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Interaction of selected flavonoids with N-acetyltransferases
Veselá, Šárka ; Hodek, Petr (advisor) ; Bořek Dohalská, Lucie (referee)
Flavonoids are natural compounds synthesized in plants as secondary metabolites. Due to broad range of their biological effects, for example we can mention antioxidant, hepatoprotective or antibacterial effects, flavonoids are increasingly used as a component in dietary supplements. Besides positive effects, some negative effects were observed, especially mutagenic and pro-oxidative properities. The biotransformation is an important process which is affected by flavonoids. Interactions between flavonoids and biotransformation enzymes in both phases of biotransformation are essential for the metabolism of drugs and carcinogenesis processes. In this bachelor thesis the influence of selected flavonoid compounds, myricetin and dihydromyricetin, on the activity of N-acetyltransferase 1 and 2, enzymes of second phase of biotransformation, was studied. Mainly the mechanism of this influence was tested. The inhibition of human recombinant and of N-acetyltransferase 1 and 2 by myricetin and dihydromyricetin was proved. The more in-depth study of interactions of N-acetyltransferase 1 and 2 with selected flavonoids shows that raising concentration of second substrate (p-aminobenzoic acid, sulfamethazine) does not lead to reduction of inhibition effect. Thus, it is highly probable that both flavonoids already...
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The relationship between splicing and posttranslational modifications of chromatin in Saccharomyces cerevisiae
Kovaľová, Libuša ; Folk, Petr (advisor) ; Čáp, Michal (referee)
Protein Prp45, the yeast ortholog of the human transcription coregulator SNW1/SKIP, has been previously associated only with the regulation of pre-mRNA splicing. However, our laboratory found that protein Prp45 genetically interacts not only with the proteins involved in pre-mRNA splicing, but also with factors important for transcription elongation and with chromatin modifying enzymes. Our data and the information about the human ortholog SNW1/SKIP suggest that Prp45 could serve as a regulator coupling splicing, transcription and chromatin state in S. cerevisiae. The main aim of this diploma thesis was to find out whether the protein Prp45, which is essential for cotranscriptional assembly of the spliceosome, affects posttranslational modifications of chromatin on transcribed genes. Using chromatin immunoprecipitation, the influence of prp45(1-169) mutation on trimethylation of histone H3 at lysine 4 and acetylation of histone H3 at lysines 9, 14 and 18 on transcriptionally active genes was not confirmed. The other aim was to analyse the behavior of cells synchronized by α-factor by using flow cytometry. According to our results, prp45(1-169) mutation leads to the prolongation of the cell cycle. For the purpose of monitoring the dynamics of nucleosomes in S. cerevisiae strains, the system of...
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Posouzení kvality lepení kombinovaných masivních materiálů na bázi dřeva
Ženatý, Petr
The idea of this work came from doc. Dr. Ing Pavel Král when some company contant him regarding research for using appropriate materials and adhesives for window frames. The goal of my work is to determine the bonding strength of various combinations of wood (oak, beech, larch, spruce, poplar and accoya) and polyurethane adhesives. The research was half focused on today less known tree species Accoya wood and its behavior in combination with other tree species. It is a modified type of wood (pine Radiata Pine), who thanks to a chemical process called acetylation becomes a high quality, durable and durable material. The practical part is a tensile stress test specimen to shear with various types of adhesives. My job was to find the right combination of wood and glue, that will have sufficient strength and concurrently resist external conditions (humidity, wind, solar radiation).
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The role of posttranslational modifications of minor proteins and acetylation of microtubules in mouse polyomavirus infection
Mariničová, Zuzana ; Horníková, Lenka (advisor) ; Saláková, Martina (referee)
Mouse polyomavirus (MPyV) capsid is composed of the main capsid protein VP1 and minor capsid proteins VP2 and VP3. Minor proteins are not essential capsid assembly, but they are key for efficient viral infection. The first part of this thesis studies the modifications of VP2 and VP3, the deamidation of Asn at 253 of VP2 (137 of VP3) and N-terminal acetylation of Ala of VP3, which could be the cause of double bands for VP2 and VP3 on SDS-PAGE. Mutated genomes of MPyV N253D (Asn to Asp) and N253E (Asn to Glu) simulating deamidation and A117V (Ala to Val) with reduced acetylation were prepared previously. We prepared three isolations of the mutant viruses and we confirmed that the deamidation is the cause of the double bands. Mutant viruses were compared to the wild type in terms of efficiency of infection, but the role of deamidation could not be proven. Virus A117V is noninfectious either due to lowered acetylation or the substitution of amino acid at this position. This thesis also studies the role of -tubulin acetylation in the infection of MPyV. The role of -tubulin acetylation in viral infection is being investigated to find new antiviral strategies. Acetylation rises after MPyV infection, but this is not due to a change in mRNA expression of tubulin acetylating (TAT1) or deacetylating enzyme...
