Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Investigation of M1a and M6a Rna Methylations in Triple Negative Breast Cancer Cells(2024) Sağlam, Buket; Akgül, Bünyamin; Akgül, Bünyamin; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyMeme 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 Identification of Long Non-Coding Rnas That Regulate Apoptosis in Human(Izmir Institute of Technology, 2015) Ahmadov, Ulvi; Akgül, Bünyamin; Akgül, Bünyamin; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyApoptosis 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, Bünyamin; Akgül, Bünyamin; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyGenome-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 Investigation of the Functions of Candidate Mirnas in Camptothecin-Induced Apoptosis in Human Cells(Izmir Institute of Technology, 2012) Demir, Şeyda; Akgül, Bünyamin; Akgül, Bünyamin; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyMicroRNAs 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.Master Thesis Analysis of Temporal and Spatial Expression of Drosophila Embryonic Small Rnas by Deep-Sequencing Method(Izmir Institute of Technology, 2012) Coşacak, Mehmet İlyas; Akgül, Bünyamin; Akgül, Bünyamin; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyThe world of small RNAs is expanding and new types of small RNAs are being identified. By using deep-sequencing techniques in addition to most abundant small RNAs; miRNA and siRNA, piRNA and tRFs were further characterized and shown to be functional. The global behavior of small RNAs during MZT and their location in the cytoplasmic complexes has not been shown. By combining polysomal fractionation and deep-sequencing technique as well as the highly regulated developmental stages in Drosophila we have shown that the temporal and spatial expression of small RNAs changes during MZT. We have shown that each small RNA group has unique behaviour in cytoplasm and is enriched in specific polysomal fractions which shows that their local function in cytoplasmic complexes is mainly translational machinery.Master Thesis Investigation of Trna-Derived Small Rnas in Drosophila Melangaster(Izmir Institute of Technology, 2011) Göktaş, Çağdaş; Akgül, Bünyamin; Akgül, Bünyamin; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyTypes of small RNAs considered as crucial players of regulation of gene expression increase gradually in number. Improvements in cloning and sequencing strategies and technologies resulted in identification of tRNA derived small RNAs which are highly expressed in the cell just like other small RNAs such as microRNAs, endosiRNA, and Piwi interacting RNAs. Depending on stress and changing physiological conditions, tRFs are originated from different positions, in different frequency and different tRNAs. However, their functions and the complexes they interact with remain largely unknown. In this thesis study, one of the aims is to characterize tRNA derived small RNAs expressed during embryonic development in Drosophila melanogaster by in-vitro and invivo experiments. This study also aimed to demonstrate the differences between embryonic tRFs and stress induced tRNA derived small RNAs. Lastly, it was aimed to obtain some clues about their biogenesis, mechanism and functions. It was shown that the tRFs expressed in 1-hour and 8-h Drosophila embryos are different from stress induced tRNA derived small RNAs in terms of both position and length. The other important result is that embryonic tRFs are associated with complexes in mRNP and 60S fractions and they are expressed temporally and selectively during Drosophila embryogenesis.