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Transcriptional regulation of miR-17-92 microRNA cluster during macrophage differentiation.
Rybářová, Jana ; Stopka, Tomáš (advisor) ; Pospíšek, Martin (referee)
miR-17-92 cluster (Oncomir1) encodes seven microRNAs (miRNA, miR) regulating many biological processes including proliferation, differentiation or apoptosis. Overexpression of microRNAs encoded by miR-17-92 cluster is found in a number of tumors including acute and chronic myeloid leukemias (Dixon-McIver et al., 2008; Li et al., 2008; Venturini et al., 2007). Myeloid progenitors express miR-17-92 cluster at a high level, while macrophage differentiation associates with its downregulation. Our laboratory found, that miR-17-92 cluster is repressed by transcription factor Early growth response 2 (Egr2) upon differentiation of primary myeloid PUER progenitors, induced with transcription factor PU.1. Aim of this thesis is to further test the abovementioned data by preparing a reporter vectors set, carrying various fragments of miR-17-92 putative promoter, which enables us to study regulation of transcription of miR-17-92 cluster. This task complicated by presence of increased GC content of the miR-17-92 promoter was successfully accomplished resulting in amplification of eight fragments containing the various parts of miR-17-92 promoter including region -3.3 to 0 kb relative to the start of miR-17-5p sequence, that were inserted into pGL3 reporter vector. Transfection of pGL3 reporter vector carrying...
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Internal ribosome entry site of the hepatitis C virus as a possible target for therapy
Roučová, Kristina ; Čáp, Michal (referee) ; Pospíšek, Martin (advisor)
Hepatitis C virus infects about 3 % of world's population. Progress in molecular biology and better knowledge of hepatitis C virus life cycle contribute to the development of specifically targeted antiviral therapies for HCV. This new treatment also targets the internal ribosome entry site (IRES), which is highly conserved and therefore an attractive target for intervention. In addition, inhibition of IRES function could disable the propagation of the virus early in the HCV life cycle. Novel therapeutics are aimed both at the HCV IRES structure and its nucleotide sequence. Small molecules and synthetic nucleic acids, e.g. antisense oligonucleotides and ribozymes, have been tested as potential therapeutic substances. In this paper particular attention is paid to small molecules interacting with HCV IRES.
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Charakter of the host cell iduced by vaccinia virus and inhibitory effects of the lipoic acid on vaccinia virus growth
Spišáková, Martina ; Mělková, Zora (advisor) ; Němečková, Šárka (referee) ; Pospíšek, Martin (referee)
Vaccinia virus is a typical member of the poxvirus family. It had been successfully used during the worldwide smallpox eradication campaign. Currently, it is used as vector for prophylactic, as well as experimental purposes. This PhD thesis stems from the findings previously published in our lab. It is partially focused on a further characterization of vaccinia virus effects on the type of host cell death. The results point to the activation of apoptosis during vaccinia virus infection, but it cannot be completed and the cell dies by necrosis. Our attempts to shift the necrotic type of cell death induced by vaccinia virus infection towards apoptosis using a pharmacological inhibition of activity of a key enzyme PARP (Poly-(ADP-ribose) polymerase) remained unsuccessful. The effects of vaccinia virus-encoded anti-apoptotic factors appear superior to the inhibition of this single enzyme. The second part of the thesis is focused on inhibitory effects of a redox-modulating compound, lipoic acid, on vaccinia virus infection. Our results demonstrated its inhibitory effects in cell lines of different embryonic origin. It appears that the lipoic acid inhibits vaccinia virus growth at the stage of late gene expression or possibly later, during virus morphogenesis. Lipoic acid could be potentially used as a supportive...
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Structure and function of IRES elements in yeast
Černý, Jiří ; Pospíšek, Martin (advisor) ; Fišer, Radovan (referee)
Translation initiation is an important step in control of gene expression. Cellular mRNAs are usually translated via classical cap-dependent pathway, but some of them can also be translated by an alternative pathway like IRES mediated one. In the first part of my work I studied a possibility of IRES dependent translation initiation of RRN3 and BAS1 mRNAs from yeast Saccharomyces cerevisiae. I inserted BAS1 and RRN3 5'UTR into the intercistronic spacer of pFGAL4h and its promotreless version and measured the activity of secondary reporters in enegineered yeast strains. My data indicates presence of cryptic promoter in tested regions. To confirm this results I first inserted the RRN3 5'UTR into the intercistronic region of another bicistronic plasmid - pRFh and measured expression of second cistron. The results were similar like in the case of pFGAL4h system. The presence of shorter RNAs transcribed from cryptic promoter within the intercistronic region was shown using real time PCR. My quest in the second part of this work was to prepare RNAs useful for next investigation of translation control of lariat capped RNAs produced by GIR1 ribozyme in a slime mold Didymium iridis. I have prepared a wide set of DNA molecules coding different versions of GIR1 in front of the firefly luciferase ORF which served as...
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