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Substrate cleavage by mammalian Dicer isoforms
Kubíková, Jana ; Svoboda, Petr (advisor) ; Pospíšek, Martin (referee)
Host organisms evolved antiviral responses, which can recognize the viral infection and deal with it. One of the frequent signs of viral infection in a cell is appearance of double-stranded RNA (dsRNA). One of the pathways responding to dsRNA is RNA interference (RNAi), which functions as the key antiviral defence system in invertebrates and plants. Mammals, however, utilize for antiviral defence a different dsRNA-sensing pathway called the interferon response. RNAi functions only in mammalian oocytes and early embryonal stages although its enzymatic machinery is present in all somatic cells, where it is employed in the microRNA pathway. A previous study indicated that the functionality of RNAi in mouse oocytes functions due to an oocyte-specific isoform of protein Dicer (DicerO ), which is truncated at the N-terminus. In my thesis, I aimed to assess whether DicerO processes RNAi substrates more efficiently in vitro than the full-length Dicer (DicerS ), which is found in somatic cells. Therefore, I developed Dicer purification protocol for obtaining both recombinant mouse Dicer isoforms of high purity. I examined their activity in a non-radioactive cleavage assay using RNA substrates with structural features characteristic of RNAi substrates. My results suggest that recombinant DicerO and DicerS do not...
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Preparation of expression vectors for the production of ZIKA and Dengue NS3 helicase
Daňhelová, Kateřina ; Konvalinka, Jan (advisor) ; Heidingsfeld, Olga (referee)
Zika and Dengue viruses have spread, due to globalisation, to all continents which lie at least in part in the subtropic and tropic climatic zones. This spread of these viruses is a reason of an increasing number of severe diseases caused by them. New drugs, which would be effective against these infections, could be an answer to this challenge. Various viral proteins, among them also viral helicase, which is the topic of this bachelor thesis, can be a suitable drug target. The task was to prepare expression constructs for production of recombinant helicases of Zika and Dengue viruses via the suitable bacterial strain Escherichia coli. Several constructs derived from plasmid pET-16b were prepared with inserted helicase of Zika and Dengue viruses. One of them was used for the preparation of recombinant purified helicase of Zika virus, that will be used for further research. [IN CZECH] Keywords: Flavivirus, Zika virus, Dengue virus, helicase, expression, purification, enzyme activity [IN CZECH]
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Role of RNA helicases in antiviral defense
Krbušek, David ; Vopálenský, Václav (advisor) ; Janovec, Václav (referee)
Hepatitis C virus is an important human pathogen against which there is no immunization yet. This virus is detected by the immune system of the eukaryotic host cell by pattern recognition receptors of the RLR receptor family, which is part of the innate immune system. These RLR receptors detect the presence of hepatitis C virus and initiate a signaling cascade triggering an antiviral immune response. In this thesis, the role of cytoplasmic PRRs involved in antiviral defense during hepatitis C virus infection of eukaryotic cells has been described and determined. Key words Helicase, RIG-I, MDA5, pattern-recognition receptors, HCV, virus
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Substrate cleavage by mammalian Dicer isoforms
Kubíková, Jana ; Svoboda, Petr (advisor) ; Pospíšek, Martin (referee)
Host organisms evolved antiviral responses, which can recognize the viral infection and deal with it. One of the frequent signs of viral infection in a cell is appearance of double-stranded RNA (dsRNA). One of the pathways responding to dsRNA is RNA interference (RNAi), which functions as the key antiviral defence system in invertebrates and plants. Mammals, however, utilize for antiviral defence a different dsRNA-sensing pathway called the interferon response. RNAi functions only in mammalian oocytes and early embryonal stages although its enzymatic machinery is present in all somatic cells, where it is employed in the microRNA pathway. A previous study indicated that the functionality of RNAi in mouse oocytes functions due to an oocyte-specific isoform of protein Dicer (DicerO ), which is truncated at the N-terminus. In my thesis, I aimed to assess whether DicerO processes RNAi substrates more efficiently in vitro than the full-length Dicer (DicerS ), which is found in somatic cells. Therefore, I developed Dicer purification protocol for obtaining both recombinant mouse Dicer isoforms of high purity. I examined their activity in a non-radioactive cleavage assay using RNA substrates with structural features characteristic of RNAi substrates. My results suggest that recombinant DicerO and DicerS do not...
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Characterization of the HelD protein from Bacillus subtilis
Sudzinová, Petra ; Krásný, Libor (advisor) ; Lichá, Irena (referee)
BACKGROUND: Bacterial RNA polymerase (RNAP) is an extensively studied enzyme required for gene expression. In our Laboratory we found a new protein named HelD. HelD copurifies with B. subtilis RNAP. HelD is a ~90 kDa protein from the UvrD/Rep helicase family, which contains protein with the 3'-5' DNA unwinding activity. The molecular role(s) HelD in cell are still unknown and its potential role in transcription has not been studied so far. OBJECTIVE: The main aim of this Diploma project was to describe HelD. APPROACHES: The characterization was carried out on three levels: (i) bioinformatics analysis in silico was used to identify HelD homologs in other bacteria; (ii) growth tests in vivo were used to determine the phenotype(s) of the HelD-null mutant strain compared to wt; and (iii) biochemical experiments in vitro were utilized to describe the effects of HelD on transcription, and to test whether HelD has DNA binding and DNA unwinding activities. RESULTS: The in silico analysis revealed that HelD is present in Firmicutes, an industrially and medicinally important group of G+ bacteria. The phenotypic experiments showed that HelD is required for rapid adaptations to nutritional changes in the environment. The biochemical experiments showed that HelD stimulates transcription despite the fact that it...
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