istovremeno dozvoljavajući indukciju imunskih mehanizama važnih za antilisterijski odgovor domaćina i stimulišući ekspresiju TGF-β iRNK u intestinalnim epitelnim ćelijama. Dodatno, modulacija MyD88 zavisna od TLR2 i TLR4 puteva od strane BGPAS1-3 ukljuĉena je u odgovor domaćina na prisustvo L. monocytogenes ATCC19111. Pokazano je da živ i toplotom ubijeni BGPAS1-3 u modelu proizvodnje jogurta smanjuje broj L. monocytogenes ATCC19111 prvih sedam dana tokom skladištenja za nešto manje od 1 log/ml. Pored celih ćelija temperaturom ubijenog BGPAS1-3, korišćene su i frakcije peptidoglikana i peptidoglikana bez teihoiĉne kiseline u cilju ispitivanja modulacije imunskog odgovora ćelija mezenteriĉnih limfnih ĉvorova (MLĈ) stimulisanim konkanavalinom A (KonA) na modelu in vitro. Pokazano je da ispitivane frakcije ne menjaju metabliĉku aktivnost i nisu citotoksiĉne u testiranim odnosima. Produkcija IFN-γ i IL-17, pro-inflamacijskih citokina ostala je nepomenjena u odnosu na netretirane KonA-stimulisane MLĈ ćelije, dok je produkcija IL-10 povećana. Uzimajući u obzir prethodne rezultate dobijene u modelima in vitro koji ukazuju na potencijalni imunosupresivni i protektivni efekat ovog soja, ispitan je efekat toplotom ubijenih ćelija na in vivo modelu inflamacijske bolesti. Kao model ispitivanja izabran je eksperimentalni autoimunski encefalomijelitis (EAE), najĉešće korišćen životinjski model multiple skleroze. Rezultati su pokazali da primena toplotom ubijenog BGPAS1-3 (109 ćelija po jednom pacovu na dan) tokom 30 dana ogleda utiĉe na kliniĉki tok bolesti ublažavajući simptome u poĉetnim fazama bolesti. S obzirom da je pokazano da imunomodulatorne terapije mogu da dovedu do kljuĉnih promena u sastavu mikrobiote, u nastavku je ispitivan efekat ćelija toplotom ubijenog BGPAS1-3 na mikrobiom imunizovanih ženki DA pacova DGGE metodom. Klonirano je i sekvencirano ukupno 14 jedinstvenih DNK fragmenata. Analizom sekvenci specifiĉnih fragmenata pokazano je da je Lactobacillus intestinalis prisutan samo na poĉetku tretmana (kod još uvek zdravih životinja), dok nije detektovan 30. dana nakon imunizacije niti u kontrolnoj grupi, kao ni u grupi koja je pila toplotom ubijeni BGPAS1-3. Interesantno je da su vrste Lb. murinus i En. hirae prisutne samo u uzorcima fecesa imunizovanih životinja koje su 30 dana tretirane toplotom ubijenim BGPAS1-3, a nisu prisutne u kontrolnoj grupi. Korišćenjem univerzalnih prajmera pokazano je da se Shuttleworthia satelles i Muribaculum intestinale pojavljuju samo 30. dan u fecesima kontrolnih imunizovanih životinja koje nisu tretirane toplotom ubijenim BGPAS1-3. Analizom sekvenci dobijenih umnožavanjem prajmerima specifiĉnim za Bifidobacteria spp. pokazano je prisustvo Bifidobacterium pseudolongum samo u fecesima kontrolnih i tretiranih životinja 30 dana nakon imunizacije. Rezultati ove doktorske disertacije pokazali su da sojevi enterokoka izolovani iz mleka i fermentisanih mleĉnih proizvoda imaju nizak patogeni potencijal i dobar probiotiĉki potencijal. Pokazano je da u in vitro modelu živ i toplotom ubijen Enterococcus faecium BGPAS1-3 ispoljava antilisterijski efekat, i istovremeno omogućava razvoj efikasnog imunskog odgovora domaćina protiv Listeria monocytogenes i podstiĉe produkciju molekula koji doprinose zaštiti intestinalne epitelne barijere. Toplotom ubijeni BGPAS1-3 ispoljava terapijski efekat in vivo na modelu eksperimentalnog autoimunskog encefalomijelitisa.

are predominantly found in the gastrointestinal tract of animals, from insects to humans and through faeces they are released into the environment, where they colonize different habitats. They have a controversial status according to the role as causative agents of hospital infections and the spread of genetic determinants of antibiotic resistance. In contrary, some strains have been used used as probiotics for animals and humans, as well as a starter cultures in the dairy industry. In order to use enterococci as probiotics, it is necessary to determine their safety, i.e. absence of hemolytic and gelatinase activity and susceptiblity to clinicaly relevant antibiotics. Enterococci synthesize antimicrobial proteins, bacteriocins, by which way they regulate the number of other bacteria and modulate the host response to the infection. They can also synthesize biomolecules that interact with the host cells, and change the different signalling pathways within host cells. Biomolecules synthesized by probiotic enterococci are called postbiotics. The use of postbiotic represents a safety alternative to the enterococci application in biomedicine. The aim of this dissertation was examination of probiotic and immunomodulatory characteristics of enterococci isolated from fermented dairy products from the Western Balkans countries. Three objectives were defined: 1. to find strains safe for use in biomedicine; 2. to examine the probiotic characteristics on in vitro models, and 3. to examine the immunomodulatory effect of the selected strain on in vitro and in vivo models. In order to analyse the safe use of enterococci as a probiotics, a total of 75 isolates from fermented dairy products were tested: Enterococcus durans (50 isolates), En. faecium (15 isolates), En. faecalis (6 isolates), En. italicus (3 isolates) and En. hirae (1 isolate). 18.7% (14/75) strains showed hemolytic activity, while 6.7% (5/75) had gelatinase activity. Based on the results of the microdilution test, 48.2% (27/56) of strains were ciprofloxacin resistance, while 10.7% (6/56) strains were resistant to gentamicin. According to the analysis of selected enterococci genomes, high incidence of adhesin encoding genes could be noticed. It was found that 30.4% of strains have three genes encoding different virulence factors, 21.7% of strains having two or four genes, 17.4% having five genes, and 4.3% of studied enterococci containing 6 or 7 genes which encode virulence factors, while the presence or absence of gene within the fsr operon varies from the analyzed strains. Hyaluronidase and cytolysin activating protein encoding genes were not detected. According to the results of biofilm formation assay, nine strains had no ability to form a biofilm and 11 strains had weak biofilm forming ability. The BGGO9-28 had a strong biofilm forming ability, while strains BGTRK4-42 and BGZLM1-5 showed very strong biofilm forming ability. An analysis of the survival of simulated conditions of the gastrointestinal tract showed that 94.4% - 98.8% ± 1.3% of tested enterococci survived gastric juice, 87.1% - 97.9% ± 2.9% survived duodenum juice and 82.7 % - 92.9% ± 3.0% survived intestinal juice. In order to achieve their probiotic effect, enterococci must bind to mucin, extracellular matrix proteins or host cells. 71.4% ± 3.1% of tested enterococci bind to mucin on average, 74.1% ± 4.1% bind to collagen and 73.9% ± 2.7% bind to fibronectin. Additionally, 86.7% ± 5.4% of enterococci adhere to HT29-MTX cell line, in average. En. faecium BGZLM1-5 and BGPAS1-3 strains showed antimicrobial effect on L. monocytogenes ATCC19111. Listeria monocytogenes, the common foodborne pathogenic bacteria, compromise the intestinal epithelial barrier, which leads to development of the listeriosis, very serious disease especially among immunocompromised individuals. Due to that, we analysed the potential of BGPAS1-3, enterococci with strong direct antilisterial effect, to modulate the response of differentiated intestinal epithelial Caco- 2 cells, in L. monocytogenes ATCC19111 infection. Considering the controversial status of enterococci, we tested the antilisterial potential of heat-killed BGPAS1-3, which could be used as potential postbiotic. It was shown that the antilisterial molecule is rotein, tied to bacterial cell wall and partially resistant to the high-temperature treatment. Additionally, heat-killed BGPAS1-3 could prevent tight junction disruption in differentiated Caco-2 monolayer infected with L. monocytogenes ATCC19111, simultaneously allowing the induction of immune mechanisms important for antilisterial host response and stimulate the expression of TGF-β in intestinal epithelial cells. Finally, we showed that the modulation of MyD88 by BGPAS1-3 was dependent on TLR2 and TLR4 pathways. In addition, we showed that live and heat-killed BGPAS1-3, in the yoghurt production model, reduced the number of L. monocytogenes ATCC19111 during the first seven days of storage for slightly less than 1 log/ml. Taking into account that the intestine is an important site for modulation of the immune system, the next goal was to investigate the effect of the heat-killed BGPAS1-3 cells, BGPAS1-3 peptidoglycan and BGPAS1-3 peptidoglycan without teichoic acid on mesenteric lymph node (MLN) cells stimulated by concanavalin A (ConA) in the in vitro model. It has been shown that the investigated fractions weren’t cytotoxic in the tested ratio and did not change the metabolic activity. The production of IFN-γ and IL-17, pro-inflammatory cytokines, remained unchanged compared to untreated ConA-stimulated MLN cells, while IL-10 production was increased. Taking into account the previous results obtained in in vitro models that indicate the potential immunosuppressive and protective effect of BGPAS1-3, the effect of the heat-killed cells in in vivo model of inflammatory disease was examined. Experimental autoimmune encephalomyelitis was chosen as model for testing the effect of heat-killed BGPAS1-3. The results showed that the application of the heat-killed BGPAS1-3 (109 cells per rat per day) during 30 days of treatment affects the clinical course of the disease alleviating the symptoms in the initial stages of the disease. Since it has been shown that immunomodulatory therapies can lead to changes in the composition of the microbiota, the effect of heat-killed BGPAS1-3 cells on the microbiota of immunized female DA rats was examined by DGGE. A total of 14 unique DNA fragments were cloned and sequenced. Sequence analysis revealed that Lactobacillus intestinalis was present only at the beginning of the treatment (healthy animals) without detection in any other experimental group of animals. It is interesting that Lb. murinus and En. hirae were present only in faeces of immunized animals that were treated for 30 days with heat-killed BGPAS1-3, but were not present in the control group. Also, it has been shown that Shuttleworthia satelles and Muribaculum intestines appeared only on the 30th day in faeces of control immunized animals that were not treated with heat-killed BGPAS1-3. Using Bifidobacteria-specific primers the presence of Bifidobacterium pseudolongum was showed in the faeces of control and treated animals 30 days after immunization. The results of this doctoral dissertation showed that enterococcal strains isolated from milk and fermented dairy products have low pathogenic potential and good probiotic potential. It has been shown that the in vitro model of the live and heat-killed Enterococcus faecium BGPAS1-3 exhibits an antilisterial effect, simultaneously allowing the development of an effective immune host response against Listeria monocytogenes and stimulates the production of molecules that contribute to the protection of the intestinal epithelial barrier. Furthermore, heat-killed BGPAS1-3 exhibits a therapeutic effect in vivo on the model of experimental autoimmune encephalomyelitis.

Efekti estradiola na regulaciju natrijum-kalijumove pumpe i morfologiju srca normalno uhranjenih i gojaznih pacova

The effects of estradiol on the regulation of sodium-potassium pump and heart morphology in normally fed and obese rat

Hormon estradiol, pored svojih efekata koje ostvaruje na reprodukciju i razviće, uključen je i u regulaciju metaboličkih procesa u različitim organima, uključujući i srce. Pored sistemskog efekta koje estradiol ostvaruje na kardiovaskularni sistem zapažen je, ali i nedovoljno razjašnjen, uticaj estradiola na srce i kardiomiocite. U literaturnim podacima koji se odnose na efekte estradiola na morfologiju srca uglavnom su izučavani efekti estradiola u patofiziološkim stanjima hipertenzije i traume, dok je uloga estradiola na morfologiju srca u patofiziološkim stanjima gojaznost i rezistencije na insulin (IR) još uvek nedovoljno izučena. Estradiol svoje efekte ostvaruje genomskim i negenomskim signalnim putevima posredstvom specifičnih receptora za estrogen (ER), pri čemu dolazi do aktivacije složene mreže međuzavisnih intracelularnih signalnih puteva uključujući i fosfatidilinozitol-3 kinazu (PI3K), protein kinazu B (Akt), kao i ekstracelularnim signalima regulisane kinaze 1 i 2 (ERK1/2). Natrijum-kalijum adenozin trifosfataza (Na+/K+-ATPaze) je membranski protein koji ima ulogu u održavanju elektrohemijskog gradijenta Na+ i K+ jona kroz ćelijsku membranu, a takođe ima i važnu ulogu u regulaciji tonusa glatkih mišića vaskulature, kao i regulaciji kontrakcija srca. Poremećaji u funkciji Na+/K+-ATPaze uočeni su u različitim patofiziološkim stanjima kao što su stanja hipertenzije, gojaznosti i IR. Signalni putevi koji su uključeni u estradiolom regulisanoj ekspresiji i aktivnosti Na+/K+-ATPaze uključuju i aktivaciju Akt i ERK1/2 kinaza. Stimulišući aktivnost Na+/K+-ATPaze, estradiol ostvaruje direktan uticaj na srce povećavajući kontraktilnost ćelija srca. Nedostatak podataka u literaturi o in vivo efektima estradiola na regulaciju Na+/K+-ATPaze i morfologiju srca, kao i mehanizmi kojima estradiol ostvaruje svoje efekte na ćelije srca u patofiziološkim stanjima (gojaznost i IR), ukazuju na značaj realizovanih istraživanja u okviru ove doktorske disertacije. U ovoj doktorskoj disertaciji izučavani su molekularni mehanizmi in vivo efekata estradiola na regulaciju Na+/K+-ATPaze i morfologiju srca, u koje su uključene Akt i ERK1/2 kinaze, kako u fiziološkim tako i u patofiziološkim uslovima kao što su stanja gojaznosti i IR. U eksperimentima su korišćeni adultni pacovi Wistar soja. Dve grupe pacova za ishranu su koristile standardnu laboratorijsku hranu za pacove (normalno uhranjeni pacovi), dok su druge dve grupe hranjene standardnom laboratorijskom hranom obogaćenom sa 42% masti (gojazni pacovi). Nakon 10 nedelja grupi normalno uhranjenih (označene kao grupa: Estradiol) i gojaznih pacova (označena kao grupa: Gojazni+Estradiol), davana je jedna doza estradiola (40µg/kg), dok je ostalim pacovima (Kontrolni i Gojazni pacovi) davana ista količina 1% etanola u fiziološkom rastvoru. Dvadeset četri sata nakon tretmana pacovi iz svih grupa su žrtvovani, izolovani su serumi i plazme, a srca su ekstrahovana. U serumu pacova određivane su koncentracije: glukoze (Glu), insulina (INS), C reaktivnog proteina (CRP), leptina, Na+, K+, ukupnog holesterola (HOL) i triglicerida (Tg), dok su u plazmi određivane koncentracije digoksinu sličnih imunoreaktivnih faktora (DLIF), slobodnih masnih kiselina (SMK) i fosfolipida. Aktivnost Na+/K+-ATPaze je određivana u frakciji plazma membrana izolovanih iz srca pacova. Korišćenjem Western blot metode određivan je nivo ekspresije i fosforilacije α1 subjedinice Na+/K+-ATPaze u frakciji plazma membrana, dok je nivo fosforilacije i ekspresije Akt i ERK1/2 kinaza određivan u lizatima ćelija. Imunohistohemijska analiza je korišćena za određivanje lokalizacije Na+/K+-ATPaze u tkivu srca pomoću antitela specifičnog na α1 subjedinicu Na+/K+-ATPaze. Histohemijske i morfometrijske analize tkiva srca su korišćene za određivanje in vivo efekata estradiola i ishrane bogate mastima na morfologiju srca. Estradiol davan normalno uhranjenim pacovima dovodi do statistički značajnog povećanja aktivnosti Na+/K+-ATPaze, ekspresije i fosforilacije α1 subjedinice Na+/K+- ATPaze na plazma membranama kardiomiocita, fosforilacije Akt i ERK1/2 kinaza, koncentracije ukupnih proteina u lizatima srca, kao i statistički značajnog smanjenja koncentracije DLIF u plazmi i koncentracije Na+ u serumu u poređenju sa netretiranim normalno uhranjenim pacovima. Ishrana bogata mastima dovodi do statistički značajnog povećanja telesne mase, mase srca, koncentracije ukupnih proteina u lizatima srca, transverzalnih dijametara kardiomiocita, koncentracije Glu, INS, leptina, CRP, HOL i Tg u serumu, koncentracije SMK i fosfolipida u plazmi, i fosforilacije ERK1/2 kinaza. Takođe, kod gojaznih pacova je uočeno statistički značajno smanjenje aktivnosti Na+/K+-ATPaze i ekspresije α1 subjedinice Na+/K+-ATPaze, aktivnosti Akt kinaze, koncentracije DLIF u plazmi i Na+ u serumu u poređenju sa normalno uhranjenim pacovima. Estradiol davan gojaznim pacovima dovodi do statistički značajnog povećanja koncentracije Na+ u serumu, aktivnosti Na+/K+-ATPaze i ekspresije α1 subjedinice Na+/K+-ATPaze, kao povećanja fosforilacije Akt kinaze. Kod gojaznih pacova tretiranih estradiolom, uočeno je statistički značajno smanjenje mase srca, odnosa mase srca/mase tela, transverzalnih dijametara kardiomiocita, koncentracije serumskog CRP i HOL. Rezultati prikazani u ovoj doktorskoj disertaciji ukazuju da estradiol u in vivo uslovima kod normalno uhranjenih pacova reguliše aktivnost i ekspresiju Na+/K+-ATPaze u srcu, molekularnim mehanizmom koji uključuje Akt i ERK1/2 kinaze. Međutim, kod gojaznih pacova kod kojih su aktivnost i ekspresija Na+/K+-ATPaze smanjene, estradiol stimuliše aktivnost i ekspresiju Na+/K+-ATPaze mehanizmom koji uključuje učešće Akt kinaze. Pored uticaja na Na+/K+-ATPazu, estradiol kod gojaznih pacova utiče i na morfologiju ćelija srca jer dovodi do smanjenja gojaznošću−indukovane hipertrofije srca. Interesantno je da dobijeni rezultati ukazuju na povezanost promena u regulaciji Na+/K+- ATPaze sa promenama u morfologiji ćelija srca normalno uhranjenih i gojaznih pacova tretiranih estradiolom. Izučavanja usmerena u pravcu rasvetljenja molekularnih mehanizma kojima estradiol ostvaruje svoje efekte na regulaciju Na+/K+-ATPaze i morfologiju srca u fiziološkim i patofiziološkim stanjima kao što je stanje gojaznosti združeno sa rezistencijom na insulin su od izuzetne važnosti kako za razumevanje uloge estradiola u kardiovaskularnim oboljenjima, tako i u cilju razvijanja novih terapeutskih strategija za tretman kardiovaskularnih oboljenja.

Hormone estradiol is also involved in the regulation of metabolic processes in various organs, including the heart. The systemic effects of estradiol on cardiovascular system, was observed, but the effect of estradiol on the heart still is not known in detail. The effects of estradiol on the morphology of the heart are mainly studied the effects of estradiol in pathophysiological conditions of hypertension and trauma, whereas the role of estradiol on the morphology of the heart in pathophysiological conditions such are obesity and insulin resistance (IR) is still insufficiently explored. Estradiol exert its effect by nongenomic and genomic signaling pathways, through specific receptors for estrogen (ER), via the activation of a complex network of dependent intracellular signaling pathways including phosphatidylinositol 3 - kinase (PI3K), protein kinase B (Akt) and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Sodium-potassium adenosine triphosphatase (Na+/K+-ATPase ) is a membrane protein that plays a role in maintaining the electrochemical gradient of Na+ and K+ ions across the cell membrane, and also plays an important role in the regulation of muscle tone of smooth muscle vasculature , as well as in the regulation of heart contraction. Disturbances in the Na+/K+-ATPase function have been observed in various pathophysiological conditions such as hypertension, obesity and IR. Estradiol regulates Na+/K+-ATPase activity and expression via the activation of Akt and ERK1/2 signaling pathways. Stimulating the activity of the Na+/K+-ATPase, estradiol has direct effect on the heart by increasing the contractility of cardiac cells. The lack of literature data about in vivo effects of estradiol on the Na+/K+-ATPase regulation, the heart morphology, and the mechanisms by which estradiol achieves its effects on heart cells in pathophysiological conditions (obesity and IR) stresses the importance of the research within the framework of this doctoral dissertation. In this doctoral thesis, the molecular mechanisms of in vivo effects of estradiol on the regulation of Na+/K+-ATPase and the morphology of the heart, via Akt and ERK1/2 kinase, both in physiological and in pathophysiological conditions such as obesity and IR, were studied and for these experiments adult Wistar rats were used. Two groups of rats were fed with standard laboratory rats’ foods (normally fed), while the other two groups were used standard laboratory food enriched with 42% fat (obese rats). After 10 weeks normally fed rats (marked as a group: Estradiol) and obese rats (marked as group: Obese + Estradiol), were treated with a single dose of estradiol (40μg/kg), while the other rats (marked as groups: Control and Obese) were given the same amount of 1% ethanol in saline. Twenty four hours after treatment, rats from each group were sacrificed, serum and plasma were isolated, and the hearts were extracted. Serum glucose, insulin, C-reactive protein (CRP), leptin, Na+, K+, total cholesterol (HOL) and triglyceride (Tg), and plasma digoxin like immunoreactive factor(s) (DLIF), free fatty acids (FFA), phospholipids were measured. Activity of Na+/K+-ATPase in plasma membrane fractions was determined using a Na+/K+-ATPase assay. Western blot analysis was used to monitor phosphorylation and expression of α1 subunit of Na+/K+-ATPase in plasma membrane fraction, and Akt and ERK1/2 in rat heart lysates. Immunohistochemical analysis was performed for detection of the Na+/K+-ATPase localisation in heart tissue. Histochemical and morphometric analysis of heart tissue were used to determine the effect of estradiol and high-fat diet on the morphology of the heart. Following administration of estradiol to normally fed rats in comparison with untreated rats, leads to a statistically significant increase Na+/K+-ATPase activity, expression and phosphorylation of α1 subunit of Na+/K+-ATPase from rat hearts, Akt and ERK1/2 kinase phosphorylation, total protein concentrations in lysates of heart, as well as a significant decrease in plasma concentration DLIF and Na+ concentrations in the serum. The high fat diet in rats leads to a statistically significant increase in body weight, heart weight, total protein concentrations in lysates of heart, the transverse diameters of the cardiomyocytes, the concentration of Glu, INS, leptin, CRP, HOL and Tg in serum, and concentrations of FFA and phospholipids in plasma, and the phosphorylation of ERK1/2 kinase. In obese rats was also observed a statistically significant decrease in Na+/K+- ATPase activity and expression of α1 subunit of Na+/K+-ATPase, activation of Akt kinase, concentration of DLIF in serum and Na+ in plasma compared to normal fed rats. Estradiol in vivo in obese rats in comparison with obese non-tretated rats leads to a statistically significant increase of Na+ concentration in serum, Na+/K+-ATPase activity and expression of α1 subunit of Na+/K+-ATPase, as Akt kinase activation. The obese rats treated with estradiol had a statistically significant reduction in heart weight, ratio of the heart weight / body weight, transverse diameters of cardiomyocytes, concentration of CRP and HOL in serum. The results presented in this doctoral thesis suggest that estradiol administrated in vivo under normal physiological conditions regulate the activity and expression of cardiac Na+/K+-ATPase activity, by molecular mechanisms that involve the participation of Akt and ERK1/2 activation. Also, obtained results show that estradiol administrated in vivo in obese/IR rats (with reduction of the Na+/K+-ATPase activity and expression), stimulates activity and expression of Na+/K+-ATPase by mechanism that involves the participation of Akt kinase. Besides the influence of the Na+/K+-ATPase, estradiol in states of obesity and IR, in vivo affects the morphology of the cardiac cells and its actions leads to a reduction in obesity- induced cardiac hypertrophy. In addition, the results indicated that there is an association between changes in the regulation of Na+/K+-ATPase with changes in the morphology of cardiac cells under the influence of estradiol in normal and in states of obesity and IR. Despite the importance of regulation of cardiac Na+/K+-ATPase under physiological and pathophysiological conditions, the molecular mechanisms of its regulation under the influence of estradiol remain unknown. Studies directed toward illuminating the molecular mechanism(s) by which estradiol achieves its effects on the regulation of cardiac Na+/K+-ATPase are of great importance for understanding its role in cardiovascular disease and for the development of new therapeutic strategies for the treatment of cardiovascular diseases.

Analiza promotorskih varijanti gena TGFB1 kao faktora rizika i modulatora odgovora na terapiju astme

Analysis of the TGFB1 gene promoter variants as risk factors and modulators of response to asthma therapy

Transformišući faktor rasta beta 1 (TGFB1) je važan inflamatorni modulator i profibrotički medijator u astmi. Promene u regulaciji ekspresije TGFB1 bi mogle biti povezane sa patogenezom astme i sa adekvatnim odgovorom na terapiju. Cilj ovog rada je bio ispitivanje promotorskih varijanti gena TGFB1 kao faktora rizika za nastanak astme i modulatora odgovora na terapiju. Studija je uključila pacijente sa astmom i zdrave ispitanike kod kojih je analiziran 5' regulatorni region gena TGFB1. Detektovane su promotorske varijante -509C>T (rs1800469) i -800G>A (rs1800468) za koje asocijativnom studijom nije pokazana povezanost sa patogenezom astme, ali je uočena potencijalna povezanost između promotorske varijante -509C>T i egzacerbacija bolesti. Uticaj ove promotorske varijante na ekspresiju gena TGFB1 pod dejstvom montelukasta, leka za astmu, je ispitivan ex vivo u ćelijama indukovanog sputuma i in vitro u ćelijama BEAS 2B. Analizom ex vivo je pokazana smanjena ekspresija gena TGFB1, pri čemu je efekat bio izraženiji kod astmatičara sa genotipom -509TT (58,9%) u odnosu na astmatičare sa genotipovima - 509CC (49,6%) i -509CT (31,8%). U ćelijama BEAS 2B je ekspresija gena bila smanjena za 27%. Nakon transfekcije ćelija BEAS 2B konstruktima sa promotorskim varijantama - 509C ili -509T i tretmana montelukastom, zapažena je povećana aktivnost promotora gena TGFB1. Odgovor varijante -509T je bio intenzivniji i za 139,9% veći u odnosu na odgovor varijante -509C. Ova studija je pokazala da promotorska varijanta -509C>T menja ekspresiju gena TGFB1 u odgovoru na montelukast, ali da bi se njena modulatorna uloga razjasnila, u daljim istraživanjima bi trebalo analizirati i druge genetičke i negenetičke faktore.

Transforming growth factor beta 1 (TGFB1) is an important inflammatory modulator and profibrotic mediator in asthma. The changes in regulation of TGFB1 expression could be associated with asthma pathogenesis and adequate response to therapy. The aim of this study was to investigate TGFB1 gene promoter variants as asthma risk factors and modulators of response to asthma therapy. The 5' regulatory region of the TGFB1 gene was analyzed in asthmatic patients and healthy individuals included in this study. The promoter variants -509C>T (rs1800469) and - 800G>A (rs1800468) were detected and the association between these variants and asthma pathogenesis was not found. The association was observed between the presence of variant -509C>T and occurence of exacerbations in patients. The influence of -509C>T variant on TGFB1 gene expression in response to montelukast, used for asthma treatment, was further investigated ex vivo in induced sputum cells and in vitro in BEAS 2B cells. Analysis ex vivo has shown the decrease in the TGFB1 gene expression, with more pronaunced effect in asthmatics with genotype -509TT (58.9%) in comparison with -509CC (49.6%) and - 509CT (31.8%) genotypes. In BEAS 2B cells the gene expression was reduced for 27%. After transfection of BEAS 2B cells with constructs carrying promoter variants -509C or - 509T and treatment with montelukast the increased activity was observed for both promoter variants. The response of variant -509T was more intensive and 139,3% higher than response of variant -509C. This study showed that promoter variant -509T alters TGFB1 gene expression in response to montelukast, but to clarify its modulatory role other genetic, and nongenetic factors as well, should be taken into consideration in further studies.

Dynamic analysis of soil-structure system using spectral element method

Динамичка анализа система тло-конструкција применом спектралних елемената.

3D numerical model for dynamic response analysis of multi-storey frame structures including soil-structure interaction has been developed using the Spectral Element Method (SEM). The structure was modeled using the SEM, while the dynamic stiffness matrix of the soil – foundation interface was determined using the Integral Transform Method (ITM). The structure consists of one – dimensional elements: beams and columns, and two – dimensional elements: plates. The soil consists of horizontal layers over the bedrock or half space. The Projection method was used to develop the dynamic stiffness matrices for the transverse and in-plane vibration of plates with arbitrary boundary conditions. The method for coupling one-dimensional and two- dimensional spectral elements was developed, as well as the coupling of the spectral elements with the soil spring – dashpot elements. Using the proposed numerical model 3D frame structures founded on soil of variable stiffness and subjected to ground vibrations induced by traffic were analyzed. Key words: dynamics of structures, spectral element method, integral transform method, soil-structure interaction, substructure approach, frequency domain, dynamic stiffness Scientific field: Civil engineering Specific scientific field: Theory of structures (Dynamic of structures) UDK: 624.04.004.92(043.3) DINAMIČKA ANALIZA SISTEMA TLO-KONSTRUKCIJA PRIMENOM SPEKTRALNIH ELEMENATA Rezime U ovom radu prikazan je 3D numerički model za dinamičku analizu višespratnih ramovskih konstrukcija uzimajući u obzir sadejstvo između tla i konstrukcije. Za modeliranje konstrukcije korišćen je Metod spektralnih elemenata, dok je dinamička matrica krutosti tla određena primenom Metode integralnih transformacija. Konstrukcija se sastoji od jednodimenzionalnih elemenata: greda i stubova, i dvodimenzionalnih elemenata: ploča. Tlo se sastoji od horizontalnih slojeva iznad krute baze ili poluprostora. Matrice krutosti ploča napregnutih na savijanje i u svojoj ravni određene su primenom Metode projekcije. Razvijen je postupak sprezanja jednodimenzionalnih i dvodimenzionalnih spektralnih elemenata, kao i sprezanje tako formiranog modela sa tlom. Primena formiranog 3D modela prikazana je na analizi 3D ramovskih konstrukcija fundiranih na tlu različite krutosti, izloženih dejstvu vibracija izazvanih saobraćajnim opterećenjem.

UNIVERSITY OF BELGRADE FACULTY OF CIVIL ENGINEERING Marija T. Nefovska-Danilović DYNAMIC ANALYSIS OF SOIL-STRUCTURE SYSTEM USING SPECTRAL ELEMENT METHOD Doctoral dissertation Belgrade, December 2012 УНИВЕРЗИТЕТ У БЕОГРАДУ ГРАЂЕВИНСКИ ФАКУЛТЕТ Марија Т. Нефовска-Даниловић ДИНАМИЧКА АНАЛИЗА СИСТЕМА ТЛО-КОНСТРУКЦИЈА ПРИМЕНОМ СПЕКТРАЛНИХ ЕЛЕМЕНАТА Докторска дисертација Београд, децембар 2012. Ментор: Др Мира Петронијевић, ванредни професор, Грађевински факултет, Универзитет у Београду Чланови Комисије: 1. __________________________________________ 2. __________________________________________ 3. __________________________________________ 4. __________________________________________ 5. __________________________________________ Датум одбране: DYNAMIC ANALYSIS OF SOIL-STRUCTURE SYSTEM USING SPECTRAL ELEMENT METHOD Abstract 3D numerical model for dynamic response analysis of multi-storey frame structures including soil-structure interaction has been developed using the Spectral Element Method (SEM). The structure was modeled using the SEM, while the dynamic stiffness matrix of the soil – foundation interface was determined using the Integral Transform Method (ITM). The structure consists of one – dimensional elements: beams and columns, and two – dimensional elements: plates. The soil consists of horizontal layers over the bedrock or half space. The Projection method was used to develop the dynamic stiffness matrices for the transverse and in-plane vibration of plates with arbitrary boundary conditions. The method for coupling one-dimensional and two- dimensional spectral elements was developed, as well as the coupling of the spectral elements with the soil spring – dashpot elements. Using the proposed numerical model 3D frame structures founded on soil of variable stiffness and subjected to ground vibrations induced by traffic were analyzed.

Funkcionalno-morfološke i etološke karakteristike šumske kornjače (Testudo hermanni)

Characteristics of functional morphology and ethology of Hermann's tortoises (Testudo hermanni)

Specifičan plan građe (čvrst koštani oklop i kratki ekstremiteti) kopnenim kornjačama postavlja znatna ograničenja u kretanju i prevazilaženju prepreka u staništu. Stoga „kompenzacija ponašanjem“ igra veoma značajnu ulogu u kretanju kopnenih kornjača. Da bi se testirale unutar- i međupopulacione razlike u agilnosti i ponašanju šumske kornjače (Testudo hermanni), vršena su tri eksperimenta. U prvom eksperimentu kornjače su postavljane na leđa i praćeno je ponašanje tokom prevrtanja u uspravan položaj. Tokom drugog eksperimenta, testa skakanja, kornjače su postavljane na „negativnu visinsku prepreku“ (horizontalnu površinu na izvesnoj visini iznad podloge) i praćeno je ponašanje tokom silaženja sa nje. Trećim eksperimentom je testirano ponašanje kornjača kada se nađu „zaglavljene“ u vegetaciji. Ovom prilikom je na prednji deo plastrona zakačena omča užeta na čijem drugom kraju se nalazio dinamometar. Praćeno je ponašanje za vreme oslobađanja od eksperimentalne aparature i merena je mišićna snaga kornjača. Parametri ponašanja dobijeni ovim testovima su analizirani u odnosu na: morfološke odlike (dužinske mere oklopa i nogu, kao i oblik i veličinu karapaksa), pol, uzrasne kategorije jedinki i odlike staništa. Pokazano je da veličina i oblik tela značajno utiču na prevrtanje kornjača. Krupnije kornjače se teže prevrću nego sitnije, pri čemu visina oklopa ima pozitivan, a dužina koštanih mostova negativan efekat na uspešnost uspravljanja. Međutim, primenom geometrijske morfometrije pokazano je da promene oblika oklopa usled povećanja veličine adultnih kornjača, među kojima je i visina oklopa, ipak nisu pozitivno asocirane sa prevrtanjem. Razlike u ponašanju i agilnosti između jedinki različitog pola su značajne samo u smislu prevrtanja, i to na polovini testiranih lokaliteta. Iako je ranije pretpostavljano da su mužjaci znatno agilniji od ženki, rezultati ovih eksperimentata navode na pretpostavku da uticaj pola nije toliko izražen koliko se ranije mislilo. Uticaj starosnih kategorija na ponašanje i agilnost je analiziran samo u testu skakanja. Adultne jedinke su bile efikasnije u ovom zadatku, što ukazuje na veliki značaj iskustva, odnosno učenja u savladavanju prepreka. U sva tri testa utvrđene su značajne razlike u ponašanju kornjača u zavisnosti od lokaliteta. Rezultati do sada sprovedenih etoloških testova ukazuju na to da su kopnene kornjače ponašanjem adaptirane na uslove staništa (topografiju terena i vegetaciju). Ovo može, osim fundamentalnog, imati i konzervacioni značaj, u smislu planiranja translokacija i uređenja privremenih prihvatilišta za kornjače. Ponašanje i funkcionalna morfologija kornjača su slabo proučene, a eksperimenti predstavljeni u ovoj tezi su prvi koji porede inter-populacione razlike u ponašanju i agilnosti kornjača u njihovim prirodnim i veštačkim staništima.

Unique body plan of terrestrial chelonians (rigid shell and short limbs) imposes significant limitations to movements of these animals. Thus “behavioural compensation” plays an important role in overcoming obstacles in their habitat, on the daily basis. To test intra- and inter-population differences in agility and behaviour of Hermann’s tortoises (Testudo hermanni) during overcoming obstacles in their habitat, three experiments were performed. In the first experiment tortoises were positioned on their backs and their behaviour was noted during overturning. For the second experiment, “jumping” test, tortoises were placed on „negative height obstacle“ (i.e. horizontal surface above substrate) and their behaviour was noted during getting off it. The third experiment tested behaviour of tortoises when they are “stuck” in dense vegetation. For this test, a non-stretchable rope was attached on the front, protuberant part of the plastron, while the dynamometer was attached on the other end of the rope. Behaviour was noted during the animals’ attempts to liberate from the experimental apparatus, and their muscular strength was measured. Behavioural parameters gathered during these tests were analysed with regard to: morphological traits (length measurements of shell and legs, and carapace shape and size), sex, age categories and habitat features. Body size and shape had significant effects on righting behaviour. Larger tortoises were less efficient in overturning than the smaller ones. Shell height showed positive, while length of bony bridges showed negative effect on the overturning efficiency. However, geometric morphometrics showed that allometric shape changes in adult tortoises (e.g. increase in relative carapace height) are not positively associated with overturning. Differences between sexes in behaviour and agility were significant only for the righting test, in which males were more successful, in half of the tested localities. Males were previously considered more agile than females, but these experiments propose that effect of sex on agility of tortoises is not as significant. Effect of age classes is analysed only in the “jumping” test. Adult tortoises were more successful in this task, which implies the importance of experience, i.e. learning. In all three tests locality had significant effect on agility performances. Results imply that tortoises are behaviourally adapted to habitat characteristics, such as topography and vegetation. These findings could have value in conservational biology, e.g. for planning of translocations and arranging of temporary shelters for tortoises. Behaviour and functional morphology of tortoises are poorly studied. Experiments presented in this thesis are the first which compare behaviour and agility among tortoise populations in their natural and artificial habitats.

Varijante promotora gena za uridin-difosfat-glukuronoziltransferazu 1A1 kao modulatori biohemijskog fenotipa i populaciono farmakogenetički markeri

Promoter variants of the gene for uridine-diphosphate-glucuronosyltransferase 1A1 as modulators of biochemical phenotype and population pharmacogenetic markers.

Uvod Familija enzima uridin-difosfat-glukuronoziltransferaza (UGT) katalizuje glukuronidaciju širokog spektra endobiotika i ksenobiotika. UGT1 familija je kodirana genskim kompleksom UGT1, koji se sastoji od 13 proksimalnih varijabilnih egzona i od 4 zajednička distalna egzona. Alternativnom obradom nastaje više transkripata, među kojima je posebno značajan onaj čiji je produkt UGT1A1 enzim, koji učestvuje u metabolizmu bilirubina i mnogih lekova. Više od 60 varijanti je detektovano u UGT1A1 genu, koje su povezane sa patološkim stanjima. Varijante u TATA boksu promotora UGT1A gena se razlikuje po broju TA ponovaka. Wild type UGT1A1*1 sadrži 6 TA ponovaka (TA6), dok su varijante koje sadrže 5, 7 ili 8 TA ponovaka označene kao UGT1A1*36 (TA5), UGT1A1*28 (TA7) i UGT1A1*37 (TA8). Transkripciona aktivnost promotora je manja što je veći broj ponovaka u odnosu na TA6. Žilberov sindrom (ŽS) je često kliničko stanje koje karakteriše hiperbilirubinemija i ikterus. Uzrokovan je smanjenom aktivnošću UGT1A1 enzima. Najčešće detektovana varijanta koja se nalazi u osnovi ŽS je UGT1A1*28. Njena učestalost varira u različitim populacijama, a kod belaca je prisutna sa 26% do 31%. ŽS je blago oboljenje, ali kao komorbiditet može dovesti do pogrešne dijagnoze bolesti jetre ili hemolitičkih procesa. U tim slučajevima je neophodno potvrditi ili isključiti dijagnozu ŽS molekularnom analizom UGT1A1 gena. Malo se zna o uticaju varijanti promotora UGT1A1 gena na scenario kod oboljenja jetre. Samo u nekoliko slučajeva je kod pacijenata sa hepatitisom C sprovedeno genetičko testiranje na UGT1A1*28, i pošto je dokazana pozitivnost objasnila detektovan nivo bilirubina, pacijenti su lečeni po standardnom protokolu i postigli su kontinuiran virusološki odgovor. Kod hemolitičkih anemija, kao što je talasemija, takođe je pokazano da je nivo bilirubina povezan sa pojavom komplikacija. Stoga se varijante u UGT1A1 genu tretiraju kao tercijarni modifikatori kod beta- talasemijskih sindroma. Pokazano je da je UGT1A1*28 alel farmakogenetički marker za brojne farmaceutike kao što su antineoplastični lekovi koji se koriste u onkologiji (irinotekan, belinostat, epirubicin, 5-fluorouracil, aksitinib i bisulfan), zatim atazanavir i ritonavir u lečenju HIV infekcije, kao i analgetik/antipiretik acetaminofen koji je u širokoj upotrebi. Postoje podaci o učestalosti ove varijante u više populacija. Nijedna studija nije izučavala učestalost UGT1A1*28 varijante u populaciji Republike Srpske i Republike Srbije. Populaciono-farmakogenetičke studije imaju klinički i ekonomski značaj. One mogu da dovedu do preporuka za uvođenje prediktivnih farmakogenetičkih testova pre uvođenja u terapiju određenih lekova, što dovodi do individualizacije terapije pacijenata i uštede u zdravstvenom sistemu jedne zemlje. Ciljevi, materijali i metode Ciljevi ove studije su bili da se utvrdi učestalost TA varijanti u promotoru UGT1A1 gena i korelacije nivoa nekonjugovanog bilirubina sa promotorskim TA genotipom UGT1A1 kod pacijenata sa ŽS, hroničnim hepatitisom C i β-talasemijom minor, da se odredi učestalost varijantnog promotorskog TA alela UGT1A1 gena u populaciji Republike Srbije i Republike Srpske, i da se proceni značaj UGT1A1*28 alela kao farmakogenetičkog markera u ispitivanim populacijama. U ovoj studiji analizirano je: 51 uzorak pacijenata sa ŽS, 22 uzorka pacijenata sa dijagnozom β-talasemije minor, 24 uzorka pacijenata sa hroničnim hepatitisom C, uzorci 100 zdravih ispitanika iz opšte populacije Republike Srbije i uzorci 121 zdravih ispitanika iz opšte populacije Republike Srpske. Za analizu TA varijanti u promotorskom regionu UGT1A1 gena primenjene su metode PCR, za umnožavanje promotorskog regiona UGT1A1 gena, a metode fragment analize i metode sekvenciranja po Sangeru, za potvrdu rezultata dobijenih reakcijom PCR.Kod 10 pacijenata kod kojih nije utvrđen rizičan genotip za razvoj ŽS (UGT1A1 7/7 TA i 7/8 TA), a imali su povišene nivoe nekonjugovanog bilirubina, izvršeno je sekvenciranje 4 stalna egzona na 3’ kraju UGT1A1 gena uključujući i intron-egzon granice. Takođe, sekvenciran je i prvi varijabilni egzon UGT1A1 gena. Rezultati su obrađeni statističkim programima. Rezultati, diskusija i zaključci U ovoj studiji detektovne su 4 vrste genotipa: UGT1A1 6/6 TA, 6/7 TA, 7/7 TA i 7/8 TA. U grupi pacijenata sa ŽS učestalost rizičnih genotipova (UGT1A1 7/7 i 7/8 TA) bila je 80%, a učestalost UGT1A1*28 alela u ovoj grupi pacijenata je 86,27%. Nivo nekonjugovanog bilirubina pri dijagnozi je bio statistički značajno veći u grupi pacijenata sa ŽS koji su imali rizični genotip (UGT1A1 7/7 i 7/8 TA) u odnosu na grupu pacijenata sa nerizičnim genotipom (UGT1A1 6/6 i 6/7 TA). Rezultati sekvenciranja kodirajućeg regiona UGT1A1 gena kod 10 pacijenata kod kojih je ŽS bio dijagnostikovan fenobarbitonskim i hipokalorijskim testom, ali kod kojih nije postojala asocijacija rizičnih promotorskih varijanti sa fenotipom ŽS, su pokazali da su samo kod jednog od 10 pacijenata, pronađene varijante dva pojedinačna nukleotida: NM_000463.2 c.997-82T>C (intron 2, pozicija 602 od 683) i c.1084+12G>A (intron 3, pozicija12 od 283). Obe varijante nisu do sada prijavljene dbSNPs. Takođe, kod jednog pacijenta koji je klinički bio diferenciran kao ŽS, a genotipizacijom nije potvrđeno prisustvo ŽS, daljom dijagnostikom je utvrđeno da jenosilac β-talasemije minor tipa. Na osnovu dobijenih rezultata ustanovljeno je da pouzdanost UGT1A1(TA)n genotipa kao dijagnostičkog markera za ŽS iznosi 80%.U grupi pacijenata sa HHC učestalost UGT1A1*28 alela je bila43,75%, što je nešto više, u odnosu na kontrolnu grupu od 40%. Nivo nekonjugovanog bilirubina kod pacijenata sa HHC je u korelaciji sa UGT1A1 genotipom i viši je kod pacijenata koji su nosioci UGT1A1*28 alela. Kod pacijenata sa β-talasemijom minor kod kojih je prisutna hemoliza, nivo nekonjugovanog bilirubina je u direktnoj korelaciji sa genotipom UGT1A1(TA)n, te je najviši kod homozigotnih nosilaca UGT1A1*28 alela. Na osnovu ovih nalaza,UGT1A1TA promotorska varijanta može da se smatra tercijernim genom modifikatorom kod β-talasemije minor. Populacione studije za populaciju Republike Srbije su pokazale da se genotipovi UGT1A1 6/6TA, 6/7TA i 7/7 TA javljaju sa učestalošću od 37%, 47% i 16% respektivno, a učestalost UGT1A1*28 alela u opštoj populaciji je 0,40. U populaciji Republike Srpske genotipovi UGT1A1 6/6TA, 6/7TA i 7/7 TA javljaju sa učestalošću od 37,19%, 37,19% i 25,60% respektivno, a učestalost UGT1A1*28 alela u opštoj populaciji je 0,44. Obzirom na izuzetno visoku učestalost UGT1A1*28 alela u opštoj populaciji Republike Srbije i Republike Srpske i na njegovu ulogu i značaj u metabolizmu brojnih lekova, došlo se do zaključka da je UGT1A1*28 alel značajan farmakogenetički marker za ove dve populacije, te se preporučuje analiza UGT1A1 (TA)n genotipa za u kliničkoj praksi pre uvođenja u terapiju lekova u čijem metabolizmu učestvuje UGT1A1 enzim.

Introduction The uridine-diphosphate-glucuronosyltransferase enzyme family (UGT) catalyse the glucuronidation of a wide range of endobiotics and xenobiotics. The UGT1 family is encoded by the UGT1 gene complex consisting of 13 proximal variable exons and 4 common distal exons. Alternative splicing gives multiple transcripts, among which the UGT1A1 enzyme product, which is involved in the metabolism of bilirubin and many drugs, is particularly important. More than 60 variantshave been detected in the UGT1A1 gene, which are associated with pathological conditions. Variants in the TATA box of the UGT1A gene promoter differ in the number of TA repeats. Wild type UGT1A1 contains 6 TA repeats (TA6), while variants with 5, 7 or 8 TA repeats are designated UGT 1A1*36 (TA5), UGT1A1*28 (TA7) and UGT1A1*37 (TA8). The transcription activity of the promoter decreased accordingly to higher number of repeats compared to TA6. Gilbert's syndrome (GS) is often a clinical condition characterized by hyperbilirubinaemia and icterus. It is caused by a reduced activity of UGT1A1 enzymes. The most commonly detected variant found in the basis of GS is UGT1A1*28. Its frequency varies in different populations, and whites are present from 26% to 31%. GS is a mild illness, but as a comorbidity it can lead to a wrong diagnosis of liver disease or hemolytic processes. In these cases, it is necessary to confirm or exclude the diagnosis of GS by molecular analysis of the UGT1A1 gene. The effect of variants of UGT1A1 gene on the development in liver disease is poorly known. In only a few cases, genetic testing for UGT1A1*28 was performed in patients with hepatitis C, and since the confirmed positivity explained the detected level of bilirubin, patients were treated by standard protocol and achieved a continious viral response. In hemolytic anemia, such as thalassemia, it has also been shown that the level of bilirubin is associated with the numerous of complications. Therefore, the TA promoter variants in the UGT1A1 gene are treated as tertiary modifiers in beta-talasemia syndromes. UGT1A1*28 allele has been shown to be a pharmacogenetic marker for numerous pharmaceuticals such as antineoplastic drugs used in oncology (irinotecan, belinostat, epirubicin, 5-fluorouracil, axitinib and bisulfan), then atazanavir and ritonavir in the treatment of HIV infection, and analgesic /antipyretic acetaminophen which is widely used. There are data about the frequency of this variant in multiple populations. There are no studies about the frequency of UGT1A1*28 variants in the population of the Republic Serbia and the Republic of Srpska. Population-pharmacogenetic studies have a clinical and economic significance. They can lead to recommendations for the introduction of predictive pharmacogenetic tests before the prescription of certain drugs, which can result in individualization of therapy and savings in the healthcare system. Aims, materials and methods. The aims of this study were to determine the frequency of promoter TA variants of UGT1A1 gene and the correlation of the level of unconjugated bilirubin with the UGT1A1(TA)n genotype in patients with GS, chronic hepatitis C and β- thalassemia minor. Then, to determine the frequency of the variant TA promoterUGT1A1 gene allele in the population of the Republic of Serbia and the Republic of Srpska. Finally, to evaluate the importance of UGT1A1*28 allele as a pharmacogenetic marker in the examined populations. In this study, 51 samples of patients with GS, 22 patients diagnosed with β- thalassemia minor, 24 patients with chronic hepatitis C, 100 healthy subjects from the general population of the Republic of Serbia and 121 healthy subjects from the general population of Republika of Srpska were analized. For the analysis of the TA variants in the UGT1A1 promoter region, PCR methodology were used for the propagation of the UGT1A1 gene promoter region, analytical fragment analysis method and Sanger sequencing method to confirm the results obtained by the PCR reaction. In 10 patients to whom did not detect a risky genotype for the development of GS (UGT1A1 7/7 TA and 7/8 TA) and which had elevated levels of unconjugated bilirubin, sequencing of 4 permanent exons at the 3'end of the UGT1A1 gene was performed, including the intron-exon regions. The first variable exon of UGT1A1 gene is also sequenced. Obtained results were processed by statistical programs. Results, discussion and conclusionIn this study, 4 types of genotypes were detected: UGT1A1 6/6 TA, 6/7 TA, 7/7 TA and 7/8 TA. In the group of patients with GS the incidence of risky genotypes (UGT1A1 7/7 TA and 7/8 TA) was 80%, and the incidence of UGT1A1*28 alleles in this group of patients was 86.27%. The level of unconjugated bilirubin at the diagnosis was statistically significantly higher in the group of patients with GS who had a risky genotype (UGT1A1 7/7 TA and 7/8 TA) compared to a group of patients with a non-risk genotype (UGT1A1 6/6 TA and 6/7 TA). The results of the sequencing of UGT1A1 gene in 10 patients who diagnosed as GS by phenobarbital and hypocaloric test, but whom not found association of risky promotor TA variants with GS phenotype, showed that only one of 10 patients had variants of two single nucleotides: NM_000463. 2 c.997-82T> C (intron 2, position 602 of 683) and c.1084 + 12G> A (intron 3, position 12 of 283). Both variants have not been reported as dbSNPs. Also, at one patient who was clinically differentiated as GS, but the diagnosis of GS was not confirmed by UGT1A1(TA)n genotyping, further diagnosis was determined presence of β-thalassemia minor type. On the basis of the obtained results, it was found that the reliability of the UGT1A1 genotype as a diagnostic marker for GS is 80%. In the group of patients with HHC, the UGT1A1*28 allele rate was 43,75%, compared to the frequency of the same allele in control group of 40%. The level of unconjugated bilirubin in patients with HHC is correlated with UGT1A1(TA)n genotype and is higher in patients carriers of UGT1A1*28 allele. In patients with β-thalassemia minor with hemolysis, the level of unconjugated bilirubin is in direct correlation with the genotype UGT1A1(TA)n, and it is the highest in homozygous carriers of UGT1A1*28 allele. Based on these findings, UGT1A1 (TA)n can be considered a gene modifier for β-thalassemia minor. Population studies for the population of the Republic of Serbia showed that genotypesUGT1A1 6/6TA, 6/7TA and 7/7 TA occur with a frequency of 37%, 47% and 16% respectively, and the frequency of UGT1A1* 28 allele in the general population is 0,40. In the population of the Republic of Srpska, genotypesUGT1A1 6/6TA, 6/7TA and 7/7 TA occur with a frequency of 37,19%, 37,19% and 25,60% respectively, and the frequency of UGT1A1*28 allele in the general population is 0,44. Considering the extremely high incidence of UGT1A1*28 alleles in the general population of Republic of Serbia and Republic of Srpska, and its vital role and importance in the metabolism of numerous drugs, it has been concluded that UGT1A1*28 allele is a significant pharmacogenetic marker in these two populations, and that UGT1A1 (TA)n genotype analysis is recommended to be performed in routine clinical practice before introducing into therapy the drugs in which metabolism UGT1A1 enzyme is involved.

Uloga hormona jajnika u involuciji timusa pacova

Role of ovarian hormones in thymic involution in rats

Timus je primarni limfoidni organ koji obezbeđuje mikrosredinu neophodnu za diferencijaciju/sazrevanje T-limfocita. Tokom ontogeneze, ovaj organ trpi značajne strukturne i funkcionalne promene koje se zbirno označavaju kao involucija timusa, i manifestuju se kao smanjena efikasnosti timopoeze i posledično, smanjen izlazak novoformiranih T-limfocita na periferiju. Involucija timusa je kod glodara najizraženija u periodu sticanja polne zrelosti. Veliki broj literaturnih podataka ukazuje na to da hormoni hipotalamo-hipofizo-gonadne (H-H-G) osovine, pre svega hormoni gonada, utiču na razvoj i involuciju timusa. Međutim, uprkos brojnim istraživanjima u proteklim decenijama, uloga hormona jajnika u inicijaciji i održavanju/progresiji involucije timusa nije do kraja rasvetljena. Cilj ove doktorske disertacije je bio da se ispita uloga hormona jajnika u inicijaciji i održavanju/progresiji involucije timusa. U tom cilju ispitivan je uticaj jednokratnog davanja testosterona u kritičnom neonatalnom uzrastu, koji dovodi do odlaganja sazrevanja H-H-G osovine i promena u obrascu sekrecije hormona jajnika kod adultnih životinja (neonatalna androgenizacija) i uklanjanja jajnika ženkama pacova u uzrastu od 10 meseci, kada su involutivne promene timusa jasno izražene, na građu timusa i timopoezu u ranom adultnom uzrastu; odnosno u uzrastu od 11 meseci. Posebno je ispitivan, mogući, indirektan uticaj promena u sekreciji hormona jajnika na aktivnost timusnog kateholaminergičkog regulatornog sistema. Neonatalna androgenizacija, delujući na različite stadijume diferencijacije/sazrevanja T-ćelija, od ulaska/diferencijacije progenitorskih ćelija do linijskog usmeravanja timocita, povećala je efikasnost timopoeze (na osnovu većeg broja svežih timusnih emigranata u perifernoj krvi i slezini), uključujući, najverovatnije, i povećano stvaranje ćelija regulatornog fenotipa (CD4+CD25+FoxP3+ i CD161+TCRαβ+ ćelije). Pored toga, kod ovih životinja je uočeno favorizovano stvaranje ćelija CD4-CD8+TCRαβ++ fenotipa (fenotipska defeminizacija/maskulinizacija procesa timopoeze). Uprkos povećanoj efikasnosti timopoeze, nepromenjena relativna zastupljenost timusnih epitelnih ćelija (TEC) kao i nivo iRNK za IL-6, a smanjen nivo iRNK za IL-7 u tkivu timusa, ukazuju, da je kod neonatalno androgenizovanih životinja, u uzrastu od 3 meseca, najverovatnije, došlo do pokretanja mehanizama negativne povratne sprege, koji ograničavaju ekspanziju TEC i timopoezu. Ovarijektomija u uzrastu od 10 meseci, nakon 30. dana, imala je za posledicu povećanje relativne zastupljenosti TEC i efikasnosti timopoeze (uključujući i povećano stvaranje ćelija regulatornog fenotipa), što se manifestovalo povećanim brojem svežih timusnih emigranata CD4+ i CD8+ fenotipa u perifernoj krvi i slezini. Međutim, uprkos regeneraciji epitelne komponente timusa i povećanoj efikasnosti timopoeze, smanjen nivo iRNK za IL-6 i IL-7 u tkivu timusa ovarijektomisani životinja sugeriše da je kod ovih životinja došlo do aktivacije intratimusnih mehanizama negativne povratne sprege koji ograničava ekspanziju TEC koje sintetišu IL-6 i efikasnost timopoeze. U zaključku, nalazi dobijeni u ovoj disertaciji ukazuju da neonatalna androgenizacija menja kinetiku postnatalnog razvoja timusa i polno specifičan obrazac diferencijacije/sazrevanja timocita i odlaže, ali vrlo verovatno ne sprečava involuciju timusa, dok uklanjanje hormona jajnika u uzrastu kada su involutivne promene timusa uznapredovale dovodi do delimične reverzije ovih promena i efikasnije timopoeze, koji su, najverovatnije, vremenski ograničenog trajanja. Pored toga, pokazano je da se uticaj hormona jajnika na ove procese odvija i indirektno, menjanjem efikasnosti modulatornog delovanja noradrenalina na timopoezu.

The thymus is a primary lymphoid organ that provides the microenvironment necessary for the differentiation/maturation of T-lymphocytes. During ontogeny, thymus undergoes significant structural and functional changes leading to reduced efficiency of thymopoiesis and, consequently, reduced output of newly generated T- lymphocytes. These changes are collectively referred to as thymic involution. The most profound thymic changes in rodents, occur around puberty. There is an accumulating body of evidence indicating that hormones of the hypothalamic-pituitary-gonadal (H-P- G) axis, particularly gonadal hormones, influence thymic development and involution. However, despite extensive research in the past decades, the role of ovarian hormones in the initiation and maintenance/progression of thymic involution is not fully understood. Bearing all aforementioned in mind, this dissertation was aimed to elucidate the role of ovarian hormones in initiation and maintenance/progression of thymic involution. To this end the effects of single injection of testosterone in critical postnatal period, which postpones H-P-G axis maturation and affects the gender-specific pattern of gonadal hormone secretion in adult animals (neonatal androgenization), and ovarian hormone removal in rats with advanced thymic involutive changes (10-month-old), on thymic structure and function, were examined in 3-month-old and 11-month-old female rats, respectively. In addition, the putative indirect, catecholamine-mediated effects of the neonatal androgenization and ovariectomy on thymopoiesis were explored. Neonatal androgenization, affecting distinct stages of thymocyte differentiation/maturation (from progenitor cell entry and differentiation to thymocyte lineage commitment), increased the efficiency of thymopoiesis and consequently thymic output (as evidenced by the increased number of recent thymic emigrants in peripheral blood and spleen), including, most likely, increased output of the cells with regulatory phenotype (CD4+CD25+FoxP3+ and CD161+TCRαβ+ cells). In addition, neonatal treatment with testosterone skewed thymocyte lineage commitment towards CD4-CD8+TCRαβ++ cells (phenotypic defeminization/masculinization of thymopoiesis). Despite the increased efficiency of thymopoiesis, unaltered relative abundance of thymic epithelial cells (TEC) and the thymic level of IL-6 mRNA, and reduced thymic level of IL-7 mRNA, indicated, most likely, activation of intrathymic negative feedback mechanisms limiting TEC proliferation and thymopoiesis in 3- month-old neonatally androgenized rats. Ovariectomy at the age of 10 months led to the expansion of TECs and greater efficiency of thymopoiesis (including generation of cells with regulatory phenotype), increasing the number of both CD4+ and CD8+ recent thymic emigrants in peripheral blood and spleen of 11-month-old rats, compared with age-matched controls. However, despite the regeneration of the thymic epithelial component and increased efficiency of thymopoiesis, reduced thymic levels of mRNA for IL-6 and IL-7 in ovariectomized rats indicated activation of intrathymic negative feedback mechanisms limiting the expansion of IL-6-synthesizing TECs and thymopoietic efficiency in these animals. In conclusion, the data obtained in this dissertation indicate that neonatal androgenization alters the kinetics of postnatal thymic development and sex-specific pattern of thymocyte differentiation/maturation and postpones thymic involution, but, most likely, does not prevent it completely. The removal of ovarian hormones in female rats with advanced thymic involutive changes leads to partial reversion of these changes, and an increase in thymopoietic efficiency of, most likely, limited duration. In addition, these data indicate that ovarian hormones might affect thymic development/involution not only directly, but also indirectly by diminishing the efficiency of noradrenaline-mediated modulation of thymopoiesis.

Molekularni markeri hronične inflamacije i apoptoze kod inflamatornih bolesti creva

Molecular markers of chronic inflammation and apoptosis in inflammatory bowel diseases

Hronične inflamatorne bolesti creva (HIBC) predstavljaju rastući globalni zdravstveni problem, posebno u belim populacijama, uključujući i srpsku populaciju. Ova studija obuhvatila je dva tipa HIBC - inflamatornu bolest creva (IBC), koju čine Kronova bolest (KB) i ulcerozni kolitis (UK); i celijačnu bolest (CB). HIBC su kompleksne bolesti u čijoj, još uvek potpuno nerazjašnjenoj etiologiji učestvuju genetički, sredinski i imunološki faktori. Razvoj IBC je rezultat poremećenog imunološkog odgovora na crevnu mikrofloru kod genetički podložnih osoba. Ustanovljen je veliki broj asocijacija između IBC i varijanti u genima čiji proteinski produkti učestvuju u nespecifičnom imunskom odgovoru. Oštećenje intestinalne mukoze kod pacijenata sa IBC dešava se kao posledica deregulacije inflamatornih i apoptotskih procesa koji utiču na dugovečnost T ćelija i integritet epitelijalne barijere. Protein NF-κB je jedan od ključnih regulatora transkripcije gena eksprimiranih tokom ovih patogenetskih promena. U mukozi pacijenata sa KB dolazi do formiranja granuloma, limfoidnih mikrostruktura karakterističnih i za druge inflamatorne bolesti, među kojima je reumatoidni artritis (RA). Zbog toga su KB i RA klasifikovane kao granulomatozne bolesti. U poređenju sa brojnim, još uvek nedovoljno definisanim genetičkim faktorima rizika za razvoj IBC, nasledna komponenta u razvoju CB je dobro okarakterisana i najviše je povezana sa specifičnim varijantama gena HLA-DQ. Jedan od ciljeva ove studije je bio da ispita asocijaciju između HIBC i određenih genetičkih varijanti. Tačnije, analizirana je povezanost IBC sa varijantama u genima NOD2, TLR4, TNF-α, IL-6, IL-1β i IL-1RN, i ispitan je njihov prediktivni značaj za pojavu IBC. Varijanta u genu IL-6 ispitana je kao faktor rizika za nastanak dve granulomatozne bolesti, KB i RA. Takođe, analizirana je distribucija genotipova HLA-DQ u grupi pedijatrijskih pacijenata obolelih od CB i procenjen je rizik za pojavu CB koju ovi genotipovi nose. Sledeći cilj je bila analiza nivoa transkripcije proinflamatornih gena IL-6 i TNF-α, i apoptotskih gena Bcl-2, Bax, Fas i FasL u intestinalnoj mukozi i perifernoj krvi pacijenata obolelih od KB, kako bi se definisali ekspresioni profili gena uključenih u patogenezu KB i ispitao njihov dijagnostički potencijal. Poslednji cilj u okviru istaživanja vezanog za IBC odnosio se na analizu DNK vezujuće aktivnosti NF-κB iz jedarnih ekstrakata intestinalne mukoze pacijenata obolelih od KB. U studiju je bilo uključeno 167 ispitanika obolelih od IBC, 73 obolelih od CB, 77 obolelih od RA i 101 kontrolni zdrav ispitanik. Genetičke varijante u svim gorepomenutim genima određene su metodama koje su bazirane na PCR analizi. Za ispitivanje nivoa ekspresije odabranih inflamatornih i apoptotskih gena korišćena je metoda qRT-PCR. Analiza DNK- proteinskih interakcija urađena je metodom usporene elektroforetske pokretljivosti. Naši rezultati su pokazali da su manje učestale varijante u genu NOD2 (Arg702Trp, Gly908Arg, Leu1007fsinsC, Arg684Trp) kao i genetičke varijante TLR4 299Gly, TNF-α - 308A, IL-6 -174C i IL-1RN VNTR A2, asocirane sa povećanim rizikom za nastanak KB, dok je genetička varijanta IL-1RN VNTR A2 povezana sa povećanim rizikom za nastanak UK. Pokazana je značajna razlika u alelskoj distribuciji TNF-α G-308A između pacijenata obolelih od KB i UK, gde je alel A povezan sa prisustvom KB, a alel G sa prisustvom UK. Prisustvo KB je asocirano sa kombinovanim efekatom pola i manje učestalih varijanti u genu TLR4. Podaci o analiziranim genotipovima su korišćeni kao ulazne varijable za nekoliko klasifikacionih algoritama u cilju dizajniranja modela za predikciju IBC. Kao mera kvaliteta ovih modela korišćena je vrednost površine ispod receiver operating krive (eng. the Area Under the receiver operating Curve, AUC). Prediktivni model je pokazao najveći kvalitet kada su za njegov dizajn upotrebljeni podaci o svim analiziranim genotipovima (AUC od 0.69 za KB i 0.60 za UK). Takođe, naši rezultati su pokazali da IL-6 G-174C može biti genetički marker dve ispitane granulomatozne bolesti, KB i RA. Što se tiče celijačne bolesti, pokazana je značajna asocijacija između kombinovanog prisustva alela HLA-DQA1*05 i - DQB1*02 sa pojavom bolesti. Takođe, uočeno je postojanje gradijenta rizika za nastanak CB koji je vezan za specifične genotipove HLA-DQ. Rezultati ekspresione analize su pokazali da postoji značajan porast nivoa IL-6 i TNF-α iRNK, i značajan pad nivoa Bcl-2 iRNK u upaljenoj ilelalnoj mukozi pacijenata sa KB. Povećanje u nivou transkripcije je izraženije kod IL-6 u odnosu na TNF-α. Rezultati su ukazali i na potencijalni dijagnostički značaj nivoa IL-6 i Bcl-2 iRNK za aktivnu fazu KB. Pored analize transkripcije u intestinalnom tkivu, odredili smo nivo transkripcije istih gena i u mononuklearnim ćelijama periferne krvi. Naši rezultati su pokazali da je nivo FasL iRNK značajno snižen kod pacijenata sa KB, ali samo u uzorku krvi muškaraca. Pored toga, rezultati pokazuju da odnos Fas/FasL ima prediktivni značaj i predstavlja potencijalni dijagnostički biomarker aktivne faze KB kod muških pacijenata. Analizom DNK vezujuće aktivnosti NF-κB iz jedarnih ekstrakata uzoraka intestinalne mukoze pacijenata sa KB, pokazano je da su snižen nivo NF-κB vezujuće aktivnosti i povišen nivo TNF-α iRNK povezani sa prisustvom fragilne intestinalne mukoze. Nasuprot tome, povišen nivo NF-κB vezujuće aktivnosti i snižen nivo TNF-α iRNK su pokazali asocijaciju sa prisustvom intestinalnih stenoza. Stoga smatramo da je status NF-κB vezujuće aktivnosti potencijalni marker apopototskih i inflamatornih procesa koji se nalaze u osnovi ova dva klinička fenotipa. Genetičke varijante ispitivane u ovoj studiji predstavljaju faktore rizika za pojavu HIBC i mogu se potencijalno koristiti u predikciji nastanka analiziranih bolesti. Takođe, ekspresioni profili odabranih proinflamatorinih i apopototskih gena, kao i nivo DNK vezujuće aktivnosti jednog od glavnih regulatora transkripcije ovih gena, NF-κB, mogu se smatrati potencijalnim dijagnostičkim i prognostičkim markerima KB. Ovo istraživanje je dalo doprinos molekularnoj karakterizaciji patoloških procesa koji se nalaze u osnovi HIBC.

Chronic inflammatory bowel diseases (CIBDs) remain an expanding global health problem, particularly in most Caucasian populations, including Serbian. The focus of this study included two types of chronic inflammatory bowel diseases - inflammatory bowel disease (IBD), which comprises Crohn’s disease (CrD) and ulcerative colitis (UC); and celiac disease (CD). These are complex diseases with various genetic, environmental and immunological risk factors, whose etiologies are not yet fully resolved. The development of IBD is a result of abnormal immune response that occurs in genetically susceptible individuals against the enteric flora. Many studies have investigated associations between IBD occurrence and variants in genes that encode proteins involved in nonspecific immune response. Intestinal mucosal damage in CrD patients occurs as a result of the deregulation of inflammatory and apoptotic processes, which influence T cell longevity and epithelial barrier integrity. Regulatory protein NF-κB has a key role in the transcription of genes whose products are involved in these pathogenic events. Another property of CrD is formation of granulomas, which can also occur in other diseases, such as rheumatoid arthritis (RA). For that reason, CrD and RA are categorized as granulomatous diseases. Compared with numerous, still insufficiently defined genetic risk factors for the development of IBD, a genetic component in CD development is well characterized and mostly associated with specific variants of HLA-DQ genes. One of the aims of this study was to evaluate disease-gene associations. More precisely, we examined IBD associations with several genetic variants - variants in NOD2, TLR4, TNF-α, IL-6, IL-1β and IL-1RN genes, and investigated their contribution in the prediction of IBD occurrence. Additionally, we analyzed variant in IL-6 gene as a risk factor for two types of granulomatous diseases, CrD and RA. Also, we inspected the distribution of HLA-DQ genotypes in the group of pediatric celiac patients and estimated the risk of CD development that these genotypes confer. Another aim was the analysis of mRNA level of pro- inflammatory IL-6 and TNF-α, and apoptotic Bcl-2, Bax, Fas and FasL genes in intestinal mucosa, as well as in the peripheral blood of CrD patients, in order to reveal the expression patterns involved in the pathogenesis of CrD. Also, our goal was to investigate the diagnostic potential of these transcriptional products. The final aim within the IBD study was the analysis of the DNA binding activity of NF-κB from the nuclear extracts of CrD patients’ intestinal mucosal samples. In this study, 167 IBD patients, 73 CD patients, 77 RA patients and 101 healthy subjects were enrolled. Genetic variants in above-mentioned genes were detected using PCR-based methodology. Gene expression levels of selected inflammatory and apoptotic genes were measured using qRT-PCR methodology. DNA-protein interactions were analyzed employing electrophoretic mobility shift assay. Our results showed that minor variants of NOD2 gene (Arg702Trp, Gly908Arg, Leu1007fsinsC, Arg684Trp), as well as TLR4 299Gly, TNF-α -308A, IL-6 -174C and IL- 1RN VNTR A2 variants, were associated with a higher risk of CrD, while the IL-1RN VNTR A2 variant was associated with a higher risk of UC incidence. CrD and UC showed a highly significant difference in the allelic distribution of TNF-α G-308A, where A allele was found to be related to CD, and G allele to UC occurrence. Among CrD patients, the combined effect of gender and TLR4 variants was observed. Using obtained genotype data as input to various classification algorithms, we designed IBD prediction models. The Area Under the receiver operating Curve (AUC) was used as a measure of their performance. When all the analyzed genotype and gender data were used, the prediction performance achieved a maximum AUC of 0.69 for CD and 0.60 for UC dataset. We revealed that IL-6 G-174C could be a genetic marker for two analyzed granulomatous diseases, CrD and RA. Considering CD-HLA relationship, the combined presence of HLA-DQA1*05 and -DQB1*02 alleles showed a highly significant association with CD. We observed a gradient of associated risk for CD occurrence determined by particular HLA-DQ genotypes. Results of the expression analyses have shown that levels of IL-6 and TNF-α mRNAs were significantly increased, while level of Bcl-2 mRNA was significantly decreased in ileal inflamed mucosa of CrD patients. The increase in the level of IL-6 transcription was more prominent compared with TNF-α. We propose that levels of IL-6 and Bcl-2 mRNAs should be considered as potential diagnostic markers of the CrD active phase. In addition to the presented analyses at local levels of inflammation, we also assessed the level of transcription of the same genes in the peripheral blood mononuclear cells of CrD patients. Our results revealed that the level of FasL mRNA was significantly decreased, but only in blood samples from male patients. We found that Fas/FasL ratio had predictive significance and therefore, it could be considered as potential diagnostic biomarker of the CrD active phase in male patients. The analysis of the DNA binding activity of NF-κB from the nuclear extracts of CrD patients’ intestinal mucosal samples revealed an association of decreased level of NF-κB binding activity and increased level of TNF-α mRNA with intestinal mucosal fragility. In contrast, increased level of NF-κB binding activity and decreased level of TNF-α mRNA were associated with intestinal strictures. Therefore, we propose the status of NF-κB binding activity as a marker of apoptotic and inflammatory processes that underlie these two types of CrD clinical phenotypes. The results of the presented study showed that examined genetic variants are genetic factors of importance in the susceptibility of analyzed CIBDs and these variants could be used as potential predictors of the CIBDs occurrence. Also, the expression profiles of selected proinflammatory and apoptotic genes, as well as the DNA binding activity status of one of the main transcription factors that is involved in the expression of these genes, NF-κB, could be considered as potential CrD diagnostic and prognostic biomarkers. This research has contributed to the molecular characterization of pathological processes that underlie chronic inflammatory bowel diseases.

Molekularni mehanizmi antiproliferativnog dejstva rutenijum(II)-arenskog kompleksa sa izohinolin-3-karboksilnom kiselinom kao ligandom u uslovima in vitro

Molecular mechanisms of the antiproliferative activity of ruthenium(II)-arene complex with isoquinoline-3-carboxylic acid as ligand under in vitro conditions

Još od otkrića cisplatine sintetisan je veliki broj kompleksa prelaznih metala, čija je antikancerska aktivnost ispitana. Naročitu pažnju zaslužuju jedinjenja zasnovana na rutenijumu kao metalu koja su pokazala antitumorsku i antimetastatsku aktivnost, sa potencijalnom primenom kod tumora rezistentnih na cisplatinu, ili kao alternative cisplatini. Cilj ove disertacije je ispitivanje molekularnih mehanizama antiproliferativne aktivnosti rutenijum(II)-arenskog kompleksa sa izohinolin-3-karboksilnom kiselinom kao ligandom. Rezultati ispitivanja citotoksičnog potencijala sedam novih jedinjenja opšte formule [Ru(η6-p-cimen)(L1-7)Cl] su pokazali da je kompleks sa L = izohinolin-3- karboksilnom kiselinom (RuT7) u odnosu na ostale komplekse iz serije, ispoljio izraženu citotoksičnu aktivnost, sa IC50 vrednostima u opsegu od 18,5 do 84,2 µM. Ovaj kompleks je pokazao dva puta veću aktivnost na HeLa ćelijama nego na normalnoj ćelijskoj liniji MRC-5, što potvrđuju i IC50 vrednosti određene nakon inkubacionog perioda od 48 h (45,4 ± 3,0 i 84,2 ± 5,7 μM). Analiza ćelijskog ciklusa HeLa ćelija koje su tretirane RuT7 kompleksom pokazala je zaustavljanje ćelija u S fazi ćelijskog ciklusa, kao i povećanje broja ćelija u sub-G1 populaciji. Aneksin V-FITC/PI esej, kao i morfološka analiza ćelija na fluorescentnom mikroskopu, pokazali su da ispitivano jedinjenje ima potencijal indukcije apoptoze. Ispitivanje akumulacije rutenijuma unutar ćelija je pokazalo prisustvo rutenijuma u ćelijskoj unutrašnjosti nakon 6 h inkubacije (8,9 ng Ru/106 ćelija). U cilju detaljnog ispitivanja mehanizma dejstva RuT7 kompleksa na HeLa ćelijama, urađena je mikroerej analiza promena u genskoj ekspresiji na celom transkriptomu HeLa ćelija. Analiza funkcionalnih kategorija i signalnih i biohemijskih puteva koji se menjaju tokom odgovora HeLa ćelija na tretman RuT7 kompleksom je pokazala da se pod uticajem tretmana aktiviraju molekularni mehanizmi koji vode ćelije u apoptozu i to preko unutrašnjeg (mitohondrijskog) signalnog puta. Do aktiviranja mitohondrijskog apoptotskog puta dolazi usled direktnog vezivanja RuT7 kompleksa za DNK molekul, kao i indirektnim oštećenjem DNK usled dejstva reaktivnih kiseoničnih vrsta. Statistička analiza promena u ekspresiji genskih setova uključenih u poznate puteve toksičnosti koje su izazvane lekovima je pokazala manju predviđenu toksičnost RuT7 kompleksa u poređenju sa cisplatinom. Sveobuhvatno, dobijeni rezultati predstavljaju osnovu za dalja istraživanja RuT7 kompleksa u okviru studija na životinjama i prekliničkim ispitivanjima kao potencijalnog kandidata za hemioterapeutika.

Since the discovery of cisplatinum, many transition metal complexes have been synthesized and assayed for antineoplastic activity. In recent years, ruthenium-based compounds have emerged as promising antitumor and antimetastatic agents with potential uses in platinum-resistant tumors or as alternatives to platinum. The aim of this thesis was to investigate molecular mechanisms of the antiproliferative activity of ruthenium(II)-arene complex with isoquinoline-3-carboxylic acid as ligand. The results of investigation of the cytotoxic potential of seven new compounds of the general formula [Ru(η6-p-cymene)(L1-7)Cl] showed that the complex with L = isoquinoline-3-carboxylic acid (RuT7) was two times as active on HeLa cells compared to normal cell line MRC-5, as indicated by IC50 values determined after 48 h of incubation (45,4 ± 3,0 vs. 84,2 ± 5,7 μM, respectively). Cell cycle analysis of HeLa cells treated with RuT7 showed S phase arrest and an increase in sub-G1 population. The apoptotic potential of the title compound was confirmed with the Annexin V-FITC/PI assay together with a morphological evaluation of cells using fluorescent microscopy. Analysis of the intracellular accumulation of ruthenium showed 8,9 ng Ru/106 cells after 6 h of incubation. To gain further insight in the molecular mechanism of action of RuT7 on HeLa cells, a whole-transcriptome microarray gene expression analysis was performed. Analysis of functional categories and signaling and biochemical pathways associated with the response of HeLa cells to treatment with RuT7 showed that it leads the cells through the intrinsic (mitochondrial) apoptotic pathway, via indirect DNA damage due to the action of reactive oxygen species, and through direct DNA binding of RuT7. Statistical analysis for enrichment of gene sets associated with known drug-induced toxicities identified fewer associated toxicity profiles in RuT7-treated cells compared to cisplatin treatment. Altogether these results provide the basis for further development of RuT7 in animal and pre-clinical studies as a potential drug candidate.

Fiziološki i molekularno-genetički pokazatelji međuvrsne hibridizacije u okviru roda Centaurium Hill (Gentianaceae)

Physiological and molecular genetic indicators of interspecific hybridization within the genus Centaurum Hill (Gentianaceae)

Vrste roda Centaurium Hill često hibridizuju u prirodi. Brojni primeri ukazuju na značaj poliploidizacije i hibridizacije tokom specijacije u okviru ovog roda. Ukrštanjem Centaurium erythraea Rafn × C. littorale (Turner) Gilmour u uslovima in vitro, dobijeno je alotetraploidno potomstvo, što ide u prilog hipotezi postojanja nedovoljno efikasnih prereproduktivnih barijera, koje bi sprečavale hibridizaciju. Primenjene metode za detekciju hibridnih jedinki u uslovima in vitro su uspešno iskorišćene i u slučaju prirodnih populacija poreklom sa Subotičko-horgoške peščare. Novi takson, nazvan Centaurium pannonicum, je heksaploid nastao iz ukrštanja C. erythraea × C. littorale ssp. compressum (Hayne) Kischner, nakon fertilizacije, tj. spajanja diploidnog i tetraploidnog jedra. De novo razvijeni EST-SSR markeri ukazuju da C. pannonicum konvergira vrsti C. erythraea, dok je prema fitohemijskim markerima hibridni takson sličniji C. littorale ssp. compressum. Razvijeni setovi EST-SSR i fitohemijskih markera, su transferabilni i na druge vrste roda Centaurium, i mogu značajno doprineti daljim istraživanjima međuvrsne hibridizacije. C. pannonicum stabilno održava svoju heksaploidnu genetičku konstitutciju u prirodnim populacijama, umnogome zahvaljujući samooplodnji i/ili drugim vidovima ukrštanja u srodstvu.

