Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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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 Systematic Computational Analysis of Potential Rna Interference Regulation in Toxoplasma Gondii(Izmir Institute of Technology, 2009) Çakır, Mehmet Volkan; Allmer, JensRNA-mediated silencing was first described in plants and became famous by studies in Caenorhabditis elegans. RNA interference (RNAi) is the mechanism through which an RNA interferes with the production of other RNAs in a sequence specific manner. MiRNAs are a type of RNA which originate from the genome with their active form being ss-RNAs of 21-23 nucleotides in length. They are being transcribed as primiRNAs then processed in the nucleus by Drosha to pre-miRNAs with a stem-loop structure and 70 nucleotides in length. This stem-loop containing pre-miRNAs is then processed in the cytoplasm to ds-RNA one strand of which will serve as interfering RNA. Toxoplasma gondii is a species of parasitic protozoa which causes several diseases. T.gondii emerges as a good candidate for computational efforts with its small genome size, publicly available genome files and extensive information about its gene structure, either based on experimental data or the prediction with several gene finders in parallel. Therefore, it seems important to establish the regulatory network composed of RNAi which may be beneficial for the Toxoplasma community. Within this context the pool of possible stem-loop constitutive transcripts are produced, further analysis of this pool for desired 2D structure is integrated and mapping of possible RNAi regulation to T.gondii.s genome is established. In connection with computational assessment and mapping, the derived information is provided as a database for quick lookup using a convenient web interface for experimental studies of RNAi regulation in Toxoplasma, thus reduce time and money costs in such studies.Master Thesis Identification and Characterization of Boron Tolerant Genes in Hordeum Vulgare (barley) by Using Mrna Differential Display and Rt-Pcr Techniques(Izmir Institute of Technology, 2007) Akıncı, Ersin; Karakaya, Hüseyin ÇağlarBoron, is a microelement that plays a role in plant development. In contrast, excess amount of boron is toxic for plants. Turkey is the second country that has the largest boron reserve in the world, thus boron is one of the major problems in agriculture in Turkey. Barley (Hordeum vulgare) is the second widely produced cereal after wheat. Because barley is used in human diet, animal feeding and beer industry it is an economically valuable crop. There are ten different barley varieties in Turkey and these varieties show different genetic variations against boron toxicity. In this study, Hamidiye (boron sensitive) and Anadolu (boron tolerant) varieties were used to identify genes responsible for boron tolerance. RT-PCR and mRNA Differential Display techniques were used from root and leaf samples of Hamidiye and Anadolu varieties grown in laboratory with or without boron conditions. Eight differentially expressed genes identified by using mRNA Differential Display technique. Sequence of these genes gave homology to an eukaryotic translation initiation factor in Arabidopsis thaliana, a chlorophyll a/b binding protein precursor in Triticum aestivum, an elongation factor in Oryza sativa, short-chain dehydrogenase/reductase family protein in Arabidopsis thaliana, a thioredoxin h isoform in Triticum aestivum, a shaggy-like kinase protein in Triticum aestivum, chloroplast genome in Hordeum vulgare, a hypothetical protein in Arabidopsis thaliana. Expression level of six of forty three antiporter genes showed differences between Anadolu and Hamidiye cultivars in Real Time PCR.Master Thesis An Integrative Data Mining Approach for Microrna Detection in Human(Izmir Institute of Technology, 2013) Saçar, Müşerref Duygu; Allmer, JensMicroRNAs (miRNAs) are single-stranded, small, usually non-coding RNAs of about 22 nucleotides in length, that control gene expression at the posttranscriptional level through translational inhibition, degradation, adenylation, or destabilization of their target mRNAs. Although hundreds of miRNAs have been identified in various species, many more may still remain unknown. Therefore, the discovery of new miRNA genes is an important step for understanding miRNA mediated post transcriptional regulation mechanisms. First attempts for the identification of novel miRNA genes were almost exclusively based on directional cloning of endogenous small RNAs and high-throughput sequencing of large numbers of cDNA clones. However, conventional forward genetic screening is known to be biased towards abundantly and/or ubiquitously expressed miRNAs that can dominate the cloned products. Hence, such biological approaches might be limited in their ability to detect rare miRNAs, and restricted to the tissues and the developmental stage of the organism under examination. These limitations have led to the development of sophisticated computational approaches attempting to identify possible miRNAs in silico. Nevertheless, the programs designed to predict possible miRNAs in a genome are not sensitive or accurate enough to warrant sufficient confidence for validating all their predictions experimentally. With this study, we aim to solve these problems by developing a new and sensitive machine learning based approach to predict potential miRNAs in the human genome.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.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, BünyaminThe 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, BünyaminTypes 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.