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Yeast isogenic system as a method for study of IFI16 protein interactions with DNA
Kratochvilová, Libuše ; Šedrlová, Zuzana (referee) ; Brázda, Václav (advisor)
This bachelor thesis deals with the binding of interferon gamma-induced protein 16 (IFI16) to the secondary local structures of the G-quadruplex (G4) and its mutations in the single-hybrid yeast system (Y1H). The IFI16 protein in the cell recognizes its own and foreign or damaged DNA, is involved in the formation of the inflammasome and induces the expression of type I interferon (IFN-I). It is also involved in the regulation of transcription and restriction of viral infection. It has been shown that the IFI16 protein binds preferentially to G-quadruplex structures and is able to stabilize them by this binding. G-quadruplexes are classified as non-canonical DNA and RNA structures formed by G-rich sequences. They are easily formed under physiological conditions and are found in a number of important regulatory structures of the genome such as telomeres or oncogene promoters. They are also part of a number of viral genomes. This makes them excellent potential targets in the treatment of cancer and viral diseases. In the first part of the work, new reporter strains of S. cerevisiae yeasts were prepared by the Delitto Perfetto method, differing only in sequence with the potential for G-quadruplex formation, which was designed and analyzed by the DNA Analyzer program. The correctness of the inserted sequences was verified by PCR and Sanger sequencing and comparison with the supplied oligonucleotide sequences by the Blast program. In the second part of the work, the newly prepared strains were transformed with vectors for the expression of p53, IFI16 proteins, and the effect of IFI16-G4 binding on the expression of the gene in connection with the tumor suppressor p53 was assessed using luciferase reporter assays. The evaluation was performed on the basis of a statistical analysis of the magnitudes of the effects obtained after normalization of the luminescence signal on the optical density of the culture at a wavelength of 600 nm. The results show that the IFI16 protein has a different effect on the trans-activation potential of the p53 tumor suppressor depending on binding to emerging structures near the reporter gene promoter, and that a G4Hunter threshold of at least 1,591 had to be reached and taken into account to successfully form a G-quadruplex the potential of the sequence to successfully form at least 2 G-tetrads.
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The metabolic role of pulmonary arterial fibroblasts in the activation of immune system during development of pulmonary arterial hypertension
Křivonosková, Monika ; Plecitá, Lydie (advisor) ; Zadražil, Zdeněk (referee)
The development of inflammation in the small distal pulmonary vessels plays an important role in the development of pulmonary arterial hypertension (PAH). One of the cell types found in the pulmonary vessels are fibroblasts, which, according to the "outside-in" theory, may be the first to respond to stimuli in the vessel, modulating remodeling of the pulmonary vessels toward the inner layers of the vessel and attracting other immune cells to the site. In addition to inflammation, the so- called Warburg effect also occurs in vessels affected by PAH, in which metabolism shifts toward glycolysis and lactate production. Among other changes, a pro-oxidative state is induced within the cell by mitochondrial metabolism and NADPH oxidase, leading to an imbalance in reactive oxygen species production. We therefore wanted to test whether calf lung fibroblasts with hypoxia-induced PAH have an active inflammasome, with which they would produce mature interleukin 1β (IL-1β) and to clarify the effect of a pro-oxidant environment on this expression. In our in vitro model we have confirmed IL-1β mRNA expression, but we were unable to detect its expression at the protein level. This was consistent with the inability to detect inflammasome activity. We believe that the inability to detect the protein form of IL-1β...
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Yeast isogenic system as a method for study of IFI16 protein interactions with DNA
Kratochvilová, Libuše ; Šedrlová, Zuzana (referee) ; Brázda, Václav (advisor)
This bachelor thesis deals with the binding of interferon gamma-induced protein 16 (IFI16) to the secondary local structures of the G-quadruplex (G4) and its mutations in the single-hybrid yeast system (Y1H). The IFI16 protein in the cell recognizes its own and foreign or damaged DNA, is involved in the formation of the inflammasome and induces the expression of type I interferon (IFN-I). It is also involved in the regulation of transcription and restriction of viral infection. It has been shown that the IFI16 protein binds preferentially to G-quadruplex structures and is able to stabilize them by this binding. G-quadruplexes are classified as non-canonical DNA and RNA structures formed by G-rich sequences. They are easily formed under physiological conditions and are found in a number of important regulatory structures of the genome such as telomeres or oncogene promoters. They are also part of a number of viral genomes. This makes them excellent potential targets in the treatment of cancer and viral diseases. In the first part of the work, new reporter strains of S. cerevisiae yeasts were prepared by the Delitto Perfetto method, differing only in sequence with the potential for G-quadruplex formation, which was designed and analyzed by the DNA Analyzer program. The correctness of the inserted sequences was verified by PCR and Sanger sequencing and comparison with the supplied oligonucleotide sequences by the Blast program. In the second part of the work, the newly prepared strains were transformed with vectors for the expression of p53, IFI16 proteins, and the effect of IFI16-G4 binding on the expression of the gene in connection with the tumor suppressor p53 was assessed using luciferase reporter assays. The evaluation was performed on the basis of a statistical analysis of the magnitudes of the effects obtained after normalization of the luminescence signal on the optical density of the culture at a wavelength of 600 nm. The results show that the IFI16 protein has a different effect on the trans-activation potential of the p53 tumor suppressor depending on binding to emerging structures near the reporter gene promoter, and that a G4Hunter threshold of at least 1,591 had to be reached and taken into account to successfully form a G-quadruplex the potential of the sequence to successfully form at least 2 G-tetrads.
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Intracellular life of pathogenic bacterium Francisella tularensis in the host.
Rädisch, Robert ; Konopásek, Ivo (advisor) ; Vopálenská, Irena (referee)
Francisella tularensis is a facultative intracellular pathogenic bacterium, which causes disease named tularemia. For the entrance to the host cells Francisella uses host's cell mechanisms by which it is incorporated into cell phagosome. Subsequently, it escapes from phagosome to cytosole where bacterial growth takes place. Some of bacteria are cleared from cytosol by autophagy, from another ones dsDNA is released. This DNA is recognized by cytosolic receptors, which form inflammasome complex. Inflammasome sets off pathway leading to the death of infected cell. Since the penetration to the cell Francisella modulates cell signallization in its own benefit to ensure enough time and nutrients for its growth. Francisella do not act only in the infected cells, where it reduces recognition of itself and clearance from cytosol, but it also induces secretion of factors, which moderate activation of adaptive immunity of the host. Key words: Francisella, tularemia, fagosome, inflammasome, autophagy, adaptive immunity
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