UNIVERZITET U BEOGRADU BIOLOŠKI FAKULTET Tijana B. Banjanac FIZIOLOŠKI I MOLEKULARNO-GENETIČKI POKAZATELJI MEĐUVRSNE HIBRIDIZACIJE U OKVIRU RODA CENTAURIUM HILL (GENTIANACEAE) Doktorska disertacija Beograd, 2016. UNIVERSITY OF BELGRADE FACULTY OF BIOLOGY Tijana B. Banjanac PHYSIOLOGICAL AND MOLECULAR GENETIC INDICATORS OF INTERSPECIFIC HYBRIDIZATION WITHIN THE GENUS CENTAURIUM HILL (GENTIANACEAE) Doctoral Dissertation Belgrade, 2016 PODACI O MENTORIMA I ČLANOVIMA KOMISIJE MENTORI: Dr Branislav Šiler, viši naučni saradnik, Univerzitet u Beogradu, Institut za biološka istraživanja „Siniša Stanković“ Dr Mihailo Jelić, docent, Univerzitet u Beogradu, Biološki fakultet ČLANOVI KOMISIJE: Dr Danijela Mišić, viši naučni saradnik, Univerzitet u Beogradu, Institut za biološka istraživanja „Siniša Stanković“ Dr Aneta Sabovljević, vanredni profesor, Univerzitet u Beogradu, Biološki fakultet Dr Marijana Skorić, naučni saradnik, Univerzitet u Beogradu, Institut za biološka istraživanja „Siniša Stanković“ Datum odbrane: Eksperimentalni deo doktorske disertacije urađen je u okviru projekta osnovnih istraživanja Ministarstva prosvete, nauke i tehnološkog razvoja Republike Srbije (OI 173024), u Institutu za biološka istraživanja „Siniša Stanković“, Univerziteta u Beogradu. Zahvaljujem svojim mentorima dr Branislavu Šileru i dr Mihailu Jeliću na pruženoj nesebičnoj stručnoj pomoći, rečima ohrabrenja, toleranciji i velikom razumevanju. Veliku zahvalnost dugujem i dr Danijeli Mišić na velikoj stručnoj pomoći, ali i uvek prisutnim rečima i delima podsticaja, usmeravanja i vođenja koje su mi pomogle da istrajem. Teško je opisati i zahvalnost koju dugujem i dr Milanu Dragićeviću koji je mnogo truda i vremena uložio pokušavajući da me uputi u tajne statistike i natera da razmišljam na neki drugi način. Dr Marijani Skorić, uvek spremnoj da svojim velikim iskustvom pomogne u rešavanju interesantnih problema i svojim smernicama uputi na ispravne zaključke, dugujem pored stručne i veliku ličnu i prijateljsku zahvalnost. Zahvaljujem se dr Ani Simonović na istrajnosti u realizaciji sekvenciranja transkriptoma kičice i profesoru Saši Malkovu za savete koji su pomogli adekvatnu bioinformatičku obradu podataka. Posebnu zahvalnost dugujem i profesoru Dmitru Lakušiću koji je sa velikim entuzijazmom pomogao oko rešavanja svih pitanja vezanih za taksonomiju. Deo istraživanja ove disertacije urađen je na Institutu za molekularnu genetiku i genetičko inženjerstvo uz podršku dr Valentine Đorđević. Takođe za deo rezultata koji se tiču nivoa ploidije zahvalnost dugujem profesoru Borutu Bohanecu sa Univerziteta u Ljubljani. Posebnu zahvalnost dugujem Nedi Đedović zbog započetih genetičkih analiza vrsta kojima se bavi ova doktorska disertacija. Za preliminarne kvalitativne analize sekundarnih metabolita zahvalna sam Urošu Gašiću sa Hemijskog fakulteta, a za stručnu savete oko postupanja sa uzorcima biljaka zahvaljujem se Urošu Buzuroviću iz Prirodnjačkog muzeja. Profesorki Aneti Sabovljević zahvaljujem na podršci u pravcu mojih istraživanja. Veliko hvala svim divnim koleginicama i kolegama sa Odeljenja za fiziologiju biljaka: dr Suzani Živković, dr Dragani Božić, dr Slađani Todorović, dr Milici Bogdanović, dr Slavici Dmitrović, dr Biljani Filipović, Milici Milutinović, Jeleni Boljević i Nedi Aničić na podršci, razumevanju i toleranciji. Posebno hvala dr Jasmini Nestorović Živković bez čije pomoći bi se eksperimentalni deo neizmerno produžio. Nabilu Galavenžiju hvala za stručnu pomoć gajenja biljaka u stakleniku i prijateljskom razgovoru. Dr Stevanu Avramovu sa Odeljanja za evolucionu biologiju se zahvaljujem na stručnim savetima i podršci na terenskim istraživanjima. Zahvalnost za divnu temu ove doktorske disertacije i ljubav prema kičicama, koju je podelio sa mnom, dugujem profesoru Dragoljubu Grubišiću. Veliko hvala suprugu Mariju Belamariću na bioinformatičkom radu, ali i na ljubavi, razumevanju i podršci bez koje ne bih mogla. Hvala mami i tati. Za Matiju i Luku Fiziološki i molekularno-genetički pokazatelji međuvrsne hibridizacije u okviru roda Centaurium Hill (Gentianaceae) REZIME Vrste roda Centaurium Hill često hibridizuju u prirodi. Brojni primeri ukazuju na značaj poliploidizacije i hibridizacije tokom specijacije u okviru ovog roda. Ukrštanjem Centaurium erythraea Rafn × C. littorale (Turner) Gilmour u uslovima in vitro, dobijeno je alotetraploidno potomstvo, što ide u prilog hipotezi postojanja nedovoljno efikasnih prereproduktivnih barijera, koje bi sprečavale hibridizaciju. Primenjene metode za detekciju hibridnih jedinki u uslovima in vitro su uspešno iskorišćene i u slučaju prirodnih populacija poreklom sa Subotičko-horgoške peščare. Novi takson, nazvan Centaurium pannonicum, je heksaploid nastao iz ukrštanja C. erythraea × C. littorale ssp. compressum (Hayne) Kischner, nakon fertilizacije, tj. spajanja diploidnog i tetraploidnog jedra. De novo razvijeni EST-SSR markeri ukazuju da C. pannonicum konvergira vrsti C. erythraea, dok je prema fitohemijskim markerima hibridni takson sličniji C. littorale ssp. compressum. Razvijeni setovi EST-SSR i fitohemijskih markera, su transferabilni i na druge vrste roda Centaurium, i mogu značajno doprineti daljim istraživanjima međuvrsne hibridizacije. C. pannonicum stabilno održava svoju heksaploidnu genetičku konstitutciju u prirodnim populacijama, umnogome zahvaljujući samooplodnji i/ili drugim vidovima ukrštanja u srodstvu. Ključne reči: Centaurium, hemotaksonomski markeri, EST-SSR, međuvrsna hibridizacija, polimorfizam, poliploidija, RAPD Physiological and molecular genetic indicators of interspecific hybridization within the genus Centaurium Hill (Gentianaceae) ABSTRACT Species of genus Centaurium Hill readily hybridize in natural habitats. Plenty of evidence reveal the importance of polyploidization and hybridization as evolutionary mechanisms responsible for speciation within this genus. Allotetraploids obtained from Centaurium erythraea Rafn × C. littorale (Turner) Gilmour cross under in vitro conditions supported hypothesis that pre-reproductive barriers existing in these species are poorly efficient in preventing hybridization. Implemented methodology for the detection of hybrid individuals were successfully applied in natural populations from Subotica-Horgoš sands. New taxon, named Centaurium pannonicum, is a hexaploid hybrid arisen from C. erythraea x C. littorale ssp. compressum (Hayne) Kischner cross, after fertilization, i.e. joining of diploid and tetraploid nuclei. De novo developed EST- SSR markers suggested that hybrid taxon converges to C. erythraea. As revealed by the selected set of phytochemical markers, C. pannonicum was more similar to C. littorale ssp. compressum. Developed sets of EST-SSR and phytochemical markers are transferable to other Centaurium species and could facilitate further investigation of interspecific hybridization within the genus. C. pannonicum maintains its hexaploidy in natural populations by self-fertilization and/or other forms of positive assortative crossing.

Bioinformatička i glikobiohemijska analiza modularne organizacije CA125 antigena čoveka

Bioinformatic and glycobiochemical analysis of human CA125 antigen modular organization

CA125 antigen, poznat kao marker seroznog karcinoma ovarijuma, predstavlja ekstracelularni deo mucina 16 (MUC16), koji nastaje njegovom proteolitiĉkom degradacijom. MUC16 pripada grupi transmembranskih proteina tipa I, i eksprimiran je u velikom broju embrionalnih, ali i adultnih tkiva. Njegovu primarnu strukturu karakteriše prisustvo tri regiona: N-terminalni region i region tandemskih ponovaka, koji su smešteni ekstracelularno, i C-terminalni region, koji se sastoji od transmembranskog dela i kratkog citoplazmatskog repa. Ekstracelularni region je intenzivno glikozilovan, a u intracelularnom regionu se nalazi potencijalno mesto fosforilacije. Fosforilacija predstavlja signal za proteolizu i oslobaĊanje ekstracelularnog dela molekula (SA125 antigen), koji se kao takav može detektovati u razliĉitim telesnim teĉnostima. Biološka uloga MUC16/CA125, još uvek nije razjašnjena. Dosadašnja ispitivanja funkcije MUC16 (membranska forma) su, uglavnom, bila bazirana na in vitro model sistemu ćelija ovarijalnog karcinoma, dok su literaturni podaci o aktivnosti CA125 antigena (solubilna forma) veoma retki. Solubilna forma predstavlja funkcionalni analog i kompetitor membranskoj formi i, u cirkulaciji, ona prva dospeva u kontakt sa razliĉitim tipovima normalnih ili patološki izmenjenih ćelija, ali ne postoje eksperimentalni dokazi o mogućem uticaju na njihovu adhezivnost, kao i imunomodulatorna svojstva. U ovom radu je ispitivana modularna organizacija MUC16/CA125 na bioinformatiĉkom i glikobiohemijskom nivou, sa ciljem da se bliže definišu biološki kapacitet i posebnosti prezentacije ovog molekula u kontekstu interaktoma ĉoveka. Bioinformatiĉka analiza, kao deo strategije za otkrivanje moguće biološke uloge MUC16/CA125, do sada nije korišćena u ispitivanjima ovog antigena. Pored toga, u kontekstu diskretnih bioloških funkcija, bez obzira na izuzetan biomedicinski znaĉaj, ne postoje eksperimentalni podaci o interakcijama MUC16/CA125 sa specifiĉnim klasama receptora na ćelijama krvnog sistema.CA125 antigen, poznat kao marker seroznog karcinoma ovarijuma, predstavlja ekstracelularni deo mucina 16 (MUC16), koji nastaje njegovom proteolitiĉkom degradacijom. MUC16 pripada grupi transmembranskih proteina tipa I, i eksprimiran je u velikom broju embrionalnih, ali i adultnih tkiva. Njegovu primarnu strukturu karakteriše prisustvo tri regiona: N-terminalni region i region tandemskih ponovaka, koji su smešteni ekstracelularno, i C-terminalni region, koji se sastoji od transmembranskog dela i kratkog citoplazmatskog repa. Ekstracelularni region je intenzivno glikozilovan, a u intracelularnom regionu se nalazi potencijalno mesto fosforilacije. Fosforilacija predstavlja signal za proteolizu i oslobaĊanje ekstracelularnog dela molekula (SA125 antigen), koji se kao takav može detektovati u razliĉitim telesnim teĉnostima. Biološka uloga MUC16/CA125, još uvek nije razjašnjena. Dosadašnja ispitivanja funkcije MUC16 (membranska forma) su, uglavnom, bila bazirana na in vitro model sistemu ćelija ovarijalnog karcinoma, dok su literaturni podaci o aktivnosti CA125 antigena (solubilna forma) veoma retki. Solubilna forma predstavlja funkcionalni analog i kompetitor membranskoj formi i, u cirkulaciji, ona prva dospeva u kontakt sa razliĉitim tipovima normalnih ili patološki izmenjenih ćelija, ali ne postoje eksperimentalni dokazi o mogućem uticaju na njihovu adhezivnost, kao i imunomodulatorna svojstva. U ovom radu je ispitivana modularna organizacija MUC16/CA125 na bioinformatiĉkom i glikobiohemijskom nivou, sa ciljem da se bliže definišu biološki kapacitet i posebnosti prezentacije ovog molekula u kontekstu interaktoma ĉoveka. Bioinformatiĉka analiza, kao deo strategije za otkrivanje moguće biološke uloge MUC16/CA125, do sada nije korišćena u ispitivanjima ovog antigena. Pored toga, u kontekstu diskretnih bioloških funkcija, bez obzira na izuzetan biomedicinski znaĉaj, ne postoje eksperimentalni podaci o interakcijama MUC16/CA125 sa specifiĉnim klasama receptora na ćelijama krvnog sistema.In silico analiza modularne organizacije MUC16/CA125, vršena je na osnovu podataka koji su preuzeti sa UniProtKB baze podataka, a koji se odnose na molekul izolovan iz ćelijske linije ovarijalnog karcinoma ĉoveka, OVCAR-3. Bioinformatiĉka analiza je obuhvatila: odreĊivanje homologije (korišćenjem BLAST, Basic Local Alignment Search Tool alatke); ispitivanje prisustva obrazaca, strukturnih motiva i konzerviranih domena (korišćenjem baza podataka kao što su Bgee: a dataBase for Gene Expression Evolution i Gene Ontology, CDD, ELM, BLOCKS, InterProScan, MyHits i iProClass); odreĊivanje fiziĉko-hemijskih osobina, globularnosti i odstupanja od pretpostavljene tercijarne strukture (korišćenjem ProtParam, ProtScale i GLOBPLOT v2.3 alatki); i predviĊanje funkcije pomoću alatki koje koriste nekoliko programa: JAFA, ProtFun 2.2 i GeneOntology. Glikobiohemijska analiza je obuhvatila ispitivanje uticaja CA125 antigena na adhezivnost i agregabilnost eritrocita, kao i moguće interakcije sa razliĉitim tipovima leukocitnih lektina tj. sigleka (Siglec - sialic acid-binding immunogobulin (Ig)-like lectin) i receptora sliĉnih lektinima tipa C (selektini, DC-SIGN - dendritic cell-specific ICAM-3-grabbing non-integrin 1, MMR - macrophage mannose receptor). Ispitivanje efekta CA125 antigena na eritrocite ĉoveka odreĊivano je u testovima na ĉvrstoj fazi ili primenom svetlosne mikroskopije. Interakcija CA125 antigena sa leukocitnim receptorima ispitivana je pomoću lektinskog- i imunoblota, kao i u testovima vezivanja i inhibicije na ĉvrstoj fazi, sa imobilisanim antigenima. Glikobiohemijskom analizom su, pored antigena iz ćelijske linije ovarijalnog karcinoma ĉoveka (clCA125), bili obuhvaćeni i antigen kancerskog porekla iz pleuralne teĉnosti (pfCA125) kao i antigen fetalnog porekla (pCA125), koji se sintetiše tokom trudnoće. Rezultati in silico analize proteinske sekvence MUC16/CA125, stavili su ovaj molekul u kontekst modularnih proteina sa anotiranom ulogom u adheziji i srodnim procesima. Uvid u sekvence koje pokazuju sliĉnost sa sekvencom MUC16/CA125, pokazao je da se one, uglavnom, nalaze u ekstracelularnom regionu MUC16, koji je bogat serinom i treoninom. Ove sliĉnosti se nisu mogle povezati sa anotiranim domenima iz dostupnih baza podataka, osim parcijalno, ali statistiĉki znaĉajno sa BLLF1, multidomenom karakteristiĉnim za glikoproteine omotaĉa virusa iz familije Herpesviridae. Bioinformatiĉka analiza je ukazala i na moguću korelaciju funkcionalnih aktivnosti, koje se pripisuju domenima bogatim serinom/treoninom kod identifikovanih proteina u okviru ispitivanih taksona, i moguće funkcije CA125 antigena. Na osnovu dobijenih rezultata, uloga CA125 antigena u procesima adhezije bi mogla ukljuĉiti i interakcije posredstvom konzerviranih proteinskih sekvenci, odgovornih za jonski transport, i interakcije sa šećernim supstratima. Polazeći od mucinske prirode CA125 antigena, kao i fizioloških stanja u kojima je njegova koncentracija u serumu povećana, u prvom delu glikobiohemijske analize, ispitan je uticaj CA125 antigena na eritrocite ĉoveka. Dobijeni rezultati su ukazali da pCA125 i pfCA125 umereno povećavaju agregaciju eritrocita, i uspešno inhibiraju njihovu adheziju. Za razliku od njih, clCA125 je pokazao neznatan uticaj na modulaciju ovih osobina eritrocita. Sumarni rezultati drugog dela analize, koja se odnosila na interakcije sa leukocitnim receptorima, pokazali su da CA125, kao ligand, može stupiti u interakcije sa razliĉitim tipovima proteina koji vezuju sijalinsku kiselinu. Pokazano je da postoje specifiĉni obrasci vezivanja sigleka za ispitivane CA125 antigene, kojima se ovi antigeni, uspešnije, razlikuju u odnosu na njihovo poreklo, nego što se to ĉini na osnovu obrazaca vezivanja biljnih lektina, takoĊe specifiĉnih za sijalinsku kiselinu. Najuoĉljivija razlika je zapažena u odnosu na ligandni kapacitet CA125 antigena fetalnog porekla prema sigleku-7, prisutnom na dendritskim ćelijama, NK (natural killer cell) ćelijama, monocitima i CD8+ T ćelijama i clCA125 antigena kancerskog porekla prema sigleku-9 i sigleku-10, prisutnim na B ćelijama, NK ćelijama, monocitima, neutrofilima i CD8+ T ćelijama. Za razliku od sigleka, sva tri ispitivana selektina: L-selektin, E-selektin i P-selektin su, na dozno-zavisan naĉin, interagovali sa pfCA125 i u manjoj meri pCA125, ali ne i clCA125. Pored toga, zapaženo je i da je P-selektin reagovao na znatno nižim koncentracijama nego L- i E-selektin. Na osnovu uoĉenog visokog afiniteta P-selektina, CA125 antigen bi pre stupio u interakcije sa aktiviranim endotelnim ćelijama ili krvnim ploĉicama, na kojima je ovaj selektin prisutan, nego sa ćelijama na kojima su prisutni drugi tipovi selektina. Ispitivanje interakcije CA125 antigena i DC-SIGN, koji je eksprimiran na površini dendritskih ćelija (DC) i koji vezuje manan i visoko-manozne glikane kao i glikane sa terminalnim Lex/Ley/Lea/Leb antigenima, pokazalo je da on prepoznaje pCA125 i pfCA125 antigen. Rezultati inhibicije su ukazali da je vezivanje DC-SIGN za pCA125 zavisno od prisustva glikana bogatih manozom, za razliku od vezivanja pfCA125, koje je nezavisno od prisustva N- ili O-glikana, što bi moglo uticati na njegove diskretne receptorske funkcije. Manozni receptor makrofaga (MMR), koji je prisutan na makrofagama i DC i koji vezuje terminalnu manozu i fukozu i SLex, nije reagovao ni sa jednim od ispitivanih CA125 antigena. Receptori sliĉni lektinima tipa C imaju važnu ulogu u imunskom sistemu, u smislu posredovanja u interakcijama tipa ćelija-ćelija, tj. u meĊusobnom kontaktu leukocita ili u njihovom kontaktu sa endotelom, kao i u vezivanju patogena. Promene u glikozilaciji liganada za receptore sliĉne lektinima tipa C, direktno utiĉu na njihovu aktivnost i specifiĉnost i imaju važne posledice na razvoj, preživljavanje i reaktivnost ćelija krvnog i imunskog sistema. Rezultati ovoga rada ukazuju da bi razlike u strukturi/glikozilaciji CA125 antigena, koje se vezuju za posebna fiziološka i patološka stanja, mogle menjati njegov uticaj na ćelije krvnog sistema ĉoveka i imati znaĉajne biomedicinske posledice u razliĉitim mikrosredinama. Dalji uvid u prirodu i mehanizme multifunkcionalnosti CA125 antigena zahteva interdisciplinarni pristup baziran na kompleksnim interakcijama koje su posredovane razliĉitim strukturnim domenima.

CA125 antigen, a well known tumour marker for serous ovarian cancer, is an extracellular part of the mucin 16 (MUC16) molecule. This antigen arises from proteolytic degradation of MUC16, a type I transmembrane protein, expressed in both embrional and adult tissue. The primary structure of MUC16 consists of three characteristic parts: an N-terminal region and a series of tandem repeats, which are located extracellularly, and a C-terminal region, which consists of the transmembrane part and a short cytoplasmic tail. The extracellular peptide is extensively glycosylated, and there is a potential phosphorylation site in the intracellular chain. Phosphorylation is a signal for proteolysis and release of the extracellular part of the molecule (CA125 antigen), which can be detected as such in various body fluids. The biological role of MUC16/CA125 is not yet understood. Previous investigations of MUC16 (membrane form) functions were generally based on in vitro cell model systems of ovarian cancer, whereas published data on the activity of CA125 antigen (soluble form) are very rare. The soluble form is a functional analog and competitor to the membrane form, and, in the circulation, it first contacts different types of normal or pathologically altered cells. However, there is no experimental evidence on its possible impact on their adhesion nor immunomodulatory properties. In this work, the modular organization of human CA125 antigen was analyzed bioinformatically and glycobiochemically, aiming at closer definition of the biological capacity and presentation of this molecule in the context of the human interactome. As part of a strategy to detect a possible biological role for MUC16/CA125, bioinformatics analysis has never been used in studies of this antigen. In addition, in the context of discrete biological functions, regardless of its great biomedical importance, there are no CA125 antigen, a well known tumour marker for serous ovarian cancer, is an extracellular part of the mucin 16 (MUC16) molecule. This antigen arises from proteolytic degradation of MUC16, a type I transmembrane protein, expressed in both embrional and adult tissue. The primary structure of MUC16 consists of three characteristic parts: an N-terminal region and a series of tandem repeats, which are located extracellularly, and a C-terminal region, which consists of the transmembrane part and a short cytoplasmic tail. The extracellular peptide is extensively glycosylated, and there is a potential phosphorylation site in the intracellular chain. Phosphorylation is a signal for proteolysis and release of the extracellular part of the molecule (CA125 antigen), which can be detected as such in various body fluids. The biological role of MUC16/CA125 is not yet understood. Previous investigations of MUC16 (membrane form) functions were generally based on in vitro cell model systems of ovarian cancer, whereas published data on the activity of CA125 antigen (soluble form) are very rare. The soluble form is a functional analog and competitor to the membrane form, and, in the circulation, it first contacts different types of normal or pathologically altered cells. However, there is no experimental evidence on its possible impact on their adhesion nor immunomodulatory properties. In this work, the modular organization of human CA125 antigen was analyzed bioinformatically and glycobiochemically, aiming at closer definition of the biological capacity and presentation of this molecule in the context of the human interactome. As part of a strategy to detect a possible biological role for MUC16/CA125, bioinformatics analysis has never been used in studies of this antigen. In addition, in the context of discrete biological functions, regardless of its great biomedical importance, there are no experimental data on the interactions of MUC16/CA125 with specific classes of receptors on cells of the blood system. The modular organization of MUC16/CA125, was analyzed in silico using data taken from the UniProtKB database, which refers to a molecule isolated from the human ovarian cancer cell line, OVCAR-3. Bioinformatic analysis included: determination of homology (using BLAST, Basic Local Alignment Search Tool Tool); investigation of patterns, structural motifs and conserved domains (using databases such as Bgee: a database for Gene Expression and Gene Ontology Evolution, CDD, ELM, BLOCKS, InterProScan and MyHits iProClass); determination of physical and chemical properties, globularity and deviations from the assumed tertiary structure (using ProtParam, ProtScale and GLOBPLOT v2.3 tools); and function prediction using tools that employ several programs: JAFA, ProtFun 2.2 and GeneOntology. Glycobiochemical analysis comprised examination of the influence of CA125 on adhesion and aggregation of erythrocytes, as well as on interactions with different types of leukocyte lectins, namely siglecs [sialic acid-binding immunogobulin (Ig)-like lectin] and C-type lectin-like receptors [selectins, dendritic cell-specific ICAM-3-grabbing non-integrin 1 (DC-SIGN) and macrophage mannose receptor (MMR)]. The effect of CA125 antigen on human erythrocytes was determined using solid phase tests and light microscopy. The interaction of CA125 with leukocyte receptors was investigated employing lectin-blot and immunoblot, as well as binding and inhibition solid phase assays. Cancer antigen isolated from human pleural fluid (pfCA125) and pregnancy-associated antigen (pCA125) were included in the glycobiochemical analysis in addition to antigen from the human ovarian carcinoma cell line (clCA125). The results of in silico analysis placed MUC16/CA125 in the context of modular proteins with an annotated role in adhesion-related processes. They pointed to similarities within extracellular serine/threonine rich regions of MUC16 to protein sequences expressed in evolutionarily distant taxa. No relation to annotated domains from available databases appeared, except for BLLF1, a multidomain characteristic of virus envelope glycoproteins of the Herpesviridae family. Bioinformatics analysis also pointed to a possible correlation between functional activities, which are attributed to serine/threonine rich domains of experimental data on the interactions of MUC16/CA125 with specific classes of receptors on cells of the blood system. The modular organization of MUC16/CA125, was analyzed in silico using data taken from the UniProtKB database, which refers to a molecule isolated from the human ovarian cancer cell line, OVCAR-3. Bioinformatic analysis included: determination of homology (using BLAST, Basic Local Alignment Search Tool Tool); investigation of patterns, structural motifs and conserved domains (using databases such as Bgee: a database for Gene Expression and Gene Ontology Evolution, CDD, ELM, BLOCKS, InterProScan and MyHits iProClass); determination of physical and chemical properties, globularity and deviations from the assumed tertiary structure (using ProtParam, ProtScale and GLOBPLOT v2.3 tools); and function prediction using tools that employ several programs: JAFA, ProtFun 2.2 and GeneOntology. Glycobiochemical analysis comprised examination of the influence of CA125 on adhesion and aggregation of erythrocytes, as well as on interactions with different types of leukocyte lectins, namely siglecs [sialic acid-binding immunogobulin (Ig)-like lectin] and C-type lectin-like receptors [selectins, dendritic cell-specific ICAM-3-grabbing non-integrin 1 (DC-SIGN) and macrophage mannose receptor (MMR)]. The effect of CA125 antigen on human erythrocytes was determined using solid phase tests and light microscopy. The interaction of CA125 with leukocyte receptors was investigated employing lectin-blot and immunoblot, as well as binding and inhibition solid phase assays. Cancer antigen isolated from human pleural fluid (pfCA125) and pregnancy-associated antigen (pCA125) were included in the glycobiochemical analysis in addition to antigen from the human ovarian carcinoma cell line (clCA125). The results of in silico analysis placed MUC16/CA125 in the context of modular proteins with an annotated role in adhesion-related processes. They pointed to similarities within extracellular serine/threonine rich regions of MUC16 to protein sequences expressed in evolutionarily distant taxa. No relation to annotated domains from available databases appeared, except for BLLF1, a multidomain characteristic of virus envelope glycoproteins of the Herpesviridae family. Bioinformatics analysis also pointed to a possible correlation between functional activities, which are attributed to serine/threonine rich domains of experimental data on the interactions of MUC16/CA125 with specific classes of receptors on cells of the blood system. The modular organization of MUC16/CA125, was analyzed in silico using data taken from the UniProtKB database, which refers to a molecule isolated from the human ovarian cancer cell line, OVCAR-3. Bioinformatic analysis included: determination of homology (using BLAST, Basic Local Alignment Search Tool Tool); investigation of patterns, structural motifs and conserved domains (using databases such as Bgee: a database for Gene Expression and Gene Ontology Evolution, CDD, ELM, BLOCKS, InterProScan and MyHits iProClass); determination of physical and chemical properties, globularity and deviations from the assumed tertiary structure (using ProtParam, ProtScale and GLOBPLOT v2.3 tools); and function prediction using tools that employ several programs: JAFA, ProtFun 2.2 and GeneOntology. Glycobiochemical analysis comprised examination of the influence of CA125 on adhesion and aggregation of erythrocytes, as well as on interactions with different types of leukocyte lectins, namely siglecs [sialic acid-binding immunogobulin (Ig)-like lectin] and C-type lectin-like receptors [selectins, dendritic cell-specific ICAM-3-grabbing non-integrin 1 (DC-SIGN) and macrophage mannose receptor (MMR)]. The effect of CA125 antigen on human erythrocytes was determined using solid phase tests and light microscopy. The interaction of CA125 with leukocyte receptors was investigated employing lectin-blot and immunoblot, as well as binding and inhibition solid phase assays. Cancer antigen isolated from human pleural fluid (pfCA125) and pregnancy-associated antigen (pCA125) were included in the glycobiochemical analysis in addition to antigen from the human ovarian carcinoma cell line (clCA125). The results of in silico analysis placed MUC16/CA125 in the context of modular proteins with an annotated role in adhesion-related processes. They pointed to similarities within extracellular serine/threonine rich regions of MUC16 to protein sequences expressed in evolutionarily distant taxa. No relation to annotated domains from available databases appeared, except for BLLF1, a multidomain characteristic of virus envelope glycoproteins of the Herpesviridae family. Bioinformatics analysis also pointed to a possible correlation between functional activities, which are attributed to serine/threonine rich domains ofproteins identified in the studied taxa, and possible CA125 antigen function. Based on these results, the role of CA125 antigen in adhesion processes could involve interactions through conserved protein sequences responsible for ion transport, and for interactions with sugar substrates. Considering the mucin nature of CA125 antigen, as well as physiological conditions in which its concentration in serum is increased, the effect of this antigen on human erythrocytes was investigated in the first part of glycobiochemical analysis. The results showed that pCA125 and pfCA125 moderately increased aggregation of erythrocytes, and successfully inhibited their adhesion. In contrast, clCA125 showed little to no modulation of these properties. The second part of the glycobiochemical analysis related to interactions with leukocyte receptors. This showed that, as a ligand, CA125 may interact with different types of proteins that bind sialic acid. Siglecs were found to have specific binding patterns, which more effectively distinguished CA125 antigens of fetal from those of cancer origin, in contrast to the binding patterns of sialic acid-specific plant lectins. The most obvious difference was the ligand capacity of pCA125 antigen towards siglec-7, expressed on dendritic cells, NK (natural killer cells), monocytes and CD8+ T cells, and clCA125 towards siglec-9 and siglec-10 on B cells, NK cells, monocytes, neutrophils and CD8+ T cells. Unlike siglecs, all three investigated selectins: L-selectin, E-selectin and P-selectin, interacted in a dose-dependent manner with pfCA125, and to a lesser extent with pCA125, but not with clCA125. Moreover, P-selectin reacted at significantly lower concentrations than L- or E-selectin. Thus, CA125 antigen would preferentially interact with activated endothelial cells or platelets expressing P-selectin, compared to cells expressing the other types of selectin. The interaction of CA125 antigen and DC-SIGN, which is specific for mannan, high-mannose glycans and glycans with terminal Lex / Ley / Lea / Leb antigens was studied. DC-SIGN is expressed on dendritic cells (DC) and recognized pCA125 and pfCA125 antigen. Inhibition of DC-SIGN binding to pCA125 was dependent on the presence of mannose-rich glycans, in contrast to the binding to pfCA125, which was not affected by the presence of N- or O-linked glycans, all which can modify their discrete receptor functions. Macrophage mannose receptor (MMR), which is expressed on macrophages and DC, and which binds terminal mannose, fucose and sialyl Lex, did not react with any of the tested CA125 antigens. C-type lectin-like receptors play an important role in the immune system, mediating in cell-cell interactions, i.e. contacts between leukocytes or between leukocytes and endothelium,and in the binding of pathogens. Glycosylation changes in ligands for C-type lectin-like receptors directly influence their activity and specificity and have important effects on the development, survival and reactivity of cells of the blood and immune systems. Based on the results of this study, differences in the structure/glycosylation of CA125 antigens associated with different physiological or pathological conditions, could alter their influence on the human blood cells, and have important biomedical implications in different microenvironments. Further insight into the nature and mechanisms of CA125 multifunctionality requires an interdisciplinary approach, based on complex interactions that are mediated by its different structural domains.

Biogeografska i ekološka studija flore briofita Srbije

Biogeographic and ecological study of bryophyte flora of Serbia

Briološka istraživanja u Srbiji su istorijski nedovoljno zastupljena. Čak 25.559 literaturnih i herbarskih podataka o briofitama zabeleženim u Srbiji, od početka istraživanja briofita u Srbiji pa do danas, sakupljeno je u novoformiranu Bryo bazu. Prikupljeni podaci poslužili su za obimne komparativne biogeografske i ekološke analize. Brioflora Srbije broji 820 taksona (1 rožnjača, 143 jetrenjače i 686 mahovina). Od toga, 17 taksona je isključeno sa liste vrsta Srbije, a još 7 su označeni kao sumnjivi taksoni čije prisustvo u Srbiji je malo verovatno ili nepotvrđeno. Za svaki takson izrađene su mape rasprostranjenja u Srbiji. Analizom podataka utvrđeno je da je skoro polovina taksona briofita u Srbiji zabeleženo na manje od 5 lokaliteta, a čak 18% ima samo lokalno potvrđeno rasprostranjenje. Najbogatiji vrstama su regioni Istočne, Zapadne i Centralne Srbije, a najsiromašniji su Bački i Pomoravski region. Analizom distribucije po administrativnim okruzima i UTM 10x10 kvadratima jasno je da prostor Srbije nije ujednačeno istraživan, a kao prioritet za dalja briološka istraživanja izdvojeno je 6 regiona i 8 okruga. Analiza spektara areal tipova pokazuje veliku sličnost sa biogeografskim spektrom briofita na Balkanskom poluostvu. Detaljnijom biogeografskom analizom uočavaju se posebnosti brioflore različitih delova Srbije. Razmatrani ekološki faktori dodatno ističu specifičnosti pojedinih oblasti Srbije, pojašnjavaju prisustvo određenog taksona, ali i omogućavaju predikciju briotaksona na neistraženim područjima Srbije. Listaste jetrenjače preferiraju donekle umerenija staništa u odnosu na grupu talusnih jetrenjača koje su češće u manje gostoljubivim uslovima. U poređenju sa mahovinama, jetrenjače preferiraju (mikro)staništa sa manjom prosečnom godišnjom temperaturom.

Bryological exploration in Serbia has been historically neglected. All together 25.559 literature and herbarium data on bryophytes recorded in Serbia, from the beginning of the exploration up to date, were collected in the newly formed Bryo database. Data collection was used for extensive comparative biogeographical and ecological analyses. The bryoflora of Serbia numbers 820 taxa (1 hornwort, 143 liverworts and 686 mosses). Out of this, 17 taxa were excluded from the checklist of Serbia, and another 7 were determined as doubtful taxa whose presence in Serbia is unlikely or unverified. For each taxa, the distribution maps in Serbia were made. Data analyses showed that almost half of the Serbian taxa were recorded in less than 5 localities, and 18% have only local occurrence. Eastern, Western and Central Serbia are the regions richest in bryophyte species, while the poorest are regions of Bačka and Pomoravlje. Distribution analyses by counties and UTM 10x10 km squares showed that the territory of Serbia is unequally bryologically investigated. Hence, 6 regions and 8 counties due to any or very few bryophyte records should be high priority for further bryological research. The analyses of the areal type spectra showed a great similarity to the biogeographical spectrum of the Balkan bryophyte flora. A more detailed biogeographical analysis revealed the specific features of the bryoflora in various parts of Serbia. The studied ecological factors additionally emphasize the peculiarities of certain areas of Serbia, also in terms of species composition. These allow prediction of bryotaxa in unexplored areas as well. Leafy liverworts prefer somewhat milder habitats compared to a group of talus liverwort species, which are more often found in harsh conditions. Compared to sister moss group, liverworts prefer (micro)habitats with the lower average annual temperatures.

Rasprostranjenje, ekologija i centri diverziteta slepih miševa (Mammalia, Chiroptera) u Srbiji

Distribution, ecology and centres of bat diversity (Mammalia, Chiroptera) in Serbia

U poslednjih šezdeset godina slepi miševi u Srbiji su veoma često bili objekti kraćih i/ili dugotrajnijih istraživanja, a rezultati su do sada samo sporadično objedinjavani i objavljivani. Cilj ove disertacije je objedinjavanje podataka o fauni slepih miševa na području Srbije, analiza i vrednovanje dominantnih ekoloških faktora koji generišu distribuciju faune, karakterizacija preferiranih tipova staništa i skloništa, analiza aktuelnog rasprostranjenja vrsta, analiza strukture faune i identifikacija centara diverziteta, procena statusa ugroženosti i predlog mera zaštite vrsta u Srbiji. Slepi miševi su po nizu ekološko-zoogeografskih pokazatelja među najugroženijim i najmanje poznatim sisarima, te je sumiranje saznanja, analiza i interpretacija podataka o rasprostranjenju preduslov za utvrđivanje ekoloških i zoogeografskih determinanti njihove raznovrsnosti kao osnova za izradu strategije očuvanja i zaštite vrsta, staništa i skloništa slepih miševa u Srbiji. U radu je izvršeno i definisanje areala svake pojedinačne vrste u Srbiji, utvrđivanje obrazaca vertikalne i horizontalne distribucije vrsta u odnosu na dominantne ekološke gradijente, definisanje karakterističnih bionomskih pokazatelja za svaku vrstu, utvrđivanje preferencije staništa i skloništa vrsta po fenološkim fazama tokom životnog ciklusa, procena brojnosti i populacionih trendova vrsta u Srbiji, analiza diverziteta slepih miševa Srbije po prostornim jedinicama, definisanje ekološkog statusa vrsta u Srbiji i nova procena statusa ugroženosti prema IUCN standardima i predlaganje mera očuvanja i zaštite vrsta, staništa i skloništa. Rasprostranjenje trideset vrsta slepih miševa, za koje je utvrđeno da žive u Srbiji, je mapirano u kvadratnim poljima osnove 10 km u okviru UTM projekcione mreže, i to: Rhinolophus hipposideros, Rh. ferrumequinum, Rh. euryale, Rh. mehelyi, Rh. blasii, Myotis daubentonii, M. dasycneme, M. capaccinii, M. brandtii, M. mystacinus, M. alcathoe, M. nattereri, M. emarginatus, M. bechsteinii, M. myotis, M. oxygnathus, Nyctalus noctula, N. leisleri, Pipistrellus pipistrellus, P. pygmaeus, P. nathusii, P. kuhlii, Hypsugo savii, Vespertilio murinus, Eptesicus serotinus, Barbastella barbastellus, Plecotus auritus, P. macrobullaris, P. austriacus i Miniopterus schreibersii. Liste nalaza su date za svaku vrstu u okviru kojih i svi prikupljeni podaci o jedinkama, kolonijama, datumu, nalazištu, staništu, skloništu, nadmorskoj visini, UTM kvadratnom polju, kolektorskom/inventarskom broju, ustanovi gde se dokazni primerak ili podatak o nalazu čuva, nalazaču, identifikatoru i eventualnoj litaraturnoj jedinici koja opisuje nalaz. Broj nalaza varira u okviru vrsta, i to od jednog (Plecotus macrobullaris) do 325 (Rhinolophus ferrumequinum). Za svaku vrstu posebno su dati opšti bionomski podaci, populacioni status, faktori ugrožavanja, zakonska zaštita, nacionalni i međunarodni status zaštite, te mere očuvanja. Tako prikazani podaci su sintetizovani, analizirani i vrednovani. Analiza rasprostranjenja nalazišta vrsta slepih miševa u Srbiji vršena je najpre po UTM kvadratnim poljima, po administrativnim celinama i po širim hipsometrijskim oblastima. Ukupno je registrovano 605 nalazišta. U UTM kvadratnim poljima je beleženo od jednog do osmadeset nalazišta. Od ukupno 2168 nalaza, među kojima dominiraju skloništa (70 %) i mesta preleta/koridori (20 %), 62 % je zabeleženo nakon 1990. godine. Na 126 nalazišta su registrovane porodiljske kolonije, na 273 zimske, a na 34 kopulatorne. Izvršena je analiza širine areala vrsta, pri čemu je nađeno da samo 4 od 30 vrsta imaju usko rasprostranjenje. Visinsko rasprostranjenje vrsta je sintetski obrađeno, pri čemu je najniža nadmorska visina nalazišta bila 40 m, najviša 1776 m, a prosečna 300 m. Oko 70 % nalazišta vrsta je grupisano u visinskom opsegu od 0 do 400 m, a oko 45 % u opsegu od 0 do 200 m. Šezdeset dva nalazišta su izdvojena kao značajna nakon vrednovanja prema tipovima i strukturi. Ukupno je prepoznato 6 tipova značajnih nalazišta, među kojima su dominantna podzemna prirodna skloništa (77 %). Utvrđena je pretežna stanišna eurivalentnost i izvršena multivarijantna analiza preferencije staništa. Utvrđena su tri dominantna gradijenta – prvi dvopolni kojeg čine termofilna otvorena staništa na karstnim formacijama i mezofilna šumska staništa sa različitim tipovima prirodnih ili veštačkih vodenih površina, drugi kojeg čine urbana, semiurbana i ruralna staništa i treći kojeg čine kombinacije parcijalnih kontrasta pojedinačnih parova staništa. Na svim gradijentima se ne vidi jasna ordinacija vrsta. Primenom dvosmerne simultane klasifikacije utvrđena su tri obrasca preferencije staništa – termofilna otvorena i poluotvorena staništa na krečnjačkim formacijama sa svojim stenotopnim vrstama, vodena staništa u šumskim i poljoprivrednim ekosistemima sa svojim setom vrsta i prirodna i veštačka vodena staništa sa mezofilnim šumskim, urbanim i semiurbanim staništima sa grupom euritopnih vrsta. Od 605 nalazišta 82 % su skloništa, od čega du dominantna antropogena (45 %), a subdominantna prirodna podzemna skloništa (32 %). Široku sklonišnu valencu pokazuju samo tri vrste, usku sedam vrsta, a ambivalentnu sve ostale. Centri diverziteta su analizirani na nivou broja vrsta po jedinici površine, na kvadratima osnove 10 km, 20 km, 50 km i 100 km, pri čemu su prikazane analize za najmanje i najveće kvadrate. Takođe je izvršena analiza diverziteta po regionima Srbije, kao i ad hoc ekspertska procena centara diverziteta. Poređenjem rezultata, utvrđeno je da regionalni pristup daje najbolju procenu centara diverziteta, kao i najbolju interpretaciju eko-geografskih determinanti faune slepih miševa Srbije. Takođe su uočene saglasnosti sa ekspertskom procenom koja je bila osnov dosadašnjih studija. Nakon primene metode divizionog klasifikovanja celokupnog skupa podataka o vrstama po regionima Srbije, iteracijom metode k-means grupisanja dobijeno je i interpretirano 7 centara diverziteta. Ovim nalazima su potvrđeni prethodni nalazi o dva glavna centra diverziteta u regionima severoistočne i severozapadne Srbije, potvrđen je i vrednovan nedavno otkriven novi centar u beogradskom mikroregiji, i otkrivena su i vrednovana dva nova centra – regioni zapadne i jugoistočne Srbije, koji nisu bili očigledni na osnovu dostupnog seta podataka. Analize faktora ugrožavanja, mera očuvanja, antropogenog stresa i neophodnih i nedostajućih istraživanja, prema IUCN klasifikacionim šemama, su upotrebljene za definisanje statusa ugroženosti vrsta. Data je i hronologija nacionalne zakonske zaštite i kategorija ugroženosti, kao i pregled međunarodnih konvencija i sporazuma relevantnih za slepe miševe. Detaljno je prikazana analiza vrsta po eko-geografskim pokazateljima stanja i ugroženosti. Na kraju su predložene neophodne mere očuvanja slepih miševa Srbije iskazane u vidu preporuka kao osnova za strategiju očuvanja i zaštite pripadnika ovog reda sisara u Srbiji.

During the last sixty years bats have often been a subject of short- and long-term research but so far the results have been synthesised and even published only sporadically. The aim of this study is to integrate data on bat fauna of Serbia, to analyse and evaluate prevailing ecological parameters determining distribution of bat fauna, to determine preferred types of habitats and roost sites, to analyse present distribution of species, to analyse the composition of bat fauna and to identify centres of diversity, to asses conservation status and to propose measures for species protection in Serbia. According to number of ecological-zoogeographic indicators, bats are among the most vulnerable and least known mammals; therefore, summation of knowledge, analysis and interpretation of data on distribution, is prerequisite for identification of the ecological and zoogeographic determinants of bat diversity as a basis for development of the strategy for conservation and protection of bat species, habitats and roosts in Serbia. This study also: defines range of each species in Serbia, identifies patterns of vertical and horizontal distribution of species in relation to the dominant ecological gradients, defines characteristic bionomic indicators for each species, identifies species’ habitat and roost preference per phenophases, assesses abundance and population trends of species in Serbia, analyses bat diversity of Serbia by spatial units, defines ecological status of species in Serbia, presents new assessment of species’ conservation status according to IUCN system, proposes measures for conservation and protection of species, habitats and roosts. Distribution of thirty bat species which have been found in Serbia is mapped by 10x10 km squares of the UTM projection grid, namely: Rhinolophus hipposideros, Rh. ferrumequinum, Rh. euryale, Rh. mehelyi, Rh. blasii, Myotis daubentonii, M. dasycneme, M. capaccinii, M. brandtii, M. mystacinus, M. alcathoe, M. nattereri, M. emarginatus, M. bechsteinii, M. myotis, M. oxygnathus, Nyctalus noctula, N. leisleri, Pipistrellus pipistrellus, P. pygmaeus, P. nathusii, P. kuhlii, Hypsugo savii, Vespertilio murinus, Eptesicus serotinus, Barbastella barbastellus, Plecotus auritus, P. macrobullaris, P. austriacus and Miniopterus schreibersii. Findings are listed for each species, containing all the data on individuals, colonies, date, locality, habitat, roost site, altitude, UTM grid square, the collector / inventory number, institution keeping a voucher specimen or information on finding, collector name, identifier name and possible literature unit describing the finding. Number of sites varies between species – from single one (Plecotus macrobullaris) to 325 (Rhinolophus ferrumequinum). General bionomical data, population status, threats, legal protection, national and international status of protection as well as conservation measures are also given for each species. All those presented data are synthesised, analysed and evaluated. Distribution of bat findings in Serbia is analysed by UTM grid squares, first of all, and then by administrative units and by broader hypsometrical areas. A total of 605 sites are registered. One to eighty sites are recorded per single UTM grid squares. 62 % of the total 2,168 findings, mostly roosts (70 %) and flight paths / commuting routes (20 %), were recorded after 1990. At 126 sites nursery colonies are registered, at 273 winter colonies while at 34 sites mating colonies are found. The width of the species distribution range is analysed and it was found that only 4 out of 30 species have a narrow distribution. Altitudinal distribution of species is synthesised, with the lowest locality elevation of 40 m, 1,776 m of the highest and an average of 300 m. About 70 % of all species sites are grouped in the elevation range of 0 to 400 m, and about 45 % in the range of 0 to 200 m. Sixty-two sites are identified as important after evaluation according to type and structure. A total of 6 types of important sites are distinguished, including predominant natural underground roost sites (77 %). The most species were found to have broad ecological amplitude regarding habitat and multivariate analysis of habitat preference were performed. Three dominant gradients were identified – the first bipolar consisting of thermophilic open habitats on karst formations and mesophilic forest habitats with different types of natural or artificial bodies of water, the other made up of urban, semiurban and rural habitats and the third consisting of a combination of partial contrasts of particular habitat pairs. In all the gradients clear ordination of species is not shown. Using simultaneous two-way classification three patterns of habitat preference are identified – thermophilic open and semi-open habitats on limestone formations with their stenotopic species, aquatic habitats in forest and agricultural ecosystems with their set of species and natural and artificial aquatic habitats within mesophilic forest, urban and semiurban habitats with eurytopic group of species. 82 % of the 605 sites are roost sites, anthropogenic being dominant (45 %) and natural underground roost sites subdominant (32 %). Only three species have broad ecological amplitude regarding roosts, it is narrow for seven of them while all the rest are ambivalent. The centres of diversity were analysed as number of species per area unit, by 10 km, 20 km, 50 km and 100 km squares, with the analysis by the smallest and by the largest squares presented. Bat diversity of the regions of Serbia is also analysed and ad hoc expert assessment of centres of diversity made. Comparing the results, it was found that a regional approach yields the best estimate of the centres of diversity, as well as the best interpretation of the eco- geographical determinants of Serbian bat fauna. Accordance with the expert assessment which was the basis of previous studies is also noted. After division classification method was applied with the entire data set of species by regions of Serbia, iteration of k-means clustering method identified 7 centres of diversity. These findings confirmed previous findings on the two main centres of diversity in the regions of Northeastern and Northwestern Serbia, they also confirmed and evaluated recently discovered new centre in Belgrade micro-region, while two new centres were discovered and evaluated - regions of Western and Southeastern Serbia, which were not apparent based on the available data set. Analyses of threats, conservation measures, anthropogenic stress and research needed, according to the IUCN classification schemes, are used to define species conservation status. Chronology of national legal protection and red list categories is also given, as well as an overview of international conventions and agreements relevant to bats. Detailed analysis of species by eco-geographical status indicators and vulnerability is given. At the end, necessary measures for conservation of bats in Serbia are proposed as recommendations and a basis of the strategy for conservation and protection of bats in Serbia.

Hipofizno-ovarijalni sistem pacova od fetalnog do peripubertalnog perioda života nakon intrauterinog izlaganja deksametazonu

Pituitary-ovarian axis of rats from fetal to peripubertal period of life after intrauterine exposure to dexamethasone

Tokom fetalnog razvića organizam prolazi kroz “kritične periode”, tokom kojih se dešavaju intenzivne ćelijske deobe, razvijanje i sazrevanje tkiva, organa i organskih sistema. Sredina u kojoj fetus raste i razvija se u velikoj meri odreñuje kvalitet razvojnog procesa, a svaka promena uslova dovodi do čitavog niza poremećaja, koji mogu biti praćeni smanjenjem fetalnog rasta ili IUGR (eng. Intrauterine growth retardation). Posledice IUGR su dugotrajne i ispoljavaju se u vidu različitih metaboličkih i kardiovaskularnih oboljenja u kasnijem životu. Fetalno okruženje na taj način odreñuje adultni fenotip, a njihova povezanost proučava se u okviru koncepta programiranja. Najčešće korišćeni eksperimentalni pristupi u proučavanju programiranja i efekta IUGR su dijeta majke koja vodi pothranjenosti, izazivanje stresne reakcije kod majke ili primena antenatalne terapije glukokortikoidima. Svaki od ovih eksperimentalnih modela različitim mehanizmima dovodi do izlaganja fetusa povećanoj koncentraciji glukokortikoida, koji u velikoj meri mogu da poremete fiziološki tok razvojnog procesa. Cilj ove studije je bio da se ustanovi da li je izlaganje fetusa pacova od 16. do 18. dana gestacije sintetičkom glukokortikoidu deksametazonu programiralo razvoj i funkciju hipofizno-ovarijalnog sistema od fetalnog do peripubertalnog perioda života. Predmet istraživanja u ovoj doktorskoj disertaciji bili su fetusi i ženke pacova koje su od 16. do 18. dana gestacije izloženi deksametazonu. Gravidne ženke pacova su tri uzastopna dana (od 16. do 18. dana gestacije) subkutano tretirane deksametazonom (Dexamethasonis phosphat - Dx, Krka, p.o., Novo Mesto) rastvorenom u fiziološkom rastvoru (0.9% NaCl), u dozi od 1.0, 0.5 i 0.5 mg Dx/kg t.m./dan. Kontrolna grupa gravidnih ženki primala je u istom periodu, adekvatnu zapreminu fiziološkog rastvora. Potomci gravidnih ženki koje su primale deksametazon (Dx), odnosno fiziološki rastvor (K) žrtvovani su u fetalnom (19. i 21. fetalni dan), neonatalnom (5. dan), infantilnom (16. dan) i peripubertalnom (38. dan) periodu života. Za obeležavanje gonadotropnih (FSH i LH) ćelija hipofize korišćeno je imunocitohemijsko bojenje, dok su preseci jajnika bojeni histološkim bojenjem hematoksilin & eozin. Obojeni preseci hipofiza i jajnika stereološki i morfometrijski su analizirani, uz pomoć newCast stereološkog softverskog paketa. Dobijene su vrednosti volumena hipofize i jajnika, kao i apsolutni broj FSH i LH ćelija, njihov volumen, volumenska i numerička gustina. Koncentracija FSH i LH u cirkulaciji odreñena je biohemijskom RIA metodom. Pored volumena, u fetalnom jajniku odreñen je i apsolutni broj zdravih i degenerisanih germinativnih ćelija, a u jajniku ženki u postnatalnom periodu broj zdravih i atretičnih primordijalnih, primarnih, višeslojnih i antralnih folikula, kao i broj žutih tela. Dobijeni rezultati su statistički obrañeni. Intrauterino izlaganje deksametazonu izazvalo je IUGR (eng. intrauterine growth retardation) koje se manifestuje značajno smanjenom (p<0.05) telesnom masom kod fetusa starih 21 dan, a značajno smanjenje (p<0.05) telesne mase zabeleženo je i u postnatalnim periodima Volumen hipofize i adenohipoze značajno su smanjeni (p<0.05) u fetalnom, neonatalnom, infantilnom i peripubertalnom periodu života. Analizirani stereološki parametri su se po istom obrascu menjali i kod FSH i kod LH ćelija, kod ženki pacova intrauterino izloženih deksametazonu u odnosu na kontrolu. Tako je broj gonadotropnih ćelija značajno smanjen (p<0.05) od fetalnog, preko neonatalnog, infantilnog do peripubertalnog perioda života, a volumen je značajno smanjen (p<0.05) samo 19. fetalnog dana. Volumenska i numerička gustina gonadotropnih ćelija značajno su smanjene (p<0.05) u neonatalnom i infantilnom periodu života. Koncentracije FSH i LH su značajno smanjene (p<0.05) u neonatalnom, infantilnom i peripubertalnom periodu života kod ženki intrauterino izloženih deksametazonu u odnosu na kontrolu.. Nakon intrauterinog izlaganja deksametazonu volumen jajnika se značajno smanjio (p<0.05) u svim ispitivanim periodima života u odnosu na kontrolne vrednosti. Jajnik ženki pacova u fetalnom periodu nema definitivnu histološku organizaciju, tako da se u njima uočava prisustvo zdravih i degenerisanih germinativnih ćelija, i somatskih ćelija. Kvantifikacijom germinativnih ćelija, ustanovljeno je da je deksametazon u jajniku 19 dana starih fetusa uticao na značajno povećanje (p<0.05) broja degenerisanih na račun zdravih germinativnih ćelija, dok ukupan broj germinativnih ćelija nije promenjen u odnosu na kontrolu. U jajniku 21 dan starih fetusa, deksametazon je uticao na značajno smanjenje (p<0.05) ukupnog broja germinativnih ćelija, kao i na značajno smanjenje broja zdravih, a povećanje broja degenerisanih germinativnih ćelija (p<0.05). U jajniku 5 i 16 dana starih ženki pacova intrauterino izloženih deksametazonu prisutne su iste klase zdravih i atretičnih folikula kao i kod kontrolnih životinja, odnosno primordijalni, primarni i višeslojni folikuli, ali u značajno smanjenom broju (p<0.05). Za razliku od jajnika kontrolnih peripubertalnih ženki, u kojima su prisutna žuta tela, u jajnicima peripubertalnih ženki intrauterino izloženih deksametazonu još uvek ih nema. Broj zdravih primordijalnih i primarnih folikula je značajno smanjen (p<0.05), dok je broj analiziranih klasa zdravih i atretičnih višeslojnih i antralnih folikula značajno povećan (p<0.05) u odnosu na kontrolne vrednosti. Rezultati ove studije nedvosmisleno pokazuju da je reproduktivni potencijal ženki koje su tokom fetalnog razvoja bile izložene deksametazonu smanjen, a polno sazrevanje odloženo. S obzirom da je deksametazon uticao u ključnom momentu razvoja hipofize i jajnika u fetalnom periodu, promene koje je izazvao su trajne, i bitno odreñuju kvalitet reproduktivnog života ženki. Ovakav rezultat ima veliki značaj i primenu u humanoj medicini, s obzirom da su uslovi koji do njega mogu dovesti deo svakodnevnog života savremene žene i buduće majke.

During fetal development an organism undergoes “critical periods” of intensive cell division as well as the growth and maturation of tissues, organs and organ systems. The fetal environment principally determines the quality of the developmental process, while variation of conditions may lead to a range of disorders followed by intrauterine growth retardation (IUGR). The consequences of IUGR are long-lasting and manifest through various metabolic and cardiovascular issues in later life. Thus, the fetal environment prejudices the adult phenotype, while their intertwining is elaborated with the programming concept. The most widely exploited experimental approaches in the field of programming and IUGR effects are maternal diet leading to offspring malnutrition, provoking stress to mother and antenatal glucocorticoid therapy. Each of these experimental approaches via different mechanisms leads to fetal exposure to elevated glucocorticoid levels, which may disrupt to a great extent the physiology of the developmental process. The aim of this study was to establish whether the exposure of rat fetuses to synthetic glucocorticoid dexamethasone, during the period between days 16 and 18 of gestation, programmed the development and function of the pituitary-ovarian system, starting from the fetal to the peripubertal period of life. The subject of this doctoral thesis investigation were rat fetuses and females exposed to dexamethasone from the 16th to the 18th gestational day. During three consecutive days (16–18 days of gestation) pregnant rat females were subcutaneously treated with dexamethasone (Dexamethasonis phosphate – Dx, Krka, p.o., Novo Mesto) dissolved in saline (0.9% NaCl), in doses of 1.0, 0.5 and 0.5 mg Dx/kg b.w./day. During the same period a control group of pregnant females received an adequate volume of saline. The offspring of pregnant rat females which received dexamethasone (Dx) or saline (K) were sacrificed during the fetal (19th and 21st fetal day), neonatal (5th day), infantile (16th day) and peripubertal (38th day) period of life. Gonadotropic (FSH and LH) pituitary cells were immunocytochemically labeled, while the ovary sections were histologically stained with hematoxylin and eosin. Thus prepared pituitary and ovary sections were stereologically and morphometrically analyzed using the newCast stereological software package. The values of pituitary and ovary volume were obtained, as well as the absolute number of FSH and LH cells, their volume, volume density and numerical density. FSH and LH blood levels were biochemically (RIA) determined. Besides volume, the absolute numbers of healthy and degenerated cells in the fetal ovary were determined, while the numbers of healthy and atretic primordial, primary, secondary and antral follicles as well as corpora lutea were determined in the postnatal female ovaries. The obtained data were statistically processed. Intrauterine exposure to dexamethasone caused IUGR, manifested with significantly (p<0.05) decreased body weight of 21 days old fetuses, but a significant (p<0.05) decrease of the same parameter was also observed during the postnatal period. Pituitary and its adeno- fraction volumes were significantly (p<0.05) decreased in the fetal, neonatal, infantile and peripubertal period of life. Compared to the controls, the analyzed stereological parameters demonstrated the same pattern of changes in both FSH and LH cells in the female rats exposed in utero to dexamethasone. Thus, the number of gonadotropic cells was significantly (p<0.05) decreased starting from the fetal, during the neonatal, infantile to the peripubertal period of life, while their volume was significantly (p<0.05) decreased only at fetal day 19. The volume and numerical density of gonadotropic cells were significantly (p<0.05) decreased in neonatal and infantile periods of life. Compared to the controls, the FSH and LH blood levels were significantly (p<0.05) decreased in neonatal, infantile and peripubertal periods of life in the female rats exposed in utero to dexamethasone. After intrauterine exposure to dexamethasone, the ovary volume was significantly (p<0.05) decreased in all the examined periods of life, compared to the control values. Female rat ovary, in the fetal period of development, does not have a definitive histological organization; therefore the presence of healthy and degenerated germinative cells, as well as somatic cells can be observed. Quantification of germinative cells showed that, in the ovaries of 19 days old fetuses, dexamethasone caused a significant (p<0.05) increase in degenerated cells number at the expense of the healthy germinative cells pool, while the total number of germinative cells was not changed in comparison with the controls. In the ovaries of 21 days old fetuses, dexamethasone caused a significant (p<0.05) decrease of germinative cells total number, as well as a significant (p<0.05) decrease of healthy and increase of degenerated cells number. In the ovaries of 5 and 16 days old female rats exposed in utero to dexamethasone the same categories of healthy and atretic follicles were present as in the controls, i.e. primordial, primary and secondary follicles, but in a significantly (p<0.05) reduced number. Unlike the control, peripubertal female ovaries which possess corpora lutea are still missing in the ovaries of peripubertal females exposed in utero to dexamethasone. The number of healthy primordial and primary follicles was significantly (p<0.05) decreased, while the number of the analyzed categories of healthy and atretic secondary and antral follicles was significantly (p<0.05) increased, compared to the control values. The results of this study definitely indicate that the reproductive potential of females exposed in utero to dexamethasone is decreased, while their sexual maturation is delayed. Considering that dexamethasone realized its effects at the crucial moment of pituitary and ovary development during the fetal period, the changes caused are permanent, and determine substantially the quality of female reproductive life. Such a result is very important and is applicable in human medicine, since the elaborated conditions are entwined with the everyday life of the modern woman and future mother.

Efekti inhibicije CXCR4 receptora i fokalne adhezione kinaze u supresiji invazije i prevazilaženju rezistencije kod nesitnoćelijskog karcinoma pluća

Effects of CXCR4 receptor and focal adhesion kinase inhibition in suppressing invasion and overcoming drug resistance in non-small cell lung carcinoma.

Rak pluća je najčešće dijagnostikovani tip malignih tumora i vodeći uzrok smrti od malignih oboljenja. Na osnovu kliničke prognoze i odgovora na terapiju čak 85% raka pluća se svrstava u tip nesitnoćelijskog karcinoma pluća (NSCLC). Iako manje agresivan od sitnoćelijskog karcinoma pluća (SCLC), NSCLC lošije odgovara na terapiju. Dva najčešća uzroka neuspeha lečenja NSCLC-a klasičnom hemioterapijom su pojave metastaza i rezistencije na citostatike. Ovi procesi su regulisani brojnim signalnim molekulima, među kojima su i CXC hemokinski receptor 4 (CXCR4) i fokalna adheziona kinaza (FAK), čije ciljano inhibiranje predstavlja obećavajući pristup u lečenju NSCLC-a. Cilj ove studije je bio da se ispita uloga CXCR4 i FAK-a u invazivnosti i rezistenciji NSCLC-a, kao i potencijal inhibicije ovih molekula u supresiji invazije i reverziji rezistencije kod ovog tipa karcinoma pluća. Konkretno, za ispitivanje uloge CXCR4 i FAK-a u invazivnosti NSCLC-a uspostavljen je in vitro sistem ćelija sa različitim funkcionalnim statusom p53 i PTEN tumor supresora, kao i in vivo ortotopni metastatski model NSCLC-a sa p53/PTEN deficijentnim tumorima. U ispitivanom in vitro sistemu istovremena inaktivacija p53 i PTEN tumor supresora dovela je do aktivacije CXCR4 i FAK molekula i nishodnih signalnih molekula AKT-a i ERK-a, što je bilo praćeno povećanjem invazivnog i migratornog potencijala ćelija. Primenom specifičnih inhibitora CXCR4 i FAK-a, WZ811 i PF-573228, pokazan je potencijal primene inhibicije ovih molekula u supresiji invazije i migracije p53-/PTEN- NSCLC ćelija, kao i metastatskog širenja agresivnih p53/PTEN deficijentnih tumora u in vivo modelu. U daljem toku studije za ispitivanje uloge CXCR4 i FAK-a u razvoju rezistencije NSCLC-a na hemioterapiju korišćene su rezistentne ćelijske linije, NCI- H460/R i COR-L23, koje se odlikuju povećanom ekspresijom ABCB1, odnosno ABCC1 transportera. Pokazano je da kombinovani tretmani inhibitorima CXCR4 i FAK-a sa doksorubicinom (DOX) uspešno senzitizuju rezistentne ćelije na pomenuti citostatik. Ispitujući mehanizam reverzije rezistencije utvrđeno je da kombinovani tretmani smanjuju fosforilaciju FAK-a i AKT-a i dovode do aktivacije senescence-a. Istovremeno sa senzitizacijom ćelija na DOX, kombinovanim tretmanima je uspešno suprimirana i invazija rezistentnih ćelija. Imajući u vidu da su rezistencija na terapiju i metataziranje dva najvažnija problema u lečenju NSCLC-a, rezultati studije pokazuju da ciljano inhibiranje CXCR4 i FAK-a, kao i njihova kombinacija sa standardnim hemioterapeuticima, može predstavljati dobar terapeutski pristup u lečenju ovog oboljenja.

Lung cancer is the most commonly diagnosed type of malignant tumor and the leading cause of cancer death. Based on clinical prognosis and terapy response, up to 85% of lung cancer is clasified as non-small cell lung carcinoma (NSCLC). Although NSCLC is less agresive than small cell lung carcinoma (SCLC), its response to terapy is worse then SCLC. Two major causes of terapy failure of NSCLC with classic chemotherapeutics are the development of metastasis and chemotherapy resistance. Among other signaling molecules, CXCR4 receptor and focal adhesion kinase (FAK) are known to regulate these processes and their targeted inhibition represents a promising approach in the treatment of NSCLC. The aim of this study was to examine the role of CXCR4 and FAK in the invasiveness and resistance of NSCLC, as well as the potential of their inhibition in the suppression of invasion and the reversal of resistance in this type of lung carcinoma. In order to examine the role of CXCR4 and FAK in the invasiveness of NSCLC, in vitro system of cancer cell lines with different functional status of the p53 and PTEN tumor suppressors was established, as well as in vivo orthoptic metastatic model of NSCLC with p53/PTEN deficient tumors. The simultaneous inactivation of p53 and PTEN tumor suppressors in in vitro system lead to significant increase in invasive and migratory potential associated with activation of CXCR4 and FAK molecules and their downstream signaling molecules, AKT and ERK. The potential for supressing invasion and migration of p53-/PTEN- NSCLC cells, as well as the metastatic spread of aggressive p53/PTEN deficient tumors in in vivo model, has been shown by the application of specific inhibitors of CXCR4 and FAK, PF-573228 and WZ811. Furthermore, resistant cell lines NCI-H460/R and COR-L23 were used to investigate the role of CXCR4 and FAK in the development of NSCLC resistance to chemotherapy. NCI-H460/R and COR-L23 cells are characterized by an increased expression of ABCB1 and ABCC1 transporters, respectively. Our results additionally indicate that inhibition of CXCR4 and FAK in combination with DOX successfully sensitized NSCLC cells to the aforementioned cytostatic. By examining the mechanism of sensitization of NSCLC cells to DOX, it has been discovered that combined treatments reduced phosphorylation of FAK and AKT and lead to activation of senescence. Simultaneously with sensitization of cells to DOX, combined treatment successfully suppressed the invasion of resistant cells. Considering that resistance to therapy and metastasis are the two most important problems in NSCLC treatment, the results of the study show that targeted inhibition of CXCR4 and FAK, as well as their combination with standard chemotherapeutics, could be a promising therapeutic approach in the treatment of this disease.

Bioindikatorske karakteristike epilitskih silikatnih algi i procena ekološkog statusa odabranih reka zapadne i centralne Srbije

Bioindicative characteristics of epilithic diatoms and ecological status assessment of selected rivers in western and central Serbia

Silikatne alge su opšte prihvaćene kao indikatori procene stanja i promena uslova u vodenim ekosistemima. Osobine koje ih čine odličnim indikatorima su mnogobrojne: globalna rasprostranjenost, naseljavanje različitih mikrostaništa, prisutnost tokom cele godine, kratko vreme generacije individualnih populacija, njihove dobro poznate ekološke valence, standardizovani načini uzorkovanja, pravljenja trajnih preparata i njihovog čuvanja, identifikacija do nivoa vrste i jasno definisan način procene brojnosti. Prvi program monitoringa ekološkog statusa površinskih voda u Republici Srbiji, koji je usklađen sa zahtevima Okvirne direktive o vodama (ODV) započet je 2012. godine. Cilj ove doktorske disertacije je utvrđivanje diverziteta i sezonske dinamike epilitskih zajednica silikatnih algi, formiranje preliminarne liste bioindikatora epilitskih silikatnih algi, predlog potencijalnih korekcija odgovarajućih dijatomnih indeksa za teritoriju Republike Srbije i procena ekološkog statusa odabranih reka zapadne i centralne Srbije. Istraživanjem su obuhvaćene tri reke u zapadnoj Srbiji (Rača, Raška i Studenica) i jedna u centralnoj Srbiji (Rasina). Sakupljanje uzoraka je obavljeno šest puta u toku 2011. i 2012. godine, sa šest lokaliteta u rekama Rači, Rasini i Studenici, odnosno sa pet lokaliteta u reci Raški. Podaci neophodni za izračunavanje indikatorskih vrednosti silikatnih algi su dobijeni iz Agencije za zaštitu životne sredine Republike Srbije. Tokom istraživanog perioda najveći broj taksona u epilitskoj zajednici silikatnih algi je identifikovan u Rasini (251), zatim u Studenici (190), Rači (171), dok je u Raški zabeležen najmanji broj taksona (122). Gomphonema, Navicula i Nitzschia su dominantni rodovi u odnosu na broj taksona u svim istraživanim rekama. Od ukupnog broja identifikovanih taksona 69 je po prvi put zabeleženo za teritoriju Srbije. Među njima, Geissleria gereckei, Navicula splendicula i Navicula moskalii su taksoni koji su retko nalaženi u Evropi. RDA analiza uticaja fizičko-hemijskih parametara na dominantne taksone pokazala je međuzavisnost nitrata i relativne brojnosti Diatoma moniliformis, Gomphonema olivaceum i vrsta roda Cocconeis (C. lineta i C. pseudolineata) u reci Rači, a u Studenici uočena je međuzavisnost nitrata i D. moniliformis i Gomphonema tergestinum. Prema Pravilniku Republike Srbije, a na osnovu rezultata fitobentosa, makroinvertebrata i fizičko-hemijskih rezultata, utvrđeno je da je ekološki status vode istraživanog toka reke Studenice dobar, Rače i Rasine umeren, a ekološki status reke Raške slab. Na osnovu podatka Agencije za zaštitu životne sredine Republike Srbije formirana je preliminarna lista indikatora silikatnih algi za Srbiju, koja obuhvata 80 taksona. Kao potencijalni indeks za procenu trofije u tekućim vodama u Srbiji predložen je trofički dijatomni indeks(TIDRS). Na osnovu naših rezultata predlaže se upotreba IPS indeksa, sa strožijim granicama klasa, i prilagođavanje novog trofičkog indeksa za procenu ekološkog statusa tekućih površinskih voda Srbije.

Diatoms are generally accepted as indicators for ecological status assessment and conditions change in aquatic ecosystems. Features that make them excellent indicators are numerous: worldwide distribution, colonization of different microhabitats, presence throughout the year, short generation time, known autecological characteristics, standardized sampling methods, making the permanent slides, their storage, identification to the level of the species and a clearly defined method of estimating the valve number. According to the Water Framework Directive (WFD), the first program for the monitoring of the ecological status of surface waters in the Republic of Serbia, started in 2012. The aim of this doctoral dissertation is determination of diversity and seasonal dynamics of epilithic diatoms, to form a preliminary list of epilithic diatom indicators, proposal of potential corrections of the appropriate diatom indices for the territory of the Republic of Serbia and ecological status assessment of selected rivers in Western and Central Serbia. The investigation includes three rivers in Western Serbia (Rača, Raška and Studenica) and one in Central Serbia (Rasina). Sampling was carried out six times during 2011 and 2012, on six sites in the rivers Rača, Rasina and Studenica and five in the Raška River. The data necessary for calculating diatom indicator values were obtained from the Serbian Environmental Protection Agency. During the investigated period, the highest number of taxa in the epilithic diatom community was recorded in the Rasina River (251), than in the Sudenica River (190), the Rača River (171), while the smallest number of taxa was recorded in the Raška River (122). The most abundant genera were Gomphonema, Navicula and Nitzschia. Of the total number of identified taxa 69 have been noted for the first time in Serbian diatom flora. Among them, Geissleria gereckei, Navicula splendicula and Navicula moskalii are taxa rarely found in Europe, too. The RDA analysis of the influence physico-chemical parameters on dominant taxa has shown interdependence of nitrates and abundance of Diatoma moniliformis, Gomphonema olivaceum, Cocconeis lineata and C. pseudolineata in the Rača River, while in the Studenica River interdependence was observed between nitrates and abundance of D. moniliformis and Gomphonema tergestinum. According to the legislation of the Republic of Serbia, and based on the results of phytobenthos, macroinvertebrates and physico-chemical results, ecological status of the water of the investigated part of the Studenica River is good, water of the Rača and Rasina rivers moderate and ecological status of the Raška River was bad. Based on the data of the Serbian Environmental Protection Agency, a preliminary list of epilithic diatom indicators for Serbia was formed, and includes 80 taxa. A trophic diatoms index (TIDRS) has been developed as a potential index for trophic assessment of running waters in Serbia. Based on our results, it is proposed use the IPS index, with more stringent class boundaries, and calibrate the new trophic index for the ecological status assessment of the surface waters in Serbia.

Uloga angiotenzina II i reaktivnih vrsta kiseonika tokom razvoja akutne bubrežne insuficijencije u eksperimentalnoj hipertenziji

The role of angiotensin II and reactive oxygen species during the acute renal failure development in experimental hypertension

Akutna bubrežna insuficijencija (ABI) se definiše kao nagli gubitak bubrežne funkcije. Uzroci nastanka ABI su mnogobrojni, a mehanizmi razvoja još uvek nedovoljno jasni. Karakterišu je iznenadna pojava, brzi tok, neizvesna i često loša prognoza krajnjeg ishoda bolesti. ABI je česta pojava kod hospitalizovanih pacijenata, a učestalost njenog nastanka na odeljenjima intenzivne nege se kreće do 30%. Udružena sa drugim oboljenjima, poput hipertenzije, uzrokuje visok stepen mortaliteta. Savremena istraživanja pokazuju da hipertenzija i oksidativni stres imaju važnu ulogu u progresiji bubrežnih oboljenja, što je usmerilo i stavilo akcenat naših istraživanja na uloge ova dva patogenetska faktora u nastanku i progresiji ABI. Sistem renin angiotenzin aldosteron (RAAS) ima značajnu ulogu u održanju krvnog pritiska i homeostatskih mehanizama bubrega. On u velikoj meri reguliše bubrežnu hemodinamiku i vaskularnu reaktivnost, a kako je intrarenalna vazokonstrikcija jedan od glavnih mehanizama razvoja ABI, očita je njegova uloga u patogenetskim mehanizmima nastanka ove bolesti. Oksidativni stres predstavlja disbalans između produkcije reaktivnih vrsta kiseonika (RVK) i sposobnosti biološkog sistema da ih uklanja ili da reparira oštećenja nastala produkcijom ovih molekula. Reaktivne vrste kiseonika su izuzetno reaktivne supstance usled postojanja nesparenih elektrona, i kao takve mogu da uzrokuju oštećenja na DNK, RNK, kao i nekim proteinima koji imaju ulogu u procesu starenja. Pokazano je da oksidativni stres ima veliku ulogu u razvoju mnogih oboljenja, kao što su Alchajmerova i Parkinsonova bolest, i neki kardiovaskularni poremećaji. Takodje je dokazano da, prilikom ishemičnih povreda organa i tkiva, nakon hipoksije, a tokom reperfuzije, dolazi do povećane produkcije molekula RVK. Iz pomenutih razloga, sasvim je izvesno da u ishemičnoj formi ABI povećana koncentracija ovih molekula, doprinosi nastanku i razvoju ovog teškog oboljenja. Kako je poslednjih godina u svetu sve veći broj hipertenzivnih pacijenata, a sa druge strane nastanak ABI predstavlja ozbiljan klinički problem, postavili smo za cilj da ispitamo ulogu angiotenzina II (AngII) i RVK u mehanizmima nastanka ishemične ABI u uslovima hipertenzijie. Time bi bar delimično doprineli razumevanju nastanka ABI u pomenutim uslovima, što posledično može dovesti do uspešnijeg terapijskog pristupa u lečenju ove bolesti kod hipertenzivnih pacijenata, koji dožive epizodu sa razvojem ABI. Radi ostvarivanja postavljenih ciljeva koristili smo pacove sa urođenom hipertenzijom (SHR – engl. Spontaneously hypertensive rat) odgajane u Institutu za medicinska istraživanja, Univerziteta u Beogradu. Životinje, muškog pola, su bile telesne mase od oko 300g, hranjene su standardnom hranom za laboratorijske životinje (Veterinarski zavod Subotica, Srbija), i imale su slobodan pristup vodi. Akutna bubrežna insuficijencija, ishemičnog tipa, izazivana je klemovanjem leve bubrežne arterije, dok je desni bubreg bio hirurški odstranjivan. Životinjama smo davali: Losartan (Antagonist AT1 receptora za angiotenzin II), u dozi od 10mg/kg t.m., rastvaran u 4 ml fiziološkog rastvora Tempol (4-hidroksi-2,2,6,6-tetrametilpiperidin-1-15N-oksil, mimetik enzima superoksid dismutaze) 40mg/kg/h t.m., rastvaran u 4 ml fiziološkog rastvora Životinje su podeljene u četiri grupe: 1. kontrolna grupa lažno operisanih pacova (SHAM); 2. kontrolna grupa sa akutnom bubrežnom insuficijencijom (ABI); 3. grupa životinja koje su dobijale infuziju losartana u toku i posle ABI (ABI+LOS); 4. grupa životinja koje su dobijale infuziju tempola za vreme i posle ABI (ABI+TEMP); Kontrolne grupe SHAM i ABI primale su infuziju 4 ml fiziološkog rastvora. Infuzija je otpočinjala 5 min pre uklanjanja netraumatske kleme i trajala narednih 175 min. Sva hemodinamska merenja kao i uzorci za biohemijska i histopatološka ispitivanja uzimana su 24 časa nakon završenog tretmana. Naši rezultati su pokazali da se kod pacova sa ABI bubrežni protok smanjuje, a bubrežni otpor naglo povećava u odnosu na grupu lažno operisanih životinja. Ovi rezultati ukazuju na snažnu intrarenalnu vazokonstrikciju u postishemičnom bubregu, što predstavlja jedan od glavnih uzroka razvoja ABI. Blokada receptora za Ang II dovela je do povećanja protoka i smanjenja otpora kroz bubrežnu arteriju. Tretman tempolom snizio je otpor protoku krvi kroz bubrežnu arteriju, što je rezultiralo blagim povećanjem protoka. Biohemijski pokazatelji bubrežne funkcije (kreatinin, urea i neorganski fosfati u plazmi), značajno su povećani u odnosu na fiziološke okvire nakon izazivanja ishemičnog tipa ABI. Tretman blokatorom AT1 receptora u značajnoj meri snižava ove biohemijske parametre, što poboljšava bubrežnu funkciju u odnosu na grupu kod koje je izazvana ishemična ABI. Uklanjanje superoksidnog anjona, sa druge strane, nije dovelo do značajnih promena u ovim parametrima u postishemičnom modelu ABI, udružene sa hipertenzijom. ABI dovodi do povećanja lipidne peroksidacije u plazmi hipertenzivnih pacova 24h nakon reperfuzije. Blokada RAAS, kao i uklanjanje RVK dovodi do smanjenja lipidne peroksidacije u plazmi SHR sa razvijenom ishemičnom ABI. Naši rezultati pokazuju da postishemično povećanje nivoa AngII u hipertenzivnim uslovima ima značajnu ulogu u nastanku tubulo-intersticijskih oštećenja. Blokada AT1 receptora dovodi do značajnog poboljšanja morfološkog izgleda postishemičnog bubrega. Sa druge strane, upotreba sakupljača superoksidnog anjona - tempola, nije imala bitan efekat na histološku sliku postishemičnog hipertenzivnog bubrega pacova. Opšti zaključak našeg rada ukazuje da postishemično povećanje nivoa AngII u hipertenzivnim uslovima, značajno utiče na razvoj renalne vazokonstrikcije i regulaciju glomerulske filtracije, kao i u nastanku tubulo-intersticijskih oštećenja u bubregu. Superoksidni anjon nema posebno izražen efekat u nastanku i održanju ABI kod hipertenzivnih pacova, ali zbog prirode ishemično-reperfuzionih oštećenja, svakako treba ispitati ulogu ostalih RVK. U cilju potpunijeg razumevanja uloge AngII i RVK kod hipertenzivnih pacijenata kod kojih dolazi do razvoja ishemične ABI, neophodno je uzeti u obzir i druge okolnosti kao što su godine života, vaskularna oboljenja, prethodno nastala bubrežna oštećenja, druge individualne karakteristike pacijenta i sprovesti mnogo detaljnija klinička istraživanja.

