Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Investigation of the Effects of Gtf2a1-Antisense Long Non-Coding Rna on Cell Fate(01. Izmir Institute of Technology, 2022) Çiftçi, Yusuf Cem; Akgül, BünyaminApoptosis is a distinct mode of programmed cell death whereby cellular contents are broken down and accumulated in the apoptotic bodies. The vast majority of the genome consists of non-coding RNAs (ncRNA). NcRNAs can be divided into groups depending on their length, for example, long non-coding RNA (lncRNA) longer than 200 nucleotides. It has been demonstrated that these have important roles in the development, and treatment of cancer and in other diseases that critically affect human life. Considering the lncRNAs’ mechanisms of action on apoptosis, they modulate activity of transcription factors, regulate miRNAs, and interact with proteins related to histone mechanisms such as chromatin modifier. In this perspective, GTF2A1-AS which is an uncharacterized and novel lncRNA was found as one of highly expressed lncRNA in transcriptomic data obtained from HeLa cells treated with cisplatin. The potential role of GTF2A1-AS within the cell was investigated through the transcriptomic data provided by GTF2A1-AS knockdown. It has been found that specific gene clusters mainly enriched in the pathway which is Defective Homology directed Repair through Homologous Recombination. In this process, double-strand breaks are repaired with the help of BRCA1/2, RAD50, RAD51, PALB2 proteins which are known as DNA damage response proteins. Thus, the genes related with DNA damage response were selected to validate the transcriptomic data. In light of this information, GTF2A1-AS knockdown has resulted in an increase in the early apoptosis in HeLa cells. Additionally, when GTF2A1-AS knockdown was combined with cisplatin, it sensitized HeLa cells against cisplatin by affecting late apoptosis, specifically. Consequently, GTF2A1-AS as a cisplatin inducible lncRNA modulates apoptosis and chemosensitivity in HeLa cells.Master Thesis Transcriptomics Profiling of M6a Rna Modifications in Tnf-Alpha Induced Apoptosis(01. Izmir Institute of Technology, 2021) Akçaöz, Azime; Akgül, BünyaminApoptosis is a form of programmed cell death that occurs as a result of physiological or pathological causes. TNF-alpha, which has a regulatory role in immune system cells, stimulates apoptosis through the external pathway. For this reason, it can be used for the treatment of various diseases. Although there are many studies on the regulatory mechanisms of TNF-alpha mediated apoptosis, the contribution of RNA modifications has not been fully elucidated. Regarding the potential role of m6A RNA modification in apoptosis, studies have focused on the effects of regulatory proteins and there is no genome-wide m6A methylation profile yet. In the present thesis, firstly, the gene expression patterns of m6A writer, eraser, and reader were examined in HeLa cells and 632 genes with differential m6A methylation pattern were identified by the miCLIP method. 99 genes involved in apoptotic pathways were determined by GO analysis. Candidates were selected based on m6A methylation fold change, intracellular expression level and apoptotic role of the relevant gene. Methylation points in IGV were confirmed and specific validation experiments were performed on these m6A points. SELECT based validation studies showed 1-2 cycle increase in the TNF-alpha group compared to the control group. This confirms the miCLIP data, which also pointed to an increase in m6A methylation. To elucidate the fate of candidate RNAs, the gene expression levels, and translational status of candidate genes were analyzed. METTL3 KD HeLa cells exposed to TNF-alpha exhibited an increase in the expression of PHLDA1, IFI6 and HRK by almost 2-fold. Polysome fractionation assay showed that translation level decreased in TNF-alpha treated METTL3 KD HeLa cells. As a conclusion, global m6A level affected RNA abundance as well as translation.Master Thesis Determination of Therapeutic Potential of Apigenin on Acute Lymphoblastic Leukemia Cells(Izmir Institute of Technology, 2019) Uzuner, Erez; Baran, YusufAcute lymphoblastic leukemia (ALL) is a hematological disorder initiating from blood-forming cells of bone marrow. ALL is characterized by the Philadelphia chromosome (Ph) arisen from a translocation between chromosome 9 and 22. This chromosome encodes