Master Degree / Yüksek Lisans Tezleri
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Master Thesis Optik Spektroskopi Teknikler Kullanarak Rna Metilasyonunun Araştırılması(2025) Akkuş, Onur; Güler, Günnur; Akgül, BünyaminN6-metiladenozin (m6A), en yaygın RNA modifikasyonlarından biridir ve RNA'nın işlenmesi, kararlılığı, nükleustan taşınması ve translasyonunun düzenlenmesinde önemli bir rol oynar. Bu tezde, m6A metilasyonun FT-IR, CD sinyali ve ikincil yapıdaki transkripsiyon sonrası modifikasyonlar incelenmektedir. FT-IR spektroskopisi, zayıflatılmış toplam yansıma (ATR) yöntemiyle birleştirilerek, metil grubunun titreşimsel davranışını analiz etmek amacıyla giderek karmaşıklaşan sistemlerde sistematik biçimde kullanılmıştır. Ana CH₃ gerilme ve bükülme modları, ilk olarak metanol gibi basit moleküllerde gözlemlenmiş sonrasında DNA, sentetik RNA ve nihayetinde hücre kaynaklı RNA gibi daha büyük sistemlerde uygulanmıştır. Özellikle, CH₃ bükülme modları yaklaşık 1478 ve 1332 cm⁻¹'de, CH₃ gerilme modları ise yaklaşık 2984 ve 2883 cm⁻¹'de ortaya çıkmıştır. Bu modlar metilasyonun göstergeleri olarak değerlendirilmiştir. Bu tayinler, metillenmiş ve metillenmemiş türler arasında net spektral ayrım yapılmasını mümkün kılarak, hücre kaynaklı RNA'larda m6A tespiti için moleküler bir parmak izi sunmuştur. Dairesel dikroizm (CD) spektroskopisi, RNA'daki lokalize epitranskriptomik yapısal modifikasyonlardan kaynaklanan baz istiflenmesi, baz eşleşmesi ve hairpin oluşumu gibi metilasyona bağlı ikincil yapı değişikliklerini gözlemlemek için kullanılmıştır. Metillenmiş ve metillenmemiş örnekler karşılaştırıldığında, 265 nm'de (baz istiflenmesi) ve 210 nm'de (sarmal asimetri) eliptiklikte önemli değişiklikler gözlemlenmiştir. Bu spektral değişiklikler, lokalize transkripsiyon sonrası konformasyonel değişiklikleri yansıtmaktadır. Bu çalışma ile, spektroskopik (IR) tabanlı RNA m6A metilasyonunun ölçülmesine dayalı alternatif bir yöntemin geliştirilebileceği ve CD spektroskopisi ile, m6A metilasyon kaynaklı transkripsiyon sonrası lokalize yapısal modifikasyonların izlenebileceğini göstermektedir. Bu bulgular ile, spektroskopik analizlerin m6A metilasyonuna dayalı yapısal biyoloji alanında destekleyici nitelikte bilgi ve yöntem sağlanması amaçlanmıştır.Master Thesis İnvaziv Lobüler Meme Kanserinde N6-metiladenozin (m6A) Silici Yağ Kütlesi ve Obeziteyle İlişkili (FTO) Proteinin Karakterizasyonu ve Hedef Genlerin Tanımlanması(2025) Gazaloğlu, Yasemin; Akgül, BünyaminMeme kanseri dünyada kadınlarda en yaygın olarak teşhis edilen bir kanser tipidir. Histolojik olarak invaziv lobüler meme kanseri memenin süt üretiminin gerçekleştiği lobüllerde meydana gelmektedir ve tüm meme kanserlerinin yaklaşık olarak %15'ini oluşturmaktadır. ER ve PR ekspresyonu yüksek HER2 ekspresyonu düşüktür ve çeşitli sinyal yolaklarında görev alan CDH1, TP53, PTEN ve AKT1, gibi genlerde mutasyon bulundurmaktadır. Kanserin regülasyonunda m6A RNA modifikasyonu gibi epitanskriptomik modifikasyonlar kanserinin biyolojik süreçlerinde görev almaktadır. m6A modifikasyonunun dinamik olarak ve geri döndürülebilir bir şekilde düzenlenmesini yazıcı, silici ve okuyucu proteinler sağlamaktadır. Bu proteinlerin ekspresyon seviyeleri kanser çeşidi ve hücre tiplerine göre farklılık göstermektedir. Bu farklılıklar kanser hücrelerinin tespiti ve tedavi edilmesi için oldukça önemlidir. Bu tezin amacı, ters genetik yaklaşımlar kullanarak FTO manipülasyonu ile MDA-MB-134 invaziv lobüler meme kanseri ilişkili fenotipler arasında bir bağlantı kurmak ve bu hücrelerde FTO'nun potansiyel hedef genlerini belirlemektir. Bu nedenle her iki hücre hattında da FTO proteini susturulmuş olup canlılığın MCF10A hücrelerinde %19,5, MDA-MB-134 hücrelerinde %16,7 oranında azaldığı; MDA-MB-134 hücrelerinin canlı hücre oranının %9,41 azaldığı ve erken apoptoz oranının %8,40 arttığı; MCF10A hücrelerinin S fazının %3,2 azalırken, MDA-MB-134 hücrelerinin S fazının %5,3 azaldığı ve G0/G1 fazında %5,1 arttığı gözlemlenmiştir. Bu nedenle MDA-MB-134 hücrelerinde bu fenotiplere sebep olabilecek ve MCF10A hücrelerinden