Acute renal failure (ARF) is defined as a sudden loss of renal function. It’s multifactorially caused, but the mechanism of pathogenesis and developement of this disease is still uncomplitely defined. ARF is characterized by sudden appearance, rapid progression of disease and very uncertain and often fatal outcome. ARF frequently occurres with hospitalized patients. The frequency of its occurrence in the intensive care units moves up to 30%. Associated with other diseases, such as hypertension, it causes high rate mortality. Recent studies show that hypertension and oxidative stress have an important role in the renal disease progression. This fact directed the focus of our study towards the influence of these two pathogenetic factors in development and progression of ARF. The system rennin angiotensin aldosterone (RAAS) has a great role in blood pressure control and kidney homeostatic mechanisms. It mostly regulates the renal hemodynamic, but considering the fact that the intrarenal vasoconstriction is one of the major mechanisms of ARF development, it obviously influences the pathogenetic mechanisms in the development of this disorders. Oxidative stress is defined as a disbalance between the production of reactive oxygen species (ROS) and an ability of biological system to remove or fix the damage made due to production of these molecules. Reactive oxygen species are highly reactive due to presence of unpaired electrons and consequently lead to damage of DNA, RNA and some other ageing related proteins. There is evidence that the oxidative stress has a great influence in the progression of numerous diseases, such as Alzheimer’s, Parkinson’s, as well as some cardiovascular disorders. Furthermore, during the ischemia/reperfusion injuries of organs and tissues, there is increased reactive oxygen species molecule production. Thus, the ischemic type of ARF is characterized by a high concentration of these molecules which contribute to the development of this devastating illnes. Considering the rising number of hypertensive patients in the world, as well as the fact that the ARF is a serious clinical problem, we believe that it is important to investigate the role of angiotensin II and reactive oxygen species in the development of ischemic ARF, during the hypertension. The goal of our research is to contribute to understanding the occurrence of ARF in these conditions which can lead to more successful therapeutic approach to treating this disease. In our study we used male adult spontaneously hypertensive rats (SHR), 24 weeks old, weighing about 300 g, which were bred in the Institute for Medical Research, Belgrade, Serbia, and fed with a standard chow for laboratory rats (Veterinary Institute JSC Subotica). The ischemic type of acute renal failure was induced by clamping the left renal artery, while the right kidney was surgically removed. Animals were given:  Losartan (angiotensin II type 1 receptor antagonist), at a dose of 10mg/kg body weight, dissolved in 4 ml saline;  Tempol (4-Hydroxy- 2,2,6,6-tetramethylpiperidine-1-15N-oxyl, superoxide dismutase mimetic) 40mg/kg/h tm, dissolved in 4 ml of saline; The animals were divided into four groups: 1- group of sham operated rats (SHAM); 2 - group with acute renal failure (ARF); 3 - group of animals that received an infusion of losartan during and after ARF (ABI+LOS); 4 - group of animals that received the infusion of tempol during and after ARF (ABI+TEMP). SHAM and group with ARF were treated by infusion of 4 ml of saline. The infusion began 5 minutes before removing nontraumatic clamps and took the next 175 min. All hemodynamic measurements and samples for biochemical and histopathological examination were taken 24 hours after completion of treatment. Twenty-four hours after ARF induction renal blood flow (RBF) was significantly reduced followed as consequence increased renal vascular resistance (RVR) in postischemic SHR. These results indicate a strong intrarenal vasoconstriction in the ischemic kidney, which is one of the main factors involved in the initiation and maintenance of ARF. Blockade of Ang II leads to an increase in RBF and reduced RVR through the renal artery. Tempol treatment reduces RVR, but only slightly affects the increasing of flow through the renal artery. Biochemical markers of renal function (creatinine, urea and inorganic phosphates in plasma), were extremely aggravated after causing the ischemic type of ARF. AT1 receptor blocker treatment significantly improved the biochemical parameters of renal function. Removal of superoxide anions, on the other hand, has not led to significant changes in these parameters in the present model of ischemic ARF, associated with hypertension. Development of ARF in SHR couses the increase of lipid peroxidation in plasma, 24 h after reperfusion. RAAS blockade and scavenging of ROS decreased lipid peroxidation in plasma of SHR rats with developed ischemic ARF. Our results indicate that postischemic increase of AngII in hypertensive conditions has a significant role in the development of tubulo-interstitial damage. AngII AT1 receptor antagonist, losartan, significantly improved patho-morphological appearance of postishemic kidney. Using scavenger of superoxide anion, the tempol, did not have a significant effect on the histology of postishemic hypertensive rat’s kidney. The general conclusion of our work would suggest that increasing levels of ischemic AngII in hypertensive conditions has a significant role in the development of renal vasoconstriction and regulation of glomerular filtration, as well as development of tubulo- interstitial damage in the kidney. On the other hand, our results indicate superoxide anion had not particularly distinguished effects in the development and maintenance of ARF in hypertensive rats, but considering the nature of the ischemic-reperfusion injury, it is needed to examine the role of other ROS form. For the understanding the full effects of AngII and ROS in hypertensive patients with the ischemic episode of ARF, we should take into account many other factors (for example, age, vascular disease, pre-formed kidney damage and other individual characteristics of patients), and conduct much more detailed clinical investigation.

Efekti anestezije indukovane propofolom na sinaptičku plastičnost, aktivnost dopaminskog sistema i ponašanje juvenilnih pacova

The effect of propofol induced anesthesia on synaptic plasticity, dopaminergic system activity and behaviour of juvenile rats

Propofol je često korišćeni anestetik u modernoj medicini. Adiktivni potencijal propofola je uočen, kao i uticaj na memorijski proces. Međutim, oba fenomena su još uvek nedovoljno istražena. Imajući u vidu da je adolescencija period izuzetne osetljivosti na dejstvo adiktivnih supstanci i intenzivne maturacije mnemoničkog potencijala, cilj ovog rada je bio da se ispita uticaj jednokratnog izlaganja propofolskoj anesteziji, što je tipičan način kliničke primene anestetika, na dopaminsku signalizaciju, sinaptičku i neuronsku aktivnost u različitim regionima mozga i ponašanje juvenilnih/peripubertetskih pacova, kao model sistema humanog periadolescentnog razvoja. Efekti su analizirani 4, 24 i 48 sati nakon tretamana, kod mužjaka Wistar pacova starih 35 dana. Dobijeni rezultati su po prvi put ukazali da izlaganje propofolskoj anesteziji izaziva promene u ekspresiji/fosforilaciji signalnih molekula koji su već prepoznati kao značajni za dejstvo adiktivnih supstanci. Od svih analiziranih dopaminoceptivnih moždanih regiona (medijalni prefrontalni korteks, strijatum i talamus) jedino su u talamusu uočene značajne promene u ekspresiji fosforilisane/aktivirane forme DARPP- 32 proteina, pokazatelja postsinaptičke dopaminske signalizacije, 4 i 24 sata nakon tretmana, i bile su praćene povećanom ekspresijom FosB/ΔFosB proteina, biohemijskog pokazatelja neuronske aktivnosti. Promene su lokalizovane u paraventrikularnom talamičkom jedru i mediodorzalnom talamusu. U strijatumu i medijalnom prefrontalnom korteksu je uočen porast u ekspresiji fosforilisane forme CaMKIIα, biohemijskog senzora sinaptičke aktivnosti koji ima važnu ulogu u pamćenju prethodnog izlaganja adiktivnim supstancama. Smanjenje u intenzitetu anksioznosti (procenjeno na osnovu rezultata dobijenih u testu svetlo/tamne kutije i uzdignutog krstastog lavirinta) je zabeleženo 24 sata nakon tretmana, kada je uočen i pad u ekspresiji FosB proteina u strijatumu, što se može tumačiti kao traženje senzacija usled smanjene aktivnosti moždanog regiona značajnog za osećaj zadovoljstva i motivisanost. Pojačan motorički odgovor na d-amfetamin i fenciklidin je uočen 24 sata nakon tretmana (ukrštena senzitizacija), kao potvrda da bez obzira na različite primarne mehanizme dejstva propofol i dve korišćene droge koriste iste neuronske puteve za ostvarivanje psihomotoričkih efekata. Rezultati studije su takođe ukazali da kod peripubertetskih pacova nakon izlaganja propofolskoj anesteziji postoje izvesne poteškoće u pozivanju memorije i memorisanju novih informacija u neaverzivnim memorijskim testovima (prostorna habituacija, prepoznavanje novog objekta), 24 sata nakon tretmana. Ove posledice su bile praćene promenama u ekspresiji molekula koji učestvuju u pozivanju i rekonsolidaciji epizodične memorije (BDNF/TrkB, Egr-1, ERK1/2, CaMKIIα, FosB/ΔFosB), u dorzalnom hipokampusu. Uočene poteškoće u prepoznavanju novine su u skladu sa malobrojnim kliničkim studijama, koje naglašavaju značaj kontrole osnovnih kognitivnih funkcija kod starije dece nakon izlaganja propofolskoj anesteziji. Takođe, u svim ispitivanim moždanim regionima osim u talamusu je zabeležen porast u ekspresiji fosforilisane forme Aducinβ proteina, što je indikacija za destabilizaciju citoskeleta i sinaptičku reorganizaciju. Gledano u celini, rezultati ove studije su ukazali da bolje razumevanje bioloških posledica primene propofola u juvenilnom/pubertetskom uzrastu može doprineti sagledavanju starosno zavisnih zdravstvenih rizika tretmana, koji su do sada intenzivno istraživani samo u ekstremima životne dobi, tj. u ranoj postnatalnoj i kasnoj životnoj fazi.

Propofol is a commonly used anesthetic in modern medicine. Addictive potential of propofol is observed, as well as the impact on the memory process. However, both phenomena are still insufficiently explored. Bearing in mind that adolescence is a period of extreme sensitivity to addictive substances and intense maturation of the mnemonic potential, the aim of this study was to examine the effect of a single exposure to propofol anesthesia, which is a typical method of its clinical application, on dopaminergic signaling, synaptic and neuronal activity in different brain regions and behavior of juvenile/peripubertal rats, as a model system of human periadolescencent development. The effects were analyzed 4, 24 and 48 hours after the treatment, in male Wistar rats aged 35 days. The obtained findings for the first time showed that exposure to propofol anesthesia caused changes in the expression/phosphorylation of signal molecules that are already recognized as significant for the action of the addictive substances. Of all the analyzed dopaminoceptive brain regions (medial prefrontal cortex, striatum and thalamus), significant changes in the expression of the phosphorylated/activated form of DARPP-32 protein, indicator of postsynaptic dopaminergic signalling, were observed only in the thalamus, 4 and 24 hours after the treatment, and were accompanied by increased expression of FosB/ΔFosB protein, a biochemical indicator of neuronal activity. The alterations were localized in the paraventricular thalamic nucleus and the mid-dorsal thalamus. An increase in the expression of the phosphorylated form of CaMKIIα, a biochemical sensor of synaptic activity that has an important role in memory on addictive substances exposure, was detected in striatum and medial prefrontal cortex. Reduction in the intensity of anxiety (estimated in accordence to the data obtained in the light/dark box and elevated plus maze tests) was observed 24 hours after the treatment, along with the decrease in the expression of FosB protein in striatum, which can be interpreted as a sensation seeking due to decreased activity of brain region important for a pleasure/motivation. An enhanced motor response to d- amphetamine and phencyclidine was observed 24 hours after the treatment (cross sensitization), as confirmation that despite the different primary mechanisms of action, propofol and these two drugs use the same neuronal pathways to achieve psychomotor effects. The results of the study also indicated that in peripubertal rats exposed to propofol anesthesia, there were some difficulties in memory retrieval and acquisition of new learning in non-aversive memory tests (spatial habituation, novel object recognition), 24 hours after the treatment. These effects were accompanied by changes in the expression of molecules involved in retrieval and reconsolidation of episodic memory (BDNF/TrkB, Egr-1, ERK1/2, CaMKIIα, FosB/ΔFosB) in the dorsal hippocampus. Detected difficulties in recognizing the novelty are consistent with a few clinical studies that emphasize the importance of controlling basic cognitive functions in older children after exposure to propofol anesthesia. Also, in all investigated brain regions, except the thalamus, an increase in the expression of the phosphorylated form of Adducinβ protein was observed, which is an indication of the cytoskelet destabilization and synaptic reorganization. Overall, the results of this study accentuated that a better understanding of the biological consequences of the use of propofol in juvenile/puberty period could contribute to the undestanding of age-dependent health risks of the treatment, which have been extensively investigated so far only at the extremes of age, i.e. during early postnatal period and in aging.

Analiza varijabilnosti mitohondrijske DNK stanovnika Republike Srbije sa populaciono genetičkog, filogenetskog i filogeografskog aspekta

Analysis of mitochondrial DNA variability of residents of the Republic of Serbia : population genetics, phylogenetic and phylogeographic perspectives.

savremenim ljudskim populacijama Evrope rezultat su veoma kompleksnih evolucionih, demografskih i kulturoloških procesa koji su se odvijali na prostoru Evrope tokom više hiljada godina. Među najvažnijim procesima izdvajaju se post-glacijalna rekolonizacija Evrope, širenje poljoprivrede tokom neolita (neolitska tranzicija) i brojne migracije ljudskih populacija, poput migracija indoevropskih naroda tokom bronzanog doba i Velike seobe naroda u ranom srednjem veku. Balkansko poluostrvo je imalo važnu ulogu tokom celokupne istorije ljudske vrste na evroazijskom kontinentu zbog toga što je predstavljalo važan migracijski koridor preko kojeg su se odvijale migracije od praistorije pa sve do savremenog doba. Takođe, tokom poslednjeg ledenog maksimuma Balkansko poluostrvo je predstavljalo jedan od glacijalnih refugijuma sa kojeg je otpočela post-glacijalna rekolonizacija Evrope. Imajući u vidu da srpska populacija naseljava centralni deo Balkanskog poluostrva, u ovoj populaciji se mogu očekivati genetički tragovi kompleksnih demografskih procesa. Predmet istraživanja ove doktorske disertacije bilo je ispitivanje varijabilnosti mitohondrijske DNK (mtDNK) u savremenoj populaciji Srbije sa populaciono genetičkog, filogenetskog i filogeografskog aspekta. U tu svrhu analizirana je varijabilnost prvog i drugog hipervarijabilnog segmenta (HVS-I i HVS-II) i polimorfizmi kodirajućeg regiona mtDNK kod 172 ispitanika čiji su preci po ženskoj liniji iz poslednje dve generacije deklarisanog srpskog etniciteta. Pored toga, odabrano je i kompletno sekvencirano 58 mitogenoma koji su analizirani u cilju boljeg razumevanja evolucije pojedinih mtDNK haplogrupa, kao i utvrđivanja doprinosa različitih migracija u oblikovanju sadašnjeg obrasca varijabilnosti mitohondrijskog genskog pula srpske populacije. U srpskoj populaciji zastupljene su većinom mtDNK haplogrupe/podhaplogrupe karakteristične za evropske populacije, a njihove učestalosti i parametri genetičke raznovrsnosti nalaze se u rasponu vrednosti detektovanih u ostalim evropskim populacijama. Srpska populacija nije genetički diferencirana u odnosu na druge južnoslovenske populacije i u svim analizama zauzima centralnu poziciju između južnoslovenskih populacija istočnog i zapadnog dela Balkanskog poluostrva. Najveći procenat HVS-I i HVS- I/HVS-II haplotipova stanovništvo Srbije deli sa geografski bliskim populacijama Balkanskog poluostrva iz južnoslovenske grupe, gde su detektovani i potencijalno privatni haplotipovi. Načelno, ispitivane evropske populacije su u većini analiza grupisane na način koji odgovara njihovoj geografskoj distribuciji. Pored toga, slovenske populacije grupisane su i u skladu sa njihovom pripadnošću odgovarajućim jezičkim porodicama. Na Balkanskom poluostrvu su detektovane visoke vrednosti parametara genetičke raznovrsnosti retkih mtDNK podhaplogrupa, što je u skladu sa njegovim ranije demonstriranim refugijalnim karakterom. Na osnovu filogenetske i filogeografske analize kompletnih mitogenoma iz srpske populacije uočene su mtDNK podhaplogrupe koje su najverovatnije nastale u južnoj Evropi, kao i one koje su potencijalno specifične za Balkansko poluostrvo. Pored toga, srpska populacija deli određeni broj mtDNK linija sa istočno- i zapadnoslovenskim populacijama, sa germanskim populacijama severne i srednje Evrope, a u manjem obimu i sa populacijama sa Bliskog istoka i istočne Azije. Ova studija je pokazala da su u oblikovanju savremenog mitohondrijskog genskog pula srpske populacije važnu ulogu imale: a) slovenske i germanske populacije iz kojih su se, tokom ranog srednjeg veka, odvijale migracije ka Balkanskom poluostrvu i b) autohtone populacije Balkanskog poluostrva iz perioda pre doseljavanja Slovena, a čiji je genski fond sadržavao genetičke tragove brojnih različitih migracija iz još drevnijih vremena (npr. nosioci jamne kulture). Stoga se postojanje izuzetno velike raznovrsnosti mtDNK savremene populacije Srbije može objasniti genetičkim doprinosom kako slovenskih/germanskih, tako i pre-slovenskih populacija.

Genetic structure and clinal distribution of genetic variation in contemporary human populations in Europe are the results of interplay between exceptionally complex evolutionary, demographic and cultural processes which took place in Europe during several thousand years. Among the most important processes are post-glacial recolonization of Europe, spread of farming during the Neolithic period (Neolithic transition) and numerous migrations of human populations such as Bronze Age migrations of Indo-Europeans and migrations during the Migration Period that occurred in the Early Middle Ages. The Balkan Peninsula played an important role during the entire history of humankind in Europe and Asia because it served as an important migration corridor starting from prehistoric to present days. Furthermore, during the last glacial maximum, this region was one of the glacial refugia from which the post-glacial recolonization of Europe was initiated. Since Serbian population inhabits the central part of the Balkan Peninsula, this population may harbour genetic traces of past complex demographic processes that were occurring in the Balkans over time. The subject of this doctoral thesis was the study of the mitochondrial DNA (mtDNA) variability in a contemporary population of Serbia from population genetics, phylogenetic and phylogeographic aspects. For that purpose, the variability of HVS-I and HVS-II regions and polymorphisms within the coding region of mtDNA have been assessed in 172 individuals whose Serbian maternal ancestry was confirmed for at least two generations. Furthermore, 58 selected mitogenomes were completely sequenced in order to provide new insights into the evolution of particular mtDNA haplogroups and to assess the contribution of various human migrations in modelling contemporary maternal gene pool of the Serbian population. Serbian population comprises mainly mtDNA haplogroups/subhaplogroups typical for European populations and their frequencies and levels of genetic diversity are mostly in the range of those detected in other European populations. Serbian population is not genetically differentiated from other south-Slavic populations and in all analyses it occupies a central position among south-Slavic populations from the eastern and western part of the Balkan Peninsula. The highest percentage of HVS-I and HVS-I/HVS-II haplotypes Serbian population shares with south-Slavic populations from the Balkan Peninsula, where potential private haplotypes have been observed as well. In general, European populations were grouped in a manner reflecting their geographic distribution. Furthermore, Slavic populations were grouped according to their linguistic affiliation. Rather high values of all parameters of genetic diversity of rare mtDNA subhaplogroups in the Balkan Peninsula are concordant with the previously demonstrated refugial character of this region. Phylogenetic and phylogeographic analyses of completely sequenced mitogenomes detected in Serbian population enabled delineation of mtDNA subhaplogroups that are probably of the southern European origin, as well as those potentially specific for the Balkan Peninsula. Furthermore, Serbian population shares a certain number of mtDNA lineages with east- and west-Slavic populations, with Germanic populations from northern and central Europe, and to a lesser extent with those from the Middle East and eastern Asia. The present study demonstrates that important roles in modelling contemporary mitochondrial gene pool of the Serbian population may be ascribed to: a) Slavic and Germanic populations that inhabited the Balkan Peninsula during the early Middle Ages, and b) autochthonous populations from the Balkan Peninsula that were present in this region prior to the migrations of Slavs and which harboured genetic traces of numerous migrations happening in even more ancient times (e.g., bearers of Yamnaya culture). Therefore, exceptionally high mtDNA diversity observed in contemporary Serbian population may be associated with the genetic impact of both Slavic/Germanic and pre-Slavic populations.

Uloga vanćelijskih proteina toplotnog stresa u inflamaciji povezanoj sa posttraumatskim stresnim poremećajem

The role of extracellular heat shock proteins on posttraumatic stress disorder-related inflammation

Posttraumatski stresni poremećaj (PTSP) je psihijatrijska bolest koja se može razviti posle izlaganja traumatskom događaju. PTSP predstavlja veliko medicinsko i ekonomsko opterećenje za društvo, zbog relativno velike učestalosti i komorbiditeta sa mnogim drugim psihijatrijskim i somatskim bolestima. Smatra se da se najbolji rezultati u razumevanju patogeneze PTSP-a i njegovoj dijagnostici i terapiji mogu postići integrisanjem psiholoških, bioloških i farmakoterapeutskih pristupa. Aktuelna istraživanja sugerišu da je PTSP povezan sa neuroendokrinim poremećajima i promenama imunskih funkcija. Smatra se da je hiperosetljivost hipotalamo-hipofizno-adrenokortikalne (HHA) ose na kortizol neuroendokrini „pečat“ PTSP-a, dok je priroda imunskih poremećaja povezanih sa PTSP-om još uvek nejasna. Većina studija pokazala je da je PTSP povezan sa pojačanom inflamacijom, ali su objavljeni i podaci o nepromenjenom inflamatornom statusu, pa čak i smanjenom nivou inflamatornih markera u cirkulaciji PTSP pacijenata. Pretpostavlja se da pojačana inflamacija kod osoba obolelih od PTSP-a može biti posledica nedovoljne imunosupresije kortizolom. Međutim, podaci o nivou kortizola i funkcionisanju kortizolskih receptora kod pacijenata sa PTSP-om nisu konzistentni. Izlaganje različitim stresorima indukuje sintezu unutarćelijskih proteina toplotnog stresa (HSP) čije funkcije potpomažu preživljavanje ćelija. Tokom mnogo godina HSP su posmatrani kao unutarćelijski proteini, ali sada se zna da ih mnoge sisarske ćelije oslobađaju u cirkulaciju, gde mogu da aktiviraju pro-inflamatorne odgovore. Uzimajući da pojačana inflamacija koja je povezana sa PTSP-om verovatno nije posledica umanjenog anti-inflamatornog delovanja kortizola, mi smo pretpostavili da ona može biti posledica indukcije i oslobađanja Hsp60 i Hsp70 psihološkom traumom. Cilj ove studije bio je da se povežu parametri inflamatornog statusa sa nivoima kortizola i HSP u cirkulaciji, kao i da se otkriju moguće veze između inflamatornih markera i vanćelijskih HSP sa izlaganjem traumi, simptomima PTSP-a, osetljivošću i otpornošću na PTSP. U tom cilju u studiju su bili uključeni pacijenia sa aktuelnim PTSP-om (N=118), osobe koje su prebolele PTSP (N=63), osobe izložene traumi koje nisu razvile PTSP (N=94) i zdrave, netraumatizovane kontrole (N=95). Izmereni parametri bili su: unutarćelijski nivoi HSP (Hsp90, Hsp70, Hsp72 i Hsp60), nivoi HSP (Hsp70, Hsp72 i Hsp60) u krvnoj plazmi, nivo kortizola u plazmi pre i posle supresije HHA ose deksametazonom i nivoi kortikosteroidnih receptora, glukokortikoidnog receptora (GR) i mineralokortikoidnog receptora (MR), u perifernim limfocitima. Nivoi proinflamatornih citokina, markera inflamacije i vanćelijskih HSP (eHsp60 i eHsp70) u krvnoj plazmi određivani su metodom ELISA, dok je semi-kvantitativni „Western blot“ korišćen za određivanje nivoa kortikosteroidnih receptora i HSP u limfocitima. Rezultati su pokazali da se promene nivoa kortizola, Hsp70 i Hsp60 u plazmi, kao i nivo MR i ravnoteža između MR i GR u limfocitima ne mogu povezati sa izlaganjem traumi, aktuelnim ili prebolovanim PTSP-om, osetljivošću i otpornošću na PTSP, niti sa oporavkom od PTSP-a. Međutim, osetljivost na PTSP karakteriše se hiperosetljivošću HHA ose na kortizol i povećanim nivoom ekspresije GR-a u perifernim limfocitima. Nivo ekspresije HSP (Hsp90, Hsp70, Hsp72 i Hsp60) u limfocitima ne razlikuje se između osoba sa aktuelnim PTSP-om, prebolovanim PTSP-om, traumatizovanih osoba bez PTSP-s i zdravih, netraumatizovanih kontrola. Interesantno je da postoji korelacija nivoa MR-a sa nivoima Hsp90 i Hsp70 u limfocitima, dok nivo GR-a koreliše samo sa nivoom Hsp90. Jačina korelacije između nivoa MR i Hsp70 u limfocitima može se povezati sa aktuelnim simptomima PTSP-a, dok izlaganje traumi pojačava korelaciju između nivoa GR-a i Hsp90. Sudeći prema opštim inflamatornim markerima (CRP, sedimentacija eritrocita, broj leukocita) i nivoima citokina (TNF-α, IL-6, IL-12p70, IL-2, IL-8, IFN-γ, IL-5, IL-4, IL-10), izlaganje traumi i PTSP se mogu povezati sa imunosupresijom, pre nego sa pojačanom inflamacijom. Nivoi nekih citokina (TNF-α, IL-6, IL-12p70, IL-2, IL-8) mogli bi se povezati sa izlaganjem traumi, pre nego sa prisustvom aktuelnih ili prošlih simptoma PTSP-a, dok se nivoi drugih citokina (IFN-γ, IL-5, IL-4, IL-10) mogu povezati sa osetljivošću na PTSP, pre nego sa izlaganjem traumi. Rezultati ovog doktorskog projekta predstavljaju originalan naučni doprinos boljem razumevanju molekularnih mehanizama koji se nalaze u osnovi patofiziologije PTSP-a. Posebno, oni doprinose razotkrivanju veze između inflamacije i traume, odnosno osetljivosti/otpornosti na PTSP, i rasvetljavanju uloge vanćelijskih HSP u inflamatornim funkcijama imunskog sistema povezanim sa PTSP-om.

Post-traumatic stress disorder (PTSD) is a debilitating psychiatric disorder that may develop following exposure to life-threatening traumatic event. PTSD represents a serious medical and economic burden for the society due to its relatively high life-time prevalence rate and comorbidity with variety of other mental disorders and somatic illnesses. General opinion is that the best results in understanding of PTSD pathogenesis, diagnosis and treatment could be achieved by integrating psychological, biological and pharmacotherapeutical approaches. Current research has suggested that PTSD is associated with neuroendocrinological disturbances and immune function alterations. The hypersensitivity of hypothalamic-pitui- tary-adrenocortical (HPA) axis to cortisol feedback inhibition is considered the neuroendocrine hallmark of PTSD, while the nature of PTSD-related immune alterations is not well understood. Most studies report that PTSD is associated with excessive inflammation, but unaltered inflammatory state and even decreased circulatory levels of inflammatory markers were also reported. It has been hypothesized that excessive inflammation in individuals with PTSD might be a consequence of insufficient immunosuppression by cortisol. However, the data on cortisol levels and cortisol receptor functioning in PTSD patients are inconsistent. Exposure to a variety of stressors will induce intracellular heat shock proteins (HSPs) with cellular functions beneficial for cell survival. For many years HSPs have been viewed as intracellular proteins, but it is now known that they can be released from various mammalian cells into the peripheral circulation, where they are capable of activating pro-inflammatory responses. Taking that PTSD-associated excessive inflammation is not likely to be caused by impaired anti-inflammatory action of cortisol, we hypothesize that it might be a consequence of Hsp60 and Hsp70 induction and release by psychological trauma. The aim of the present study was to relate the parameters of inflammatory status with circulatory levels of HSPs and cortisol, and to uncover possible associations between inflammatory markers and extracellular HSPs with trauma-exposure, PTSD symptoms, vulnerability and resilience to PTSD. Toward that end the relevant parameters were determined in patients with current PTSD (N=118) and life-time PTSD (N=63), in trauma-exposed individuals without PTSD (N=94) and in non-traumatized healthy subjects (N=95). The measured parameters were: circulatory levels of a number of cytokines and C-reactive protein, as inflammatory markers; intracellular levels of HSPs (Hsp90, Hsp70, Hsp72 and Hsp60); plasma levels of HSPs (Hsp70, Hsp72 and Hsp60); plasma level of cortisol before and after suppression of HPA axis by dexamethasone and the level of corticosteroid receptors, glucocorticoid receptor (GR) and mineralocorticoid (MR), in peripheral blood mononuclear cells (PBMCs). Plasma levels of pro-inflammatory cytokines, markers of inflammation and extracellular HSPs (eHsp60 and eHsp70) were determined by ELISA, while semi-quantitative Western blot was performed for determination of lymphocyte level of corticosteroid receptors and HSPs. The results demonstrate that the alterations in plasma levels of cortisol, Hsp70s and Hsp60, as well as the MR level and MR/GR balance in peripheral lymphocytes are not associated with trauma exposure, current or life-time PTSD symptoms, resilience to PTSD, vulnerability to PTSD and/or with remission of the disorder. However, vulnerability to PTSD is characterized by hypersensitivity of HPA axis to cortisol and by increased level of GR expression in PBMCs. The levels of expression of HSPs (Hsp90, Hsp70, Hsp72 and Hsp60) in the lymphocytes do not vary between current PTSD, life-time PTSD, trauma control and healthy control groups of subjects. Interestingly, MR level in peripheral lymphocytes is correlated with the levels of both Hsp90 and Hsp70, while the GR level is correlated only with that of Hsp90. The strength of correlation between the lymphocyte levels of MR and Hsp70 is related to current PTSD, while war trauma-exposure may improve correlation between the levels of GR and Hsp90. Judging by the levels of general inflammatory markers (CRP, erythrocytes sedimentation rate, leukocyte count) and cytokines (TNF-α, IL-6, IL-12p70, IL-2, IL-8, IFN-γ, IL-5, IL-4, IL-10), trauma-exposure and PTSD could rather be linked to immunosuppression than to enhanced inflammation. The levels of some cytokines (TNF-α, IL-6, IL-12p70, IL-2 and IL-8) might be connected with trauma-exposure rather than with the presence of current or life-time PTSD symptoms, while the levels of others (IFN-γ, IL-5, IL-4 and IL-10) could be related to vulnerability to PTSD rather than to trauma-exposure. The results of the present PhD project constitute an original scientific contribution to better understanding of the molecular mechanisms underlying pathophysiology of PTSD. Specifically, they add to unraveling the relationship between inflammation and trauma or vulnerability/resilience to PTSD, and shed a new light on the role of extracellular HSPs in PTSD-related inflammatory actions of the immune system.

Prediktivni značaj prisustva aleksitimije u proceni anksioznosti i depresivnosti kod studenata obolelih od bronhijalne astme

Predictive role of alexithymia in the assessment of anxiety and depression in students with bronchial asthma

Astma kao jedna od najčešćih hroničnih bolesti utiče ne samo na fizičko nego i na mentalno zdravlje pacijenta. U mlađoj populaciji, uključujući i studente univerziteta, astma je od posebnog značaja, zbog više prevalencije u poređenju sa drugim hroničnim bolestima. Adolescenti sa astmom su u dva puta većem riziku za psihološke poremećaje kao što su anksioznost i depresija i u povećanom riziku za smrtni ishod zbod nje. Anksioznost i depresivnost su nezavisni faktori rizika za lošu kontrolu astme. Uloga aleksitimije, kao posebne specifične psihološke karakteristike opisane kao smanjene mogućnosti u prepoznavanju i opisivanju emocija, i povezanost sa nivoom anksioznosti i depresivnosti u astmi, takođe je prepoznata. Prevalencija aleksitimije je procenjena na 8- 19% u opštoj populaciji, ali je primećena i kod obolelih od hroničnih bolesti. Aleksitimija može biti povezana sa rekurentnim veoma teškim egzacerbacijama kod obolelih od astme. Postoji mali broj istraživanja koja su pratila specifičnu ulogu aleksitimije u populaciji univerzitetskih studenta sa astmom. Cilj ove studije je bio da se odredi prevalencija aleksitimije kod studenata sa astmom, i njena relacija sa plućnom fumkcijom, anksioznošću i depresivnošću. Takođe, cilj je bio i validacija srpske verzije testa za kontrolu astme (ACT) kod obolelih od astme. Metod. Studija preseka je sprovedena u Zavodu za zdravstvenu zaštitu studenata u Beogradu, Srbija, i uključila je 210 studenata univerziteta sa astmom i 201 zdravog studenta kao kontrolnu grupu. Toronto aleksitimija skala (TAS-20) je korišćena za procenu aleksitimije i poređenje skorova tri glavna faktora aleksitimije: teškoće u prepoznavanju osećanja (Faktor 1), teškoće u opisivanju osećanja (Faktor 2) i eksterno orijentisano mišljenje (Faktor 3). Svi ispitanici su podvrgnuti spirometrijskom testiranju, procenjena im je kontrola astme korišćenjem Testa za kontrolu astme i na osnovu preporuka Globalne inicijative za astmu (GINE) i psihometrijski parametri su prezentovani Bekovom skalom depresivnosti (BDI) i Bekovom skalom anksioznosti (BAI). Psihometrijski skorovi izmereni ovim upitnicima su sumirani u Totalni psihometrijski skor (TPS) sa ciljem da se definiše odgovarajući psihološki poremećaj. Rezultati. Aleksitimija utvrđena TAS-20 upitnikom je bila mnogo češća kod studenata sa astmom u odnosu na zdrave studente (11% vs 4%, p<0,05) bez razlike u distribuciji po polu. Značajno viši prosečni TAS-20 skorovi su detektovani za Faktor 1 i Faktor 2 kod studenata sa astmom u odnosu na kontrolnu grupu zdravih (p<0,05). Značajna pozitivna korelacija TAS-20, BDI i BAI skora pokazana je u celoj grupi studenta sa astmom, bez obzira na status aleksitimije (p<0,001). ACT test je završilo 98,8% pacijenata a srednje vreme rešavanja testa je bilo 4,5 minuta. Korelacija rezultata ACT testa i parametra plućne funkcije (FEV1 i FVC) je bila signifikantna (p= 0,016, p= 0,002). Promene u ACT skoru između bazalnih vrednosti i nakon 6 meseci nisu bile povezane sa promenama vrednosti FEV1 i FVC. ACT skor je imao odličnu dijagnostičku tačnost prema klasifikaciji kontrole astme lekara i čak izuzetnu tačnost prema klasifikaciji ispitanika sa astmom. ACT skor je bio najniži u grupi sa najvišim vrednostima TPS skora, BDI i BAI skora, kao što su i sva tri faktora TAS-20 bila u porastu sa porastom TPS. Važni prediktori statusa aleksitimije su bili: ACT skor (p= 0,030) i MEF25% (p= 0,019) kao mera plućne funkcije, i parametri psihometrijskog testiranja za procenu aleksitimije, depresije i nivoa anksioznosti. Zaključak: Aleksitimija ima visoku prevalenciju kod studenata sa astmom i dobro koreliše sa njihovim psihološkim statusom. Nesposobnost da pokažu osećanja mogu voditi ka losim prepoznavanjem pogoršanja bolesti i lošim ishodima bolesti. Aleksitimija bi trebalo da se razmatra i sagledava od strane zdravstvenih profesionalaca, posebno kod pojedinaca u ovoj starosnoj grupi, u cilju boljeg razumevanja i lečenja astme. Rezultati ove studije su potvrdili pouzdanost, validnost i tačnost srpske verzije Testa za kontrolu astme, doprinoseći već utvrđenoj vrednosti originalnog ACT testa, i u skladu su sa istraživanjima validnosti verzija ACT na drugim jezicima. Zbog toga bi ga trebalo više koristiti u svakodnevnoj kliničkoj praksi, kao korisno sredstvo za procenu kontrole astme.

Asthma as one of the most prevalent chronic disease has impact not only on physical but also on mental health of the patient. In younger population, including university students, asthma is of special importance, having higher prevalence compared to other chronic diseases. Adolescents with asthma are at two-fold higher risk of psychological disorders such as anxiety and depression and are at increased risk for asthma death as well. Anxiety and depression are independant risk factors for poor asthma control. The role of alexithymia, as separate specific psychological feature described as lack of capability to identify and verbalize emotions, and association with levels of anxiety and depression in asthma has also been recognized. Prevalence of alexithymia is estimated to be 8-19% in general population, observed also in chronic diseases. Alexithymia may be related to recurrent very severe asthma exacerbations in patients with asthma. Limited research was published specifically at the role of alexithymia in university student population with asthma. The aim of this study was to determine prevalence of alexithymia in students with asthma, and its relationship with pulmonary function, anxiety and depression. Also, the aim was to validate Serbian version of the Asthma control test (ACT) in patients with athma. Method. The cross-section study was conducted in the Institute for Students Health Care in Belgrade, Serbia, including 210 university students diagnosed with asthma and 201 healthy students as control group. Toronto Alexithymia Scale (TAS-20) was used for alexithymia assessment and compared with scores for the three main alexithymia factors: difficulty identifying feelings (Factor 1), difficulty describing feelings (Factor 2) and externally-oriented thinking (Factor 3). All subjects underwent spirometry. Asthma control was assessed by Asthma Control test (ACT) score and Global Initiative for Asthma (GINA) guidelines and psychometric parameters presented by Beck’s Depression Index (BDI) and Beck’s Anxiety Index (BAI). All these parameters were analyzed for correlation. Psychometric scores obtained by these questionnaires were summed (Total Psychometric Score-TPS) with the aim to define an appropriate psychological disorder. Results. Alexithymia assessed by TAS-20 was more frequent in students with asthma vs healthy control (11% vs 4%, p<0,05) without difference in gender distribution. Significantly higher average TAS-20 score was detected for Factor 1 and Factor 2 in asthma compared to healthy students (p<0,05). Significant positive correlation between TAS-20, BDI and BAI was found in a whole group of asthma patients, disregarding alexithymia status (p<0,001). ACT was completed by 98,8% of patients with mean time of completion of 4,5 minutes. The correlation of ACT score and lung function parameters (FEV1 and FVC) was significant (p= 0,016, p= 0,002, respectively). The change in ACT scores between baseline and 6-months visit was nor associated with tha change in FVC and FEV1. ACT score had exellent diagnostic accuracy according to physicians asthma control classification and even outstanding accuracy according to patients classification. ACT score was the lowest in a group with the highest Total Psychometric score values. Total TAS-20 score, BDI and BAI scores, so as three main elements of TAS-20 score (Factor 1-3) were increased with TPS increasing. Important predictors of alexithymia status were: ACT score (p= 0,030) and MEF25% (p= 0,019) values, as measures of pulmonary function, and psychometric tests for estimating alexithymia, depression and anxiety levels. Conclusion. Alexithymia is highly prevalent in students with asthma and well correlated with their psychological status. Inability to express the feelings could lead to low recognition of disease worsering and poor outcomes. Alexithymia should be considered and analyzed by healthcare profesionals, especialy in patients in this age group, in order to better management of asthma. The results od this study confirm reliability validity and accuracy of Serbian version of the ACT, contributing to established value of original ACT test and with consistent findings as previously reported validity of ACT in other languages. Therefore it should be utilized more in everaday clinical practice, as a useful and reliable tool of asthma control assessment.

Tumačenje prava u okviru Betijeve hermeneutike kao univerzalne metode duhovnih nauka

Legal interpretation in the context of Betti's hermeneutics as universal method of Geisteswissenschaften

Poslednje decenije XX veka su i u okviru filozofije, čiji je osnovni problem jezik, dovele do preokreta koji se naziva interpretativnim okretom. U mnoštvu uticaja koje je filozofija prava pretrpjela od analitičke i kontinentalne tradicije u filozofiji jednim su dijelom ostali zanemareni oni uticaji koji potiču od same teorijske jurisprudencije, ona pravila koja proizilaze od same praktičke jurisprudencije, te se u neku ruku i izgubila samosvojnost koju je Kelsen, izgrađujući čistu teoriju prava, želio da podari pravnoj nauci. Centralna tema najvećeg dijela doktorske disertacije jeste upravo hermenautika uopšte i hermenautika jednog italijanskog pravnika posebno.

The so-called interpretative turn has been a major development in the last two decades in philosophy. Among the variety of influences that the philosophy of law has experienced from analytical and phenomenological tradition, the influences derived from theoretical jurisprudence have remained neglected resulting in the dissolving of the self-contained character of legal science that Kelsen established by building his a pure theory of law. The core topic of this doctoral thesis is hermeneutics in general, with an emphasis on hermeneutics and legal hermeneutics developed by an Italian jurist and philosopher of law - Emilio Betti.

Fitohemijska i morfološka diferencijacija prirodnih populacija Satureja montana L., S. kitaibelii Wierzb. ex Heuff. i S. subspicata Bartl. ex Vis. (Lamiaceae)

Phytochemical and morphological differentiation of natural populations of Satureja montana L., S. kitaibelii Wierzb. ex Heuff. and S. subspicata Bartl. ex Vis. (Lamiaceae)

Analizirana je morfološka i fitohemijska varijabilnost, kao i diferencijacija prirodnih populacija taksona roda Satureja (Lamiaceae) sa prostora Balkana. Morfološki karakteri su izmereni kod 366 individua iz 27 prirodnih populacija S. montana ssp. montana, S. montana ssp. variegata, S. montana ssp. pisidica, S. kitaibelii, S. subspicata ssp. subspicata, S. subspicata ssp. liburnica. Proučavano je 12 morfoloških karaktera (šest karaktera lista i šest čašice). Karakteri čašice su pokazali mnogo manju intrapopulacionu varijabilnost i bolje su odslikavali razlike između taksona, naročito podvrsta unutar vrste S. subspicata. Primenjene univarijante i multivarijantne statističke metode su pokazale različite stepene diferencijacije na različitim nivoima u zavisnosti od vrste. Diferencijacije populacija je pokazana kod S. kitaibelii i delimično kod S. subspicata. Dve podvrste S. subspicata se na osnovu morfoloških karaktera jasno odvajaju. U slučaju S. montana nisu uočene diferencijacije, ni na jednom nivou, zbog velike intrapopulacione varijabilnosti. Uočene su korelacije morfoloških karaktera i pojedinih bioklimatskih parametara, uglavnom vezanih za temperaturu i padavine. Takođe, kod S. subspicata postoji i pozitivna korelacija između morfoloških karaktera i geografskog regiona, što nije bio slučaj kod druge dve vrste. Određen je sastav i relativni udeo komponenti površinskih flavonoida izolovanih sa celih listova kod 108 individua iz 25 prirodnih populacija sa Balkana. Detektovano je 11, a identifikovano 10 jedinjenja. Kod populacija S. subspicata ssp. subspicata je bilo prisutno svih 11 jedinjenja, dok je u zavisnosti od populacije broj jedinjenja kod druge dve vrste varirao 5-10 (S. montana) i 7-10 (S. kitaibelii). U profilu S. montana u većini slučajeva dominiraju 5,6- diOH-7,3'4'-triMe flavon i cirzimaritin, kod S. kitaibelii ksantomikrol i cirzimaritin, a unutar S. subspicata ksantomikrol i cirzimaritin (ssp. liburnica), odnosno ksantomikrol i 8- metoksicirzilineol (ssp. subspicata). Udeo ksantomikrola je najveći kod populacija S. subspicata ssp. liburnica (skoro 60 %). Površinski flavonoidi, analizirani statističkim metodama, na celom setu podataka, pokazuju diferencijaciju između vrsta, uključujući i podvrste. Najviše se odvaja tipska podvrsta S. subspicata od svih ostalih taksona. Iako podvrsta S. montana ssp. montana pokazuje najveću varijabilnost, kontinentalne populacije ove podvrste se preklapaju sa populacijama S. kitaibelii s jedne, dok se primorske preklapaju sa populacijama S. montana ssp. variegata, s druge strane. Populacije S. subspicata ssp. liburnica se po sastavu flavonoida nalaze između ovih primorskih i kontinentalnih populacija. Uočena je korelacija sastava površinskih flavonoida i geografske distribucije populacija, jaka kod S. subspicata i S. kitaibelii, a slaba kod S. montana. Relativni udeo pojedinačnih n-alkana u epikutikularnim voskovima određen je kod 316 individua iz 26 prirodnih populacija. U svim uzorcima su bili prisutni alkani u nizu od 19 do 35 ugljenikovih atoma. Postoji razlika u profilima između vrsta na osnovu dominantnih komponenti. Kod S. kitaibelii je dominantna komponenta C31 pa sledi C33, dok su kod S. montana i S. subspicata to C29 i C31. Međutim, ove dve vrste se razlikuju po relativnom udelu dominantnih komponenti u profilu alkana: kod S. montana udeo C29 je bio između 25,5 i 41,5 % a udeo C31 između 28,4 i 46,3 %, a kod S. subspicata udeo C29 je bio između 41,0 i 66,1 % a udeo C31 između 18,7 i 41,5%. Univarijante i multivarijante statističke analize su pokazale diferencijaciju populacija kod S. kitaibelii, a podvrsta kod S. subspicata. Međutim, zbog velike varijabilnosti populacija unutar vrste S. montana na osnovu profila alkana se ne mogu razlikovati podvrste. Generalno, najviše su diferencirane vrste S. kitaibelii i S. subspicata, posebno podvrsta S. subspicata ssp. subspicata. Utvrđena je korelacija dva alkana sa bioklimatskim podacima (temperaturom) kod S. montana, dok je kod S. kitaibelii više bioklimatskih parametara i nadmorska visina bilo u korelaciji sa većim brojem alkana. Za razliku od njih, kod S. subspicata je uočena blaga korelacija alkana i nadmorske visine, jaka korelacija sa ekspozicijom i geografskim regionom. Sastav etarskog ulja, izolovanog iz nadzemnog dela, određen je kod 333 individue iz 23 prirodne populacije. Identifikovano je preko 150 jedinjenja, najviše u etarskom ulju S. subspicata (146) a najmanje u ulju S. kitaibelii (100). Monoterpeni su dominantna grupa kod S. montana (69,8 – 86,9 %) i S. kitaibelii (52,1 – 84,6 %), dok su kod S. subspicata to bili seskviterpeni (50,4 – 68,8 %) kod većine populacija. U profilu etarskog ulja od monoterpena dominiraju oksidovani kod S. montana (23,5 – 62,0 %) i S. kitaibelii (31,1 – 52,4 %). Seskviterpenski ugljovodonici, od seskviterpena, su bili najzastupljenija grupa u sastavu etarskog ulja kod većine populacija S. subspicata (13,8 – 48,1 %). Profilom etarskog ulja kod jedinki S. montana dominirala su tri monoterpena (p-cimen, karvakrol i timol), uz izuzetak populacije Gacko (linalol), kod S. kitaibelii je najzastupljeniji bio limonen kod većine jedinki. Sastav etarskog ulja S. subspicata se odlikovao većim brojem dominantnih komponenti, kod svih populacija bar jedna od glavnih komponenti je bila (E)-kariofilen, kariofilen oksid ili α-pinen. Uočena je diferencijacija proučavanih vrsta na osnovu profila etarskog ulja. Najviše se međusobno odvajaju S. montana i S. subspicata. Takođe, primećena je korelacija distribucije vrsta i sastava etarskog ulja. Vrste sa jako do umereno fragmentisanim arealima (S. kitaibelii i S. subspicata) pokazuju manju varijabilnost sastava etarskog ulja u okviru populacija i veću diferenciranost populacija u odnosu na vrstu sa gotovo kontinuiranim arealom (S. montana). Međutim, kod S. montana su uočena tri do četiri hemotipa u zavisnosti od koriščene statističke metode, bez obzira na njenu veliku varijabilnost sastava etarskog ulja. Uticaj genetičkih faktora na morfološke i fitohemijske karaktere analiziran je kod populacija S. montana i S. subspicata koje žive u simpatriji. Pokazano je da se jedinke jasno razdvajaju po pripadnosti vrsti na svim analiziranim karakterima (morfološkim karakterima, kao i po sastavu flavonoida, alkana i etarskih ulja). Fitohemijski i morfološki karakteri, analizirani u ovom radu, su pokazali različite stepene diferencijacije proučavanih taksona. Morfološki karakteri, posebno oni vezani za čašicu, bili su informativniji kod vrsta koje imaju manje areale S. kitaibelii i S. subspicata. Analize profila alkana su pokazale diferencijaciju vrsta. Međutim, fitohemijski karakteri koji su pokazali najveću infraspecijsku, intra- i interpopulacionu diferencijaciju proučavanih vrsta su sastav flavonoida i etarskih ulja, posebno kod S. subspicata. Uporedne analize svih taksona i svih fitohemijskih i morfoloških kraktera su pokazale veću diferencijaciju unutar S. subspicata nego između S. montana i S. kitaibelii, što je u saglasnosti sa dosadašnjim analizama koje su ukazivale na sestrinski odnos S. montana i S. kitaibelii i potencijalno na njihovo skorije razdvajanje.

This thesis deals with morphological and phytochemical variability and differentiation of natural populations of taxa belonging to genus Satureja L. (Lamiaceae) (S. montana ssp. montana, S. montana ssp. variegata, S. montana ssp. pisidica, S. kitaibelii, S. subspicata ssp. subspicata and S. subspicata ssp. liburnica) from the Balkans. Morphological characters were measured in 366 individuals from 27 natural populations of selected taxa. In total 12 morphological characters were studied (six leaf and six calyx characters). Calyx characters varied less at the intrapopulation level and better reflected differences between the taxa, particularly between S. subspicata subspecies. Applied statistical methods (univariate and multivariate) showed diverse differentiation levels depending on the species studied. The separation between populations was observed in S. kitaibelii (complete) and in S. subspicata (partial). However, two S. subspicata subspecies were clearly differentiated. In the case of S. montana due to the high intrapopulation variability, no separation was found. Correlations between some of the morphological characters and some of the bioclimate parameters mostly related to temperature and precipitation were observed. Positive correlation between the morphological characters and the geographic region was shown only in S. subspicata. The surface flavonoids composition has been determined by HPLC and LC-MS analysis in 108 individuals from 25 natural populations. In total, 11 compounds were detected and 10 identified. All of them were present in S. subspicata ssp. subspicata, while in other two species number of compounds varied depending on the population studied (5-10 compounds in S. montana and 7-10 in S. kitaibelii). Surface flavonoids profile of S. montana, in most cases, was dominated by 5,6-diOH-7,3'4'-triMe flavone and cirsimaritin, while xanthomicrol and cirsimaritin dominated profiles of S. kitaibelii and S. subspicata ssp. liburnica. In S. subspicata ssp. subspicata xanthomicrol and 8-methoxycirsilineol were dominant. Xanthomicrol percentage was the highest in the S. subspicata ssp. liburnica (almost 60%). Flavonoids analyzed by statistical methods, in the whole dataset, showed differentiation among species including subspecies, particularly S. subspicata ssp. subspicata. Although, S. montana ssp. montana showed the highest variability of flavonoid composition, based on composition, continental populations of this subspecies overlap with S. kitaibelii populations, on one side, while coastal populations overlap with S. montana ssp. variegata populations, on the other. S. subspicata ssp. liburnica populations, based on flavonoid composition, were between those coastal and continental populations. Also, data showed a correlation between flavonoid composition and populations' geographical distribution – strong for S. subspicata and S. kitaibelii populations, and weak for S. montana. The composition of the epicuticular leaf n-alkanes has been determined by GC-FID and GC/MS analysis in 316 individuals from 26 natural populations. The chain length of detected and identified n-alkanes ranged from C19 to C35. n-Alkane profile showed differences among studied taxa. Hentriacontane (C31) followed by tritriacontane (C33) were dominant in S. kitaibelii, whereas nonacosane (C29) and hentriacontane (C31) were dominant in S. montana and S. subspicata. However, these two species differentiate based on the relative proportion of dominant compounds in n-alkane profiles. In S. montana C29 ranged from 25.5 to 41.5 % and C31 from 28,4 to 46,3 %, while in S. subspicata C29 proportion was much higher ranging from 41.0 to 66.1 % and C31 from 18.7 to 41.5 %. Univariate and multivariate statistical analysis had shown population and subspecies differentiation, in S. kitaibelii and S. subspicata, respectively. Due to high intrapopulation variability found in S. montana, no subspecies differentiation was observed. Generally, based on the n-alkane profile, S. kitaibelii and S. subspicata were the most differentiated, particularly S. subspicata ssp. subspicata. Correlation between n-alkanes and bioclimatic parameters was found in S. montana (two alkanes and temperature) and in S. kitaibelii (several alkanes and bioclimatic parameters and altitude). Unlike previous species in S. subspicata correlation between alkanes and altitude was weak, while it was strong for exposition and geographic region. Essential oil composition, extracted from the areal parts was determined by GC-FID and GC/MS analysis in 333 individuals from 23 natural populations. In the essential oil composition, little over 150 compounds were detected and the largest number of compounds were found in S. subspicata (146) and the smallest was in S. kitaibelii populations (100). Monoterpenes dominated essential oil composition in S. montana (69.8 – 86.9 %) and S. kitaibelii (52.1 – 84.6 %), wheres in most S. subspicata populations sesquiterpenes were dominant (50.4 – 68.8 %). In both S. montana and S. kitaibelii oxygenated monoterpenes represented 23.5 – 62.0 % and 31.1 – 52.4 %, respectively. Sesquiterpene hydrocarbons represented 13.8 to 48.1 % of the total oil, were the dominant type found in essential oil from most S. subspicata populations. Three monoterpenes, p-cymene, carvacrol and thymol, dominated S. montana essential oil profile, with exception of population Gacko (linalool dominated). Although limonene was present in all S. kitaibelii individuals in higher procentage, it was not a dominant component in all populations. S. subspicata essential oil composition was characterized by a larger number of dominant components in all samples, with at least one of them being (E)-caryophyllene, caryophyllene oxide and/or α-pinene. Studied taxa could be differentiated based on the essential oil composition. The highest separation was observed between species S. montana and S. subspicata. Also, the essential oil profile and geographical distribution of taxa was correlated. Species with highly to moderately fragmented distribution (S. kitaibelii and S. subspicata) showed less variable essential oil composition, compared to the species with almost continuous range (S. montana). However, regardless of the very variable essential oil composition found in S. montana, three to four chemotypes could be differentiated, depending on the statistical methods used. Influence of genetic factors on morphological and phytochemical characters was analyzed in four sympatric S. montana and S. subspicata populations. Individuals from these populations were clearly separated on all of the studied characters according to species to which they belong. Analyzed phytochemical and morphological characters demonstrated different levels of taxa differentiation. Morphological characters, particularly those related to calyx were more useful at differentiation of populations and subspecies in taxa with smaller ranges (i.e. S. kitaibelii and S. subspicata). n-Alkane profiles showed species differentiation with a lower resolution than the other two phytochemical markers. Surface flavonoids and essential oils composition showed the highest intra- and interpopulation differentiation of the studied taxa, particularly in S. subspicata. Comparative analysis of all studied taxa and all phytochemical and morphological characters showed higher differentiation between S. subspicata subspecies than between S. montana and S. kitaibelii, which is in concordance with previously available data that suggested sister relation of two species and potentially their more recent separation.

Komparativna analiza agenasa za biološku kontrolu patogenih gljiva izolovanih sa lekovitih biljaka

Comparative analysis of agents for biological of pathogenic fungi isolated from medicinal plants

Primena lekovitog bilja i njihovih preparata u prevenciji i lečenju različitih poremećaja u ljudskom organizmu može biti ograničena njihovom mogućom kontaminacijom fitopatogenim gljivama i mikotoksinima. Saznanja o riziku pri primeni hemijskih fungicida po rukovaoca, potrošača i životnu sredinu, dovela su do povećanja interesa za uvođenje alternativnih mera u zaštiti bilja, gde posebno mesto pripada preparatima prirodnog porekla tzv. agensima biološke kontrole. Biološka kontrola podrazumeva primenu korisnih mikroorganizama (bakterija, kvasaca, gljiva) ili produkata njihovog metabolizma, kao i primenu biljnih ekstrakata i etarskih ulja u zaštiti biljaka. Ispitivanjem preko 40 vrsta lekovitog bilja najlošiji mikrobiološki kvalitet utvrđen je za sledeće droge: kukuruznu svilu, list i herbu nane, list koprive, herbu rastavića i cvet nane. Iako su na svim biljnim drogama utvrđene mešovite infekcije gljivama iz različitih rodova, većina izolovanih vrsta gljiva pripada rodu Fusarium, a potom Aspergillus i Alternaria. Osim pomenutih, identifikovani su i predstavnici rodova: Penicillium, Phoma, Cephalosporium, Nigrospora, Cladosporium, Epicoccum, Gliocladium, Myrothecium, Cercospora, Phomopsis, Verticillum, Dreschlera (=Bipolaris), Rhizoctonia, Septoria, Trichoderma, Curvularia, Stahybotrys, Trichotecium, Puccinia, Botrytis, Mucor i Rhizopus sp., u zavisnosti od biljne droge. U cilju pronalaženja efikasnog biokontrolog agensa ispitivali smo mogućnost primene etarskih ulja i izolata Bacillus sp. u kontroli odabranih identifikovanih gljiva. U tom smislu, odabrali smo sledeće vrste gljiva: Fusarium solani, F. equiseti, F. oxysporum (izolovani sa kukuruzne svile i nevena), F. tricinctum, F. semitectum, F. sporotrichioides, F. subglutinans, Aspergillus flavus, A. niger, Alternaria alternata, Penicillium sp., Chaetomium sp., Curvularia lunata, Trichoderma viride, Trichotecium roseum, Gliocladium roseum, Myrotechium verrucaria, Phoma sp., Phomopsis sp. i Verticillium dahliae. . ii Ispitivan je kvalitativni i kvantitativni sasatav, kao i antifungalna aktivnost 22 etarska ulja (čubra, origana, timijana, ruže, anisa, čajnog drveta, zdravca, lavande, korijandera, bergamota, ljubičice, ruzmarina, bibera, cimeta, bosiljka, dalmatinske i španske žalfije, eukaliptusa, limuna, narandže, kamilice i vetivera). Antifungalna aktivnost etarskih ulja određivana je u in vitro testu, mikrodilucionom metodom na mikrotitracionim pločama. Određivane su minimalne inhibitorne i minimalne fungicidne koncentracije (MIC i MFC). Sva testirana etarska ulja su pokazala antifungalnu aktivnost na sve fitopatogene gljive izolovane sa biljnih droga, ali sa različitim stepenom inhibitorne aktivnosti. Etarska ulja čubra, origana i timijana, sa fenolima kao dominantnim komponentama, pokazala su najveći antifungalni potencijal na sve testirane gljive, pri najnižim koncentracijama. Etarska ulja vetivera i kamilice, sa seskviterpenima kao dominantnim komponentama, su pokazala najslabiju antifungalnu aktivnost, kao i etarska ulja eukaliptusa, limuna i narandže u kojima preovladavaju monoterpenski ugljovodonici. Ostala ulja ispoljila su od veoma dobre do umerene antifungalne aktivnosti. Među vrstama roda Fusarium, F. subglutinans, F. semitectum i F. sporotrichioides, generalno su najotpornije gljive na većinu analiziranih etarskih ulja, a od predstavnika drugih rodova to su T. viride, A. alternata, A. flavus i Chaetomium sp. Najosetljivije gljive na većinu etarskih ulja su F. oxysporum izolovan sa kukuruzne svile, kao i M. verrucaria i T. roseum. Prema rezultatima ispitivanja sinergističke aktivnosti odabranih etarskih ulja može se reći da su kombinacije etarskih ulja ispoljile bolju antifungalnu aktivnost nego kada su ulja ispitivana pojedinačno. U in situ ispitivanjima, najveću efikasnost u redukciji ukupnog broja gljiva u uzorcima biljne droge, ispoljila su ulja timijana, origana i ruže. Primenom in vitro testa dvojne kultivacije ispitivana je antagonistička aktivnost 14 izolata Bacillus sp. prema odabranim gljivama. Testirani izolati ispoljili su različit stepen inhibicije prema različitim vrstama testiranih gljiva. Najbolji antagonizam prema odabranim fitopatogenim gljivama ispoljili su izolati SS-12.6, SS-13-1, kao i SS-27.7, SS-38.3, SS-38.4 i SS-40.2. Izolati SS-39.1 i SS-39.3 su ispoljili najslabiju inhibitornu aktivnost na gotovo sve testirane gljive. Slabiji antagonizam u odnosu na većinu gljiva ispoljili su i Bacillus sp. izolati SS-10.7 i SS-21.7. Ostali izolati (SS-6.2, SS-35.4, SS- . iii 38.2, SS-40.6) ispoljili su različite antifungalne potencijale u zavisnosti od testirane gljive. Uporednom analizom dobijenih rezultata može se reći da su Fusarium vrste nešto otpornije na Bacillus sp. izolate od vrsta drugih rodova gljiva. Ispitivanjem antifungalne aktivnosti lipopeptidnih ekstrakta izolovanih iz Bacillus sp. SS-12.6 i SS-13.1, može se uočiti da je ekstrakt izolata SS-12.6 veoma efikasan u inhibiciji rasta većine testiranih gljiva, dok ekstrakt izolovan iz izolata SS- 13.1 nije ispoljio značajniji antagonizam na većinu testiranih gljiva, izuzev Penicillium sp. Ispitivanjem potencijalnog sinergizma između Bacillus sp. izolata SS-12.6 i SS- 13.1 i etarskih ulja timijana, čubra i ruže, na vrste roda Fusarium utvrđeno je da su izolati uglavnom jače inhibirali rast većine ispitivanih gljiva u prisustvu ovih etarskih ulja, nego kada su ispitivani sami kao antagonisti, uz pojedine izuzetke. Na osnovu rezultata dobijenih u ovom radu može se reći da etarska ulja čubra, origana, timijana i ruže, kao i Bacillus sp. izolat SS-12.6 i njegov lipopeptidni ekstrakt, predstavljaju dobru osnovu za potencijalnu formulaciju preparata sa efikasnošću u kontroli fitopatogenih gljiva, bilo prevencijom kontaminacije u polju, ili dekontaminacijom u zatvorenom prostoru.

The application of medicinal plants and their preparations for the prevention and treatment of various disorders in humans may be limited by the possible contamination with phytopathogenic fungi and mycotoxins. Risk of using chemical fungicides for the operator, the consumer and the environment, have led to increasing interest in the introduction of alternative measures in plant protection. Lately, preparations of natural origin, so-called biological control agents are in the focus of investigation. Biological control involves the use of beneficial microorganisms (bacteria, yeasts, fungi) or the products of their metabolism, as well as the application of plant extracts and essential oils in plant protection. Examining over 40 stored dried medicinal plant species the lowest microbial quality were determined for next herbal drugs: Maydis stigmata (corn silk), Mentha leaf and herb (mint herb and leaf), Urtica leaf (nettle leaf), Equisetum herb (horsetail herb) and Calendula flower (marigold flower). Although mixed infections was recorded with different types of fungus the Fusarium was noted as the most dominant genera for most tested drugs, followed by Aspergillus and Alternaria. Twelve species of the genus Fusarium was identified. In addition, species from the following genera were identified: Phoma, Cephalosporium, Nigrospora, Cladosporium, Epicoccum, Gliocladium, Myrothecium, Cercospora, Phomopsis, Verticillum, Dreschlera (=Bipolaris), Rhizoctonia, Septoria, Trichoderma, Curvularia, Stahybotrys, Trichotecium, Puccinia, Botrytis, Mucor and Rhizopus sp. depending on plant species. In order to find an effective biological control agent, we investigated the possibility of applying the essential oils and isolates of Bacillus sp. in the control of selected identified fungi. In this regard, we chose the following fungal species: Fusarium solani, F. equiseti, F. oxysporum (isolated from corn silk and marigold flower), F. tricinctum, F. semitectum, F. sporotrichioides, F. subglutinans, Aspergillus flavus, A. niger, Alternaria alternata, Penicillium sp., Chaetomium sp., Curvularia . v lunata, Trichoderma viride, Trichotecium roseum, Gliocladium roseum, Myrotechium verrucaria, Phoma sp., Phomopsis sp. and Verticillium dahliae. The antifungal activity and the qualitative and quantitative content of 22 essential oils were investigated (savory, oregano, thyme, rose, anise, tea tree, geranium, lavender, coriander, bergamot, violet, rosemary, pepper, cinnamon, basil, dalmatian and spanish sage, eucalyptus, lemon, orange, chamomile and vetiver). Microdilution method on microtitration plates was used for monitoring the antifungal activity of essential oils and for determination of their minimum inhibitory and fungicide concentration (MIC and MFC) in vitro. All tested essential oils showed antifungal activity on pathogenic fungi isolated from herbal drugs, but with varying degrees of inhibitory activity. Essential oils of savory, oregano and thyme, with phenols as the dominant component, were the most effective, at the lowest concentration, while chamomile and vetiver oils, and those from Citrus species, with dominant monoterpen hydrocarbons, were the least effective against pathogenic fungi. Other oils had expressed from very good to moderate antifungal activity. Among all Fusarium species, F. subglutinans, F. semitectum and F. sporotrichioides, and T. viride, A. alternata, A. flavus i Chaetomium sp. among other genera, showed very good resistance to all oils investigated. F. oxysporum (from corn silk), M. verrucaria and T. roseum were the most susceptible pathogenic fungi. According to the results of tested synergistic activity of selected essential oils, combination of essential oils exhibited better antifungal activity than when they were tested individually. Using in situ tests, we showed that the highest efficacy in reducing the total number of fungi in samples of herbal drugs exhibited essential oils of thyme, oregano and rose. Using in vitro, dual cultivation assay, we have investigated antagonistic activity of 14 Bacillus sp. isolates, against selected fungal species. Tested isolates showed various degree of inhibition against different types of plant pathogenic fungi. The best antagonism against selected phytopathogenic fungi showed isolates SS-12.6, SS-13-1, as well as SS-27.7, SS-38.3, SS-38.4 and SS-40.2. Bacillus sp. isolates SS-39.1 and SS- 39.3 manifested the weakest antagonistic effect against almost all fungi tested. Minor antagonism against most fungi exhibited Bacillus sp. isolates SS-10.7 and SS-21.7. The . vi rest of isolates (SS-6.2, SS-35.4, SS-38.2 and SS-40.6) exhibited different antifungal potential depending on the test fungi. According to the comparative analysis of the obtained results it can be said that Fusarium species were more resistant to Bacillus sp. isolates from other genera of fungi. Evaluation of lipopeptide extracts isolated from Bacillus sp. SS-12.6 and SS- 13.1 isolates showed that the lipopeptide extract from SS-12.6 was very effective in inhibition of the growth of most fungi tested, while the lipopeptide extract from isolate SS-13.1 did not exhibit a significant antagonism against the majority of tested fungi, except Penicillium sp. Examining the potential synergism between Bacillus sp. isolates SS-12.6 and SS-13.1 and the essential oils of thyme, savory and roses, against the Fusarium species, showed that the isolates were more effective in the presence of essential oils, than when tested individually, with particular exceptions. Based on the results obtained in this work we conclude that the essential oils of savory, oregano, thyme and roses, as well as Bacillus sp. isolate SS-12.6 and its lipopeptide extract, represent a good basis for the formulation of products with potential efficacy in the control of plant pathogenic fungi, either in preventing the contamination in the field, or via indoors decontamination.

Procena antigenotoksičnog potencijala etanolnog ekstrakta lista masline (Olea europaea L.) u prisustvu hormona tiroksina, adrenalina, estradiola i dietilstilbestrola u leukocitima periferne krvi in vitro kod čoveka

Evaluation of antigenotoxic potential of ethanolic olive leaf extract (Olea europaea L.) on the effect of thyroxine, adrenaline, estradiol and diethylstilbestrol in human peripheral blood leukocytes in vitro

Hormoni su organska jedinjenja različite hemijske prirode koji svojim dejstvom utiču na rast, funkciju i metabolizam organizma. Povišene koncentracije hormona dovode do stvaranja slobodnih radikala, što može izazvati oksidativni stres i oštećenja DNK molekula. Poznato je da komponente tradicionalne mediteranske ishrane imaju pozitivne efekte na smanjenje oksidativnog stresa i prevenciju mnogih bolesti. Brojne in vitro i in vivo studije su potvrdile korisne efekte suvog ekstrakta lista masline (DOLE) i njegovih sastojaka, kao i zaštitni potencijal u odnosu na oksidativna oštećenja DNK molekula. Stoga, glavni cilj ove doktorske disertacije je bio da se ispita antigenotoksični potencijal spektra koncentracija suvog ekstrakta lista masline u leukocitima periferne krvi čoveka u prisustvu hormona tiroksina, adrenalina, estradiola i dietilstilbestrola in vitro. Ispitivanje je obavljeno primenom alkalnog komet testa, osetljive i brze metode za određivanje i analizu primarnih oštećenja DNK u pojedinačnim ćelijama. Rezultati ove studije su pokazali da su tiroksin, adrenalin, estradiol i dietilstibestrol sposobni da budu medijatori značajnog povećanja oštećenja DNK molekula. Suvi ekstrakt lista masline je u svim testiranim koncentracijama ispoljio značajan antigenotoksični potencijal u oba eksperimentalna protokola, u pretretmanu i posttretmanu. Praćenje kinetike reparacije DNK u prisustvu DOLE u odnosu na oštećenja izazvana hormonima je pokazalo da ekstrakt nije značajno uticao na stimulaciju reparacije oštećenja DNK. Sumirajući rezultate ove disertacije, može se zaključiti da suvi ekstrakt lista masline poseduje izražen potencijal smanjenja primarnih oštećenja DNK izazvanih tiroksinom, adrenalinom, estradiolom i dietilstilbestrolom. Predstavljeni in vitro model na leukocitima periferne krvi čoveka pružio je podatke koji su korisni za buduće in vivo studije i klinička ispitivanja suvog ekstrakta lista masline.

Hormones are organic compounds of different chemical nature that affect cell growth, function and metabolism. Elevated concentrations of hormones lead to the formation of free radicals, which can cause oxidative stress and damage to the DNA molecule. It is known that the components of traditional Mediterranean food have positive effects on the reduction of oxidative stress and the prevention of many diseases. Numerous in vitro and in vivo studies have confirmed the beneficial effects of dry olive leaf extract (DOLE) and its constituents, as well as protective potential on the oxidative damage of DNA molecules. Therefore, the main aim of this doctoral dissertation was to investigate the antigenotoxic potential of the spectrum of concentrations of dry olive leaf extract in peripheral blood leukocytes in the presence of hormone thyroxine, adrenaline, estradiol, and diethylstilbestrol in vitro. The study was performed using an alkaline comet assay, a sensitive and fast method for determining and analyzing primary DNA damage in individual cells. The results of this study have shown that thyroxine, adrenaline, estradiol, and diethylstibestrol are capable of being mediators of significant increase of DNA damage. Dry olive leaf extract demonstrated significant ability to reduce primary DNA damage at all tested concentrations and in both experimental protocols, in pretreatment and posttreatment. Monitoring the kinetics of DNA repair in the presence of DOLE in relation to damage induced by homones has shown that the extract did not significantly affect the stimulation of DNA damage repair. Summarizing the results of this dissertation, it can be concluded that the dry olive leaf extract possesses a strong potential to reduce primary DNA damage induced by thyroxine, adrenaline, estradiol, and diethylstilbestrol. The presented in vitro model on human peripheral blood leukocytes provided data that are useful for future in vivo studies and clinical trials of dry olive leaf extract.

Ekspresija gena za inhibitor serinskih proteinaza (BvSTI) šećerne repe (Beta vulgaris L.) i uloga u otpornosti na insekte

Expression of sugar beet (Beta vulgaris L.) serine proteinase inhibitor gene (BvSTI) and the role in insect resistance

Biljni inhibitori proteinaza aktivno učestvuju u odbrani biljaka od insekata štetočina inhibirajući insekatske digestivne proteinaze. In planta analiza ekspresije BvSTI gena koji kodira inhibitora serinskih proteinaza urađena je sa ciljem otkrivanja uloge ovog inhibitora u otpornosti biljaka šećerne repe na insekte, kao i radi utvrđivanja potencijala ovog gena kao pogodnog kandidat-gena koji bi se biotehnološkim metodama mogao uvesti u osetljive biljne genome, čime bi se povećala njihova otpornost prema insektima štetočinama. Ekspresija BvSTI gena praćena je kod tri genotipa šećerne repe koji se odlikuju umerenom otpornošću prema larvama štetočine korena Tetanops myopaeformis Roder, F1016, F1015 i UT-8, kao i kod jednog osetljivog genotipa, F1010. Kod svih otpornih genotipova mehaničko povređivanje indukovalo je ekspresiju ovog gena, a nivo transkripcije bio je povišen u poređenju sa nivoom kod osetljivog genotipa. Najintenzivniji odgovor na povređivanje zabeležen je kod otpornih genotipova F1016 i UT-8. U listovima osetljivog F1010, ali i trećeg otpornog genotipa F1015, registrovan je samo neznatni porast u intenzitetu BvSTI transkripcije, dok je u korenovima ova dva genotipa mehaničko povređivanje dovelo do blage početne supresije u aktivnosti BvSTI gena. Akumulacija BvSTI transkripata u listovima i korenovima otpornog F1016 i osetljivog F1010 kojima su se hranile larve Spodoptera frugiperda J.E. Smith pokazala je sličan obrazac ekspresije kod oba genotipa. U poređenju sa transkripcionim obrascima dobijenim nakon mehanilčkog povređivanja, ishrana insekata dovela je do znatno sporije indukcije i slabijeg intenziteta transkripcije. Analize na proteinskom nivou pokazale su da nakon povređivanja listova dolazi do akumulacije proteina veličine 30 kDa za koji su se vezala poliklonalna BvSTI specifična antitela kako kod otpornog F1016, tako i kod osetljivog F1010 genotipa. Aktivnost BvSTI inhibitora protiv tripsina pokazana je kod F1016 korenova i listova, kao i kod F1010 listova. U F1010 korenovima aktivnost BvSTI inhibitora nije detektovana. U biotestu u kome je ispitivana otpornost pojedinačnih genotipova na larve S. frugiperda korišćena su sva četiri genotipa šećerne repe kod kojih je pokazano da povređivanje utiče na ekspresiju BvSTI gena. Larve koje su se hranile listovima sva tri otporna genotipa bile su statistički značajno lakše od larvi koje su hranile osetljivim F1010 listovima. Prelaskom larvi u stadijum lutke statistički značajne razlike u masi mađu larvama koje su se hranile listovima različitih genotipova su se izgubile i sve lutke su bile sličnih masa. Larve koje su se hranile korenovima šećerne repe bile su u proseku dvostruko lakše od larvi sa listova, a lutke koje su se razvile iz njih osim što su bile značajno lakše bile su i obavijene hitinskim omotačem znatno svetlije boje. U nekoliko slučajeva zabeležene su i morfološke abnormalnosti kod lutki razvijenih iz larvi koje su se hranile rezistentnim sa F1016 i F1015 listovima. Obrazac indukcije promotorskog regiona BvSTI gena praćen je u transgenim biljkama duvana kod kojih se pod kontrolom ovog promotora nalazio reporter GUS gen. Kod mladih in vitro gajenih biljaka nezavisno razvijenih T2 linija duvana ekspresija GUS gena bila je više konstitutivna. S druge strane, kod starijih biljaka gajenih u staklari mehaničko povređivanje, kao i povređivanje nastalo ishranom S. frugiperda larvi, indukovali su lokalizovanu ekspresiju GUS gena samo na mestu povređivanja u listovima i korenovima. Pokazana korelacija između stepena otpornosti genotipova šećerne repe i nivoa ekspesije BvSTI gena upućuje na zaključak da je ovaj gen učestvuje u mehanizmima odbrane protiv herbivornih insekata, te se smatra da može biti korišćen u biotehnološkim procesima unapređivanja otpornosti na insekte kod biljaka. Takođe, promotorski region BvSTI gena pokazao je inducibilnu prirodu ekspresije i može biti pogodan za kontrolisanu ekspresiju transgena u listovima i korenovima biljaka kao prva linija odbrane od insekata štetočina.

Plant proteinase inhibitor genes are among the prime candidates suitable for insect resistance improvement in plants. Expression pattern of a sugar beet serine proteinase inhibitor gene, BvSTI, was characterized in response to mechanical and fall armyworm (Spodoptera frugiperda J.E. Smith) induced wounding. BvSTI expression was analyzed in three breeding lines moderately resistant to sugar beet root maggot (Tetanops myopaeformis Roder.) and in a susceptible line, F1010. Increased mechanical wounding induced levels of BvSTI expression were observed in all resistant lines as compared to F1010. The most intensive response to wounding was observed in resistant lines, F1016 and UT-8, with a maximum up to 4- and 2,5-fold increase of BvSTI transcript levels over non-wounded roots and leaves, respectively. In contrast, slight increase of BvSTI transcript levels in leaves and even an initial decrease in roots were observed in sensitive F1010, but also in the third resistant line, F1015. BvSTI transcript accumulation in F1016 and F1010 tissues wounded by FAW showed a similar gene expression pattern, but it was delayed and less intense than the response incited by abiotic wounding. On the protein level, BvSTI-specific polyclonal antibodies confirmed increased accumulation of the 30 kDa BvSTI protein in wounded leaves but not in roots of F1016 and F1010. Using trypsin inhibition assays, the activity of BvSTI was confirmed in F1016 roots and leaves and F1010 leaves. In F1010 roots BvSTI activity was completely lacking. To confirm the potential role of the BvSTI gene in defending mechanisms to insect pests in sugar beet the same analyzed germplasm were bioassayed for resistance to fall armyworm insects. Larvae fed sugar beet leaves from all three resistant germplasms (F1016, F1015 and UT-8) had significant reductions in larval weights as compared to larvae fed on sensitive F1010 leaves. The observed daily weight increase was also the highest in larvae from sensitive vs. resistant leaves. As the larvae entered the pupal stage, pupal sizes did not reflect the overall larval weights and all developed pupae were similar. Larvae fed on roots were almost double lighter than larvae from leaves for all analyzed gemplasms. Some developmental abnormalities of the pupae fed on F1016 and F1015 leaves were noted. Since identification of gene promoters that are specifically activated in response to wounding or pest attack will facilitate regulated transgene expression in plants, we examined the induction pattern of a sugar beet promoter derived from the BvSTI proteinase inhibitor gene. BvSTI promoter was previously fused to the β-glucuronidase (GUS) reporter gene and transferred to Nicotiana benthamiana plants. GUS activity driven by BvSTI promoter was evaluated in independently derived BvSTI transgenic tobacco T2 homozygous progeny. Mechanical wounding and fall armyworm larval feeding induced GUS gene expression in tobacco leaves and roots that was localized at the wound site in mature tobacco tissues. Based on our results we can conclude that BvSTI gene expression was wound induced in the insect resistant germplasm suggesting that this gene can be used in biotechnological approaches or in breeding programs for improving insect resistance. Observation of GUS gene activity at the wound site in transgenic tobacco indicates that the BvSTI promoter is inducible in these tissues and, therefore, should prove useful for expressing resistance transgenes in leaves and roots as a first line of defense against plant pests and pathogens.

