Miracle-dz/UniFAR-FTUs huggingface dataset:
Establishing Vertebrate Model Systems for the Study of Gle1 Mediated Motor Neuron Disease

Gle1 is an evolutionary conserved protein involved in both mRNA export and translation. A recent study linked mutations in Gle1 with recessive and fatal motor neuron diseases characterized by ventral horn motor neuron degeneration before birth. This is particularly interesting in light of a growing pool of evidence indicating that a common denominator in many motor neuron disorders is defects in mRNA regulation. To investigate the role of Gle1 in motor neuron development, we used chick and mouse models to develop tools for examining Gle1s expression, localization, biochemical interactions, and altered activity when mutated. We have confirmed the presence of Gle1 transcripts in a variety of cell types and stages of development, including during motor neuron development, and we generated Gle1-Flag fusion constructs to study Gle1 localization and biochemistry following failure of commercial antibodies to reliably detect Gle1 through immunohistochemistry and immunoblotting. Overexpressing mutant Gle1 does not appear to noticeably affect the development of motor neurons in chick, suggesting that the mutant protein does not cause disease through a gain of function. To study the effects of Gle1 loss on development, we generated a mouse line expressing a Gene-trap allele in which Gle1 is created as a truncated protein linked to [Beta]-Geo. As animals homozygous for this allele appear nonviable, we selected a rescue strategy in which we will express Gle1 from the ROSA26 locus in a Gene-trap background, allowing us to examine the specific effects of Gle1 mutation on motor neuron survival and function
Microglia Respond to Both Permanent and Temporary Deaferentation in the Adult Zebrafish Olfactory Bulb

The major goal of this study was to investigate a microglial response following deafferentation in the adult zebrafish, Danio rerio, olfactory bulb. Microglia were identified in the olfactory bulb using the 4c4 antibody and phagocytic-dependent labeling (DiA). Microglia responded to both methods of deafferentation namely, permanent (cautery) and temporary (Triton X-100). On one and three days following both permanent and temporary deafferentation there was a significant increase in microglia in the treated olfactory bulb. At five and seven days after permanent and temporary deafferentation there was no longer a significant difference between the treated and untreated olfactory bulbs, indicating that a microglia response occured following permanent and temporary deafferentation. This study has established the foundation for future microglia studies in the degeneration and reinnervation processes in the adult zebrafish olfactory bulb.
Physiological aspects underpinning recombinant protein production in Escherichia coli

Many biopharmaceutical projects require the production of recombinant protein in a bacterial host. Conventional procedures used for recombinant protein production (RPP) involve the rapid synthesis of the target protein. This results in the accumulation of unfolded protein, the induction of the heat shock stress response and bacterial growth arrest. More importantly, the target protein accumulates in inclusion bodies and hence is useless to determine its structure. The immediate impact of this is that both the yield and quality of the target protein are compromised. This thesis reports two generically successful approaches that were developed to overcome this series of stress-induced events in Escherichia coli. Both strategies were developed during the production of a cytoplasmic protein and outer membrane lipoproteins using the pET expression system in the bacterial host, E. coli strain BL21(DE3)*. First, the induction protocol was modified to minimise the stress on the host bacterium. This method relies on the induction of very low levels of the T7 RNA polymerase in BL21* and thus the correspondingly slow synthesis of the target protein. Using this approach, growth and productivity of different types of correctly folded target proteins were sustained for at least 70 h. Secondly, mutant hosts that significantly improve recombinant protein production during conventional protocols were isolated. These improved hosts are resistant to IPTG-induced stress and continue to accumulate high levels of the correctly folded target protein. Key to the stress resistance is the presence of mutations that downregulate the synthesis of T7 RNA polymerase. However, different improved hosts were able to enhance the production of different types of target protein, such as those requiring extensive post-translational modification. The potential for isolating a plethora of improved bacterial hosts that are tailored for the production of different types of recombinant protein is discussed in light of the challenges faced by bioindustry. Procedures enabling the isolation of mutant hosts during the production of GFP-tagged and untagged proteins are reported.
Responses of monocotyledonous crops to crude extracts of Cucumis myriocarpus fruits as a pre-emergent bio-nematicide.

