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The effect of Vps34p in yeast colony
Červenka, Jakub ; Schierová, Michaela (advisor) ; Převorovský, Martin (referee)
The phosphatidylinositol-3-kinase (PI3K) signalling pathway is evolutionarily conserved in all eukaryotes and its main function is the regulation of autophagy and protein sorting to the vacuole/lysosome. In the pathogenic yeast species Candida albicans and Cryptococcus neoformans the PI3K signalling pathway is required for virulence. In the yeast Saccharomyces cerevisiae the PI3K signalling pathway consists of two proteins - phosphatidylinositol-3-kinase, Vps34p and its regulator Vps15p. In this diploma thesis I analyse the role of the PI3K signalling pathway in the growth and development of colonies of natural and laboratory strains. I proved that VPS34 or VPS15 deletion in haploid laboratory strains has a significant influence on colony size and invasive growth (in strain ΣSh vps15Δ). Deletion of VPS34 or VPS15 also increases sensitivity of cells to oxidative stress and detergents. Attempts to delete VPS34 in natural strains were unsuccesful, probably because VPS34 is essential in these strains. Constitutive expression of VPS34 does not affect cell resistance in inhibitory tests, the size and differentiation of colonies or ammonia signalling but differences are notable in giant colony morphology and in patterns of invasiveness of the medium. Tagging of the C-terminal of Vps34p with GFP affects...
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Cell wall integrity signalling pathway and yeast colony morphology
Reslová, Gabriela ; Schierová, Michaela (advisor) ; Seydlová, Gabriela (referee)
In the yeast Saccharomyces cerevisiae, stress on the cell wall is caused by various external influences (e.g. exposure to chemicals, oxidative stress, osmotic changes, pH changes or heat shock) which trigger the cell wall integrity signalling pathway (CWI). The aim of my work was to investigate the effect of the CWI pathway on yeast colony morphogenesis. Using strains with deletions in genes of the CWI pathway derived from two parental strains BR-F-Flo11p-GFP and PORT, I have found that differences in genetic background influences the process and activation of this pathway. Among the strains derived from BR-F-Flo11p-GFP, only the strain with the deletion of MID2 affects the appearance of colonies. MID2 encodes a cell-surface sensor of CWI pathway. In all deletion strains derived from PORT, the disruption of the CWI pathway causes a slower development of colonies growing on glycerol medium supplemented with 0,05 mM selenate inducing fluffy colony morphology. The largest effect has deletion of gene MTL1 which also encodes a cell-surface sensor with homology to Mid2. I have confirmed that strains with deletions in genes of CWI pathway have altered sensitivity to inhibitors disrupting cell wall integrity (Calcofluor white, Congo red, zymolyase). By means of zymolyase assay, I have confirmed the...
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Phospholipid metabolism in the formation of structured yeast colonies
Pavlíčková, Martina ; Schierová, Michaela (advisor) ; Heidingsfeld, Olga (referee)
Yeasts in their natural environment form structured colonies. This allows them to better adapt to environmental conditions, but also to more easily resist various types of yeast infection inhibitors. The metabolism of phospholipids is closely related to the morphology of colonies. An important gene involved in phospholipid metabolism is INO1, which encodes inositol-3- phosphate synthase. Expression of the INO1 gene is regulated by the Opi1p negative transcription factor, which also affects a number of other genes for phospholipid metabolism enzymes, is also necessary for the expression of the FLO11 gene, encoding Flo11p, which is essential to the formation of a structured colony. The main aim of my work was to investigate the correlation between colony morphology of a natural strain of Saccharomyces cerevisiae and phospholipid metabolism. I have found that changes in INO1 gene expression and colony morphology are influenced by carbon source, selenate activity and the inhibitor of β-oxidation, 2-bromooctanoic acid. Although the INO1 gene is not essential for cell viability, its deletion or overexpression causes changes in phospholipid metabolism and colony morphology. Selenate and 2-bromooctanoic acid also alter expression of the FLO11 gene, which is reflected in colony structure. Thus, 2-...
