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Analysis of essentiality of glmM gene coding for phosphoglucosamine mutase of Streptococcus pneumoniae.
Krupička, Jiří ; Branny, Pavel (advisor) ; Čáp, Michal (referee)
Phosphoglucosamine mutase (GlmM) is an enzyme of bacterial cell wall biosynthesis. The main aim of this thesis was to find out, whether gene glmM is essential for viability of Streptococcus pneumoniae. Therefore, we prepared merodiploid strain containing two copies of glmM; the genomic gene and ectopic copy under control of zinc inducible promoter. Subsequently, depletion strain was prepared by deletion of genomic copy of glmM. This strain was further used for analysis of viability and phenotype features in the medium containing various concentrations of zinc ions, an inducer of ectopic glmM expression. We found out, that the viability of this strain was strictly dependent on the concentration of inducer and further, that depletion of GlmM resulted in remarkable morphological defects. The rescue of mutant strain was observed after addition of inducer up to the level of the control sample. These results have provided the evidence of glmM essentiality for S. pneumoniae viability. Furthermore, we analyzed, whether phosphorylation of key amino acid residues, S99 and S101, is essential for GlmM functionality. Four different strains were prepared by means of site-directed mutagenesis expressing glmM with substitutions of key serine residues for alanine or glutamic acid. Since deletion of chromosomal locus in...
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mTORC1 complex function in regulation of translation initiation
Holásková, Lucie ; Feketová, Zuzana (advisor) ; Čáp, Michal (referee)
My bachelor thesis deals with the effect of mTOR pathway to different processes in the cell. In particular, it focuses on the influence of translation initiation. mTOR protein is part of two complexes, which occur in different organisms - mTORC1 and mTORC2. Eukaryotic initiation factor 4E (eIF4E) plays an important role in controlling translation initiation. The activity of eIF4E protein is regulated by family of repressor 4E-binding proteins (4E-BPs). Linking these proteins to eIF4E is regulated by their phosphorylation state. For the release of 4E-BP1 from eIF4E, phosphorylation must occur at four phosphorylation sites (Thr37, Thr46, Ser65 and Thr70). The study also covers some of the other events that occur in the mTOR pathway.
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Properties and function of middle T antigen of the murine polyomavirus
Fabiánová, Anna ; Forstová, Jitka (advisor) ; Čáp, Michal (referee)
Polyomaviruses are small DNA viruses, which are able to induce a broad variety of tumors. The main oncoprotein of the mouse polyomavirus (MPyV) is middle T antigen (MT antigen) which is able to transform cells. MT antigen has not an enzymatic activity of its own. It is able to activate signal transduction of host cells through its interactions with certain cellular proteins. These proteins include protein phosphatase 2A (PP2A), Src kinase, phosphatidylinositol 3 kinase (PI3K), Shc protein, 14-3-3 protein and phospholipase Cγ1 (PLCγ1). This work is focused on interaction between MT antigen and cellular proteins and on the impact of this interaction on cell transformation. Since MT antigen is a potent oncogene, the work also deals with the character of transformed cells and tumor development in mouse mammary epithelium. Keywords: polyomaviruses, MT antigen, PP2A, PI3K, PLCγ1, Shc protein, 14-3-3 protein
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Bacteriophages - current knowledge and possibilities for their therapeutic use
Glendová, Kristýna ; Lichá, Irena (advisor) ; Čáp, Michal (referee)
Bacteriophages, as viruses of bacteria, are the most abundand entity, populate every biotope where also bacteria live. One of the alternatives to combat infections caused by resistant strains of bacteria currently appear bacteriophage therapy, consists in the application of lytic bacteriophage, or only bacteriophage enzymes to inhibit bacterial growth. Thesis mentions the history of phage therapy, a crucial part of the thesis deals with a summary of current trends in bacteriophage therapy, beginning to develop in recent years. Many studies are dedicated to the possibilities of treatment of bacterial infections by phage lysates, including genetically modified bacteriophages and also application of bacteriophage enzymes themselves - endolysins, or a combination of the phage lysates and endolysins with antibiotics. The main interests in studies are the efficiency, specificity and safety of therapy. The effectiveness of bacteriophage therapy was already proved by many studies, both in vitro and in vivo. The safety of therapy for clinical usage needs to be prove by in vivo experiments. Key words: bacteriophage, bacteriophage therapy, endolysins, enzybiotics, multiresistence
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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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Differentiation in Saccharomyces cerevisiae colonies
Čáp, Michal ; Palková, Zdena (advisor) ; Pichová, Iva (referee) ; Demnerová, Kateřina (referee)
Yeast colonies are, alongside to bacterial biofilms, multicellular communities formed by unicellular microorganisms. These specific communities differ in many ways from populations cultivated in planktonic cultivations. Gradients of nutrients, metabolic by- products and other factors are formed and preserved within these structures, which provides a basis for cellular differentiation. Current literature concerning these issues with emphasis on yeast colonies and biofilms is summarised in the Introduction of this work. Section Results then describes my contribution to the knowledge on the differentiation of the colonies of Saccharomyces cerevisiae as a model system for studying microbial multicellular structures. Previously described horizontal differentiation, i.e. differentiation between colony centre and margin, is further characterised with respect to ammonia signalling and stress resistance. The importance of genes conferring the cell`s oxidative stress resistance in colony differentiation was studied and it was concluded that not stress resistance but rather metabolic and other changes promoted by ammonia signal are important for colony differentiation and survival. A new type of colony differentiation - the horizontal, i.e. differentiation between upper and lower part of the colony, is...