The southern root-knot nematode, Meloidogyne incognita, remains a devastating plant-parasitic nematode in crop production, particularly in tropical areas with sandy soils. Extracts of ground Cucumis myriocarpus fruits consistently suppress Meloidogyne incognita race 2 when used as a post-emergent bio-nematicide. The suitability of extracts of ground C. myriocarpus fruits as a pre-emergent bio-nematicide was investigated on maize (Zea mays), finger millet (Eleusine coracana), sorghum (Sorghum bicolor) and onion (Alliums cepa). The seven levels of aqueous crude extracts of C. myriocarpus fruits, arranged in a completely randomized design, with 4 replications. The germination percentage had significant polynomial relationships with concentrations of crude extracts, explaining 93, 83, 96 and 79% of the total treatment inhibition in germination of maize, millet, sorghum and onion, respectively. Results suggested that extracts of ground C. myriocarpus fruits were not suitable for use as a pre-emergent bionematicide in monocotyledonous seeds.
OBESITAS UMUM BERDASARKAN INDEKS MASA TUBUH DAN OBESITAS ABDOMINAL BERDASARKAN LINGKAR PINGGANG TERHADAP KEJADIAN PREDIABETES

Prediabetes adalah masalah kesehatan global. Prevalensi prediabetes meningkat signiikan di seluruh dunia dan umumnya tinggi di masyarakat serta merupakan keadaan risiko tinggi untuk DM. Obesitas memiliki peranan penting dalam patoisiologi prediabetes. Penelitian ini bertujuan untuk mengetahui apakah obesitas umum dan obesitas abdominal secara bersama-sama berhubungan terhadap kejadian prediabetes pada kelompok usia 20-65 tahun di Kecamatan Bogor Tengah berdasarkan faktor riwayat DM keluarga, jenis kelamin, umur, merokok, hipertensi, aktiitas isik dan stress. Desain studi yang digunakan adalah potong lintang dengan Cox Regression untuk analisis multivariable. Data analisis merupakan data baseline dari studi kohort faktor risiko penyakit tidak menular tahun 2012. Ada 3244 responden yang dipilih dengan teknik sampel acak. Hasil analisis menunjukkan bahwa obesitas terhadap kejadian prediabetes setelah mengendalikan faktor umur adalah obesitas umum PR 1,56 (95% CI: 1,15-2,11), obesitas abdominal PR 1,43 (95% CI; 1,09-1,85) dan obesitas umum dan obesitas abdominal secara bersama-sama PR 1,93 (95% CI; 1,62-2,28). Untuk itu, obesitas umum dan obesitas abdominal secara bersama-sama berkontribusi paling besar terhadap peningkatan prevalensi prediabetes. Peningkatan kesadaran dan skrining prediabetes pada kelompok risiko tinggi berdasarkan pengukuran IMT bersama dengan lingkar pinggang penting untuk dipertimbangkan sebagai bagian dari upaya untuk mengurangi epidemi prediabetes di masyarakat. Kata Kunci: obesitas abdominal, obesitas umum, prediabetes
Tissue and organelle targeted transgene expression in plants.

This research develops systems for tissue and organelle targeted transgene expression in commercial strawberry. Two approaches were taken to achieve this. I) the development of a root-specific expression system for nuclear transgenes and II) the development of plastid transformation methodologies for strawberry. FavRB7, a root-specific, RB7 tonoplast intrinsic protein homologue was isolated from strawberry root tissue using reverse transcnptase-polymerase chain reaction (RT-PCR). A 2.8 kb region of promoter sequence associated with the FavRB7 gene was isolated and cloned upstream of the β-glucuronidase (gusA) reporter gene. Transgenic strawberry and tobacco were produced harbouring the gusA gene under control of the FavRB7 gene promoter. Promoter activity in these lines was characterised using histochemical, fluorometric and RNA molecular assays. FavRB7 promoter activity was shown to be strong and near root-specific in strawberry. However in contrast, in the heterologous species tobacco, FavRB7 promoter activity was shown to be constitutive. A series of novel plastid transformation vectors, harbouring selectable and scorable marker genes under control of strawberry plastome regulatory elements, were created to enable plastid transformation in strawberry and tobacco. Plastid transformation methodologies were developed for strawberry utilising the aminoglycoside 3'-adenyltransferase (aadA) gene and spectinomycin selection. Additionally, plastid transformation using a non-antibiotic selection system was investigated utilising the xylose isomerase (xylA) gene and D-xylose selection. Heteroplasmic plastid transformed strawberry lines were recovered using both selection systems and expression of the soluble modified green fluorescent protein (smGFP) scorable marker gene visualised specifically in strawberry chloroplasts of three individual lines. Additionally, heteroplasmic plastid transformed tobacco lines harbouring transgenes under control of strawberry plastome regulatory elements were generated using spectinomycin selection.
Hair follicle germinative epidermal cells : a molecular study