BCR-ABL oncogene that is a driver regulator. BCR-ABL based studies improved tyrosine kinase inhibitors (TKI) including imatinib, dasatinib, nilotinib, and ponatinib to eliminate this disease. However, the studies on Ph+ ALL patients showed that bioactive sphingolipids have crucial roles in the elimination of the positive effects of these drugs by activating the proliferation-associated pathways, inhibition of apoptosis and increasing drug resistance of the cells treated with these drugs. In this study, therapeutic potential of apigenin, which is a natural flavonoid obtained from celery, parsley and chamomile was investigated on Ph+ ALL cell line, SD-1, and non-cancerous lung cell line Beas-2B. The cytotoxic effects of apigenin on SD-1 and Beas-2B cells were determined by MTT cell proliferation assay. The cell viability analyses on SD-1 cells were conducted by Trypan blue dye exclusion assay following apigenin treatment. Cell cycle and apoptosis analyses including Annexin V/PI-dual staining and JC-1 dye-based mitochondrial membrane potential were examined by flow cytometry. Expression levels of bioactive sphingolipids were determined by RT-PCR and western blot. The cytotoxic analyses indicated that apigenin selectively inhibits the expression of SD-1 cells whereas the IC50 value of apigenin for SD-1 cells has the anti-apoptotic roles in Beas-2B cells. SD-1 cells experience cell death via apoptosis-related pathways and apigenin might arrest the cells at G2/M phases. Indeed, the changes in the expression levels of bioactive sphingolipids genes indicated their roles in apigenin-induced apoptosis in SD-1 cells. This study investigated the cytotoxic and apoptotic effects of apigenin on SD-1 cells and the roles of apigenin in bioactive sphingolipid metabolism for the first time.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 Indentification of Circular Ribonucleic Acids Differentially Expressed in Apoptotic Hela Cells(Izmir Institute of Technology, 2018) Yaylak, Bilge; Akgül, BünyaminApoptosis is a mechanism of programmed cell death that is essential for survival, homeostatis and development. Various protein coding genes and non-coding RNAs were reported as apoptosis regulators. However, the potential roles of circular RNA in the regulation of apoptosis are still unknown. In this study, we have performed transcriptomics study to reveal differentially expressed, pathway-drug specific and/or global circRNAs in apoptotic HeLa cells. Cisplatin (CP), doxorubicin (DOX), Fas mAb(FAS) and TNF-alpha (TNF-a) were used to trigger apoptosis in HeLa cells. Apoptosis rates of three replicates of treatment and control cells were measured by flow cytometry and differentially expressed circular RNAs were identified by deep RNA sequencing. Circular RNA candidates were firstly sorted based on their significance according to pad j value, further classified based on fold change, pathway-drug specificity and source genes. Then, circular RNA candidates were analysed bioinformatically to obtain their coding potential, potential miRNA binding sites and involvement in possible apoptotic pathways. Furthermore, divergent primers were designed to validate backsplicing junction sequence of circular RNA candidates. RNAse R treatment was used to eliminate linear transcripts and enrich circular RNAs. The expression of candidate circular RNAs was analysed RNAse R treated samples. Backsplicing junctions of positive circular control circ-HIPK3 was validated by TA cloning and sequencing. Differential expression of positive control (circ-HIPK3), candidate-8 and candidate-6 were validated by quantitative PCR.Master Thesis Molecular Characterization of the Gtf2a-1 Antisense Long Non-Coding Rna(Izmir Institute of Technology, 2017) Yarımçam, Murat Caner; Akgül, BünyaminOne of the essential events in cell regulation and normal development of an organism is apoptosis. The dysregulation of apoptosis is associated with diseases such as cancer. Apoptosis induction can kill cancer cells without harming the individual. For this purpose, new methods are developed to fight the cancer cells. One of the novel approaches is based on long non-coding RNAs (lncRNAs). LncRNAs are differentially expressed in cancer cells and they regulate and interact essential pathways. The ones related to apoptosis are the targets. In this study, target lncRNA was determined based on RNA-Seq data. Then apoptosis was induced