Uticaj trombina na regulaciju proliferacije glatkih mišićnih ćelija aorte pacova

Effects of trombin in the regulation of rat's aortic smooth muscle cells proliferation

Kardiovaskularne bolesti predstavljaju najveći uzrok smrtnosti ljudske populacije, a njihova glavna i osnovna patološka komponenta je ateroskleroza. Proliferacija ili deoba glatkih mišićnih ćelija krvnog suda (VSMC) ključni je događaj u nastanku raznih vaskularnih oboljenja, uključujući aterosklerozu i hipertenziju. U procesu diferencijacije i abnormalne deobe VSMC povezanih sa hipertenzijom i aterosklerozom uključen je i trombin. Stimulisanje VSMC trombinom dovodi do aktivacije ekstracelularnim signalima regulisanih kinaza 1 i 2 (ERK1/2), preko transaktivacije receptora za epidermalni faktor rasta (EGFR). U ranijim studijama Isenović i saradnici potvrdili su na osnovu inhibicije ERK1/2 od strane PD9805 inhibitora, učešće ERK1/2 u regulaciji proliferacije VSMC izazvanoj trombinom. U nastavku, faktor rasta sličan epidermalnom faktoru rasta koji vezuje heparin (HB-EGF), protein kinaza C delta (PKCδ) i matriksne metaloproteinaze (MMP), nađene su u VSMC i pokazano je da je i njihova aktivnost regulisana trombinom. ADAM (engl. “A Disintegrin And Metalloproteinase”) transmembranske su metaloproteinaze koje pripadaju adamalizinskoj grupi i razlikuju se od matriksnih metaloproteinaza po tome što imaju vanćelijski dizintegrinski i citoplazmatski domen. C terminalni domen može da stupa u interakciju sa unutarćelijskim proteinima. Uvidom u literaturu, do sada ne postoji istraživanje koje ukazuje na prisustvo ADAM metaloproteinazne aktivnosti vezane za proliferaciju VSMC stimulisane trombinom. Predmet ove doktorske teze jeste proučavanje uloge EGFR, ERK1/2, HB-EGF, PKCδ, ukupnih metaloproteinaza, kao i specifičnih MMP-2 i ADAM 12, u posredovanju proliferativnog efekta trombina na VSMC pacova. Inkubacija VSMC pacova sa trombinom (1 U/ml) u periodu od 5 minuta rezultirala je u značajnom povećanju: fosforilacije ERK1/2 od 8.7 ± 0.9 puta (p<0.001), fosforilacije EGFR od 8.5 ± 1.3 puta (p<0.001) i sinteze DNK od 3.6 ± 0.4 puta (p<0.001). Prethodni pojedinačni tretmani ovih ćelija u trajanju od 30 minuta sa 10 µM PD169540 (PD), ireverzibilnim inhibitorom EGFR, i 20 µM antitelom protiv HB-EGF značajno su smanjili trombinom stimulisanu fosforilaciju EGFR za 81 % i 72 % i ERK1/2 za 48 % i 61 %, respektivno. Istim ovakvim pretretmanima sa PD i HB-EGF antitelom smanjena je značajno proliferacija VSMC stimulisana trombinom za 44 % i 45%. Takođe, polučasovni pretretmani VSMC u toku 30 minuta sa 10 µM KB-R7785 (KB), specifičnim ADAM 12 inhibitorom i 10 µM specifičnim MMP-2 inhibitorom značajno su smanjili fosforilacije izazvane trombinom: EGFR za 47 % i 78 % i ERK1/2 za 25 % i 25 %, respektivno. U nastavku, istim ovim pretretmanima sa KB ili MMP2 inhibitorom, smanjena je trombinom stimulisana proliferacija za 27 % i 45 %. Prethodni polučasovni tretman VSMC sa 5 µM rotlerinom, specifičnim inhibitorom za PKC delta, značajno je smanjio fosforilaciju EGFR i ERK1/2 i proliferaciju VSMC stimulisanu trombinom za 58 %, 55% i 32%, respektivno. Dobijeni rezultati pokazuju da je proliferacija VSMC pod delovanjem trombina pretežno zavisna od nivoa aktivacije ERK1/2 kinaze nego od nivoa aktivacije EGFR. Takođe, ovi rezultati ukazuju da trombin deluje preko EGFR i ERK1/2 signalnih puteva koji uključuju PKCδ, MMP-2 i ADAM 12, stimulišući proliferativni proces VSMC pacova. U zaključku, saznanje o učešću ADAM 12 molekula u proliferaciji VSMC pod delovanjem trombina je od izuzetne važnosti za bolje razumevanje uloge trombina u kardiovaskularnoj biologiji i medicini. Pošto su MMP-2 i ADAM 12 potvrđeni kao medijatori migracije i hipertrofije VSMC, oni predstavljaju potencijalne ključne „mete” u novim terapeutskim intervencijama kojima bi se smanjilo ireverzibilno oštećenje tkiva povezano sa aterosklerozom i hipertenzijom. Zasigurno bi jedna od poželjnih opcija, kao in vivo regulisane sinteze MMP-2 i ADAM 12, doprinela redukciji patološke proliferacije VSMC.

ardiovascular disease is the largest single cause of mortality and its major underlying pathology is atherosclerosis. The proliferation of vascular smooth muscle cells (VSMC) is a key event in the pathogenesis of various vascular diseases, including atherosclerosis and hypertension. Thrombin is involved in the differentiation and abnormal proliferation of VSMC associated with atherosclerosis and hypertension. Thrombin stimulation results in extracellular signal-regulated kinase (ERK 1/2) activation through transactivation of the epidermal growth factor receptor (EGFR). Based on the studies in which PD98059 used to inhibit ERK1/2, we have shown previously that ERK1/2 was involved in the regulation by thrombin of VSMC’s proliferation. In addition, heparin-binding EGF-like growth factor (HB-EGF) and matrix metalloproteinases (MMPs) have also been detected in VSMC and shown to be regulated by thrombin. ADAMs (A Disintegrin And Metalloproteinase) are transmembrane metalloproteinases, belonging to adamalysin group, that are distinct from matrix metalloproteinases (MMPs) in that, they have an extracellular disintegrin domain and cytoplasmic domain that can associate with intracellular proteins. To the present knowledge there is no study that indicates the activation of an ADAM member in thrombin-induced VSMC proliferation. In this dissertation, the role of EGFR, ERK1/2, HB-EGF, general metalloproteinases, MMP-2 and ADAM 12, as well as PKCδ in mediating the mitogenic action of Thrombin in rat VSMC was investigated. Incubation of rat VSMC with Thrombin (1 U/ml) for 5 minutes resulted in significant increase of ERK1/2 phosphorylation by 8.7 ± 0.9 fold (p<0.001), EGFR phosphorylation by 8.5 ± 1.3 fold (p<0.001) and DNA synthesis by 3.6 ± 0.4 fold (p<0.001). Separate pretreatments for 30 minutes with EGFR tyrosine kinase irreversible inhibitor, 10 µM PD169540 (PD), and 20 µM anti-HB-EGF antibody, significantly reduced thrombin- stimulated EGFR and ERK1/2 phosphorylation by 81 %, 72 % and by 48 % and 61%, respectively. Furthermore, same pretreatments with PD and anti-HB-EGF antibody reduced Thrombin-induced VSMC’s proliferation by 44% and 45%, respectively. In addition, pretreatments for 30 minutes with 10 µM KB-R7785 (KB), a specific ADAM 12 inhibitor or 10 µM specific MMP2 inhibitor significantly reduced thrombin-stimulated EGFR and ERK1/2 phosphorylation by 47 %, 47 % and by 25 % and 25 %, respectively. Furthermore, same pretreatments with either KB or MMP2 inhibitor reduced thrombin-induced VSMC’s proliferation by 27 % and 45 % respectively. Pretreatment of VSMC with 5 µM rottlerin, a specific inhibitor of PKCδ, for 30 min significantly reduced EGFR and ERK1/2 phosphorylation by 58 % and 55%, respectively and reduced significantly thrombin-induced VSMC’s proliferation by 32%. These results show that thrombin-induced DNA synthesis correlates with the level of ERK1/2 activation rather than EGFR activation. Further, these results suggest that thrombin acts through EGFR and ERK 1/2 signaling pathways involving PKCδ, MMP-2 and ADAM 12, to up-regulate proliferation of VSMC. Therefore, this thesis examines recent findings in signaling mechanisms employed by thrombin in modulating the regulation of proliferation of VSMC with particular emphasis on involvement of ADAM 12 and MMP2 that have been identified as important mediators of VSMC hypertrophy and vascular diseases. These findings are critical for understanding the role of thrombin in vascular biology and vascular diseases. Since these two molecules have been acknowledged as important mediators of VSMC migration and hypertrophy, MMP-2 and ADAM 12 are identified as one of key targets for novel therapeutic interventions to minimize irreversible tissue damage associated with hypertension and atherosclerosis. A safe option to regulate in vivo synthesis of MMP-2 and ADAM 12 would be welcome.

Analiza ekspresije eNOS i angiogenih faktora HIF-1alfa i VEGF u mijeloproliferativnim neoplazmama: veza sa prisustvom mutacija u genima za JAK2 i CALR

Analysis of the expression of eNOS and angiogenic factors HIF-1alfa and VEGF in myeloproliferative neoplasms: a connection with the presence of mutations in the genes for JAK2 and CALR

Mijeloproliferativne neoplazme (MPN) predstavljaju klonalne bolesti matičnih ćelija hematopoeze koje karakteriše poremećaj proliferacije jedne ili više mijeloidnih ćelijskih linija. MPN obuhvataju 3 entiteta: policitemiju veru (PV), esencijalnu trombocitemiju (ET) i primarnu mijelofibrozu (PMF). Najzastupljeniji poremećaj kod MPN je JAK2V617F mutacija koja je prisutna kod 97% pacijenata sa dijagnozom PV i 50-60% pacijenata sa dijagnozama ET i PMF. JAK2V617F mutacija indukuje konstitutivnu aktivaciju nishodnih signalnih puteva (JAK2/STAT3, PI3K/AKT, MAPK) koji su uključeni u mijeloproliferaciju. Druga najzastupljenija somatska mutacija, koja je prisutna kod većine JAK2V617F negativnih ET i PMF pacijenata, povezana je sa genom koji kodira kalretikulin (CALR) prisutna kod 31,9% ET i 31,7% PMF pacijenata. Kod različitih hematoloških maligniteta, uključujući i MPN, kao odraz pojačane angiogeneze, opisano je prisustvo povišene ekspresije faktora rasta vaskularnog endotela (VEGF). U novijim istraživanjima pokazana je veza između ekspresije VEGF gena i nivoa hipoksija- inducibilnog faktora-1 (HIF-1) azot monoksida (NO). Nekoliko studija do sada je pokazalo da NO može indukovati produkciju VEGF putem PI3K/Akt/PKB/HIF- 1α puta, ali i da deluje kao inhibitor HIF-1α ekspresije. Cilj istraživanja ove disertacije bilo je ispitivanje stepena ekspresije i korelacija ključnih angiogenih faktora - HIF-1α, VEGF i endotelne NO sintaze (eNOS) u kostnoj srži, CD34+ ćelijama i granulocitima MPN pacijenata prema JAK2 i CALR statusu i terapiji. Pored toga, koristili smo proinflamatorni IL-6 da indukujemo ekspresiju HIF-1α, VEGF i eNOS u humanoj HEL 92.1.7 ćelijskoj liniji sa JAK2 mutacijom, kao i u diferentovanim makrofagama. Takođe ispitivano je učešće različitih signalnih puteva (JAK2/STAT3, PI3K/Akt, MAPK) putem kojih IL-6 ostvaruje svoje efekte, na modelu HEL ćelijske linije kao i kod granulocita MPN pacijenata. Rezultati ove studije su pokazali da angiogeni faktori imaju izraženiju ekspresiju u granulocitima nego u CD34+ i ćelijama kostne srži, što ukazuje na značaj inflamatornih granulocita u razvoju angiogeneze. Pokazana je i značajna negativna korelacija između proteinske ekspresije HIF-1α i VEGF, kao i HIF-1α i eNOS, dok između VEGF i eNOS postoji značajna pozitivna korelacija kod granulocita MPN pacijenata. Standardno lečenje primenom hidroksiureje kod MPN pacijenata ima antiangiogeni potencijal u smislu smanjenja sva tri angiogena faktora – HIF-1α, VEGF i eNOS u granulocitima. IL-6 povećava ekspresiju sva tri angiogena faktora u JAK2V617F mutiranim HEL ćelijama kao i diferenciranim makrofagama. Pored toga, JAK1/2 inhibitor ruksolitinib pokazuje jaču i stabilniju stimulaciju angiogenih faktora nego JAK2 specifični inhibitor 1,2,3,4,5,6- heksabromocikloheksan i kod HEL ćelija i kod diferenciranih makrofaga. IL-6 pokazuje JAK2V617F zavisnost u aktivaciji MAPK signalnog puta kod MPN, dok ruksolitinib snažno aktivira MAPK put kod ET i PMF pacijenata, nezavisno od JAK2 mutacije. IL-6 i oba JAK1/2 inhibitora redukuju broj HEL ćelija u G2/M fazi ćelijskog ciklusa i takođe dovode do pojačane apoptoze Kod MPN pacijenata, IL-6 zaustavlja granulocite u S fazi ćelijskog ciklusa, pojačavajući DNK replikaciju i sprečavajući apoptozu preko JAK1/2 stimulacije. Ćelijski ciklus u granulocitima kod PV pacijenata je u kontrastu u odnosu na ET i PMF pacijente, bez uticaja JAK2 mutacije, izuzev apoptoze kod ET pacijenata. Oba JAK1/2 inhibitora pojačavaju apoptozu u granulocitima kod PV pacijenata. Faktori angiogeneze stimulisani inflamacijom su povećani kod MPN i smanjuju se primenom hemioterapije. Inflamacija stimuliše DNK replikaciju i JAK1/2 zavisnu proliferaciju, karakteristično pojačanu kod MPN.

hematopoiesis characterized by a disorder of proliferation of one or more myeloid cell lines. MPNs include 3 entities: polycythemia (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The most common disorder in MPN is the JAK2V617F mutation present in 97% of patients diagnosed with PV and 50-60% of patients with ET and PMF diagnoses. The JAK2V617F mutation induces the constituent activation of the innate signal pathways (JAK2 / STAT3, PI3K / AKT, MAPK) involved in myeloproliferation. The second most common somatic mutation, which is present in most JAK2V617F negative ET and PMF patients, is associated with the calreteculin (CALR) encoding genome present in 31.9% ET and 31.7% of PMF patients. In the case of various hematological malignancies, including MPN, as a reflection of increased angiogenesis, the presence of elevated expression of vascular endothelial growth factor (VEGF) has been described. Recent studies have shown the relationship between the expression of the VEGF gene and the levels of hypoxia- inducible factor-1 (HIF-1) and nitrogen monoxide (NO). Several studies have so far shown that NO can induce the production of VEGF via PI3K / Akt / PKB / HIF-1α times, but also acts as an inhibitor of HIF-1α expression. The aim of the study was to investigate the degree of expression and correlation of key angiogenic factors - HIF-1α, VEGF and endothelial NO synthase (eNOS) in the bone marrow, CD34+ cells and granulocytes of MPN patients according to JAK2 and CALR status and therapy. In addition, we used proinflammatory IL-6 to induce the expression of HIF-1α, VEGF and eNOS in the human HEL 92.1.7 cell line with a JAK2 mutation, as well as in differentiated macrophages. Also studied was the participation of various signaling pathways (JAK2 / STAT3, PI3K / Akt, MAPK) through which IL-6 achieved its effects on the HEL cell line model as well as in granulocyte of MPN patients. The results of this study have shown that angiogenic factors have a more pronounced expression in granulocytes than in CD34+ and bone marrow cells, which indicates the importance of inflammatory granulocytes in the development of angiogenesis. A significant negative correlation between HIF-1α and VEGF protein expression, as well as HIF-1α and eNOS, was shown, while there was a significant positive correlation between VEGF and eNOS in granulocytes of MPN patients. Standard treatment with hydroxyurea in MPN patients has antiangiogenic potential in terms of reducing all three angiogenic factors - HIF-1α, VEGF and eNOS in granulocytes. IL-6 increases the expression of all three angiogenic factors in JAK2V617F mutated HEL cells as well as differentiated macrophages. In addition, the JAK1/2 inhibitor ruxolitinib exhibits a stronger and more stable stimulation of angiogenic factors than the JAK2 specific inhibitor 1,2,3,4,5,6-hexabromocyclohexane, both in HEL cells and in differentiated macrophages. IL-6 shows the JAK2V617F dependence on the activation of the MAPK signaling pathway in MPN, while ruxolitinib actively activates the MAPK pathway in ET and PMF patients, independently of the JAK2 mutation. IL-6 and both JAK1/2 inhibitors reduce the number of HEL cells in the G2/M phase of the cell cycle and also lead to increased apoptosis. In MPN patients, IL-6 stops granulocytes in the S phase of the cell cycle, enhancing DNA replication and preventing apoptosis through JAK1/2 stimulation. The cell cycle in granulocytes in PV patients contrasts with ET and PMF patients, without the influence of the JAK2 mutation, except for apoptosis in ET patients. Both JAK1/2 inhibitors enhance apoptosis in granulocytes in PV patients. Inflammation-stimulated angiogenesis factors have been increased in MPN and decreased by the use of chemotherapy. Inflammation stimulates DNA replication and JAK1/2 dependent proliferation, characteristically enhanced by MPN.

Rasprostranjenje, ekologija i predviđanje distribucije invazivnih taksona roda Reynoutria Houtt. (Polygonaceae) na području Srbije i jugoistočne Evrope

Distribution, ecology and prediction of potential ranges of invasive Reynoutria Houtt. taxa (Polygonaceae) in Serbia and southeastern Europe

Iako su invazivni predstavnici roda Reynoutria proučavani od strane više autora, prevashodno na nivou pojedinačnih država, pojedinačnih staništa ili zaštićenih područja, nema naučnih radova u kojima se celovito analizira njihova ekologija, ukupno rasprostranjenje, kao i predviđanje buduće distribucije na području Srbije i jugoistočne Evrope. Takođe, evidentna je nedovoljna istraženost mehanizama adaptacije i strategije preživljavanja ovih taksona u antropogeno zagađenim staništima. Zbog svega, osnovni ciljevi ove disertacije su utvrđivanje prisustva i distribucije invazivnih taksona roda Reynoutria na području Srbije i jugoistočne Evrope, ekološka analiza u odnosu na klimatske parametre i tip staništa, procena invazibilnosti zaštićenih područja Srbije i jugoistočne Evrope, kao i analiza bioakumulacionih potencijala za makronutrijente i teške metale taksona R. x bohemica. Istraživanja su obavljena u periodu 2006. – 2016. godina. Zabeleženo je ukupno 4081 lokaliteta, u okviru tri različita tipa staništa. Najčešći taksoni su R. japonica i hibrid R. × bohemica. Primenom metode MaxEnt modelovanja utvrđeno je da padavine najtoplijeg kvartala (BIO18) imaju najveći uticaj na formiranje ekološke niše ovih taksona. Modelovana niša taksona R. × bohemica ukazuje na njegovu toleranciju visokih temperatura u različitim periodima godine, uz istovremeno niže količine padavina (i do 60 mm u odnosu na roditeljske vrste). Hemijskom analizom biljnih tkiva R.× bohemica, utvrđena je slaba akumulacija teških metala sa strategijom njihovog isključivanja, uz veoma efikasnu akumulaciju makronutrijenata. Rezultati upućuju na potrebu sprečavanja osnivanja novih populacija u blizini zaštićenih područja koja imaju pogodne sredinske faktore za razvoj ovih taksona.

Although the invasive Reynoutria taxa have been studied by several authors, primarily at country, habitat or protected areas level, there are no scientific papers in which their ecology, distribution and future distribution predictions in Serbia and Southeastern Europe are completely analyzed. Also, there is lack of scientific research of adaptation mechanisms and survival strategies of these taxa in anthropogenically polluted soils. Hence, the main objectives of this dissertation are determination the presence and distribution of invasive Reynoutria taxa in Serbia and southeastern Europe, ecological study of bioclimatic parameters in different habitats and protected areas invasibility assessment in Serbia and Southeastern Europe, as well as R. × bohemica‘s bioaccumulation potential of macronutrients and heavy metals. Research was carried out in the period 2006-2016. A total of 4081 localities were recorded in seven SE Europe countries, in three types of habitats. The most frequent taxa are R. japonica and hybrid R. × bohemica. The precipitation of the warmest quarter (BIO18) had a highest influence on the potential distribution modelling of these taxons according to the MaxEnt. Potential distribution modelling revealed R. × bohemica's high temperature tolerance in different seasons with high durability of drought (up to 60 mm per year less precipitation than parental species). Weak heavy metal accumulation accompanied by exclusion, with very efficient macronutrients accumulation, seems to be the survival and expansion strategy of Reynoutria × bohemica according to the chemical analysis results. The results indicate the need to prevent the establishment of new populations near protected areas with suitable bioclimatic parameters for their development.

Diverzitet aerofitskih cijanobakterija i algi u biofilmu odabranih pećina u Srbiji

Diversity of aerophytic cyanobacteria and algae in biofilm from selected caves in Serbia

Predmet ove doktorste disertacije je analiza zajednice aerofitskih cijanobakterija i algi iz biofilma sa stenovitog supstrata ulaza petnaest odabranih pećina iz Srbije kao i nekoliko tačaka u blizini veštačkog osvetljenja. Akcenat istraživanja bio je na fototrofnim mikroorganizmima iz biofilma, dok su kao dodatak endolitski predstavnici identifikovani iz nekoliko uzoraka stene. Analizirana je i morfologija, stepen razvijenosti biofilma, kao i udeo sadržaja vode, organske i neorganske materije u biofilmu. U svim pećinama, mereni su ekološki parametri i određene su primarna produkcija (preko koncentracije hlorofila a) i petrografska analiza stenovitog supstrata. Na odabranim uzorcima rađena je kvantitativna i sezonska analiza cijanobakterija i algi. Statističke analize su poslužile kao alat za sagledavanje odnosa zabeleženih fototrofnih mikroorganizama i različitih ekoloških i opisnih parametara. Zabeleženo je prisustvo ukupno 290 taksona cijanobakterija i algi koje su svrstane u 4 razdela: Cyanobacteria (134 taksona, od kojih 82 taksona pripada kokoidnim formama), Bacillariophyta (129 taksona), Chlorophyta (26 taksona) i Xanthophyta (1 takson). Najveći broj taksona Cyanobacteria zabeležen je u rodovima Gloeocapsa, Chroococcus, Gloeothece, Leptolyngbya i Scytonema. U razdelu Bacillariophyta se izdvajaju rodovi Luticola i Humidophila, a u razdelu Chlorophyta rod Cosmarium. Iz biofilma Božanine pećine opisana je nova vrsta cijanobakterije za nauku, Nephrococcus serbicus S. Popović, G. Subakov Simić i J. Komárek, sp. nov. pri čemu je sam rod po prvi put zabeležen na teritoriji Evrope. Cyanobacteria po broju taksona dominiraju na svim lokalitetima, osim Rćanske i Bogovinske pećine. Najveći broj pripadnika Bacillariophyta i Chlorophyta zabeležen je u Rćanskoj (usled prisustva vode na biofilmu) i pećini Samar (zbog sezonskog uzorkovanja). Upravo se značaj sezonskog uzorkovanja biofilma u pećinama Samar i Jezava pre svega ogleda kroz znatno veći diverzitet aerofitskih cijanobakterija i algi u odnosu na druge pećine. Predstavnici Cyanobacteria dominirali su u pećinama čiji je supstrat rekristalisali i organogeni krečnjak, a Bacillariophyta na klastičnom i mikritskom krečnjaku. U dobro razvjenim, želatinoznim biofilmovima dominirale su cijanobakterije. U tankom sloju biofilma na stenovitom supstratu dominirale su zelene alge ukoliko su biofilmovi bili suvi, odnosno silikatne alge ukoliko su bili vlažni. Vrednosti ekoloških parametara na svakoj tački uzorkovanja dovedeni su u vezu sa lokacijom pećine, morfologijom i veličinom pećinskog ulaza, godišnjim i dnevnim variranjima meteoroloških činilaca. Vrednosti temperature i relativne vlažnosti vazduha bile su prilično ujednačene na nivou pojedinačne pećine, dok su vrednosti intenziteta svetla pokazale varijacije i zavisnost u odnosu na blizinu tačke uzorkovanja ulazu, kao i njene ekspoziciju. Koncentracija hlorofila a, kao i sadržaj vode, organske i neorganske materije bili su korelisani sa dobro razvijenim biofilmom na ulazima pećina i biofilmom na horizontalnoj površini. Sezonska analiza biofilma u odabranim pećinama pokazala je da odnos vrednosti parametara u biofilmu zavisi od sezone, što se najviše odrazilo na sadržaj vode. Vrednosti hlorofila a bile su više u hladnijim mesecima. Najveća vrednost biomase je u Degurićkoj pećini izračunata na mestu gde je visok sadržaj vode, kao i viša koncentracija hlorofila a, a u pećini Vernjikica na mestu gde je zabeležen i visok sadržaj organske materije.

The aim of this PhD thesis is the analysis of the aerophytic cyanobacterial and algal community from biofilm developed on stone substrate at the entrance of fifteen selected caves from Serbia and several sites near the artificial light in caves. The focus of the study was on phototrophic microorganisms from the biofilm, while endolithic representatives were additionaly identified from several stone samples. Morphology, degree of biofilm development, as well as the content of water, organic and inorganic matter in biofilm were also observed. At all sampling sites, ecological parameters, primary production (through concentration of chlorophyll a), as well as petrographic analysis of rocky substrates were measured and determined. Quantitative and seasonal analyzes of cyanobacteria and algae were assessed in selected samples. Statistical analyzes have served as a tool for examining the relationship between recorded phototrophic microorganisms and various ecological and descriptive parameters. A total of 290 cyanobacterial and algal taxa were recorded and classified into 4 divisions: Cyanobacteria (134 taxa, of which 82 taxa belong to the coccoid forms), Bacillariophyta (129 taxa), Chlorophyta (26 taxa) and Xanthophyta (1 taxon). The highest number of cyanobacterial taxa was documented within the following genera: Gloeocapsa, Chroococcus, Gloeothece, Leptolyngbya and Scytonema. Luticola and Humidophila were the most diverse genera within Bacillariophyta division, while in Chlorophyta Cosmarium had the highest number of species. A new cyanobacterial taxon, Nephrococcus serbicus S. Popović, G. Subakov Simić & J. Komárek, sp. nov., was described from a biofilm sample of Božana Cave. This also represents the first record of the genus Nephrococcus in Europe. Cyanobacteria were dominant in all caves, except Rćanska and Bogovinska cave. The largest number of Bacillariophyta and Chlorophyta was recorded in the Rćanska cave (due to the presence of water on samplins sites) and in the Samar Cave (due to seasonal sampling). The significance of the seasonal biofilm sampling in Samar and Jezava caves is primarily reflected through the higher diversity of aerophytic cyanobacteria and algae in comparison to other caves. Representatives of Cyanobacteria dominated in caves in which substrate was characterized as recrystallized and organogenic limestone, while representatives of Bacillariophyta prevailed in biofilm developed on clastic and micritic limestone. In well-developed, gelatinous biofilms, Cyanobacteria were dominant. In thin biofilm layer on stone substrate, green algae were dominant if the biofilm was dry, while diatoms prevailed if water was present on biofilm. The values of the ecological parameters that were measured at each sampling site were related to the cave location, the morphology and the size of the cave entrance, annual and daily fluctuations of meteorological parameters. The values of temperature and relative humidity were fairly uniform in each cave, while the light intensity varied and was dependant on the proximity of the sampling site to the cave entrance, as well as its exposure. The concentration of chlorophyll a, as well as the biofilm parameters (water content, content of organic and inorganic matter) was correlated with well-developed biofilms at the cave entrances and biofilm on horizontal surfaces. Seasonal analysis of biofilms in selected caves showed that the ratio of the biofilm parameters was dependent on the season, which mostly affected the water content. The values of chlorophyll a were higher in colder months of the year. The largest cyanobacterial and algaa biomass in Degurić cave was at the sampling site where high content of water in the biofilm and higher concentration of chlorophyll a were recorded, and for Vernjikica cave, the largest biomass was recored at sampling site with high content of organic matter.

Sastav, animikrobna i antioksidativna aktivnost etarskih ulja Thymus algeriensis, Th. capitatus, Salvia fruticosa i Satureja thymbra iz Libije

Composition, antimicrobial and antioxidant activity of the essential oils of Thumus algeriensis, Thymus capitatus, Satureja thymbra and Salvia fruticosa from Libya

U ovom radu analiziran je sastav etarskih ulja nekih biljnih vrsta iz familije Lamiaceae koje rastu kao samonikle u Libiji (Thymus capitatus, Th. algeriensis, Satureja thymbra i Salvia fruticosa) korišćenjem GC i GC-MS tehnika. Ulja Thymus capitatus, Th. algeriensis i Salvia fruticosa karakterišu se prisustvom oksigenovanih monoterepena, koji su zastupljeni sa 87.60%, 54.67% i 64.89%. Monoterpenski ugljovodonici su glavna grupa jedinjenja kod vrste Satureja thymbra , čineći 58.57% od ukupnog ulja. Glavna jedinjenja etarskog ulja Thymus capitatus su karvakrol (68.19%) i timol (12.29%), dok etarsko ulje Th. algeriensis karakteriše timol (38.50%) koji je glavna komponenta. Ulje Satureja thymbra kao vodeće komponente sadrži γ- terpinen (39.23%), timol (25.16%) i p-cimen (7.17%). Visok sadržaj 1.8- cineola (49.34%) i kamfora (7.53%) je prisutan u ulju Salvia fruticosa. Takođe je ispitivano antioksidativno dejstvo ulja i glavnih komponenata etarskih ulja korišćenjem DPPH testa. Najviši antioksidativni potencijal pokazalo je ulje Satureja thymbra (IC50 = 0.0967 mg/ml rastvora), zatim Thymus capitatus (IC50 = 0.119 mg/ml rastvora), Th. algeriensis sa IC50 od 0.299 mg/ml, a najniža aktivnost dobijena je sa uljem Salvia fruticosa (IC50 = 15.53 mg/ml rastvora). Dobijene IC50 vrednosti za timol i karvakrol su 0.403 i 0.105 mg/ml, i 0.0717 mg/ml rastvora za BHA. Korišćenjem metode mikrodilucije ispitivana je antimikrobna aktivnost na osam bakterija i osam gljiva. Kao test organizmi korišćeno je četiri vrste Gram-negativnih bakterija - Escherichia coli (ATCC 35210), Pseudomonas aeruginosa (ATCC 27853), Salmonella typhimurium (ATCC 13311), Proteus mirabilis (humani izolat) i četiri Gram- pozitivne bakterije – Listeria monocytogenes (NCTC 7973), Bacillus cereus (klinički izolat), Micrococcus flavus (ATCC 10240), i Staphylococcus aureus 6 (ATCC 6538). Najbolju aktivnost među uljima analiziranih biljnih vrsta, pokazalo je ulje Thymus capitatus, gde je minimalna inhibitorna aktivnost na testirane bakterije bila 0.001-0.002 mg/ml, dok je baktericidna aktivnost (MBC) postignuta u koncentraciji 0.001-0.04 mg/ml. Ulje Th. algeriensis imalo je vrednosti MIC od 0.001-0.05 mg/ml, dok je MBC postignuta sa vrednostima ulja od 0.0025-0.05 mg/ml. Ulje Satureja thymbra je imalo bakteriostatsko delovanje u koncentraciji od 0.001-0.1 mg/ml i baktericidno od 0.002-0.2 mg/ml. Etarsko ulje Salvia fruticosa bilo je efikasno u rasponu od 0.125-1.5 mg/ml za MIC i od 0.5-2.0 mg/ml za MBC. Za utvrđivanje antifungalne aktivnosti etarskih ulja, testirane su sledeće mikromicete: Aspergillus flavus (ATCC 9643), Aspergillus fumigatus (humani izolat), Aspergillus niger (ATCC 6275), Aspergillus ochraceus (ATCC 12066), Penicilliim funiculosum (ATCC 36839), Penicillium ochrochloron (ATCC 9112), Trichoderma viride (IAM 5061) i kvasac Candida albicans (humani izolat). Thymus capitatus je i sa ovog aspekta pokazao najveću aktivnost, gde se minimalna inhibitorna koncentracija kretala od 0.0002 do 0.001 mg/ml i minimalna fungicidna koncentracija od 0.002-0.025 mg/ml, u poređenju sa MIC za Th. algeriensis od 0.0005 do 0.025 mg/ml i MFC 0.001-0.05 mg/ml. Ulje Satureja thymbra je pokazala fungistatičku aktivnost od 0.001-0.025 mg/ml i fungicidnu od 0.001-0.1 mg/ml. MIC za Salvia fruticosa ulje kretala se od 0.125 do 1.0 mg/ml, a MFC u rasponu od 0.125 do 1.5 mg/ml.

The essentiasl oils composition of some essential oils from Lamiaceae family wild growing in Libya (Thymus capitatus, Thymus algeriensis, Satureja thymbra and Salvia furticosa) have been analyzed using GC and GC-MS. The oils of Thymus capitatus, Th. algeriensis and Salvia fruticosa were characterised with domination of oxygenated monoterepenes with percentage 87.60%, 54.67% and 64.89 % respectively. Monoterpene hydrocarbons was the major group of the compounds of the oil of S. thymbra represented 58.57% of the total oil. The main compounds of Th. capitatus essential oil were carvacrol (68.19%) followed by thymol (12.29%), while Th. algeriensis essential oil was characterized by thymol (38.50%) as the major component. The oil of S. thymbra was demonstrated by γ- terpinene (39.23%), thymol (25.16%) and p-cymene (7.17%) as the major constituents. S. fruticosa oil was characterized by contained 1,8-cineole (49.34%) and camphor (7.53%) as the main compounds. The oils were also, screened for antioxidant activity by DPPH assay, and compared with their main compounds. The highest antioxidant activity showed S. thymbra oil with IC50 = 0.0967 mg/ml of solution, followed by Th. capitatus (IC50 = 0.119 mg/ml, Th. algeriensis with the IC50 of 0.299 mg/ml and the lowest activity was found for S. fruticosa (IC50 = 15.53 mg/ml of solution), comparing with 0.403 and 0.105 mg/ml for thymol and carvacrol, and 0.0717 mg/ml of solution for BHA. Furthermore, antimicrobial activity was tested using microdilution method against eight bacteria and eight fungal species. Gram- negative from bacteria: Escherichia coli (ATCC 35210), Pseudomonas aeruginosa (ATCC 27853), Salmonella typhimurium (ATCC 13311), Proteus mirabilis (human isolate) and from Gram-positive bacteria: Listeria monocytogenes (NCTC 7973), Bacillus cereus (clinical isolate), Micrococcus flavus (ATCC 10240), and Staphylococcus aureus (ATCC 6538) were used. Among the oils, Th. capitatus oil showed the best activity, where the inhibitory activity against tested bacteria was at 0.001-0.002 mg/ml, while bactericidal activity (MBC) was achieved at 0.001- 0.04 mg/ml. Th. algeriensis oil showed MIC at 0.001-0.05 mg/ml, while bactericidal activity (MBC) was achieved at 0.0025-0.05 mg/ml, oil of Satureja thymbra showed 8 bacteriostatic activity at 0.001–0.1 mg/ml and bactericidal at 0.002–0.2 mg/ml. The essential oil of S. fruticosa was efficient with MIC range from 0.125-1.5 mg/ml and MBC from 0.5-2.0 mg/ml. For antifungal assay the oils were tested against the following fungi: Aspergillus flavus (ATCC 9643), Aspergillus fumigatus (human isolate), Aspergillus niger (ATCC 6275), Aspergillus ochraceus (ATCC 12066), Penicillium funiculosum (ATCC 36839), Penicillium ochrochloron (ATCC 9112), Trichoderma viride (IAM 5061) and yeast C. albicans (human isolate). Again Th. capitatus was the most powerful with MICs ranged from 0.0002-0.001 mg/ml and MFC 0.002-0.025 mg/ml, compared with activity MICs for Th. algeriensis, ranged 0.0005-0.025 mg/ml and MFC 0.001-0.05 mg/ml. S. thymbra showed fungistatic effects at 0.001–0.025 mg/ml and fungicidal effects at 0.001–0.1 mg/mL and S. fruticosa exhibited fungistatic (MIC) at 0.125-1.0 mg/ml and fungicidal effect (MFC) at 0.125-1.5 mg/ml.

Bioinformatički modeli za automatsko mapiranje interakcija između proteina kod čoveka

Bioinformatics models for automatic prediction of human protein-protein interaction

Interakcije između bioloških makromolekula imaju ključnu ulogu u osnovnim procesima u živim organizmima, posreduju u metaboličkim putevima, putevima prenosa signala, transkripciji, translaciji i drugim ćelijskim i sistemskim procesima. Veliki broj oboljenja uzrokovan je mutacijama proteina u regionima odgovornim za interakciju sa drugim proteinima koje mogu dovesti do ometanja interakcije protein-DNK, promene u obrascima savijanja proteina, novih nepoželjnih interakcija i omogućavanje interakcije protein-patogen. Mapiranje interaktoma, odnosno kompletne mape interakcija protein- protein (IPP) unutar organizma, je od suštinske važnosti za razumevanje kompleksnih molekularnih odnosa unutar živih sistema, kao i za rasvetljavanje raznih patoloških stanja ljudskog organizma. Bioinformatičke metode za automatsko predviđanje IPP, kao suplementi eksperimentalnim metodama za analizu IPP, omogućavaju bolje razumevanje bioloških procesa i funkcija, lakše otkrivanje potencijalnih meta za ciljanu terapiju i smanjenja vremena i troškova razvoja novih terapeutika. U ovoj studiji razvijeni su modeli i metode za automatsko predviđanje IPP bazirane na mašinskom učenju i proteinskoj sekvenci, koja predstavlja univerzalnu, visoko kvalitetnu i eksperimentalno potvrđenu informaciju o proteinu. Generisani su modeli za predviđanje IPP za specijalne slučajeve: (i) između transkripcionih regulatora, odnosno proteina koji učestvuju u kompleksnom procesu transkripcione regulacije koji kontroliše ekspresiju gena i značajan je za normalnu fiziologiju ćelije, i (ii) proteina sa neuređenom tercijarnom strukturom, koji su kao takvi uključeni u ključne biološke procese interakcijom sa višestrukim partnerima, imaju fleksibilnu strukturu, višestruke funkcije, centralnu ulogu u regulaciji signalnih puteva, procesu prepoznavanja i vezivanja za male molekule, i čine većinu proteina povezanih sa neprenosivim bolestima. Pored toga, kreirane su tri nove vrste atributa za predstavljanje proteina: (i) atributi zasnovani na primarnoj strukturi proteina, (ii) evolutivni atributi i (iii) mrežni atributi, kao i metode bazirane na genetskom algoritmu za (i) automatsko generisanje i selekciju atributa i (ii) za automatsko formiranje i optimizaciju ansambla modela zasnovanim na mašinskom učenju, u svrhu proširenja prostora atributa i povećanja efikasnosti predviđanja IPP. Kao glavni rezultat studije, na osnovu toga, razvijen je opšti model HP-GAS za automatsko mapiranje IPP na nivou proteoma čoveka. Opsežna evaluacija i poređenje sa state-of-the-art metodama pokazali su da HP-GAS predstavlja trenutno najefikasniji metod za mapiranje IPP na nivou proteoma čoveka, sa efikasnošću predviđanja AUC= 0.93 i 0.85 tačnosti. HP-GAS metod

Interactions between biological macromolecules have a critical role in essential processes in living organisms, mediate the metabolic pathways, signaling pathways, transcription, translation and other cellular processes and systems. A large number of diseases caused by mutations in the regions of the protein responsible for the interactions with other proteins which can lead to interference of protein-DNA interaction, changes in the patterns of protein folding, new undesirable interaction and facilitate interaction of the protein-pathogen. Interactome mapping, i.e. mapping of complete network of protein- protein interactions (PPI) within the organism, is essential to an understanding of complex molecular relationships within the living system, as well as to elucidate the various human pathological conditions. Bioinformatics methods for automated PPI prediction, as addition to experimental methods for the analysis of PPI, allow a better understanding of biological processes and functions, easier detection of potential therapeutic targets, and reduce the time and cost of drug development. In this study, models have been developed and methods for the automated prediction of PPI based on machine learning and the protein sequence, which is a universal, high-quality and experimentally confirmed information on the protein. Models for the PPI prediction were generated for special cases: (i) between human transcriptional regulators, i.e. the proteins involved in the complex process of transcriptional regulation that controls the gene expression and they are important for normal cell physiology, and (ii) intrinsically disorder proteins, characterized by the lack of a fixed tertiary structure, which are as such involved in the regulation of key biological processes via binding to multiple protein partners, are malleable adapting to structurally different partners, have multiple functions, play a central roles in the regulation of signaling pathways, the process of molecular recognition and binding of small molecule, and are the prevailing protein class associated with noncommunicable diseases. In addition, three novel types of features for the representation of the proteins were created: (i) the features based on the protein sequence, (ii) the evolutionary features, and (iii) the graph features, as well as methods based on the genetic algorithm for (i) automatic feature-engineering process and (ii) automatic ensembling of different machine learning algorithms, in order to expand the feature space and to improve the PPI prediction performance. Based on that, as a main result of the study, the general model HP-GAS for automatic mapping of PPIs at the human proteome level was created. Extensive evaluation and comparison with the state-of-the-art methods, show that the HP-GAS represents currently the most efficient method for proteome-wide forecasting of protein interactions, with prediction efficacy of AUC = 0.93 and 0.85 accuracy. HP-GAS method and PPI prediction models for special cases were implemented as free, time-efficient and easy-to-use software tools.

Antitumorski efekat steroidnih tetraoksana na maligno transformisane ćelijske linije čoveka

Antitumor effect of steroidal tetraoxanes on malignantly transformed human cell lines

Uvod: Pronalaženje agenasa sa potencijalnim antitumorskim dejstvom je imperativ u modernoj onkologiji. Pri tome se sve više zapaža da određene hemijske strukture imaju specifičnije toksično dejstvo na maligne ćelije. Otkriće artemizinina je označilo početak istraživanja peroksida kao potencijalne zamene za tradicionalne antimalarijske lekove, a iz ovih istraživanja nastala je i strukturno jednostavna klasa peroksida - 1,2,4,5-tetraoksani, za koje je ubrzo pokazano da pored antimalarijskog pokazuju i snažan antiproliferativni efekat. Maligne ćelije imaju poremećaje u regulatornim mehanizmima koji upravljaju ćelijskom proliferacijom i homeostazom. Sposobnost tumorskih ćelijskih populacija da povećaju broj ćelija je određena ne samo intenzitetom ćelijske proliferacije, već i brzinom uklanjanja ćelija. Programirana ćelijska smrt - apoptoza, predstavlja glavni izvor ovog uklanjanja. Cilj rada je bio da se odredi nivo citotoksičnog dejstva grupe mešovitih tetraoksana prema različitim humanim malignim ćelijama, kao i da se odredi koeficijenat selektivnosti u njihovom dejstvu u odnosu na zdrave imunokompetentne ćelije. U cilju dobijanja uvida u mehanizam dejstva ispitivanih jedinjenja odrediće se tip ćelijske smrti koju indukuju ispitivani tetraoksani, kao i produkcija reaktivnih kiseoničnih vrsta u Hela ćelijama. Cilj 3D QSAR studije o antiproliferativoj aktivnosti trideset tri 1,2,4,5-tetraoksanska derivata prema Hela i Fem-x tumorskim ćelijskim linijama, je bio da se utvrdi koje su najvažnije farmakofore steroidnih tetraoksana koje utiču na potenciju ispitivanih jedinjenja prema HeLa i Fem-x tumorskim ćelijskim linijama. Materijal i metode: Citotoksično dejstvo tetraoksana DO-122 - DO-124 i DO-126 - DO-128, prema pet tumorskih ćelijskih linija je ispitivano standardnim MTT testom. U cilju određivanja tipa ćelijske smrti indukovane tretmanom ispitivanim tetraoksanima načinjena je morfološka analiza HeLa ćelija obojenih smešom akridin oranža i etidijum bromida. Analiza određivanja distribucije faza ćelijskog ciklusa HeLa ćelija obojenih propidijum jodidom, urađena je na protočnom citometru. Produkcija intraćelijskih reaktivnih vrsta kiseonika (ROS) je merena fluorometrijski pomoću fluorescentne boje 2’,7’-dihlorodihidrofluorescein diacetata. Zavisnost strukture i funkcije trideset tri 1,2,4,5-tetraoksanskih derivata prema HeLa i Fem-x ćelijskim linijama pokazana je 3D QSAR studijom. Rezultati: Ispitivani tetraoksani pokazuju dozno-zavisnu antiproliferativnu aktivnost u mikromolarnim koncentracijama prema ciljnim tumorskim ćelijama, uz dobru selektivnost u aktivnosti prema tumorskim ćelijama, u odnosu na normalne imunokompetentne ćelije. 24-časovni tretman HeLa ćelija sa svim ispitivanim tetraoksanima indukuju tipične morfološke karakteristike kasne apoptoze (kondenzovana i/ili fragmentisana jedra). Posle 24 i 48 časova inkubacije sa tetraoksanima, broj HeLa ćelija u G1 fazi se značajno uvećava. Nakon tretmana HeLa ćelija ispitivanim tetraoksanima, nivo reaktivnih kiseoničnih vrsta je značajno porastao ukazujući na mogući oksidativni stres. Korišćenjem GRIND metodologije utvrđeno je da su farmakofore kojima se može pripisati najzna?ajnije dejstvo, amidni NH primarnih ili sekundarnih amida i acetoksi fragmenti na pozicijama 7 i 12 steroidnog jezgra, koji su zajedno sa tetraoksanskim prstenom, zajednički za sva ispitivana jedinjenja. Zaključci: Ispitivani tetraoksani pokazuju dozno-zavisnu antiproliferativnu aktivnost u mikromolarnim koncentracijama prema ciljnim tumorskim ćelijama. Rezultati pokazuju i da ispitivani tetraoksani indukuju apoptozu i zaustavljaju HeLa ćelije u G1 fazi ćelijskog ciklusa i potentno generišu ROS ukazujući na mogući oksidativni stres. U 3D QSAR studiji utvrđeno je da su farmakofore steroidnih tetraoksana kojima se može pripisati najznačajnije dejstvo, amidni NH primarnih ili sekundarnih amida i acetoksi fragmenti na pozicijama 7 i 12 steroidnog jezgra, koji su zajedno sa tetraoksanskim prstenom, zajednički za sva ispitivana jedinjenja.

Introduction: Finding agents with potential anticancer effect is imperative in modern oncology. It is increasingly noted that certain chemical structures have specific toxic effect on cancer cells. Artemisinin discovery marked the beginning of peroxide research as a potential replacement for traditional antimalarial drugs, and the structurally simple class of peroxides that emerged from these studies was the 1,2,4,5-tetraoxanes, which was soon shown that in addition to antimalarial show strong antiproliferative activity . Malignant cells have a disruption in the regulatory mechanisms that govern cellular proliferation and homeostasis. The ability of tumor cell populations to increase the number of cells is determined not only by the intensity of cell proliferation, but also with rate of the removal of cells. Programmed cell death - apoptosis, is the main source of this removal. The aim of this study was to determine the level of the cytotoxic effect of six mixed tetraoxanes to various human malignant cells, as well as to determine the coefficient of selectivity in their activity in comparison to healthy immunocompetent cells. In order to gain insight into the mechanism of action of investigated compounds the type of cell death induced by tetraoxanes will be examined, as well as production of reactive oxygen species in HeLa cells. The goal of 3D QSAR studies on antiproliferative activities of thirty three 1,2,4,5-tetraoxane derivatives against HeLa and Fem-x tumor cell lines has been to determine which are most important pharmacophore of steroid tetraoxanes that affect the potency of the compounds against HeLa and Fem x-tumor cell lines. Material and Methods: The cytotoxic effect of tetraoxanes DO-122 - DO- 124 and DO-126 - DO-128, against five tumor cell lines were examined with a standard MTT assay. In order to determine the mode of HeLa cell death induced by the investigated compounds, morphological analysis by microscopic examination of acridine orange and ethidium bromide stained cells was performed. Analysis to determine the cell cycle phase distribution of HeLa cells stained with propidium iodide was performed on the flow cytometer. Intracellular production of reactive oxygen species (ROS) was measured using the fluorescent color 2,7- Dichlorodihydrofluorescein diacetate. The dependence of the structure and function of the thirty-three 1,2,4,5-tetraoxane derivatives against HeLa and Fem-x cell lines was demonstrated with a 3D QSAR study. Results: The tested tetraoxanes show dose-dependent antiproliferative activity in micromolar concentrations to target tumor cells with good selectivity in activities to tumor cells, compared to normal immunocompetent cells. treatment of HeLa cells for 24-hour with all tested tetraoxanes induce the typical morphological features of late apoptosis (condensed and/or fragmented nuclei). After 24 and 48 hours of incubation with tetraoxanes, the number of HeLa cells in G1 phase was significantly increased. After treatment of HeLa cells with examined tetraoxanes, the level of reactive oxygen species increased significantly indicating a possible oxidative stress. Using GRIND methodology revealed that the pharmacophore which may account for the most significant effect was amide NH of primary or secondary amides and acetoxy fragments at positions 7 and 12 of the steroid nucleus, which together with the tetraoxane ring moiety is common to all compounds. Conclusions: The tested tetraoxanes show dose-dependent antiproliferative activity in micromolar concentrations to the target tumor cells. The results show that the tested tetraoxanes induce apoptosis in HeLa cells, stop cell cycle in the G1 phase, and potently generate ROS pointing to possible oxidative stress. 3D QSAR study revealed that the pharmacophore of steroid tetraoxanes which may account for the most significant effect is amide NH of primary or secondary amides and acetoxy fragments at positions 7 and 12 of the steroid nucleus, which together with the tetraoxane ring moiety is common to all compounds.

Ekspresija gena za inhibitore cisteinskih proteinaza (OCI i OCII) u transformisanim biljkama krompira (Solanum tuberosum L.)

Expression of cysteine proteinase inhibitor genes (OCI and OCII) in transformed potato (Solanum tuberosum L.) plants

Kombinovanje ili “slaganje” različitih gena u transgenim biljkama radi postizanja uspešnije kontrole patogena i štetočina i/ili većeg prinosa predstavlja jednu od glavnih oblasti istraživanja savremene biotehnologije. Orizacistatini I i II (OCI i OCII), proteinazni inhibitori različitih specifičnosti, pokazali su potencijal u kontroli štetočina koje koriste cisteinske proteinaze za digestiju proteina. Da bi se pojačao njihov inhibitorni potencijal i, eventualno, povećala efikasnost ovih inhibitora u kontroli štetočina, oba cistatina su koeksprimirana u transformisanim biljkama tri sorte krompira. “Slaganje” orizacistatinskih gena kod sorti Dragačevka i Dezire ostvareno je postupkom ko-transformacije i zabeležena je frekvenca kointegracije OCI i OCII gena od 20-22%. Kod sorte Jelica sekvencijalna re-transformacija se pokazala kao efikasniji pristup: frekvenca integracije OCII gena nakon re-transformacije OCI-transformisane linije iznosila je 91%. Istovremeno, “slaganje” dva orizacistatnska gena, bilo postupkom ko- ili re- transformacije, postignuto je upotrebom nptII gena kao jedinog selekcionog markera. Ekspresija OCI i OCII gena indukovana povređivanjem i akumulacija biološki aktivnih rekombinantnih OCI i OCII proteina potvrđena je kod svih analiziranih OCI/OCII transformisanih linija krompira. OCI/OCII linije krompira nisu ispoljavale značajna odstupanja od normalnog fenotipa, što ukazuje na nizak nivo somaklonalnih varijacija i odsustvo uticaja rekombinantnih OCI i OCII na metabolizam biljke domaćina. Iako nije uticala na preživljavanje, ishrana larvi krompirove zlatice (Leptinotarsa decemlineata Say) listovima krompira koji eksprimiraju oba orizacistatina imala je značajan uticaj na različite osobine performanse rasta i razvića larvi. Larve hranjene transformisanim listovima su se presvlačile ranije, i tokom L2 i L3 stupnja uvećavale masu do 29,7% brže i konzumirale listove do 29,1% brže u odnosu na one hranjene netransformisanim listovima. Istovremeno, larve na OCI/OCII listovima su do tri dana ranije dostizale maksimum mase i ranije “usporavale” sa ishranom ulazeći u prepupalnu fazu razvića. Uprkos povećanju performansi rasta i ishrane, pri istoj efikasnosti ishrane, L4 larve na transformisanim listovima nisu u potpunosti uspele da kompenzuju negativne efekte prisustva orizacistatina u hrani. U odnosu na larve hranjene netransformisanim listovima, maksimalna masa na kraju larvenog razvića i ukupan stepen oštećenja listova bili su do 19,4% i do 18,5% manji kod larvi krompirove zlatice hranjenih OCI/OCII transformisanim listovima krompira. Smanjenje mase larvi na OCI/OCII listovima dovelo je i do pojave adulta krompirove zlatice sa do 26,3% redukovanom telesnom masom. Analiza ukupne proteinazne aktivnosti kod larvi krompirove zlatice pokazala je inicijalno smanjenje digestivnog kapaciteta L3 larvi do 56%, koje je praćeno inhibicijom specifične aktivnosti cisteinskih proteinaza do 62% (akutni efekat). Sa druge strane, pri hroničnoj ingestiji OCI/OCII listova krompira, ukupna i aktivnost cisteinskih proteinaza kod L3 larvi ne pokazuje značajna odstupanja od kontrolnog nivoa, ukazujući na kompenzatorne odgovore proteaza larvi na prisustvo rekombinantnih orizacistatina u ishrani. Uočene promene u ishrani, rastu i razviću larvi krompirove zlatice mogu biti tumačene kao regulatorni odgovor kojim se postiže maksimum telesne mase uprkos prisustvu rekombinantnih inhibitora. Time se pokreću složene interakcije između ishrane, digestivnih procesa i regulatornih mehanizama rasta i razvića koje mogu da kompenzuju potencijalno smanjenje adaptivne vrednosti usled ishrane transformisanim listovima.

The combination or stacking different genes in transgenic plants to achieve disease and pest control and/or higher crop yield is one of a major method of contemporary biotechnology. Oryzacystatins I and II (OCI and OCII), inhibitors with different specificity, show potential in controlling pests that utilize cysteine proteinases for protein digestion. To strengthen this inhibitory range and, possibly, achieve an additive effect in the overall efficiency of these proteins against pests, both cystatins were co-expressed in three potato cultivars. Oryzacystatin genes pyramiding in Dragačevka and Desiree cultivars were achieved by co-transformation with OCI and OCII genes co-integration frequency of 20-22%. For Jelica cultivar sequential re-transformation was more efficient approach: OCII gene integration frequency following re-transformation of an OCI-expressing line was 91%. Additionally, pyramiding of different oryzacystatin genes, by co- or re-transformation approach, were achieved using the nptII gene as the only selection marker. Wounding induction of OCI and OCII gene transcripts and accumulation of biologically active OCI and OCII recombinant proteins was confirmed in all analyzed OCI/OCII transformed lines. OCI/OCII potato lines did not exhibit morphological abnormalities, indicating low level of somaclonal variation or interference of the recombinant OCI or OCII with host plant metabolism. In the absence of significant mortality, feeding Colorado potato beetle larvae (Leptinotarsa decemlineata Say) on OCI/OCII-expressing foliage had an impact on various aspects of the growth and developmental performances of larvae. Larvae feeding on transformed potato leaves tended to molt earlier and, especially during L2-L3 stages, gain weight up to 29.7% faster and consume leaf material up to 29.1% faster, compared to those on untransformed foliage. Larvae on OCI/OCII foliage were also reach maximum weight gained three days earlier and slow down earlier in preparation for pupation. Despite their faster growth and feeding, with similar efficiencies of conversion of ingested food, L4 larvae reared on transformed foliage were not compensating presence of the recombinant oryzacystatins in the diet. Compared to those on untransformed foliage, maximum weight gained and amount of foliage consumed were up to 19.4% and 18.5%, respectively, lower for the larvae fed on OCI/OCII potato foliage. Larval weight reduction on OCI/OCII foliage resulted in adult emergence with up to 26.3% reduced body mass. Analysis of total digestive proteinases activity showed initially, up to 56%, reduction in digestive capacity of L3 potato beetle larvae, accompanied with inhibition of cysteine proteinase specific activity up to 62% (acute effect). However, by continual ingestion of OCI/OCII potato foliage total and cysteine proteinases specific activities were at control level, suggesting compensatory responses of larvae protease system to the presence of recombinant oryzacystatins in the diet. The observed alterations in larval feeding and growth performance can be interpreted as regulatory responses aimed at stabilizing the final body weight despite presence of the recombinant inhibitors. These changes can trigger complex interactions between feeding, food processing and growth regulatory mechanisms, which tend to compensate for the potential fitness loss caused by feeding on transformed foliage.

Metaboličke karakteristike sindroma policističnih jajanika u visceralnom masnom tkivu i leptinska rezistencija u hipotalamusu pacova tretiranog 5alfa-dihidrotestosteronom: uloga glukokortikosteroida

Metabolic characteristics of polycistic ovary syndrom in the visceral adipose tissue and leptin resistance in the hypothalamus of the rat treated with 5alfa-dihydrotestosterone: role of glycocorticoids

Sindrom policističnih jajnika (PCOS) je najčešća endokrinopatija žena u reproduktivnom životnom dobu i predstavlja heterogeno oboljenje, koje u osnovi karakterišu hiperandrogenizam, hronična anovulacija i policistični jajnici, a sa njim su povezane i karakteristike metaboličkog sindroma, kao što su insulinska rezistencija, gojaznost, dislipidemija i hipertenzija. Patofiziologija i etiologija PCOS-a, odnosno uzročno-posledična povezanost njegovih pojedinačnih simptoma, nisu do kraja razjašnjene. Generalno se smatra da reproduktivni, endokrini i metabolički poremećaji u PCOS-u formiraju začarani krug u čijem centru je hiperandrogenemija, koju stimulišu visceralna gojaznost, narušen lipidni metabolizam, inflamacija i insulinska rezistencija. Glukokortikoidni hormoni utiču na pojavu visceralne gojaznosti i, delujući antagonistički sa insulinom, mogu da doprinesu patofiziologiji metaboličkog sindroma. Glukokortikoidni hormoni tkivno-specifičnim efektima podstiču nastanak insulinske rezistencije na nivou organizma. Imaju i generalno antiinflamatorna dejstva, ali u gojaznosti podstiču ekspresiju nekih proinflamatornih činilaca. Leptin je hormon masnog tkiva, čija je najvažnija uloga centralno anoreksigeno dejstvo u hipotalamusu tokom kontrole unosa energije. Pri tome, leptin deluje sinergistički sa insulinom i antagonistički sa glukokortikoidnim hormonima. U gojaznosti dolazi do hiperleptinemije i nemogućnosti leptina da ostvari svoje efekte, to jest razvoja leptinske rezistencije. Glukokortikoidi kroz interakcije sa leptinskim signalnim putem mogu da podstaknu njen nastanak. Izučavanje PCOS-a olakšano je uspostavljanjem životinjskih modela koji razvijaju glavne karakteristike ovog sindroma. Za ovu studiju odabran je pacovski model uspostavljen kontinuiranim tromesečnim tretmanom ženki potentnim androgenom 5α-dihidrotestosteronom (DHT), počevši od puberteta. Cilj ove studije bila je detaljna morfološka, biohemijska, fiziološka i molekularno-biološka karakterizacija ovog modela PCOS-a, naročito sa aspekta uticaja glukokortikoidne signalizacije na metaboličke probleme povezane sa sindromom, kao što su visceralna gojaznost, dislipidemija i metabolička inflamacija, insulinska i leptinska osetljivost. Posebno je analiziran prereceptorski metabolizam i uticaj glukokortikoidnih hormona na lipidni metabolizam u visceralnom masnom tkivu i leptinsku signalizaciju u hipotalamusu. Rezultati ove studije pokazuju da primenjeni tretman dovodi do razvoja reproduktivnih i metaboličkih promena koje se sreću kod žena sa PCOS-om. Među njima treba posebno istaći atreziju folikula jajnika i anovulaciju, dislipidemiju, visceralnu gojaznost sa hipertrofičnim rastom adipocita, sistemski smanjenu osetljivost na insulin, kao i hiperleptinemiju. Rezultati ukazuju da povećana lokalna koncentracija glukokortikoida utiče na pomeranje lipidnog metabolizma u pravcu lipogeneze u visceralnom masnom tkivu. Insulinska osetljivost tog tkiva nije se promenila, ali je uočena lokalna inflamacija. S druge strane, u hipotalamusu DHT-om tretiranih pacova nisu uočene promene komponenti leptinskog signalnog puta na koje utiču glukokortikoidni hormoni, kao ni parametri lokalne inflamacije. Reproduktivne i metaboličke promene dobijene u ovoj studiji modela PCOS-a generisanog tretmanom ženki pacova DHT-om ukazuju na adekvatnost tog modela za ispitivanja raznih aspekata PCOS-a kod žena. Promene uočene na molekularnom nivou impliciraju uključenost glukokortikoida u promene metabolizma lipida u visceralnom masnom tkivu kod DHT-om tretiranih životinja. Na nivou hipotalamusa odabranog modela PCOS-a, interakcija između glukokortikoidne i leptinske signalizacije nije uočena.

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy of women of reproductive age. It is a heterogenous disorder, with hyperandrogenism, chronic anovulation and polycystic ovaries as basic characterisctics, and features of the metabolic syndrome, such as insulin resistance, obesity, dislipidemia and hypertension as associated metabolic characteristics. Pathophysiology and aetiology of PCOS and interlinks between its symptoms are yet to be clarified. It is generally considered that the reproductive, endocrine and metabolic features of PCOS create a vicious circle in the centre of which lies hyperandrogenemia, stimulated by visceral obesity, disturbed lipid metabolism, inflammation and insulin resistance. By functioning antagonistically with insulin, glucocorticoids influence the genesis of visceral obesity and contribute to the pathophysiology of the metabolic syndrome. Their tissue-specific effects stimulate systemic insulin resistance. In addition, glucocorticoids generally exhibit antiinflamatory actions. In obesity, however, they show an ability to stimulate proinflammatory factors. Leptin is a hormone with anorexigenic effects in the hypothalamus. In the course of energy intake and consumption control it works synergistically with insulin, but antagonizes the actions of glucocorticoids. Increased secretion of leptin - hyperleptinemia and leptin resistance are common consequences of obesity. By modulating leptin signaling components in the hypothalamus, glucocorticoids can contribute to the development of leptin resistance. The studies of PCOS have been facilitated by the introduction of animal models exhibiting the principal features of this syndrome. In this study, a rat model of PCOS was established by continual, three-month long treatment of pubertal female rats with a potent androgen, 5α-dihydrotestosterone (DHT). The aim of the study was a detailed morphological, biochemical, physiological and molecular characterization of this model, particularly focused on the influence of glucocorticoid signaling on the metabolic features of PCOS, such as visceral obesity, dislipidemia, metabolic inflammation, and insulin and leptin resistance. A particularly detailed analyses of lipid metabolism in the visceral adipose tissue and hypothalamic leptin signaling were performed. The results of this study show that this treatment leads to the development of the reproductive and metabolic features seen in women with PCOS, of which folicular atresia and anovulation, dislipidemia, visceral obesity with hypertrophic adipocytes, systemically low insulin sensitivity and hyperleptinemia are particularly important. The increased local regeneration of glucocorticoids influences the molecular changes that shift the lipid metabolism toward lipogenesis in the visceral adipose tissue of the model. Insulin sensitivity of the visceral adipose tissue, was not changed. However, markers of local inflammation were increased. On the other hand, the changes of leptin signaling components influenced by glucocorticoids could not be seen in the hypothalamus of DHT treated rats, which was also the case with local inflammation parameters. Reproductive and metabolic changes gained in this study of the DHT induced rat model of PCOS point to the adequacy of the use of this model in studies concerning the different aspects of PCOS in women. The observed molecular changes imply the involvement of glucocorticoids in the changes of lipid metabolism in the visceral adipose tissue of the DHT treated animals. In the hypothalamus of the chosen PCOS model, however, the interactions between glucocorticoid and leptin signaling were not observed.

Potencijalna uloga gubara (Lymantria dispar, L.) kao bioindikatora zagađenosti životne sredine: uticaj hroničnog izlaganja kadmijumu na razviće, aktivnosti digestivnih enzima srednjeg creva, mehanizme detoksifikacije i stepen oštećenja DNK

Potential role of gypsy moth (Lymantria dispar, L.) as a bioindicator of environmental pollution: the impact of cadmium chronic exposure on the development, midgut digestive enzyme activities, detoxification mechanisms and DNA damage level

Razvoj industrije i poljoprivrede, kao i globalna urbanizacija, doveli su do pojave velikog broja polutanata u životnoj sredini, među kojima je i izuzetno toksični teški metal kadmijum. Cilj ove disertacije bila je procena larvi gubara (Lymantria dispar, L.) kao potencijalnog bioindikatora zagađenosti kadmijumom. Ispitani su odgovori izabranih fizioloških parametara na različitim nivoima biološke organizacije u prisustvu dve koncentracije metala (50 i 100 μg Cd/g suve hrane). Radi ispitivanja efekta porekla populacije, legla gubara sakupljena su na dve nezagađene lokacije (Kosmaj i Homoljske planine) i dve zagađene (pored Ibarske magistrale i borskog rudnika). Larve sa Kosmaja odlikuje veća osetljivost digestivnih enzima srednjeg creva (proteaza, alkalnih i kiselih fosfataza i nespecifičnih esteraza) na tretmane, naročito na nižu koncentraciju kadmijuma, u odnosu na one uz Ibarsku magistralu. Od ispitanih detoksifikacionih mehanizama u crevu, indukcija Hsp70 značajnija je kod kosmajskih larvi, a sinteza metalotioneina kod ibarskih. Trend sniženja aktivnosti antioksidativnih enzima, superoksid dismutaze i katalaze, uočen je kod obe populacije. U svim grupama tretiranim kadmijumom najveća količina ovog metala eliminiše se putem fecesa, ali intenzivnije u ibarskoj populaciji u odnosu na kosmajsku nakon tretmana sa 50 μg Cd/g suve hrane. Kod homoljskih gusenica tretmani kadmijumom doveli su do povećanja nivoa oštećenja DNK u hemocitama i dozno-zavisnog sniženja njihove vijabilnosti, dok je u populaciji iz Bora zabeleženo samo smanjenje vijabilnosti nakon primene više koncentracije metala. Štetni efekti kadmijuma i visoka cena detoksifikacionih mehanizama odrazili su se i na osobine fitnesa u vidu promena mase larvi, relativne brzine rasta i trajanja razvića kod svih populacija u različitom stepenu. Primena većine ispitanih parametara (specifične aktivnosti tripsina, nelizozomskih kiselih fosfataza i nespecifičnih esteraza, nivoa ekspresije Hsp70, vijabilnosti hemocita i nivoa oštećenja DNK) kao biomarkera zagađenosti životne sredine kadmijumom ograničena je na osetljive populacije gubara koje prethodno nisu bile izložene polutantima, dok bi koncentracija metalotioneina mogla biti podobna kao biomarker kod populacija sa lokacija kontaminiranih tokom dužeg perioda.

considerable increase in the environmental pollution including extremely toxic heavy metal cadmium. The aim of this dissertation was to evaluate gypsy moth (Lymantria dispar, L.) larvae as bioindicators of cadmium contamination. Parameters at different levels of biological organization were assessed after chronic exposure of larvae to the metal (50 and 100 μg Cd/g dry food). To investigate the effects of population origin, gypsy moth egg-masses were collected from two unpolluted localities on Kosmaj and Homolje mountains and two polluted sites near the busy Ibar highway and Bor copper mine. Midgut digestive enzymes (proteases, alkaline and acid phosphatases and nonspecific esterases) of Kosmaj larvae were more sensitive to cadmium, especially to lower metal concentration, compared to those of Ibar larvae. Hsp70 induction was the most prominent midgut detoxification mechanism against cadmium in the population from the unpolluted forest, whereas metallothionein synthesis was the most significant in larvae from the polluted site. Activities of antioxidative enzymes, superoxide dismutase and catalase, exhibited a decreasing trend in both populations. The primary route of cadmium elimination was via feces in all metal-fed groups, but after an exposure to 50 μg Cd/g dry food metal content was significantly higher in feces of Kosmaj larvae compared to those from the highway locality. Exposure of caterpillars from the Homolje mountains to the metal led to the higher level of DNA damage in the hemocytes and a dose-dependent decrease in cell viability, whereas in Bor population only hemocyte viability was reduced after the treatment with 100 μg Cd/g dry food. Devastating cadmium effects and energetically expensive detoxification mechanisms affected the fitness traits (larval mass, relative growth rate or development parameters) in all populations. The application of selected parameters (specific activities of trypsin, nonlisosomal acid phosphatases and nonspecific esterases, Hsp70 expression, hemocyte viability and DNA damage level) as biomarkers of environmental cadmium pollution is limited mostly to sensitive populations that have not been previously exposed to pollutants. Metallothionein concentration might be suitable as a biomarker for the populations at locations that have been contaminated for a long period.

Uticaj biljke domaćina na kriptičnu diferencijaciju populacija vektora Hyalesthes obsoletus Signoret, 1865 (Hemiptera: Cixiidae) i epidemiološke puteve prenošenja 'Candidatus Phytoplasma solani'

Cixiidae) and on epidemiological transmission routes of 'Candidatus Phytoplasma solani'

Hyalesthes obsoletus, vektor biljnog patogena 'Candidatus (Ca.) Phytoplasma solani', smatra se polifagnom štetočinom od ekonomskog značaja u centralnoj Evropi i Mediteranu. Istraživanjima sprovedenim u jugoistočnoj Evropi, koja gravitira centru arela vrste i gde je zabeležen značajan broj 'Ca. P. solani'- uslovljenih biljnih bolesti, utvrđeno je postojanje četiri asocijacije vektora sa biljkama dualnim domaćinima: Convolvulus arvensis (Ca), Urtica dioica (Ud), Vitex agnus-castus (Vac) i Crepis foetida (Cf). Populaciono-genetičkim analizama mitohondrijskih i nuklearnih markera (mikrosatelita) utvrđena je kriptična diferencijacija simpatričkih (uključujući sintopske) i geografski udaljenih H. obsoletus populacija na tri filogenetičke linije uslovljene biljkom domaćinom: (1) Ca-Ud, (2) Vac i (3) Cf. Pored jasne diferencijacije na mitohondrijskim genima (1.1% - 1.5%) i mikrosatelita koji potvrđuju izdvajanje populacija u jedan od tri klastera prema biljci domaćinu (pripadnost >90%), podršku za izdizanje genetičke divergencije H. obsoletus sensu lato na nivo kriptične specijacije daje registrovana distanca u odnosu na morfološki najsrodniju vrstu - Hyalesthes thracicus (2.1% - 3.3%). Posledice specijalizacije i diferencijacije H. obsoletus sensu lato prema biljkama domaćinima na epidemiologiju 'Ca. P. solani' procenjene su multigenskom karakterizacijom izolata fitoplazme detektovanih (1) in situ u prirodnim epidemiološkim sistemima mediteranskih vinograda i (2) eksperimentima unakrsne inokulacije biljaka putem prirodno inficiranih populacija vektora. Potvrđena je osnovna epidemiološka divergencija 'Ca. P. solani' na tuf-a/-ab i tuf-b cikluse vezane za Ud i Ca uz ukrštanje sa Vac-uzrokovanom tuf-b epidemiologijom i utvrđena kompleksnost tuf-b epidemiologije u kojoj učestvuje i treća genetička linija vektora, H. obsoletus ex Cf, kao i sama biljka dualni domaćin - C. foetida.

The cixiid planthopper Hyalesthes obsoletus is generally considered as a polyphagous vector of 'Candidatus (Ca.) P. solani' associated with numerous wild and cultivated plants in central Europe and Mediterranean. Research conducted in southeastern Europe, the distribution centre of H. obsoletus and the area of most 'Ca. P. solani'-inflicted crop diseases, points toward specific dual host-plant associations with: Convolvulus arvensis (Ca), Urtica dioica (Ud), Vitex agnus-castus (Vac) and Crepis foetida (Cf). Population-genetic analysis of mitochondrial and nuclear genetic markers (microsatellites) have revealed cryptic differentiation between sympatric (including syntopic) and geographically distant H. obsoletus populations into three phylogenetic lineages delineated by host-plant preference: 1) Ca-Ud, 2) Vac i 3) Cf. Cryptic species diversification within H. obsoletus sensu lato is supported by the genetic divergence of mitochondrial genes (1.1% - 1.5%), microsatellites that confirm population clustering into three genetic groups associated with specific host-plant (average assignment >90%) and genetic distances among host-associated H. obsoletus metapopulations comparable to the most closely related, morphologically distinguishable species - Hyalesthes thracicus (2.1% - 3.3%). Consequences of the H. obsoletus sensu lato differentiation on the epidemiology of 'Ca. P. solani' are assessed by multilocus genotyping of the phytoplasma isolates detected (1) in situ in natural epidemiological systems of the Mediterranean vineyards and (2) plant inoculation experiments using naturally infected vector populations. Basic 'Ca. P. solani' epidemiological divergence into tuf-a/-ab and tuf-b cycles, each associated with Ud and Ca, respectively, is confirmed with evidences of intermixing Vac-induced tuf-b epidemiology, while complexity of the tuf-b epidemiology is further evidenced through influence of the third genetic lineage of the vector H. obsoletus ex C. foetida and its dual host-plant .

Srpska memorijalna arhitektura 1918-1955

Serbian memorial architecture 1918-1955

Radom Srpska memorijalna arhitektura 1918-1955 obuhvaćen je relativno dug vremenski period u okviru koga su predstavljeni raznovrsni vidovi memorijalnog graditeljstva, pod kojim se u širem smislu podrazumevaju obeležja, objekti, groblja, nadgrobnici, prostorne površine, istorijski objekti i celine, koji čuvaju uspomenu na istorijske događaje, ličnosti i pale ratnike. Razvoj srpskog memorijalnog graditeljstva ispraćen je kroz dva različita društveno-politička sistema, dve države- Kraljevinu Jugoslaviju i socijalističku Jugoslaviju, u čijem sastavu se nalazila Srbija u periodu 1918-1955. Period okupacije 1941-1944 obuhvaćen je kroz jedino reprezentativno delo memorijalnog graditeljstva podignuto u ovom periodu, Spomen-kosturnicu u Smederevu. Kraljevina SHS - Jugoslavija u čiji sastav su ušle slovenske zemlje nekadašnji delovi austrougarske svoju politiku zasnivala je na nacionalnoj i verskoj ravnopravnosti svih naroda. Uređenje srpskih vojničkih grobalja, podizanje memorijala posvećenih srpskoj vojsci, i pored velikih zasluga koje je srpska vojska stekla u Prvom svetskom ratu, država je sprovodeći politiku ravnopravnosti svih jugoslovenskih naroda prepustila srpskim nacionalnim udruženjima, organizacijama i posebno formiranim odborima. Država je pokazala inicijativu za uređenje pojedinih vojničkih grobalja u inostranstvu i podigla nekoliko memorijala. U međuratnom periodu srpsko memorijalno graditeljstvo, i pored nedovoljnog angažovanja Države, doživljava ekspanziju. U ovom periodu dolazi do intezivnog uređivanja vojničkih grobalja i podizanja kosturnica sa crkvama, kapelama, mauzolejima ili arhitektonskim spomenicima nad njima. Uređenje vojničkih grobalja je po prvi put kod nas zakonski regulisano što je dalo poseban značaj ovom vidu memorisanja. U ovom periodu kroz podizanje spomenika Neznanom junaku i nameru da se podigne spomen-hram tipa Panteona, prihvaćeni su novi vidovi memorisanja iz evropske memorijalne arhitekture. Pored reprezentativnih arhitektonskih memorijala podignutih na Zebrnjaku, Ceru, Gučevu, Spomenika braniocima Beograda na Novom groblju i drugih, poseban nacionalni značaj po brojnosti imaju i drugi vidovi memorisanja; obelisci, česme, spomen-obeležja, koje je obično podizala lokalana zajednica. U međuratnom periodu po prvi put memorijalna arhitektura je prihvaćena od strane arhitekata i društva kao značajan vid stavralaštva, što su potvrdile izložbe studenata arhitekture i Saloni arhitekture. U memorijalnom graditeljstvu posebno su se istakle arhitekte koje su radile pri Ministarstvu građevina, Momir Korunović, Nikolaj Krasnov, Vasilij Androsov, kao i samostalne arhitekte Roman Verhovskoj i Aleksandar Deroko, dok je značajan opus koji se sastoji od idejnih projekata ostvario arhitekta Milutin Borisavljević. Memorijali su izvođeni pretežno u srpsko-vizantijskom stilu ili pod uticajima akademizma, mada su primetni i uticaji romantičarskog ekspresionizma. U periodu posle Drugog svetskog rata promena političke situacije, pojava socijalističkog realizma pod uticajem SSSR-a, donošenje rezolucije Informbiroa i razlaz sa Sovjetima, dovela je do pojave novih formalnih rešenja memorijala. Promenjena politička, društvena i kulturna situacija, kao i internacionalni karakter NOB-a i žrtava rata uslovili su da se spomenici ne posvećuju srpskim vojnicima. Pod uticajem nove ideologije uspostavljaju se novi amblemi, a iz memorijalnog graditeljstva nestaju crkve i spomenici u formi krsta. Pored starih uvode se novi vidovi memorijalnog graditeljstva, pre svega kroz spomen-grobalja koja se uređuju u vidu spomen- parkova, i podizanje kosturnica narodnih heroja i palih boraca po trgovima i parkovima. U arhitekturi je prioritet preuzela mlađa predratna generacija arhitekata modernističke orjentacije što je pored promenjene kulturne politike uticalo i na formalna rešenja. Na memorijalima su pored uticaja predratnih stilskih tendencija, preko amblema prilagođenih ideologiji socijalizma, primetni jači uticaji modernizma. Pedesetih godina, prilikom proslave stopedesetogodišnjice Prvog srpskog ustanka, došlo je do kratkotrajnog oživljavanja srpske tradicije u memorijalnom graditeljstvu, posvećivanjem spomenika ustanicima i podizanjem jednog od najznačajnijih srpskih memorijala, Spomenika Kosovskim junacima na Gazimestanu.

The work Serbian memorial architecture 1918-1955 covers a relatively long period of time, during which various forms of memorial construction were undertaken. In the broadest sense of the word, this covers emblems, buildings, cemeteries, graves, areas of land, and historical buildings and entities, all preserving the memory of historical events, historical figures and fallen soldiers. The development of Serbian memorial construction is presented through two differing socio-political systems, in two states – the Kingdom of Yugoslavia and socialist Yugoslavia, which Serbia was a part of in the period 1918-1955. The period of occupation (1941-44) is represented by the only grandiose work of this time, the Memorial Vault in Smederevo. The Kingdom of Slovenes, Croats and Serbs – Yugoslavia, comprising Slavic countries that were once part of Austria-Hungary, based its politics on the national and religious equality of all peoples. Despite the enormous praise the Serbian army earned during the First World War, the state, applying its policy of equality of all Yugoslav peoples, left the restoration of Serbian military cemeteries and the erecting of memorials dedicated to the Serbian army to Serbian national associations, organisations and specially formed committees. The state however did show willing to restore certain military cemeteries abroad and erected several memorials. During the inter-war period, Serbian memorial construction increased, despite the lack of state involvement. This period was marked by the intensive restoration of military cemeteries and the building of vaults with churches, chapels, mausoleums or architectural memorials above them. The restoration of military cemeteries was regulated by law for the first time in the country’s history, which gave special significance to this form of memorial. During this period, with the erection of memorials to the Unknown Soldier and the intention to build a memorial temple like the Pantheon, new forms of memorialising from European memorial architecture were accepted. Besides the grandiose architectural memorials erected at Zebrnjak, Cer, and Gučevo, the Monument to the Defenders of Belgrade at New Cemetery and others, other forms of memorial are of special national significance due to their number: obelisks, drinking fountains, and memorial emblems, which were often erected by local organisations. It was in the inter-war period that memorial architecture was accepted for the first time by architects and society as a significant form of creation, confirmed by the exhibitions of architecture students and the Salons of Architecture. Architects who worked for the Ministry of Civil Engineering - Momir Korunović, Nikolaj Krasnov, and Vasilij Androsov - excelled in particular in memorial architecture, as well as the freelance architects, Roman Verhovskoy and Aleksandar Deroko, while the architect Milutin Borisavljevic compiled a significant oeuvre comprising concept designs. Memorials were created mainly in the Serbian-Byzantine style or influenced by academism, although influences of Romantic Expressionism are also evident. In the period after the Second World War, a change in the political situation, the rise of socialist realism under the influence of the USSR, the passing of the Cominform Resolution and the split from the Soviets, led to the emergence of new formal designs of memorials. The altered political, social and cultural situation, as well as the international character of the Yugoslav Resistance Movement and the victims of the war, meant that monuments were not dedicated to Serbian soldiers. Influenced by the new ideology, new emblems were used, and churches and monuments in the form of a cross disappeared from memorial construction. In addition to the old forms, new forms of memorial construction were introduced, above all through memorial cemeteries, which were organised in the form of memorial parks, and the building of vaults to national heroes and fallen soldiers on squares and in parks. In architecture, a younger pre-war generation of architects of modernist orientation came to the fore, which, along with different cultural politics, had an influence on the final designs. In addition to the influence of pre-war stylistic tendencies, through emblems corresponding to socialist ideology, strong influences of modernism could also be noted on memorials. In the fifties, on the occasion of celebrations marking the 150th anniversary of the First Serbian Uprising, Serbian tradition in memorial construction experienced a brief revival, through the dedication of a monument to the rebels and the building of one of the most significant Serbian memorials, the Monument to the Kosovo Heroes at Gazimestan.

