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 Expression of Aquaporin 1, 3 and 4 in T Cell Activation and Apoptosis(Izmir Institute of Technology, 2018) Gelmez, Ayşe Bengisu; Nalbant Aldanmaz, AytenAquaporins (AQPs) are membrane proteins responsible for transporting water, some gases and small solutes such as CO2 and glycerol. Until now, it has been shown that AQP1, 3 and 5 expressed in both B and T lymphocytes of mice, regulate cell volume. However, aquaporin expression involved in activation, proliferation, and differentiation as well as apoptosis of T cells are not well known yet. The goal of this study is to detect the expression level of AQP1, AQP3, and AQP4 in activated and apoptotic T cells. In order to do that, two types of T cells cultured in both condition were utilized. Peripheral Blood Mononuclear Cells (PBMCs) were isolated from Human peripheral blood drawn from healthy donors by ficoll density gradient Centrifugation method. Naive CD4+ T cells were sorted from PBMC. The stimulants generating Th17 were chosen for activation and differentiation of naïve CD4 T cells. Jurkat cell line as a second cell type were activated by PMA/Ionomycin as well as treated by camptothecin for apoptotic processing. Th17 and Jurkat cell cultures were analysed by flow cytometry to measure the rate of both activation and apoptosis. Western Blot was performed to identify expression of AQP 1, 3 and 4. We found a significance between increased expression level of AQP1, 3, and 4 in activated T cells as well as decreased expression level of each three AQPs in apoptotic T cell populations. According to our findings, tested aquaporin proteins may play roles in T cell activation, differentiation, and apoptosis. The scientific significance of this research is that it can fill the gaps about these three functional processes of T cells. Besides, all findings can contribute to treatment of many autoimmune disease like MS which Th17 cells involve in pathogeny.Master Thesis Nucleofection Effects on Human T Cells as a Non-Viral Transfection Technique(Izmir Institute of Technology, 2016) Güler, Seminay; Nalbant Aldanmaz, AytenIn times of quickly developing ways of immunological approaches applying on cells, nucleic acid delivery becomes a fundamental process. The ways of delivering nucleic acids mainly affects the consequence of immunological response. The nucleofection technology which is a modified form of electroporation is reliable method for transfection of hard to transfect cells. In the current study, we compared naive CD4 T cells and Jurkat cells in terms of nucleofection efficiency and cell viability. Naive CD4 T cells and Jurkat cells were transfected with pmax GFP at different time points. Out of 5 different nucleofection programs, one for naive CD4 T cells was identified. Jurkat cells were also transfected with one nucleofection program. Flow cytometric analyses of nucleofected naive CD4 T cells and Jurkat cells indicated that transfection efficiency significantly increased at 48h post transfection. We also differentiated naive CD4 T cells into T helper 17 cell phenotype by using certain stimulants and we activated Jurkat cells by using PMA and Ionomycin. We found that GFP was efficiently taken up by both T helper 17 cells and activated Jurkat cells. Therefore, nucleofection significantly holds important place in transfecting of hard to transfect cells including primary human CD4 T cells.Master Thesis Investigation of the Effects of Il-7 on the Th-17 Cell Apoptosis(Izmir Institute of Technology, 2015) Aydınlı, Fatmagül İlayda; Nalbant Aldanmaz, AytenTh17 cells known as Interleukin-17 (Inflammatory Cytokine) producing cells are differentiated subsets from naïve CD4+ T cells and have crucial roles in regulation of inflammation, host defense and autoimmunity. TCR (T Cell Receptor) activation is triggered under Th17 cell culture conditions and resulting naïve CD4+ T cells are induced to differentiate through Th17 cells. In the life time of activated T cells, the activation process also induces an apoptotic mechanism which is called activation-induced cell death (AICD) for elimination of activated cells from the environment for maintenance of homeostasis. AICD is known as the main programmed cell death mechanism for T cells by Fas-FasL signaling