farklı metile olan aday genler ZFP36L1, CDKN2B, SSTR2 ve AR olarak belirlenmiş ancak ekspresyon seviyelerinde FTO'ya bağlı bir değişim gözlemlenmemiştir.Master Thesis Investigation of M1a and M6a Rna Methylations in Triple Negative Breast Cancer Cells(2024) Sağlam, Buket; Akgül, BünyaminMeme kanseri dünya çapında kadınlarda en sık görülen kanser türüdür ve iki alt gruba ayrılır: invaziv lobuler ve invaziv duktal meme kanseri. Bunlardan invaziv duktal meme kanseri, %80 oranında dünya çapındaki meme kanserleri arasında yerini almaktadır. Üçlü negatif meme kanseri ise östrojen reseptörü, progesteron reseptörü ve insan epidermal büyüme faktörü reseptör 2'nin çoğaltılmasını gerçekleştiremeyen agresif bir meme kanseri alt türüdür. Kanser çalışmalarında RNA metilasyonları da hücrenin kaderine etkilerini kanıtlamış önde gelen modifikasyonlardır. Bütün metilasyonlarda olduğu gibi, m6A ve m1A de yazıcı, okuyucu ve silici proteinlerin yardımı ile gerçekleştirilen dinamik bir düzenleme mekanizmasına sahiptir. Bu proteinler, kanser türlerine göre farklı ifadelenme seviyelerine sahiptirler. Bu karakteristik özellikleri ile tedavi ve tespit amaçlı kullanılmaları hedeflenmektedir. Mevcut tez çalışmasında, yazıcı proteinleri olan METTL3 ve TRMT61A proteinlerinin susturulması sonrası, m6A ve m1A metilasyonlarının etkilerinin ve karşılaştırılmasının üçlü negatif meme kanseri hücrelerinden biri olan HCC1143 hücre hattında incelenmesi amaçlanmıştır. Öncelikle, METTL3 susturulması sonrası 72 saatte, maksimum düzey olan %41,2 oranında m6A miktarında azalma gözlenmiştir. Ardından fenotipik etkileri incelemek amaçlı gerçekleştirilen canlılık deneylerinde, METTL3 ve TRMT61A'nın susturulması ile sırasıyla %40,1 ve %27,4 azalma gözlemlendi. Ek olarak, TRMT61A'nın yıkılmasının aksine, yalnızca m6A metilasyonunun azalması sonucu G2/M fazında duraksama gözlenmiştir. m6A miktarının azaltılması sonucu 585 artan/azalan gen ve m1A metilasyonun azaltılması sonucu 687 artan/azalan gen tespit edilmiştir. Bunlardan 151 gen ortak olarak değişkenlik göstermiştir. Gen Ontolojisi zenginleştirme analizleri sonucunda METTL3 yıkımında hücre migrasyonu ve hücre motilite yolakları yoğun olarak gözlenmiştir. m1A azalması sonucu ise bağışıklık sistemi ve canlılığı negatif yönde etkileyen yolaklarda değişkenlik gözlenmiştir.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 Examination of Stable Intronic Sequence Rna Profile Under Apoptotic Conditions(Izmir Institute of Technology, 2022) Kara, Merve; Akgül, BünyaminApoptosis is a process of programmed cell death. Cisplatin, a chemotherapeutic drug, activates intrinsic pathway of apoptosis while TNF-alpha, a death ligand, activates the extrinsic pathway of apoptosis. Noncoding RNAs involve in regulation of apoptotic pathways at post-transcriptional level. Stable intronic sequence RNAs (sisRNAs) are the novel class of non-coding RNAs which can be generated by splicing- dependent and independent mechanisms. sisRNAs transcribed from their intronic promoter may contain 5’ cap and polyA tail. Despite the reports of several studies about sisRNAs in Xenopus and Drosophila, a genome-wide profile of sisRNAs in human is lacking. Therefore, we aimed to identify sisRNAs profile that are transcribed from their intronic promoter under cisplatin- and TNF-alpha- mediated apoptosis conditions. In this thesis study, the deep sequencing of total RNA, polyA + and polyA eliminated fractions from cisplatin-, TNFalpha-, DMSO-treated cells were performed. Differentially expressed intronic transcripts were analysed by DE-kupl algorithm. The intronic transcripts both in total RNA and polyA + RNA fractions but not in polyA eliminated fractions were screened visually on Integrated Genome Viewer (IGV) and selected as sisRNA candidateS. 