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Role of yeast WSS1 protease in DNA repair.
Adámek, Michael ; Grantz Šašková, Klára (advisor) ; Čáp, Michal (referee)
Sustaining the integrity of DNA throughout the lifetime is critical for every living organism. Therefore organisms evolved numerous ways to detect and repair different types of DNA damage caused by various endogenous and exogenous factors resulting in replication stress. Defects in these repair mechanisms can lead to severe human diseases such as neurological disorders, familial cancers or developmental syndromes. In presented master thesis, we investigated the function of a yeast protein named Wss1, a metalloprotease that participates in a recently discovered DNA repair pathway that proteolytically removes DNA-protein crosslinks. Wss1 shows strong negative interaction with another DNA repair protease, Ddi1, in which case was discovered, that double-deleted yeast strain lacking WSS1 and DDI1 is hypersensitive to hydroxyurea. Hydroxurea is a ribonucleotide reductase inhibitor that, in the end, arrests cells in the S-phase of cell-cycle. Based on previous studies, we performed rescue experiments with various deletions and single-site mutants of Wss1p to assess the involvement of particular yeast Wss1p domains in the replication stress response to hudroxyurea.
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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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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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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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Reflection of Carl von Clausewitz's work in anglo-american military historiography
Cáp, Michal ; Šedivý, Ivan (advisor) ; Beneš, Zdeněk (referee)
This bachelor's thesis is concerned with reflection of Carl von Clausewitz's work in anglo-american military historiography. Carl von Clausewitz's work On war, is generally considered one of the most important and most well known books devoted to the problematic of armed conflict.. That is why it was chosen as a phenomenon, through which development and transformations of anglo-american military historiography is followed. In the first part of this theses, problematics of military history in general and legacy of Carl von Clausewitz in particular are described. In the second part gained knowledge is applied on historiographical survey.
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Characterization of DNA binding of CSL transcription factors in fission yeast
Jordáková, Anna ; Převorovský, Martin (advisor) ; Čáp, Michal (referee)
Cbf11 and Cbf12 proteins, the members of the CSL transcription factors family, are involved in a wide range of cellular processes in the fission yeast Schizosaccharomyces pombe - among other things they regulate cell adhesion and they have also been implicated in maintenance of genome integrity. At the level of the whole genome we previously identified target loci bound by CSL proteins in vivo. Many of them do not contain any consensus CSL-binding element. There are probably different DNA binding modes of the Cbf11/12 proteins and it has not been known what specific biological function is associated with the particular way of DNA binding. For the purpose of studying CSL DNA binding modes we have worked in this project on the implementation of the DNA binding mutation (DBM), which prevents direct DNA binding of CSL proteins to canonical motif in vitro, into the chromosomal locus of the cbf11 and cbf12 genes. Using the "ura4 selection system" we have successfully constructed the scar-less Cbf12-TAP and Cbf12DBM-TAP knock-ins, i.e. the strains without/with DBM in the open reading frame of Cbf12 where Cbf12 is C- terminally TAP-tagged and contains the intact 3'UTR. In our laboratory we have established the CRISPR/Cas9 system by which we have been able to prepare the Cbf11- TAP strain. We have failed to...
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The Role of DNA Repair in the Onset and Therapy of Ovarian Cancer
Tomášová, Kristýna ; Vodička, Pavel (advisor) ; Čáp, Michal (referee)
DNA repair and DNA damage response are very important biological systems, inevitable to maintain genomic stability and fidelity of the genetic information, for the onset of ovarian cancer. Further, DNA repair is also substantially involved in the response to the therapy, since many chemotherapeutics act as DNA damaging agents. This literary analysis is intended to survay the relevance of DNA repair to ovarian carcinogenesis. Special emphasis is placed on repair defects, as it is inextricably associated with the onset of cancer and treatment outcome. Apart from well-known alternations in ovarian cancer susceptibility genes, such as BRCA1 and BRCA2 involved in homologous recombination repair, ample space will be dedicated to less common gene mutations across different repair pathways. Research confirms that abnormalities in the proteins responsible for homologous recombination repair are the leading cause of ovarian cancer. The majority of authors also suggested that targeting DNA repair pathways, especially base excision repair, can improve chemotherapy efficiency in a synergic manner. The same applies to nucleotide excision repair, which repairs platinum-DNA adducts and thus contibutes to platinum drugs resistance emerging. By way of contrast, mismatch repair in ovarian cancer is rather poorly...
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Implementation of New Methods for Studying the Molecular Genetic Basis of the CADASIL Disease
Hrubá, Monika ; Vlášková, Hana (advisor) ; Čáp, Michal (referee)
CADASIL is a neurodegenerative autosomal dominant hereditary disease with late onset. Main symptoms are migraines with aura, cerebral ischemic events, cognitive impairment and dementia. The disease is caused by a mutation in the NOTCH3 gene. The major mutation type changes the number of cysteine residues in the EGF-like repeats of the Notch3 protein. In Czech Republic, currently used methods for molecular genetic analysis of the CADASIL disease are Sanger sequencing and MLPA. But there are patients with CADASIL-like symptoms who were not confirmed by these methods. Therefore, the aim of this thesis was to implement transcript analysis by Sanger sequencing of cDNA PCR products and quantitative real-time PCR (qPCR) to analyze gross deletions and duplications to clarify the molecular genetic basis of the disease. By transcript analysis, the existence of the transcript variant X1 was experimentally confirmed in control samples. Moreover, the results from transcript analysis showed that non-typical missense mutation c.1725G>A (p.T575=) which does not directly change the number of cysteine residues, can cause the CADASIL disease via missplicing and subsequent causing deletion including cysteine residues. The other tested variants did not show any changes in the transcript level. The qPCR method did not...
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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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