At the base of the hair follicle epidermal matrix is a population of germinative epidermal (GE) cells that is in close communication with the dermal papilla. These GE cells are at the core of activities that comprise the fundamental processes of cell signalling and differentiation in the hair follicle. Since it is in the germinative region that the signals that produce hair are being received and transcribed, identification of genes expressed in the GE cells will be important for our understanding of hair growth control and the molecular mechanisms operating at the site of epidermal proliferation and differentiation. This study describes the production of a series of cDNA libraries, both by conventional means from rat vibrissa follicles and follicle end bulbs, and by PGR from the GE cells and the tissues of the upper end bulb. These libraries were then used for a variety of screening approaches to isolate cDNA clones, firstly for molecules which are known to be involved in the control of hair growth, and secondly for molecules which are differentially expressed in the follicular germinative epidermis. In order to identify such preferentially expressed genes, a dual labelling differential screen of the vibrissa follicle end bulb cDNA library was performed, using probes derived from the germinative epidermal and upper end bulb PGR generated libraries. Nine putative differentially expressed clones were isolated and sequenced. RNase protection analysis and non radioactive in situ hybridisation was then performed to confirm that these clones were expressed in the germinative epidermis of rat vibrissa follicles. Further characterisation by northern blotting revealed that several of the clones were expressed in multiple tissues. Nucleotide sequence analysis revealed that six of the clones had a concensus BG1 repeat sequence at the end of their 3'UTR. This has been implicated in post-transcriptional control of intracellular mRNA localisation. Three of these clones were related to genes implicated in induction and vesicle trafficking. These clones may therefore be involved in the signal transduction pathways operating in the germinative epidermis in response to primary signalling molecules received from the dermal papilla.
A new blood clotting inhibitor.

On attempting to purify thromboplastin, various investigators1 have obtained mat,erials which possess decreased thromboplastic activity. In our work we have confirmed these observations.2 The question arises as t#o what causes this decrease in tjhromboplastzic act,ivity. Is it that a component inherent to the thromboplastin molecule has been denatured, inactivated, or removed, thus inactivating the molecule? Or is it due to the presence of a.n inhibitor, the concentration of which has been increased by the attempted purification procedure, thus causing a decresse in tzhe observed thromboplast)in a.ctivity? It is our belief that’ the reduction in t,hromboplastic activity is due to an increase in concentration of t,hc thromboplastin inhibitor. Chemical isolation and identification of such a substance might indirectly point to the chemical structure of thromboplastin, since it is a known fact that, most, biologically a,ctive compounds ha\vc! a structure similar t,o their inhibibors. We have investigated the possibility of isolating a thromboplastin inhibitor. By chemical fractionation, a material possessing inhibitory properties was obtained from the phosphatide fraction of dried beef brain and rabbit lung thromboplastins, and also from soy bean phosphatides and human pla.sma. 3 In our preliminary work our most active inhibitory fraction had chemical properties similar to inositol phosphatides isolated from brain and soy beans. On chemical analysis, t,he following data were obtained: brain inositol phosphat.ide, C 53.3, I’ 4.3, K 1.0, ash 17.1; soy bea.n inositol phosphatide, C 54.1, 1’ 2.9, N 1.1, ash 13.2. The analytical dat.a compare favorably with the analysis given by Folch4 on brain inositol phosphatide, and by Woolley5 on soy bean inositol phospha,tide. To verify t’he identity of our thromboplastin inhibitor preparation, we t)hen isolat,ed brain and soy bean inositol phosphatides by the methods of Folch4 and Woolley.6 The material isolated in t,his manner had the same properties as our t’hromboplastin inhibitory fraction, indicating that it is identical with the inositol phosphatide fraction. Samples of inositol phosphatide
Requirements for Regenerative Mechanisms in Tissue Growth and Homeostasis in Adult Zebrafish