Učestalost i tipovi mutacija u genima BRCA u porodicama sa pozitivnom istorijom za karcinom dojke i/ili jajnika u Srbiji

Frequency and types of mutations in BRCA genes in families with positive history of breast/ovarian cancer in Serbia

Uvod: Proteinski produkti gena BRCA1 i BRCA2 uključeni su u važne ćelijske procese, kao što su kontrola ćelijskog ciklusa i popravka oštećenja DNK. Mutacije u jednom od ovih gena predstavljaju korak bliže gubitku kontrole nad genomskom stabilnošću i ćelijskom deobom, pa osobe koje naslede mutacije u ovim genima češće oboljevaju od karcinoma dojke i/ili jajnika. Geni BRCA su veliki, mutacije su raspoređene duž celih gena bez grupisanja, što dodatno otežava njihovu detekciju. Spektar mutacija u genima BRCA različit je u različitim etničkim grupama. U nekim populacijama sa visokom učestalošću zastupljene su mutacije koje su u drugim populacijama retke. U isto vreme, veliki deo mutacija u genima BRCA detektovane su samo jednom, pa se može reći da većina porodica pod rizikom ima svoju sopstvenu porodičnu mutaciju. Mutacija 5382insC u 20. egzonu gena BRCA1 karakteristična je za slovenske populacije i njena učestalost opada od istoka ka zapadu Evrope. Oko 6-7% svih karcinoma dojke i oko 10% svih karcinoma jajnika je nasledno, povezano sa mutacijama germinativnih ćelija u genima BRCA1 i BRCA2. Nosioci mutacija u genima BRCA nose 5 do 8 puta veći životni rizik za oboljevanje od karcinoma dojke i 10 do 20 puta veći životni rizik za karcinom jajnika. Cilj istraživanja je bio da se otkriju najučestalije mutacije u genima BRCA1/2 u našoj populaciji i utvrdi koje se od njih mogu smatrati osnivačkim mutacijama, kako bi se ubrzalo, pojednostavilo i pojeftinilo BRCA testiranje. Da bi se ovaj cilj ostvario, bilo je neophodno utvrditi tipove i učestalosti mutacija u genima BRCA1 i BRCA2 kod osoba sa pozitivnom porodičnom istorijom za karcinom dojke i/ili jajnika u Srbiji. Osim toga, u sistematskom uzorku karcinoma dojke bilo je potrebno utvrditi učestalost mutacije 5382insC u genu BRCA1 kao potencijalne osnivačke mutacije u našoj populaciji. U ispitivanoj grupi osoba koje su testirane na prisustvo mutacija u genima BRCA1/2 procenjivana je i efikasnost predviđanja BRCAPRO programa. Materijal i metode: Analizirano je 85 uzoraka periferne krvi osoba sa povećanim rizikom za karcinom dojke i/ili jajnika koje potiču iz 69 porodica. Takođe, analiziran je i sistematski uzorak karcinoma dojke koji se sastoji od 257 uzoraka krvi obolelih. Prisustvo mutacija u genima BRCA analizirano je automatskim sekvenciranjem, dok je prisustvo mutacije 5382insC u genu BRCA1 u sistematskom uzorku određivano PCR-om specifičnim za alel. Za statističku obradu podataka korišćeni su testovi neparametrijske statistike: χ2 test i Fišerov test. Rezultati: Učestalost oštećujućih mutacija u uzorku porodica sa naslednim karcinomom dojke i/ili jajnika iznosi 10,59% (9/85). Sve detektovane oštećujuće mutacije su po tipu frameshift mutacije. Detektovane su i 3 nove porodično-specifične mutacije (1 u genu BRCA1 i 2 u genu BRCA2). Učestalost mutacije 5382insC u genu BRCA1 u sistematskom uzorku karcinoma dojke je 0,39% (1/257). Podizanjem granice BRCAPRO verovatnoće na 40% moguće je uočiti razliku između benignih polimorfizama i oštećujućih mutacija. Nije bilo moguće subgrupisanje neklasifikovanih varijanti na osnovu vrednosti BRCAPRO verovatnoće. BRCAPRO verovatnoća, sem sa srodnicima prvog i drugog stepena srodstva, koreliše i sa brojem obolelih srodnika u široj porodici. U odnosu na anatomsku lokalizaciju, BRCAPRO verovatnoća koreliše sa brojem srodnika obolelih od karcinoma dojke, ali ne i od karcinoma jajnika. Zaključak: Učestalost mutacija u genima BRCA1/2 kod ispitanika pod rizikom za nastanak naslednog karcinoma dojke i/ili jajnika u Srbiji slična je učestalostima dobijenim u drugim populacijama. Osim već poznatih mutacija u genima BRCA1/2 pokazane su i nove porodično-specifične mutacije. Nisu detektovane mutacije koje bi se mogle okarakterisati kao osnivačke mutacije za našu populaciju. BRCAPRO program se pokazao kao koristan pri odabiru osoba za BRCA testiranje, ali pri odabiru kandidata za testiranje, usled nedostataka BRCAPRO programa, treba analizirati i rodoslov. Podizanje granice BRCAPRO verovatnoće prilikom odabira ispitanika za BRCA testiranje sa 10% na 40% moglo bi da poveća efikasnost BRCA testiranja.

Background: Protein products of BRCA1 and BRCA2 genes are included in important cellular processes, such as cell cycle control and DNA repair. Mutations in one of these genes is a step thowards losing control over genomic stability and cell division, and individuals who inherit mutations in these genes develop breast and/or ovarian cancer more frequently. BRCA genes are large, mutations are scattered throughout whole genes without clustering, which makes mutation detection even more difficult. The spectrum of BRCA mutations is different for each ethnic group. Mutations that are highly frequent in some populations are rare in other populations. At the same time, large proportion of BRCA mutations have been detected only once, and it can be said that majority of families under risk have their own family mutation. Mutation 5382insC in BRCA1 exon 20 is characteristic for Slavic populations and its frequency decreases from east to west of Europe. About 6-7% of all breast cancer cases and about 10% of all ovarian cancer cases are hereditary, associated to germ line mutations in BRCA1 and BRCA2 genes. BRCA mutation carriers have 5 to 8 times higher lifetime risk for breast cancer and 10 to 20 times higher lifetime risk for ovarian cancer. Aim of this study was to identify the most frequent BRCA1/2 mutations in our population and to find out which of them may be regarded as founder mutations, in order to make BRCA testing faster, easier and less expensive. In order to achieve this goal, it was necessary to identify types and frequencies of BRCA1/2 mutations in individuals with positive family history of breast and/or ovarian cancer in Serbia. In addition, it was necessary to identify the frequency of BRCA1 mutation 5382insC in breast cancer consecutive sample, as possible founder mutation in our population. In analysed group of individuals tested for the presence of BRCA1/2 mutations the efficiency of BRCAPRO software prediction was assessed. Material and methods: Eighty five peripheral blood samples from high risk individuals for breast and/or ovarian cancer from 69 families was analyzed. In addition, consecutive sample that included 257 blood samples from breast cancer patients was analyzed. The presence of BRCA gene mutations was analyzed by automatic sequencing, while the presence of 5382insC mutation in BRCA1 gene was determined by alel-specific PCR. For statistic analyses, non-parametric tests were used: χ2 test and Fisher test. Results: The frequency of deleterious mutations in sample of families with hereditary breast and/or ovarian cancer is 10.59% (9/85). All detected deleterious mutations are frameshift mutations. Three novel family-specific mutations have been detected (one in BRCA1 and two in BRCA2 gene). The frequency of BRCA1 mutation 5382insC in breast cancer consecutive sample is 0.39% (1/257). By raising the treshold of BRCAPRO probability to 40% it is possible to observe the difference between benign polymorphisms and deleterious mutations. Subgrouping of unclassified variants according to the value of BRCAPRO probability was not possible. BRCAPRO probability, in addition to the first and second degree relatives, correlates also with the number of more distant relatives who developed cancer. In relation to anatomic localisation, BRCAPRO probability correlates with the number of relatives who developed breast cancer, but not with the number of those who developed ovarian cancer. Conclusion: The frequency of BRCA1/2 mutations in probands under rsk for hereditary breast and/or ovarian cancer in Serbia is similar to those determined in other populations. In addition to already known BRCA1/2 mutations, new family-specific mutations have been detected. Mutation that could be qualified as founder mutations for our population have not been detected. BRCAPRO software has been shown to be useful in selection of probands for BRCA testing, but in this selection, due to BRCAPRO limitations, the pedigree should be analysed too. Raising the treshold for BRCAPRO probability in proband selection for BRCA testing from 10% to 40% could raise the efficiency of BRCA testing.

Uticaj fiskalne politike na budžetsku, spoljnu i cenovnu ravnotežu u Republici Srbiji

The impact of fiscal policy on budgetary, external and price equilibria in the Republic of Serbia

U disertaciji se analizira uticaj fiskalne politike na budžetsku, spoljnu i cenovnu ravnotežu u Republici Srbiji nakon 2000. godine. Rezultati iz I poglavlja teze upućuju na neodrživost fiskalne pozicije Republike Srbije u analiziranom periodu, pri čemu je globalna finansijska kriza, zajedno sa izbornim političkim ciklusom, delovala u pravcu produbljivanja fiskalne neravnoteže, dok su aranžmani sa MMF-om delovali u pravcu stabilizacije javnih finansija Republike Srbije. Budžetska neravnoteža nakon 2000. godine je formirana kao posledica nesinhronizovanih diskrecionih mera na obe strane budžeta, što je u skladu sa hipotezom o institucionalnoj separaciji. Određeni empirijski dokazi iz II poglavlja, međutim, ukazuju da se putem kontrole javnih prihoda može uticati na dinamiku diskrecionih primarnih javnih rashoda, što je u skladu sa Fridmanovom oporezuj-troši hipotezom. Rezultati iz II poglavlja, takođe, odbacuju hipotezu o Rikardijanskoj ekvivalenciji, i upućuju da je neto efekat pozitivnih fiskalnih inovacija povezan sa: 1) aprecijacijom realnog i nominalnog efektivnog deviznog kursa; 2) smanjenjem privatne štednje domaćinstava; i 3) rastom bruto investicija. Opisani transmisioni mehanizmi deluju, zatim, u pravcu pogoršanja spoljnotrgovinske pozicije Republike Srbije. Konačno, rezultati iz III poglavlja teze pokazuju da fiskalna reakcija javnog duga na rast u primarnom fiskalnom bilansu postaje statistički značajna, u proseku, tek dve godine od početka sprovođenja programa fiskalne konsolidacije, što je u skladu sa teorijskim postavkama rikardijanskog, tj., monetarno-dominantnog, režima formiranja cena. Pojava statistički značajne negativne autokorelacije u stohastičkim procesima za primarni i strukturni primarni fiskalni bilans nakon isteka perioda od 3 godine upućuje, međutim, na potencijalnu relevantnost nerikardijanskog, tj., fiskalno- dominantnog, režima formiranja cena u slučaju Republike Srbije nakon 2000. godine.

This thesis explores the impact of fiscal policy on budgetary, external and price equilibria in the Republic of Serbia after the year 2000. The results from the I thesis chapter point to unsustainable fiscal practices, with a particular emphasis on the destabilising effects of the Great Recession and the electoral political cycle, and the stabilising effects of the “stand-by” arrangements with the IMF. Budgetary disequilibrium in Serbia emerged as a consequence of ad-hoc discretionary measures on both sides of the budget, which is in accordance with the institutional separation hypothesis between government revenues and expenditures. Certain econometric estimates from the II thesis chapter, however, imply how policy makers can curtail primary government expenditures via government revenues control, giving support to Friedman’s tax-spend hypothesis. Other results from the II thesis chapter, which are concerned with the impact of fiscal policy on external equilibrium, reject the Ricardian equivalence hypothesis, and show how the net effect of unanticipated positive budgetary changes is connected with: 1) real and nominal exchange rate appreciation; 2) lower private saving; 3) higher gross fixed capital formation. Aforementioned transmission mechanisms lead, consequently, to the contraction of net exports. Finally, the III thesis chapter shows how fiscal reaction of public debt to an increase in primary fiscal balance becomes statistically significant, on average, after 2 years of fiscal consolidation, an empirical result consistent with the Ricardian, i.e., monetary-dominant, price formation regime. The occurrence of statistically significant negative autocorrelation in the stochastic processes for primary fiscal balance and structural primary fiscal balance after a 3 year period points, however, to potential relevance of nonricardian, i.e., fiscally dominant, price formation regime in the Republic of Serbia after the year 2000.

Analiza govorno-jezičkog, socio-emocionalnog i kognitivnog razvoja bolesnika sa heterozigotnom mikrodelecijom regiona q11.2 na hromozomu 22

Analysis of speech and language, social-emotional and cognitive development of patients with heterozygous microdeletion of the region q11.2 of chromosome 22.

Sindrom delecije 22q11.2 (22q11.2DS) karakteriše prisustvo heterozigotne mikrodelecije regiona q11.2 na hromozomu 22. Pokazano je da deca sa 22q11.2DS često kasne na polju govorno-jezičkog, socio-emocionalnog i kognitivnog razvoja. Kako u literaturi nema podataka o ovim sposobnostima kod bolesnika sa 22q11.2DS govornika južno-slovenskih jezika, u okviru ove doktorske disertacije analizirane su govorno-jezičke, kognitivne i socio-emocionalne sposobnosti bolesnika sa 22q11.2DS izvornih govornika srpskog jezika. Dijagnoza 22q11.2DS postavljena je na osnovu prisustva najmanje dve od pet najčešćih fenotipskih karakteristika 22q11.2DS (urođene srčane malformacije konotrunkalnog tipa (KSM), facijalna dismorfija, aplazija/hipoplazija timusa, rascep nepca, hipokalcemije). Za detekciju mikrodelecije 22q11.2 primenjene su fluorescentna in situ hibridizacija (FISH) i metoda višestrukog umnožavanja proba koje je zavisno od ligacije (MLPA). Na osnovu rezultata FISH i/ili MLPA metoda, bolesnici sa kliničkom dijagnozom 22q11.2DS svrstani su u eksperimentalne grupe E1 (kod kojih je detektovana mikrodelecija 22q11.2) i E2 (kod kojih mikrodelecija 22q11.2 nije detektovana). Familijarna forma 22q11.2DS utvrđena je kod četiri porodice. Kako su kod svih bolesnika grupa E1 i E2 detektovane KSM i kako one mogu da utiču na razvoj govorno-jezičkih, kognitivnih i socio-emocionalnih sposobnosti istraživanjem su bila obuhvaćena i bolesnici sa nesindromskim KSM kod kojih mikrodelecija 22q11.2 nije detektovana FISH metodom (grupa E3). Govorno-jezičke, kognitivne i socio- emocionalne sposobnosti bolesnika grupa E1, E2 i E3 su poređene sa ovim sposobnostima vršnjaka urednog govorno-jezičkog, socio-emocionalnog i kognitivnog razvoja, dobrog zdravstvenog stanja bez prisutnih hroničnih bolesti (grupa K). Rezultati istraživanja su pokazali da bolesnici grupe E1 imaju lošije govorno-jezičke sposobnosti u poređenju sa bolesnicima grupa E2 i E3 i ispitanicima grupe K. Niži nivo psihofizioloških sposobnosti detektovan je kod bolesnika grupe E1 u poređenju sa bolesnicima grupa E2 i E3 i ispitanicima grupe K. Procenjivanjem kognitivnih sposobnosti uočeno je da su ove sposobnosti kod 38,9% bolesnika grupe E1, 18,8% bolesnika grupe E2 i 25% bolesnika grupe E3 bile ispod očekivanih za njihov hronološki uzrast. Analizom bolesnika sa familijarnom formom 22q11.2DS uočena je inter- i intrafamilijarna varijabilnost u nivou govorno-jezičkog razvoja i interfamilijarna varijabilnost u nivou kognitivnog i socio-emocionalnog razvoja. Takođe, rezultati ukuzuju da na osnovu veličine mikrodelecije 22q11.2 nije moguće predvideti nivo govorno-jezičkog, senzomotornog i socio-emocionalnog razvoja. Dobijeni rezultati ukazuju da bolesnici sa mikrodelecijom 22q11.2, izvorni govornici srpskog jezika, predstavljaju rizičnu kategoriju za pojavu govorno-jezičke patologije i neophodno ih je uključiti u program rane stimulacije govorno-jezičkog, kognitivnog i socio-emocionalnog razvoja u cilju poboljšanja njihovih komunikativnih, kognitivnih i socio-emocionalnih sposobnosti.

22q11.2 deletion syndrome (22q11.2DS) is caused by a heterozygous microdeletion of region q11.2 of chromosome 22. It has been shown that speech and language, cognitive and socio-emotional impairments are very common in children with 22q11.2DS. To the best of our knowledge, there are no published data about these abilities in children with 22q11.2DS, native speakers of South-Slavic languages. Therefore, the main goal of this Doctoral Dissertation is to analyze speech and language, cognitive and socio-emotional abilities of children with 22q11.2DS, monolingual native speakers of the Serbian language. Enrollment of patients was based on the presence of at least two out of the five major characteristics of 22q11.2DS (congenital heart malformations (CHM), facial dysmorphism, thymic aplasia/hypoplasia, palatal clefts and hypocalcemia). Fluorescent in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA) have been applied in order to detect 22q11.2 microdeletion. Based on FISH/MLPA results, patients with phenotypic features of 22q11.2DS were divided into experimental groups E1 (with 22q11.2 microdeletion) and E2 (without the 22q11.2 microdeletion). A familial form of 22q11.2DS was detected in four families. Patients with non-syndromic CHM (group E3) were included in the study since literature data implies that these children may exhibit a speech and language, cognitive and socio- emotional impairments. Applying FISH, 22q11.2 microdeletion was not detected in these patients. Speech-language, cognitive and socio-emotional abilities of patients from the groups E1, E2 and E3 were compared to their age peers with proper speech- language, cognitive and socio-emotional development, and good general health condition without chronic diseases (group K). Obtained results revealed that patients from group E1 have less developed speech and language skills and psychophysiological abilities compared to patients from group E2 and E3 and children from group K. Also, cognitive abilities of 38.9% of patients from group E1, 18.8% of patients from group E2 and 25% of patients from group E3 did not reach levels expected for their calendar age. Results obtained by analyzing families with the inherited form of 22q11.2DS revealed inter- and intrafamilial variability in the level of speech and language development and interfamilial variability in the level of cognitive and socio-emotional development. Also, presented results indicated no correlation between the level of speech-language, cognitive and socio-emotional development and the size of 22q11.2 microdeletion. Obtained results imply that patients with the 22q11.2 microdeletion, native speakers of Serbian language, should be considered at risk for speech and language delay and, should be included in an early stimulation of speech-language, cognitive and socio-emotional development in order to improve their communication, cognitive and socio-emotional skills.

Neuroinflamatorni procesi mozga u amiotrofičnoj lateralnoj sklerozi : studija na modelu hSOD1 G93A pacova

Neuroinflammatory processes in the amyotrophic latera sclerosis brain : study of hSOD1 GV3A pacova

Amiotrofična lateralna skleroza (ALS) je neurodegenerativno oboljenje koje pogađa motorne neurone u kičmenoj moždini i višim moždanim centrima. Bolest karakteriše vrlo brza progresija i kratak životni vek od pojave simptoma. Predloženo je više mehanizama koji opisuju patogenezu bolesti, ali je uzrok ALS i dalje nepoznat. Cilj ove studije je da okarakteriše promene u višim moždanim centrima u modelu hSOD1G93A pacova i pokuša da dodatno razjasni mehanizme bolesti. Primenom tehnike magnetno-rezonantnog oslikavanja lokalizovana su neurodegenerativna žarišta u mozgu hSOD1G93A pacova. Istom metodom je in vivo praćena infiltracija T-limfocita pomoću posebno dizajniranih antitela obeleženih ultra malim paramagnetnim partikulama gvožđe oksida. Pokazano je da se CD4+ T-limfociti infiltriraju u tkivo oko moždanih komora, a CD8+ T-limfociti u moždano stablo hSOD1G93A pacova. Praćenje stanja krvno-moždane barijere pomoću Gd-DTPA kontrastnog agensa je ukazalo na njenu narušenost u moždanim regionima gde je uočena infiltracija T-ćelija. Prisustvo aktivirane mikroglije i reaktivnih astrocita je uočeno u moždanom stablu hSOD1G93A pacova. Pored toga, reaktivna mikroglija je uočena u moždanom tkivu oko lateralnih komora i hipokampusu. Nastavci aktivirane mikroglije ostvaruju kontakt sa telom neurona što ukazuje na mogućnost njihove interakcije. Primenom Western blot metode je pokazano da je ekspresija akvaporina-4 (AQP4) povećana, a ulaznog kalijumovog ispravljača (Kir4.1) smanjena u moždanom stablu i moždanoj kori hSOD1G93A pacova. Imunohistohemija na zamrznutim moždanim presecima je pokazala povećanu AQP4 i smanjenu Kir4.1 imunoreaktivnost u nc. facialis, nc. trigeminus i motornoj kori hSOD1G93A pacova. Kada su ispitivani kortikalni astrociti u kulturi, uočena je povećana AQP4 i smnjena Kir4.1 imunoreaktivnost u ALS. Proučavanje funkcionalnih osobina Kir kanala je ukazalo na smanjenu gustinu struja i smanjenu specifičnu membransku provodljivost u ALS astrocitima u kulturi. Pored toga, blokiranje ovih struja dodavanjem 1 mM CsCl ili 100 µM BaCl2 u vanćelijski rastvor je pokazalo da su Cs+-senzitivne odnosno Ba2+-senzitivne struje značajno manje u ALS astrocitima. U ovoj studiji su lokalizovana žarišta neurodegenerativnih i neuroinflamatornih promena u mozgu hSOD1G93A pacova, uočeno je oštećenje krvno-moždane barijere, a posebno su istaknute promene u ekspresiji i funkciji astrocitnih proteina koji doprinose stabilnosti krvno-moždane barijere. Dobijeni podaci su pružili dodatna objašnjenja koja doprinose razumevanju mehanizama ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting lower and upper motor neurons. The disease shows fast progression and short lifetime following the disease onset. Several mechanisms contributing to ALS pathogenesis have been proposed, but the cause of disease remains unclear. The aim of this study was to characterize changes in the upper CNS regions of the hSOD1G93A rat model and to try to clarify disease mechanisms. Foci of neurodegeneration and neuroinflammation were localized in the brain of hSOD1G93A rat using magnetic resonance imaging. The same method was applied for in vivo monitoring of T-lymphocytes infiltration using specifically designed antibodies conjugated with ultrasmall superparamagnetic iron oxide particles. It was shown that CD4+ T-lymphocytes were infiltrated into the brain tissue around lateral ventricles while CD8+ T-lymphocytes were infiltrated into the brainstem of the hSOD1G93A rat. Using Gd-DTPA contrast, it has been shown that the blood-brain barrier was compromised in the brain regions invaded with T-cells. Activated microglia and reactive astrocytes were observed in the brainstem of the hSOD1G93A rat. In addition, reactive microglia was shown in the brain tissue around lateral ventricles and in the hippocampus. Processes of activated microglia were in contact with neuronal somata indicating possible interaction. Western blot results showed increased aquaporin-4 (AQP4) and reduced inwardly rectifying potassium channel (Kir4.1) expression in the brainstem and cortex of the hSOD1G93A rat. Immunohistochemistry on the frozen brain slices showed increased AQP4 and decreased Ki4.1 immunoreactivity in the nc. facialis, nc. trigeminus and motor cortex of the hSOD1G93A rat. Increased AQP4 and decreased Kir4.1 immunoreactivity were retained in cortical ALS astrocytes in culture. Examination of the functional properties of Kir channels revealed a decreased current density and diminished specific membrane conductance in ALS astrocytes in culture. Addition of 1 mM CsCl or 100 µM BaCl2 to the extracellular solution revealed significantly lower Cs+-sensitive and Ba2+-sensitive Kir currents in ALS. In this study we localized and characterized neurodegenerative nad neuroinflammatory changes in the brain of hSOD1G93A rat, confirmed the blood-brain barrier compromise and observed changes in the astrocytic proteins involved in the maintenance of the blood-brain barrier. Obtained data may contribute to further understanding of the mechanisms underlying ALS pathology.

Karakterizacija antimikrobnih supstanci prirodnih izolata Bacillus sp. za primenu u biološkoj kontroli fitopatogenih bakterija i gljiva

Characterization of antimicrobial substances from natural isolates of Bacillus sp. for application in biological control of plant pathogenic bacteria and fungi

Bolesti biljaka izazvane infekcijama patogenim bakterijama i gljivama mogu dovesti do smanjenja kapaciteta biljnog rasta ili naneti mnogo ozbiljniju štetu dovodeći do smrti biljaka i značajnih gubitaka u proizvodnji hrane. Veliki broj istraživanja je u poslednje vreme posvećen proučavanju roda Bacillus kao producenta sekundarnih metabolita u kontroli različitih biljnih patogena. Analizom diverziteta gena odgovornih za produkciju neribozomalno sintetisanih lipopeptida kolekcije od 205 Bacillus sp. izolata utvrđeno je da je za najveći broj izolata (171) ustanovljen DNK fragment očekivane dužine, koji odgovara bacilomicin D sintetazi, dok su u sličnom broju izolata dobijeni fragmenti očekivanih dužina za kurstakinske (154) i surfaktinske operone (142), a prisustvo iturinskog operona ustanovljeno je kod 84, odnosno 59 izolata u slučaju gena za fengicin sintetazu. Prisustvo svih pet biosintetičkih operona je potvrđeno kod 11 izolata. Najveća produkcija antimikrobnih jedinjenja za pet izabranih izolata je pokazana u eksponencijalnoj ili ranoj stacionarnoj fazi rasta. Prikazan je jak antifungalni efekat lipopeptidnih ekstrakata prema više od 30 testiranih gljiva, sa najnižim zabeleženim minimalnim inhibitornim koncentracijama od 0,008 mg/ml protiv Fusarium semitectum, kao i izražen antibakterijski potencijal, naročito protiv fitopatogenih vrsta rodova Xanthomonas i Pseudomonas, sa najnižom minimalnom inhibitornom koncentracijom od 0,06 mg/ml. Proučavanje interakcija pojedinačnih lipopeptidnih ekstrakata u međusobnim, kao i u kombinacijama sa etarskim uljima, ukazalo je na postojanje sinergističkog efekta za neke kombinacije u in vitro, odnosno aditivnog efekta u in situ uslovima, na bakterijskim i fungalnim patogenima. Utvrđivanjem direktnog antagonističkog efekta lipopeptidnih jedinjenja in planta, ustanovljena je značajna redukcija nekroze tkiva listova Arabidopsis thaliana L. i Beta vulgaris L., tretiranih bakterijskim patogenima. Takođe, u in situ uslovima postignuta je značajna redukcija nekroze tkiva ploda jabuke izazvana fungalnom infekcijom. Strukturna karakterizacija lipopeptidnih jedinjenja iz različitih ekstrakata pokazala je njihovu raznolikost i dominantno prisustvo članova iturinske i surfaktinske familije. Takođe, masenom analizom je utvrđeno da etil-acetatna ekstrakcija daje najbolji prinos, a primenom bioautografije, došlo se do zaključka da su za antimikrobni efekat lipopeptidnih ekstrakata najverovatnije odgovorna jedinjenja iz iturinske familije. Filogenetska rekonstrukcija odabranih Bacillus sp. izolata bazirana na sekvenci gena za 16S rRNK je pokazala da izolati SS-12.6, SS-13.1, SS-27.2 i SS-38.4 pokazuju najviše sličnosti sa Bacillus amyloliquefaciens, odnosno Bacillus pumilus u slučaju izolata SS-10.7.

Plant diseases caused by infection with pathogenic bacteria and fungi can lead to the reduction in the capacity of plant growth or can cause far more serious damage, leading to the death of plants and significant losses in food production. Numerous recent studies are devoted to the genus Bacillus as a producer of secondary metabolites in the control of different plant pathogens. In the analysis of diversity of genes responsible for the nonribosomal synthesized lipopeptides in collections of 205 Bacillus sp. isolates, for the largest number of isolates (171) the DNA PCR fragment of the expected length, corresponding to bacillomycin D synthases was obtained, while in a similar number of isolates fragments of the expected length for kurstakin (154) and surfactin operons (142) were obtained. The presence of iturin operon in the 84 or 59 isolates in the case of the gene for fengycin synthetase, was also found. The presence of all five biosynthetic operons in the 11 isolates was confirmed as well. The maximum production of antimicrobial compounds for five selected isolates in exponential or early stationary phase of growth was determined. Strong antifungal effect of lipopeptide extracts towards more than 30 fungi tested, with lowest recorded minimal inhibitory concentrations of 0,008 mg/ml against Fusarium semitectum, as well as expressed antibacterial potential, in particular against phytopathogenic species of the genera Pseudomonas and Xanthomonas, with the lowest minimum inhibitory concentration of 0,06 mg/ml was determined. In the analysis of the interaction of individual lipopeptide extracts mutually, as well as in combination with essential oils, the existence of a synergistic effect in vitro respectively additive effect in situ, on bacterial and fungal pathogens, for some combinations were shown. Establishing a direct antagonistic effect of lipopeptide compounds in planta, significant reduction in necrotic lesions of leaves of Arabidopsis thaliana L. and Beta vulgaris L. treated with bacterial pathogens was obtained. Also, a significant reduction in necrotic lesions of apple fruit caused by fungal infection in situ was determined. Structural characterization of lipopeptide compounds from different extracts showed their diversity and dominant presence of members of iturin and surfactin families. Also, mass analysis revealed that the ethyl acetate extraction has been most efficient procedure with the highest yield of isolated lipopeptides. Simultaneously, applying bioautography, it was concluded that compounds from iturin family were most likely responsible for the antimicrobial effect of lipopeptide extracts. Phylogenetic reconstructions of selected Bacillus sp. isolates based on sequence of 16S rRNA gene showed that isolates SS-12.6, SS-13.1, SS-27.2 and SS-38.4 were the most related to Bacillus amyloliquefaciens, or Bacillus pumilus for SS-10.7 isolate.

Dinamička analiza posebnih klasa linearnih singularnih sistema sa kašnjenjem na konačnom i beskonačnom vremenskom intervalu

Dynamical analysis of particular classes of linear time-delay singular control systems defined over finite and infinite time interval

U disertaciji su razmatrani problemi dinamičke analize posebnih klasa singularnih sistema sa čistim vremenskim kašnjenjem prisutnim u stanju sistema, kao i njihovo ponašanje na konačnom i beskonačnom vremenskom intervalu. Pružen je presek savremenih koncepata stabilnosti, prednosti jednih nad drugima i posebno su obrađeni tzv neljapunovski koncepti: stabilnost na konačnom vremenskom intervalu i koncept praktične stabilnosti. Nadograđene su osnovne definicije stabilnosti. Isrpno je izložen hronološki sistematičan pregled osnovnih koncepata stabilnosti, polazeći od ljapunovske metodologije, kao osnove na kojoj se zasniva dinamička analiza sistema. Ukazano je na istorijski razvoj i nastanak ideja i rezultata u ovoj oblasti i na taj način su izvedene i smernice daljih istraživanja otvorenih problema. U disertaciji su sistemi tretirani sa stanovišta dva savremena pristupa: deskriptivnog i LMI, odnosno sa pozicija linearnih matričnih nejednakosti, koja se svodi na metode konveksne optimizacije. Izvedeni su i saopšteni novi rezultati. Izložen je prilaz koji se bazira na kvaziljapunovskim funkcijama za dobijanje uslova praktične i stabilnosti na konačnom vremenskom intervalu posebne klase singularnih sistema sa čistim vremenskim kašnjenje, u stanju sistema. Pokazano je da, polazeći od pretpostavke da agregacione funkcije ne moraju da budu određene po znaku i da njihovi izvodi duž trajektorija sistema ne moraju da budu negativno odrređene funkcije, uz pomoć deskriptivnog prilaza se mogu dobiti novi kriterijumi za ocenu neljapunovske stabilnosti. Kombinovanjem rezultata sa ljapunovskim prilazom, izvedeni su o uslovi atraktivne praktične stabilnosti. Drugi doprinos je određivanje dovoljnih uslova stabilnosti na konačnom vremenskom intervalu iste klase sistema pomoću savremenih LMI metoda. Dobijeni i prezentovani rezultati imaju praktičnu primenu u savremenoj teoriji i praksi upravljanja i mogu se primeniti na sve klase proučavanih sistema, pod uslovom da su dostupni verodostojni matematički modeli. Verifikacija rezultata je izvedena kroz numeričke primere

In this thesis the problems of dynamical analysis of particular class of singular control systems with time delays are considered, as well as their behavior on finite and infinite time intervals. Emphasis has been put on the peculiar properties of singular ad descriptor systems, concerning the existence and uniqueness of the solutions, the problems of impulsive behavior, consistent initial conditions and causality of the system itself. On overview of the modern stability frameworks has been presented, starting from the classical Lyapunov ideas and extending through so called non-lyapunov concepts: finite time stability and practical stability in particular. A historical overview of ideas, concepts and results has been presented and the key contributions have been highlighted through key papers from the modern literature. This dissertation follows two main lines of research: the descriptive approach and the LMI (linear matrix inequalities) methodology, the latter being known to reduce control tasks to convex optimization problems, thus making them easily solvable by numerical computation. New results are presented. A new approach, based on lyapunov-like functions, is used in order to establish new sufficient conditions of practical and finite time interval stability of a particular class of singular time delay systems. Another new result is based on the modern LMI approach and gives new sufficient conditions for finite time stability. The obtained results are numerically verified and have great practical value, as they are easy to compute and less restrictive and conservative than their predecessors.

Derivati ramnolipida i 4-aminohinolina kao inhibitori virulencije kod vrsta Pseudomonas aeruginosa i Serratia marcescens

Derivatives of rhamnolipids and 4-aminoquinoline as Pseudomonas aeruginosa and Serratia marcescens virulence inhibitors

Rezistencija bakterija na antibiotike je rastući problem globalnih razmera tako da su pronalaženje i uvođenje novih terapijskih opcija u borbu protiv bakterijskih infekcija postali prioriteti i fundamentalnih i primenjenih istraživanja. U potrazi za jedinjenjima prirodnog porekla sa anti-virulentnom aktivnošću izdvojen je nepatogeni soj iz rizosfere biljke dobračice (Glechoma hederacea), Lysinibacillus sp. BV152.1, čiji su metaboliti inhibirali formiranje biofilmova P. aeruginosa PAO1. Hemijske analize su pokazale da aktivnu komponentu etil acetatnog ekstrakta čini smeša ramnolipida. Uporednom analizom anti-biofilm aktivnosti utvrđeno je da su di-ramnolipidi soja Lysinibacillus sp. BV152.1 bolji inhibitori procesa formiranja biofilmova (adhezije i sazrevanja) kod P. aeruginosa PAO1 od komercijalno dostupnih ramnolipida, poreklom iz P. aeruginosa. Amidnom derivatizacijom di- ramnolipida poboljšana je njihova anti-biofilm aktivnost, gde je najaktivniji derivat di- ramnolipida iz Lysinibacillus sp. BV152.1 imao morfolinsku grupu i inhibirao je formiranje biofilmova P. aeruginosa PAO1 za 80% u koncentraciji od 100 μg/ml, a isti derivat u koncentraciji od 50 μg/ml inhibirao je formiranje biofilmova S. marcescens za 88%. Hinolini, jedinjenja sa širokim spektrom bioloških aktivnosti, a takođe i autoinduceri PQS signalnog puta međubakterijske komunikacije P. aeruginosa predstavljaju dobru osnovu za razvoj anti-virulentnih jedinjenja. Derivati aminohinolina sintetisani u ovoj studiji nisu pokazali značajnu antibakterijsku aktivnost prema Gram negativnim patogenima. Najveću inhibitornu aktivnost na formiranje biofilmova kod P. aeruginosa i S. marcescens imala su jedinjenja koja su sadržala atom hlora ili CF3 grupu na poziciji C(7) i alifatični lanac sa 12 C atoma na poziciji C(4) (jedinjenja 5 i 10), sa BFIC50 koncentracijama od 69 μM odnosno 63 μM. Ova jedinjenja su prvi derivati hinolina za koje je utvrđena sposobnost da inhibiraju biofilmove S. marcescens. Detaljnom analizom odnosa strukture i aktivnosti jedinjenja 5 i 10 je pokazano da dužina alifatičnog lanca i lipofilnost jedinjenja imaju najveći uticaj na formiranja biofilomova kod S. marcescens. Jedinjenje 10 je izdvojeno kao najpotentniji inhibitor proizvodnje piocijanina kod P. aeruginosa hinolinske prirode opisan do sada. 7 Dalja derivatizacija je išla u pravcu modifikacije jedinjenja AQ8 (N- oktanamino-4-aminohinolin) benzil grupama radi identifikovanja strukturnih zahteva za poboljšanje njegove anti-virulentne aktivnosti. Jedinjenje sa benzofuranom (jedinjenje 34) se izdvojilo kao najpotentniji inhibitor formiranja biofilmova P. aeruginosa PAO1 i PQS signalnog puta. Prediktivni model ukazuje da se jedinjenje 34 vezuje za PqsR receptor za isto mesto kao i prirodni autoinduceri. Dodatne interakcije sa aminokiselinskim ostacima receptora preko amino alkil bočnog lanca i N-benzil supstituenta omogućavaju inhibiciju PqsR receptora jakim i stabilnim vezama. Ovi rezultati su ukazali da su amino alkil bočni lanci 4-aminohinolina pogodno mesto za njihovu dalju modifikaciju u pravcu dobijanja potentnijih inhibitora PQS sistema međubakterijske komunikacije.

Bacterial resistance to antibiotics is a growing problem on a global scale, so finding and introducing new therapeutic options to combat bacterial infections has become a priority in both fundamental and applied research. In a search for structurally new compounds with anti-virulent activity, novel non-pathogenic strain was isolated from the rhizosphere of a Glechoma hederacea plant, named BV152.1, whose metabolites inhibited the formation of P. aeruginosa PAO1 biofilms. Chemical analyzes have shown that the active compound of ethyl acetate extract is a mixture of rhamnolipids. Comparative analysis of their anti-biofilm activity revealed that the di-rhamnolipids from the strain Lysinibacillus sp. BV152.1 are better inhibitors of P. aeruginosa PAO1 biofilm adhesion and maturation, than commercially available rhamnolipids, originated from P. aeruginosa. The amide derivatization of di-rhamnolipids enhanced the anti-biofilm activity of these compounds, where the most active di-rhamnolipid derivative from Lysinibacillus sp. BV152.1 had a morpholine group and inhibited the formation of P. aeruginosa PAO1 biofilms by 80% at a concentration at 100 μg/ml. The same derivative at 50 μg/ml inhibited formation of S. marcescens biofilm by 88%. Quinolines, compounds with a wide range of biological activities, and autoinducers of the quorum sensing PQS signaling pathway of P. aeruginosa represent a good basis for the development of compounds with anti-virulent activity. The aminoquinoline derivatives synthesized in this study did not show significant antibacterial activity against Gram-negative pathogens, making them suitable chemical structures for the development of anti-virulent agents. The highest inhibitory activity of P. aeruginosa and S. marcescens biofilm formation showed compounds containing a chlorine atom (compound 5) or CF3 group (compound 10) at position C(7) and an aliphatic chain of 12 C atoms at position C(4) with BFIC50 concentrations of 69 μM and 63 μM, respectively. These compounds are the first quinoline derivatives identified to have the ability to inhibit S. marcescens biofilms formation. Detailed analysis of the structure and activity relationships of compounds 5 and 10 showed that the aliphatic chain length and lipophilicity of the compounds had the greatest influence on inhibition of biofilm formation in S. marcescens. 9 Compound 10 was the most potent inhibitor of P. aeruginosa pyocyanin production of a quinoline nature described so far (IC50 = 2.5 μM). Further derivatization of long-chained aminoquinolines went towards structural modification of the compound AQ8 (N-octanamino-4-aminoquinoline) with benzyl groups, with the aim to identify structural parameters required for improving its anti- virulent activity. The benzofuran-substituted compound (compound 34) appeared as the most potent inhibitor of P. aeruginosa PAO1 biofilm formation and PQS signaling pathway. According to the model predicting the binding orientation of compound 34 for PqsR receptor, its quinoline ring binds to the same site as native autoinducers. Amino alkyl side chain and N-benzyl substituent of 34 form additional strong and stable bonds with amino acid residues of the receptor causing inhibition of PqsR receptor. These results suggested that the side chains are preferred target for further modification for obtaining more potent inhibitors of the PQS signaling pathway. The results of this study indicate that long-chained 4-aminoquinolines are promising chemical structures for the further development of potent inhibitors of PQS signaling pathway.

Uticaj dugotrajne dijetalne restrikcije na insulinski signalni put u mozgu pacova tokom starenja

The effect of long-term dietary restriction on insulin signaling pathway in rat brain during aging

Starenje je normalan, fiziološki proces koji obuhvata sve organe i organske sisteme i tokom koga se organizam suočava sa nizom strukturnih i funkcionalnih promena. Starenje mozga je izrazito kompleksan proces, predstavljen nizom sukcesivnih događaja koji postepeno rezultiraju gubitkom kognitivnih i motornih funkcija. Postoji niz farmakoloških i sredinskih faktora koji su u stanju da odlože i/ili uspore mnoge od starosno-zavisnih procesa, a dijetalna restrikcija (DR) je jedna od najviše i najduže istraživanih. Mnogobrojni su eksperimentalni i epidemiološki podaci koji govore u prilog korisnih efekata restriktivnog režima ishrane, bez obzira da li se radi o svakodnevnoj, ili intermitentnoj dijeti, smanjenju količine hrane ili kalorija. Međutim, u poslednje vreme se sve više pojavljuju podaci koji dovode u pitanje univerzalnost korisnog dejstva dijete. Da bi ovakva intervencija postigla svoj optimalni efekat, neophodno je povesti računa o određenim specifičnostima prilikom uspostavljanja restriktivnog režima ishrane. Pre svega, pokazano je da stepen restrikcije igra bitnu ulogu u efektima koje ona proizvodi i da previše restriktivan režim ishrane može imati negativne posledice po organizam. Takođe, postoje indicije da nisu svi organski sistemi podjednako podložni procesima starenja, kao ni dejstvu dijete. Efekat dijete se može veoma razlikovati u zavisnosti od stepena i tipa restrikcije, pola i organa gde se efekat očekuje, kao i od starosti jedinke na koju se restriktivni režim primenjuje. Prvi korak u istraživanju korisnih efekata restrikcije hrane koja je iznosila 60% prosečnog dnevnog unosa hrane, bio je ispitati efekat različitih dijetalnih režima, odnosno efekte DR koja je različito trajala i koja je započeta u različitim životnim dobima kod mužjaka Wistar soja pacova. Praćen je čitav niz parametara u aktivnosti životinja i detektovane su značajne razlike u njihovim fizičkim i kognitivnim preformansama, kako tokom starenja, tako i pod uticajem različitih režima ishrane. Dugotrajna dijetalna restrikcija otpočeta u adultnom dobu je dovela do poboljšanja u izvođenju motoričkih i kognitivnih testova i do sveukupnog poboljšanog stanja organizma, što se ogledalo u smanjenoj krhkosti ovih životinja. Nasuprot tome, restriktivni režimi istog tipa, ali kasnijeg početka i/ili kraćeg trajanja, pokazali su se kao neuspešni u pokušaju da se izazovu korisni efekti i poboljša stanje organizma. Dijeta sa izrazito kasnim početkom i kratkog trajanja je dovela čak i do negativnih posledica po organizam, izazivajući dodatne poteškoće u habituaciji životinja i povečavajući njihovu krhkost. Shodno detektovanim rezultatima, za dalji nastavak istraživanja je izabrana dijeta za koju je pokazano da daje nedvosmisleno dobar efekat i dalje je ispitivan njen uticaj na energetski metabolizam mozga. Smatra se da je narušavanje homeostaze energetskog metabolizma jedan od vodećih uzroka koji leži u osnovi brojnih neurodegenerativnih bolesti koje se javljaju tokom starenja. Ključnu ulogu u metabolizmu glukoze i održavanju energetske homeostaze imaju proteini insulinskog signalnog puta koji su odgovorni za regulaciju unosa hranjivih materija, zatim AMPK kao glavni energetski senzor ćelije i glukozni transporteri koji omogućavaju ulazak glukoze u mozak i nervne ćelije. Novija istraživanja ukazuju da pomenuti proteini imaju važnu ulogu u procesima koji leže u osnovi efekata DR na starosno-zavisne promene. Upravo zato, važan aspekt ove doktorske disertacije predstavljao je ispitivanje promena u ekspresiji glukoznih transportera, AMPK proteina, Neuropeptida Y, insulina, insulinskog receptora, supstrata insulinskog receptora i protein kinaze B. Ispitivanja su sprovedena na nivou kore prednjeg mozga, hipokampusa i hipotalamusa, struktura u kojima je pokazano da dolazi do znatnog narušavanja energetske homeostaze tokom starenja. Dobijene promene su sagledane u odnosu na promene relevantnih biohemijskih parametara u serumu, kao što su glukoza, insulin, holesterol i trigliceridi. Zaključeno je da dugotrajna dijetalna restrikcija dovodi do niza promena, koje uključuju povećanje nivoa NPY proteina i insulina, utišavanje insulinskog signalnog puta u hipotalamusu i hipokampusu, kao i do bolje snabdevenost kore velikog mozga i hipokampusa dovoljnom količinom energije. Sve ove promene su zajedno doprinele poboljšanju kognitivnih i motoričkih performansi životinja u dubokoj starosti. Rezultati ove doktorske disertacije upućuju na korisnost restriktivnog režima ishrane, ali istovremeno nameću limite u pogledu trenutka uvođenja ovakvog režima ishrane i dužine trajanja, uzimajući u obzir negativne efekte do kojih DR može dovesti u određenim slučajevima.

Aging is a normal, physiological process that involves all organs and organic systems, and during which the organism faces a number of structural and functional changes. Brain aging is an extremely complex process, represented by a series of successive events that gradually result in loss of cognitive and motor functions. There are a number of pharmacological and environmental factors that are able to postpone and / or slow down many of the age-dependent processes, and dietary restriction (DR) is one of the most investigated and most widely used experimental intervention in aging research. There are numerous experimental and epidemiological data that support the beneficial effects of a restrictive diet, regardless of the type: a daily reduction or intermittent diet, reduction in food or in calories, all of those approaches seem beneficial. Nevertheless, there is a body of data that challenges current premise about comprehensive DR usefulness. In order for DR to achieve its optimal effect, it is necessary to take certain specificities into account while establishing a restrictive dietary regime. First of all, it has been shown that the percentage of restriction plays an important role in the effects it induces, and that a very restrictive diet may have negative consequences for the organism. In addition, not all the body systems are equally susceptible to aging processes, nor to the beneficial effects of DR. The effect of the restrictive diet can vary greatly, depending on the degree and type of restriction, gender and organs examined, as well as the age of the individual to which the restrictive regimen is applied. In order to investigate the effect of 60% DR on the energy homeostasis and insulin signaling in the brain, we first examined the effects of three different types of dietary restriction that varied in length and onset, on rat behavior during aging. The experiments were performed on 12-, 18- and 24-month-old male Wistar rats exposed to AL or DR type of feeding (60% of AL daily intake). A variety of parameters were monitored in the activities of animals and significant differences in their physical and cognitive performance were detected, both during aging and under the influence of different dietary regimes. Long-term dietary restriction that started in young adulthood led to the improvement of motor and cognitive performances and to the reduced frailty. By contrast, restrictive regimes of the same type, but with late-onset and/or the shorter duration, have a less pronounced positive impact on motor and cognitive functions during aging. A diet that started at old age and had the shortest duration has even led to negative consequences, causing additional difficulties in the habituation of animals, and increased their frailty score. Based on results mentioned above and the most favorable outcome of DR, we continued to investigate the longest dietary regime and its effect on brain metabolism during aging. Disruption of homeostasis in the brain energy metabolism is considered to be one of the leading causes that lie behind many age-related neurodegenerative diseases. The key proteins that regulate processes of glucose metabolism and energy homeostasis are: AMPK, as the main energy cell sensor, glucose transporters that facilitate glucose transport across the blood-brain-barrier and plasma membrane, and proteins involved in insulin signaling pathway, responsible for the regulation of nutrient intake. These proteins play an important role in processes involved in the effects of DR on age-dependent changes. For this reason, we examined changes in the expression of glucose transporters, AMPK protein, Neuropeptide Y, insulin, Insulin Receptor, Insulin Receptor Substrate, and Protein Kinase B. The analysis was performed in cerebral cortex, hippocampus and hypothalamus, structures most severely affected by process of aging and also with important role in energy homeostasis. Relevant biochemical parameters in the serum were also investigated, such as glucose, insulin, cholesterol, and triglycerides. The most pronounced changes elicited by long-term restrictive regime were related to increased levels of insulin and NPY proteins, as well as attenuation of the insulin signal pathway in the hypothalamus and the hippocampus, and improved energy supply of cortex and hippocampus. All the above changes resulted in a significant improvement of cognitive and motor performances of 24 month-old animals. The results of this doctoral dissertation point to the usefulness of a restrictive diet, but at the same time impose certain limits, regarding the time point when the diet should be applied and also the duration of diet, taking into account the negative effects that DR may have in certain cases.

Biološki potencijal i analiza metabolita jestivih i lekovitih gljiva iz rodova Agrocybe, Laetiporus, Pleurotus i Polyporus sa teritorije Srbije

Biological potential and metabolite analysis of edible and medicinal fungi from genera Agrocybe, Laetiporus, Pleurotus i Polyporus from the territory of Serbia

krupna plodonosna tela, dovoljno velika da se vide golim okom i uberu rukom. U ovoj doktorskoj disertaciji makromicete iz rodova: Agrocybe, Laetiporus, Pleurotus i Polyporus, sakupljene na teritoriji Srbije, detaljno su analizirane. Ispitivan je sadržaj makronutrijenata i odabranih metabolita u makromicetama i utvrđen je njihov biološki potencijal. Identifikacija i određivanje sadržaja nutrijenata (ukupnih ugljenih hidrata, proteina, masti i pepela), šećera, tokoferola, masnih, fenolnih i organskih kiselina izvedeno je putem HPLC-a sa različitim detektorima u zavisnosti od jedinjenja od interesa. Radi detaljne procene biološkog potencijala ekstrakata makromiceta, izvedeni su testovi in vitro antioksidativne, antimikrobne, anti-quorum sensing i citotoksične aktivnosti, a iz bazidiokarpa Laetiporus sulphureus 1 izolovan je i okarakterisan lektin i procenjen njegov antibakterijski potencijal. Rezultati analize makronutrijenata pokazali su da testirane makromicete imaju povoljan profil nutrijenata zbog čega se mogu preporučiti u ishrani. Karakteriše ih znatna količina ugljenih hidrata i proteina kao i nizak sadržaj masti. Analizom šećera identifikovane su: ramnoza, fruktoza, manitol, glukoza i trehaloza koja je bila i najzastupljeniji šećer sa 16,50 g/100 g suve mase kod Pleurotus ostreatus K i 32,60 g/100 g suve mase kod P. ostreatus S. U uzorcima testiranih vrsta makromiceta određen je sadržaj četiri izoforme tokoferola (α-, β-, γ- i δ- tokoferol), dok je analizom masnih kiselina potvrđeno prisustvo 27 masnih kiselina, među kojima je nutritivno poželjna linolna kiselina najviše zastupljena u uzorku Agrocybe aegerita 1 (78,40%). Među prostim organskim kiselinama utvrđeno je prisustvo: oksalne, kininske, jabučne, limunske i fumarne kiseline, dok su analizom organskih kiselina sa fenolnom grupom identifikovane protokatehinska, p-hidroksibenzoeva, p-kumarna i cinaminska kiselina. Izoloacijom i karakterizacijom lektina iz bazidiokarpa L. sulphureus 1 pokazano je da je lektin multimerne građe, pI vrednosti ~ 4,6. Rezultati antioksidativne aktivnosti (redukciona sposobnost, DPPH· aktivnost, β-karoten/linolna kiselina i TBARS test) pokazali su da su testirane makromicete moćni in vitro antioksidativni agensi više ili manje efikasni u neutralizaciji slobodnih radikala, inhibiciji lipidne peroksidacije i redukcionoj sposobnosti (EC50/ EC0.5 vrednosti su u opsegu 0,25 - 23,78 mg/mL). Etanolni i metanolni ekstrakti bazidiokarpa testiranih makromiceta pokazali su visok antimikrobni potencijal (u odnosu na vodeni ekstrakt) na testirane mikroorganizme, a utvrđeno je da pri testiranim koncentracijama lektin ne deluje na bakterije, što ide u prilog tvrdnji da supstance prirodnog porekla biološki efekat ostvaruju zahvaljujući aditivnim i sinergističkim interakcijama većeg broja prisutnih jedinjenja. Metanolni ekstrakti testiranih makromiceta su na subinhibitornom nivou efikasno ometali međućelijsku komunikaciju oportunističkog patogena Pseudomonas aeruginosa PAO1, što je zaključeno nakon smanjenja dijametra njegove kolonije, struktura za kretanje i sinteze pigmenta piocijanina koji je ujedno i faktor virulentnosti. Najbolji antibiofilm efekat postigao je uzorak metanolnog ekstrakta Polyporus squamosus 1 (88,30%). Umereni citotoksični potencijal na tumorske ćelijske linije humanog porekla HeLa, NCI-H460 i MCF-7 pokazao je samo polisaharidni ekstrakt uzorka L. sulphureus 1. Rezultati preliminarne analize hepatotoksičnosti predočili su da ekstrakti nisu toksični za primarne svinjske hepatocite čime je otvoren put za sigurnu primenu preparata na bazi testiranih makromiceta kod ljudi. Rezultati sprovedenih istraživanja su pokazali da testirane makromicete imaju potencijal funkcionalne hrane zahvaljujući produkciji biološki aktivnih jedinjenja koja mogu pozitivno delovati na zdravlje, a zahvaljujući sposobnosti eradikacije patogenih mikroorganizama, ekstrakti makromiceta su važni pri formulaciji novih antimikrobnih agenasa. Upotreba makromiceta ima značajan potencijal u unapređivanju zdravlja, dugovečnosti i kvaliteta života i predstavlja primer jedinstva tradicije i nauke. Dalja proučavanja makromiceta ukazuju na pravce za njihov napredak od funkcionalne hrane do dostupnih medicinskih preparata, a kako su neke od testiranih vrsta uspešno kultivisane, veliki su izgledi da se ovako nešto zaista i dogodi u bliskoj budućnosti.

Macromycetes are fungi from subdivisions Ascomycotina and Basidiomycotina that are large enough to be seen with naked eye and to be picked by hand. In this thesis, selected species from the genera: Agrocybe, Laetiporus, Pleurotus and Polyporus were harvested in Serbia and chemically characterized. Their nutrients profile and selected metabolites as well as their biological potential was evaluated. Quantification of macronutrients (carbohydrates, proteins, fat and ash), selected sugars, tocopherols, fatty, phenolic and organic acids was determined by means of HPLC coupled with various detectors, depending on the type of desired compound. Regarding the evaluation of biological potential, detailed assesments of antioxidant, antimicrobial, anti-quorum sensing and cytotoxic activities were performed. A lectin was extracted from the fruiting body of Laetiporus sulphureus 1 as a compound of interest, and was evaluated for its antibacterial activity. Results of the macronutrient profile suggest that macromycetes are a rich source of carbohydrates and proteins, with low fat content which makes them excellent candidates for food. Sugar analysis revealed the presence of: rhamnose, fructose, mannitol, glucose and trehalose which was the most abundant sugar with 16,50 g/100 g dry weight in Pleurotus ostreatus K and 32,60 g/100 g dry weight in P. ostreatus S. Four isoforms of tocopherols (α-, β-, γ- and δ- tocopherol) were quantified in samples, while the fatty acid analysis revealed the presence of 27 fatty acids with linoleic acid as the most abundant polyunsatturated fatty acid in sample Agrocybe aegerita 1 (78,40%). Amongst simple organic acids, the following were detected in the samples: oxalic, quinic, malic, citric and fumaric acid. Phenolic group containing organic acids analysis indicated the presence of: protocatechuic, p-hydroxybenzoic, p-coumaric and cinnamic acid. Isolation and characterization of lectin from the L. sulphureus 1 basidiocarp revealed that lectin is a multimer with pI value of ~ 4,6. Results of antioxidant activity which was evaluated using four protocols (reducing power, DPPH· scavenging activity, β- caroten/linoleic acid and TBARS test) indicate that selected macromycetes are potent antioxidant agents that showed ability to neutralize free radicals, inhibit lipid peroxidation and reducing power (EC50/EC0.5 is in range of 0,25 – 23,78 mg/mL). Ethanolic and methanolic extracts of mushroom fruiting bodies revealed potent antimicrobial potential against the tested pathogenic microorganisms (compared to the aqueous extracts), while lectin didn't show antibacterial activity at the tested concentrations, which supports the claim that natural sourced substances achieve their full biological potential thanks to the additive and synergistic interactions of all the present compounds. Methanolic extracts of the selected macromycetes were tested at subinhibitory level for their ability to interfere with bacterial cell-cell communication using Pseudomonas aeruginosa PAO1 as a model system. Obtained results suggest that mushroom extract very efficiently reduced the diameter of bacterial colony, structures that enable its dispersion, as well as the synthesis of pigment pyocyanin – its virulent factor. The best antibiofilm effect was observed for the methanolic extract of Polyporus squamosus (88,30%). Moderate cytotoxic potential was exclusive for polysaccharide extract of L. sulphureus 1 against the following carcinogenic cell lines: HeLa, NCI-H460 and MCF-7. In addition, results of the preliminary hepatotoxicity analysis indicate the extracts are non-toxic for the porcine primary cells in tested concentrations, providing the opportunity for safe human consumption of these mushrooms. All the obtained results indicate that the selected macromycetes have potential as functional foods thanks to the production of biologically active compounds which have beneficial health effects and in addition, given their ability to eradicate pathogenic microorganisms, mushrooms are important in the formulation of new available antimicrobial agents. Macromycetes are a true example of tradition and novelty unity and they have significant potential in maintaining and improving health, longevity and quality of life. Their current status and future prospects open up new avenues for improving their status from functional foods to proper medicinal products. Since the tested mushrooms have been successfully cultivated, something like this may actually happen in near future.

Analiza ekspresije i uloge humanog gena SOX14 u neuralnoj diferencijaciji in vitro i regulaciji malignog fenotipa

Analysis of the expression and role of the human gene SOX14 in neural differentiation in vitro and in the regulation of malignant phenotype

Visok stepen očuvanosti tokom evolucije i činjenica da do danas nije opisan nijedan klinički sindrom povezan sa mutacijama u genu SOX14, ukazuju na značajnu ulogu ovog gena tokom embrionalnog razvića. Iako je prošlo dvadeset godina od otkrića gena SOX14, funkcija ovog gena još uvek nije dovoljno istražena. U ovoj doktorskoj disertaciji analizirana je ekspresija i uloga gena SOX14 u procesu neuralne diferencijacije in vitro i regulaciji malignog fenotipa ćelija poreklom od karcinoma grlića materice. Tokom neuralnog razvića kod miša i pileta, gen Sox14 je eksprimiran u određenom setu neuralnih progenitorskih ćelija i u određenim tipovima zrelih neurona u mozgu. Za članove SOXB1 podgrupe, kojoj pripadaju geni SOX1, SOX2 i SOX3, poznato je da doprinose održavanju neuralnih progenitorskih ćelija u stanju proliferacije, sprečavajući proces neuralne diferencijacije, dok se za članove SOXB2 podgrupe, koja obuhvata gene SOX14 i SOX21, pretpostavlja da imaju ulogu u određivanju sudbine neuralnih progenitora, indukujući proces neurogeneze. Smatra se da nivo ekspresije SOX14 i SOX21 proteina tokom neuralnog razvića određuje da li će ćelija ostati neuralni progenitor ili će započeti proces neurogeneze. Iako je okarakterisan kao marker neurona, u ovoj tezi je pokazano da se gen SOX14 eksprimira u pluripotentnim ćelijama, neuronima, kao i u drugim tipovima ćelija koje se dobijaju nakon neuralne diferencijacije pluripotentnih NT2/D1 i P19 ćelija in vitro. Takođe, u ovoj tezi je pokazano da je profil ekspresije SOX14 proteina tokom neuralne diferencijacije sličan profilu ekspresije markera postmitotičkih neurona β-III-Tubulina kod NT2/D1 i P19 ćelija. Rezultati prikazani u ovoj doktorskoj disertaciji su pokazali da izlaskom ćelija iz stanja pluripotentnosti i ulaskom u proces neuralne diferencijacije dolazi do povećanja nivoa ekspresije SOX14 proteina. Pokazano je da SOX14 protein ima preklapajući profil ekspresije sa SOXB1 proteinima tokom procesa neuralne diferencijacije in vitro, a dobijeni rezultati su ukazali na specifičnu regulaciju ekspresije gena SOX14 na post- transkripcionom nivou u pluripotentnim ćelijama. Novija istraživanja ukazuju da je ekspresija gena SOX14 izmenjena u maligno transformisanim ćelijama. Naime, istraživanja su pokazala da je gen SOX14 metilovan u tkivima poreklom od tumora, i to naročito u tkivima poreklom od karcinoma grlića materice. Dok studije metilacionog statusa na uzorcima tkiva pacijentkinja obolelih od ovog karcinoma ukazuju na odsustvo ekspresije gena SOX14 i njegovu potencijalnu ulogu tumor supresora, in vitro studije na ćelijama poreklom od karcinoma grlića materice ukazuju na onkogeni potencijal ovog gena. Upravo je kontradiktornost ovih podataka otvorila nove pravce istraživanja usmerene na detaljnu analizu ekspresije i uloge gena SOX14 u regulaciji malignog fenotipa ćelija poreklom od karcinoma grlića materice. Rezultati prikazani u ovoj doktorskoj disertaciji su pokazali da SOX14 ostvaruje funkciju tumor supresora u HeLa ćelijama aktivacijom p53 signalnog puta koji se smatra jednim od najvažnijih signalnih puteva u ćeliji. Povećana ekspresija SOX14 proteina dovodi do stabilizacije p53 proteina povećanjem nivoa njegove fosforilisane forme, koja se vezuje za promotorske sekvence ciljnih gena i reguliše njihovu ekspresiju. U ovoj disertaciji pokazano je da usled povećanja nivoa SOX14 proteina i stabilizacije p53, dolazi do indukcije ekspresije p53 ciljnih gena uključenih u regulaciju apoptoze i ćelijskog ciklusa, kao što su pro-apoptotski gen BAX i ciklin zavisna kinaza CDKN1A/p21Waf1/Cip1. Pokazano je da usled aktivacije p53 signalnog puta u uslovima povećane ekspresije SOX14 proteina dolazi do smanjenja proliferacije, zastoja u ćelijskom ciklusu i programirane ćelijske smrti. Imajući u vidu da je p53 signalni put kompromitovan prisustvom HPV virusa i da povećana ekspresija SOX14 aktivira ovaj važan signalni put, indukcija ekspresije SOX14 proteina u ćelijama poreklom od grlića materice može imati terapeutski značaj. Rezultati ove doktorske disertacije doprinose boljem razumevanju uloge jednog od evolutivno najočuvanijih SOX proteina tokom neuralne diferencijacije in vitro i u regulaciji malignog fenotipa. Prikazani rezultati pokazali su da je još jedan član familije gena SOX uključen, kako u regulaciju procesa razvića i diferencijacije ćelija, tako i u regulaciju procesa koji su povezani sa malignim svojstvima ćelija. .

SOX14 might have an essential role during development due to the high evolutionary conservation and lack of any known mutated phenotype associated with this gene. Although the first SOX14 gene in vertebrates was cloned and characterized two decades ago, the function of this gene is largely unknown. This doctoral dissertation provides an insight into SOX14 expression and its roles during neural differentiation in vitro and in regulation of malignant phenotype of cervical carcinoma cells. During the neural development, the expression of Sox14 gene is restricted to a defined set of neural progenitors and the precise subset of neurons in the brain. Because of its specific expression in the brain, SOX14 is recognised as a marker of neurons. While SOXB1 subgroup of genes, including SOX1, SOX2 and SOX3 genes, maintains neural progenitors' identity, it is proposed that SOXB2 subgroup, comprising SOX14 and SOX21 genes, are important for induction of neurogenesis. It is suggested that the fine balance between expressions of these proteins during early stages of neural development determines neural progenitor identity. Results presented in this thesis show that SOX14 is expressed in pluripotent cells, as well as in neurons and non-neuronal differentiated cells. Also, it is shown that the expression profile of SOX14 correlates to the expression profile of neuronal marker β-III-Tubulin and that the exit of cells from the pluripotent state toward neural differentiation is accompanied with the increased expression of SOX14. It is also shown that the expression of SOX14 overlaps with the expression of SOXB1 proteins during neural differentiation in vitro. The presented results also imply the specific post-transcriptional mechanism of regulation of SOX14 expression in pluripotent cells. Recently it became evident that SOX14 has a role during malignant transformation of the cells. Previous studies showed that SOX14 is methylated in tissues originating from several tumors, including cervical carcinoma. Results obtained by the analysis of the methylation status of SOX14 gene in tissues from cervical carcinoma suggested its potential role as a tumor suppressor, while, on the other hand, in vitro studies suggested that SOX14 exerts oncogenic potential in cervical carcinoma cells. These contradictory data opened up the possibility for further study of the role of SOX14 gene in cervical carcinoma. Results presented in this thesis show that SOX14 exerts tumor suppressor activity in HeLa cells through activation of p53 signaling pathway, one of the most important signaling pathways in cell. In particular, increased expression of SOX14 leads to stabilization of p53 protein by increasing the level of phosphorylated form of p53 protein in HeLa cells. Phosphorylated p53 acts as a transcriptional regulator of target genes that have an important role in tumor growth prevention. The increased expression level of SOX14 and stabilization of p53 are followed by induction of the expression of p53 target genes, mainly involved in apoptosis and cell cycle regulation, such as pro-apoptotic BAX gene and cyclin-dependent kinase inhibitor CDKN1A/p21Waf1/Cip1. Increased expression of SOX14, through activation of a p53 signaling pathway, leads to decreased proliferation, cell cycle arrest and induction of apoptosis. Obtained results imply that SOX14 induces proper function of p53 tumor suppressor in cervical carcinoma cells and prevents the malignant phenotype of HeLa cells. Presented results indicate that SOX14 has an important role in suppression of cervical carcinoma. Its ability to activate p53 signaling pathway, which is commonly inactivated with by HPV in cervical carcinoma, could have potential therapeutic application. Obtained results contribute to a better understanding of the roles of one of the most conserved SOX proteins and show that one more member of SOX gene family is implicated in the regulation of processes of development and differentiation, as well as in regulation of malignant phenotype.

Molekularna karakterizacija humanih izolata laktobacila rezistentnih na antibiotike

Molecular characterization of human vaginal lactobacilli resistant to antibiotics

^ ' : ' rj M O L E K U L A R N A K A R A K T E R IZ A C IJA H U M A N ÏH - ' ^ ' - : - t _ •' _ , . . ' ? IZOLATA LAKTOBACILA REŽISTENTMIH ' | - ' A ' i N A A N T ÏB ÏO T IK E , ' | JELENA BEGOVIĆ DOKTORSKA DISERTACIJA _ _-s ' ' %■ -V. r ‘ BEOGRAD, 2008 X A UN IV ERZ ITE T U BEO. lA D U BIOLOŠKIFAKULTET MOLEKÜL ARN A K A RA K TERim CïJA KUMANIH IZOLATA LAKTOBACILA REZISTENTNÎH NA. ANTÏMO TIKE JELENA BEGOVIĆ DOKTORSKA DISERTACUA BEOGRAD, 2008 MENTORI: dr Ljubiša Topisirović, redovni profesor Biološki fakultet, Beograd dr Ivana Strahinić, viši naučni saradnik Institut za molekulamu genetiku i genetičko inženjerstvo, Beograd ČLANOVIKOMISIJE: dr Ljubiša Topisirović, redovni profesor Biološki fakultet, Beograd dr Ivana Strahinić, viši naučni saradnik Institut za molekulamu genetiku i genetičko inženjerstvo, Beograd dr Đorđe Fixa, docent Biološki fakultet, Beograd dr Milan Kojić, naučni savetnik Institut za molekularnu genetiku i genetičko inženjerstvo, Beograd DATUM ODBRANE: DATUM PROMOCIJE: DOKTORAT NAUKA: Ovaj rad je realizovan u Laboratoriji za fnolekularnu genetiku industrijskih mikroorganizama, Instituta za molekularnu genetiku i genetičko inzenjerstvo. Ovom prilikom želim da se zahvalim: Prof, dr Ljubisi Topisiroviću na ukazanom poverenju, strpljenju i velikoj slobodi u toku mog rada, podrsci u mnogobrojnim idejama i lutanjima kao i brojnim korisnim diskusijama koje su doprinele efikasnoj izradi ove teze; dr Geert Huys-u iz Laboratorije za mikrobiologiju (Faculty of Sciences, Ghent University, Gent, Belgium), za veliku pomoć i sugestije u tumačenju dobijenih rezultata, kao i za povezivanje sa dr Mayo Baltasar-om (Instituto de Productos Lacteos de Asturias, Asturias, Spain) kome se zahvaljujem što je doprineo finalizaciji ove teze; dr Ivani Strahinić za pomoć i za izuzetno korisne sugestije tokom pisanja ove teze kao i kritičku ocenu rada; dr Milanu Kojiću za pomoć i mnogobrojne savete tokom eksperimentalne izrade ovog rada; dr Đorđu Firi za kriticku ocenu rada; dr Nataši Golić za pomoć tokom izrade ovog rada; dr Vesni Cvetković koja je obezbedila humane bakterijske izolate korišćene u ovoj tezi; Branku za razumevanje svih mojih nerazumnih ideja i teorija, i za sjneh koji je bio neophodan da se ovaj posao uspešno zavrsi. Jelki za unošenje reda u nas sistem, za iskrenost i opet naravno smeh i prijateljsku atmosferu koja vlada u ovoj laboratoriji. Amareli, Kaći, Maji, Milici pre svega na razumevanju, razgovorima i svakom lepom trenutku provedenom u ovoj laboratoriji. APSTRAKT Humani vaginalni izolati izolovani su iz 39 žena poreklom iz Srbije. Analizom profila traka dobijenih pomoću (GTG)5-PCR-a ovi izolati identifikovani su kao Lactobacillus rhamnosus (n=17), Lactobacillus fermentum (n=ll) i Lactobacillus plantarum (n=ll). Među izolovanim sojevima, 19 pokazuje antimikrobnu aktivnost kada se kao indikator koristi soj Salmonella enterididas, dok svi deluju antimikrobno kada se kao indikator sojevi koriste Lactococcus lactis subsp. cremoris NS1 i Lb. paracasei subsp. paracasei BGBUK2-16. Takođe za 11 izolata pokazano je da sintetiše H2O2. Rezultati određivanja minimalnih inhibitomih koncentracija (MIK) za šest antibiotika (ampicilin, eritromicin, tetraciklin, streptomicin, gentamicin i vankomicin) otkrili su različite stepene fenotipske rezistencije medu sojevima. Takode, rezultati ovih testiranja pokazuju zavisnost od medijuma koji su korišćeni za testiranje (MRS i LSM). Medutim, u sojevima rezistentnim na eritromicin i tetraciklin PCR-om nije utvrdeno prisustvo ermA, ermB, ermC, tetK i tetL gena kao ni tet gena iz familije proteina za protekciju ribozoma (RPP). Dalja istraživanja obuhvatila su PCR- RFLP analizu i sekvenciranje gena za 23 S rRNK poreklom iz pet Lb. rhamnosus sojeva koji su pokazivali visok stepen rezistencije na eritromicin (>256 pg/pl). Za sojeve BGHVl’, BGHV20, BGHV29, BGHV389, i BGHV719 pokazano je prisustvo tranzicione A-^G mutacije na poziciji 2058 u okviru V petlje 23S rDNA. Stepen heterozigotnosti mutacije medu ovim sojevima se razlikovao. Na osnovu rezultata “Southern blot” hibridizacije soj Lb. rhamnosus BGHV719 poseduje najmanje šest kopija gena za 23S rRNK. Promenom sastava MRS medijuma kao i dodavanjem različitih šećera u modifikovani MRS medijum dolazi do značajnih promena nekih osobina Lb. rhamnosus sojeva. Prvo, promene u MRS medijumu utiču na hidrofobnost površine ćelija sojeva Lb. rhamnosus BGHV719 i Lb. rhamnosus BGHV954 kao i na rezultate testiranja rezistencije na eritromicin i gentamicin.. Drugo, u soju BGHV719 dolazi i do promena u obliku ćelija, u formiranju agregata kao i u sastavu proteina ćelijskog zida Na kraju, promenom sastava MRS medijuma kao i dodavanjem različitih šećera u modifikovani MRS medijum, dolazi i do promene u mukoidnosti kolonija proizvođača egzopolisaharida- soja Lb. rhamnosus BGHV954.

^ ' : ' rj M O L E K U L A R N A K A R A K T E R IZ A C IJA H U M A N ÏH - ' ^ ' - : - t _ •' _ , . . ' ? IZOLATA LAKTOBACILA REŽISTENTMIH ' | - ' A ' i N A A N T ÏB ÏO T IK E , ' | JELENA BEGOVIĆ DOKTORSKA DISERTACIJA _ _-s ' ' %■ -V. r ‘ BEOGRAD, 2008 X A UN IV ERZ ITE T U BEO. lA D U BIOLOŠKIFAKULTET MOLEKÜL ARN A K A RA K TERim CïJA KUMANIH IZOLATA LAKTOBACILA REZISTENTNÎH NA. ANTÏMO TIKE JELENA BEGOVIĆ DOKTORSKA DISERTACUA BEOGRAD, 2008 MENTORI: dr Ljubiša Topisirović, redovni profesor Biološki fakultet, Beograd dr Ivana Strahinić, viši naučni saradnik Institut za molekulamu genetiku i genetičko inženjerstvo, Beograd ČLANOVIKOMISIJE: dr Ljubiša Topisirović, redovni profesor Biološki fakultet, Beograd dr Ivana Strahinić, viši naučni saradnik Institut za molekulamu genetiku i genetičko inženjerstvo, Beograd dr Đorđe Fixa, docent Biološki fakultet, Beograd dr Milan Kojić, naučni savetnik Institut za molekularnu genetiku i genetičko inženjerstvo, Beograd DATUM ODBRANE: DATUM PROMOCIJE: DOKTORAT NAUKA: Ovaj rad je realizovan u Laboratoriji za fnolekularnu genetiku industrijskih mikroorganizama, Instituta za molekularnu genetiku i genetičko inzenjerstvo. Ovom prilikom želim da se zahvalim: Prof, dr Ljubisi Topisiroviću na ukazanom poverenju, strpljenju i velikoj slobodi u toku mog rada, podrsci u mnogobrojnim idejama i lutanjima kao i brojnim korisnim diskusijama koje su doprinele efikasnoj izradi ove teze; dr Geert Huys-u iz Laboratorije za mikrobiologiju (Faculty of Sciences, Ghent University, Gent, Belgium), za veliku pomoć i sugestije u tumačenju dobijenih rezultata, kao i za povezivanje sa dr Mayo Baltasar-om (Instituto de Productos Lacteos de Asturias, Asturias, Spain) kome se zahvaljujem što je doprineo finalizaciji ove teze; dr Ivani Strahinić za pomoć i za izuzetno korisne sugestije tokom pisanja ove teze kao i kritičku ocenu rada; dr Milanu Kojiću za pomoć i mnogobrojne savete tokom eksperimentalne izrade ovog rada; dr Đorđu Firi za kriticku ocenu rada; dr Nataši Golić za pomoć tokom izrade ovog rada; dr Vesni Cvetković koja je obezbedila humane bakterijske izolate korišćene u ovoj tezi; Branku za razumevanje svih mojih nerazumnih ideja i teorija, i za sjneh koji je bio neophodan da se ovaj posao uspešno zavrsi. Jelki za unošenje reda u nas sistem, za iskrenost i opet naravno smeh i prijateljsku atmosferu koja vlada u ovoj laboratoriji. Amareli, Kaći, Maji, Milici pre svega na razumevanju, razgovorima i svakom lepom trenutku provedenom u ovoj laboratoriji. APSTRAKT Humani vaginalni izolati izolovani su iz 39 žena poreklom iz Srbije. Analizom profila traka dobijenih pomoću (GTG)5-PCR-a ovi izolati identifikovani su kao Lactobacillus rhamnosus (n=17), Lactobacillus fermentum (n=ll) i Lactobacillus plantarum (n=ll). Među izolovanim sojevima, 19 pokazuje antimikrobnu aktivnost kada se kao indikator koristi soj Salmonella enterididas, dok svi deluju antimikrobno kada se kao indikator sojevi koriste Lactococcus lactis subsp. cremoris NS1 i Lb. paracasei subsp. paracasei BGBUK2-16. Takođe za 11 izolata pokazano je da sintetiše H2O2. Rezultati određivanja minimalnih inhibitomih koncentracija (MIK) za šest antibiotika (ampicilin, eritromicin, tetraciklin, streptomicin, gentamicin i vankomicin) otkrili su različite stepene fenotipske rezistencije medu sojevima. Takode, rezultati ovih testiranja pokazuju zavisnost od medijuma koji su korišćeni za testiranje (MRS i LSM). Medutim, u sojevima rezistentnim na eritromicin i tetraciklin PCR-om nije utvrdeno prisustvo ermA, ermB, ermC, tetK i tetL gena kao ni tet gena iz familije proteina za protekciju ribozoma (RPP). Dalja istraživanja obuhvatila su PCR- RFLP analizu i sekvenciranje gena za 23 S rRNK poreklom iz pet Lb. rhamnosus sojeva koji su pokazivali visok stepen rezistencije na eritromicin (>256 pg/pl). Za sojeve BGHVl’, BGHV20, BGHV29, BGHV389, i BGHV719 pokazano je prisustvo tranzicione A-^G mutacije na poziciji 2058 u okviru V petlje 23S rDNA. Stepen heterozigotnosti mutacije medu ovim sojevima se razlikovao. Na osnovu rezultata “Southern blot” hibridizacije soj Lb. rhamnosus BGHV719 poseduje najmanje šest kopija gena za 23S rRNK. Promenom sastava MRS medijuma kao i dodavanjem različitih šećera u modifikovani MRS medijum dolazi do značajnih promena nekih osobina Lb. rhamnosus sojeva. Prvo, promene u MRS medijumu utiču na hidrofobnost površine ćelija sojeva Lb. rhamnosus BGHV719 i Lb. rhamnosus BGHV954 kao i na rezultate testiranja rezistencije na eritromicin i gentamicin.. Drugo, u soju BGHV719 dolazi i do promena u obliku ćelija, u formiranju agregata kao i u sastavu proteina ćelijskog zida Na kraju, promenom sastava MRS medijuma kao i dodavanjem različitih šećera u modifikovani MRS medijum, dolazi i do promene u mukoidnosti kolonija proizvođača egzopolisaharida- soja Lb. rhamnosus BGHV954. Ključne reči: vaginalni laktobacili, (GTG)5-PCR, MIK, antibiotska rezistencija, mutacija A2058, površinske osobine ABSTRACT Human vaginal isolates were isolated from 39 Serbian women. According to analysis of (GTG)5-PCR fingerprints, the isolates were found to belong to Lactobacillus rhamnosus (n=17), Lactobacillus fermentum (n=ll) and Lactobacillus plantarum (n=ll). Among these isolates, 19 showed antimicrobial activity against Salmonella enterididas, while all human vaginal isolates inhibited growth of Lactococcus lactis subsp. cremoris NSi and Lb. paracasei subsp. paracasei BGBUK2-16. Among human vaginal isolates, 11 were found to produce H20 2.MIC results obtained for six antibiotics (ampicillin, erythromycin, tetracycline, streptomycine, gentamicin and vancomycin) revealed different levels of phenotypic resistance among strains. In addition, the results of susceptibility testing differed when two media, MRS and LSM were used. Erythromycin-resistant and tetracycline- resistant isolates were screened for the presence of ermA, ermB, ermC, tetK and tetL genes and tet genes of the ribosomal protection (RP) family, but no positive signal was obtained in PCR reactions with respective primers. Subsequent research was focused on PCR-RFLP and sequencing analysis of 23S rRNA gene from five highly erythromycin resistant (>256 pg/pl) Lb. rhamnosus strains. In strains BGHV1’, BGHV20, BGHV29, BGHV389 and BGHV719 the presence of A-^G transition mutation at the position 2058 in 23S rRNA gene was detected. Among the strains, different levels of mutation heterozigosity were encountered. Results of Southern blot hybridization revealed the presence of at least six 23S rRNA gene copies for Lb. rhamnosus BGHV719. The changes in MRS composition as well as the addition of different sugars to modified MRS significantly influence some features of Lb. rhamnosus strains. First, these changes reduce hydrophobicity of Lb. rhamnosus BGHV719 and Lb. rhamnosus BGHV954 and also influence their survival rate in the presence of erythromycin and gentamycin. In addition, for strain BGHV719, changes in cell shape, aggregate formation as well as differences in cell wall protein composition were observed. Additionally, MRS composition and sugar source in modified MRS induce changes in colony mucoidity of exopolysaccharide producer strain Lb. rhamnosus BGHV954.