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CRISPR/Cas9-based genome editing in mice: state of the art and future perspectives
Eliáš, Jan ; Kašpárek, Petr (advisor) ; Čáp, Michal (referee)
Mutant mice are crucial tools for understanding gene functions in vivo. Recently, generation of mouse mutants was revolutionized by rapid developement of programmable nucleases, predominantly by the CRISPR/Cas9 system. Genome editing based on introduction of CRISPR/Cas9 components into early stage mouse embyros allows fast and inexpensive generation of gene-deficient animal models, especially when compared to the traditional techniques based on modification of embryonic stem cells (ESCs). The ability of CRISPR/Cas9 to induce double-strand break (DSB) at a given location of genomic DNA enables effective gene-ablation by random modification of the coding sequences or by complete ablation of the gene. However, precise modification of the gene sequences, such as incorporation of a DNA fragment into specific loci, are still difficult to make. In this work, I present a review of CRISPR/Cas9 system, its use in production of mutant mice and possible modifications of the system to increase the efficiency of precise gene-targeting. Keywords: CRISPR/Cas9, mouse, transgenesis, homologous recombination
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Translational control in immune response.
Hlaváček, Adam ; Valášek, Leoš (advisor) ; Čáp, Michal (referee)
Immune reaction often requires a prompt modification of gene expression that in turn alters cellular physiology. There are an increasing number of articles supporting a critical role of translational control in this aspect of cellular biology. The aim of this work is to present some of cellular and molecular mechanisms that connect translational control and immune reaction in immune and somatic cells and can be possibly misused by some viruses. Perhaps not surprisingly, many immunologically relevant translational control mechanisms are similar to those acting during the stress response. Over the years it has been documented that the T cells, dendrocytes, Natural killer cells and macrophages utilize translational control for their immunological activation following stimulation. Combination of general and gene-specific translational control mechanisms enables fast changes in proteome and physiology that are characteristic for immune cell activation. The overall impact of translational control on immune response is further illustrated by the fact that it acts upon each stage of life of immune cells - from their activation, through survival, to a programmed cell death. Even in some non-immune cells the translational control plays an important role with respect to immunity, as these cells are known to have an...
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Oxidative stress in bacteria - with an emphasis on the model organism of Escherichia coli
Moravcová, Andrea ; Lichá, Irena (advisor) ; Čáp, Michal (referee)
Most bacterial species encounter aerobic conditions during their life, which can be toxic. This leads to a state of oxidative stress. Toxicity of aerobic environment results from the chemical properties of molecular oxygen and its reactive species (ROS). Bacteria had to adapt to these conditions in the past to ensure preservation and prosperity. This thesis is preferably focused on oxidative stress adaptations in the most elaborated bacterial model - Escherichia coli. Regulation of adaptations at the regulation of transcription, translation and metabolism level are described with emphasis on molecular mechanisms. The main adaptation mechanism against oxidative stress is the deactivation of ROS, as well as the repair of damaged cell structures (macromolecules). These enzyme activities are regulated by several transcriptional regulators. The transcriptional regulators OxyR and SoxRS have been well studied in E. coli. Even though these regulators are conserved across the bacterial spectrum, they may not have the same function in all organisms. For this reason, also other, more or less studied bacterial species - Bacillus subtilis, Streptomyces coelicolor, Pseudomonas putida, Pseudomonas aeruginosa - were included in this thesis. The various strategies of how these bacteria use not only OxyR and SoxRS...
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