48 sisRNA candidates were detected in cisplatin-treated data while 33 sisRNA candidates were detected in TNF-alpha- treated data. 5’ and 3’ RACE PCRs were performed for determination of transcriptional units of sisRNA candidates. Overexpression of sisRDOCK7-IT1 caused 8.09% increase in total apoptosis of HeLa cells in 48 hours. sisRDOCK7-IT1 triggers the activation of apoptosis but the mechanism of its induction of apoptosis is still unknown.Master Thesis Investigation of Long Non-Coding Rna and Chromatin Interactions in Hela Cells(Izmir Institute of Technology, 2022) Atbinek, Melis; Akgül, BünyaminThe DNA in the cells is surrounding histone proteins to form nucleosomes. The structure is packed further into chromatin. The chromatin structure is dynamic and flexible. It is regulated by many factors including long non-coding RNAs (lncRNAs). LncRNAs are a class of non-coding RNAs, transcripts that do not encode protein. They are longer than 200 nucleotides and might contain a polyA tail and a 5’ cap. Thus, they are localized in the nucleus. lncRNAs interact with chromatin in two ways, indirect and direct. Direct interaction occurs via two mechanisms: R-loop and triplex formation. These interactions affect the folding of chromatin inducing gene expression under various cellular conditions. LncRNAs interacting with chromatin regulating genes are found in HEK cells. Thus, it is hypothesized that lncRNA – chromatin interactions may differ in cancerous cells as well. In this study, the iMARGI method is optimized to be used in adenocarcinoma HeLa cells. The chromatin digestion and incubation conditions are adjusted to give optimal results for HeLa cells. iMARGI is a recently developed method employed to investigate such interactions in a genome-wide manner. iMARGI allows the isolation of all lncRNAs interacting with the whole genome. The interacting RNA – DNA molecules are pulled down with streptavidin conjugated beads after linker ligation. The chimeric molecules are amplified with PCR forming lncRNA – chromatin libraries of HeLa cells. In the future, new libraries can be formed after inducing apoptosis in HeLa cells. Identification of lncRNAs involved in chromatin remodeling in apoptotic conditions can facilitate new therapeutic methods for fighting tumor initiation and development.Master Thesis Investigation of the Interaction Between Dr5-As Long Noncoding Rna and Caprin1 Protein(Izmir Institute of Technology, 2022) Kaçar, Vahide İlayda; Akgül, BünyaminCell proliferation is the crucial process for many physiological incidents such as tissue and organ development, wound healing, and immune system reactions. It is achieved by the growth and division of cells in a multicellular organism. Investigation of molecules involved in the regulation of cell cycle mechanism provides insight into reasons and treatments of the diseases such as cancer. In recent years, information that acquired from deep sequencing reveals that several proteins and non-coding RNAs have crucial role in the regulation of cell cycle and proliferation. Death receptor 5 antisense (DR5-AS) is a novel long non-coding RNA (lncRNA) transcript that is cisplatin inducible and is involved in modulation of cell proliferation and cell cycle in HeLa cells. When DR5-AS lncRNA was knocked down, the morphology of HeLa cells became spherical without inducing apoptosis. Although this lncRNA reduces cell proliferation via a cell cycle arrest at S and G2/M phases, mechanism behind this cell cycle arrest is not known. lncRNAs work in complexes with RNA, DNA, and protein interactions in the cell. There are several experimental and bioinformatical approaches to investigate RNA: protein interactions such as PAR-CLIP. In this approach, proximal protein and RNAs are covalently bonded with UV radiation. Then this complex is immunoprecipitated with specific antibodies. According to PAR-CLIP data of DR5-AS lncRNA, CAPRIN1 is a cell cycle associated protein that has the highest interaction score. The results suggest that CAPRIN1 and DR5-AS work reversely in cell proliferation although under the cisplatin treatment, CAPRIN1 enhances the expression of DR5-AS lncRNA. All these observations were confirmed by many quantitative experiments. Conclusively, this study provides a clue about how DR5-AS lncRNA might regulate cell cycle and proliferation through CAPRIN1 protein.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.