Modelovanje proboja u gasovima Monte Karlo tehnikom

Modeling of a gas breakdown by Monte Carlo technique

Zbog velike zastupljenosti radiofrekvencijskih (RF) plazmi u industriji javila se potreba za definisanjem osnovnih fizičkih principa koji određuju proboj u RF električnim poljima. Uslovi, u vidu probojnog napona i pritiska, mogu se prikazati naponskom probojnom krivom, predstavljenom u V-p ravni. Oblik i položaj krive definisani su kako prirodom pozadinskog gasa tako i karakteristikama spoljašnjeg RF električnog izvora. Ova disertacija ima za cilj da nedvosmisleno definiše fizičke procese koji su odgovorni za proboj pri RF električnim poljima. Za to je korišćena Monte Karlo tehnika. Odabirom slučajnih brojeva, uz poznavanje preseka za određene procese, moguće je verno (čak se može tvrditi egzaktno) opisati kretanje roja naelektrisanih čestica između dve elektrode koje su pobuđene RF generatorom. Izgradnja modela je započeta posmatranjem kretanja elektrona u argonu pri frekvenciji od 13,56 MHz. Na primeru argona objašnjeni su osnovni mehanizmi proboja koji definišu oblik naponske probojne krive. Nadogradnja modela, kao verodostojne predstave RF proboja, uključuje efekte na površinama elektroda (refleksiju i emisiju sekundarnih elektrona), uticaj promene frekvencije i dvofrekvencijskih izvora, kao i teških čestica. Model je primenjen na različite gasove: helijum, kiseonik, sintetički vazduh i atmosfera Marsa.

The wide range of radiofrequency (RF) plasma applications in industry rely on research of fundamental physical processes responsible for the RF breakdown. Conditions, such as the breakdown voltage and pressure, can be presented by breakdown voltage curves in the V-p plane. The shape and coordinates of the curve are defined by the nature of the background gas and characteristics of the applied RF electric field. The goal of this dissertation is to define physical processes responsible for the RF breakdown. For that purpose a Monte Carlo computational code has been used. If one knows cross sections for different processes by employing a random number generator, the transport of electrons between two electrodes can be determined. The development of the RF breakdown model begun by observing the motion of electrons in argon at the field frequency of 13.56 MHz. Argon was used to explain the basic mechanisms that define the shape of the breakdown voltage curve. Further upgrade of the model involved inclusion of the surface effects on electrodes (reflection and secondary electron emission), the influence of frequency and dual frequency RF power supplies, as well as the influence of heavy particles on the breakdown. The model was applied to different gases: helium, oxygen, and synthetic air, as well as the atmosphere of Mars.

Epilitske zajednice silikatnih algi - sezonska dinamika i procena ekološkog statusa odabranih reka istočne i jugoistočne Srbije

Epilithic diatom communities - seasonal dynamics and ecological status assessment of selected rivers from the eastern and southeastern Serbia

Analizom zajednice silikatnih algi moņe se dobiti slika opńteg stanja, ekolońkog integriteta i kvaliteta vode odreĊenog vodenog ekosistema. U cilju implementacije Okvirne direktive o vodama, u Srbiji je doneta zakonska regulativa na osnovu koje je obavezna procena ekolońkog statusa povrńinskih voda na osnovu bentosne zajednice silikatnih algi. Predmet ove doktorske disertacije je floristiĉka studija epilitskih zajednica silikatnih algi, njihova sezonska dinamika i primena dijatomnih indeksa kao relevantnih parametara u proceni ekolońkog statusa odabranih reka istoĉne i jugoistoĉne Srbije. Algolońki i fiziĉko-hemijski uzorci su sakupljeni ńest puta u periodu od dvanaest meseci sa ńest lokaliteta duņ toka reke Vrle i reke Crnice, odnosno sa pet lokaliteta duņ toka reke Mlave i Radovanske reke. Pri izboru lokaliteta vodilo se raĉuna o poloņaju pastrmskog ribnjaka i njegovom uticaju kao potencijalnom izvoru zagaĊenja ovih reka. Tokom istraņivanog perioda identifikovano je ukupno 375 taksona epilitskih silikatnih algi u Vrli, Mlavi, Crnici i Radovanskoj reci. Pored ugroņenih taksona, identifikovani su i taksoni koji se smatraju retkim. Od ukupnog broja identifikovanih, 50 taksona je prvi put zabeleņeno u flori algi Srbije. Rod Decussata predstavlja novi rod za floru algi Srbije. CCA analiza uticaja ekolońkih parametara na distribuciju taksona epilitske zajednice silikatnih algi iz 4 istraņivane reke je pokazala da ribnjaci utiĉu na fiziĉko-hemijske osobine vode, a samim tim na sastav epilitske zajednice. Kao znaĉajni fiziĉko-hemijski faktori izdvojili su se NH4+, NH3 i EP. Na osnovu većine dijatomnih indeksa voda sve ĉetiri istraņivane reke je dobrog ekolońkog statusa (II klasa kvaliteta). Prema zakonskoj regulativi Srbije, na osnovu biolońkih (fitobentos) i fiziĉko-hemijskih elemenata, ekolońki status vode istraņivanog dela reke Vrle je dobar (II klasa), a reke Mlave umeren (III klasa). Konaĉan ekolońki status Crnice i Radovanske reke je procenjen kao slab (IV klasa)...

By analyzing the benthic diatom community, a picture of the general condition, ecological integrity and water quality of a particular aquatic ecosystem can be obtained. In order to implement the Water Framework Directive, a legislation has been adopted in the Republic of Serbia that requires an assessment of the ecological status of surface waters based on the benthic diatoms. The aim of this PhD thesis was the floristic analysis of the epilithic diatom communities, their seasonal dynamics and the application of diatom indices as relevant parameters in the ecological status assessment of the selected rivers from the Eastern and Southeastern Serbia. Algological and physico-chemical samples were collected six times over a period of twelve months from six sites along the Vrla and Crnica rivers, and from five sites along the Mlava and Radovanska rivers. In selecting the sites taken into account the position of a trout ponds and its impact as a potential sources of pollution of the rivers. A total of 375 epilithic diatom taxa in Vrla, Mlavi, Crnica and Radovanova rivers were identified during the investigated period. Some endangered and rare taxa were identified. Of the total number of identified taxa, 50 taxa were new records for the Serbian algal flora. The genus Decussata, with the rare species D. hexagona, was recorded for the first time in Serbia. The CCA analysis of the impact of ecological parameters on the distribution of the diatom taxa from the investigated rivers has shown that the fish ponds affect the water physico-chemical properties and, consequently, the composition of the epilithic diatom communities. NH4+, NH3 and EP have been identified as important physical-chemical factors...

Ekološka analiza zajednice makrozoobentosa reke Nišave

Ecological analysis of macrozoobenthos communities in the Nišava River

Doktorska disertacija obuhvata ekološku analizu zajednica makrozoobentosa reke Nišave i njihovu zavisnost od biotičkih i abiotičkih faktora. Istraživanja su obavljena mesečnom dinamikom na dvanaest lokaliteta od maja 2006. do aprila 2007. godine. U disertaciji se diskutuje zavisnost koncentracije hlorofila a i mase perifitona od abiotičkih faktora. Sa druge strane, razmatran je pritisak (na osnovu brojnosti) filtratornih vrsta zajednice makroinvertebrata na količinu fitoplanktona (izraženu preko hlorofila a) i pritisak grebača na masu perifitona. Dobijen je predikcioni model za izračunavanje koncentracije hlorofila a korišćenjem multiple linearne regresije. Model ima značajnu predikcionu moć (sig=0,003). Na osnovu Pirsonovog koeficijenta korelacije konstatovano je da postoji značajna povezanost koncentracije hlorofila a sa mutnoćom (p=0,000), sa ukupnim fosforom (p=0,029) i sa kombinovanom (zbirnom) koncentracijom ukupnog azota i fosfora (p=0,036). Nije utvrđena značajna povezanost sa brojnošću filtratornih organizama, što govori da njihov pritisak nije dovoljno jak da bi limitirao količinu fitoplanktona. Taksonomskom analizom makroinvertebrata reke Nišave utvrđeno je prisustvo 132 vrste iz 4 različita filuma. Filum Platyhelminthes je predstavljen sa 3 vrste iz 2 familije. Filum Annelida obuhvata 23 vrste iz 6 familija. Najveći broj vrsta ovog filuma pripada potklasi Oligochaeta (16 vrsta). Filum Mollusca obuhvata 13 vrsta iz 5 familija, od kojih 11 pripada klasi Gastropoda, a 2 klasi Bivalvia. Najveći broj vrsta faune makroinvertebrata pripada filumu Arthropoda (92 vrste iz 33 familije), od kojih 3 pripadaju subfilumu Crustacea, a sve ostale podfilumu Hexapoda, odnosno klasi Insecta. Rezultati taksonomske analize pojedinačnih grupa u okviru zajednice makroinvertebrata pokazuju da najveći broj vrsta pripada grupi Ephemeroptera (28 vrsta), zatim slede Diptera (26 vrsta) i Trichoptera (20 vrsta). Ostale grupe su zastupljene sa manjim brojem vrsta. Diskutovana je sezonska i longitudinalna dinamika grupa makroinvertebrata i njihova uslovljenost faktorima sredine. Razmatrani su indeksi diverziteta i adekvatnost njihovog korišćenja kao pokazatelja zagađenosti ekosistema, posebno za svaku grupu...

This doctoral dissertation comprises the ecological analysis of macrozoobenthos communities in the Nišava River and their dependence on biotic and abiotic factors. The studies followed the monthly dynamics at 12 sites, from May 2006 to April 2007. The dissertation is discussing the dependence of chlorophyll a concentration and periphyton mass on abiotic factors, as well as the pressure (based on abundance) of filtrating species in the macroinvertebrate community on the amount of phytoplankton (expressed as chlorophyll a) and pressure of scraper species on periphyton mass. The prediction model for calculating chlorophyll a concentration was derived from the multiple linear regression. This model has a significant prediction power (sig=0.003). The Pearson's correlation coefficient was used to determine the correlation of chlorophyll a concentration with water turbidity (p=0.000), total phosphorus (p=0.029) and combined (summative) concentration of total nitrogen and total phosphorus (p=0.036). No significant connection with the abundance of filtrating organisms was determined, which indicates that their pressure was not strong enough to limit the quantity of phytoplankton. The taxonomic analysis of macroinvertebrates in the Nišava River yielded the presence of 132 species from 4 different phyla. The phylum Platyhelminthes was represented with 3 species from 2 families. The phylum Annelida was represented with 23 species from 6 families. Most recorded species from this phylum belong to the subclass Oligochaeta (16 species). The phylum Mollusca was represented with 13 species from 5 families, of which 11 were from the class Gastropoda and 2 from the class Bivalvia. Most species of the macroinvertebrate fauna belong to the phylum Arthropoda (92 species from 33 families), of which 3 were from the subphylum Crustacea, while all others belong to the subphylum Hexapoda and class Insecta. The results of the taxonomic analysis of individual groups within the macroinvertebrate community have shown that most species belong to the group Ephemeroptera (28 species), followed by Diptera (26 species), and Trichoptera (20 species). Other groups were represented with a smaller number of species. The study also includes the discussion on seasonal and longitudinal dynamics of macroinvertebrate groups and the effect of environmental factors on their presence. The diversity indices and their suitability as indicators of ecosystem pollution were discussed for each group separately...

Značaj razlika nivoa imunoglobulina specifičnih za melanin i tirozinazu u antitumorskoj imunosti bolesnika sa melanomom

The importance of the differences in immunoglobulin levels specific for melanin and tyrosinase in antitumor immunity in patients with melanoma

Melanom je visoko imunogeno maligno oboljenje. Melanomski antigeni imaju sposobnost imunizacije i stimulacije sinteze specifičnih antitela. Antitela specifična za iste, melanomske antigene, koji su prisutni kako na neoplastično transformisanim, tako i na neizmenjenim melanocitima, pronađeni su i kod bolesnika sa vitiligom. Pojava hipopigmentacija sličnih vitiligu kod bolesnika sa melanomom utiče na bolju prognozu preživljavanja, što ukazuje na značaj veze između tumorske imunosti i autoimunosti kod ovih bolesnika. Ćelije melanoma karakteriše povećano prisustvo melanina i tirozinaze, a u serumu bolesnika sa metastatskim melanomom uočena je tirozinazna aktivnost. Dipeptidil peptidaza IV (DPPIV/CD26) je transmembranski glikoprotein koji se eksprimira na površini limfocita, i predstavlja važan marker aktivacije ćelija imunskog sistema. Upravo su limfociti jedan od najznačajnijih izvora solubilne forme ovog proteina u serumu. DPPIV ima značajnu ulogu u regulaciji imunskih funkcija i procesu neoplastične transformacije. Osnovni cilj ovog istraživanja je bio da se ispitaju razlike u nivoima IgG, IgA i IgM klasa antitela specifičnih za melanin i tirozinazu u bolesnika sa melanomom u odnosu na bolesnike sa vitiligom i zdrave osobe. Od značaja je bilo i da se ispita postojanje razlika u humoralnom imunskom odgovoru između bolesnika sa melanomom bez metastaza i bolesnika sa melanomom i metastazama. S ciljem dodatne karakterizacije imunskog odgovora, određivan je procenat CD16+ i CD16+CD56+ limfocita, i CD89+ granulocita kod bolesnika sa melanomom, kao i bolesnika sa vitiligom i zdravih osoba, a zatim je ispitivana povezanost nivoa antitela i CD16/CD89+ ćelija u grupi bolesnika sa melanomom. Kako bi se ispitala uloga DPPIV u patogenezi melanoma i mogući klinički značaj kao dijagnostičkog markera, od važnosti je bilo da se odredi enzimska aktivnost dipeptidil peptidaze IV u serumu, procenat CD26+ limfocita i ekspresija CD26 antigena na limfocitima kod bolesnika sa melanomom, bolesnika sa vitiligom i zdravih osoba. Rezultati istraživanja su pokazali da bolesnici sa melanomom bez metastaza imaju statistički značajno viši nivo anti-melaninskih IgG antitela u odnosu na bolesnike sa vitiligom i zdrave osobe, i što je još bitnije u odnosu na bolesnike sa melanomom i metastazama. Kod bolesnika sa melanomom sa i bez metastaza, i bolesnika sa vitiligom pronađen je niži nivo anti-tirozinaznih IgG antitela u poređenju sa nivoom ovih antitela kod zdravih osoba. Nizak nivo anti-tirozinaznih IgG antitela kod bolesnika sa melanomom u skladu je sa smanjenom IgG posredovanom citotoksičnošću, kao i sa dobijenim nižim procentom CD16+ limfocita i CD16+CD56+ limfocita, kod ovih bolesnika u odnosu na zdrave osobe i bolesnike sa vitiligom. Nivoi anti-melaninskih i anti-tirozinaznih IgM antitela su bili statistički značajno niži u bolesnika sa melanomom u odnosu na bolesnike sa vitiligom i zdrave osobe. Većina bolesnika sa melanomom koji su imali niske nivoe anti-tirozinaznih i anti- melaninskih IgM antitela imali su metastaze. Ovaj podatak ističe značaj anti- melanomskih IgM antitela u kontroli melanoma i destrukciji melanocita. Nivoi anti-melaninskih i anti-tirozinaznih IgA antitela bili su viši u bolesnika sa melanomom u odnosu na bolesnike sa vitiligom i zdrave osobe. Takođe, bolesnici sa melanomom imali su i viši procenat CD89+ granulocita i povećan procenat ukupnih granulocita. Postojanje pojačanog imunskog odgovora posredovanog IgA klasom antitela u bolesnika sa melanomom ukazuje na moguću imunosupresivnu, odnosno blokirajuću ulogu anti-melanomskih antitela IgA klase. Određivanje ekspresije CD26 antigena na površini limfocita, kao i enzimske aktivnosti DPPIV, pokazali su da bolesnici sa melanomom imaju statistički značajno niži procenat CD26+ limfocita u populaciji ukupnih leukocita, kao i statistički značajno nižu aktivnost DPPIV u serumu u poređenju sa zdravim osobama i bolesnicima sa vitiligom. Statistički značajno smanjen procenat ukupnih limfocita kod bolesnika sa melanomom predstavlja još jednu potvrdu slabijeg imunskog odgovora ove grupe bolesnika. Izmenjeni nivoi antitela IgG, IgA i IgM klase specifičnih za melanomske antigene, melanin i tirozinazu, kod bolesnika sa melanomom u poređenju sa zdravim osobama ukazuju na značaj specifičnog imunskog odgovora u patogenezi melanoma. Od posebne važnosti je podatak da nivoi anti-melaninskih IgG, IgA i IgM antitela, i anti-tirozinaznih IgA i IgM antitela imaju statistički značajnu moć diskriminacije između bolesnika sa melanomom i zdravih osoba. Statistički značajna moć diskriminacije između bolesnika sa melanomom i zdravih osoba koju imaju nivo enzimske aktivnosti DPPIV u serumu, i procenat CD26+ limfocita u populaciji ukupnih leukocita, potvrđuje vezu DPPIV i imunskog sistema i ukazuje na moguću značajnu ulogu DPPIV u mehanizmima patogeneze melanoma.

Melanoma is a highly immunogenic malignancy. Melanoma antigens are capable of immunization and stimulation of the synthesis of specific antibodies. Antibodies specific for same, melanoma antigens, which are present both on the neoplastically transformed, as well as on the non-transformed melanocytes, were also found in patients with vitiligo. The presence of vitiligo-like hypopigmentations in patients with melanoma is associated with a better survival prognosis, indicating the importance of the link between tumor immunity and autoimmunity. The increased presence of melanin and tyrosinase is characteristic of melanoma cells, while the tyrosinase activity was observed in the sera of patients with metastatic melanoma. Dipeptidyl peptidase IV (DPPIV/CD26) is a transmembrane glycoprotein which is expressed on the surface of lymphocytes, and represents an important marker of activation of immune system cells. Lymphocytes represent one of the major sources of soluble form of this protein in the serum. DPPIV is implicated in the regulation of immune functions and neoplastic transformation. The main goal of this study was to examine the differences in the levels of IgG, IgA and IgM classes of antibodies specific for melanin and tyrosinase in patients with melanoma, compared with the group of patients with vitiligo and the group of healthy individuals. The examination of the presence of differences in the humoral immune response between patients with melanoma without metastases and patients with melanoma and metastases was also significant for our research. To further characterize the immune response, it was important to determine the percentage of CD16+ and CD16+CD56+ lymphocytes and CD89+ granulocytes in patients with melanoma, as well as patients with vitiligo and healthy persons, and also to investigate the connection between the levels of antibodies and CD16/CD89+ cells in the group of patients with melanoma. In order to evaluate the role of DPPIV in the pathogenesis of melanoma and its possible clinical significance as a diagnostic marker, we determined the enzymatic activity of dipeptidyl peptidase IV in the serum, the percentage of CD26+ lymphocytes and the expression of CD26 antigen on the lymphocytes in patients with melanoma, patients with vitiligo and healthy persons. The results of our research showed that patients with melanoma without metastases had significantly higher levels of anti-melanin IgG antibodies than patients with vitiligo and healthy persons, and what is even more important they had significantly higher levels of anti-melanin IgG antibodies than patients with melanoma and metastases. In melanoma patients who had metastases and patients without metastases, and patients with vitiligo, lower levels of anti-tyrosinase IgG antibodies were found compared with the levels of these antibodies in healthy people. Low levels of anti-tyrosinase IgG antibodies in patients with melanoma is in accordance with reduced IgG-mediated cytotoxicity, as well as obtained lower percentage of CD16+ lymphocytes and CD16+CD56+ lymphocytes in these patients than in healthy persons and patients with vitiligo. The levels of IgM antibodies specific for both melanin and tyrosinase were significantly lower in patients with melanoma than in patients with vitiligo and in healthy people. Most of the patients with melanoma who had low levels of anti- melanin and anti-tyrosinase IgM antibodies belong to the group of patients with metastases. These data highlights the importance of anti-melanoma IgM antibodies in the control of melanoma and destruction of melanocytes. The levels of anti-melanin and anti-tyrosinase IgA antibodies were higher in patients with melanoma in comparison to patients with vitiligo and healthy control persons. Also, patients with melanoma had a higher percentage of CD89+ granulocytes and an increased percentage of total granulocytes. The existence of increased IgA antibodies-mediated immune response in melanoma patients suggest the immunosuppressive, or the blocking role of anti-melanoma antibodies of IgA class. Determination of the expression levels of CD26 on the surface of lymphocytes and the enzymatic activity of DPPIV in the sera of patients with melanoma, showed that the group of patients with melanoma had a significantly lower percentage of CD26+ lymphocytes in population of overall leukocytes, in addition to significantly lower serum DPPIV enzymatic activity when compared to healthy persons and patients with vitiligo. A statistically significant reduction in the percentage of lymphocytes in patients with melanoma represents another confirmation of weaker immune response in this group of patients. Altered levels of IgG, IgA and IgM classes of antibodies specific for melanoma antigens, melanin and tyrosinase, in melanoma patients when compared to healthy individuals indicate the importance of the specific immune response in the pathogenesis of melanoma. The finding that the levels of anti-melanin IgG, IgA and IgM antibodies, and anti-tyrosinase IgA and IgM antibodies possess a significant power of discrimination between melanoma patients and healthy persons is of crucial importance. The significant power of discrimination between melanoma patients and healthy individuals which have DPPIV serum enzymatic activity and the percentage of CD26+ lymphocytes in the population of overall leukocytes confirms the relationship between DPPIV and the immune system and suggest the possible important role of DPPIV in the mechanisms of melanoma pathogenesis.

Ispitivanje senzitivnosti genoma kod pacijenata sa bolestima hromozomske nestabilnosti .

Genome sensitivity in patients with chromosomal instability disorders

U cilju ispitivanja senzitivnosti genoma i potvrde klinički postavljene dijagnoze bolesti hromozomske nestabilnosti, korišćeni su uzorci dve grupe pacijenata sa kliničkom slikom: Fankonijeve anemije (FA) i Nijmegenovog sindroma nestabilnosti (NSN) (eng. Nijmegen Breakage syndrome, NBS). Specifična hipersenzitivnost ćelija obolelih od FA na diepoksibutan (DEB), odnosno kod obolelih od NSN na bleomicin (BLC), iskorišćena je u diferencijalno dijagnostičke svrhe. Studija je obuhvatila ukupno 100 pacijenata: 90/100 sa kliničkom slikom FA i 10/100 pacijenata sa kliničkim znacima NSN, koji su dijagnostikovani na Institutu za zdravstvenu zaštitu majke i deteta Srbije „Dr Vukan Čupić“. U detekciji DEB-om indukovane hromozomske nestabilnosti obolelih od FA primenjene su standardne citogenetičke metode. Za potvrdu ili isključenje mozaičnog fenotipa FA korišćene su dodatne metode: analiza oštećenja DNK komet testom i protočno citometrijska analiza zastoja ćelija u G2 fazi ćelijskog ciklusa. Hromozomska osetljivost ćelija obolelih od NSN je analizirana primenom bleomicinovog testa (BLC test). Molekularna analiza prisustva ili odsustva c.657_661del5 mutacije u NBN genu obolelih od NSN, urađena je korišćenjem modifikovane PCR metode uz analizu heterodupleksa. Kod 11,11% (10/90) pacijenata pod sumnjom da boluju od FA (FA grupa), otkrivena je povećana hromozomska osetljivost na DEB u odnosu na kontrolnu grupu (Mann-Whitneyev test: p < 0,05), dok su kod 3,33% (3/90) pacijenata dobijene granične vrednosti iste analize (FA* grupa). U cilju preciznijeg klasifikovanja pacijenata u FA grupi, primenjen je indeks hromozomske fragilnosti. Na ovaj način je u FA grupi, kod 40% (4/10) pacijenata otkriven ćelijski fenotip FA nemozaičnog tipa, dok je kod 60% (6/10) pacijenata dobijen mozaični odgovor na DEB. Kod svih pacijenata sa mozaičnim fenotipom FA, kao i kod dva pacijenta sa graničnim vrednostima diepoksibutanovog testa (DEB testa), daljim analizama primenom komet testa i analizom zastoja ćelija u G2 fazi, potvrđena je dijagnoza FA. U drugoj grupi od 10 bolesnika sa sumnjom da boluju od NSN analizirana je BLC-om indukovana hromozomska nestabilnost, koja je bila povećana kod 40% (4/10) pacijenata (NBS grupa). Kod 30% (3/10) pacijenata dobijeni rezultati su se preklapali sa rezultatima kontrolne grupe (ne-NBS grupa), dok je kod preostala tri pacijenta analiza bila neuspešna zbog slabog mitotskog indeksa. Molekularnim analizama je otkriveno homozigotno prisustvo c.657_661del5 mutacije u NBN genu kod sva četiri pacijenta iz NBS grupe, kao i kod tri pacijenta kod kojih je citogenetička analiza bila neuspešna, i tako potvrđena konačna dijagnoza NSN. Na osnovu dobijenih rezultata može se zaključiti da su analize korišćene u ovoj studiji omogućile potvrdu klinički postavljene dijagnoze FA i NSN, što opravdava njihovu dalju primenu u diferencijalnoj dijagnostici ovih oboljenja. .

In order to determine the sensitivity of the genome and to confirm a clinical diagnosis of chromosomal instability disorders, blood samples from patients with clinical suspicion of Fanconi’s anemia (FA) and Nijmegen Breakage syndrome (NBS), were collected for chromosome fragility evaluation by the diepoxybutane (DEB) and bleomycin (BLC) tests. The study considered a total of 100 patients: 90/100 with the hematological and/or congenital phenotypic symptoms reminiscent of FA and 10/100 patients with clinical features of NBS, all diagnosed at the Mother and Child Health Care Institute of Serbia "Dr Vukan Čupić”. The DEB-induced chromosomal fragility analysis in patients with FA symptoms was carried out by using standard cytogenetic methods. The additional methods, such as: DNA damage analysis by comet assay and flow cytometric analysis of G2 phase cell cycle arrest, were used in order to confirm FA phenotype in patients with mosaic cytogenetic response to DEB. Chromosomal sensitivity of cells in patients with NBS symptoms was analyzed using a BLC test. Molecular analysis for the presence of the c.657_661del5 mutation in NBN gene in patients with NBS was carried out using modified PCR method and heteroduplex analysis on PAGE gel. In this study 11.11% (10/90) of patients with clinical suspicion of FA were found to have an increased chromosomal sensitivity to DEB (FA group), comparing to healthy controls (Mann-Whitney test: p < 0.05), while 3.33% (3/90) of patients showed borderline results of the same analysis (FA* group). The chromosome fragility index was used in order to provide a clear cut-off diagnostic level distinguishing FA mosaic from other patients in the FA group. In this group, 40% (4/10) of FA patients revealed non-mosaic FA phenotype, while the remaining 60% (6/10) of FA patients showed a mosaic response to DEB. In all of FA mosaic patients, and two patients with borderline DEB-sensitivity, further analyses were performed using comet assay and analysis of G2 phase cell cycle arrest, in order to confirm the diagnosis of FA. In the second group of 10 patients with clinical suspicion of NBS, the cytogenetic analysis revealed an increased chromosomal sensitivity to BLC in 40% (4/10) of them (NBS group). The BLC-induced chromosome fragility values in 30% (3/10) of these patients were overlapping those in the control group (non-NBS group). In the remaining of three patients the cytogenetic analysis was unsuccessful, due to the low mitotic index. Molecular analysis confirmed the presence of homozygous c.657_661del5 mutation in all seven NBS patients, including three patients with unsuccessful cytogenetic analysis, which confirmed the final diagnosis of NBS. Based on these results it can be concluded that the analyses used in this study are useful in confirmation of clinical diagnosis of FA and NBS, which justifies their further application in the differential diagnosis of these two diseases. .

Molekulski mehanizmi strukturnog remodeliranja mrkog masnog tkiva pacova indukovanog insulinom

Molecular mechanisms of insulin-induced rat brown adipose tissue structural remodelling

U svetlu sve većeg broja dokaza koji ukazuju na prisustvo metabolički aktivnog mrkog masnog tkiva (BAT – brown adipose tissue, engl.) kod odraslih ljudi, kao i na njegovu potencijalnu ulogu u sprečavanju razvoja gojaznosti, insulinske rezistence i metaboličkog sindroma uopšte, sve je više studija o rasvetljavanju metaboličkog značaja ovog tkiva, kao i njegovoj termogenoj stimulaciji u svrhu terapije navedenih poremećaja. Insulin se, kao važan anabolički hormon, smatra značajnim modulatorom strukturne organizacije i funkcije BAT. Cilj ovog istraživanja bio je rasvetljavanje molekulskih osnova insulinom- indukovanog strukturnog remodeliranja BAT putem identifikacije mehanizama koji regulišu insulinom-stimulisanu proliferaciju i diferencijaciju ćelija, kao i identifikacije mehanizama koji učestvuju u oštećenjima ćelija i njihovom umiranju. Pacovi soja Wistar su jednom dnevno tretirani fiziološkom (0.4 IU/kg telesne mase) i suprafiziološkom (4 IU/kg telesne mase) dozom insulina, jedan (akutan tretman) ili tri (hroničan tretman) dana. Kao fiziološka kontrola, dve grupe pacova su u istom trajanju tretirane fiziološkim rastvorom (akutna i hronična kontrola). Tri sata nakon poslednje doze, životinje su žrtvovane, a interskapularni depo BAT je izolovan, izmeren i pripremljen za tehnike svetlosne i elektronske mikroskopije. Analiza tkiva izvršena je korišćenjem metoda histohemijskog i imunohistohemijskog bojenja, metoda konfokalne i transmisione elektronske mikroskopije i elektron-disperzivne analize X-zracima (EDX) hemijskog sastava tkiva kao i metoda stereološke i morfometrijske analize. Rezultati su pokazali postojanje strukturnog i funkcijskog remodeliranja BAT, gde dominiraju procesi hipertrofije i hiperplazije ćelija, praćene povećanjem termogenog kapaciteta mrkih adipocita (raste ekspresija dekuplujućeg proteina 1 (UCP1 – uncoupling protein 1, engl.), udeo mitohondrija, procesi mitohondriogeneze). S druge strane, primetno je da se, naročito pri hroničnim i tretmanima visokom dozom insulina, javljaju citotoksični i inhibitorni efekti na pojedine ćelije ili klastere ćelija: raste učestalost ćelijske smrti, ekstravazacija eritrocita i njihovo uklanjanje od strane mrkih adipocita i makrofaga, (evidentno toksično po adipocite sudeći po rastu: ekspresije enzima antioksidativne odbrane; lipidne peroksidacije i nitrozilacije proteina, učestalosti lipofuscinogeneze). Takođe se pri hronično visokoj hiperinsulinemiji smanjuje ekspresija regulatora termogeneze (PGC-1α - peroxisome proliferator-activated receptor – γ coactivator - 1α, engl.) ukazujući da produženo izlaganje visokim dozama insulina, može da inhibira termogeni odgovor tkiva, o čemu svedoči i povećana učestalost mitohondrijalnih oštećenja. Povećana ekspresija proinflamatornih citokina (TNF-α - tumor-necrosis factor – α, engl., interleukina 6), nukleusnog faktora – kB (NF-kB) i makrofagnog inflamatornog proteina - 3β (MIP-3β) u pojedinačnim zrelim, multilokulusnim adipocitima, preadipocitima i unilokulusnim adipocitima ukazuje na njihovu potencijalnu ulogu u lokalnom smanjenju insulinske senzitivnosti. U vezi sa hiperplazijom ćelija, primećeno je da su adipogeneza i angiogeneza u BAT vremenski i prostorno povezane, tj. da postoji udruženost navedenih procesa, koja ukazuje na potrebu udruženog nastanka adipocita i endotelskih ćelija, u svrhu efikasnog strukturno-funkcijskog remodeliranja tkiva. Sumarno, može se zaključiti da hiperinsulinemija ima značajan efekat na BAT – potvrđeno je anaboličko dejstvo insulina i važnost njegove uloge u stimulaciji BAT, ali je i pokazana toksičnost visoke doze i hroničnog izlaganja na ćelije BAT; što je sve doprinelo rasvetljavanju molekulskih mehanizama u osnovi strukturnog remodeliranja ovog tkiva. Takođe, pokazano je da se visoka plastičnost BAT ogleda u postojanju funkcionalnih klastera ćelija, u kojima se odvijaju svi važni tkivni procesi (adipo/angiogeneza, ćelijska smrt, eritrofagocitoza), što ukazuje na pravilnu uređenost tkivne organizacije i strukturnog remodeliranja, kao preduslova za pravilno funkcionisanje tkiva, kako u fiziološkim, tako i u uslovima izmenjene tkivne homeostaze, što je pokazano na modelu hiperinsulinemije.

There is an increasing amount of evidence that indicates presence of brown adipose tissue (BAT) in adult humans, and its potential role in prevention of obesity, insulin resistance and metabolic syndrom, in general. That is why number of studies on elucidation of the metabolic importance of this tissue, along with its thermogenic stimulation in order to treat these disorders, increases. As an important anabolic hormone, insulin is considered as a major modulator of BAT structure and function. The aim of this study was to elucidate the molecular basis of insulin-induced structural remodeling of BAT through the identification of mechanisms that regulate insulin-stimulated proliferation and differentiation and through the identification of mechanisms involved in cell damage and death. Wistar strain rats were treated intraperitoneally with physiological (0.4 IU/kg BW) or supraphysiological (4 IU/kg BW ) dose of insulin for one (acute treatment) or three days (chronic treatment), respectively. Two groups of rats served as physiological controls, and were treated with saline (acute and chronic control). Three hours after the last injection the animals were sacrificed and the interscapular portion of BAT was removed, its weight was measured and it was processed for electron and light microscopic examinations. Methods of histochemical staining, immunohistochemical labeling, confocal and transmission electron microscopy, tissue electron-dispersive X-ray microanalysis (EDX) and stereological and morphometric analyses were performed. The results showed the existence of structural and functional remodeling of BAT, based on adipocyte hypertrophy and hyperplasia which are followed by increasing of thermogenic capacity (expression of uncoupling protein 1 (UCP1), cellular proportion of mitochondria, mitochondrial biogenesis). On the other hand, it is obvious that, especially after chronic and high-dose treatments, insulin cytotoxic and inhibitory effects on individual cells or cell clusters occur: incidence of cell death increases, as well as red blood cell extravasation and their removal by brown adipocytes and macrophages (which is evidently toxic for the adipocyte, since expression of antioxidative defense enzymes, lipid peroxidation, protein nitrosilation and lipofuscin formation were increased). Also, during chronically high hyperinsulinemia, expression of peroxisome proliferator-activated receptor - γ coactivator - 1α (PGC-1α), regulator of thermogenesis decreases, indicating that prolonged exposure to high doses of insulin can inhibit thermogenic response of BAT, as evidenced by the increased frequency of mitochondrial damage in brown adipocytes. Increased expression of proinflammatory cytokines (tumor- necrosis factor - α (TNF-α), interleukin 6), nuclear factor – kB (NF-kB) and macrophage inflammatory protein - 3β (MIP-3β) in individual mature, multilocular adipocytes, preadipocytes and unilocular adipocytes, demonstrates their potential role in local decrease of insulin sensitivity. Regarding hyperplasia of cells, it was noticed spaciotemporal association between processes of adipogenesis and angiogenesis, which means that there is a connection between these processes, demonstrating the need for the coupled differentiation of adipocytes and endothelial cells, for the purpose of efficient structural and functional remodeling of the tissue. In summary, it can be concluded that hyperinsulinemia has a significant impact on BAT – we confirmed the anabolic effect of insulin, and the importance of its role in BAT stimulation, but also demonstrated the toxicity of high dose and chronic exposure to insulin on BAT cells, all of which contribute to understanding the molecular mechanisms underlying structural remodeling of the tissue. In addition, it was shown that the high plasticity of BAT is reflected in the existence of functional clusters of cells, which take place in all the important processes in the tissue (adipo/angiogenesis, cell death, erythrophagocytosis), indicating the proper arrangement of tissue organization and structural remodeling as a prerequisite for the proper functioning of the tissue under physiological conditions and during altered tissue homeostasis, as shown on the model of hyperinsulinemia.

Micromorphological and cytological analysis of trichomes and biological effects of extracts of Salvia aegyptiaca L., S. fruticosa Mill. and S. lanigera Poir. (Lamiaceae) from Libya

Mikromorfološka i citološka analiza trihoma i biološki efekti ekstrakata Salvia aegyptiaca L., S. fruticosa Mill. i S. lanigera Poir. (Lamiaceae) iz Libije

Tri vrste žalfije iz Libije su podvrgnute sveobuhvatnoj analizi mikroskopskih karakteristika lisnih trihoma (dlačica) i bioloških aktivnosti njihovih ekstrakata, s obzirom da nisu ranije proučavane. Mikromorfologija, ultrastruktura i histohemija lisnih trihoma S. aegyptiaca, S. fruticosa i S. lanigera je proučavana svetlosnom, skenirajućom i transmisionom elektronskom mikroskopijom. Listovi na licu i naličju nose brojne negranate neglandularne trihome i dva tipa glandularnih trihoma – peltatne i kapitatne. S obzirom na korišćenje vrsta žalfije u lekovite i kozmetičke svrhe, zahvaljujući etarskim uljima koja se sintetišu u glandularnim trihomama, u fokusu istraživanja je bila analiza glandularnih trihoma. Na listovima S. aegyptiaca peltatne trihome su sastavljene od bazalne ćelije, kratke cilindrične ćelije vrata i široke glave od 8 do 12 ćelija u jednom krugu, ili više njih u dva kruga. Kapitatne trihome se sastoje od jednoćelijske žlezdane glavice na dršci sastavljenoj od varijabilnog broja ćelija, klasifikovane u dva tipa: kapitatne trihome tipa I ili kratke kapitatne trihome i kapitatne trihome tipa II ili duge kapitatne trihome. Histohemijski testovi su pokazali da je sekretovani materijal u svim tipovima trihoma kompleksne prirode. Pozitivne reakcije na lipide su zapažene u svim tipovima trihoma, sa obilnom sekrecijom u peltatnim i kapitatnim trihomama tipa II. Kod S. fruticosa peltatne trihome se sastoje od bazalne ćelije, vrlo kratke drške i krupne okrugle glave od osam ćelija kružno postavljenih. Kapitatne trihome se mogu podeliti u dve grupe, sa kratkim i sa dugim vratom, a zatim u pet podtipova, prema broju ćelija u vratu, morfologiji i broju ćelija u glavici. Digitiformne trihome se sastoje od jedne bazalne ćelije, jedne do dve ćelije drške i jedne apikalne sekretorne ćelije sličnog dijametra i dužine. Brojne i raznovrsne glandularne trihome i duge drške kapitatnih trihoma ukazuju na kseromorfnu prirodu ove vrste koja je otporna na sušu. S. lanigera ima peltatne trihome sastavljene od jedne bazalne ćelije, kratke cilindrične ćelije vrata sa kutiniziranim zidovima i krupne okrugle ili malo ravnije sekretorne glave od osam ćelija u krugu. Kapitatne trihome se sastoje od jednoćelijske žlezdane glavice i jednoćelijske drške, a određene su kao kratke kapitatne trihome. Može se zaključiti da su ispitivane libijske vrste Salvia pokazale razlike u tipovima, gustini i rasporedu trihoma, sa najvećom brojnošću i razlikama žlezdanih trihoma kod S. fruticosa. S obzirom na rastuće interesovanje za supstance prirodnog porekla koje pokazuju različite biološke aktivnosti, kao i porast zabrinutosti zbog mogućih štetnih efekata sintetičkih aditiva, proučavana je potencijalna farmakološka vrednost ovih vrsta. Analiziran je sastav dihlometanskih, etil acetatnih, metanolnih, acetonskih, hloroformskih, etanolnih i vodenih ekstrakata ovih vrsta, kao i njihova antioksidativna aktivnost pomoću četiri testa. Za antimikrobno, citotoksično dejstvo i antineurodegenerativnu aktivnost odabrani su vodeni i etanolni ekstrakti, s obzirom na njihovu široku upotrebu u fitoterapiji. Dobijeni rezultati su potvrdili visok antioksidatvni kapacitet S. aegyptiaca. Etanolni ekstrakt je pokazao najjaču aktivnost u poređenju sa ostalim ekstraktima, u DPPH, ABTS i FRAP testovima (IC50 20.74 µg/ml, 1.98 mg AEE/g, 1500.38 μmol Fe(II)/g, respektivno), dok je u ß-CB testu etanolni ekstrakt pokazao nisku aktivnost (9.04% inhibicije). Najveći sadržaj totalnih fenola je izmeren u etanolnom ekstraktu (150.71 mg GAE/g), dok je najviše flavonoida bilo u etil acetatnom ekstraktu (14.64 mg QE/g). Svi testovi su dobro korelisani sa totalnim sadržajem fenola (r vrednost od ±0.806 do ±0.993, respektivno). Etanolni ekstrakt S. fruticosa je pokazao najjaču antioksidativnu aktivnost u svim testovima (DPPH: 29.55 IC50, µg/ml, ABTS: 2.31 mg AEE/g, FRAP: 834.10 μmol Fe(II)/g, ß-CB: 85.11%). Pokazalo se da libijska S. fruticosa ima značajan sadržaj fenolnih komponenti koje se smatraju za jake antioksidanse (154.18 mg GAE/g suvog etanolnog ekstrakta). Najviši sadržaj fenolnih komponenti, pre svega kempferolnih glikozida je bio prisutan u vodenom, metanolnom i etanolnom ekstraktu. DPPH aktivnost je bila dobro korelisana sa sadržajem fenola. Etanolni ekstrakt je pokazao citotoksičnu aktivnost na ćelijsku liniju HCT-116 (375.96 μg/ml), dok je vodeni ekstrakt pokazao jaču aktivnost na K562 ćelije (386.00 μg/ml), što se smatra niskom aktivnošću. Etanolni ekstrakt pokazuje jaču antibakterijsku aktivnost od vodenog ekstrakta, naročito protiv gram pozitivnih bakterija (MIC vrednosti 10-15 mg/ml). Antifungalno dejstvo ekstrakata S. fruticosa je ocenjeno kao slabo u poređenju sa ketokonazolom. U AChE testu inhibicije, ekstrakti su pokazali slabiju aktivnost od galantamina, dok su pokazali jaču aktivnost na testiranim koncentracijama (57.89- 74.66%) u testu inhibicije tirozinaze u poređenju sa kojičnom kiselinom. Među uzorcima S. lanigera metanolni i vodeni ekstrakti su pokazali najveći nivo fenolnih komponenti, posebno kempferolnih glikozida. Vodeni ekstrakt je pokazao bolju antioksidativnu aktivnost u primenjenim testovima, osim FRAP testa, gde je metanolni ekstrakt bolji od vodenog (DPPH: 205.45 IC50, µg/ml, ABTS: 1.77 mg AEE/g, FRAP: 200.11 μmol Fe(II)/g). U ß-CB testu vodeni i etanolni ekstrakti su ispoljili aktivnost (77.66% i 76.06%, respektivno) veću od BHA i BHT standarda. Antioksidativni testovi su snažnije korelisani sa sadržajem totalnih fenola nego flavonoida. Ekstrakti su pokazali vrlo slabu citotoksičnost na HCT-116 liniju (IC50 vrednosti veće od najveće koncentracije od 500 μg/ml), dok je vodeni ekstrakt nešto jače delovao na K562 ćelije (383.29 μg/ml). Etanolni ekstrakt je pokazao jači inhibitorni efekat na bakterijski rast (vrednosti MIC 15-35 mg/ml) u poređenju sa vodenim ekstraktom (vrednosti MIC od 30 do preko 50 mg/ml). Etanolni ekstrakt je pokazao bolju aktivnost na Aspergillus glaucus, dok je vodeni ekstrakt pokazao najajči efekat na Trichophyton mentagrophytes. Etanolni i vodeni ekstrakti su na testiranim koncentracijama pokazali inhibiciju AChE od oko 30%. Oba ekstrakta su na testiranim koncentracijama pokazala jaču inhibiciju (55.26-70.20%) od standarda kojične kiseline (33.73-51.81%) u testu inhibicije tirozinaze. Inhibitorni efekat vodenih ekstrakata je jači u poređenju sa etanolnim ekstraktom. Ispitivane libijske vrste Salvia su pokazale razlike u testiranim biološkim aktivnostima. S. aegyptiaca and S. frutocosa su pokazale visoku antioksidativnu aktivnost, ali su antimikrobna i citotoksična aktivnost uglavnom niske. Vodeni i etanolni ekstrakti S. fruticosa and S. lanigera su na testiranim koncentracijama u testu inhibicije tirozinaze pokazali jaču aktivnost u poređenju sa kojičnom kiselinom kao standardom.

Three Libyan Salvia species were subjected to comprehensive study of microscopic characteristics of trichomes and biological activities of their extracts, while the selected species have not been studied previously. The micromorphology, ultrastructure and histochemistry of S. aegyptiaca, S. fruticosa and S. lanigera leaf trichomes were investigated using light, scanning and transmission electron microscopy. The leaves bear abundant unbranched non-glandular and two types of glandular trichomes - peltate and capitate, on both leaf surfaces. Since Salvia species are used for medicinal and cosmetic purposes, due to essential oils secreted in the glandular trichomes, the focus was on the analyses of the glandular trichomes. In S. aegyptiaca leaves the peltate trichomes were composed of basal cell, short cylindrical stalk cell and broad head of eight to twelve secretory cells arranged in a single circle, or more in two circles. Capitate trichomes (hairs) consisted of one-celled glandular head, subtended by a stalk of variable length, and classified into two types: capitate type I or short-stalked capitate and capitate type II or long-stalked capitate trichomes. Histochemical tests showed that the secreted material in all types of S. aegyptiaca glandular trichomes was of a complex nature. Positive reactions to lipids for all types of glandular trichomes were obtained, with especially abundant secretion observed in peltate and capitate trichomes type II. In S. fruticosa peltate hairs consisted of basal epidermal cell, very short stalk cell and large round head of eight secretory cells arranged in a circle. Capitate trichomes can be divided into two main types, short stalked and long stalked, and further into five subtypes according to the number of stalk cells, the morphology and the number of the glandular head cells. Digitiform trichomes consisted of one basal cell, one or two stalk cells and one apical secretory cell, which are of similar diameter and approximately equal length. The abundant and diverse glandular trichomes and long stalks of the capitate trichomes of S. fruticosa show the xeromorphic character of the investigated plant species which is very drought resistant. S. lanigera possessed peltate trichomes consisted of one basal epidermal cell, short cylindrical stalk cell with cutinized lateral walls and large round or slightly flattened secretory head of eight secretory cells arranged in a single circle. Capitate trichomes consisted of one-celled glandular head, subtended by a stalk of one cell, determined as short-stalked capitate trichomes. It can be concluded that the investigated Libyan Salvia species showed differences in types, density and distribution of trichomes, with the most abundant and diverse glandular trichomes in S. fruticosa. Considering the growing interest in substances of natural origin possessing various bioactivities, as well as the increasing concern about potentially harmful effects of synthetic additives, the potential pharmacological value of the investigated species was studied. In the present study dichloromethane, ethyl acetate, methanol, acetone, chloroform, ethanol and water extracts prepared from three Libyan Salvia species were analyzed for their composition and the antioxidant activity measured by four parallel assays. For the antimicrobial, cytotoxic and enzyme inhibitory activities, water and ethanol extracts were chosen, because of their wide usage in phytotherapy. Obtained results confirmed the high antioxidant capacity of S. aegyptiaca. Ethanol extract showed the strongest activity compared to other extracts in DPPH, ABTS and FRAP assays (IC50 20.74 µg/ml, 1.98 mg AEE/g, 1500.38 μmol Fe(II)/g, respectively) while in the ß-CB assay, ethanol extract showed low activity (9.04% of inhibition). The highest content of total phenolics was measured for ethanol extract (150.71 mg GAE/g), while flavonoids were the most abundant in the ethyl acetate extract (14.64 mg QE/g). All of assays were strongly correlated to total phenolic content (r value from ±0.806 to ±0.993, respectively). The ethanol extract of S. fruticosa showed the strongest antioxidant activity in all applied assays (DPPH: 29.55 IC50, µg/ml, ABTS: 2.31 mg AEE/g, FRAP: 834.10 μmol Fe(II)/g, ß-CB: 85.11%). Our study revealed that Libyan S. fruticosa have significant amount of phenolic compounds which are considered as powerful antioxidants (154.18 mg GAE/g dry ethanolic extract). The highest content of phenolic compounds, mainly kaempferol glycosides, was present in the water, methanol and ethanol extracts. DPPH activity was strongly correlated with total phenolic content. In our study S. fructicosa ethanolic extract showed cytotoxic activity against HCT-116 cell line (375.96 μg/ml), while water extract performed stronger activity on K562 cells (386.00 μg/ml), which is considered as low cytotoxic activity. Ethanol extract showed stronger antibacterial activity than water extract, particularly against Gram-positive bacteria (MIC values 10-15 mg/ml). The antifungal activity of S. fruticosa extracts was estimated as poor comparing to values obtained for ketoconazole. In AChE inhibition assay, extracts exhibited weaker activity (16.28-26.82%) than standard galanthamine, while extracts performed stronger activity (57.89-74.66%) in tyrosinase inhibition assay in comparison with standard kojic acid at tested concentrations. Methanol and water extracts showed the highest content of phenolic components, particularly kaempferol glycosides. Among S. lanigera extracts water extract exhibited better antioxidant activity in the applied assays, except in FRAP assay where methanol was better than water extract (DPPH: 205.45 IC50, µg/ml, ABTS: 1.77 mg AEE/g, FRAP: 200.11 μmol Fe(II)/g). In ß-CB assay, water and ethanol extracts exhibited activity (77.66% and 76.06%, respectively) higher than standards BHA and BHT. Antioxidant assays were more strongly correlated to total phenolic than to flavonoid content. S. lanigera extracts possessed very poor cytotoxic activity against HCT-116 cell line (IC50 values higher than the highest investigated concentration of 500 μg/ml), while water extract was stronger against K562 cells (383.29 μg/ml) Ethanol extract showed stronger inhibitory activity on bacterial growth (MICs 15-35 mg/ml) comparing to water extract (MICs from 30 to over 50 mg/ml). Ethanol extract showed better activity to Aspergillus glaucus, while the water extract showed the strongest effect on Trichophyton mentagrophytes. At tested concentrations, ethanol and water extracts exhibited inhibition of AChE about 30%. Both extracts performed stronger inhibition (55.26-70.20%) than standard kojic acid (33.73-51.81%) at tested concentrations in tyrosinase inhibition assay. The inhibitory effects of water extract was stronger compared to ethanol extract. The investigated Libyan Salvia species showed differences in tested biological activities. In the cases of S. aegyptiaca and S. frutocosa antioxidant activity was high, but antimicrobial and cytotoxic activities were generally low. The water and ethanol extracts of S. fruticosa and S. lanigera at tested concentrations performed stronger activity in tyrosinase inhibition assay compared to standard kojic acid.

Razvoj atletike na paraolimpijskim igrama pod uticajem novih tehnologija

Development of athletics at the paralympic under the influence of new technologies

Naučno-tehnološka revolucija bazirana na ubrzanim društvenim promenama je doprinela popularizaciji i razvoju Paraolimpijskog pokreta, odnosno, vrhunskog sporta osoba sa invaliditetom. Težnja ka ostvarivanju sve boljih rezultata u vrhunskom sportu ima za potrebu da se ulažu veliki materijalni i nematerijalni resursi. Napredak tehnologije i predanost sportista postigli su to da nekada nedostižni rezultati danas budu stvarnost. Na Paraolimpijskim igrama, kao i na Olimpijskim igrama, od svih sportova, atletske discipline privlače najveći broj sportista i gledalaca. Na atletskim takmičenjima učestvuju i muškarci i žene u svim kategorijama invaliditeta. Problem koji se odnosi na razvoj atletike na Paraolimpijskim igrama pod uticajem novih tehnologija nije bio predmet naučnih istraživanja do sada. Najveći broj istraživanja u oblasti sportskih aktivnosti osoba sa invaliditetom se odnosi na trenažni deo u skladu sa stepenom invalidnosti. Pitanja koja se odnose na uticaj novih tehnologija na razvoj atletike na Paraolimpijskim igrama zahtevaju odgovarajuće naučne odgovore. Činjenica je da je vizija razvoja Paraolimpijskog pokreta u budućnosti u direktnoj sprezi sa razvojem nove tehnologije. U cilju postignuća što boljih rezultata ovih sportista, pored kvalitetnog trenažnog procesa, otvorena je mogućnost za poboljšanje kvaliteta njihovih pomagala. Kvalitet pomagala kojima se služe sportisti sa invaliditetom se razvijao i usavršavao paralelno sa samim Paraolimpijskim pokretom. Zahvaljujući provajderima protetičkih i ortopedskih pomagala i tehničkoj servisnoj podršci, ovi sportisti mogu da se usredsrede na samo takmičenje. Predmet istraživanja se odnosi na fenomen atletike koja postoji u programu Paraolimpijskih igara – na nastanak i razvoj atletike pod uticajem novih tehnologija. Cilj istraživanja je bio da se kroz razmatranje više aspekata Paraolimpijskih igara utiče na razvoj naučne misli iz iii oblasti atletike, koja bi prevashodno uticala na interesovanje i poboljšanje kvaliteta tehnologije pripreme i uslova treniranja osoba sa invaliditetom, usavršavanje stručnih kadrova iz ove oblasti, kao i mogućnosti daljeg razvoja ove oblasti. U istraživanju se koristila istorijska i metoda teorijske analize, kao metode kojima su se logički doveli u sklad prikupljeni podaci o fenomenu atletike na Paraolimpijskim igrama. Koristila se još kauzalna i deskriptivna metoda istraživanja. Rezultati istraživanja su potvrdili postavljene pretpostavke: da su osobe sa telesnim invaliditetom predstavljale uslov nastanka sporta/atletike na Paraolimpijskim igrama; da je razvoj atletike na Paraolimpijskim igrama značajno bio uslovljen nastankom paraolimpijskog pokreta; da postoji težnja da se atletika na Paraolimpijskim igrama značajno približi vrednostima Paraolimpijskog pokreta i da postoji značajan uticaj novih tehnologija na razvoj atletike na Paraolimpijskim igrama. Razvoj inovacione tehnologije u sportu uticao je na to da su kod paraolimpijaca koji se takmiče u nekim atletskim disciplinama dostignuti rezultati koji na određeni način predstavljaju „nemoguća ostvarenja“. Ovim naučnim istraživanjem sagledalo se više aspekata Paraolimpijskih igara koji su imali uticaj na razvoj naučne misli iz oblasti atletike. On se ogledao u poboljšanju kvaliteta tehnologije pripreme i uslova treniranja osoba sa invaliditetom, usavršavanju stručnih kadrova, kao i mogućnosti daljeg razvoja ove oblasti.

The scientific and technological revolution based on the rapid social changes has contributed to the popularization and dissemination of the Paralympic Movement, i.e., top-level sports for persons with disabilities. Thanks to the better results achieved in top-level sports and consequently because of the increasing expectations there is a large investment of tangible and intangible resources in the development of top-level sports. Technological progress and commitment of athletes have achieved so much that some of the unattainable results are today a reality. At the Paralympic Games, as well as at the Olympic Games, out of all sports, track and field disciplines attract the largest number of athletes and spectators. Men and women of all categories of disability take part in track and field competitions. The problem that relates to the development of track and field at the Paralympics under the influence of new technologies has not been the subject of scientific research nor of special expert analysis. Most of the research in the field of sports activities of persons with disabilities refers to the training part according to the degree of disability. The development and the impact of new technologies on the development of track and field at the Paralympics require appropriate scientific answers. The vision of the development of the Paralympic Movement in the future is directly linked to the development of new technologies. In order to achieve the best possible results of these athletes, in addition to the quality training process, the possibility of improving the quality of their supplies has also increased. Quality of tools used by the athletes with disabilities has been developing and perfected in parallel with the very Paralympic Movement. Thanks to the provider of prosthetic and orthopedic aids and technical service support, these athletes can focus on the competition and competition only. The subject of this research refers to the phenomenon of track and field that exists at the Paralympic Games - to the creation and development of track and field under the influence of new technologies. The aim of this research is to take the v consideration of multiple aspects of the Paralympic Games and affect the development of the scientific thought in the field of track and field, which would primarily affect the interests and improving the quality of preparation technology and training conditions of persons with disabilities, training of professional staff in this field, as well as the possibilities for further development of the field. In this study, we used historical and theoretical analysis methods as the methods used to logically attune the data collected on the phenomenon of track and field at the Paralympics. We also used causal and descriptive research methods. Research results confirmed the set assumptions: that people with physical disabilities were a condition of origin of track and field at the Paralympics; that the development of track and field at the Paralympics was significantly conditioned on the emergence of the Paralympic Movement; that there is a tendency to bring track and field at the Paralympics as close as possible to the values of the Paralympic Movement, and that there is a significant impact of new technologies on the development of track and field at the Paralympics. The development of innovative technology in sports has influenced the fact that the Paralympic athletes who compete in some track and field disciplines have achieved results that are in a certain way "impossible to achieve". This research has looked at the multiple aspects of the Paralympic Games that affect the development of the scientific thought in the field of track and field, which would primarily affect the interests and improving the quality of preparation technology and training conditions of persons with disabilities, training of professional staff in this field, as well as the possibilities for further development of the field.

Biologija kozice Parapenaeus longirostris (Lucas, 1846) (Crustacea, Decapoda) na šelfu crnogorskog primorja

Biology of the deep water pink shrimp Parapenaeus longirostris (Lucas, 1846) (Crustacea, Decapoda) on the Montenegrin shelf

Jedna od najvažnijih komercijalnih vrsta dekapodnih rakova u kočarskom ribolovu na šelfu Crnogorskog primorja je kozica, Parapenaeus longirostris (Lucas, 1846). Istraživanje je obavljeno tokom dvije godine, od novembra 2006. do oktobra 2007. i od juna 2009. do maja 2010. godine pri čemu su uzorci uzimani sa kočarskih brodova mjesečnom dinamikom. Tokom prve godine je ukupno obrađeno 1979 jedinki, od kojih su 1421 (71.80%) bile ženke a 558 (28.20%) mužjaci. Odnos polova je i u drugoj godini bio u korist ženki tokom koje je analizirano 1967 jedinki, od kojih su 1304 (66.30%) bile ženke a 663 (33.70%) mužjaci. Raspon dužina karapaksa svih ulovljenih jedinki zajedno je bio manji u prvoj godini i kretao se od 15 do 37 mm (25.23 ± 4.48) dok je u drugoj taj raspon bio 11-37 mm (24.85 ± 4.48). Uočen je polni dimorfizam: ženke su u prosjeku i duže i teže od mužjaka, i u dužinskim grupama >28- 30 mm CL su 100% prisutne u ulovu. Mužjaci su u boljoj kondiciji od ženki. Vrijednosti alometrijskog koeficijenata b dužinsko-težinskog odnosa u prvoj godini iznose b = 2.58 za ukupan uzorak, b = 2.51 za ženke i b = 2.34 za mužjake; dok te vrijednosti za drugu godinu iznose b = 2.59 za ukupan uzorak, b = 2.57 za ženke i b = 2.33 za mužjake. Prva polna zrelost ženki je nastupila pri dužini karapaksa od 20.48 mm u prvoj godini, odnosno od 19.89 mm CL u drugoj godini...

Parapenaeus longirostris (Lucas, 1846) is one of the most economically important decapod species in the trawl fishery on the Montenegrin shelf (southern Adriatic Sea). Despite its importance, there were no biological studies on the species for that area. Data were obtained from monthly samples taken from commercial catches realized in the southern Adriatic during the period from November 2006 to October 2007 and from June 2009 to May 2010. During the first year among the 1979 specimens, 71.80% were females and 28.20% males, while during the second year of investigation, among the 1967 specimens, 66.30% were females and 33.70% males. The overall sex ratio was in favour of females. The carapace length of all specimens ranged from 15 to 37 mm, with a mean of 25.23 ± 4.48, while during the second year the range of carapace length of all specimens was wider and ranged from 11 to 37 mm, with a mean of 24.85 ± 4.48. A marked sexual dimorphism was observed between males and females: females have a wider size range, greater size and weight. Percentage of females reached 100% in the 28-30 mm size class and beyond. Males were in better condition than females. The calculated allometric coefficient of length-weight relationships was b= 2.58 for the total sample of 2006-2007 year, b= 2.51 for females and b= 2.34 for males; while during the 2009-2010 values were b=2.59 for the total sample, b=2.57 for females and b=2.33 for males. The size at which 50% of the female population reached maturity was at a carapace length of 20.48 mm in the first year and 19.89 mm during the second year...

Asocijacija genskih varijanti na hromozomu 9p21 i transkripcije gena za CDKN2B i HACD4 sa nastankom ateroskleroze i njenim kliničkim komplikacijama kod čoveka

Association of genetic variants in chromoseome 9p21 and transcription of CDKN2B and HACD4 genes with the onset of atherosclerosis and its clinical complications in human.

Ateroskleroza je kompleksna, hronična i progresivna bolest arterijskih krvnih sudova koja se razvija tokom života i najčešće se klinički manifestuje u kasnijem životnom dobu. Dve najznačajnije kliničke komplikacije su ishemijski moždni i srčani udar (IM), na čiji nastanak utiču sredinski i nasledni faktori (genetska predispozicija). U ovoj studiji ispitana je asocijacija četiri genetičke varijante prisutne u hromozomskom regionu 9p21 (rs10757278 i rs518394 u genu ANRIL, i rs36212560 i rs2275888 u genu HACD4), sa rizikom za nastanak IM, karotidnog plaka (KP) i kliničkih komplikacija KP (ultrasonografski definisan tip plaka, simptomatska karotidna bolest, prisustvo ulceracije na plaku). Ispitana je transkripcija gena CDKN2B i HACD4 kao i nivo iRNK u tkivu karotidnog aterosklerotskog plaka i mononuklearnim ćelijama periferne krvi (MĆPK) bolesnika koji su doživeli prvi IM, 6 meseci nakon infarkta. Nivo iRNK ispitan je u odnosu na genotipove i tip karotidnog plaka. Utvrđena je značajna asocijacija rs10757278 sa rizikom za nastanak KP kod žena. Utvrđeno je da je haplotip genetičkih varijanti rs36212560 i rs2275888 značajno asociran sa rizikom za nastanak KP i IM. Rezutlat je pokazao polno specifičnu asocijaciju (kod muškaraca) kao i značajnu asocijaciju varijante rs36212560 testirane van haplotipa. Nije detektovana značajna asocijacija genetičkih varijanti rs2275888 i rs518394 sa rizikom za razvoj karotidnog plaka, ali je ustanovljena značajna, nezavisna asocijacija varijante rs2275888 sa rizikom za nastanak IM. Varijanta rs10757278 je značajno asocirana sa tipom karotidnog aterosklerotskog plaka. Ustanovljeno je da je haplotip varijanti rs36212560 i rs2275888 značajno asociran sa rizikom za razvoj ulceracije karotidnog plaka, i sa simptomatskom karotidnom bolešću u grupi bolesnika sa KP, kao i da polno specifična asocijacija sa haplotipom postoji kod muškaraca. Razlika u nivoima ekspresije merena je na nivou iRNK u odnosu na genotipove ispitivanih varijanti. U tkivu KP nije detektovana razlika u nivou iRNK za CDKN2B u odnosu na genotipove ispitivanih varijanti, ali je ustanovljen statistički trend u asocijaciji sa genotipom varijante rs1075278. Varijanta rs2275888 je prethodno okarakterisan kao eQTL (eng. expression Quantitative Trait Locus) za gen FOCAD gen u tibijalnoj arteriji. Da bi se ispitao efekat varijante rs2275888 na nivo ekspresije obližnjih gena urađena je replikacija ekspresije gena FOCAD u tkivu karotidnog plaka i dobijena je značajna asocijacija, rezultat prethodno dobijen od strane GTEx konzorcijuma u tibijalnoj arteriji. U MĆPK je detektovana značajna asocijacija varijante rs10757278, i statistički trend u asocijaciji varijante rs518394 i relativnim nivoom ekspresije iRNK za CDKN2B. U MĆPK nije detektovana značajna asocijacija u nivou iRNK gena HACD4 sa genotipovima ispitivanih varijanti. Detektovan je snižen nivo iRNK gena CDKN2B u MĆPK bolesnika sa IM (6 meseci nakon prvog infarkta miokarda) u odnosu na zdrave kontrole.

Atherosclerosis is a complex, chronic and progeressive disease of arterial blood vessels that develops during the life and usualy with clinical manifestations at later age. Two most singnifficant clinical manifestations are stroke and myocardial infarction (MI), which development is affected by the median and hereditary factors (genetic predisposition). In this study, association of four genetic variants from chromosome region 9p21 have been investigated (rs10757278 and rs518394 in the ANRIL gene, and rs36212560 and rs2275888 in the HACD4 gene) with the risk for development of MI, the carotid plaque (CP) and its clinical complications (ultrasonographicaly deffined plaque type, symphtomatic disease, presence of carotid plaque ulceration). The transcription of genes CDKN2B and HACD4 at the mRNA level was examined in the CP tissue and peripheral blood mononuclear cells (PBMC) of patients who sustained first MI, six months after the infarction. Level of mRNA was examined in relation to genotypes and CP type. Signifficant association was determined for rs10757278 with the risk for CP development among females. It was determined that haplotype of gene variants rs36212560 and rs2275888 is significantly associated with the risk for CP and MI development. Result has shown gender specific association (among males) as well as association of rs36212560 with CP and MI when tested independently. It wasn’t detected signifficant association of gene variants rs2275888 and rs518394 with the risk for the development of CP, but, the signifficant idependant association of rs2275888 variant with the risk for MI. Variant rs10757278 is signifficantly associated with the carotid atherosclerotic plaque type. Haplotype of rs36212560 and rs2275888 variants is signifficantly associated with the carotid plaque ulceration, as well as with simpthomatic carotid disease in the group of CP patients., and in addition, the gender speciffic association with the haplotype exist in males. Diference in expression levels was measured at the level of mRNA in relation to genotypes of tested variants. In CP tissue difference in CDKN2B mRNK level wasn’t detected in relation to tested variants according to genotype, but statistical trend was detected in association with rs10757278 genotypes. Variant rs2275888 was previously characterized as eQTL (expression Quantitative Train Locus) for gene FOCAD in tibial arthery. To investigate effect of variant rs2275888 on proksimal genes expression level, the validation of the gene FOCAD expression was conducted in CP tissue and the signifficant association was detected, the result previously obtained by GTEx consortia in tibial artery. In PBMC, signifficant association of variant rs10757278 and statistical trend in association of variant rs518394 with the level of CDKN2B mRNA expression was detected. It wasn’t detected signifficant association of HACD4 mRNA level with the genotypes of investigated variants in PBMC. A significantly lower CDKN2B mRNA level was detected in PBMC of MI patients 6 months after first MI in relation to healthy controls.

Efekat estradiola na regulaciju endotelne i inducibilne azot-monoksid-sintaze u srcu gojaznih pacova

Effect of estradiol on regulation of endothelial and inducible nitric oxide synthase in the heart of obese rats

Estradiol ostvaruje pozitivan efekat na kardiovaskularni sistem (KVS), sprečavanjem nastanka ateroskleroze i endotelne i/ili vaskularne disfunkcije, a deluje i direktno na srce smanjujući njegovu hipertrofiju. Estradiol ostvaruje pozitivne efekte u KVS regulacijom azot-monoksid (NO)-sintaza (NOS), endotelne (eNOS) i inducibilne (iNOS) forme, koje su odgovorne za sintezu NO, aktivacijom sloţene mreţe unutarćelijskih signalnih puteva, u koje su uključeni signalni molekuli supstrat receptora za insulin 1 (IRS-1), fosfatidil- inozitol-3-kinaza (PI3K) i protein kinaza B (Akt). Estradiol utiče na signalnu transdukciju angiotenzina II (Ang II), delujući na ekspresiju receptora za Ang II tipa 1 (AT1R) i tipa 2 (AT2R), kao i na nivo RhoA proteina. Estradiol utiče i na transport energetskih supstrata regulišući translokazu masnih kiselina (CD36) i transportere za glukozu tipa 1 (GLUT1) i tipa 4 (GLUT4). Kontrolni i gojazni muţjaci pacova soja Wistar tretirani su estradiolom (40µg/kg) ili 1% etanolom 24 sata pre ţrtvovanja. Spektrofotometrijskim metodama određivane su koncentracije NO u plazmi i koncentracije L-Arginina (L-Arg), NO i slobodnih masnih kiselina (SMK) u lizatu srca. Western blot metodom određivani su nivoi proteina eNOS, iNOS, NF-κB-p65, pIRS-1/IRS-1, p85 i p110 subjedinice PI3K, pAkt/Akt, RhoA, AT1R, AT2R, GLUT1, GLUT4 i CD36 proteina, kao i asocijacija IRS-1 sa p85-PI3K proteinom u srcu pacova, dok je imunohistohemijskom metodom određivana ekspresija i lokalizacija eNOS i iNOS. Metodom qRT-PCR određivan je nivo iRNK za eNOS i iNOS u srcu pacova. Rezultati su analizirani korišćenjem Studentovog t-testa. Rezultati prikazani u ovoj doktorskoj disertaciji pokazuju da tretman estradiolom kod kontrolnih pacova povećava nivo p85 subjedinice PI3K i fosforilaciju Akt na Thr308, dok smanjuje ekspresiju gena za iNOS. Estradiol smanjuje ekspresiju AT1R i RhoA proteina i reguliše transport energetskih supstrata u srce kod kontrolnih ţivotinja, povećavajući nivo GLUT4, a smanjujući nivo CD36 proteina na plazma membrani kardiomiocita. Estradiol kod gojaznih ţivotinja smanjuje ekspresiju gena za iNOS kao i fosforilaciju IRS-1 na Ser307. Sa druge strane estradiol povećava asocijaciju IRS-1 sa p85 subjedinicom PI3K, kao i nivoe p85, p110 i Akt proteina i dovodi do povećanja ekspresije gena za eNOS. Estradiol kod gojaznih ţivotinja smanjuje nivo AT1R i AT2R na plazma membrani kardiomiocita, kao i nivo RhoA proteina u lizatu srca. Takođe, smanjenje nivoa CD36 i povećanje nivoa GLUT4 na plazma membrani kardiomiocita zabeleţeno je kod gojaznih pacova nakon tretmana estradiolom. Na osnovu dobijenih i prikazanih rezultata u okviru ove doktorske disertacije moţemo zaključiti da estradiol učestvuje u regulaciji ekspresije eNOS i iNOS u srcu gojaznih pacova preko IRS-1/PI3K/Akt signalne kaskade. Pozitivni efekti estradiola u patologiji gojaznosti ogledaju se i u smanjenju nivoa lipida akumuliranih u srcu. Istraţivanja obuhvaćena ovom doktorskom disertacijom su doprinela sagledavanju molekulskih mehanizama kojima estradiol utiče na regulaciju ekspresije eNOS i iNOS, kao i na metabolizam energetskih supstrata u srcu normalno uhranjenih i gojaznih pacova i predstavljaju značajan doprinos razumevanju mehanizama koji se nalaze u osnovi patologije gojaznosti.

Estradiol has a positive effect on the cardiovascular system (CVS) by preventing the development of atherosclerosis and endothelial and/or vascular dysfunction. Estradiol exerts many of its effects on the CVS by regulation of nitric oxide synthase (NOS), endothelial (eNOS) and inducible (iNOS) forms that are responsible for the synthesis of nitric oxide (NO), through activation of the complex network of intercellular signal pathways that include signaling molecules: insulin receptor substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt.) Estradiol also affects the signal transduction of angiotensin II (Ang II) by affecting the expression of Ang II receptors: type 1 (AT1R) and type 2 (AT2R), as well as the level of RhoA protein. Finally, estradiol indirectly regulates the transport of energy substrates in heart, by regulating transporters of free fatty acids (FFA) and glucose, CD36 and glucose transporters type 1 (GLUT1) and type 4 (GLUT4). Control and obese male Wistar rats were treated with one dose of estradiol (40 µg/kg) or with the same amount of 1% ethanol in saline 24 hours before sacrificed. The concentration of NO was measured in the plasma and in lysates, while the concentrations of L-Arginine and FFA were determined in the lysates of the rats‘ hearts. The Western blot method was used to determine the level of eNOS, iNOS, NF-kB p65, IRS-1, p85 and p110 subunits of PI3K, Akt, AT1R, AT2R, RhoA, CD36, GLUT1 and GLUT4 in the rats‘ heart, as well as association of IRS-1 with p85 subunit of PI3K. Immunohistochemical analysis was used for localization and expression of eNOS and iNOS proteins in heart. qRT-PCR was used to determen the level of iRNA of eNOS and iNOS in rats‘ heart. Results were analyzed using Student‘s t test. The results presented in this doctoral dissertation show that estradiol treatment in controls increased the level of the p85 subunit of PI3K and phosphorylation of Akt on Thr308 while reducing the expression of the iNOS gene. Also, estradiol reduces the AT1R expression and the level of RhoA protein in control rats. Estradiol regulates the transport of glucose to the heart by increasing the GLUT4 level on the plasma membranes of cardiomyocytes. Estradiol in obese rats decreases the phosphorylation of IRS-1 on Ser307 while increasing the association of IRS-1 with the p85 subunit of PI3K, as well as levels of p85, p110, and Akt proteins. Estradiol increased the expression of the eNOS gene and decreased the expression of iNOS gene. Estradiol in obese animals decreased the AT1R and AT2R levels, as well as RhoA protein level. Also, a decrease of the CD36 and increase of GLUT4 on the plasma membranes of cardiomyocyte was noticed in obese rats after estradiol treatment. Based on the findings of this doctoral dissertation, we can conclude that estradiol is involved in regulation of eNOS and iNOS expression in the heart of obese rats via IRS-1/PI3K/Akt signaling pathway. Positive effects of estradiol in the pathology of obesity are also reflected in the reduction of lipids accumulated in the heart. The research findings included in this doctoral dissertation contributed to better overview of molecular mechanisms of estradiol regulation of eNOS and iNOS expression as well as the metabolism of energetic substrates in the hearts of control and obese rats. The results of this doctoral dissertation represent a significant contribution to better understanding of the mechanisms that underlie the pathology of obesity.

Efekti i mehanizmi delovanja interleukina-17 na funkcije mezenhimskih i endotelskih ćelija

Effects of interleukin-17 on mesenchymal and endothelial cell functions and mechanisms involved

Interleukin (IL) 17 familija citokina se smatra najmanje istraženom citokinskom familijom sa značajnom ulogom u procesu inflamacije. Ubikvitarna rasprostranjenost receptora za IL-17 omogućava ovom citokinu mnogobrojne biološke efekte u organizmu, poput važne uloge u imunskom odgovoru i regulaciji procesa hematopoeze, ali i u progresiji različitih bolesti, uključujući inflamatorna, autoimunska i maligna oboljenja. IL-17 ima ulogu u ćelijskim procesima poput proliferacije i diferencijacije, i to prvenstveno na hematopoetskim matičnim i progenitor ćelijama, pri čemu IL-17 ispoljava različite efekte na proliferaciju i diferencijaciju zavisno od tipa ćelijske loze i stepena diferencijacije ćelija. Takoñe, zbog ubikvitarne rasprostranjenosti svog receptora, IL-17 je uključen u interakcije izmeñu imunskog sistema i somatskih tkiva. Novija istraživanja su pokazala da IL-17 ima i ulogu finog regulatora diferencijacije mezenhimskih matičnih ćelija (MSC, Mesenchymal stem cells), s obzirom na to da stimuliše osteogenu diferencijaciju dok, s druge strane, inhibira adipogenu diferencijciju humanih MSC. Savremena istraživanja biologije MSC ukazala su na širok opseg njihove potencijalne terapijske primene, prvenstveno u regenerativnoj medicini, jer ove multipotentne adultne matične ćelije koje predstavljaju nediferencirane ćelije sa sposobnošću samoobnove, imaju sposobnost diferenciranja u ćelije tkiva mezenhimskog porekla, poput osteoblasta, hondroblasta i adipocita, a prema saznanjima koja imamo danas, i u ćelije tkiva ektodermalnog i endodermalnog porekla. Matične ćelije različitog stepena multipotentnosti izolovane su iz raznih adultnih tkiva, kao i perinatalnih tkiva koja se poslednjih godina sve češće pominju kao povoljan izvor MSC. Meñutim, i pored velikog napretka u ovoj oblasti, nema konačnih saznanja kako o mehanizmima odgovornim za regulaciju višestrukih funkcija MSC, tako i o njihovom ukupnom terapijskom potencijalu. Kako biološke funkcije MSC umnogome zavise od konteksta mikrosredine, ispitivanje interakcija MSC i IL-17 u održavanju multipotentnosti, mobilizaciji i usmerenoj diferencijaciji MSC je od posebnog značaja. Pored mezenhimskih ćelija, IL-17 ostvaruje svoje efekte delovanjem na endotelske ćelije. Jedan od terapijskih ciljeva u regenerativnoj medicini, koji je još uvek nedovoljno objašnjen, je i podsticanje revaskularizacije oštećenog tkiva. Stimulacija postojećih, in situ prisutnih endotelskih ćelija na angiogenezu je jedan od mogućih mehanizama. Efekti IL-17 na endotelske ćelije nisu do sada u potpunosti opisani, i poznato je samo da IL-17 u prisustvu drugih poznatih angiogenih faktora stimuliše sposobnost endotelskih ćelija za angiogenezu. Pored toga, poznato je da se funkcije ćelija kao i delovanje citokina značajano razlikuju u zavisnosti od procenta kiseonika prisutnog u mikrosredini ćelija. Efekat delovanja IL-17 na endotelske ćelije u prisustvu različitih koncentracija kiseonika do sada nije istraživan. Takoñe je još uvek nepoznato da li IL-17 može uticati na diferencijaciju MSC u endotelske ćelije. Da bi se sprovela istraživanja u ovom pravcu, neophodno je uspostaviti pogodan model za diferencijaciju MSC u endotelske ćelije. Cilj istraživanja ove disertacije bilo je ispitivanje efekata IL-17 na različite ćelijske funkcije mezenhimskih i endotelskih ćelija u specifičnim uslovima mikrosredine. Analizirano je dejstvo IL-17 na proliferaciju, migraciju, angiogenezu i diferencijaciju ćelija. Istraživani su i molekularni mehanizmi koji omogućavaju finu regulaciju ćelijskih funkcija od strane IL-17, analizom aktivacije signalnih molekula, kao i ekspresije gena i proteina neophodnih za specifične funkcije. Posebna pažnja u istraživanjima bila je posvećena izolaciji, karakterizaciji i endotelskoj diferencijaciji primarnih MSC iz tkiva pupčanika (UC-MSC, Umbilical cord-mesenchymal stem cells). U okviru razjašnjavanja efekata i mehanizama delovanja IL-17 na diferencijaciju mezenhimskih ćelija, primenjena je multipotentna mišja C2C12 ćelijska linija. Uticaj IL-17 na diferencijaciju analiziran je na osnovu ekspresije gena i proteina specifičnih za odreñeni tip diferencijacije: teškog lanca miozina (MyHC, Myosin heavy chain) i miogenina za miogenu, a alkalne fosfataze (ALP, Alkaline phosphatase), Runx2/Cbfa1 (Runt-related transcription factor 2/Core-binding factor subunit alpha-1) transkripcionog faktora i ciklooksigenaze 2 (Cox-2, Cyclooxygenase 2) za osteogenu diferencijaciju. Pored toga, utvrñivano je učešće mitogenom aktiviranih protein kinaza (MAPK) i morfogenetskih proteina kosti (BMP, Bone morphogenic proteins) u IL-17- usmerenoj diferencijaciji C2C12 ćelija. Rezultati su pokazali da IL-17 inhibira miogenu i stimuliše osteogenu diferencijaciju C2C12 ćelija, s obzirom na to da je IL-17-zavisna inhibicija miogeneze povezana sa redukcijom ekspresije iRNK za miogenin, redukcijom ekspresije MyHC i izostankom formiranja miotuba, dok je IL-17-zavisna indukcija osteogeneze povezana sa indukcijom ekspresije iRNK za Runx2/Cbfa1, indukcijom ekspresije Cox-2 i povišenom aktivnošću ALP. Rezultati su takoñe pokazali da IL-17 ostvaruje ove efekte putem aktivacije ERK1,2 MAPK signalnog puta, nezavisno od BMP-Smad signalnog puta. U drugom delu ove studije ispitivan je efekat IL-17 na funkcije endotelskih ćelija na modelu humane endotelske ćelijske linije, EA.hy 926, praćenjem njihove proliferacije, migracije i tubulogeneze u uslovima normoksije i hipoksije. Analizirana je i ekspresija gena za endotelsku azot monoksid sintazu (eNOS, Endothelial nitric oxide synthase) i Cox-2, kao i njihovih proteinskih produkata, budući da ovi molekuli imaju važnu ulogu u procesu angiogeneze. Dobijeni rezultati su potvrdili da IL-17 stimuliše ključne funkcije uključene u proces angiogeneze EA.hy 926 ćelija, poput ćelijske migracije i tubulogneze, kao i ekspresiju eNOS i Cox-2 u ovim ćelijama. Pro-angiogeni efekti IL-17 su pokazani u uslovima normoksije (20% O2). Uslovi hipoksije (3% O2) pokazali su toksično dejstvo na ovu ćelijsku liniju, dok je IL-17 u ovakvim uslovima umanjio broj apoptotičnih ćelija. U okviru trećeg dela ove disertacije MSC su izolovane iz vezivnog tkiva pupčanika, tzv. Vartonove sluzi, umnožene i okarakterisane prema važećim preporukama i kriterijumima Meñunarodnog društva za ćelijsku terapiju. Zatim je ispitan i odreñen optimalni protokol za njihovu efikasnu diferencijaciju u endotelske ćelije, u cilju uspostavljanja novog ćelijskog modela za buduća istraživanja. Princip diferenciranja UC-MSC u endotelske ćelije zasnivao se na inhibiciji signalizacije faktora transformacije rasta (TGF-β, Transforming growth factor beta), i to njegovog receptora, aktivinu-nalik kinaze 5 (ALK5, Activin-like kinase). Na osnovu dobijenih rezultata, zaključeno je da se UC-MSC odlikuju visokom sposobnošću samoobnove i visokim kapacitetom za diferencijaciju kao i da se mogu diferencirati u endotelske ćelije putem inhibicije ALK5 receptora.

