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Mapping of regulatory elements within 5' region of the Disp3 locus
Oltová, Jana ; Bartůněk, Petr (advisor) ; Krásný, Libor (referee)
Dispatched 3 (Disp3), a thyroid hormone-regulated gene, is studied extensively in our laboratory. Phenotype of cells with overexpressed Disp3 and its expression pattern make it a perfect candidate for a molecular link between thyroid hormone action and cholesterol homeostasis in the brain. Moreover, we hypothesize that it might play a role in certain neurodegenerative disorders and brain tumours. This thesis is aimed at the process of regulation of this gene via thyroid hormone receptor (TR), specifically identification of responsive elements of the thyroid hormone receptor that are necessary for the regulation. Also, we searched for elements recognized by liver X receptor (LXR), as LXR binds to the same arrangement of repeats as TR and there are a number of genes regulated by both of them. We combined in silico analysis of the Disp3 locus with reporter luciferase assays. A cluster of six elements identified around the first exon with two of them being conserved among human and mice draw our attention. In order to analyze this sequence in more detail, reporter vectors of various truncations of 3 kb region around exon 1 were constructed and tested in reporter assays. Reporter assays did not reveal any substantial element activated by TR or LXR; on the other hand, region containing repressor element(s)...
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The role of Disp3 gene in cell proliferation
Ditrychová, Karolína ; Zíková, Martina (advisor) ; Pospíchalová, Vendula (referee)
Dispatched 3 (DISP3), sterol - sensing domain (SSD) - containing protein, is a key focus of our laboratory. It was described as a gene regulated by thyroid hormone and its expression is mainly localized within neural tissue. Our preliminary data suggested increased DISP3 expression in medulloblastoma, a highly common pediatric cerebellar tumour, therefore we wanted to examine DISP3 role in human cancer cells. The aim of this thesis is to perform DISP3 overexpression and downregulation in human medulloblastoma cell lines and in mouse neural progenitors and analyse its effect on cell proliferation and differentiation. For this purpose, we chose DAOY and D341, human medulloblastoma cell lines with low and high expression of DISP3 and mouse multipotent neural progenitor cell line, C 17.2, with low DISP3 expression. We showed, that DISP3 ectopic expression leads to increase in cell proliferation in both DAOY and C 17.2 cells. Next, we examined the ability of C 17.2 cells to differentiate into neurons and astrocytes and observed, that cells overexpressing DISP3 reveal delay in differentiation, what we proved by analysis of cell specific markers. Using CRISPR-Cas9 targeting system, we reduced DISP3 expression within D341 cells and observed decrease in their proliferation. Finally, we analysed cell cycle...
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Mapping of regulatory elements within 5' region of the Disp3 locus
Oltová, Jana ; Bartůněk, Petr (advisor) ; Krásný, Libor (referee)
Dispatched 3 (Disp3), a thyroid hormone-regulated gene, is studied extensively in our laboratory. Phenotype of cells with overexpressed Disp3 and its expression pattern make it a perfect candidate for a molecular link between thyroid hormone action and cholesterol homeostasis in the brain. Moreover, we hypothesize that it might play a role in certain neurodegenerative disorders and brain tumours. This thesis is aimed at the process of regulation of this gene via thyroid hormone receptor (TR), specifically identification of responsive elements of the thyroid hormone receptor that are necessary for the regulation. Also, we searched for elements recognized by liver X receptor (LXR), as LXR binds to the same arrangement of repeats as TR and there are a number of genes regulated by both of them. We combined in silico analysis of the Disp3 locus with reporter luciferase assays. A cluster of six elements identified around the first exon with two of them being conserved among human and mice draw our attention. In order to analyze this sequence in more detail, reporter vectors of various truncations of 3 kb region around exon 1 were constructed and tested in reporter assays. Reporter assays did not reveal any substantial element activated by TR or LXR; on the other hand, region containing repressor element(s)...
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