Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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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 Molecular Analysis of Mammalian Neu4 Sialidase Gene Promoter(Izmir Institute of Technology, 2011) Delman, Murat; Seyrantepe, VolkanThere are four different mammalian sialidases that have been described; lysosomal (Neu1), cytoplasmic (Neu2), plasma membrane (Neu3), lysosomal/mitochondrial (Neu4). The activity of sialidase Neu4 enzyme against sialic acid containing ganglioside GM2 has been demonstrated. Biological role of sialidase Neu4 enzyme has been shown by the transfection of neuroglia cells from a Tay-Sachs patient with a Neu4-expressing plasmid showed clearance of accumulated ganglioside GM2. It has been also shown that sialidase Neu4 enzyme is responsible for degradation reactions of another ganglioside such as GD1a in brains of Neu4-/- mice. Aim of our study is to identify minimal promoter region of human Neu4 gene and demonstrate binding of transcription factors to this region. In our study, we used bioinformatic approaches to predict the sequence motifs where several specific transcription factors bind using TESS (Transcription Element Seach System) tool. We amplified seven different DNA fragments from human Neu4 promoter region, cloned into luciferase expression vector and performed reporter assay. We also performed electrophoretic mobility shift assay to demonstrate binding of transcription factors to candidate promoter region. We demonstrated that 187 bp upstream of Neu4 gene is minimal promoter region to control transcription from Neu4 gene. Electrophoretic mobility shift assay showed that 187 bp upstream region recruits several transcription factors. Our results demonstrated the minimal promoter region revealing several putative transcription factors such as Sp-1 and c-myc which might be responsible mainly for regulation of Neu4 gene transcription. The data we obtained might be useful to discover small molecules which can control Neu4 gene expression. High expression of Neu4 gene might be controlled using drugs or small molecules and the accumulated GM2 ganglioside in lysosomes of Tay-Sachs patients can be reduced.