Interleukin (IL) 17 cytokine family is considered the least explored cytokine family with an important role in inflammation. Due to the ubiquitous expression of its receptor, IL-17 has been implicated in interactions between the immune system and somatic tissues. In this context, recent findings have shown that IL-17 acts as a fine regulator of mesenchymal stem cell (MSC) differentiation. Having in mind that behaviour of MSC depends on the surrounding microenvironment, it is important to analyze the interactions between MSC and IL-17 involved in the maintenance of their multipotency, mobilization and directed differentiation. In addition to mesenchymal cells, IL-17 exerts its roles by acting on endothelial cells as well. Even though IL-17’s effects on endothelial cells have not been fully discovered yet, it is well known that IL- 17, in the presence of other angiogenic factors, has the ability to stimulate endothelial cells to undergo angiogenesis. Furthermore, it is well known that cellular functions, as well as cytokine effects can be significantly modulated by the percentage of oxygen present in their microenvironment. However, effects of IL-17 on endothelial cells in the context of different O2 concentrations have not been explored yet. Also, it is still unknown whether IL-17 can affect endothelial differentiation of MSC, but to address this issue, it is necessary to create a valid model of endothelial differentiation of MSC. The objective of this study was to investigate the effects of IL-17 on different functions of mesenchymal and endothelial cells in specific microenvironmental conditions. The influence of IL-17 on proliferation, migration, angiogenesis and differentiation was analyzed, as well as the molecular mechanisms involved. The activation of various signaling molecules and the expression of genes and proteins involved in specific functions were examined. Yet another objective was to establish an appropriate model for the mesenchymal-endothelial transdifferentiation. For this task primary MSC from umbilical cord tissue (UC-MSC) were isolated and characterized with emphases on their endothelial differentiation. In order to elucidate the effects and mechanisms involved in IL-17 acting on mesenchymal cell differentiation, a multipotent mouse C2C12 cell line was used. The influence of IL-17 on cell differentiation was analyzed based on the expression of genes and proteins specific for the certain type of differentiation: Myosin heavy chain (MyHC) and Myogenin for myogenic, and Alkaline phosphatase (ALP), Runt-related transcription factor 2/Core-binding factor subunit alpha-1 (Runx2/Cbfa1) and Cyclooxygenase-2 (Cox-2) for osteogenic differentiation. Additionaly, the involvement of Mitogen-activated protein kinases (MAPK) and Bone morphogenetic proteins (BMP) in IL-17-directed differentiation of C2C12 cells was investigated. Results obtained demonstrated that IL-17 inhibits myogenic and stimulates osteogenic differentiation of C2C12 cells, by down-regulating the Myogenin mRNA expression, MyHC expression and myotube formation, while up-regulating the Runx2/Cbfa1 mRNK expression, Cox- 2 expression and ALP activity. IL-17 exerted these effects by activating ERK1,2 MAPK signaling pathway, which in turn regulated the expression of relevant genes and proteins to inhibit myogenic differentiation and induce osteogenic differentiation. It was also shown that the induction of osteogenic differentiation by IL-17 is independent of the BMP-Smad signaling pathway. In the second part of this study the effect of IL-17 on endothelial cell functions was investigated using human endothelial cell line EA.hy 926 as a model. The changes in cells’ proliferation, migration and tubulogenesis were analyzed, along with the expression of genes for endothelial Nitric oxide synthase (eNOS) and Cox-2, and their corresponding protein products, since these molecules have important roles in angiogenesis. The influence of the environmental stimuli (normoxia vs. hypoxia) was also investigated. Results obtained demonstrated that IL-17 stimulates crutial events involved in angiogenesis of EA.hy 926 cells, such as cell migration and tubulogenesis, along with the expression of eNOS and Cox-2. However, the effects of IL-17 were dependent on the O2 concentration, since pro-angiogenic effects of IL-17 were noticed in the presence of 20% O2, while 3% O2 exerted toxic effect on this cell line, and IL-17 in these conditions decreased the number of apoptotic cells. In the final task, MSC were isolated from the umbilical cord Wharton’s jelly, expanded in culture and characterized according to the recomendations from the International Society for Cell Therapy. Following this, an optimal protocol for the endothelial diffrentiation of UC-MSC was established. The endothelial-directed differentiation was based on the inhibition of the Transforming growth factor beta (TGF-β) signaling, specifficaly its receptor activin-like kinase 5 (ALK5). Based on the results gained, it was concluded that UC-MSC have a high ability to selfrenew and high diferentiation capacity, as well as that these cells can be differentiated into endothelial cells by inhibiting ALK5 receptor.

Farmakogenomski i farmakotranskriptomski markeri odgovora na terapiju glukokortikoidnim lekovima kod dece sa akutnom limfoblastnom leukemijom: molekularni mehanizmi delovanja, klinički i populacioni aspekti

Pharmacogenomic and pharmacotranscriptomic markers in glucocorticoid treatment of pediatric acute lymphoblastic leukemia: molecular mechanism of action, clinical and population aspects

Personalizovana medicina, medicina XXI veka, nastoji da individualizuje terapiju za svakog pacijenta, kako bi lečenje bilo što efikasnije i sa što manje rizika od toksičnosti. Personalizovana medicina se danas najviše oslanja na farmakogenomiku i farmakotranskriptomiku, koje su već dale svoj doprinos unapređenju lečenja mnogih bolesti, a posebno maligniteta. Akutna limfoblastna leukemija (ALL) predstavlja najčešći hematološki malignitet pedijatrijskog uzrasta. Mada stopa izlečenja dostiže 80-90%, ALL je i dalje glavni uzrok mortaliteta u ovih pacijenata. Terapija izaziva neželjene efekte u 75% pacijenata. Pored toga, u 1-3% pedijatrijskih pacijenata sa ALL smrtni ishod nije posledica bolesti, već je uzrokovan terapijom. Lečenje pedijatrijske ALL nije napredovalo uvođenjem novih lekova, već nastojanjem da se smanje neželjena dejstva onih lekova koji su već sastavni delovi postojećih terapijskih protokola, zbog čega su farmakogenomika i farmakotranskriptomika dobile ključno mesto u ovoj oblasti. Glukokortikoidni lekovi (GK) se koriste u početnoj fazi lečenja ALL u dece, u fazi indukcije remisije. Od početka lečenja do 8. dana se primenjuju isključivo GK. Ipak, farmakogenomske i farmakotranskriptomske studije za glukokortikoidne lekove još uvek nisu dovele do algoritma koji bi mogao biti primenjen u lečenju dečje ALL. Stoga je izuzetno značajno nastaviti sa istraživanjima farmakogenomskih i farmakotranskriptomskih markera relevantnih za uspešnost GK terapije u dece sa ALL. Razumevanje molekularnog mehanizma dejstva GK vodi ka otkrivanju novih markera koji mogu biti iskorišćeni za optimizaciju GK terapije. Nove tehnologije, kao što je sekvenciranje nove generacije (eng, next generation sequencing, NGS) su omogućile dizajniranje panela za farmakogenomske i farmakotranskriptomske markere za različite lekove...

Personalized medicine, the medicine of the XXI century, aims to individualize therapy for each patient, in order for the treatment to be as efficient and safe as possible. Today, personalized medicine is the most reliant on pharmacogenomics and pharmacotranscriptomics, which have already given their contribution to enhancing treatment of many diseases, especially malignancies. Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy of childhood. Even though the percentage of cured patients reaches 80-90%, ALL is still the main cause of mortality in this group of patients. Therapy causes side effects in 75% of patients. Aside from that, 1-3% of pediatric ALL patients die because of therapy side effects rather than the disease itself. Treatment of pediatric ALL wasn’t improved by the introduction of new drugs, but by decreasing the side effects of the drugs which are already included in existing protocols. Due to this fact, pharmacogenomics and pharmacotranscriptomics have gained key positions in this field. Glucocorticoid drugs (GC) are used in the initial phase of childhood ALL treatment, in the phase of remission induction therapy. From the beginning of the treatment until day 8, GCs are exclusively applied. Pharmacogenomic and pharmacotranscriptomic studies for GC drugs have yet to produce an algorithm that could be applied in childhood ALL treatment. Therefore, it is of extreme importance to continue researching pharmacogenomic and pharmacotranscriptomic markers relevant to the success of the GC therapy of children with ALL. Understanding the molecular mechanism of action of GC can lead to discovery of new markers that could be used for the optimization of GC therapy. New technologies, such as next generation sequencing (NGS) have created a possibility for designing panels for pharmacogenomic and pharmacotranscriptomic markers of response to different drugs. Utilization of these panels in population pharmacogenomic studies can lead to new knowledge that could open wide the doors to predictive pharmacogenomic testing...

Analiza prirode rezistencije na hinolone i molekularna tipizacija odabranih serotipova Salmonella enterica subspecies enterica

Analysis of nature of resistance to quinolones and molecular typing of selected serotypes of Salmonella enterica subspecies enterica

Salmoneloze spadaju među najznačajnije zoonoze u svetu. S obzirom da se ljudi zaražavaju najčešće putem konzumacije hrane životinjskog porekla, nadzor nad salmonelama neophodan je u svakom stadijumu proizvodnje hrane od farme do viljuške. Širenje rezistencije na hinolone među salmonelama predstavlja veliki javno zdravstveni problem zbog uticaja na lečenje ljudi i životinja. Cilj istraživanja je bio da se ispita mogućnost brze i efikasne tipizacije Salmonella enterica subspecies enterica serotip Enteritidis (S. Enteritidis) kao najčešćeg serotipa kod ljudi i učestalost i molekularni mehanizmi rezistencije na hinolone kod salmonela. U kolekciji izolata S. Enteritidis poreklom od ljudi, pilića i iz namirnica živinskog porekla, kombinacijom RAPD-PCR tipizacije sa četiri prajmera, rezistotipizacije i polimorfizma mutacija u regionu gyrA gena koji kodira rezistenciju na hinolone (QRDR), diskriminisano je 22 genetičke grupe sa indeksom diskriminacije 0.828. Svi izolati bili su visoko srodni, a tipizacija je dokazala nasumičnu raspoređenost tipova u sve tri kategorije izolata, odnosno cirkulaciju sojeva kroz razne stadijume lanca ishrane. RAPD-PCR tipizacija uspešno je izdvojila epidemijski soj među tri izolata S. Enteritidis iz jednog restorana brze hrane. Analizom uzoraka iz fabrike biskvita ustanovljena je kontaminacija sa najmanje četiri soja S. Enteritidis i povezanost serotipa Enteritidis sa jajima kao izvorom kontaminacije. Analizom većeg uzorka izolata poreklom od namirnica i ljudi sa teritorije Južnobačkog okruga u šestogodišnjem periodu ustanovljena je mala učestalost rezistencije S. Enteritidis na antibiotike, retka pojava višestruke rezistencije na antibiotike i mala učestalost rezistencije na hinolone (2.9%). Nađena su tri tipa mutacija odgovornih za rezistenciju na hinolone u QRDR regionu: Asp87Asn, Asp87Gly i Ser83Phe. Dominirale su mutacije na 87. poziciji i to Asp87Asn supstitucija. Nisu nađeni dokazi o klonalnom širenju sojeva rezistentnih na nalidiksičnu kiselinu. Rezistencija na fluorohinolone nije otkrivena. Dokazana je smanjena osetljivost na fluorohinolone kod S. Enteritidis rezistentnih na nalidiksičnu kiselinu. Kod drugih serotipova salmonela rezistencija na nalidiksičnu kiselinu i udruženost sa više markera rezistencije bila je učestalija nego kod S. Enteritidis, naročito kod S. Hadar. Rezistencija na ampicilin i nalidiksičnu kiselinu bile su najčešće kod salmonela bez obzira na serotip. Diskriminativna moć i jednostavnost RAPD-PCR metode sa četiri prajmera udružene sa rezistotipizacijom preporučuju ih kao efikasan sistem tipizacije prilikom nadzora nad kretanjem salmonela u humanoj i životinjskoj populaciji. Ispitivanje rezistencije na antibiotike, a naročito na hinolone kod salmonela neophodno je u cilju upozoravanja kliničara na terapijske rizike, a sa epidemiološkog aspekta, radi detekcije pojave i širenja opasnih sojeva.

Salmonelloses are among most significant zoonoses in the world. Since people are most often infected by consumption of food of animal origin, surveillance of salmonella is necessary in every stage of food production from farm to fork. Emergence of resistance to quinolones among salmonella is a significant public health problem, considering implications on the treatment of humans and animals. The aims of the investigation were to analyze the possibility of fast and efficient typing of Salmonella enterica subspecies enterica serotype Enteritidis (S. Enteritidis) as the most frequent serotype in humans and to investigate frequency and molecular mechanisms determining resistance to quinolones in salmonella. In a collection of S. Enteritidis isolates originating from humans, poultry and marketed poultry products and eggs, a combination of RAPD-PCR typing with four primers, resistotyping and polymorphism of mutations in quinolone resistance determining region (QRDR) of gyrA gene revealed 22 genetic groups with discrimination index of 0.828. All isolates were closely related and typing has proved random distribution of types in all three categories of isolates, ie. circulation of strains through subsequent stages of food chain. RAPD-PCR typing succesfully discriminated epidemic strain among 3 S. Enteritidis isolates from one fast food restaurant. Analysis of samples from one biscuit factory showed contamination with at least four strains of S. Enteritidis and strong connection of serotype Enteritidis with eggs as a source of contamination. Analysis of larger collection of isolates from food and humans in Southern Bačka county in a six-year period revealed low frequency of resistance of S. Enteritidis to antibiotics, rare occurence of multiple resistance to antibiotics and low frequency of resistance to quinolones (2.9%). Three types of mutations determining resistance to quinolones in QRDR were discovered: Asp87Asn, Asp87Gly and Ser83Phe. Mutations at position 87 are dominant, with Asp87Asn as the most frequent. There was no evidence of clonal spread of strains resistant to quinolones. Resistance to fluoroquinolones was not detected. Reduced susceptibility to fluoroquinolones among S. Enteritidis resistant to nalidixic acid was proved. Resistance to nalidixic acid and association with multiple antibiotic resistance were more common among other serotypes of salmonella, especially in S. Hadar, than among S. Enteritidis. Resistance to ampicillin and nalidixic acid were the most common in all investigated salmonella serotypes. Discriminative power and simplicity of RAPD-PCR method with four primers together with resistotyping recommend them as the efficient typing system for monitoring of salmonella in human and animal population. Investigation of resistance to antibiotics, especially to quinolones among salmonella is essential for alerting clinicians for therapy risks, and from epidemiological aspect, for detection of emergence and spread of dangerous strains.

Prirodne vrednosti kao osnova unapređenja turističke ponude severozapadne Crne Gore

Prirodne vrednosti kao osnova unapređenja turističke ponude severozapadne Crne Gore

U ovoj disertaciji su studiozno sagledane mogućnosti unapređenja turističke ponude severozapadne Crne Gore (opštine Žabljak, Pljevlja, Plužine, Šavnik) na bazi nedovoljno iskorišćenih prirodnih turističkih vrednosti. Upravo raznovrstan, horizontalno i vertikalno raščlanjen reljef i veoma složen geološki sastav, specifične klimatske karakteristike, potom reke i brojna planinska jezera, te bogat biljni i životinjski svet, predstavljaju one prirodne vrednosti koje potencijalno čine osnovu unapređenja turističke ponude ovog dela Crne Gore baziranog i na zaštićenim prirodnim dobrima (Nacionalni park „Durmitor“, Regionalni park „Piva“). Poseban akcenat je dat u sagledavanju i oceni nivoa iskorišćenosti prirodnih turističkih vrednosti, kao primarnih turističkih motiva u granicama istraživanog prostora. Istovremeno su uz procenu i vrednovanje ovih resursa predložene pojedine aktivnosti za njihovo kvalitetnije uklapanje u turističku ponudu severozapadne Crne Gore. Realizacija takvih aktivnosti bi trebala uticati na unapređenje turističke ponude koje se može postići, generalno samo razvojem turizma koji je u saglasnosti sa ekološkim normama, naročito ako se imaju u vidu postojeća zaštićena prirodna dobra na teritoriji područja. Takođe je u razmatranju navedene problematike prezentovano aktuelno stanje postojeće ukupne turističke ponude istraživanog područja koja uključuje ili bi trebala uključivato pojedine antropogene vrednosti. One praktično nisu uključene u ponudu lokalnih turističkih agencija, s obzirom na to da su agencije gotovo isključivo usmerene na programe valorizacije prirodnih vrednosti (rafting, planinarenje i dr.). Ove vrednosti jesu osnova unapređenja turističke ponude područja, ali su antropogene vrednosti njena potencijalno adekvatna dopuna koja uz odgovarajuće aktivnosti nadležnih subjekata može uticati jednim delom na sadržajnu diverzifikaciju turističke ponude područja. SWOT analizom se kroz njene sastavne faktore ukazalo na Mr Mićo Srdanović Doktorska disertacija 392 značajne pretpostavke u formiranju turističke ponude istraživanog područja, kao što se ukazalo i na pragove ograničenja, ali i na mogućnosti koje mogu poslužiti za umanjenje ili prevazilaženje nekih od njih. Za realizaciju tih mogućnosti je potrebna svest lokalne zajednice o tome da se rezultati pojedinih negativnih procesa na delu područja (izraziti primer NP „Durmitor“) koji su motivisani kratkoročnim potrebama moraju sa aspekta kvaliteta prirodnih vrednosti, odraziti na iste kroz dugoročne implikacije. Osim ovom analizom, značajna saznanja su dobijena i iz anketnog istraživanja sprovedenog na uzorku od 200 slučajno izabranih turista na više lokacija unutar područja. Rezultati ovog istraživanja su poslužili kao veoma validan indikator prednosti i nedostataka aktuelne turističke ponude, ali i nivoa zadovoljstva anketiranih turista čime je takođe ukazano na smernice unapređenja pojedinih segmenata turističke ponude. Naglašenim aplikativnim karakterom ovaj deo istraživanja pruža odgovarajući informativni doprinos nadležnim turističkim subjektima kojim se ukazuje na anketnim istraživanjem utvrđen stepen afirmativnosti mesta, odnosno područja u celini. Istovremeno, rezultati pomenutog istraživanja pružaju, između ostalog, i značajne informacije koje se odnose na ostvaren kvalitet, kao i na izraženije preferiranje nekih segmenata turističke ponude pri čemu isto može poslužiti delom i kao polazna osnova u prilagođavanju pojedinih segmenata ponude potrebama turista. Istraživanjem u okviru izrade disertacije je konstatovatovana prisutnost određene diskoordinacije usled različitosti u nivoima nadležnosti i neujednačenosti u pogledu centralizacije/decentralizacije upravljanja nad pojedinim organizacionim subjektima. U vezi sa tim, a na osnovu rezultata proisteklih iz celokupno sprovedenog istraživanja kroz interdisciplinarni pristup došlo se do sagledavanja potrebe realizacije više konkretnih mera među kojima je jedna od njih potreba formiranja Regionalne turističke organizacije što je jedna od predispozicija koje su od značaja u prosecu boljeg pozicioniranja istraživanog područja kao turističke destinacije.

The dissertation deals with the advancement possibilities of tourist offer in north western part of Montenegro (municipalities of Žabljak, Pljevlja, Plužine, Šavnik) based on insufficiently utilised natural tourism values. Natural tourism values such as diverse, horizontally and vertically segmented relief, extremely complex geological formation, specific climate features, rivers and numerous mountain lakes, bio-diversity of flora and fauna form the prospective ground for tourism offer advancement in this part of Montenegro jointly with protected natural assets (National Park Durmitor and Regional Park Piva). There is special emphasis on comprehension and evaluation of utilization level of natural tourism assets, as primary tourist motives within the boundaries of the researched area. Simultaneous to estimation and evaluation of the resources, certain activities have been proposed for their qualitative integration into the tourist offer of north western part of Montenegro. Realisation of the activities should impact the tourist offer advancement that can be achieved only by means of tourism development that is in concordance with ecology principles, especially related to existing natural assets of this area. Moreover, the study of the issues describes the current condition of the total tourist offer which implies or should imply certain anthropogenic values of the researched area. In practice anthropogenic values were not included into the offer of the local travel agents. Instead, there was exclusive orientation towards the valorisation programmes of natural values (rafting, mountaineering, etc.). Natural values do create the basis for tourist offer advancement in the area, but anthropogenic values create prospective adequate complement to the tourist offer which together with appropriate activities of the subjects in charge may in return partly impact the content diversification of the tourist offer in the area. SWOT analysis by means of its constituent factors specifies the presuppositions in tourist offer creation of the researched area, identifies restriction factors and also the possibilities that may help to alleviate or surpass some of them. Furthermore, the realisation of the possibilities demands higher awareness of the local community on the issue that the results of certain negative processes in the part of the researched area (prominent example National Park Durmitor) motivated by short-termed needs must have Мр Мићо Срдановић Докторска дисертација 395 long-termed impact on natural values in terms of quality. Apart from this analysis, important findings have been obtained by the questionnaire conducted on the sample of 200 randomly selected tourists on several locations within the area. The results of the research serve as a valid indicator of strengths and weaknesses of the current tourist offer and the level of satisfaction of the respondents, which also indicates the directions for future improvement of certain segments within the tourist offer. This part of the research offers appropriate informative contribution to the tourism subjects by means of its highlighted applicative potential and defined level of affirmative features of the total area. At the same time the results of the research offer significant information on the achieved quality level and preferred segments of tourist offer that may partly serve as the basis for adaptation of certain segments within the offer to tourist needs. The research conducted for doctoral thesis highlighted certain discoordination due to differences between the levels of responsibility and unevenly centralised/decentralised management of organisational subjects. Related to this and based on the results of the research based on interdisciplinary approach several measures have been highlighted, among which the need to form Regional tourist organisation as one of the predispositions for the process of better positioning of the researched area as a tourist destination.

Uticaj hroničnog intenzivnog treninga na parametre oksidativnog stresa i masnokiselinski profil plazme i eritrocita kod sportistkinja

The effect of chronic intensive training on oxidativ stress parameters and fatty acid profiles of plasma and erythrocytes in female athletes

Intenzivna fizička aktivnost utiče na metabolizam lipida i masnih kiselina (MK), dovodi do povećane potrošnje kiseonika u mišićima i drugim tkivima što vodi povećanoj produkciji reaktivnih kiseoničnih vrsta i može uticati na funkciju imunog sistema. Uticaj hroničnog intenzivnog vežbanja na parametre oksidativnog stresa i masnokiselinski profil fosfolipida kod aktivnih sportistkinja nije dovoljno ispitivan. Zbog toga je ova studija imala za cilj da ispita MK profil fosfolipida plazme i eritrocita, parametre oksidativnog stresa, aktivnost enzima antioksidativne zaštite, kao i aktivnost monocita kao dela imunog sistema, kod dve grupe aktivnih sportistkinja: vaterpolistkinja (n=15) i fudbalerki (n=19). Kontrolnu grupu (n=14) su činile zdrave devojke istih godina starosti koje se ne bave fizičkom aktivnošću. Uprkos sličnom dijetarnom unosu, MK profil plazme pokazao je znatno veći udeo stearinske, oleinske i mononezasićenih masnih kiselina (MUFA) i značajno niži procenat ukupnih i n-6 polinezasićenih MK (PUFA) kod fudbalerki u odnosu na vaterpolo i kontrolnu grupu. Sa druge strane vaterpolistkinje su imale višu procentualnu zastupljenost palmitoleinske i arahidonske kiseline u odnosu na kontrolnu grupu. MK profil eritrocita pokazao je značajno veći procenat oleinske kiseline i MUFA kod fudbalerki u odnosu na kontrolnu grupu, i smanjeni procenat stearinske kiseline uz viši udeo palmitinske i palmitoleinske kiseline kod vaterpolistkinja u odnosu na druge dve grupe. Obe grupe sportistkinja su imale značajno niži procenat n-6 PUFA i ukupnih PUFA u poređenju sa kontrolnom grupom. Procenjena aktivnost desaturaza i elongaza u eritrocitima takođe je promenjena kod sportistkinja. Analiza parametara oksidativnog stresa pokazala je značajno veći ukupni antioksidativni status u plazmi i veći stepen lipidne peroksidacije u eritrocitima kod sportistkinja u odnosu na kontrolnu grupu. Koncentracija H2O2 u plazmi bila je veća kod sportistkinja u odnosu na neaktivne žene, ali su fudbalerke imale viši nivo H2O2 i u odnosu na vaterpolistkinje. Sa druge strane, koncentracija O2 .- bila je najniža kod vaterpolistkinja a najviša kod fudbalerki, značajno viša i u odnosu na kontrolnu grupu. Koncentracija NO2 - u plazmi bila je značajno viša kod fudbalerki u odnosu na kontrolnu grupu. Aktivnost enzima antioksidativne zaštite u eritrocitima razlikovala se između sportistkinja i kontrolne grupe. Tako je aktivnost katalaze kod sportistkinja bila značajno viša u odnosu na kontrolnu grupu, dok se aktivnost glutation- peroksidaze nije razlikovala između ispitivanih grupa. Aktivnost superoksid-dismutaze bila je najviša kod vaterpolistkinja, i nije se značajno razlikovala između fudbalerki i kontrolne grupe. Analiza metaboličke aktivnosti mononuklearnih ćelija periferne krvi pokazala je veću aktivnost mononuklearnih ćelija kod sportistkinja, dok se produkcija O2 .- i NO2 - u mononuklearnim ćelijama nije razlikovala između ispitivanih grupa. Naši rezultati su pokazali da dugotrajna intenzivna fizička aktivnost značajno utiče na masnokiselinski profil fosfolipida plazme i eritrocita, parametre oksidativnog stresa, aktivnost enzima antioksidativne zaštite, menja aktivnost mononuklearnih ćelija periferne krvi ali ne utiče na produkciju O2 .- i NO2 - u mononuklearnim ćelijama. Zapažene razlike između grupa vaterpolistkinja i fudbalerki ukazuju da tip sporta igra značajnu ulogu u promenama u metablizmu kod sportistkinja.

Intensive physical activity affects metabolism of lipids and fatty acids (FA), increases consumption of oxygen in muscles and other tissues that leads to increased production of reactive oxygen species and can affect immune system. The effect of chronic intensive exercise on parameters of oxidative stress and phospholipids FA composition in elite female athletes has not been studied extensively so far. Thus the aim of this study was to investigate FA profiles in plasma and erythrocytes phospholipids, parameters of oxidative stress, activity of antioxidative enzyme system and activity of monocytes as a part of immune system in two groups of active female athletes: water polo (n=15) and football (n=19) players. The control group was composed of 14 apparently healthy age-matched sedentary women. In spite of similar dietary patterns, plasma FA profile in the football players showed significantly higher proportion of stearic acid, oleic acid and monounsaturated FA (MUFA), and significantly lower proportion of total and n-6 polyunsaturated FA (PUFA) than the water polo and control group. The water polo players had higher percentages of palmitoleic acid and arachidonic acid than the control subjects. Erythrocyte FA profile differed among groups. We found significantly higher proportion of oleic acid and MUFA in the football group than in the controls, and decreased stearic acid and elevated palmitic and palmitoleic acid in the water polo players than in the other two groups. Both groups of athletes had significantly lower percentages of n-6 PUFA and total PUFA compared to the controls. The estimated activities of elongase and desaturases in erythrocytes were also altered in the athletes. Analysis of oxidative stress parameters showed significantly higher plasma total antioxidant status and higher lipid peroxidation in erythrocytes of the athletes than in the controls. Concentration of H2O2 in plasma was higher in the athletes compared with sedentary women, but also higher in football than in the water polo players. On the other hand, level of O2 .- was the lowest in water polo players and the highest in the football group. Concentrations of both O2 .- and NO2 - were significantly higher in the football than in the control group. Catalase activity was significantly higher in the athletes that in sedentary women, while glutation peroxidase activity was similar in all groups. Superoxide-dismutase activity was higher in the water polo group than in the other two groups. Furthermore, metabolic activity of peripheral blood mononuclear cells was higher in the athletes, and production of O2 .- i NO2 - in mononuclear cells did not differ among the studied groups. Our results have shown that long-term, intense physical training significantly affected FA status of plasma and erythrocyte phospholipids, oxidative stress and antioxidant enzyme activity in women. It also changes metabolic activity of mononuclear cells but not the production of O2 .- i NO2 - in mononuclear cells. The observed differences between the water polo and the football players suggest that the type of regular training may contribute to the altered metabolism in athletes.

Uloga inaktivisane vakcine protiv gripa u regulaciji autoimunskih procesa u eksperimentalnom autoimunskom encefalomijelitisu

Role of inactivated influenza vaccine in regulation of autoimmune processes in experimental autoimmune encephalomyelitis

Influenca je infektivna respiratorna bolest uzrokovana virusima influence. Iako je sama infekcija virusom influence retko bila povezivana sa različitim organ-specifičnim i sistemskim autoimunskim bolestima, neurološke autoimunske manifestacije posle primene influenca vakcine su bile dokumentovane tokom vremena. Multipla skleroza (MS) je hronična, u osnovi autoimunska neurološka bolest koja se najčešće dovodi u vezu sa vakcinacijom. Eksperimentalni autoimunski encefalomijelitis (EAE) je najintenzivnije izučavan mišji model MS, a mijelin oligodendrocitnim glikoproteinom (MOG) indukovan EAE je mnogo sličniji MS kod ljudi nego druge varijante EAE. S obzirom da je rasprava o dobrobiti vakcinacije bolesnika sa MS još uvek otvorena cilj naše studije je bio davanje doprinosa u rasvetljavanju uloge vakcine protiv gripa i njenog efekta na regulaciju EAE kroz praćenje kliničkih znakova, titra serumskih antitela ELISA testom, ekspresije molekula glavnog histokompatibilnog kompleksa (MHC) I i II klase na presecima moždanog tkiva imunohistohemijskim metodama i efekat na kognitivne funkcije (prostorno učenje i pamćenje određivano Morisovim testom vodenog lavirinta; MWM). C57BL/6 miševi su prvo vakcinisani split virion inaktivisanom influenca vakcinom i tri dana kasnije je indukovan EAE sa MOG35-55 peptidom. Ukupno dobijeni rezultati predstavljeni u ovoj disertaciji ukazuju da influenca vakcina nije uticala na produkciju anti- MOG antitela i na razvoj kliničkih znakova EAE. I pored toga što je poremećaj na nivou (auto)imunskog odgovora kod MS bolesnika evidentan, imunski odgovor na većinu uobičajenih virusnih i bakterijskih infekcija izgleda da je očuvan. Rezultati ove studije su pokazali da je titar anti- influenca antitela kod zdravih vakcinisanih miševa i kod onih kod kojih je nakon vakcinacije indukovan EAE, četiri nedelje nakon vakcinacije, bio identičan, što ukazuje na očuvanost humoralnog imunskog odgovora i kod miševa kod kojih je indukovan EAE...

Influenza is a contagious respiratory illness caused by influenza viruses. Influenza infection per se has seldom been associated with various organ-specific and systemic autoimmune diseases but neurological autoimmune phenomena have been reported following influenza vaccine. Multiple sclerosis (MS) is the most common chronic neurological disease of an autoimmune nature that has been viewed in conjunction with vaccination. Experimental autoimmune encephalomyelitis (EAE) is the most extensively studied mouse model of MS and myelin oligodendrocyte glycoprotein (MOG) induced EAE more closely resembles MS than other EAE variants. Since the debate about vaccine safety in patients with MS is still wide open, the objective of our study was to shed more light on and supply additional information about the influenza vaccination effects on regulation of EAE through monitoring of clinical signs, serum antibody titers by using ELISA, expression of MHC (Major Histocompatibility Complex) class I and II molecules in brain tissue by immunohistochemical detection, and its effect on cognitive functions (spatial learning and memory measured by Morris Water Maze test). C57BL/6 mice were first vaccinated with influenza split virion inactivated vaccine and three days later EAE was induced with MOG35-55 peptide. The overall data presented in this study indicate that influenza vaccine had no effect on the production of anti-MOG autoantibodies and the development of clinical signs. Although the unbalanced immune system is evident in patients with MS, immune defense against common viral and bacterial infections appears to be preserved. In this study anti-influenza antibody titers in healthy vaccinated mice and in MOG induced EAE-vaccinated mice, four weeks after vaccination with influenza vaccine have indicated preserved long-lasting antibody response in MOG induced EAE mice. High expression of MHC II and mild-to-low expression of MHC I was detected in mice with EAE...

Karakterizacija i putevi sinhronizacije perifernog biološkog časovnika i steroidogeneze u Lajdigovim ćelijama pacova

Characterization and pathways of synhronization of peripheral biological clock and steroidogenesis in rat Leydig cells

Biološki časovnik organizuje metabolizam i fiziološke procese u cirkadijalne ritmove. Na nivou ćelije, on se sastoji od grupe gena koji preko negativnih povratnih sprega održavaju ritam sopstvene transkripcije, ali regulišu i ritmičnost transkripcije mnogih drugih gena. Iako je poznato da su određeni geni časovnika neophodni za sintezu testosterona i fertilnost mužjaka, još uvek nema preciznih podataka o cirkadijalnoj fiziologiji testosteron produkujućih Lajdigovih ćelija. Ova studija je dizajnirana da definiše (1) cirkadijalni obrazac endokrine funkcije Lajdigovih ćelija uključujući i eksprimiranje gena perifernog časovnika i (2) upletenost LH-cAMP signalizacije u sinhronizaciju ritma Lajdigovih ćelija korišćenjem in vivo modela poremećene cAMP homeostaze (hipogonadotropni hipogonadizam, starački hipogonadizam, pinealektomija) i in vitro stimulacije Lajdigovih ćellija. Rezultati su pokazali cirkadijaln ritam funkcije Lajdigovih ćelija koji se ogleda u vremenski koordinisanoj oscilatornoj produkciji testosterona i intracelularnog cAMP, cirkadijalnom eksprimiranju regulatora (Nur77 i Arr19), steroidogenih elementa (Star/StAR, Cyp11a1 i Cyp17a1), kao i elementa časovnika (Bmal1/BMAL1, Per1/2/3, Cry1/2, Rev-erba/b/REV-ERBA, Rorb, Dec1/2, Dbp i E4bp4). Ritam transkripcije osnovnih gena časovnika kao i ključnog elementa steroidogeneze (Star) se održava i u primarnoj kulturi ovih ćelija. Redukcija cAMP detektovana u Lajdigovim ćelijama pacova sa hipogonadotropnim hipogonadizmom stimuliše transkripciju većeg broja gena časovnika: Per2, Rorb, Rev-erbb, Dec1/2, E4bp4, Ck1e/d, i inhibiše Npas2. Sa druge strane, in vitro stimulacija cAMP-signalizacije povećava transkripciju Per1, Dec1/2, Rorb, Npas2 i E4bp4, i smanjuje transkripciju Rev-erba. Starenje, dovodi do opadanja robusnosti cirkadijalne funkcije Lajdigovih ćelija koja se ogleda u smanjenju oscilacija intracelularnog cAMP, smanjenja amplitude eksprimiranja najvažnijih gena časovnika (Bmal1/BMAL1, Per1/2, Rev-erba/REV-ERBA), gena uključenih u metabolizam holesterola (Lipe, Soat2, Scarb1) i steroidogenih gena, (Star/StAR, Cyp11a1, Cyp17a1, Hsd3b1/2/HSD3B, Hsd17b4)...

Biological clock organizes metabolic and physiological processes in circadian rhythms. At cell level, it consists of group of genes that regulate its own transcription by negative feedback loop, also regulating transcription rhythmicity of other genes. Although, it is known that some clock genes are necessary for testosterone synthesis and male fertility, there is no precise data about circadian physiology of testosterone-producing Leydig cells. This thesis was design to define (1) circadian pattern of endocrine function of Leydig cells, including expression of clock genes, and (2) involvement of LH-cAMP signaling in synchronization of Leydig cells rhythm using in vivo model of disturbed cAMP homeostasis (hypogonadotropic hypogonadism, hypogonadism in aging, pinealectomy) and in vitro Leydig cell stimulation. Results confirmed circadian rhythmicity of Leydig cell function represented by temporal coordination of cyclic testosterone production and intracellular cAMP, circadian expression of regulators (Nur77, Arr19), steroidogenic (Star/StAR, Cyp11a1 i Cyp17a1) and clock elements (Bmal1/BMAL1, Per1/2/3, Cry1/2, Rev-erba/b/REV-ERBA, Rorb, Dec1/2, Dbp, E4bp4). Rhythm in transcription of core clock genes as well as key steroidogenic element (Star) was preserved in primary Leydig cell culture. Reduction in cAMP, detected in Leydig cells from hypogonadotropic hypogonadal rats, stimulated transcription of some clock genes: Per2, Rorb, Rev-erbb, Dec1/2, E4bp4, Ck1e/d, but inhibited Npas2. On the other hand, in vitro stimulation of cAMP signaling increased transcription of Per1, Dec1/2, Rorb, Npas2 and E4bp4, and reduced transcription of Rev-erba. Aging dulled robustness of circadian function of Leydig cells, represented by decline in intracellular cAMP oscillations and amplitude of expression of core clock genes (Bmal1/BMAL1, Per1/2, Rev-erba/REV-ERBA), genes involved in cholesterol metabolism (Lipe, Soat2, Scarb1) and steroidogenic genes (Star/StAR, Cyp11a1, Cyp17a1, Hsd3b1/2/HSD3B, Hsd17b4). Abolishment of melatonin, a main cue that spread information of light regime via cAMP signaling, stimulated expression of clock (Bmal1/BMAL1, Per1/2) and steroidogenic (Star/StAR, Hsd3b/HSD3B) elements...

Funkcionalna analiza interakcija TET-posredovane oksidacije 5-metilcitozina i PARP-zavisne ADP-ribozilacije u procesu demetilacije DNK

Functional analysis of interactions between TET-mediated oxidation 5-methylcytosine and PARP-dependent ADP-ribosylation in the process of DNA demethylation

Važan aspekt u rasvetljavanju procesa demetilacije DNK predstavlja identifikacija faktora koji regulušu aktivnost TET proteina. Cilj ove doktorske disertacije bio je da se ispita funkcionalna veza između TET-posredovane oksidacije 5mC i PARP-zavisne PARilacije u procesu demetilacije DNK, na globalnom i lokalnom nivou. Rezultati ovog istraživanja ukazuju da PARP-1 interaguje sa TET1 i TET2. Pokazano je i da oba proteina podležu in vitro PARilaciji. PARP-1-zavisna PARilacija TET1 proteina negativno utiče na kinetiku aktivnosti TET1 in vitro. Rezultati in cellulo eksperimenata pokazali su da u odsustvu PARP-1 dolazi do porasta ekspresije Tet1 i Tet2, a da prilikom inhibicije PARilacije i u odsustvu PARP-1 dolazi do pada globalnog nivoa metilacije i porasta hidroksimetilacije DNK. Za analizu lokalnih efekata TET-PARP interakcije izabran je gen za hemokin CXCL12 koji učestvuje u brojnim fiziološkim i patološkim procesima pa je ispitivanje regulacije njegove ekspresije važno za buduće terapijske pristupe. Pokazano je da u odsustvu PARP-1 dolazi do povećane ekspresije kao i do pada u metilaciji Cxcl12. Nakon tretmana aktivatorom/inhibitorom TET aktivnosti, u odsustvu PARP-1, ekpresija Cxcl12 je povećana/smanjena. Prisustvo TET1 i TET2 detektovano je na promotoru Cxcl12 uz povećano regrutovanje TET2 u odsustvu PARP-1. U ovoj disertaciji pokazan je inhibitorni uticaj PARP-1 i PARilacije na aktivnost TET enzima u procesu demetilacije DNK na globalnom nivou, a funkcionalnost lokalnih efekata povezanosti TET i PARP enzima u procesu (de)metilacije ustanovljena je na primeru Cxcl12. Demetilacija DNK nalazi se u osnovi brojnih fizioloških i patoloških stanja, zato rasvetljavanje mehanizama regulacije ovog procesa predstavlja važan korak u potencijalnoj primeni stečenih saznanja u medicini.

elucidating the process of DNA demethylation. The aim of this doctoral dissertation was to investigate the functional relationship between TET-mediated oxidation of 5mC and PARP-dependent PARylation in the process of DNA demethylation at both global and local levels. This thesis shows that PARP-1 interacts with TET1 and TET2, and that both TET proteins can undergo in vitro PARylation. PARP-1-dependent PARylation of TET1 negatively affected the kinetics of TET1 activity in vitro. The results of in cellulo experiments revealed that the expression of Tet1 and Tet2 increased in the absence of PARP-1, whereas when PARylation was inhibited or in the absence of PARP-1, there was a decrease in the global level of DNA methylation and an increase in DNA hydroxymethylation. The Cxcl12 gene was selected for the analysis of the local effects of TET-PARP interaction since chemokine CXCL12 participates in numerous physiological and pathological processes, and exploring the regulation of expression of this gene is important for potential clinical intervention. Increased expression and decreased methylation of Cxcl12 were observed in the absence of PARP-1. After treatment with an activator or inhibitor of TET activity in the absence of PARP-1, Cxcl12 expression was respectively increased or decreased. The presence of TET1 and TET2 was detected on the Cxcl12 promoter, with enhanced recruitment of TET2 in the absence of PARP-1. This dissertation describes the inhibitory effects of PARP-1 and PARylation on the activity of TET enzymes in the process of DNA demethylation at the global level, and the local effects of TET-PARP interplay on DNA (de)methylation of Cxcl12 gene. DNA demethylation is at the root of numerous physiological and pathological conditions, and elucidating the mechanisms that regulate this process is an important step in the potential application of acquired knowledge in medicine.

Analiza uloge gena SOX1 i SOX3 u promovisanju malignog fenotipa ćelija glioblastoma

Analysis of the role of SOX1 and SOX3 genes in promotion of malignant phenotype of glioblastoma cells

Glioblastom, glioma tumor gradusa IV, je najčešći maligni tumor mozga kod odraslih i jedan od najsmrtonosnijih tipova tumora. I pored agresivne terapije koja obuhvata hirurško uklanjanje tumora, radio- i hemio-terapiju, prosečno preživljavanje bolesnika sa ovim tipom tumora je oko 15 meseci. Glioblastom (GBM), pored tumorskih ćelija, sadrži i populaciju samo-obnavljajućih tumor-inicirajućih matičnih ćelija (matične ćelije glioblastoma) koje se smatraju odgovornim za nastanak, progresiju, metastaziranje i rezistenciju na terapiju. Geni SOXB1 podgrupe (SOX1, SOX2 i SOX3) kodiraju regulatorne proteine koji imaju značajne uloge u mnogim procesima u toku razvića, kao što su održavanje pluripotentnosti matičnih ćelija i održavanje populacije neuralnih progenitora u pluripotentnom i proliferišućem stanju. Ovi geni imaju i značajne funkcije u procesu karcinogeneze. Ekspresija gena ove podgrupe detektovana je u GBM. Funkcija gena SOX2 je dobro proučena kod ovog tipa tumora; pokazano je da ovaj gen promoviše maligni potencijal ćelija GBM i neophodan je za održavanje tumorogenog potencijala matičnih ćelija glioblastoma. Za razliku od gena SOX2, uloga gena SOX1 i SOX3 u ćelijama GBM još uvek nije dovoljno istražena. Stoga, u okviru ove doktorske disertacije analizirana je uloga ovih gena u ćelijama glioblastoma. Dobijeni rezultati pokazuju da ćelijske linije GBM eksprimiraju gen SOX1. Pored toga, u uslovima utišane ekspresije proteina SOX1 detektovano je smanjenje proliferativnog kapaciteta, vijabilnosti i migratornog potencijala U251 ćelija GBM, kao i povećanje broja ovih ćelija u senescenciji. Nakon dediferencijacije ćelijskih linija GBM detektovano je povećanje ekspresije gena SOX1 u poređenju sa ekspresijom uočenom u njihovim parentalnim ćelijama. Nivo ekspresije gena SOX1 povećan je u kulturama matičnih ćelija glioblastoma u poređenju sa ekspresijom ovog gena u imortalizovanim U87 i U251 ćelijama; pri diferencijaciji ovih kultura uočeno je smanjenje ekspresije gena SOX1. Utišavanje ekspresije gena SOX1 u GNS166 kulturi matičnih ćelija GBM dovodi do smanjenja proliferativnog kapaciteta i vijabilnosti ovih ćelija. Rezultati dobijeni u ovoj disertaciji pokazuju da ćelijske linije GBM i GBM tumori eksprimiraju gen SOX3. Ekspresija gena SOX3 povećana je kod većine uzoraka GBM tumora u odnosu na ekspresiju detektovanu u netumorskom moždanom tkivu. Nivo ekspresije ovog gena ne korelira sa preživljavanjem obolelih. U uslovima povećane ekspresije proteina SOX3 uočeno je povećanje proliferativnog kapaciteta, vijabilnosti, migratornog i invazivnog potencijala ćelija GBM. Povećanje ekspresija proteina SOX3 ne utiče na aktivnost WNT signalnog puta, povećava aktivnost Hedgehog signalnog puta i ima inhibitorni efekat na proces autofagije u ćelijama GBM. Radioterapija γ zračenjem ne dovodi do promene ekspresije gena SOX3 u ćelijama GBM, dok efekat temozolomida na ekspresiju ovog gena zavisi od koncentracije (doze) temozolomida i tipa ćelija GBM. Detektovano je da je dediferencijacija ćelijskih linija poreklom od GBM praćena povećanjem ekspresije gena SOX3, kao i da je nivo ekspresije ovog gena povećan u kulturama matičnih ćelija glioblastoma u poređenju sa ekspresijom u imortalizovanim U87 i U251 ćelijama GBM. Pri diferencijaciji ovih kultura uočeno je smanjenje ekspresije gena SOX3. Na osnovu dobijenih rezultata može se zaključiti da geni SOX1 i SOX3 promovišu maligni fenotip ćelija GBM, kao i da visoka ekspresija ova dva gena korelira sa nediferenciranim stanjem matičnih ćelija GBM, što ukazuje na potencijalnu ulogu gena SOX1 i SOX3 u održavanju populacije matičnih ćelija glioblastoma. Jedna od perspektivnih strategija u lečenju malignih bolesti je diferencijaciona terapija koja ima za cilj diferencijaciju ćelija tumora što vodi ka smanjenju njihovog malignog potencijala. Stoga, u okviru ove disertacije analiziran je i efekat all-trans retinoične kiseline (RK), jednog od najčešće primenjivanih agensa u diferencijacionoj terapiji, na osobine U251 ćelija GBM. Rezultati ove analize su pokazali da RK smanjuje vijabilnost, menja način migracije i dovodi do promene morfologije ćelija GBM, ali ne indukuje neuralnu diferencijaciju ovih ćelija. Takođe, efekat RK na sposobnost adhezije U251 ćelija GBM za komponente ekstraćelijskog matriksa zavisi od primenjene koncentracije ovog agensa. Na osnovu dobijenih rezultata može se zaključiti da su potrebna dodatna istraživanja pre potencijalne primene RK u terapiji GBM.

one of deadliest cancers. Despite aggressive treatment, including surgical resection, chemotherapy and radiation, the median survival of patients with glioblastoma is 15 months. A growing body of evidence indicates that GBM contains a population of self- renewing tumor-initiating cells (glioblastoma stem cells - GSCs) that drive tumor initiation, propagation, metastasis and therapy resistance. SOXB1 genes (SOX1, SOX2 and SOX3) encode transcription regulators with important roles in embryonic development and carcinogenesis. Literature date revealed that SOXB1 genes are expressed in GMB tumor samples. The role of one member of this group, SOX2 gene, is well documented in GBM. It was shown that SOX2 gene promotes malignant potential of GBM cells and it is mandatory for maintenance of tumorogenicity of GSCs. Since the function of SOX1 and SOX3 genes in GBM still remains to be established, the aim of this thesis was to analyze the role of these genes in GBM. Obtained results demonstrated that all analyzed GBM cell lines express SOX1. Downregulation of this gene expression decreases proliferation, viability and migration, and induces senescence of U251 cells. Furthermore, dedifferentiation of GBM cells is accompanied by increase of SOX1 level compared to that in parental cells. Expression of this gene was significantly increased in patient-derived GSC cultures compared to that in U87 and U251 cells; downregulation of SOX1 gene expression was seen upon differentiation of GSCs. In addition, knock-down of this gene expression reduced proliferation and viability of GSCs. Results obtained in this thesis reveal SOX3 expression in GBM cell lines and tumor tissue; the expression of this gene was elevated in the most of analyzed GBM samples compared to expression levels detected in non-tumoral brain tissues. A high SOX3 expression was not associated with the overall survival of GBM patients. Ectopic overexpression of this gene increased proliferation, viability, migration and invasion of GBM cells. Moreover, it was found that SOX3 protein overexpression had no influence on WNT signalling but increased activity of Hedgehog signaling pathway and decreased autophagy in GBM cells. Radiotherapy had no impact on SOX3 expression in GBM cells, while effect of temozolomide is dose- and cell type-specific. Dedifferentiation of GBM cells was accompanied by increase of SOX3 level compared to that in parental cells and expression of this gene was increased in patient-derived GSC cultures compared to that in U87 and U251 cells. Moreover, the expression of SOX3 was decreased upon differentiation of GSC cultures. Overall, it could be concluded that SOX1 and SOX3 genes promote malignant phenotype of GBM cells and that a high-level of expression of these genes is associated with undifferentiated state of GBM cells and it is important for maintaining of GSCs. One of the new therapeutic approaches for treating cancer is differentiation therapy which aims to differentiate cancer cells in order to suppress their malignant potential. Therefore, the one of the aims of this thesis was to analyze the effects of all-trans retinoic acid (ATRA), the prototype of a differentiation therapy agent, on features of human glioblastoma U251 cells. Obtained results demonstrated that ATRA treatment reduced viability, affected the mode of migration and induced morphological changes of U251 cells. The effect of ATRA on the cell-matrix adhesion capability of U251 cells was dose- dependent. However, this agent had no impact on neural differentiation of GBM cells. Based on obtained results it could be concluded that further studies are warranted before ATRA could be considered for the treatment of glioblastoma.

Evolucija starenja kod laboratorijskih populacija Acanthoscelides obtectus: uloga mitohondrija i oksidativnog stresa

Evolution in laboratory populations of Acanthoscelides obtectus: role of mitochondria and oxidative stress

Ključna pretpostavka evolucione teorije starenja jeste da opadanje uzrasno-specifičnog preživljavanja organizama predstavlja rezultat smanjenja intenziteta prirodne selekcije nakon početka reprodukcije. U osnovi evolucije starenja nalaze se dva populaciono- genetička mehanizma: akumulacija mutacija (AM) i antagonistička plejotropija (AP). U skladu sa pretpostavkama evolucione teorije starenja, kod eksperimentalnih populacija pasuljevog žiška (Acanthoscelides obtectus) selektovanih više od 170 generacija za ranu (E režim) ili kasnu (L režim) reprodukciju, uočena je divergencija u dužini života i drugim preadultnim i adultnim osobinama životne istorije. Evolucija veće dužine života u L režimu u saglasnosti je sa pretpostavkama AP modela, dok u evoluciji kraćeg života E žižaka važnu ulogu ima akumulacija mutacija. Tretiranjem E i L žižaka pesticidima (parakvat i tebufenpirad) koji generišu slobodne radikale, potvrđena je pozitivna korelacija između dužine života i otpornosti na oksidativni stres. Budući da mitohondrija predstavlja glavno mesto sinteze energije i slobodnih radikala, u ovoj tezi je istraženo da li je uzrasno-specifična selekcija oblikovala varijabilnost mtDNK, nDNK i njihovih epistatičkih interakcija. Genotipizacijom mitohondrijskog COI gena i mikrosatelitskih lokusa pokazana je efikasnost uzrasno-specifične selekcije u međupopulacionoj divergenciji mitohondrijskog i jedarnog genskog pula kod pasuljevog žiška. Odvijanje procesa mito-jedarne koevolucije analizirano je korišćenjem mito-jedarnih linija u kojima su E i L mitohondrijski genomi kombinovani sa E i L jedarnim genomima. Pokazano je da su linije sa narušenim mito-jedarnim interakcijama imale niže aktivnosti mitohondrijskih kompleksa elektron transportnog lanca i lošiju preadultnu performansu.

According to the evolutionary theory of ageing, age-specific decline in survival is the result of decreasing intensity of natural selection after the onset of reproduction. The evolution of ageing may be explained by two different, but not mutually exclusive, genetic mechanisms: antagonistic pleiotropy (AP) and mutation accumulation (MA). In accordance with evolutionary theory of ageing, two sets of the seed beetle (Acanthoscelides obtectus) experimental lines selected for more than 170 generations for early (E regime) or late (L regime) reproduction, show divergence in longevity and ageing patterns, as well as in other preadult and adult life history traits. Evolution of long life in L regime is predominantly based on antagonistic pleiotropy gene effects, while mutation accumulation plays important role in evolution of short life in E regime. By exposure of E and L beetles to two pesticides (paraquat and tebufenpyrad) that generate free radicals, it was confirmed that extended longevity in L beetles (especially in females) was associated with higher oxidative stress resistance. Additionally, since mitochondria are main site of free radical and energy production, effects of age-specific laboratory selection on mtDNA, nDNA and their epistatic interaction were assessed. Genotyping of mitochondrial COI gene and nuclear microsatellite loci showed that selection in E and L regimes led to divergent evolution of mtDNA and nDNA gene pools. Mitonuclear coevolution was investigated using mitonuclear introgression lines in which E and L mitochondrial genomes were expressed in both E and L nuclear background. It was found that lines with disrupted mitonuclear interactions had lower activity of the mitochondrial electron transport chain complexes and lower preadult performance.

Hibridizacija, morfološka varijabilnost i stabilnost razvića velikih mrmoljaka (Triturus spp.)

Hybridization, morphological variability and developmental stability of large-bodied newts (Triturus spp.)

Hibridizacija može imati različite evolucione i ekološke ishode. Veliki mrmoljci (Triturus spp.) formiraju hibridne zone u kojima učestvuju vrste različitog stepena filogenetske srodnosti i predstavljaju pogodan model sistem za ispitivanje procesa i mehanizama hibridizacije i njenih ishoda. Vrste T. ivanbureschi i T. macedoncius hibridizuju u centralnoj i istočnoj Srbiji formirajući specifičnu zonu u kojoj dolazi do zamene vrsta i asimetrične introgresije mitohondrijske DNK uz postojanje vijabilnih hibridnih populacija. Relativno sličan scenario sreće se u zapadnoj Francuskoj gde T. cristatus i T. marmoratus hibridizuju i produkuju sterilne hibride. Ciljevi ove doktorske disertacije su određivanje parametara koji su doprineli formiranju i održavanju hibridne zone, kao i procena uticaja hibridizacije na vijabilitet, morfološku varijabilnost (sa posebnim osvrtom na složeni životni ciklus i metamorfozu) i stabilnost razvića hibrida i roditeljskih vrsta. Podaci su prikupljani tokom tri godine intra- i interspecijskog eksperimentalnog ukrštanja, kao i analizom materijala iz prirodnih populacija. Praćeni su parametri rane životne istorije, ontogenija i disparitet oblika tela i uticaj hibridizacije na stabilnost razvića. Morfološka varijabilnost i stabilnost razvića procenjeni su metodama geometrijske morfometrije. Triturus macedonicus odlikuje se većim reproduktivnim potencijalom u odnosu na T. ivanbureschi, što je moglo da omogući početak širenja areala. Hibridizacija smanjuje vijabilitet jaja, ali ne i embriona. Hibridi se morfološki razlikuju od obe roditeljske vrste i uglavnom su intermedijarni. Larveno razviće je kanalisano, dok su stupnjevi kada jedinke dolaze u kontakt sa novom sredinom (izvaljivanje larvi i metamorfoza) izuzetno varijabilni. Hibridizacija ne utiče na stabilnost razvića velikih mrmoljaka.

Hybridization can have various evolutionary and ecological outcomes. Large- bodied newts (Triturus spp.) hybridize mutually regardless of the degree of genetic similarity. This characteristic makes them a good model system to explore hybridization, as well as hybridization outcomes. Triturus ivanbureschi and T. macedonicus have specific species displacement scenario which includes hybridization in Serbia, wide zone of asymmetric mtDNA introgression and viable hybrid populations. Relatively similar species displacement scenario is found in France, where T. cristatus and T. marmoratus hybridize, but their hybrids are sterile. The main aims of this doctoral dissertation are to establish parameters which enabled formation and maintenance of hybrid and introgressive zone and to estimate effects of hybridization on viability, morphological variation (with special emphasis on complex, biphasic life cycle and metamorphosis) and developmental stability. The data was collected during a three-year hybridization experiment, as well as from natural populations. The early life history parameters, ontogeny and disparity of body shape, and the effects of hybridisation on developmental stability were recorded. Morphological variability and developmental stability were analysed by geometric morphometrics. Triturus macedonicus have higher reproductive output compared to T. ivanbureschi which could be an advantage at the initial species contact. Hybridization reduces egg viability, but not the viability of embryos. Hybrids are morphologically divergent from parental species, with mostly intermediate position relative to parental species. Larval development is highly canalized, while the ontogenetic stages when newts confront new environment (hatching and metamorphosis) are highly variable. Hybridization does not affect developmental stability.

Taksonomsko-faunistička studija leptira (Insecta : Lepidoptera) Fruške Gore

Taxonomic-faunistical study of butterflies and moths (Insecta : Lepidoptera) of mt. Fruška Gora

U studiji je dat prikaz jedanaestogodišnjih istraţivanja insekata iz reda Lepidoptera na Fruškoj gori. Registrovane su 934 vrste leptira i moljaca. Za 382 vrste Lepidoptera ili 40,89% od ukupnog broja vrsta izvršena je taksonomska verifikacija analizom hitinskih armatura genitalnih aparata. U periodu od 2001. do 2011. godine sakupljani su leptiri i moljci uglavnom uz pomoć svetlosne klopke (ţivine sijalice TEŢ WTF od 250 W, “Philips Ml“ od 100, 160, 250 i 400 W i petromaks lampe od 400 W), iza koje je postavljeno belo pamuĉno platno. 934 zabeleţene vrste su svrstane u 22 superfamilije, 47 familija i 564 rodova. Broj od 934 vrste Lepidoptera predstavlja u odnosu na evropsku faunu leptira (8478 vrsta) 11,01%. Sve vrste leptira i moljaca Fruške gore svrstane su u 7 zoogeografskih kategorija. PreovlaĊuju evroazijske vrste (449 vrsta ili 48,07% od ukupnog broja vrsta). Evropskih vrsta je 236 (25,27% od ukupnog broja vrsta). Mediteransko-azijski elementi su zastupljeni sa 162 vrste ili 17,34% od ukupnog broja vrsta. Holarktiĉki elementi zastupljeni su sa 44 vrste ili sa 4,71% od ukupnog broja vrsta, a palearktiĉki sa 25 vrsta ili 2,68% od ukupnog broja vrsta. Paleotropsko-suptropski elementi zastupljeni su sa 10 vrsta ili sa 1,07% od ukupnog broja vrsta. Kosmopolitskih vrsta je 8 vrsta ili 0,86% od ukupnog broja vrsta. Sve evidentirane vrste u ovoj studiji grupisane su u pet skupova: mali moljci, veliki moljci, dnevni leptiri, zemljomerke i sovice. Ukupno 287 vrsta malih moljaca svrstano je u 16 superfamilija i 31 familiju. Više od 5% vrsta malih moljaca Evrope (5,16%) je zastupljeno na Fruškoj gori. Veliki moljci (Lasiocampidae, Saturniidae, Sphingidae, Drepanidae, Notodontidae, Lymantriidae i Arctiidae) su zastupljeni na Fruškoj gori sa 77 vrsta . Više od ĉetvrtine vrsta velikih moljaca Evrope (27,5%) je zastupljeno na Fruškoj gori. Dnevni leptiri (Hesperiidae, Papilionidae, Pieridae, Lycaenidae i Nymphalidae) su zastupljeni na Fruškoj gori sa 115 vrsta. Više od ĉetvrtine vrsta dnevnih leptira Evrope (26,13%) je zastupljeno na Fruškoj gori. Zemljomerke su zastupljene na Fruškoj gori sa 187 vrsta. Oko petine vrsta zemljomerki Evrope (20,43%) je zastupljeno na Fruškoj gori. Sovice (Noctuidae, Pantheidae, Nolidae) su zastupljene na Fruškoj gori sa 268 vrsta. Više od petine vrsta sovica Evrope (20,87%) je zastupljeno na Fruškoj gori. Najbrojnija je grupa malih moljaca sa 287 zabeleţenih vrsta ili sa 30,73% od ukupnog broja utvrĊenih vrsta. Zatim sledi grupa sovica sa 268 zabeleţenih vrsta ili 28,69% od ukupnog broja utvrĊenih vrsta. Na trećem mestu po zastupljenosti nalazi se grupa zemljomerki sa 187 zabeleţenih vrsta ili sa 20,02% od ukupnog broja utvrĊenih vrsta. Slede dnevni leptiri (sa 115 vrsta ili 12,31% od ukupnog broja vrsta) i veliki moljci (sa 77 vrsta ili 8,24% od ukupnog broja vrsta). Na Fruškoj gori je zabeleţeno 129 vrsta Lepidoptera (13,80% od ukupnog broja vrsta) koje mogu naneti štetu lišćarskom i ĉetinarskom drveću. Istraţivanjima na Fruškoj gori identifikovana je 61 vrsta Lepidoptera iz grupe migratornih vrsta, tj. selica. Sezonskih selica prvog reda ima 7 vrsta, sezonskih selica drugog reda 1 vrsta, selica iseljenika (lokalnih selaca prvog i drugog reda) 36 vrsta, selica raseljenika (povremenih selaca) 10 vrsta, selica raseljenika (vrsta koje proširuju svoj areal) 2 vrste, dok mogućih selica ima 5 vrsta. U odnosu na zabeleţeni broj od 934 vrste leptira, procenjuje se da je poznato svega oko jedne trećine vrsta koje bi se mogle otkriti na Fruškoj gori. PronaĊene su 133 nove vrste iz reda Lepidoptera za faunu Srbije. Prvi put u Srbiji, ali i u zemljama u sastavu bivše SFRJ je kao prilog poznavanju faune Lepidoptera u ovoj studiji dat detaljan prikaz hitinskih armatura genitalnih aparata sa opredeljujućim taksonomskim karakteristikama za 250 vrsta (26,76% od ukupnog broja vrsta) uglavnom iz grupe malih moljaca i za nekoliko izdvojenih taksona ostalih grupa. Dat je uporedni prikaz brojnosti vrsta po familijama i superfamilijama za Frušku goru, Timoĉku krajinu, Evropu, Rumuniju, rumunski Banat i MaĊarsku. Lepidoptera su veoma slabo istraţeni na pojedinim podruĉjima Fruške gore. Ovo se prvenstveno odnosi na podruĉje juţno od Dunava oko Neština, Suseka, Banoštora i Ĉerevića, deo od Sremskih Karlovaca do Petrovaradina i Ĉortanovaca, od Krušedola i Neradina do Iriga sa juţne strane Fruške gore i od Vrdnika do Leţimira sa jugozapadne strane (izuzimajući podruĉje oko grgurevaĉkog lovaĉkog doma, selo Grgurevci). Neophodno je da se faunistiĉka i ekološka istraţivanja nastave. Veliki broj lokaliteta je nedovoljno istraţen, na pojedinim mikrolokalitetima istraţivanja nisu ni otpoĉela, a potencijalno su na njima se mogu naći zanimljivi nalazi.

This thesis presents results of an eleven-year research on insects belonging from order Lepidoptera on Mt. Fruška Gora. Total of 934 species were recorded. For 382 species Lepidoptera, or 40.89% of the total number of species, a taxonomic verification has been performed by the analysis of chitinous armatures of genital apparatus. In the 2001-2011 period, the specimens were collected mostly by light trap (mercury bulb TEŢ WTF of 250W, “Philips Ml“ of 100, 160, 250 and 400W, and Petromax lamp of 400W), with a white cotton sheet propped in behind. 934 recorded species of Lepidoptera belong to 22 superfamilies, 47 families and 564 genera. The number of 934 species represents 11,01% of European fauna of Lepidoptera (8,478 species). The Lepidoptera species from Mt. Fruška Gora are classified into seven zoogeographical categories. Eurasian species are prevailing (449 species or 48.07% of the total number of species). We recorded 236 European species (25.27% of the total number of species) and 162 Mediterranean-Asian species (17.34% of the total number of species). Holarctic elements are represented by 44 species (4.71% of the total number of species), Palearctic elements by 25 species (2.68% of the total number of species), while Paleotropical-subtropical elements are represented by 10 species (1.07% of the total number of species). Finally, cosmopolitan elements are represented by eight species (0.86% of the total number of species). Within the research, all registered species were classified into five groups: Microlepidoptera (micromoths), macromoths, butterflies, geometrid moths and owlet moths. 287 species of micromoths are classified into 16 superfamilies and 31 families. More than 5% of European species of micromoths (5.16%) are present on Mt. Fruška Gora. Macromoths (Lasiocampidae, Saturniidae, Sphingidae, Drepanidae, Notodontidae, Lymantriidae and Arctiidae) are represented on Mt. Fruška Gora with 77 species. More than one quarter of European species of macromoths (27.5%) are recorded on Mt. Fruška Gora. Butterflies (Hesperiidae, Papilionidae, Pieridae, Lycaenidae and Nymphalidae) are represented on Mt. Fruška Gora with 115 species, which is more than a quarter of the butterfly species of Europe (26.13%). Geometrid moths are represented by 187 species on Mt. Fruška Gora, which is about one fifth of European species of Geometridae (20.43%). Owlet moths (Noctuidae, Pantheidae and Nolidae) are represented on Mt. Fruška Gora by 268 species, which is more than a fifth of the European owlet moth species (20.87%). The micromoths are the most numerous group with 287 recorded species, or 30.73% of the total number of species recorded on Mt. Fruška Gora. They are followed by the group of owlet moths with 268 recorded species (28.69% of the total number of species), geometrid moths with 187 recorded species (20.02% of the total number of species), butterflies (with 115 species or 12.31% of the total number of species), and macromoths (with 77 species or 8.24% of the total number of species). Of all Lepidoptera species recorded in Mt. Fruška Gora, 129 (or 13.80% of the total number of species) may be harmful for deciduous and coniferous trees. Research also revealed 61 species of Lepidoptera belonging to the group of migratory species. Seven species are first-order seasonal migratory species (eumigrators), one seasonal species is second-order migratory species (paramigrator), and 36 species are emigrants (first- and second-order local migrants). Finally, among the dismigrators there are two invasive species that expand their areas and five species that are possibly migratory. Regarding the recorded number of 934 species of Lepidoptera, it is estimated that this is just one third of the species that might exist on Mt. Fruška Gora and the rest could be recorded in the future. From Lepidoptera species recorded, 133 are new for the fauna of Serbia. For the first time in Serbia, but also in other former Yugoslav countries, a detailed analysis of chitinous armatures of genital apparatus with presenting specific taxonomic characteristics for 250 species (26.76% of the total number of recorded species), mostly for micromoths and some selected taxa. A comparative overview is given of species population number within families and superfamilies in investigated Fruška Gora area, as well as for Timoĉka Krajina, Europe, Romania, Romanian Banat, and Hungary. The order Lepidoptera is not sufficiently investigated in some areas of Mt. Fruška Gora. This primarily refers to the area south of the Danube River around Neštin, Susek, Banoštor, and Ĉerević, to the part between Sremski Karlovci, Petrovaradin and Ĉortanovci, from Krušedol and Neradin to Irig on the south slope of Mt. Fruška Gora, and from Vrdnik to Leţimir on the southwest slope (except the area around hunter’s house in the village of Grgurevci). Therefore, it is necessary to continue further faunistic and ecological research. A large number of localities are not explored enough, while at some microlocalities investigations have not even started, although they could be very interesting with possible new findings.

Molekularni mehanizmi antioksidativnog dejstva ekstrakta kičice (Centaurium erythraea RAFN) u eksperimentalnom modelu dijabetesa pacova

Molecular mechanisms of the antioxidative effect of centaury (Centaurium erythraea RAFN), extract in the experimental model of diabetes in rats

Kičica se u Srbiji tradicionalno koristi za lečenje različitih oboljenja, uključujući i dijabetes. Cilj ovog rada bio je rasvetljavanje mehanizama antioksidativnog i protektivnog dejstva ekstrakta kičice (EK) koji doprinose očuvanju strukture i funkcije β-ćelija pankreasa i ublažavanju komplikacija u dijabetesu. Efekti EK analizirani su na eksperimentalnom modelu dijabetesa pacova izazvanom streptozotocinom (STZ) kao i na Rin-5F β-ćelijama izloženim oksidativnom stresu primenom vodonik peroksida, natrijum nitroprusida ili STZ-a. EK je ispoljio antidijabetogeno dejstvo uočeno kao sniženje nivoa glukoze/glikohemoglobina, poboljšanje lipidnog statusa i povećanje nivoa insulina u cirkulaciji dijabetičnih pacova što se može pripisati detektovanom uticaju EK na oc uvanje strukture Langerhansovih ostrvaca i povec anju mase β-c elija koje produkuju insulin. ntioksidativni efekat EK na β-c elije ogledao se u redukovanju oksidativnih os tec enja, modulisanju aktivnosti i ekspresije enzima antioksidativne zaštite (C T, SOD, GPx i GR) i uravnotežavanju aktivnosti redoks-senzitivnih faktora (NFκB, Nrf-2, Sp1, FOXO3 ) uključenih u regulaciju transkripcije gena za antioksidativne enzime. Dodatni doprinos EK boljem preživljavanju β-ćelija i sekreciji/ekspresiji insulina ostvaren je finim promenama u aktivnosti Akt, ERK i p38 kinaza kao i PDX-1 i MafA proteina. Pored toga, antioksidativni efekat EK kod dijabetičnih životinja ogledao se u zaštiti eritrocita, jetre i bubrega od gliko- oksidativnih oštećenja što je doprinelo poboljšanju njihovih funkcionalnih parametara. Opisani efekti EK ukazuju na značaj daljeg razjašnjavanja mehanizama antioksidativnog i protektivnog dejstva EK i njegovih komponenti u cilju potencijalnog terapijskog delovanja u dijabetesu.

Centaury is traditionally used in Serbia for treating different diseases, including diabetes. This study aimed to elucidate the mechanisms of the antioxidant and protective effects of the centaury extract (CE) that preserve the structure and function of pancreatic β-cells and alleviate diabetic complications. The effects of the CE were analyzed on the experimental model of streptozotocin (STZ)-induced diabetic rats and in Rin-5F β-cells exposed to oxidative stress after exposure to hydrogen peroxide, sodium nitroprusside or STZ. The CE exhibited an anti-diabetic effect, observed as a decrease in hyperglycemia and glycohemoglobin concentration, an improved lipid status, and as increased insulin level in the circulation of diabetic rats. These effects can be attributed to the detected impact of the CE on the preservation of the structure of the islets of Langerhans, and on the increase in insulin producing β-cell mass. The antioxidant effect of the CE on β- cells was manifested as a reduction in oxidative damage, changed activities and expression of antioxidant enzymes, CAT, SOD, GPx and GR, and as a readjustment of the activities of redox-sensitive factors, NFκB, Nrf-2, Sp1, FOXO3A, involved in the regulation of gene transcription of antioxidant enzymes. Additional contributions of the CE to improved β-cell survival and insulin secretion/expression were achieved by the fine-tuning of the activities of Akt, ERK and p38 kinases and transcription regulators, PDX-1 and MafA. The antioxidant effect of the CE in diabetic animals was also reflected on increased protection of erythrocytes, liver and kidneys from glyco-oxidative damage, which contributed to the improvement of their functional parameters in diabetic rats. The described effects of the CE point to the importance of further clarification of the mechanisms of the antioxidant and general protective effects of the CE and its components in view of its potential use for therapeutic intervention in diabetes.

Odgovor hipotalamo-hipofiznog sistema pacova na izoflavone soje : morfofunkcionalna studija

Effects of soy isoflavones on the hypothalamic-pituitary system in rats: a morphofunctional study

Izoflavoni soje, genistein i daidzein, se sve više koriste u preventivi i alternativnoj terapiji različitih patoloških stanja. Uticaj izoflavona soje na neuroendokrini sistem bio je, a i danas je, predmet istraživanja različitih studija. Veliki broj literaturnih podataka govori o uticaju izoflavona soje na reproduktivni sistem, s obzirom na njihovu estogensku i anti-estrogensku aktivnost. Ipak, podaci o njihovom uticaju na somatotropni i adrenokortikotropni sistem oskudni su, a neophodni, imajuću u vidu značaj ovih sistema za normalan razvoj i funkcionisanje organizma. Takoñe, mnogobrojne in vitro studije obezbeñuju samo limitirane informacije o potencijalnim efektima in vivo. Cilj ove studije je bio da se ispita uticaj hroničnih tretmana estradiol- diproprionatom, genisteinom i daidzeinom na histološke, stereološke i biohemijske parametre somatotropnog i adrenokortikotropnog sistema orhidektomisanih adultnih pacova. Na početku eksperimenta adultni pacovi Wistar soja, stari 3 meseca, su podeljeni u dve eksperimentalne grupe: lažno orhidektomisanu (Sham) i orhidektomisanu (Orx) grupu. Dve nedelje nakon sham operacije ili orhidektomije pacovi su podeljeni u sedam eksperimentalnih grupa. Prvu (Sham1) grupu su sačinjavale sham operisane životinje subkutano (s.c.) tretirane odgovarajućom zapreminom sterilnog maslinovog ulja, medijumom estradiol-diproprionata (medijum I). Drugu (Orx1) grupu su sačinjavale orhidektomisane životinje s.c. tretirane odgovarajućom zapreminom medijuma I. U trećoj (Orx1+E) grupi orhidektomisane životinje su s.c. tretirane estradiol- dipropionatom u medijumu I (0,6 mg/kg t.m.). Četvrta (sham operisana - Sham2) i peta (orhidektomisana - Orx2) grupa s.c. su tretirane odgovarajućom zapreminom mešavine alkohola i sterilnog maslinovog ulja, medijumom genisteina i daidzeina (u odnosu 1:2; medijum II). Šestu (Orx2+G) grupu su sačinjavale orhidektomisane životinje s.c. tretirane genisteinom u medijumu II (30 mg/kg t.m.), dok je sedma (Orx2+D) grupa orhidektomisanih pacova s.c. tretirana daidzeinom u istom medijumu (30 mg/kg t.m.). Sve grupe životinja su dobijale tretman tokom tri nedelje (hroničan tretman), a žrtvovane su 24 h nakon poslednje primljene doze. Pre početka i nakon završenog tretmana izmerene su telesne mase životinja svih eksperimentalnih grupa, dok su mase hipofiza izmerene nakon žrtvovanja. Za lokalizaciju arkuatnog (Arc) i periventrikularnog (Pe) jedra, paravenrikularnog jedra (PVN) i eminencije medijane (ME) korišćeno je histološko bojenje krezil-ljubičastom bojom. Za obeležavanje oslobañajućih hormona hipotalamusa (oslobañajućeg hormona hormona rasta–GHRH, somatostatina-SS, kortikotropnog oslobañajućeg hormona-CRH) i ćelija adenohipofize (somatotropnih–GH i adrenokortikotropnih-ACTH) korišćeno je imunohistohemijsko bojenje. Histološki i imunohistohemijski obojeni preseci hipotalamusa, kao i imunohistohemijski obojeni preseci hipofiza stereološki su analizirani, uz pomoć newCAST stereološkog softverskog paketa. Dobijene su vrednosti: volumena Arc i Pe jedra i PVN, volumenske gustine SS neurona, volumena hipofize, volumenske i numeričke gustine GH i ACTH ćelija, apsolutnog broja i volumena GH i ACTH ćelija. Koncentracija GH i ACTH u cirkulaciji odreñena je biohemijskim metodama. Dobijeni podaci su statistički obrañeni. Orhidektomija je dovela do značajnog smanjenja (p<0.05) telesne mase adultnih pacova u odnosu na Sham grupu pacova. Hroničan tretman estradiol-diproprionatom je doveo do značajnog smanjenja (p<0.05) telesne mase u odnosu na Orx1 grupu pacova. Hroničan tretman genisteinom ili daidzeinom nije doveo do promene telesnih masa u odnosu na Orx2 grupu pacova. Orhidektomija adultnih pacova je dovela do smanjenja (p<0.05) apsolutne mase hipofize, dok je volumen hipofize ostao nepromenjen u odnosu na Sham grupu pacova. Tretman estradiol-diproprionatom je doveo do povećanja (p<0.05) apsolutne mase i volumena hipofize u odnosu na Orx1 grupu pacova. Tretman genisteinom je povećao (p<0.05) apsolutnu masu i volumen hipofize u odnosu na Orx2 grupu pacova. Tretman daidzeinom nije doveo do promena mase i volumena hipofize u odnosu na Orx2 grupu pacova. Orhidektomija je dovela do značajnog povećanja (p<0.05) volumena Arc jedra; i do značajnog smanjenja (p<0.05): imunoobojenosti GHRH neurosekreta u ME, volumenske gustine SS neurona u Pe jedru, imunoobojenosti SS neurosekreta u ME kao i volumenske i numeričke gustine GH ćelija; a nije uticala na volumen Pe jedra, apsolutni broj, volumen i intenzitet obojenosti GH ćelija i nivo GH u cirkulaciji, u odnosu na odgovarajuće parametre Sham grupe pacova. Nakon tretmana estradiol- diproprionatom došlo je do povećanja (p<0.05) volumena Arc i Pe jedra, volumenske gustine SS neurona Pe jedra, intenziteta obojenosti SS neurosekreta u ME, inteziteta obojenosti GH ćelija i nivoa GH u cirkulaciji; i do smanjenja (p<0.05) volumenske i numeričke gustine GH ćelija; dok tretman nije uticao na intenzitet obojenosti GHRH neurosekreta u ME, apsolutni broj i volumen GH ćelija; sve u odnosu na odgovarajuće parametre Orx grupe. Hroničan tretman genisteinom je doveo do povećanja (p<0.05): volumena Arc jedra, intenziteta obojenosti GHRH neurosekreta u ME, volumenske gustine SS neurona Pe jedra, količine SS neurosekreta u ME, volumenske i numeričke gustine GH ćelija, apsolutnog broja i intenziteta obojenosti GH ćelija i nivoa GH u cirkulaciji; a nije uticao na volumen Pe jedra i volumen GH ćelija; sve u odnosu na odgovarajuće parametre Orx grupe. Hroničan tretman daidzeinom je doveo do povećanja (p<0.05): intenziteta obojenosti GHRH neurosekreta u ME, volumenske gustine GH ćelija, volumena i intenziteta obojenosti GH ćelija; do smanjenja (p<0.05) numeričke gustine i apsolutnog broja GH ćelija; a nije uticao na volumen Arc i Pe jedra, volumensku gustinu SS neurona Pe jedra, intenzitet obojenosti SS neurosekreta u ME i nivo GH u cirkulaciji; sve u odnosu na odgovarajuće parametre Orx2 grupe. Orhidektomija je dovela do značajnog povećanja (p<0.05): volumena PVN, volumenske gustine CRH neurona i duplo obeleženih CRH/cFos neurona u PVN, intenziteta obojenosti CRH neurosekreta u ME i nivoa ACTH u cirkulaciji; do značajnog smanjenja (p<0.05) volumenske i numeričke gustine ACTH ćelija i intenziteta obojenosti ACTH; dok apsolutni broj i volumen ACTH ćelija nisu promenjeni; sve u odnosu na odgovarajuće parametre Sham grupe pacova. Hroničan tretman estradiol-diproprionatom doveo do značajnog povećanja (p<0.05) volumenske gustine CRH/cFos neurona, intenziteta obojenosti CRH neurosekreta u ME, apsolutnog broja ACTH ćelija i nivoa ACTH u cirkulaciji; do značajnog smanjenja (p<0.05) volumenske i numeričke gustine i intenziteta obojenosti ACTH ćelija; dok nije uticao na volumen PVN, volumensku gustinu CRH neurona i volumen ACTH ćelija; sve u odnosu na odgovarajuće parametre Orx1 grupe. Hroničan tretman genisteinom je doveo do povećanja (p<0.05): volumenske gustine CRH/cFos neurona PVN, volumenske gustine i apsolutnog broja ACTH ćelija i nivoa ACTH u cirkulaciji; do smanjenja (p<0.05) numeričke gustine i intenziteta obojenosti ACTH ćelija; dok nije uticao na volumen PVN, volumensku gustinu CRH neurona PVN, intenzitet obojenosti CRH neurosekreta u ME i volumen ACTH ćelija; sve u odnosu na odgovarajuće parametre Orx2 grupe. Hroničan tretman daidzeinom je doveo do povećanja (p<0.05) volumenske gustine CRH/cFos neurona i nivoa ACTH u cirkulaciji; smanjenja (p<0.05) volumenske gustine CRH neurona; dok nije uticao na volumen PVN, intenzitet obojenosti CRH neurosekreta u ME, volumensku i numeričku gustinu, apsolutni broj, volumen i intenzitet obojenosti ACTH ćelija; sve u odnosu na odgovarajuće parametre Orx2 grupe. Rezultati dobijeni u ovoj studiji ukazuju na stimulatorni efekat genisteina na histološke, stereološke i biohemijske parametre somatotropnog i adrenokortikotropnog sistema, dok daidzein pokazuje nešto manje izražen stimulatorni efekat na pomenute sisteme. Ova studija predstavlja solidnu osnovu za dalja istraživanja mehanizama delovanja genisteina, odnosno daidzeina na somatotropni i adrenokortikotropni sistem, kao i za njihovu potencijalnu terapijsku korisnost.

