Moleküler Biyoloji ve Genetik

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Browsing Moleküler Biyoloji ve Genetik by Author "Akiva, İzzet."

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    Characterization of novel Wnt/β-catenin pathway targets
    (Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2016., 2016.) Akiva, İzzet.; İyison, Necla Birgül.
    The Wnt/β-catenin signaling pathway is an evolutionary conserved pathway which has important functions in vertebrate early development such as axis formation, cellular proliferation and morphogenesis. The activation of this pathway leads to translocation of the transcriptional activator β-catenin into the nucleus where it activates T-cell factor/Lymphoid enhancer factor (Tcf/Lef) family of transcription factors, which regulate expression of developmental and cell cycle-related genes. Apart from its roles in various cellular processes, Wnt/β-catenin signaling pathway is also one of the most important intracellular pathways implicated cancer progression. A significant number of identified target molecules of this pathway, are known to have tumorigenic characters when mutated. Previous studies confirmed BRI3 (Brain protein I3) gene to be one of the transcriptional target genes of Wnt/β-catenin pathway. We used various approaches for the functional characterization of its novel targets. As a first step in our study, IFITM3 and MGAT1 proteins were confirmed as interaction partners for BRI3 by Yeast-two Hybrid and Co-IP techniques. BRI3 was found to be upregulated in response to TNF-α treatment and overexpression of BRI3 resulted in an increase in NFkB promoter activity. On the other hand, MGAT1 is a putative novel target of Wnt/β-catenin signaling pathway and determined to be upregulated in response to β-catenin activation. Cell proliferation and migration assays showed that, Huh7 cells stably expressing each of the BRI3 and MGAT1 genes have greater proliferative and invasive capabilities compared to control Huh7 cells. Furthermore, in vivo xenograft experiments were performed and it was determined that the stable overexpression of both of these genes in Huh7 cell lines lead to increased rate of tumor growth in NUDE/SCID mice. The resulting tumors were subjected to transcriptomic analyses using RNA-Sequencing technique in order to determine which pathways and biological processes take part in the cancer initiation process.
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    Identification of novel proteins that interact with BR13, a putative transcriptional target of the Wnt/β-catenin signaling pathway
    (Thesis (M.S.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2009., 2009.) Akiva, İzzet.; İyison, Necla Birgül.
    The Wnt/β-catenin signaling pathway is an evolutionary conserved pathway which has important functions in vertebrate early development, axis formation, cellular proliferation and morphogenesis. The activation of this pathway leads to translocation of the transcriptional activator β-catenin into the nucleus where it activates T-cell factor/Lymphoid enhancer factor (Tcf/Lef) family of transcription factors, which regulate expression of developmental and cell cycle-related genes. The results of SAGE (Serial Analysis of Gene Expression) performed recently in our lab indicated that BRI3 (Brain Protein I3) is one of the genes displaying increased expression in the presence of mutant -and thus more stable- form of β-catenin. In previous studies, BRI3 expression was found to be upregulated in Huh7 cell lines upon lithium treatment. Moreover, with regard to previous literature, BRI3 was found to have a very important role in the TNF-α mediated cell death pathway. Also, its promoter activity was analyzed by luciferase reporter assays and found to have been increased due to overexpression of β-catenin. In this study, we screened a human liver cDNA library by yeast two-hybrid assay using BRI3 as bait, with the aim to find novel binding partners of BRI3 and provide clues for the functional study of BRI3 with respect to the Wnt/β-catenin pathway. Library screening by yeast mating resulted in the identification of 2 candidate positive clones, which have been confirmed by cotransformation to the competent yeast cells, but the results still need to be verified by co-immunoprecipitation using mammalian cells. In conclusion, this study mainly will lead to the discovery of novel BRI3-interacting proteins which is essential for identification of the action mechanism of BRI3 in Wnt/β-catenin signaling. Furthermore, any possible interaction between Wnt/β-catenin pathway and TNF-α mediated cell death pathway can be revealed ultimately.