Capsid of mouse polyomavirus (MPyV) is composed from three structural proteins: major structural protein VP1 and minor structural proteins VP2 and VP3. Posttranslational modifications may affect functions of proteins. This work deals with acetylation of MPyV structural proteins and its impact on the viral replication cycle. First part of the thesis is focused on acetylation of VP1. We showed that the VP1 protein is acetylated in viral particles and that interaction of VP1 with minor proteins supports VP1 acetylation. Further, we showed that cytoplasmatic deacetylase, histone deacetylase 6 (HDAC6), is important for virus infectivity. Overexpression of HDAC6 decreased MPyV infectivity, also decreased infectivity was exhibited by virus isolated from HDAC6 knock out cells. In addition, VP1 protein of virus from HDAC6 knock out cells was more acetylated in comparison with virus from parental cell line. These data suggest that VP1 is substrate for HDAC6. Second part of the thesis is focused on the characterization of N-terminal acetylation of VP3 minor structural protein. It has been previously shown that VP3 protein is N-terminally acetylated and MyPV with mutated (unacetylated) form of VP3 protein is non-infectious. The main aim of this part is to prove the hypothesis that N-terminal acetylation is...

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...

Alternative splicing is an important cellular mechanism. It allows to produce multiple protein isoforms from a limited number of genes. Regulation of alternative splicing involves cis-acting elements on pre-mRNA and trans-acting splicing factors (SR and hnRNP proteins). Because splicing occurs co-transcriptionaly, chromatin structure appears to have a role in the regulation of alternative splicing. We have studied the effect of histone acetylation on alternative splicing. We have prepared splicing reporter for alternative EDB exon, which is part of the fibronectin gene. We have shown, that the inhibition of histone deacetylases affects splicing pattern of EDB exon from the reporter in the same way as the splicing of the endogenous EDB exon. Furthermore, we have shown, that the structure of the promoter affects splicing of alternative EDB exon from splicing reporter. Currently we have found out, that the structure of the promoter influences the degree of histone H4 acetylation. Inclusion of alternative EDB exon in mRNA was inversely proportional to histon acetylation on the reporter. This work might explain why various promoters have different splicing patterns of alternative exons.

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...

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...

Wood impregnation is one of the oldest and the most frequently used techniques to protect the wood. This treatment offers a wide range of protection depending on used impregnants (preservatives). Impregnation can make the wood more resistant to decay, less flammable, more dimensionally stable, harder, stronger, more UV stable resistant and many more. Generally, the aims of this thesis is to give a new and modern approaches to already known technologies. The experimental part have been carried out using laboratory vacuum-pressure impregnation plant and many other devices. Almost all treatments and measurement have been done in laboratories of Research centre in Útěchov. The different wood species e.g. European beech (Fagus sylvatica L.) and hybrid poplar (Populus hybrids) have been studied. The patented solution shows the method of the treatment and colouring of wood containing tannins, characterized by impregnation with a product containing iron oxide nanoparticles, in a concentration of 0.4 g/l to 42 g/l, an average particle size in the range of 1 to 100 nm, and a liquid medium. Impregnation can be performed as pressure impregnation, followed by soaking, or the product can be applied by brushing or spraying. This method is an alternative to wood ammonification. The tests (Paper I) shows following results. Both nanoiron and ammonia treatments tested induced darker colouring of oak wood, more intensive darkening was observed in case of the nano-iron treatment. The native oak showed a steep drop in lightness ca 25-50 hours of exposure followed by gradual re-increase during further exposure to the artificial sunlight. The darker surfaces generated by tannin reacting chemically with iron and ammonia evidently faded in the course of light exposure. In case of chromatic parameters a* and b*, the AT (ammonia treatment) oak more or less copied the colour change progress of native oak. On the contrary, the NIT (nanoiron treatment) wood showed a double increase in both parameters. The colour of NIT wood after light exposure was more or less equivalent to the original colour of the ammonium-treated. Distinct reddening of NIT oak may be caused by oxidation of non-precipitated iron particles. The antifungal effects of copper and silver nanoparticles against two wood-rotting fungi were investigated with following results (Paper II). The highest value of retention was observed for pine sapwood (~ 2 kg/m3) for both nanoparticle solutions. The amount of nanoparticles in the wood did not increase proportionally with an increasing concentration, but only 1.5-2 times increase was reached. An average leaching of 15% to 35% was observed for copper nanoparticles, depending on used wood species and concentration. Significantly lower leaching (max. 15 %) was observed for pine sapwood impregnated by silver nanoparticles with a concentration of 3 g/l. The highest antifungal effect (under 5 % of mass loss) against both tested fungi was found for nano-copper treatment at the concentration of 3 g/l. However, this effect of treatment seems to be almost negligible after the leaching test. Therefore, this study aims to present fundamental material properties of wood treated with copper and silver nanoparticles, and provide groundwork for further research (e.g. fixation of substances in the wood structure, etc.). In this study (Paper III), selected physical and mechanical properties, i.e., density profile, bending strength, hardness and moisture absorption were investigated for Lignamon (i), which was obtained from the Czech industrial production. Selected properties were also investigated using steam-densified beech (ii) and native beech (iii) and compared with each other. Densitometry of Lignamon showed a large variability of the density profile compared to the density profile of only densified beech. It is affected by the degree of densification, temperature and moisture gradients, and their relationship to the glass transition of the wood cell wall. Modulus of elasticity, hardness, moisture exclusion and anti-swelling efficiency of Lignamon are enhanced compared to densified beech. The enhanced dimension stability and lower hygroscopicity of Lignamon are probably caused by heat treatment during the process. Further investigation will be carried out with self-produced Lignamon samples. The paper IV deals with the effect of vacuum-pressure impregnation of poplar wood (Populus alba L.) by aqueous solutions of sucrose and sodium chloride on its physical properties. The most satisfying final properties were achieved in impregnation of sucrose with concentration of 6.25 g/100 ml H2O. The retention was 31 kg/m3 (WPG around 8 %). The values of ASE (anti-swelling efficiency) reached to 36 % and MEE (moisture exclusion efficiency) was reduced by 33 %. In Paper V, MW and conventional acetylation of wood was carried out to determine its efficacy on the material properties. Both MW and conventional heating positively affected the selected material properties. The results showed that no significant differences were found between MW and conventional heating; therefore, MW heating can be used as a valid replacement in the acetylation process. MW power of 2 kW and 0.1 m/min conveyor speed were the optimum conditions for MW acetylation. These process parameters resulted in 39.4 % ASE (T) and 35.2 % ASE (R) for beech and 38.0 % ASE (T) and 16.3 % ASE (R) for poplar samples. This work provides insight into the details of wood acetylation using MW heating. The study (Paper VI) aims to evaluate the antifungal activity of extractive compounds obtained with fexIKA accelerate extraction process. Results showed that the extractive compounds obtained from black locust heartwood were able to increase the native durability of European beech from class 5 (i.e. not durable with an average mass loss of 43.6 %) to class 3 (i.e. moderately durable with an average mass loss of 12.7 %). The final Paper VII shows following results. After impregnation with residual liquids and leaching, high amounts of TT (thermal treatment) and HTC (hydrothermal carbonisation) solution were washed out, whereas Pyrolysis liquids stayed with 25 % to 40 % remaining in the wood. Volumetric swelling in 65 % relative humidity at 20 °C was reduced from 6.5 % in untreated poplar to 5 % in impregnated samples with process residues of thermal treatment using 180 °C or 200 °C. The processes using HTC liquids from Miscanthus sp. (Misc.) or Sawdust showed reduction to 3-3.5 % and Pyrolysis liquid treatment to 2.5 %. After exposure to fungi, the mass loss of untreated Beech (30-35 %) and Poplar (40-50 %) was reduced to 2 % in case of Pyrolysis- and 4 % in case of TT-treatment.