The soy isoflavones genistein and daidzein are being used increasingly as preventive or alternative therapeutics in various pathological conditions. The effects of soy isoflavones on the neuroendocrine system were, and still are, the subject of numerous studies. Literature data predominantly focus on their effects on the gonadotrophic axis, considering the soy isoflavones established estrogenic and antiestrogenic mode of action. However, data about their potential effects on the somatotrophic and adrenocorticotrophic systems are rather scarce, but nevertheless very meaningful, considering the importance of these systems for the normal development and function of an organism. Furthermore, in vitro studies provide only limited data, that cannot be fully extrapolated to in vivo conditions. The aim of this study was to investigate the effects of chronically applied estradiol dipropionate, genistein and daidzein on the histological, stereological and biochemical parameters of somatotrophic and adrenocorticotrophic systems in orchidectomized adult rats. Initially, adult (three months old) Wistar rats were divided into two groups: sham operated (Sham) and orchidectomized (Orx). Two weeks after the surgery, the rats were subdivided into seven experimental groups. The first group (Sham1) included sham operated rats subcutaneously (s.c.) treated with an adequate volume of sterile olive oil, the medium for estradiol dipropionate (medium I). In the second group (Orx1) were orchidectomized rats s.c. treated with an adequate volume of medium I. The third group (Orx1+E) was comprised of orchidectomized rats s.c. treated with estradiol dipropionate in medium I (0.6 mg/kg b.w.). The fourth (sham operated – Sham2) and fifth (orchidectomized – Orx2) group of rats were s.c. treated with adequate volumes of absolute ethanol and sterile olive oil mixture, the medium for genistein or daidzein (mixing ratio was 1:2; medium II). In the sixth group (Orx2+G) were orchidectomized rats s.c. treated with genistein in medium II (30 mg/kg b.w.), while the seventh group (Orx2+D) included orchidectomized rats s.c. treated with daidzein in the same medium (30 mg/kg b.w.). All the groups of animals received the treatment during three weeks (chronic treatment), and were sacrificed 24 h after the last injected dose. All the animals were weighed before and after the treatment, while the pituitary weights were measured after sacrificing. For the localization of arcuate (Arc), periventricular (Pe) and paraventricular (PVN) nuclei, as well as for median eminence (ME) cresyl violet histochemical staining was used. Hypothalamic releasing hormones (growth hormone releasing hormone – GHRH, somatostatin – SS, corticotrophin releasing hormone – CRH) and pituitary cells (somatotrophic – GH and adrenocorticotrophic – ACTH) were immunohistochemically (peroxidase-antiperoxidase) labeled. Histochemically stained and immunohistochemically labeled hypothalamic sections, as well as immunohistochemically labeled pituitary sections were stereologically analysed, using newCast stereological software package. The following values were obtained: the Arc, Pe and PVN nuclei volumes, volume density of SS neurones, volumes of the pituitaries, the volume and numerical density of GH and ACTH cells, as well as the absolute numbers and volumes of GH and ACTH cells. The circulating GH and ACTH were determined biochemically. The obtained data were statistically processed. Orchidectomy significantly (p<0.05) decreased the body weight of adult rats in comparison to Sham1 operated group. Chronic treatment with estradiol dipropionate caused a significant (p<0.05) decrease in body weight compared to the Orx1 group of rats. There were no changes in the same parameter after chronic treatments with genistein or daidzein, in comparison to Orx2 group. Orchidectomy of adult rats led to a decrease (p<0.05) in absolute pituitary weight, while the pituitary volume remained unchanged compared to Sham1 group. Treatment with estradiol dipropionate increased (p<0.05) absolute pituitary weight and volume, in comparison to Orx1 group of rats. Genistein treatment increased (p<0.05) absolute pituitary weight and volume compared to Orx2 group, while the same parameters remained unchanged after daidzein treatment. Orchidectomy provoked a statistically significant enlargement (p<0.05) of the Arc nucleus volume; a decrease (p<0.05) of: the GHRH and SS amounts in ME, the volume density of SS neurons in Pe nucleus, the volume and numerical density of GH cells, while orchidectomy did not affect: the volume of Pe nucleus, the absolute number, volume and intensity of staining of GH cells and GH level in blood, all corresponding to the appropriate parameters of Sham group. Administration of estradiol dipropionate to orchidectomized rats provoked an enhancement (p<0.05) of: Arc and Pe nucleus volume, the volume density of SS neurons in Pe nucleus, the SS amount in ME, the intensity of staining of GH cells and GH level in blood; and a decrease (p<0.05) of the volume and numerical density of GH cells; while the treatment did not affect: the GHRH amount in ME, the absolute number and volume of GH cells, all corresponding to the appropriate parameters of Orx group. Genistein treatment caused an increase (p<0.05) in: Arc nucleus volume, the GHRH and SS amounts in ME, the volume density of SS neurons in Pe nucleus, the volume and numerical density of GH cells, the absolute number and intensity of staining of GH cells, GH level in blood; while the treatment did not affect: the Pe nucleus volume and volume of GH cells, all corresponding to the appropriate parameters of Orx group. Daidzein treatment caused an increase (p<0.05) in: the GHRH amount in ME, the volume density of GH cells, volume and intensity of staining of GH cells; and a decrease (p<0.05) of: numerical density and absolute number of GH cells, while the treatment did not affect: Arc and Pe nucleus volume, the volume density of SS neurons in Pe nucleus, the SS amount in ME and GH level in blood, all corresponding to the appropriate parameters of Orx group. Orchidectomy provoked a statistically significant enlargement (p<0.05) of: the PVN volume, the volume density of CRH and CRH/cFos neurons in PVN, the CRH amount in ME and ACTH level in blood; a decrease (p<0.05) of: the volume and numerical density and intensity of staining of ACTH cells; while the treatment did not affect the absolute number and volume of ACTH cells, all corresponding to the appropriate parameters of Sham group. Administration of estradiol dipropionate to orchidectomized rats provoked an enhancement (p<0.05) of: the volume density of CRH/cFos neurons in PVN, the CRH amounts in ME, the number of ACTH cells and ACTH level in blood; a decrease (p<0.05) of : the volume and numerical density and intensity of staining of ACTH cells; while the treatment did not affect: PVN volume, the volume density of CRH neurons in PVN and volume of ACTH cells, all corresponding to the appropriate parameters of Orx group. The treatment with genistein caused an increase (p<0.05) in: the volume density of CRH/cFos neurons in PVN, the volume density and absolute number of ACTH cells and ACTH level in blood; a decrease (p<0.05) of: the numerical density and intensity of staining of ACTH cells; while the treatment did not affect: PVN volume, the volume density of CRH neurons in PVN, the CRH amounts in ME and volume of ACTH cells, all corresponding to the appropriate parameters of Orx group. Daidzein treatment caused an increase (p<0.05) in: the volume density of CRH/cFos neurons in PVN and ACTH level in blood; a decrease (p<0.05) of the volume density of CRH neurons in PVN; while the treatment did not affect: PVN volume, the CRH amounts in ME, the stereological parameters of ACTH cells, all corresponding to the appropriate parameters of Orx group. These results indicate stimulatory effects of genistein on the majority of examined histological, stereological and biochemical parameters of the somatotropic and adrenocorticotrophic systems, while daidzein has less pronounced stimulatory effects on the examined parameters. This study represents a solid basis for some further investigations aimed at establishing the precise mechanisms of genistein/daidzein actions on the somatotropic and hypothalamic–pituitary–adrenal systems and their potential therapeutic usefulness.

Fluktuirajuća asimetrija kao pokazatelj stabilnosti razvića odabranih biljnih taksona u uslovima nenarušene životne sredine i antropogeno indukovanog stresa

Fluctuating asymmetry as an indicator of developmental stability of selected plant taxa in unpolluted environment and under anthropogenically induced stress

Promene u životnoj sredini od industrijske revolucije do danas su bez presedana u istoriji živog sveta i svojim razmerama prete da nadmaše adaptivni potencijal mnogih živih organizama, naročito biljaka, koje kao sesilni organizmi imaju samo ograničene mehanizme za izbegavanje stresa. Razumevanje kako biljke reaguju na ovakve promene, od subcelularnog do nivoa zajednica, je od krucijalnog značaja. U ovom radu smo ispitivali mogućnosti korišćenja analiza fluktuirajuće i radijalne asimetrije kao pokazatelja stabilnosti razvića vegetativnih i reproduktivnih organa kod dve zeljaste monokotile (Iris pumila i Iris germanica) i jedne drvenaste dikotile (Robinia pseudoacacia), u uslovima nenarušene životne sredine i u uslovima antropogeno uzrokovanog stresa (zagađenja u industrijskim i urbanim zonama). Analizirali smo mogućnosti korišćenja ovih analiza kao primarnih indikatora u sredinama sa negativnim antropogenim uticajem, kao i prednosti i mane ovog i nekih drugih metoda detekcije zagađenja. Stabilnost razvića procenjena putem fluktuirajuće i radijalne asimetrije se razlikuje između izučavanih sredina u slučaju sve tri vrste. Kod vrste R. pseudoacacia kao sredina u kojoj je detektovan najveći stepen razvojne nestabilnosti se izdvojio Kostolac (antropogeno degradirana sredina). Kod vrste I. pumila su biljke poreklom iz sredine pod antropogenim uticajem pokazale veću razvojnu nestabilnost kako u slučaju fluktuirajuće asimetrije, tako i u slučaju radijalne asimetrije. Kod vrste I. germanica su detektovane značajne razlike između zagađenja u slučaju indeksa radijalne asimetrije gde su biljke poreklom sa zagađenog staništa pokazale značajno veću razvojnu nestabilnost od biljaka poreklom sa nezagađenog staništa. Kod sve tri vrste je utvrđena značajna interakcija individue i asimetrije osobina, tj. da ekspresija razlika u fluktuirajućoj i radijalnoj asimetriji između ispitivanih osobina zavisi od klona tj. individue. Slično je i za interakciju lokaliteta i asimetrije osobina, što Rezime/Summary znači da su se različite osobine sve tri vrste razlikovale u stepenu detekcije razlika između lokaliteta. Koncentracija hlorofila a, hlorofila b, ukupnog hlorofila, karotenoida, kao i odnos hlorofila a i karotenoida u listu I. pumila je bio značajno manji u zagađenoj sredini , dok je odnos hlorofila a i hlorofila b u ovoj sredini bio veći u odnosu na nezagađenu sredinu. Za teške metale značajne razlike su detektovane između zagađenih i nezagađenih sredina. Arsenik je u značajno većoj koncentraciji detektovan u zagađenim lokalitetima- Obrenovcu i Kostolcu, a olovo i nikl na lokalitetu Autoput, dok za Cd nisu detektovane značajne razlike. Iako je analiza koncentracije teških metala pokazala jasnu diskriminaciju između sredina pod antropogenim uticajem i onih koje to nisu, kao i koncentracija fotosintetičkih pigmenata, analiza razvojne stabilnosti ima prednosti u odnosu na ove metode jer se može jednostavno primeniti na sve taksone, ne zahteva posebne laboratorijske i druge preduslove i ispituje već unapred poznat optimum tj. simetriju. Može se zaključiti da iako je povezanost asimetrije, razvojne stabilnosti i stresa neosporna, analize radijalne i fluktuirajuće asimetrije ne detektuju uvek efekte zagađenja na svim vrstama i svim osobinama jednoznačno, da bi se na osnovu ovih razlika moglo sa sigurnošću tvrditi da li je izučavana populacija pod stresom ili ne. Najpreciznije su se pokazale analize koje su uključivale više osobina tj. multikomponentni i kompozitni indeksi, kao i multivarijantne analize koje mogu bolje detektovati razlike na nivou individue tj. populacije.

Changes in the environment from the industrial revolution till present are unprecedented in the history of the life on Earth and their magnitude can overpower the adaptive potential of many living organisms, especially plants, that are sessile and have only limited mechanisms for avoiding stress. Understanding how plants respond to these changes, from subcellular to the community level, is crucial. In this study we investigated the possibility of using analysis of fluctuating and radial asymmetry as an indicator of developmental stability of vegetative and reproductive organs in two herbaceous monocots (Iris pumila and Iris germanica) and one wooden dikotile (Robinia pseudoacacia), in unpolluted environment, as well as in environment characterized by anthropogenically induced stress (industrial and urban zones pollution). We have analyzed the possibility of using this analysis as a primary indicator of pollution in areas with negative human impact, and the advantages and disadvantages of these and some other methods of pollution detection. Developmental stability estimated by fluctuating and radial asymmetry differed between the studied habitats for all three species. For R. pseudoacacia plants from Kostolac (polluted environment) had the highest degree of developmental instability. I. pumila plants originated from the environment under the influence of anthropogenic stress showed greater developmental instability in the case of fluctuating asymmetry, as well as radial asymmetry. In I. germanica species we detected significant differences between polluted and unpolluted habitats estimated by radial asymmetry where the plants originated from contaminated habitats showed significantly greater developmental instability. In all three species we detected significant interaction between individuals and trait asymmetries, e.g. expression of the difference in the radial and fluctuating asymmetry between the studied traits depended on the individual (clone). Similar situation is with Rezime/Summary the habitat x trait assymetry interaction, where different traits of all the three species differed in the degree of discrimination between the habitats studied. Concentration of chlorophyll a, chlorophyll b, total chlorophyll, carotenoids, and the chlorophyll a and carotenoids ratio in the I. pumila leaf had significantly lesser values in a polluted environment, while the ratio of chlorophyll a and chlorophyll b in this environment had higher values compared to unpolluted environment. Significant differences in heavy metal concentration were detected between polluted and unpolluted areas. Arsenic was detected in significantly higher concentrations in leaf samples from polluted sites-Obrenovac and Kostolac, lead and nickel were detected in significantly higher concentrations on the site Autoput, while no significant differences were detected for Cd. Although the analysis of heavy metal concentrations showed a clear discrimination between the environments, similar to discrimination obtained in analysis of photosythetic pigments, developmental stability analysis has overall advantage because it is easily applied to all taxa, demands no specific laboratory testings and is based on already known optimum-symmetry. It can be concluded that although the positive relationship between asymmetry, developmental instability and stress is indisputable, analysis of radial and fluctuating asymmetry does not always detect pollution effects on all species and all traits, unequivocally, so one could with certainty conclude if the studied population is under stress or not. The most accurate analysis were those involving more traits-analysis of multicomponent and composite indices, and multivariate analysis that can better detect differences at the individual as well as population level.

Morfološka, fitohemijska i molekularna istraživanja karpatsko - balkanskog kompleksa Centaurea atropurpurea (Asteraceae) - filogenetski i taksonomski aspekt

Morphological, phytochemical and molecular studies of Carpathian-Balkan complex Centaurea atropurpurea (Asteraceae) - phylogenetic and taxonomic implications

Predstavnici kompleksa Centaurea atropurpurea (Acrocentron, Asteraceae) naseljavaju Balkan i južne Karpate. Varijabilnost taksona ovog kompleksa navodi na potrebu detaljnog istraživanja, kako bi se razjasnio njihov taksonomski status i bolje razumela filogenija grupe. Morfološke, fitohemijske, citološke i molekularne analize obuhvatile su 23 populacije vrste Centaurea calocephala sa celog areala, kao i 16 populacija ostalih taksona iz kompleksa Centaurea atropurpurea. Na osnovu horoloških podataka urađene su mape distribucije pojedinačnih taksona i istraživanog kompleksa. Opisana je nova vrsta za nauku – Centaurea zlatiborensis, endemična vrsta za Srbiju. Urađena je lektotipifikacija C. gjurasinii i C. crnogorica. Rezultati morfoloških analiza pokazali su veću varijabilnost karaktera stabljike i listova nego karaktera cvasti. Klaster analiza i CDA populacija C. calocephala pokazale su izdvajanje populacija: 1 (Lika) i 17 (Cluj-Napoca) na osnovu karaktera cvasti, dok su se populacije: 17 (Cluj-Napoca) i 7 (Đerdap) izdvojile na osnovu karaktera stabljike i listova. Fitohemijska ispitivanja isparljivih komponenti taksona kompleksa Centaurea atropurpurea pokazala su prisustvo dominantnih komponenti: germakrena D, E-kariofilena i kariofilen oksida. Na osnovu fitohemijskih markera u okviru taksona C. calocephala izdvojile su populacije 1 (Lika) i 20 (Cheile Turzii), dok su se taksoni C. zlatiborensis i C. crnogorica izdvojili od ostalih takona kompleksa. ETS analiza pokazala je monofiletsko poreklo Balkanske klade sek. Acrocentron. Uočena je introgresija i retikulacija koje su posledice homoploidne hibridizacije između taksona tokom ledenog doba u refungijumima na Balkanu. Na osnou ETS i AGT1 izdvojile su se grupe Calocephala A i B, kao i Grbavacensis grupa. Citološke analize pokazale su da svi istraživani taksoni iz kompleksa imaju diploidan broj hromozoma: 2n=20 i 2n=22.

Representatives of Centaurea atropurpurea complex (Acrocentron, Asteraceae) are distributed on the Balkans and the southern Carpathians. The variability of the taxa of this complex indicates the need for detailed research, in order to clarify their taxonomic status and phylogeny. Morphological, phytochemical, cytological and molecular analyses included twenty-three populations of Centaurea calocephala and sixteen populations of other taxa from Centaurea atropurpurea complex. Based on the horological data, maps of the distribution for each taxon and the investigated complex were made. A new species for science has been described ‒ Centaurea zlatiborensis, an endemic species for Serbia. C. gjurasinii and C. crnogorica are lectotypified. The results of morphological analyses showed a greater variability of the stem and the leaves characters in comparison to the capitula characters. In cluster analysis and CDA population of C. calocephala separated from populations: 1 (Lika) and 17 (Cluj-Napoca) based on the characters of capitula, while the populations: 17 (Cluj-Napoca) and 7 (Djerdap) were separated based on the characters of the stem and leaves. Phytochemical analysis of the volatile compounds of the Centaurea atropurpurea complex showed the presence of the dominant components: germacrene D, E-caryophyllene and caryophyllene oxide. On the basis of the phytochemical markers, the populations of 1 (Lika) and 20 (Cheile Turzii) were separated within the C. calocephala, while the C. zlatiborensis and C. crnogorica separated from other taxa of the complex. ETS analysis showed the monophyletic origin of the Balkan clade of the sect. Acrocentron. Introgression and reticulation were observed, which are the consequences of homoploid hybridization between taxa during the ice age in Balkan refugia. ETS and AGT1 showed forming the groups Calocephala A and B, as well as the Grbavacensis group. Cytological analyses have shown that all investigated taxa from the complex have a diploid chromosome number: 2n = 20 and 2n = 22.

Studies on structural and functional changes of mouse choroid plexus in the initiation of neuroinflammation

Значај структурних и функционалних промена хороидног плексуса миша у иницијацији неуроинфламације

Smatra se da je neuroinflamacija jedan od zajedničkih imenitelja i ključnih faktora u procesu neurodegeneracije kod različitih poremećaja u funkcionisanju centralnog nervnog sistema (CNS), koji mogu biti izazvani traumatskom povredom mozga, moždanim udarom, neurodegenerativnim bolestima, sepsom, itd. Kod ovih patoloških stanja, pojavljuje se sličan obrazac dogadjaja, koji uključuje lokalnu sintezu medijatora inflamacije, otvaranje barijera CNS-a, ulazak leukocita, kao i aktivaciju ćelija mikroglije. Horoidni pleksus (HP) je struktura koja čini jednu od tri barijere CNS-a, krvno-likvornu barijeru (KLB). Nalazi se u sve četiri moždane komore i jedinstvene je strukture. Sastoji se iz epitelijalnih ćelija horoidnog pleksusa (EĆHP) spojenih čvrstim vezama koje okružuju fenestrirane kapilare. HP je odgovoran za većinsku proizvodnju cerebrospinalne tečnosti (CST), a na površini EĆHP postoje brojni transporteri i receptori, koji utiču na održavanje homeostaze i nesmetani rad mozga. Zbog svoje pozicije, strukture i funkcije HP je prepoznat kao ključni organ koji je uključen u nadzor imunskog odgovora u mozgu i kao važan faktor u komunikaciji između periferije i CNS-a u procesu inflamacije. Glavni cilj ove teze je bio da se ispita doprinos HP inicijaciji neuroinflamacije, kroz promenu morfologije i funkcionalnosti EĆHP. U tu svrhu korišćena su dva mišija modela bolesti povezanih sa neuroinflamacijom: a) model sistemske inflamacije, lipopolisaharidom (LPS) indukovan model sepse i b) novouspostavljeni model Alchajmerove bolesti, indukovan intracerebroventrikularnom (i.c.v) aplikacijom oligomerima amiloida beta (AβO). Najvažniji rezultati ovog istraživanja pokazuju da je propusnost krvno-likvorne barijere (KLB) povećana kod AβO modela, a da je sam uzrok toga gubitak tipične kuboidne morfologije EĆHP, kao i smanjenje ekspresije komponenti čvrstih veza, kao i da je ova promena prolaznog karaktera i da se kroz nedelju dana barijera vraća na fiziološki nivo propustljivosti. Ispitivanje propustljivosti krvno- moždane barijere nije pokazalo nikakve značajne promene. Što se tiče promena funkcionalnosti HP, uočena je povećana ekspresija gena za ispitivane proinflamatorne citokine u HP i u hipokampusu, kao i njihov povišeni nivo u CST. Takođe, zabeleženo je povećanje ekspresije gena za određene matriksne metaloproteinaze (MMP) u HP, kao i njihova povišena aktivnost u CST. Nakon istovremenog injeciranja MMP inhibitora širokog spektra sa AβO, primećen je nivo propustljivost barijere kao kod intaktnih životinja. Slično, smanjenje povećane propustljivosti KLB uzrokovane i.c.v. aplikovanjem AβO je uočeno i kod MMP3 deficijentnih miševa. Kada je ispitana aktivacija mikroglije, kao rezidentne imunske ćelije CNS-a, nije primećena značajna promena kod AβO modela. Ustanovljeno je takodje i povećanje broja ekstracelularnih vezikula (EV) u CST, kao i povećanje ekspresije markera za EV, kao i mikroRNK-155, koja je prepoznata kao jedna od ključnih mikroRNK koje dovode do promene ekspresije gena uključenih u inflamatorni odgovor, a samim time i do povećanja odgovora na inflamatorni stimulus. Primećena je i prolaznost odgovora EĆHP kroz povećanje ekspresije EV i mikroRNK-155, nedelju dana nakon primene inflamatornog stimulusa. Sličan obrazac promena u HP nađen je i u modelu LPS-om indukovane sepse. Dobijeni rezultati su ukazali na značajnu ulogu HP u inicijaciji neuroinflamacije, kroz strukturne i funkcionalne promene EĆHP, u dva različita modela bolesti povezanih sa neuroinflamacijom. Ovi rezultati ukazuju na HP kao potencijalni terapeutski target u poremećaju funkcionisanja CNS-a usled inflamatornih procesa.

Neuroinflammation has been considered a common denominator and crucial player in neurodegeneration observed in various central nervous system (CNS) disorders. Considering the increasing evidence on the role of choroid plexus (CP) in neuroinflammatory processes through alterations in morphology and functionality of the choroid plexus epithelial (CPE) cells, the main goal of this thesis was to test the contribution of the CP in the initiation of neuroinflammation in two animal models of neuroinflammation-associated diseases: a model of Alzheimer’s disease, induced by intracerebroventricularly (i.c.v.) injected amyloid beta oligomers (AβO) and a lipopolysaccharide (LPS) induced sepsis animal model of systemic inflammation. Main findings from this research conclude that blood cerebrospinal fluid barrier (BCSFB) permeability is increased upon AβO injection, resulting from the loss of typical cuboidal morphology of CPE cells and a decrease in expression of tight junctions components. In the CP, upregulation of gene expression for various cytokines was observed, as well as their increased levels in the cerebrospinal fluid (CSF). Also, increase in gene expression for several matrix metalloproteinases (MMP) in the CP, and in MMP activity in the CSF was noted. After i.c.v. injection of broad spectrum MMP inhibitor with AβO, prevention of AβO-induced BCSFB permeability was found. In accordance with this, increase in BCSFB permeability upon AβO injection was observed in MMP3 deficient mice, but to a lesser extent. Furthermore, an increase in the number of particles in the CSF and an increase in gene expression of extracellular vesicles (EV) markers and miR-155 was found in the CP. A similar pattern of changes in the CP was observed in response to LPS injection compared to AβO injection. The results of this study revealed a significant role for the CP in the initiation of neuroinflammation, through structural and functional changes, in two different models associated with neuroinflammation.

Variranje odnosa polova, polnog dimorfizma i komponenti adaptivne vrednosti u populacijama Mercurialis perennis L. (Euphoriaceae) duž gradijenta nadmorske visine

Variation in sex ratio, sexual dimorphism, and fitness components in populations of Mercurialis perennis L. (Euphorbiaceae) along the altitudinal gradient

Vrste roda Mercurialis su se pokazale kao atraktivan objekat za raznovrsna ekološka, genetička i istraţivanja u evolucionoj biologiji biljaka. Šumski prosinac, Mercurialis perennis L., je višegodišnja zeljasta anemofilna i dvodoma vrsta, karakteristična za primarna ili manje narušena šumska staništa. Analiza populacija ove vrste sa različitih nadmorskih visina pruţila je mogućnost da se na relativno malom geografskom prostoru istraţi uticaj velikog raspona variranja ekoloških faktora na morfološke karakteristike i reproduktivnu alokaciju (RA) kod pripadnika različitih polova, kao i na brojčani odnos polova u populaciji. Istraţivanje je obuhvatilo populacije sa 14 lokaliteta u Srbiji, koje su uzorkovane u periodu 2006–2009. Statistički značajno odstupanje od pretpostavljenog ravnoteţnog odnosa polova ustanovljeno je u 69,5% populacionih uzoraka. Broj uzoraka sa neravnoteţnim odnosom polova varirao je meĎu godinama, a u većini istraţenih populacija neravnoteţa je bila u korist muških biljaka. Neravnoteţa u korist muških biljaka ustanovljena je i u istraţivanjima na drugim populacijama ove vrste, a ovakav obrazac neravnoteţe polova se smatra uobičajenim za višegodišnje dvodome biljke. Indeks polnog dimorfizma je kod većine analiziranih osobina imao male do umerne vrednosti, dok su izrazito i dosledno dimorfne bile one osobine koje su direktno povezane sa reprodukcijom – broj cvetova i masa cvasti. Smer polnog dimorfizma razlikuje se kod ovih osobina. Muški pol produkuje više cvetova, što ukazuje na pojačanu intraseksualnu selekciju u muškom polu za veću produkciju polena. S druge strane, masa ţenskih cvasti je višestruko veća od mase muških cvasti, što se preslikava i u veću reproduktivnu alokaciju ţenskih jedinki. U ţenskom polu je detektovan i obrazac smanjenja ulaganja u reprodukciju sa povećanjem nadmorske visine na kojoj biljka raste. Analize polnog dimorfizma u visini biljke pokazale su da je u većini populacionih uzoraka muški pol viši, što se uklapa u pretpostavke hipoteze disperzije polena. Generalizovanom metodom najmanjih kvadrata kreirani su statistički regresioni modeli prostornog i vremenskog variranja odnosa polova i reproduktivne alokacije, meĎu kojima su najbolji odabrani savremenim metodama statističkog zaključivanja. U celokupnom uzorku, kao i u uzorcima iz pojedinačnih godina, sa porastom nadmorske visine smanjivala se učestalost muških biljaka u populaciji. Smer neravnoteţe se time pomerao od populacija sa više muških biljaka na lokalitetima sa malom nadmorskom visinom ka populacijama sa više ţenskih biljaka na lokalitetima sa većom nadmorskom visinom. Stres na staništu je izraţeniji na većim nadmorskim visinama, što bi po teoriji reproduktivne alokacije dovelo do manjeg učešća ţenskih biljaka. Veće učešće ţenskih biljaka u analiziranim populacijama M. perennis na većoj nadmorskoj visini moţe biti rezultat adaptivnog odgovora. Statistički model ukazuje na dvojaku značajnost nadmorske visine i vegetacione sezone na odnos polova u analiziranim populacijama kroz uticaj na srednju vrednost udela muških jedinki u populaciji, kao i kroz uticaj na varijansu ovog udela. Faktori koji su u statističkim modelima ustanovljeni kao značajni za veličinu i variranje ulaganja u reprodukciju ukazuju na različite selekcione pritiske koji deluju na polove. Na razlike u odnosu polova u populaciji osetljive su jedino ţenske jedinke, koje sa smanjenjem udela muških biljaka u populaciji manje ulaţu u reprodukciju. Kod vrste M. perennis je veoma izraţen citogenetički polimorfizam – do sada je opisano više od 40 različitih citotipova, sa opsegom broja hromozoma od 42 do 112. U analiziranim populacijama u Srbiji utvrĎena je velika citogenetička varijabilnost: prisutna su 23 od ukupno 47 citotipova opisanih u Evropi i skoro potpun opseg broja hromozoma. U populacijama na većim nadmorskim visinama prisutni su viši nivoi ploidnosti. Filogenetskim i istorijsko- biogeografskim analizama ustanovljeno je vreme divergencije roda Mercurialis od ostatka potfamilije Acalyphoideae na pre oko 65–66 miliona godina, definisan je centar porekla roda – Indomalajska oblast i potvrĎena je pleziomorfnost dvodomosti kao seksualnog sistema u ovom rodu.

The plant species of genus Mercurialis are very attractive object for various ecological, genetical and evolutionary researches. Dog’s mercury, Mercurialis perennis L., is a perennial anemophilous and dioecious herb, which usually grows in old and undisturbed forests. The analyses of populations of dog’s mercury from various altitudes gave us opportunity to investigate the effect of great environmental variation on morphological traits, reproductive allocation in different sexes, as well as on sex ratios in populations. This research included populations from 14 localities in Serbia sampled in 2006–2009. Statistically significant bias in sex ratio was found in 69.5% of population samples. Number of samples with biased sex ratio varied among years, and the majority of samples were male-biased. The male-biased populations of dog’s mercury were found elsewhere, and this pattern of bias is regarded to as the common one in dioecious perennials. Sexual dimorphism index for greater part of analysed traits had small to moderate values, while reproductive traits (number of flowers, mass of inflorescences) were markedly and consistently dimorphic. The range and direction of dimorphism differed in these reproductive traits. Males produced more flowers, and this information indicates pronounced intrasexual selection in males for greater pollen production. On the other hand, mass of female inflorescences was manifold bigger than mass of male inflorescences, and female reproductive allocation was bigger in a similar manner. In females, we also found pattern of decrease in reproductive allocation with the increase of altitude. The analyses of sexual dimorphism in plant size showed that males were the higher sex in majority of population samples. This is in agreement with pollen-dispersal hypothesis. The statistical regression models of spatial and temporal variation of sex ratio and reproductive allocation were created by generalized least squares method. Among these models, the best-fitting ones were selected by contemporary statistical inference. The proportion of male plants in population decreased with increasing altitude in the whole sample, as well as in the samples from different years. The bias direction therefore changed from male-biased in the lowland populations to female-biased in the higher altitudes. The environmental stress is more pronounced in the higher altitudes, and according to sex allocation theory this would lead to male-bias in the population. The female-biased sex ratio in populations of M. perennis in higher altitudes could be the adaptive response. Statistical models showed the significance of altitude and vegetation season in two ways: through affecting the mean of male plant proportion, as well as through affecting the variance of this proportion. The factors that showed significance in statistical models of reproductive allocation indicate the existence of difference in selective pressures on sexes. Only female plants were sensitive to sex ratio, with reducing reproductive allocation when there is smaller proportion of males in the population. Cytogenetic polymorphism is very expressed in M. perennis – more then 40 cytotypes were described so far, ranging from 42 to 112 chromosomes. In analysed populations from Serbia, the great cytogenetic variability was also found. There were 23 out of 47 cytotypes described in Europe and almost whole range of reported chromosome numbers. Populations from higher altitudes exert higher ploidy levels. Phylogenetic and historical biogeographic analysis indicated divergence time of genus Mercurialis from the rest of the subfamily Acalyphoideae to approximately 65-66 Mya, centre of the genus origin was defined in Indomalaya and plesiomorphism of dioecious sex system was confirmed.

Tkivna specifičnost efekata 17ß-estradiola na signalni put insulina

Tissue specificity of 17ß-estradiol effects on insuli signaling pathway.

Insulin i estradiol imaju vaţnu ulogu u regulaciji metabolizma ugljenih hidrata i lipida. Razliĉita kliniĉka istraţivanja i eksperimentalni podaci ukazuju da varijacije u koncentraciji estrogena utiĉu na insulinsko delovanje. Cilj ove studije je bilo poreĊenje uticaja estradiola na poĉetne molekule insulinskog signalnog puta u glavnim ciljnim tkivima za insulin (jetra) i estradiol (uterus), kao i u srcu, u kome oba hormona ostvaruju znaĉajne efekte. Ovarijektomisane ţenke pacova su tretirane estradiolom 6 h pre analize sadrţaja proteina i iRNK molekula signalnog puta insulina. Da bi se istakli efekti estradiola na fosforilacije i asocijacije molekula relevantne za insulinski signalni put, ţivotinje su dodatno injecirane insulinom 30 min pre eksperimenta. Tretman estradiolom nije promenio nivo insulina i glukoze u plazmi, ali je doveo do znaĉajnog smanjenja nivoa slobodnih masnih kiselina i povećanja teţine uterusa. U jetri, tretman estradiolom je doveo do smanjenja fosforilacije IR, kao i smanjenja proteinskog sadrţaja IRS-1, što navodi na zakljuĉak da je estradiol suprimirao efekte insulina preko IR/IRS-1 puta i verovatno ih usmerio na alternativni put, što je potkrepljeno i povećanjem proteinskog sadrţaja IRS-2. U uterusu je tretman estradiolom nakon 6 h doveo do statistiĉki znaĉajnog povećanja proteinskog sadrţaja skoro svih ispitivanih molekula signalnog puta insulina. U srcu je estradiol uzrokovao povećanje asocijacije IRS-1/p85, povećanja sadrţaja proteina i iRNK p85, kao i povećanje fosforilacije Akt na Ser473. S druge strane, tretman estradiolom je u srcu izazvao i smanjenje fosforilacije tirozina IR, smanjenje proteinskog sadrţaja IRS-2 i iRNK oba IRS proteina. Rezultati ove studije ukazuju na to da tretman estradiolom indukuje tkivno specifiĉne promene u insulinskom signalnom putu. Posledice tretmana estradiolom na molekule insulinskog signalnog puta su oĉiglednije u uterusu, ali je njihov fiziološki znaĉaj za insulinsko delovanje verovatno veći u jetri. S druge strane, rezultati dobijeni u srcu sugerišu veoma kompleksnu ulogu estradiola u fiziologiji srca, koja se manifestuje i kombinacijom pozitivnog i negativnog, genomskog i negenomskog delovanja ovog hormona na molekule signalnog puta insulina.

Insulin and estradiol play important role in regulation of carbohydrate and lipid metabolism. Various clinical observations and experimental data suggest that variations in the concentration of estrogens affect insulin action. The aim of the present study was to compare the impact of estradiol on early steps of insulin signaling in main target tissues of insulin (the liver) and estradiol (the uterus), and in the heart, where both hormones realize important effects. Ovariectomized female rats were treated with estradiol 6 h prior to analysis of protein and mRNA content of insulin signaling molecules. To delineate estradiol effects on phosphorylations and molecular associations relevant for insulin signaling, animals were treated additionally with insulin 30 min before the experiment. Treatment with estradiol did not change the levels of plasma insulin and glucose, but it significantly decreased the free fatty acid level and increased uterine weight. In liver, estradiol treatment decreased IR phosphorylation and IRS-1 protein content, suggesting that estradiol suppressed insulin action through IR/IRS-1 and probably redirected it to alternative pathway. This conclusion is supported with increase of IRS-2 protein content. In uterus, estradiol treatment resulted in significant increase of protein content of almost all analyzed molecules. In heart, estradiol increased IRS-1/p85 association, p85 protein and mRNA level, and Ser473Akt phosphorylation. On the other hand, estradiol treatment decreased tyrosine phosphorylation of cardiac IR, protein content of IRS-2, and mRNA of both IRS proteins. These results suggest that estradiol treatment induces tissue-specific changes in insulin signaling. Consequences of estradiol treatment on insulin signaling molecules are more apparent in uterus, but their physiological relevance for insulin action is probably more important in liver. On the other hand, results obtained in heart suggest very complex role of estradiol in heart physiology, represented by, amongst other effects, combination of positive and negative, genomic and nongenomic actions of this hormone on insulin signaling molecules.

Funkcionalna analiza genskih varijanti FIIc.1787G>A (protrombin, Beograd) i FIIc.*64_*66del i njihova povezanost sa trombofolijom

Functional analysis of FIIc.1787G>A (protrombin, Belgrade) and FIIc.*64_*66del gene variants and their assotiation with trombophilia

Protrombin predstavlja zimogen trombina (koagulacinog faktora II), koji ima centralnu ulogu u održanju hemostazne ravnoteže. Zahvaljujući alosteričnoj regulaciji, trombin može da vrši i prokoagulantnu i antikoagulantnu funkciju. Region 3' kraja gena za protrombin ima nekanonsku organizaciju i podložan je nastanku genskih varijanti koje mogu imati značajnu ulogu u regulaciji ekspresije i funkcije protrombina. Do sada opisane varijante u ovom regionu su povezivane sa povišenom ekspresijom protrombina koja može dovesti do hiperkoagulacije i povećane sklonosti ka trombozama, odnosno trombofiliji. U Laboratoriji za molekularnu biologiju, Instituta za molekularnu genetiku i genetičko inženjerstvo, u 3' kraju gena za protrombin opisane su FIIc.1787G>A i FIIc.*64_*66del varijante čiji mehanizmi do sada nisu rasvetljeni. Varijanta FIIc.1787G>A (protrombin Beograd) se nalazi u poslednjem egzonu gena za protrombin i dovodi do aminokiselinske zamene Arg596Gln u proteinu. Ova varijanta se nalazi u regionu za koji se vezuju joni natrijuma, neophodni za prokoagulantnu aktivnost trombina, kao i prirodni trombinski inhibitor- antitrombin. Varijanta FIIc.*64_*66del se nalazi u 3' netranslatirajućem regionu gena i predstavlja deleciju 3 nukleotida. Imajući u vidu pozicije u različitim strukturnim elementima 3' kraja gena, pretpostavlja se da su mehanizmi delovanja ovih varijanti različiti. Prvi deo studije je imao za cilj određivanje učestalosti FIIc.1787G>A i FIIc.*64_*66del varijanti kod 353 pacijenta sa različitim trombotičkim poremećajima i 250 zdravih ispitanika u srpskoj populaciji. Pacijenti su u zavisnosti od tipa trombotičkog poremećaja podeljeni u 4 grupe: tromboze dubokih vena (94 pacijenta), izolovani plućni embolizam (104 pacijenta), kombinovani trombotički poremećaji (49 pacijenata) i spontani pobačaji (106 pacijentkinja). Drugi deo studije je obuhvatio ex vivo analize, s ciljem da se ispita uticaj navedenih varijanti na funkcionalnost i raspoloživu količinu protrombina u plazmi ispitanika. U trećem delu studije ispitivan je uticaj FIIc.1787G>A i FIIc.*64_*66del varijanti na ekspresiju gena za protrombin na nivou iRNK i proteina u in vitro uslovima. Učestalost FIIc.1787G>A varijante u ukupnoj grupi pacijenata sa različitim trombotičkim poremećajima iznosila je 0,57%, dok u zdravoj populaciji ova varijanta nije detektovana. Varijanta FIIc.1787G>A je detektovana u heterozigotnoj formi kod dva pacijenta sa kombinovanim trombotičkim poremećajima. Ex vivo analize u uzorcima plazme ispitanika su pokazale da FIIc.1787G>A dovodi do statistički značajno smanjene prokoagulantne aktivnosti protrombina kod nosilaca (49%±4,1) u odnosu na nenosioce (96%±2,2), ali da ne utiče na nivo protrombina u plazmi. Endogeni trombinski potencijal je kod nosilaca FIIc.1787G>A varijante 2,2 puta veći u odnosu na nenosioce (3645 nM/min vs. 1653 nM/min). Takođe je pokazano da FIIc.1787G>A varijanta dovodi do rezistencije na antitrombin i trombomodulin u plazmi nosilaca. In vitro analize su utvrdile da FIIc.1787G>A ne utiče na promenu ekspresije gena za protrombin ni na nivou iRNK ni na nivou proteina. U ukupnoj grupi pacijenata FIIc.*64_*66del varijanta je bila prisutna kod 1,13% ispitanika, dok nijedan ispitanik u zdravoj populaciji nije bio nosilac. Varijanta FIIc.*64_*66del je detektovana u heterozigotnoj formi kod jednog pacijenta sa izolovanim plućnim embolizmom i tri pacijentkinje sa spontanim pobačajima. Na osnovu ex vivo analiza, FIIc.*64_*66del varijanta ne utiče na promenu aktivnosti i nivoa protrombina u plazmi nosilaca, a dovodi do blago povišenog nivoa endogenog potencijala trombina. Rezultati in vitro analiza su pokazali da FIIc.*64_*66del varijanta dovodi do statistički značajno niže ekspresije gena za protrombin na nivou iRNK i proteina. Efekat FIIc.*64_*66del varijante je modifikovan nizvodnim regulatornim elementom (DSE) u smeru izraženijeg smanjenja ekspresije. Takođe, pokazano je da u regionu FIIc.*64_*66del postoji mesto vezivanja male RNK, koja se vezuje nezavisno od ispitivane varijante. Deo sekvence u okviru koje je eksperimentalno potvrđeno mesto vezivanja, preklapa se sa mestom vezivanja hsa-mir-2052 dobijenog in silico predikcijom. Rezultati ove studije pokazuju da varijanta FIIc.1787G>A predstavlja novi trombofilni marker koji treba uvrstiti u panel testova za trombofiliju kod pacijenata sa izrazitim trombotičkim fenotipom koji imaju smanjenu prokoagulantnu aktivnost protrombina. Sa druge strane, na osnovu dobijenih rezultata, FIIc.*64_*66del varijanta ne predstavlja trombofilni marker.

Prothrombin is a zymogen of thrombin (coagulation factor II), which plays a central role in maintaining hemostatic balance. Due to the allosteric regulation, thrombin has dual function: procoagulant and anticoagulant. The 3' end of prothrombin gene has noncanonical organization, which is susceptible to the genetic variants that might have a significant role in the regulation of prothrombin expression and function. Variants reported within this region lead to increased prothrombin expression which is associated with hypercoagulability and increased tendency to thrombophilia. In the Laboratory for Molecular Biology at the Institute of Molecular Genetics and Genetic Engineering, we have reported FIIc.1787G>A and FIIc.*64_*66del variants in 3' end of prothrombin gene, but their mechanisms of action have not been elucidated so far. The FIIc.1787G>A (prothrombin Belgrade) is located in the last exon of the prothrombin gene, leading to amino acid substitution Arg596Gln in the protein. This variant is positioned in a region of antithrombin (thrombin inhibitor) binding site, as well as in Na+ binding loop, which is significant for procoagulant activity of thrombin. The FIIc.*64_*66del is located in the 3' untranslated gene region and represents a deletion of three nucleotides. Taking into account the position of these two variants in different structural elements of 3' end of prothrombin gene, it is assumed that they differ in the mechanisms of action. In the first part of the study, we determined the frequency of FIIc.1787G>A and FIIc.*64_*66del variants in 353 patients with thrombotic disorders and 250 healthy subjects in Serbian population. Patients were divided into 4 groups according to the type of thrombotic disorders: deep vein thrombosis (94 patients), isolated pulmonary embolism (104 patients), combined thrombotic disorders (49 patients) and fetal loss (106 patients). The second part of the study included the ex vivo analyses, with aim to investigate the impact of these variants on the functionality and available amount of prothrombin in the subjects plasma samples. 2 In the third part of the study, the impact of FIIc.1787G>A and FIIc.*64_*66del variants on gene expression was assessed in vitro by determining the prothrombin mRNA and protein level. The frequency of FIIc.1787G>A variant in total group of patients was 0.57%, whereas in healthy population this variant was not detected. Among patients, two FIIc.1787G>A heterozygous carriers were detected in combined thrombotic disorders group. Ex vivo analyses of subjects plasma samples showed that the presence of FIIc.1787G>A variant results in significantly reduced prothrombin activity in carriers (49±4.1%) compared to non-carriers (96%±2.2), without effect on prothrombin plasma level. Endogenous thrombin potential was 2.2 times higher in FIIc.1787G>A carriers compared to those with wild-type genotype (3645 nM/min vs. 1653 nM/min, respectfully). In carriers’ plasma samples it was shown that FIIc.1787G>A variant leads to impaired inhibition of mutant thrombin by antithrombin (antithrombin resistance) and altered interaction with thrombomodulin (thrombomodulin resistance). In vitro analyses have revealed that FIIc.1787G>A does not affect the prothrombin gene expression on mRNA and protein levels. In the total group of patients, FIIc.*64_*66del was present in 1.13%, while this variant was not observed among healthy subjects. The FIIc.*64_*66del variant was detected in the heterozygous form in one patient with isolated pulmonary embolism and three patients with fetal loss. Based on ex vivo analyses in carriers plasma samples, the FIIc.*64_*66del variant does not change the prothrombin activity and protein level and leads to slightly increased endogenous thrombin potential. Results of in vitro assays shown that FIIc.*64_*66del variant leads to significantly decreased gene expression by affecting the prothrombin mRNA and protein levels. This effect of FIIc.*64_*66del variant was modulated by regulatory downstream sequence element (DSE). By modified Footprinting Primer-PCR assay we detected the binding of small RNA within the region of FIIc.*64_*66del variant, which overlaps with the hsa-mir-2052 binding site obtained by in silico prediction. The results of our study indicate that FIIc.1787G>A variant represents a new thrombophilic marker which should be included in the thrombophilia testing panel for patients with severe thrombotic phenotype and decreased prothrombin activity. On the 3 other hand, our results indicate that FIIc.*64_*66del variant does not represent thrombophilic marker.

Uticaj restriktivnog režima ishrane na plastičnost neurona i glije nakon povrede senzomotorne kore mozga pacova

Effect of dietary restriction on meuronal and glial plasticity following injury to the rat somatosensory cortex

Traumatska povreda mozga (engl. traumatic brain injury, TBI) predstavlja povredu moždanog tkiva uzrokovanu mehaničkom povredom glave. TBI je jedan od vodećih uzroka smrtnosti i invaliditeta u svetu, u populaciji ljudi do 45 godina starosti. Posledice TBI zavise od lokalizacije i količine oštećenja moždanog tkiva i mogu varirati od blažih motoričkih i kognitivnih smetnji, pa sve do težih oblika invaliditeta i smrti. Traumatska povreda mozga dovodi do narušavanja krvno-moždane barijere, što rezultuje tačno određenim sledom događaja u povređenom tkivu CNS-a. Naime, akutna faza, označena kao primarna povreda, nastaje direktnim delovanjem mehaničke sile na moždano tkivo i javlja se u trenutku povrede. Događaji koji slede nakon toga označeni su kao sekundarna povreda koja nastaje kao posledica inicijalnog oštećenja, a obuhvata procese koji dovode do daljeg oštećenja tkiva u danima i nedeljama nakon povrede. To su pre svega zapaljenski procesi koji dovode do smrti neurona koji prvobitno nisu bili zahvaćeni mehaničkom povredom. Smatra se da je broj neurona koji umre usled širenja sekundarne povrede daleko veći od broja neurona koji strada usled primarne povrede. Činjenica da se većina procesa u okviru sekundarne povrede odigrava relativno kasno (satima i danima nakon povrede) čini ih podložnim različitim terapeutskim intervencijama. Već decenijama je poznato da restrikcija hrane povoljno deluje na čitav organizam tako što odlaže starosno-zavisne fiziološke promene i smanjuje incidencu različitih obolenja (kancer, autoimunske bolesti, Parkinsonova i Alchajmerova bolest, itd.) Međutim, tek u poslednjih desetak godina intenzivno se istražuje uticaj smanjenog unosa hrane na procese oporavka nakon povrede. Dosadašnja istraživanja na ovom polju su pokazala da restrikcija hrane u trajanju od nekoliko meseci pre povrede CNS-a ima neuroprotektivno dejstvo i promoviše funkcionalni oporavak u nekoliko različitih modela povrede. Efekti dijetalne restrikcije na različite aspekte sekundarne povrede, kao i na sekundarnu ćelijsku smrt nakon traumatske povrede mozga još uvek nisu dovoljno ispitani. Stoga je cilj ovog istraživanja bio je da se utvrdi da li i u kojoj meri dijetalna restrikcija (DR) može uticati na procese koji se u mozgu dešavaju nakon povrede, a pre svega, da li može da utiče na procese sekundarne povrede. Za potrebe eksperimenta, mužjaci pacova Wistar soja stari 3 meseca su podeljeni u dve grupe, ad libitum životinje (AL) i životinje na restriktivnom režimu ishrane (DR). Životinje iz AL grupe imale su neograničen pristup hrani tokom čitavog vremena trajanja eksperimenta, dok su životinje iz DR grupe dobijale 50% normalnog dnevnog unosa hrane. Sa navršenom starošću od 6 meseci životinje su podvrgnute ubodnoj leziji senzomotorne kore prednjeg mozga, a zatim su žrtvovane u različitim vremenskim tačkama: kontrola, 2, 7, 14. i 28. dan nakon lezije. Kako bi se utvrdio efekat dijetalne restrikcije na oporavak nakon traumatske povrede mozga praćeni su procesi inflamacije, sekundarne neurodegeneracije i neuralne plastičnosti. Primenom metoda Western blot analize, RT- i PCR-a u realnom vremenu, histološkog bojenja i imunohistohemije ispitivane su promene u vremenskoj i prostornoj ekspresiji ciljnih iRNK i proteina, markera ovih procesa. Rezultati dobijeni u okviru ove disertacije pokazuju da dijetalna restrikcija u značajnoj meri menja odgovor tkiva mozga na povredu, utičući na brojne procese nakon povrede. Naime, dobijeni rezultati su pokazali da dijetalna restrikcija utiče na procese inflamacije suprimirajući aktivaciju mikroglijskih ćelija, kao i indukciju solubilnog TNF-α proteina nakon povrede. Takođe, DR deluje neuroprotektivno suprimirajući indukciju aktivne kaspaze-3 i sekundarnu smrt neurona u ranoj fazi oporavka nakon povrede mozga. Dijetalna restrikcija povećava ekspresiju proteina sinaptičke plastičnosti GAP-43 i sinaptofizina oko samog mesta povrede, uz istovremenu supresiju aktivacije astrocita i smanjenje sinteze inhibitornog proteoglikana neurokana. Takođe, DR dovodi do izrazitog povećanja koncentracije kortikosterona, kao i posledične promene u fosforilaciji GR-a, i u nivou 11β-HSD1 proteina nakon povrede. Dijetalna restrikcija je uticala i na ekspresiju transkripcionog faktora NF-κB, kao i na ekspresiju iRNK za Bcl-2 i Bcl-xL, antiapoptotskih gena koje reguliše NF-κB. Rezultati ove studije su pokazali da dijetalna restrikcija ima kapacitet da u značajnoj meri oblikuje odgovor povređenog tkiva mozga direktnim uticajem na procese sekundarne povrede, ali i plastičnosti. Takođe, ovi rezultati ukazuju na mogućnost primene dijetalne restrikcije i u kliničkoj praksi kod povreda mozga.

Traumatic brain injury (TBI) represents a brain tissue injury caused by mechanical head injury. TBI is one of the leading causes of death and disability in the world human population, up to 45 years of age. Consequences of brain injury depend on the location and amount of brain tissue damage, and can range from mild motor and cognitive impairment, to severe forms of disability and death. Traumatic brain injury leads to impairment of blood-brain barrier, resulting in a precisely defined sequence of events in injured CNS tissue. Two main phases could be distinguished: acute phase, which is called the primary injury that occurs by direct action of mechanical forces on the brain tissue and occurs at the time of injury. The events that followed thereafter are called the secondary injury. Secondary injury occurs as a result of the initial damage, and includes the processes that lead to further tissue damage in the days and weeks following injury. These are primarily inflammatory processes that lead to the death of neurons that initially were not affected by mechanical injury. It is believed that the neuronal cell death caused by secondary injury is far greater than the one caused by primary injury. The fact that most of the processes within the secondary injury occur relatively late (hours and days following injury) makes them subject to various therapeutic interventions. For decades, it is known that food restriction has a beneficial effect on the entire organism by delaying age-dependent physiological changes and reducing the incidence of various diseases (cancer, autoimmune disease, Parkinson's and Alzheimer's, etc). However, only in the last ten years has been to investigate the influence reduced food intake in the recovery process following injury. Previous research in this field has shown that several months long food restriction prior to CNS injury has neuroprotective effects and promotes functional recovery in several different models of injury. The data considering effects of DR on different aspects of secondary injury, as well as secondary neuronal cell death following TBI are still scarce. Therefore, the aim of this study was to determine whether dietary restriction (DR) can affect the processes that occur in the brain after injury, primarily, whether it can influence the process of secondary injury. For the purpose of the experiment, male Wistar rats 3 months old were divided into two groups, the ad libitum animals (AL) and the dietary restricted animals (DR). Animals from the AL group had unlimited access to food throughout the duration of the experiment, whereas the animals DR groups received 50% of normal daily food intake. Upon reaching the age of 6 months, the animals were subjected to stub lesion to the somatosensory cortex, and then were sacrificed at different time points: control, 2, 7, 14 and 28 day following lesion. In order to determine the effect of dietary restriction on processes following TBI following processes were examined: inflammatory processes, secondary neurodegeneration and neural plasticity. Changes in the temporal and spatial expression of target mRNA and proteins were examined by using Western blot analysis, RT-PCR and real-time RT-PCR, histological and immunohistochemical staining. To determine the effect of dietary restriction on recovery following TBI genes and proteins involved in the processes of inflammation, secondary neurodegeneration, as well as those who participate in the processes of plasticity, were investigated. The results obtained in this thesis show that dietary restriction significantly changes the brain tissue response to injury, affecting numerous processes following injury. Namely, dietary restriction affects the inflammatory process by suppression of microglial activation, and TNF-α protein induction following injury. Also, DR acts neuroprotective by suppressing active caspase-3 induction and secondary neuronal death in the early phase of recovery following brain injury. Dietary restriction increases the expression of synaptic plasticity proteins GAP-43 and synaptophysin around the site of the injury, with simultaneous suppression of the activation of astrocytes and reduction of synthesis of inhibitory proteoglycan - neurocan. Also, DR leads to a prominent increase in the concentration of corticosterone, and consequent changes in the GR signaling pathway. Dietary restriction affected the expression of NF-kappaB transcription factor, as well as the expression of mRNA for Bcl-2 and Bcl-xL, antiapoptotic genes regulated by mentioned transcription factor. The results of this study showed that dietary restriction has the capacity to significantly shape the response of damaged brain tissue by direct impact on the processes of secondary injury and neural plasticity. Also, these results point to the possibility of using dietary restriction in clinical practice for patients who have suffered a brain injury.

Paleoekologija karbonskih drvenastih prečica reda Lepidodendrales sa posebnim osvrtom na region Balkanskog poluostrva

Paleoecology of carboniferous arboreous Lycopsids from order Lepidodendrales with special consideration of region of Balkan peninsula

Tokom perioda karbona, od pre 320 do pre 306 mliona godina, u području tropskog pojasa (paleoekvatora) Evroameričkog kopna, razvijale su se močvarne šume čiji su edifikatori bile drvolike prečice reda Lepidodendrales. Prečice generalno pripadaju razdelu Lycopodiophyta i predstavljaju jednu od najstarijih grupa biljaka, čija evolutivna linija traje sve do današnjih dana. Njihovi najstariji predstavnici Asteroxylon, .Drepanophycus i Baragwanathia pojavili su se pre oko 400 miliona godina i spadaju u grupu prvih kopnenih biljaka. Pored toga Lycopodiophyta su evolitivno bile i prve biljke koje su formirale fanerofitnu životnu formu (rodovi Cyclostigma i Archaeosigillaria u gornjem devonu pre 370 miliona godina). Tokom karbona, drvolike prečice su dostigle svoj maksimalan razvoj. U to vreme veliki prostori paleotropskog pojasa bili su prekriveni močvarnim šumama u kojima su dominirale Lycopodiophyta čija su stabla prevazilazila 40 metara visine. Najveći broj karbonskih prečica pripadao je redu Lepidodendrales i rodovima Lepidodendron, Synchysidendron, Diaphorodendron, Lepidophloios i Sigillaria. Krajem karbona, pre oko 300 miliona godina sve drvolike prečice su izumrle, i koliko je za sada poznato, u okviru ovog razdela više se nisu pojavljivale drvolike životne forme. Danas predstavnici Lycopodiophyta predstavljaju marginalnu grupu biljaka, minorne uloge i značaja u okviru različitih vegetacija, čiji predstavnici (Lycopodium, Selaginella, Isoetes i dr.) nemaju gotovo nikakvih morfoloških sličnosti sa predstavnicima iz reda Lepidodendrales. Najveći deo uporedne analize izmedju Lepidodendrales i recentne denroflore bio je baziran prvenstveno na principima aktuelizma i zakonitostima koje se javljaju prilikom fosilizacije biljaka. Aktuelističke metode su primenjene zato što je utvrdjeno da postoje bazične sličnosti u razvoju vegetacija gornjeg karbona i neogena/kvartara. Tu prvenstveno treba pomenuti pojedine uticaje/dogadjaje koji su doveli do formiranja i diferenciranja različitih vegetacijskih pojaseva, kao što su pojava Karbonskog i Pleistocenskog ledenog doba, nastanak intenzivnih orogeneza (Hercinsko/Varscinska i Alpijska), formiranje sličnih močvarnih staništa, postojanje tropskog vegetacijskog pojasa, kao i činjenica da su karbonske kao i neogene močvarne šume ostavile za sobom izrazito velike količine uglja. Paleotropske šume u kojima su dominirale drvolike prečice reda Lepidodendrales predstavljaju izuzetno značajnu i na osnovu fosila veoma dobro dokumentovanu šumsku vegetaciju čiji se kompleksni ekološki značaj može sagledati u sledećem: 1. Neobnovljivi energetski resursi sadržani u ogromnim količinama karbonskog uglja koji je nastao uglavnom od stabala Lepidodendrales. 2. Konvergentne morfo-anatomske karakteristike sa filogenetski udaljenim kopnenim (vaskularnih) semenim biljakama (drvoliki habitus, tipovi grananja, strukture slične semenima, pojava monokarpije). To ukazuje da su se mnoge krakteristike biljaka, kao i životne forme (u ovom slučaju fanerofitna) pojavljivale nezavisno i više puta u filogenetski udaljenim evolutivnim linijama. 3. Tek sa formiranjem i razvojem bujnih paleotropskih šuma došlo je, zahvaljujući povoljnoj šumskoj mikroklimi, pogodnim staništima i gotovo neograničenom izvoru hrane, do ubrzanog razvoja kopnenih životinja. U cilju objašnjenja razvoja i dinamike ovih šuma analizirane su paleoekološke osobine pojedinih rodova koji su bili njihovi edifikatori. Kao referentni rodovi uzeti su oni za koje postoji najviše fosilnih i literaturnih podataka i koji su već ranije utvrdjeni kao morfotipovi, kao što su Lepidodendron, Synchysidendron, Diaphorodendron, Lepidophloios i Sigillaria. Ovi rodovi su svojim morfoanatomskim karakteristikama, prvenstveno opštim izgledom habitusa, tipom grananja, visinom stabala i odlikama korenskog sistema uporedjivani sa strukturnim karakterisitkama recetnih predstavnika dendroflore, pre svega sa drvećem i drvenastim semenim biljkama. Na osnovu pojave i uloge sličnih ili različitih morfo- anatomskih karakteristka kod ispitivanih fosilnih Lepidodendrales i referentnih predstavnika recentne dendroflore utvrdjene su brojne konvergetne strukturne karakteristike koje objašnjavaju paleoekološki karakter i značaj izumrlih Lepidodendrales. Prilikom analize opšteg izgleda habitusa i visine stabala utvrdjeno je da su monokarpne vrste, posebno rod Lepidodendron, mogle da budu visoke i preko 40 m (koliko je za sada utvrdjena najveća visina ovog roda), što ukauje da su neki predstavnici Lepidodendrales visinom približavali recentnim četinarima, odnosno da su bili viši od mnogih današnjih drvenastih cvetnica. Konstatovane su analogne heteroblastične i monokarpne odlike vegetativnih i reproduktivnih adultnih stadijuma predstavnika drevnih rodova Lepidodendron, Synchysidendron i Lepidophloios i recentnih monokarpnih vrsta iz familija Agavaceae, Bromelidaceae i Asteraceae. Monokarpni Lepidodendrales pokazuju određenu sličnost sa nekim džinovskim rozetastim (zeljastim) monokarpnim vrstama roda Agave koje upadljivo, heteroblastično, menjaju izgled habitusa tokom reproduktivnog stadijuma. Pored toga, monokarni predstavnici Lepidodendrales se opstajanjem na staništu i posle reproduktivne faze mogu porediti sa drvenastim monokarpnim vrstama roda Tachigali kod kojih nema heteroblastičnih promena, ali koje se, takođe zadržavaju na staništu još izvesno vreme posle reproduktivne faze, obezbedjujući svojim stablom prostor mladim jedinkama iste vrste. Uočena je i sličnost heteroblastičnih promena tokom smenjivanja vegetativnog u reproduktivni stadijum kod Lepidodendrales i prelaza iz juvenilnog u adultni stadijum recentnih polikarpnih vrsta roda Pseudopanax. Uporedne analize bazirane na gradji i ulozi listova i lisnih jastučića dovele su do zaključka da su listovi Lepidodendrales, koji periodično opadaju ostavljajući za sobom lisne baze (lisne jastučiće), jedinstvena karakteristika reda Lepidodendrales i da se ne mogu definitivno porediti sa recentnim biljkama. Nasuprot tome, postoje izvesne analogne morfološke odlike osnovnih delova lista, pre svega lisne ploče i ligule Lepidodendrales i nekih recentnih biljaka. Na poprečnom preseku lisna ploča Lepidodendrales slična je lisnoj ploči nekih vrsta roda Pinus iz grupe Diploxylon, odnosno vrstama iz grupe Haploxylon. Donekle se i ligula Lepidodendrales može porediti sa ligulom kod familije Poaceae, pre svega u pogledu sličnog položaja - u pazuhu lista, tj na prelazu lisne sare u lisnu ploču. Rizomorfni ili korenski sitem Lepidodendrales označen kao stigmarija (rod Stigmaria) može se uporediti sa korenovima nekih drvenastih recentnih biljaka sa vlažnih i močvarnih staništa. Naime, može se pretpostaviti da su bočni korenski izdanci stigmarija imali aeracionu ulogu slično pneumatoforama na korenovima današnjih vrsta iz rodova Taxodium, Nyssa, ili mangrova vegetacija (Rhizophora spp.). Na osnovu velikog broja fosilnih ostataka zaključeno je da su stigmarije bile površinski organi ili površinski deo korenskog sistema čiji se podzemni deo, poput drugih podzemnih biljnih organa, nije sačuvao u fosilnom obliku. U doktorskoj disertaciji je obavljena i uporedna analiza paleotropskih šuma Lepidodendrales i odgovarajućih recentnih šumskih zajednica. Utvrdjeno je da paleotropske šume najviše sličnosti pokazuju sa suptropskim močvarnim šumama u kojima dominira močvarni četinar Taxodium spp. Obe ove, vremenski veoma udaljene, zajednice odlikuje slično močvarno stanište, kao i sličan nasumičan raspored i opšti izgled edifirkatorskih vrsta, najveći deo fitomase se nalazi u stablu, dok je veličina krošnji zanemarljiva. Paleotropske močvarne šume se mogu porediti i sa recentnim tropskim kišnim šumama, pre svega stoga što se obe zajednice razvijaju u uslovima vlažne tropske klime. Međutim, mora se uzeti u obzir da struktura recentnih tropskih šuma daleko složenija zahvaljujući raznovrsnim životnim formama koje su razvile različite vrste cvetnica. Diskutovana je i izvesna analogija paleotropskih šuma Lepidodendrales i borealnih četinarskih šuma - tajgi, koja se ogleda u maloj taksonomskoj raznovrsnosti i malom broju edifikatorskih vrsta. U disertaciji su po prvi put iscrpno komentarisana dosadašnja istraživanja Lepidodendrales na Balkanskom poluostrvu. Na osnovu dostupnih podataka florističkih spiskova i paleofitocenoloških podataka većeg broja različitih paleotropskih zajednica Lepidodendrales na području zapadnog i centralnog Balkana, na teritoriji današnjih država Slovenije, Bosne i Hercegovine i Srbije, uradjena je rekonstrukcija mogućeg/pretpostavljenog staništa odgovarajućih paleofitocenoza.

During the Carboniferous period, 320-306 million years ago, swamp forests with tree- like lycopsids named Lepidodendrales as edificatory species have developed in the region of tropical belt (paleoequator). The lycopsids belong to the general division of Lycopodiophyta, representing one of the oldest plant groups, and their evolution continues to this day. The oldest lycopsids have appeared about 400 million years ago and their representatives from genera Asteroxylon, Drepanophycus and Baragwanathia belong to the group of first land plants. Lycopodiophyta were also the first land plants to form the phanerophyte life form (genera Cyclostigma and Archaeosigillaria in Upper Devonian, 370 million years ago). During the Carboniferous period, the arboreous lycopsids have reached their maximal development. At that time there were large areas of paleotropical belt covered in swamp forests, where the edificatory species were Lycopodiophyta over 40 m tall. The Carboniferous arboreous lycopsids belonged to order Lepidodendrales and genera Lepidodendron, Synchysidendron, Diaphorodendron, Lepidophloios and Sigillaria. By the end of Carboniferous (about 300 million years ago) all the arboreous lycopsids became extinct, and according to present knowledge the tree-like life forms never appeared again in this group. Today the representatives of Lycopodiophyta are a marginal group of plants, with a minor role and importance in various vegetation lists. Their representatives (Lycopodium, Selaginella, Isoetes etc.) have almost no morphological similarities with the arboreous lycopsids of order Lepidodendrales. The comparative analysis of Lepidodendrales and the recent dendroflora was mostly based on principles of actualism and rules of plant fossilization. The actualistic methods were applied as some basic similarities were noted in development of vegetation in Upper Carboniferous and Neogene/Quaternary. It is particularly important to mention certain factors/events that led to formation and differentiation of different vegetation belts. They include appearance of Carboniferous and Pleistocene Ice Ages, appearance of intensive orogeny (Hercynian/Variscan and Alpine), formation of similar wetland habitats, presence of a tropical vegetation belt, as well as the fact that Carboniferous and Neogene swamp forests have both left behind prominent large amounts of coal. The paleotropical forests dominated by tree-like lycopsids of order Lepidodendrales are representative of particularly important forest vegetation, well-documented by fossils. Its complex ecological importance may be summed in a following way: 1. Unsustainable energetic resources of enormous amounts of Carboniferous coal, mostly originating from stems of Lepidodendrales. 2. Morpho-anatomic characteristics convergent with those of phylogenetically distant land (vascular) seed plants (tree-like habit, branching types, seed-like structures, appearance of monocarpy). This indicates that many of the plant characteristics and life forms (in this case phanerophytes) have appeared independently several times in phylogenetically distant evolutionary lines. 3. The accelerated development of land animals took place only after the formation and development of lush paleotropical forests and appearance of favorable forest microclimates, suitable habitats and almost infinite sources of food. In order to explain development and dynamics of these forests, paleoecological characteristics of certain edificatory genera were analyzed. The reference genera were chosen by number of fossil and literature data and by being previously determined as morphotypes, for example Lepidodendron, Synchysidendron, Diaphorodendron, Lepidophloios and Sigillaria. These genera were compared with structural characteristics of recent representatives of dendroflora, primarily trees and woody seed plants, regarding the general habit, type of branching, height of habit/stem and characteristics of root system. Appearance and role of similar or different morpho-anatomical characteristics in studied fossil Lepidodendrales and referenced representatives of recent dendroflora were used to determine numerous convergent structural characteristics explaining the paleoecological character and importance of extinct Lepidodendrales. During the analysis of general habit and height of trees, it was determined that the monocarpous species, particularly the genus Lepidodendron, could reach over 40 m in height (that is the greatest determined height of this genus so far), indicating that certain representatives of Lepidodendrales could reach the height of recent conifers and were taller than many present-day arboreous flowering plants. Analogous heteroblastic and monocarpous characteristics of vegetative and reproductive adult stages were recorded in representatives of ancient genera Lepidodendron, Synchysidendron and Lepidophloios and in recent monocarpous species from families Agavaceae, Bromelidaceae and Asteraceae. The monocarpous Lepidodendrales are showing certain similarities with some giant rosette-bearing (herbaceous) monocarpous plants of genus Agave, which have a pronounced heteroblastic change in habitus during the reproductive stage. As monocarpous representatives of Lepidodendrales remain in the habitat after the reproductive phase, they may also be compared with arboreous monocarpous species of genus Tachigali, which lack the heteroblastic changes but also remain in the habitat for some time after the reproductive phase, providing shelter for young individuals of its species with its stem. Also noted was similarity of heteroblastic changes during the shift from the vegetative to the reproductive stage in Lepidodendrales and shift from the juvenile to the adult stage in recent polycarpous species of genus Pseudopanax. The comparative analyses based on structure and role of leaves and leaf cushions led to conclusion that leaves of Lepidodendrales, periodically excised while leaving leaf bases (cushions) on the stem, are a unique characteristic of order Lepidodendrales and may not be definitively compared with recent plants. On the other hand, there are certain analogous morphological characteristics of main flower parts, primarily the leaf plate and ligula in Lepidodendrales and certain recent plants. The transversal cut of leaf plate of Lepidodendrales is similar to that of certain species of Pinus from Diploxylon group or species from Haploxylon group. The ligula of Lepidodendrales may be compared to some extent to ligula in family Poaceae, primarily due to the similar position – between the leaf and the stem, at the transition between the leaf sheath and blade. The rhizomorph or root system of Lepidodendrales named stigmaria (genus Stigmaria) may be compared with roots of certain recent woody plants from wetland and marsh habitats. It may be assumed that the lateral root shoots of stigmaria had a role in aeration, similar to pneumatophores in roots of present-day species from genera Taxodium, Nyssa, or mangrove vegetation (Rhizophora spp.). In relation to the large number of fossil remains it was concluded that stigmaria were aerial organs or aerial parts of root system, while the underground part, like many other underground plant organs, was not preserved in fossil form. The Ph.D. thesis also includes a comparative analysis of paleotropical forests of Lepidodendrales and corresponding recent forest associations. It was determined that the paleotropical forests show most similarities with the subtropical swamp forests dominated by swamp conifer Taxodium spp. Both these associations, although very distant from each other in time, are characterized by similar swamp soil and similar random distribution and general habitus of edificatory species – the greatest part of phytomass is situated in the bole while the canopy size is negligible. The paleotropical swamp forests may also be compared to the recent tropical rainforests, as both associations develop in conditions of humid tropical climate. However, it must be considered that the structure of recent tropical forest is far more complex due to diverse life forms developed by various species of flowering plants. Also discussed was certain analogy between the paleotropical forests of Lepidodendrales and boreal conifer forest (taiga), expressed in low taxonomic diversity and a small number of edificatory species. This dissertation is the first paper with detailed analysis of all studies on Lepidodendrales performed at Balkan Peninsula. The available data in floristic lists and paleophytocoenological data on a larger number of different paleotropical associations of Lepidodendrales in western and central parts of Balkans (the territories of present-day Slovenia, Bosnia-Herzegovina and Serbia) were used for reconstruction of possible/assumed habitat of appropriate paleophytocoenoses.

Metabolička raznovrsnost bakterija izolovanih iz površinskog rečnog sedimenta pod uticajem petrohemijskih efluenata: Pseudomonas sp. tn301 kao proizvođač polihidroksialkanoata

Metabolic varsality of bacteria isolated from surgace river sediment influenced by petrochemical effluentes: the case of Pseudomonassp. TN301 as polyhydroxyalkanoate producer

Mono- i poliaromatična jedinjenja predstavljaju široko rasprostranjene zagađivače životne sredine. Ova jedinjenja veoma dugo opstaju u datoj sredini, a mnoga od njih imaju i toksična, kancerogena i mutagena svojstva. Sredine zagađene aromatičnim jedinjenjima su najčešće kontaminirane i teškim metalima, a odlikuju se i neravnotežnim odnosom esencijalnih nutrijenata. Sa druge strane, ovakve sredine su dobar izvor bakterija sa sposobnošću preživljavanja u izuzetno teškim uslovima. Cilj ovog rada je bila izolacija robustnih bakterija sa potencijalnom biotehnološkom aplikacijom iz sedimenata koji su pod uticajem petrohemijskih efluenata. Upotrebom dve različite strategije izolovanja, toplotnog pretretmana i medijuma za rast i sporulaciju Actinobacteria, i predobogaćivanja uzoraka naftalenom u kombinaciji sa odabirom brzo rastućih bakterija sa sposobnošću rasta na naftalenu kao jedinom izvoru ugljenika i energije izolovane su 34 Gram-pozitivne i 4 Gram-negativne bakterije. Izolati sa sposobnošću degradacije od 9 do 15 najčešćih aromatičnih polutanata poput benzena, bifenila i naftalena pripadali su rodovima Bacillus, Arthrobacter, Rhodococcus, Gordonia, Streptomyces, Staphylococcus, Actinobacteria, Sinorhizobium i Pseudomonas. Rhodococcus sp. TN105, Gordonia sp. TN103 i Arthrobacter sp. TN221 su identifikovani kao novi sojevi. Od 11 izolata sa sposobnošću rasta u prisustvu bar jednog od testiranih teških metala (živa, kadmijum, nikl) u 100 mM koncentracijama, 8 je imalo sposobnost degradacije 15 različitih aromatičnih jedinjenja. Rezistenciju na bar jedan od testiranih antibiotika pokazalo je 60% izolata. Međutim, nije primećena visoka učestanost multirezistencije na antibiotike. Trećina izolata je imala sposobnost da akumulira bar jedan od biopolimera. Dvanaest izolata, uglavnom pripadnika rodova Bacillus i Arthrobacter su akumulirali polifosfate, 3 Bacillus sp. izolata su akumulirala polihidroksibutirat (PHB), Pseudomonas sp. TN301 je akumulirao polihidroksialkanoate srednje duzine niza (mcl-PHA), dok su 4 Grampozitivna izolata akumulirala egzopolisaharide. U ovom radu je po prvi put pokazana sposobnost pripadnika roda Bacillus da konvertuju toluen u PHB. Pseudomonas sp. TN301 se istakao zbog svoje sposobnosti da akumulira mcl-PHA do 22% suve ćelijske mase iz poliaromatičnog jedinjenja naftalena. Razvijen je metod za dodavanje izrazito hidrofobnih poliaromatičnih jedinjenja u kultivacioni medijum. Najveća masa i najviši nivo akumuliranog mcl-PHA postignuti su dodavanjem sintetičkog surfaktanta Tweena 80 (0.5 g l-1). Pokazano je da Pseudomonas sp. TN301 ima sposobnost akumulacije mcl-PHA iz širokog opsega poli- i monoaromatičnih jedinjenja, kao i njihovih smeša. Ovaj rad predstavlja prvu sveobuhvatnu studiju metabolički raznovrsnih, u prvom redu Gram-pozitivnih bakterija sa velikim aplikacionim potencijalom u procesu bioremedijacije sredina zagađenih mešovitim polutantima, ali i sa drugim biotehnološkim aplikacijama kao sto je konverzija otpadnog materijala u biopolimere. U ovom radu je po prvi put pokazana sposobnost bakterijskog soja da konvertuje različita poliaromatična jedinjenja i njihove smeše u biodegradabilni polimer (mcl- PHA). S obzirom na to da ima odlike biodegradabilnog termoelastomera, mcl-PHAsve više dobija na važnosti, pa je izolacija novih sojeva sa sposobnošću njegove produkcije od izuzetnog značaja.

Mono- and polyaromatic compounds are among the most prevalent and persistent pollutants in the environment. They have been reported as toxic, carcinogenic and mutagenic. Contaminated sites usually contain complex mixture of aromatic compounds and heavy metals, and are characterized by essential nutrients imbalance. On the other hand, polluted sites proved to be valuable sources of bacteria capable of surviving in these harsh environments. The aim of this study was to obtain robust bacteria with potential biotechnological application from the river sediments affected by the proximity of a petrochemical industrial site. Using two different isolation strategies, heat pretreatment and media for growth and sporulation of Actinobacteria and naphthalene enrichment combined with the selection of fast growing bacteria capable of using naphthalene as the sole source of carbon and energy, 34 Gram-positive and 4 Gram-negative bacterial strains were isolated. Isolates that were able to use 9 or more common aromatic pollutants, such as benzene, biphenyl, naphthalene etc. as a sole source of carbon and energy included members of Bacillus, Arthrobacter, Rhodococcus, Gordonia, Streptomyces, Staphylococcus, Actinobacteria, Sinorhizobium, and Pseudomonas genus. Rhodococcus sp. TN105, Gordonia sp. TN103 and Arthrobacter sp. TN221 were identified as novel strains. Out of 11 isolates able to grow in the presence of one or more metals (mercury, cadmium, nickel) at high concentration (100 mM), eight could degrade 15 different aromatic compounds. Antibiotic resistance to at least one of the tested antibiotics was found among 60% of the isolates. However, high incidence of multiresistance was not observed. One third of the isolates could accumulate at least one biopolymer. Twelve isolates (mainly Bacillus sp. and Arthrobacter sp.) accumulated polyphosphate, three Bacillus sp. accumulated polyhydroxybutyrate (PHB), and Pseudomonas sp. TN301 accumulated medium chain length polyhydroxyalkanoate (mcl-PHA), while four Gram-positive isolates could accumulate exopolysaccharides. This study is the first report of a Bacillus strain capable of converting toluene to PHB. Pseudomonas sp. TN301 was distinguished by its ability to accumulate mcl-PHA up to 22% of cell dry weight from the naphthalene. A method of supplying highly hydrophobic polyaromatic hydrocarbons to a culture medium was developed. The best biomass and mcl-PHA yields were achieved with the addition of synthetic surfactant Tween 80 (0.5 g l-1). It was shown that Pseudomonas sp. TN301 can accumulate mcl-PHA from a wide range of poly- and monoaromatic hydrocarbons, and mixtures thereof. This is the first comprehensive study on the metabolically diverse, primarily Grampositive bacteria with great application potential for mixed pollution bioremediation studies, as well as in other biotechnological applications such as waste conversion to biopolymers. Also, this is the first report of the ability of a bacterial strain to convert a range of polyaromatic hydrocarbon compounds to the biodegradable polymer (mcl- PHA). Mcl-PHA is gaining importance as a promising biodegradable thermoelastomer and therefore isolation of new producing strains is highly significant.