in HeLa cells with cisplatin and qRT-PCR was performed with isolated RNAs from the cells to validate the data with regard to upregulation of GTF2A-1 anti-sense lncRNA in apoptosis. Then GapmeR specific to target lncRNA was designed and transfected into HeLa cells in order to induce apoptosis. After induction of apoptosis, total RNA and protein were isolated from the cells. qRTPCR was performed to validate the RNA-Seq data. Western blotting was performed in order to characterize the target lncRNA by controlling its effects on different apoptosis pathways. Western blotting results are showing resemblance between GTF2A-1 antisense lncRNA silencing-induced apoptosis and cisplatin-induced apoptosis. The western blotting result of Cytochrome c is interesting because its amount is decreased in GTF2A- 1 anti-sense lncRNA silencing-induced apoptosis. The candidate, GTF2A-1 anti-sense lncRNA, is directly regulating the apoptosis in HeLa cells and in this study, some of the pathways that are regulated with this lncRNA were shown.Master Thesis Identification of Long Non-Coding Rnas That Regulate Apoptosis in Human(Izmir Institute of Technology, 2015) Ahmadov, Ulvi; Akgül, BünyaminApoptosis is essential for cellular homeostasis and normal development. Aberrant apoptosis (too much or too less) is associated with many important diseases such as autoimmune diseases and cancer. Studies have led to the identification of a number of proteins and microRNAs involved in the regulation of apoptosis. However, the role of long non-coding RNAs (lncRNAs) is still unclear. In this study, two cancer therapeutics drugs, cisplatin and doxorubicin, and two ligands, Fas mAb and TNF-alpha, were used in identification of differentially expressed pathway-drug specific and/or global lncRNAs in apoptotic HeLa cells. Following dose-kinetics experiments the level of apoptosis was measured by Flow Cytometry and was further verified by Fluorescence Microscopy and Western Blotting via measurement of Caspase 3, 8 and 9 protein levels. Three replicates of total RNAs (control and drug/ligand-treated cells) were sent to deepsequencing using the Illumina platform. The resulting reads matched to the human genome greater than 95%. Under our experimental setting, treatments with cisplatin, doxorubicin, Fas mAb and TNF-alpha led to the differential expression of 1644, 506, 584 and 807 lncRNAs, respectively (2-fold or higher, P < 0.01). Two of identified lncRNAs common for all inducers was in antisense position to TRAIL-R2 receptor and FasR associated factor which play directly in apoptosis. Results suggest that many lncRNAs are differentially expressed upon treatment with the indicated agents. Functional characterization of candidates might provide an interesting insight into regulation of apoptosis.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 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 Investigation of the Functions of Candidate Mirnas in Camptothecin-Induced Apoptosis in Human Cells(Izmir Institute of Technology, 2012) Demir, Şeyda; Akgül, BünyaminMicroRNAs are non-coding 19-25nt long, small RNAs that regulate expression of about 30% of human genes by inhibiting mRNA translation or inducing its degradation. MicroRNAs play important role in cell growth, differentiation, apoptosis. miRNAs regulate apoptosis by targeting genes involved in apoptotic pathway as a pro or anti-apoptotic genes. This study has aimed to identify whether candidate miRNAs ( miR-17* and miR-425) have a regulatory role in camptothecin induced apoptosis or not in Human cells and Hela cells that derived from cervical cancer were used as a model cell line. These candidates were selected based on deep sequencing data that showed some miRNAs differentially expressed after camptothecin treatment as compared with non-treated control group. To show candidate miRNAs whether have a role or not in regulation of camptothecin induced apoptosis, first Hela cells were transfected with candidate miRNAs then candidate miRNA over-expressed cells were treated with camptothecin eventually level of apoptosis was measured by flow cytometry and the results were evaluated by comparing miRNA over-expressed cell group with un-transfected control group. Active caspase-3 level also was measured by using flow cytometry and the data showed miR-17* and miR-425 function as pro-apoptotic regulator in camptothecin induced apoptosis in Hela cells.