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Characterisation of the mechanisms regulating 53BP1 nuclear transport
Liďák, Tomáš ; Macůrek, Libor (advisor) ; Brábek, Jan (referee)
Tumor suppressor p53-binding protein 1 (53BP1) is an integral part of a sophisticated network of cellular pathways termed as the DNA damage response (DDR). These pathways are specialized in the maintenance of genome integrity. Recently, it was reported that nuclear import of 53BP1 depends on importin ß. Here, I used fluorescence microscopy and co-immunoprecipitation experiments to identify its nuclear localization signal (NLS). Clusters of basic amino acids 1667-KRK-1669 and 1681-KRGRK- 1685 were required for 53BP1 interaction with importin ß and for its nuclear localization. Short peptide containing these two clusters was sufficient for interaction with importin ß and targeting EGFP to the nucleus. Additionally, the effect of 53BP1 phosphorylation at S1678 on its nuclear import was examined. Mimicking the phosphorylation in the 53BP1-S1678D mutant decreased the binding to importin ß and resulted in a mild defect in 53BP1 nuclear import. However, 53BP1 entered the nucleus continuously during the cell cycle, suggesting that CDK-dependent phosphorylation of S1678 probably does not significantly contribute to the regulation of 53BP1 nuclear transport. Taken together, 53BP1 NLS meets the attributes of a classical bipartite NLS. Although no cell cycle-dependent regulation of its import was observed, the...
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The role of acetylation in the RNA recognition motif of SRSF5 protein
Icha, Jaroslav ; Staněk, David (advisor) ; Šenigl, Filip (referee)
Acetylation is emerging as an important posttranslational modification, which is found in thousands of proteins in eukaryotes, as well as prokaryotes. Global proteomic studies implicated acetylation in regulation of various processes like metabolism, gene expression, cell cycle or aging to name a few. In this work I set out to investigate the role of acetylation of a splicing regulatory protein SRSF5 by creating mutations in its acetylation site. I tested the hypothesis that acetylation influences SRSF5 interaction with RNA. I expressed acetylation-mimicking (Q) or non-acetylable (R) mutant of SRSF5 in HeLa cells and measured their interaction with RNA by RNA immunoprecipitation or in vitro by fluorescence anisotropy. Both approaches agreed that mutants interact with RNA less than the wild type protein and Q mutant bound RNA weaker than R mutant. I did not detect further difference in localization or dynamics among the proteins in vivo, which suggests that difference caused by weakened interaction of mutants with RNA was outweighed by other factors influencing SRSF5 behaviour, probably protein-protein interactions. I also found out that mutant SRSF5 proteins do not have a dominant effect on splicing of fibronectin alternative EDB exon. The data obtained give an indirect evidence for the hypothesis that...
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Preparation of organocatalysts derived from monosaccharides
Nekvinda, Jan ; Veselý, Jan (advisor) ; Trnka, Tomáš (referee)
This bachelor thesis is focused on the synthesis of organocatalysts derived from monosaccharides, in particular D-glucose and D-glucosamine, with various protecting groups. Synthesis of various thiourea catalysts and the attempt to prepare new squaramide catalysts is described. ABSTRAKT Tato bakalářská práce je zaměřena na syntézu organokatalyzátorů odvozených od monosacharidů, zejména z D-glukózy a D-glukosaminu, s různými chránícími skupinami. Dále je popsána syntéza různých thiomočovinových katalyzátorů a pokus o přípravu katalyzátoru obsahující derivát kyseliny čtvercové.
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Differences in histone acetylation in normoxia and hypoxia
Čepek, Pavel ; Poljaková, Jitka (advisor) ; Eckschlager, Tomáš (referee)
Histones and their N and C terminal tails undergo different covalent modifications that regulate gene transcription. Among these histone modifications are methylation, ubiquitinilation, SUMOylation, ADP- ribosylation, phosphorylation, proline izomerization, deimination and acetylation. Histone acetylation is regulated by histonacetyltransferases (HATs) and histondeacetylases (HDACs). The balance between acetylation/deacetylation influences chromatin condensation and thus regulates gene transcription. Acetylation balance is disrupted in many human cancers and this fact can contribute to the development of malignant diseases. Histondeacetylase inhibitors (HDACi) can restore this acetylation imbalance. One of these HDACi is valproic acid (VPA) which has been used in treatment of epilepsy for decades. VPA shows antitumour effect in many studies. Decreased expression of n-myc oncoprotein, inhibition of tumour growth and angiogenesis are one of these anticancer effects observed in neuroblastoma cell lines after treatment with VPA. Despite the fact that exact mechanism of antitumour effect of VPA remains unclear, one of the most important mechanisms is hyperacetylation of histone H3 and H4. It is shown in this work that VPA increases acetylation of histones H3 and H4 in human neuroblastoma cell lines...
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