resulting activation of early and late apoptotic caspase proteins such as caspase-3 and caspase-8. Moreover, Interleukin-7, which is a member of Interleukin-2 family, has a survival mechanism in T cells by the activation and maintenance of anti-apoptotic proteins mainly Bcl-2 and inhibition of pro-apoptotic proteins such as Bax and Bim. This research analyzes apoptosis mechanism in Th17 cells in terms of AICD and the effects of IL-7 on that apoptosis signaling pathway. Our results showed that IL-7 did not have any effect to AICD throughout Fas-FasL signaling and activation of caspase-3 and caspase-8 protein.Master Thesis Cytokine Expression Pattern of Human T Lymphocytes in Response To A. Actinomycetemcomitans Groel Protein(Izmir Institute of Technology, 2009) Saygılı, Tahsin; Nalbant Aldanmaz, AytenActinobacillus (Aggregatibacter) actinomycetemicomitans is a bacterium that plays important role in Periodontitis. Recent publications have correlated AaGroEL (A.actinomycetemcomitans GroEL Protein) protein with periodontal pathologies however, how does this protein affect T lymphocytes and the profile of T lymphocytes. immune response, are not reported yet. Within this thesis project, AaGroEL protein is produced as its recombinant form. The effects of AaGroEL protein, after it is obtained, have been investigated on naive T lymphocytes in peripheral blood. In this research, the proliferative and activating effects of AaGroEL protein on T lymphocytes were determined by investigating the expression of CD25 and CD69 cell surface molecules. AaGroEL protein has an effect on CD4+ T lymphocytes and depending on time, it increases CD25 and CD69 expression. Additionally, it was found that, CD4+CD25+T cells are negative for FOXP3. The cytokines of CD4+ T lymphocytes were profiled. Firstly, the cytokines that were released by PBMC.s on culture supernatant were determined. Following AaGroEL stimulation, IL-6, IL-10, IFNg ve TNF. were secreted to the supernatant. Intracellular cytokine staining was applied to determine the source of those cytokines which was found that CD4+ T cells produce those cytokines as well as nonlymphocytes and B or/and CD8+ cells also secrete them. IL-2 was secreted by CD4+ T lymphocytes only. These findings have demonstrated us; AaGroEL protein activates naive CD4+ T cells towards Th1 response. In conclusion, AaGroEL protein has an antigenic effect on T lymphocytes, regulates immune response and play important role on periodontal pathology.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 Determination of Apoptotic Effects of Clinoptilolite on Human T Lymphocytes(Izmir Institute of Technology, 2008) Uslu, Mehmet Emin; Nalbant Aldanmaz, AytenZeolites are defined as aluminum silicates that have made up of oxygen, aluminum and silicon. SiO4 and Alo4 tetrahedrals are the smallest units that give the specific shape to the molecule. There are more than 40 types of natural and over 150 types of synthetic zeolites occurs and those zeolites are used in agriculture, animal husbandry, architecture, pharmaceuticals and metallurgy. Recent years in literature it was shown that these zeolites can have regulatory effects on immune system. Also it was shown that they can influence the development of cancer and have a role on the expression of tumor suppressor genes. However there is no evidence can be found on how these molecules affect the function of specific cell types in the molecular level and also the mechanism of this effect.In this study the apoptotic effects of clinoptilolite which is a natural zeolite found in Gördes region of Turkey, on human T lymphocytes were studied. T cells were chosen in this study because they are the main players in the immune system. These cells can establish immune regulation and organize immune response. As a result, they are important in pathology and therapy. Peripheral blood mono nuclear cells (PBMCs) in which T cells can be found were isolated from the healthy donors blood by Ficoll Hypaque Gradient Method and then these cells were incubated with clinoptilolite in the RPMI 1640 media. Apoptosis were measured in FacsArray after appropriate immunofluorescent labeling and by agarose electrophoresis technique after DNA fragmentation assay done.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.