Cancer cells are able to adapt to different stress factors such as hypoxia, which is caused by insufficient tumor vascularization. An increased acetylation status of histones H3 and H4 in UKF-NB-3 and UKF-NB-4 neuroblastoma cell lines was found to be a mechanism of adaptation of these cells to hypoxia. An increase in acetylation of histones H3 and H4 is suggested to cause changes in the structure of chromatin that lead to activation of gene transcription. In addition, cultivation of tested neuroblastoma cells under hypoxic conditions changes expression of proteins of a transcription factor N-myc, which is essential for development of neuroblastomas. This transcription factor is also responsible for a metabolic adaptation of neuroblastoma cells, increases their aggressiveness and its expression leads to a worse prognosis of the disease. Inhibitors of histone deacetylases (HDAC) are suggested to be the promising agents exhibiting various anticancer effects. They can induce cell cycle arrest, differentiation or programmed cell death in sensitive tumors. In this study, the effect of one of inhibitors of HDACs, valproate, on expression of proteins of transcription factors N-myc and hypoxia inducible factor 1α (HIF-1α) was investigated. Valproate decreases protein levels of both transcription factors in...

The phenotype-linked fertility hypothesis predicts that both, male carotenoid-based sexual ornamentation and their spermatozoa are phenotypically plastic and may be co-affected by the environment. One of the factors affecting their phenotype may be oxidative stress and the ability of organism to eliminate its effect. Oxidative stress may reduce sperm quality because sperm lack the ability to repair DNA, but it can also affect spermatogenesis itself. However, some substances may function as antioxidants, and thus eliminate effect of reactive oxygen species (oxidative stress) in the body. In this study, adult zebra finch males (Taeniopygia guttata) originating from the domesticated and recently wild-derived populations were exposed to the diquat (D), which enhances the oxidative stress, and carotenoid lutein (L), which could have an antioxidant function. Experimental design had factorial character 2x2 with a control (group L, D, LD, control). Neither oxidative stress, carotenoids, nor their interactions affected sperm morphology or velocity and it also did not increase abnormal sperm proportion in the ejaculate. However, the differences were observed at the molecular level, where by inducing the oxidative stress, the sperm had reduced signal intensity of acetylated α-tubulin in the sperm tails....

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...

5 Abstract Neuroblastoma is one of the most common cancer diseases diagnosed in children. This rapidly growing solid tumor is usually formed by hypoxic areas which arise as a consequence of inefficient and disorganized neovascularization. The cells stressed by hypoxia triggers transcription of many genes necessary for their survival, and conversely stop the production of proteins which are not necessarily needed for the survival in these severe conditions. The adaptation of cells to hypoxic conditions may appear due to the epigenetic regulation of metabolism associated with chromatin remodeling which involves the DNA methylation and also the posttranslational modifications of histones. Among the most important of these, there is the acetylation of lysine residues of histones associated with the DNA strands loosening, facilitated binding of transcription factors and the activation of gene expression. Thus, the first part of this study is concerned with changes in the acetylation of histones H3 and H4 of human neuroblastoma cell lines UKF-NB-3, UKF-NB-4, SH-SY5Y and SK-N-AS, cultured in parallel under standard culture conditions and in the absence of oxygen (hypoxia, 1% O2) for 24 hours, which are studied by Western blot analysis. Thereupon, the activity of histone deacetylases and histonacetyltransferases,...