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Phospholipid metabolism in the formation of structured yeast colonies
Pavlíčková, Martina ; Schierová, Michaela (advisor) ; Heidingsfeld, Olga (referee)
Yeasts in their natural environment form structured colonies. This allows them to better adapt to environmental conditions, but also to more easily resist various types of yeast infection inhibitors. The metabolism of phospholipids is closely related to the morphology of colonies. An important gene involved in phospholipid metabolism is INO1, which encodes inositol-3- phosphate synthase. Expression of the INO1 gene is regulated by the Opi1p negative transcription factor, which also affects a number of other genes for phospholipid metabolism enzymes, is also necessary for the expression of the FLO11 gene, encoding Flo11p, which is essential to the formation of a structured colony. The main aim of my work was to investigate the correlation between colony morphology of a natural strain of Saccharomyces cerevisiae and phospholipid metabolism. I have found that changes in INO1 gene expression and colony morphology are influenced by carbon source, selenate activity and the inhibitor of β-oxidation, 2-bromooctanoic acid. Although the INO1 gene is not essential for cell viability, its deletion or overexpression causes changes in phospholipid metabolism and colony morphology. Selenate and 2-bromooctanoic acid also alter expression of the FLO11 gene, which is reflected in colony structure. Thus, 2-...
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Cell wall integrity signalling pathway and yeast colony morphology
Reslová, Gabriela ; Schierová, Michaela (advisor) ; Seydlová, Gabriela (referee)
In the yeast Saccharomyces cerevisiae, stress on the cell wall is caused by various external influences (e.g. exposure to chemicals, oxidative stress, osmotic changes, pH changes or heat shock) which trigger the cell wall integrity signalling pathway (CWI). The aim of my work was to investigate the effect of the CWI pathway on yeast colony morphogenesis. Using strains with deletions in genes of the CWI pathway derived from two parental strains BR-F-Flo11p-GFP and PORT, I have found that differences in genetic background influences the process and activation of this pathway. Among the strains derived from BR-F-Flo11p-GFP, only the strain with the deletion of MID2 affects the appearance of colonies. MID2 encodes a cell-surface sensor of CWI pathway. In all deletion strains derived from PORT, the disruption of the CWI pathway causes a slower development of colonies growing on glycerol medium supplemented with 0,05 mM selenate inducing fluffy colony morphology. The largest effect has deletion of gene MTL1 which also encodes a cell-surface sensor with homology to Mid2. I have confirmed that strains with deletions in genes of CWI pathway have altered sensitivity to inhibitors disrupting cell wall integrity (Calcofluor white, Congo red, zymolyase). By means of zymolyase assay, I have confirmed the...
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The effect of Vps34p in yeast colony
Červenka, Jakub ; Schierová, Michaela (advisor) ; Převorovský, Martin (referee)
The phosphatidylinositol-3-kinase (PI3K) signalling pathway is evolutionarily conserved in all eukaryotes and its main function is the regulation of autophagy and protein sorting to the vacuole/lysosome. In the pathogenic yeast species Candida albicans and Cryptococcus neoformans the PI3K signalling pathway is required for virulence. In the yeast Saccharomyces cerevisiae the PI3K signalling pathway consists of two proteins - phosphatidylinositol-3-kinase, Vps34p and its regulator Vps15p. In this diploma thesis I analyse the role of the PI3K signalling pathway in the growth and development of colonies of natural and laboratory strains. I proved that VPS34 or VPS15 deletion in haploid laboratory strains has a significant influence on colony size and invasive growth (in strain ΣSh vps15Δ). Deletion of VPS34 or VPS15 also increases sensitivity of cells to oxidative stress and detergents. Attempts to delete VPS34 in natural strains were unsuccesful, probably because VPS34 is essential in these strains. Constitutive expression of VPS34 does not affect cell resistance in inhibitory tests, the size and differentiation of colonies or ammonia signalling but differences are notable in giant colony morphology and in patterns of invasiveness of the medium. Tagging of the C-terminal of Vps34p with GFP affects...
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