Requirements for Regenerative Mechanisms in Tissue Growth and Homeostasis in Adult Zebrafish by Airon Alease Wills Department of Cell Biology University Program in Cell and Molecular Biology Duke University Medical Center Date:___________________ Approved: _______________________ Kenneth D. Poss, Advisor _______________________ Brigid L.M. Hogan _______________________ John Klingensmith _______________________ Howard Rockman _______________________ Michel Bagnat An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell Biology in the Graduate School of Duke University 2009
The roles of the Rb and p53 tumor suppressor pathways in an intestinal tumorigenesis model system

Cancer is characterized by unregulated growth. SV40 large tumor (T) antigen interacts with cellular proteins to cause transformation and tumors in rodents. Interaction with the retinoblastoma (Rb) family of tumor suppressors allows T antigen to inhibit growth arrest, while T antigen interaction with the p53 tumor suppressor blocks apoptosis.Transgenic mice that express T antigen (TAgwt) in intestinal enterocytes develop hyperplasia that progresses to dysplasia. Expression of a mutant of T antigen (TAg3213) that is unable to interact with Rb family members in enterocytes does not result in an intestinal phenotype. Thus, T antigen interaction with Rb family members (pRb, p130 and p107) is required for T antigen-induced intestinal neoplasia. pRb performs the majority of its tumor suppressive properties through repression of E2Fs, a family of transcription factors that regulate the expression of many genes required for S phase. T antigen binds Rb family members, disrupts p130/E2F complexes and induces E2F2 and E2F3a in villi. These E2Fs are bound to the promoters of up-regulated E2F-responsive genes, supporting a role for them in T antigen-induced hyperplasia. Expression of an amino-terminal truncation mutant of T antigen (TAgdl1137) in villi enterocytes is sufficient to induce hyperplasia, but not progression to dysplasia. We hypothesized the T antigen interaction with p53 is required for the progression to dysplasia. However, T antigen does not bind and stabilize p53 in villi enterocytes. Furthermore, TAgdl1137/p53-/- mice do not progress to dysplasia. Therefore, we propose that the p53 is not active in intestinal enterocytes and its inactivation is not required for T antigen-induced progression to dysplasia.These results suggest that distinct tumor suppressor proteins and pathways function in specific cell types to regulate normal or abnormal mechanisms of proliferation.
Virus Induced Formation Of Extracellular Microvesicles During Coxsackievirus Dissemination In The Host

OF THE THESIS Virus-Induced Formation of Extracellular Microvesicles during Coxsackievirus Dissemination in the Host by Vrushsali Mangale Master of Science in Biology with a Concentration in Molecular Biology San Diego State University, 2014 Coxsackievirus (CVB) is a relatively common enterovirus belonging to the picornaviridae family that is capable of causing aseptic meningitis, pancreatitis and myocarditis in humans. Coxsackievirus B3 (CVB3) preferentially targets neural progenitor and stem cells (NPSCs) with lasting consequences in the central nervous system (CNS). We genetically engineered a recombinant coxsackievirus expressing “fluorescent timer” protein (Timer-CVB3). ”Fluorescent timer” protein undergoes a slow conversion from green to red over time, and thus, Timer-CVB3 can be utilized to detect the progression of virus infection. Upon infection with Timer-CVB3, partially differentiated neural progenitor and stem cells (NPSCs) and C2C12 myoblast cells released extracellular microvesicles (EMVs) containing matured “fluorescent timer” protein and infectious virus. Purified EMVs isolated from the supernatants of Timer-CVB3 infected cells were positive for the exosomal marker flotillin1, the autophagosomal marker LC3-II, and viral capsid protein VP-1. EMVs isolated by iodixanol isopycnic density purification were found in low density fractions consistent with membrane association distinct from high density viral fractions representing infectious virions. Detection of the lipidated form of LC3 protein (LC3-II) in EMVs observed within infected cell culture supernatants suggests involvement of the autophagy pathway during the release of EMVs. Also, viral protein co-localized with LC3 protein within EMVs identified in infected cultured cells, indicating that autophagy is an important process involved in release of shed microvesicles harboring infectious CVB3. Clarifying the role of these infectious EMVs is important for understanding virus immune evasion and for the development of new antiviral therapies and vaccines.
Evaluation of New Strawberry Cultivars for their Potential Use in Organic Farming and in Verticillium-infested Soils

