Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Investigation of Fas/Fas-ligand Interaction in Helper T 17 Cell Functions(01. Izmir Institute of Technology, 2021) Çalışkan, Tufan Utku; Nalbant Aldanmaz, Ayten; Nalbant Aldanmaz, AytenTh17 cells are key players of the adaptive immune system. They mainly take part in responses to extracellular parasites and neutrophil recruitment. They can infiltrate into the inflammation sites and survive for long periods of time. Their malfunction leads to the manifestation of several autoimmune diseases, such as Multiple Sclerosis and Rheumatoid Arthritis. The main reason behind their roles in autoimmune pathogenicity is thought to be their longevity and resistance to apoptosis. One of the main players of apoptosis, Fas, has been found to have non-apoptotic roles and is a candidate for the survival mechanisms of Th17 cells. This study aims to discover possible non-apoptotic roles of the Fas signaling pathway in Th17 cell functions. For this purpose, buffy coats of healthy individuals were used to isolate PBMCs and CD4+CD45RA+ naive T cells were sorted from the PBMCs. Obtained naive T cells were cultured under Th17 polarizing conditions and the expressions of Fas, FasL, TNFR1, and TNF-α have been monitored along with apoptosis. The expression of Fas has been found to significantly increase in the cells cultured under Th17 polarizing conditions. However, there were no FasL and TNFR1 expressions observed. The expression of TNF-α was observed on both the negative culture and Th17 polarizing culture, however, there was no significant difference found. In addition, there was no increase in apoptosis in neither culture. In summary, Fas expression has been found to increase in the cells cultured under Th17 polarizing conditions. Further investigation of possible survival mechanisms, such as NFkB, in these cells can shed light on the effects of the Fas signaling pathway on the longevity of Th17 cellsMaster Thesis Investigation of Bcl-2 Proteins in Th17 Cell Functions(01. Izmir Institute of Technology, 2021) Çimen, Tuğçe; Nalbant Aldanmaz, AytenInterleukin 17 producing T helper 17 cells are the distinct subset of CD4⁺ T cells. Th17 cells are an important part of the immune response of host defense. Dysregulation of Th17 cells plays a role in various pathologies including autoimmune diseases and cancer types. Bcl-2 family proteins are mostly known regulators of apoptotic cell death. The apoptotic and survival mechanisms of Th17 cells are not well known yet. Therefore, this study aims to investigate Bcl-2 protein family functions in Th17 cell survival and to understand the regulation network of apoptotic mechanisms of Th17 cells. To do that, Peripheral Blood Mononuclear Cells were isolated from a healthy buffy coat by Ficoll separation. Naive T cells were sorted from PBMC and cultured under Th17 polarizing conditions. Th17 cells were phenotypically characterized by flow cytometry. Afterward, cell lysates were obtained from Th0 and Th17 cells at different time points. The expressions of human transcription factor RORC2, proapoptotic Bik, Bid, Puma and Bim and, antiapoptotic Mcl-1 and Bcl-xL at cell groups were detected by Western blotting. The increased expressions of Bcl-xL and Mcl-1 were detected where the diminished expressions of Bim and Puma were detected in proportional with Th17 differentiation by increased RORC2 and elevated RORC/IL17A levels. Bik was undetectable in both cell groups while non-truncated isoform of Bid was barely decreased among cell groups. Outputs of this study allow us to understand the dynamics of Bcl-2 family proteins in human Th17 cell survival. The understanding roles of Bcl-2 proteins in Th17 cells may help to develop different therapeutics for Th17 associated diseases in the future.Master Thesis Identification of Micrornas Involved in Camptothecin-Induced Apoptosis in Jurkat T Cell Line(Izmir Institute of Technology, 2008) Erdoğan, İpek; Akgül, BünyaminMicroRNAs which are non-coding RNAs 19-25 nt in length regulate gene expression at post-transcriptional and translational stages. Although it is known that they play a role in critical processes such as development and differentiation of T cells a major component of the immune system, the function of miRNAs in T cell apoptosis is unknown. This study has aimed to identify miRNAs. involvement in camptothecininduced T cell apoptosis in the Jurkat T cell leukemia cell line model. Following the enrichment of the apoptotic population by magnetic seperation, the negative and apoptotic fractions were profilled and compared according to the expression levels of microRNAs. Out of the 866 miRNAs in the miRBASE, 37 and 58 of them were downand up-regulated in the apoptotic fraction, respectively. 7 miRNAs were members of the clusters that have predicted targets as anti-apoptotic genes and tumour suppressor proteins. 66 miRNAs have no known function. Candidate miRNAs, selected based on their higher differential expression levels with predicted apoptotic/antiapoptotic targets, will be verified by qPCR. These candidate then will be further characterized by overxpression and knock-down studies.Master Thesis Determination of Human T-Lymphocyte Apoptosis Mediated by Bacterial Heat Shock Protein(Izmir Institute of Technology, 2009) Dinç, Melis; Nalbant Aldanmaz, AytenPeriodontal diseases are the most common inflammatory disease worldwide which caused by the pathogenic organism living in biofilm. Aggregatibacter Actinomycetemcomitans (Aa) is the main player of the periodontitis disease pathology. Although some of the virulence factors of Aa has been identified up to now, its cytotoxic mechanism has not been clearly known yet. Although known virulence factors of Aa; ltx and cdt has been knocked out, the mutant Aa strains have retained the ability to induce apoptosis. Depending on the literature there must be another important virulence factor. 64kDa GroEL protein which is a molecular chaperone and a heat shock protein can be the potential candidate for being a virulence factor. AaGroEL protein has not been studied in terms of apoptosis up to now and it is not known how AaGroEL mediate immune regulation of T cells. In this study AaGroEL protein has been purified by using ATP Affinity chromatography and electroelution methods. After the purification step lps contamination has been removed by detoxi-gel endotoxin removal gel and detected by LAL Assay. Peripheral Blood Mononuclear Cells (PBMCs) were isolated by Ficoll Hypaque Density Gradient Centrifugation method. It was found that AaGroEL protein induces T cell apoptosis in dose and a time dependent manner. AaGroEL protein mediated T cell apoptosis has been detected by plasma membrane changes, activation of caspase-3 and DNA fragmentation. In conclusion, AaGroEL has antigenic properties that effect T lymphocytes by regulating immune response that would play important role in periodontal pathology.Master Thesis Differentiation of Human Naive Cd4+t Cells Into Th17 Cell Phenotype(Izmir Institute of Technology, 2011) Akıncılar, Semih Can; Nalbant Aldanmaz, AytenTh17 cells are recently identified CD4+ T cell subset and best defined as their secretion of IL17, an inflammatory cytokine, and their role in host defense and autoimmunity. Further information on Th17 cell subset is strongly correlated with the differentiation and maintenance of these cells in culture. Although the master regulators and culture conditions in mouse Th17 differentiation are defined, the requirements and maintenance of human Th17 cell cultures remain unclear. Here, we suggest a new culture condition that maximizes IL17 expression and gives minimum IL17+IFNg+ and IL17+Foxp3+ among human studies. Our data define the doses and combinations of various cytokines that rise IL17 expression, as well as regulatory molecules in human Th17 cell differentiation. Various combinations of cytokines reveal that IL1β is the most important cytokine that primes Th17 differentiation. Also it was observed that TGFβ positively regulates Th17 differentiation in a dose-dependent manner by inhibiting IFNγ expression thus represses Th1 differentiation and impairs Treg polarization at transcription factor state by tightly regulating Foxp3. Although it was suggested that Th17 cells must express RORC2, the master regulator, a minor cell population that expresses IL17, but does not co-express RORC2 was observed.