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Linking transcriptional regulation of lipid metabolism with catastrophic mitosis in fission yeast
Marešová, Anna ; Převorovský, Martin (advisor) ; Čáp, Michal (referee) ; Veverka, Václav (referee)
Linking regulation of lipid metabolism with nuclear division has recently become a thoroughly investigated phenomenon. It has been shown that an undisturbed lipid metabolism is required for proper mitotic division from yeasts to mammals. In my doctoral project, I focused on the transcriptional regulation of lipid metabolism genes with respect to mitotic fidelity in the fission yeast Schizosaccharomyces pombe. Cbf11 and Mga2 are two transcriptional regulators of lipid metabolism genes. For a long time, it was assumed that they act as two independently functioning units. However, we have identified a novel regulatory partnership between Cbf11 and Mga2. Our results show that Cbf11 and Mga2 work closely together. In addition to regulating lipid metabolism, Cbf11 was known to be involved in cell cycle progression and a range of other processes. We have now determined that all known functions of Cbf11 require its canonical DNA-binding ability. Interestingly, we have identified novel roles for both Cbf11 and Mga2 and found that Mga2 mirrors the functions of Cbf11, including cell cycle control. Furthermore, we have shown that lipid metabolism has a pronounced impact on chromatin structure and the proper regulation of gene expression, and we have suggested how fatty acid synthesis contributes to mitotic...
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Feasibility Study of Benefits of Étoilé User Environment
Čáp, Michal ; Zemčík, Pavel (referee) ; Eysselt, Miloš (advisor)
Tato práce popisuje cíle projektu Étoilé a analyzuje jejich proveditelnost. Étoilé je open-source projekt, který se snaží odstranit zastaralé koncepty ovládání počítače, často stále používané v současných operačních systémech jako je Microsoft Windows nebo Mac OS X. Práce obsahuje tyto části: popis koncept uživatelského rozhraní navržených v rámci projektu Étoilé ztvárnění fiktivního prostředí, které ukazuje možnou budoucí podobu projektu Étoilé rozbor úkonů prováděných při práci v tomto fiktivním prostředí - psaní dopisu a kolaborativní úpravy dokumentu průzkum, jak uživatelé přijímají tyto nové koncepty stručný popis systémové architektury projektu Étoilé odhad náročnosti implementace projektu
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Characterization of Cbf11 and Mga2 interactions in the fission yeast
Grulyová, Michaela ; Převorovský, Martin (advisor) ; Čáp, Michal (referee)
Transcription factor Cbf11 belongs to the CSL protein family. The CSL protein family is well known for its function in Notch signalling pathway, however representatives in Notch- less fission yeast were discovered. Mga2 protein is a transcription regulator of triacylglycerol and glycerophospholipid metabolism. A crosstalk between Cbf11 and Mga2 was found. Cbf11 and Mga2 share target genes, and both are required for mitotic fidelity. This thesis aims to validate and characterize relationship between these transcription regulators. We show here that protein levels of Cbf11 and Mga2 change in response to presence of the other protein, as well as in response of nitrogen source. We also determine phylogenetic distribution of Cbf11 and Mga2 among Fungi, pointing to its connection. Using proteomic analysis of mga2 and cbf11 deletion strains we found that there is an overlap between proteins up/downregulated in these strains. Together, these results acknowledge crosstalk between Cbf11 and Mga2 proteins, bringing a novel connection between CSL protein family member and a functional analogue of mammalian SREBP-1 protein Mga2.
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Searching for interaction partners associated with the transcriptional apparatus of yeast linear plasmids
Ľalíková, Kristýna ; Vopálenský, Václav (advisor) ; Čáp, Michal (referee)
The pGKL plasmids are a type of yeast linear double-stranded DNA plasmids found in the cytoplasm of yeast Kluyveromyces lactis. The plasmid gene transcription process involves the plasmid transcription apparatus, which shows similarity to the transcription apparatus of poxviruses. Poxviruses cause a range of serious diseases. Thus pGKL plasmids offer a safe way to study some of the mechanisms of these viruses without the risk of infection. The plasmid transcription apparatus comprises non-canonical RNA polymerase, helicase, and capping enzyme as its main components. Although RNA polymerase has been thoroughly characterized and described, little focus has been on the interactions between proteins within the transcription apparatus. Therefore, this study aims to shed light on this fundamental aspect of the gene expression of pGKL plasmids. The main goal of the thesis was to prepare a system that would facilitate the search for interaction partners of the pGKL transcription apparatus and verify the interactions between chosen proteins in yeast cells. The foundation of this system relies on the use of optimized genes and the yeast two-hybrid system method. The development of the experimental method has begun successfully, and it's expected to conclude soon, although further optimizations are still...
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The impact of nucleophosmin gene mutations on its interaction potential
Šašinková, Markéta ; Brodská, Barbora (advisor) ; Čáp, Michal (referee) ; Starková, Júlia (referee)
Nucleophosmin 1 (NPM1) is predominantly localized in the nucleolus and occurs mainly in oligomers formed through its N-terminal domain (NTD). As a transport facilitator and chaperone, NPM1 has a wide range of interacting partners including tumor suppressors p53 and p14Arf. Characteristic C-terminal mutations in NPM1 are reported in approximately 30 % of acute myeloid leukemia (AML) cases and cause aberrant cytoplasmic localization of mutated (mut) NPM1. As a result, many NPM1-interacting proteins, including wild type (wt) NPM1, are relocalized to the cytoplasm. In order to analyze interactions and the oligomeric state of NPM1, we have introduced and optimized several in vitro techniques - native and semi-native polyacrylamide gel electrophoresis and immunoprecipitation - as well as in vivo confocal microscopy and time-resolved fluorescence approaches. Using these methods, we revealed that mutations at the C-terminal domain of NPM1 prevent it from binding nucleolar protein nucleolin (NCL), which has previously been shown to interact with the central part of NPM1, and that drug-induced relocation of mutNPM1 to close proximity of NCL does not induce mutNPM1-NCL complex formation. We proved a lowered stability of mutNPM1-formed oligomers as compared to the wtNPM1 ones, which could be useful for NPM1...
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Role of Ccr4 deadenylase in cell cycle regulation in yeast colonies
Daumová, Lenka ; Čáp, Michal (advisor) ; Převorovský, Martin (referee)
Regulation of cellular processes is of key importance for survival of cells. Many regulations are mediated by the CCR4-Not complex, a highly conserved protein complex, which is present in eucaryotic cells, from yeast to mammals. In this work I study mostly the role of the Ccr4 subunit on yeast survival in the aging colony of Saccharomyces cerevisiae, mainly during cell cycle progression. Ccr4 si a deadenylase, and its main function is cleavage of poly(A) tail of mRNA molecules, and by doing so, shortening the mRNA life-time. It is very important during strictly regulated cellular processes, because it is essential that gene expression of specific genes happens only at specific time. Saccharomyces cerevisiae BY 4742 is a haploid yeast strain, which can easily be used for making deletion mutants in specific genes. In this diploma thesis I focus on studying a deletion mutant of CCR4, along with deletion of other genes, which influence yeast cell cycle. By comparing the phenotype of these mutants with wild type, it is possible to identify changes in phenotype, caused by these deletions, and their influence on yeast cell survival. Key words: cell processes regulation, CCR4-Not complex, Ccr4 subunit, cell cycle, yeast, Saccharomyces cerevisiae, gene deletion
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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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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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