gants is forbidden and other methods of reducing Cold stored plants of 12 cultivars ('Alice', 'Alba', 'Clery', soil-borne pathogens effectively are not found yet. 'Elsan- 'Daroyal', 'Darselect', 'Divine', 'Dora', 'Eva', 'Record', ta', the main early-mid season strawberry cultivar in parts 'Queen Elisa', 'Salsa', 'Sonata') and of 'Elsanta' as control of Europe, though convincing because of good fruit char- were planted in April-June 2005 at 11 sites on 9 farms in acteristics and high yield, is highly susceptible to soil-borne 5 different regions of Eastern Austria (20-48 plants each pathogens (Barth et al. 2002, Spornberger et al. 2005). cultivar and site, 1-4 replications per site). 2 of the farms The aim of this study was to find new cultivars which were 'organic', all others were managed conventionally, combine tolerance to V. dahliae and other diseases with a The content of microsclerotia of V. dahliae in the different high yield, a high percentage of marketable fruits and soils was determined using the wet-sieving method de with consumer acceptance, which should be achieved by scribed by Harris et al. (1993). From each site (area on carrying out trials on farms with different farming sys- which the trial was planted) a bulked sample of 25 cores terns and climate conditions in the main Austrian straw- (to a depth of 20 cm) was taken using a soil borer. The in berry regions. To find early-ripening cultivars with these festation risk for susceptible cultivars was estimated us qualities is of particular interest for farmers because ing the scheme of Harris et al. (1993): <0.3 vital strawberries in the beginning of strawberry season gain microsclerotia g"1 soil = very low risk, 0.3-0.6 = low risk,
ATM mutant cellular phenotype in B-cell chronic lymphocytic leukaemia : clinical consequences and therapeutic implications

ATM germ-line mutations have been identified in a proportion of patients with chronic lymphocytic leukaemia (CLL) but their role in the development of this tumour remains unknown. In the course of this study it was established that ATM pathogenic mutations were increased among patients with chromosome 11q deletion/loss of one ATM allele when compared to healthy control individuals but not in those who did not acquire this deletion in their leukemic clone. The results indicate that ATM germ-line heterozygosity does not play a role in CLL but may influence disease progression through complete ATM loss and clonal expansion. The analysis of the clinical outcome among CLL patients from phase III LRF UK CLL4 trial identified a distinctive subgroup with a particularly poor prognosis. Those patients had bi-allelic inactivation of ATM gene and showed significantly reduced progression-free survival (PFS) compared to those with ATM wild-type or mono-allelic ATM defects. Furthermore, they had equally poor PFS as those with mono- and bi-allelic TP53 abnormalities. The clinical implication of this finding might include re-consideration of the treatment strategies for this particular subgroup of patients. The improved understanding of the biological background of progressive and resistant CLL tumours, such as those with TP53 or ATM defects, results in a development of further targeted treatment strategies. The assessment of their efficacy and toxicity could be facilitated by CLL xenograft models. The optimization strategies overcoming the limitations of existing xenograft model were investigated during the course of this study. The results showed that partial depletion of patient CD3+, CD4+ or CD25+ cells prior to injection into NOG mice can prolong the engraftment of CLL cells within xenogenic microenvironment hence, providing expanded period of time for testing new therapeutic agents.
The mammalian herbivore stomach : comparative anatomy, function, and evolution

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