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 Investigation of the Effect of Dr5-As Long Non-Coding Rna on Cell Proliferation(Izmir Institute of Technology, 2020) Gürer, Dilek Cansu; Akgül, BünyaminCell proliferation is the process of increasing cell number in a multicellular organism. In literature, there are numerous proteins and non-coding RNAs reported as regulators of cell proliferation, yet, many of others are waiting to be explored. Unravelling the mechanism behind the regulation of cell proliferation is crucial to develop new strategies for fighting numerous diseases such as cancer, immune diseases, or neurodegenerative diseases. Long non-coding RNAs (lncRNAs) are known to regulate various cellular processes. To determine which ones are related to cell proliferation and apoptosis in HeLa cells, a transcriptomics study was performed under cisplatin, doxorubicin, TNF-? and Anti-Fas treatments. DR5-AS is a novel lncRNA transcript selected from this transcriptomics study as a promising regulatory lncRNA candidate due to its overlap with DR5 protein-coding gene which is known to regulate apoptosis and proliferation. Several phenotypic characterization methods were performed to understand the function of DR5-AS lncRNA. These studies showed that DR5-AS knockdown causes a significant decrease in cell proliferation, an alteration in the normal HeLa cell morphology, a shift through S and G2/M phases in cell cycle profile, and significant accumulation of cells in the metaphase phase. A second transcriptomics study was performed with DR5-AS knockdown HeLa cells to uncover which pathways are responsible for these changes. The results suggest that DR5-AS lncRNA regulates expression of numerous key proteins in cell cycle regulation. This observation was confirmed by several qPCR experiments. In conclusion, this study provides the first evidence that DR5-AS lncRNA modulates cell cycle and proliferation in HeLa cells.Master Thesis Analysis of Tnfrsf10b-As Long-Noncoding Rna's Effects on Various Cancer Cell Properties(Izmir Institute of Technology, 2019) Alkan, Ayşe Hale; Akgül, BünyaminLong noncoding RNAs (lncRNAs) being longer than 200 nucleotides constitute a different class of RNA molecules. Several studies indicated that they have regulatory role in cellular processes including cancer development. Some of them have exclusively high expression in particular cancer types and regulate certain cancer cell properties. This renders them potential biomarker or therapeutic target in cancer. In this study, effects of a candidate lncRNA TNFRSF10B-AS and lncCAMTA1 on cancer cell properties were investigated. Candidate lncRNAs from Doxorubicin, Fas mAB, TNF-alpha and Cisplatin treated HeLa cell line were chosen and their expression level was measured in different cell lines including healthy (BEAS2B and MCF10A), metastatic (H1299 and MDA-MB- 231) and non-metastatic cell lines (A549 and MCF-7) by qPCR. From a few candidates lncCAMTA1 and TNFRSF10B-AS were selected for further analysis. qPCR results obtained from comparison of different cancer cell lines showed that their expression differs at least in one comparison of cell lines. TNFRSF10B-AS silencing decreased proliferation of HeLa cells. lncCAMTA1 was silenced or overexpressed in HeLa cells but phenotypic effect couldn’t be detected by apoptosis and cell proliferation assay. Additionally, phenotypic effect also couldn’t be observed in other cell lines when TNFRSF10B-AS was silenced.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 Cloning of Polysome-Associated Small Rnas in Drosophila Melanogaster Embryos(Izmir Institute of Technology, 2009) Yiğit, Hatice; Akgül, BünyaminGenome-encoded regulatory small RNAs are classified into 3 groups; microRNAs (miRNAs), endogeneous small interfering RNAs (endo siRNAs) and piwi interacting RNAs (piRNAs). miRNAs, 17-21 nucleotide in size, are involved in posttranscriptional gene regulation via precise or imprecise base pairing with target mRNAs resulting in either target mRNA degradation or translational inhibition. Endo siRNAs ,on the other hand, may function transposon regulation but their precise regulatory function and mechanism have not been elucidated yet. piRNAs are mainly involved in transposon silencing in spermatogenesis. Despite their discovery, biological roles and modes of functions of small RNAs remain to be elucidated. The aim of this thesis was to identify polysome-associated small RNAs in Drosophila melanogaster embryos by deep sequencing and investigate their role in translational regulation. Deep sequencing and microarray results determined stage and fraction specific distribution of genome encoded small RNAs. Surprisingly, the results implied that mRNAs may be posttranscriptionally regulated by antisense transcripts in polysome.Master Thesis Genomic Profiling of Micrornas Regulating Translation in Drosophila Melanogaster Embryos(Izmir Institute of Technology, 2008) Tüncel, Özge; Akgül, BünyaminAmong the small RNAs, microRNAs are a particular class of 21 to 23 nucleotide RNAs that negatively regulate translation and play a pivotal role in posttranscriptional gene expression. microRNAs are found in many phyla that control such diverse events as metabolism, cell fate, cell death and development.The aim of this study is to investigate molecular mechanism of miRNAmediated translational regulation by profiling developmentally important microRNAs according to their translational status and to identify new microRNAs that have roles in translational regulation during the embryogenesis of Drosophila. Following RNA purification from different embryonal stages the fractionated RNAs were analyzed by microRNA microarray. Preliminary results show that 9 miRNAs were expressed in both stages whereas 60 miRNAs were accumulated in RNA fractions of 8 hour embryos.Also there are 2 miRNAs in all fractions of both stages in Drosophila embryos. It can be concluded that most of them were expressed in late embryonal development and there does not appear to be a switch in microRNA profiles in fractions for different stages of embryos. The preliminary results suggest that microRNAs may suppress protein synthesis at pre-initiation and initiation phases based on the microarray data.Further studies are required to solidify the preliminary findings.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.