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Vacuolar proteins in development of yeast colonies
Trubitsyna, Yana ; Palková, Zdena (advisor) ; Heidingsfeld, Olga (referee)
The laboratory strains of yeast Saccharomyces Cerevisiae form colonies which can differentiate into two main cell subpopulations. U and L cells demonstrate different morphology, metabolism and stress-resistance. It was also proved that some of metabolic pathways in U cells are a similar to ones in tumor cells. The unique metabolism is activated in U cells; the TORC1 is active in these cells together with autophagy and glycogen accumulation, which are characteristic for cells with inactivated TORC1. CORVET and HOPS complexes together with vacuolar ATPase are involved in processes related to vacuolar fusion and trafficking. Also, these complexes contribute to the regulation of TORC1 activity. Vam6p is a subunit of HOPS complex and it is also involved in regulation of TORC1 acting as GEF for Gtr1p GTPase, which activates TORC1. The aim of this study was to outline whether selected subunits of mentioned complexes affect TORC1 activity in U cells. Further aim was to confirm the effect of Vam6p on selected proteins production. These proteins were chosen based on results of proteomic analysis performed in our laboratory. In order to investigate possible effects of proteins of interest absence on colonies' morphology, strains deleted in selected genes were prepared (VPS3, VPS8, VPS33, VPS41, VPH2, VAC7 a...
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Ammonia production by colonies of mutants and aging of wrinkled colonies of Saccharomyces cerevisiae
Nedbálková, Jana ; Janderová, Blanka (advisor) ; Heidingsfeld, Olga (referee)
Production of ammonia by the colonies of mutants and aging of wrinkled colonies of Saccharomyces cerevisiae The aim of this diploma thesis is to observe the development, respectively the aging of cells in yeast colonies Saccharomyces cerevisiae. Yeast cells S. cerevisiea form multicellular organized structures on a solid substrate, i.e. colonies, which the intercellular interactions occur in. These interactions influence forming, morphology and aging of yeast colonies. This diploma thesis is focused partly on the changes in ammonia production by giant colonies of deletion mutants and partly on the aging of colonies with the wrinkled morphology. I characterized mutant strains of S. cerevisiae with the deletion in RTG1, RTG2, RTG3, FIS1, CIT2 genes. Their products play an important role in the colony development. The transcription of these genes changes during the transition from the acidic to alkali phase during developmental process of the colonies. I have found out that the ammonium production rate was in accordance with the results of the alkalization in giant colonies surroundings and mentioned mutants derived from the BY strain has been producing ammonia since the 15th day. The rate of the ammonia production by rtg3∆ strain was comparable to the parental strain. Compared to parental strain, lower...
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CUG Codon in Pathogenic Yeasts of the Genus Candida
Marečková, Lucie ; Heidingsfeld, Olga (advisor) ; Půta, František (referee)
2. Abstract In many Candida species the standard leucine CUG codon is translated as a serine, although not in 100% cases. This dual specifity of the CUG codon has evolved through a mechanism that required codon ambiguity mediated by a unique tRNACAG, which is in vitro aminoacylated more often by serine than by leucine. This codon ambiguity has been tolerated for more than 170 million years. The explanation at least for now is that the CUG codon reassignment could have generated genetic diversity that facilitated occurrence of new phenotypes resistant to stress. Beside this, an important step was to reduce negative impact of the codon ambiguity by crucial mutations in the structure of the ser-tRNACAG. Candida species became a valuable experimental model for elucidation of the genetic code changes. While consequences of the CUG codon reassignment have been extensively studied in Candida albicans, this topic has not yet been addressed in Candida parapsilosis. Solving the structure of C. parapsilosis secreted proteinase Sapp1p provided a tool to carry out a "case study" of possible effects of the CUG codon ambiguity. The SAPP1 gene contains one CUG codon, and the respective serine is located on the loop in the close proximity of the active site of the proteinase.
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Antifungal Drug Discovery: Focus on Incrustoporin Derivatives
Silva, Luis André Vale ; Buchta, Vladimír (advisor) ; Heidingsfeld, Olga (referee) ; Hamal, Petr (referee)
1 CONCLUSIONS In the context of Medical Mycology today, the development of new more effective antifungal agents is a priority. In fact, there are still only about six drugs in use to treat invasive fungal infections, concretely the polyene amphotericin B (including its new lipidic formulations), the antimetabolite flucytosine, the triazoles fluconazole, itraconazole, and voriconazole, and the echinocandin caspofungin. Simultaneously, the incidence of invasive opportunistic mycoses has been increasing steadily with the increasing number of immunocompromised patients, caused both by HIV (human immunodeficiency virus) infection and AIDS and the development of medical techniques, particularly referring to oncology or transplant patients. In this setting, the relevance of the development of new antifungal agents and, hence, our work presented here, is easily understandable. The group of the acyloxymethylated incrustoporin derivatives is now in a higher stage of development, after being previously studied concerning structure-antifungal activity relationship and tuning of antifungal activity. Our work has shown the best derivatives from the group, compounds LNO6-22, LNO15-22, and LNO18-22, to have broad spectrum in vitro antifungal activity and high potency, inhibiting growth of a variety of pathogenic yeasts and...
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Transcription factors CSL and their role in the yeast Schizosaccharomyces pombe
Oravcová, Martina ; Převorovský, Martin (advisor) ; Heidingsfeld, Olga (referee) ; Krásný, Libor (referee)
Proteins of the CSL family (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) act as effectors of the Notch signalling pathway in metazoan organisms. They function as repressors or activators of gene transcription in the framework of this pathway and influence many developmental processes. Metazoan CSL proteins can regulate gene expression Notch-independently as well. Notch-independent functions of CSL proteins might be evolutionarily ancestral and in cells and organisms may be important equally as Notch-dependent functions. Presence of CSL proteins was identified in several fungal species, organisms lacking the Notch signalling pathway components and most of known metazoan interacting partners of CSL proteins. CSL paralogs of the fission yeast Schizosaccharomyces pombe, cbf11 and cbf12, are non-essential genes encoding proteins localized in the nucleus of the cell. They exert antagonistic effects on regulation of processes like coordination of nuclear and cellular division and cell cycle progression, ploidy maintenance, cell adhesion and other. In this study, we have proved that both CSL paralogs are able to sequence-specifically bind the CSL-response element DNA in vitro and Cbf11 in vivo as well. Both proteins could activate gene expression in vivo and perform the function of transcription factors....
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Formation of protein granules in differentiated cells of yeast colonies
Kočířová, Eliška ; Palková, Zdena (advisor) ; Heidingsfeld, Olga (referee)
Saccharomyces cerevisiae is a unicellular eukaryotic organism capable of forming organized multicellular communities - colonies and biofilms. During development, colonies of laboratory strains differentiate into specifically localized cell subpopulations - U and L cells, located in the upper and lower part of the colony, respectively. The U and L subpopulations of cells vary in morphology, metabolic processes and stress resistance. Protein granules are membrane-less "organelles" found in both unicellular and multicellular eukaryotic organisms. The formation of protein granules is related to the physiological state of the cell (e.g. chronological and replicative aging), but also to changing environmental conditions and to cellular responses to stress factors. A relatively large fraction of proteins relocalizes to some type of protein granule during the lifespan of the cell. Granule formation can increase fitness of cells, help them to cope with limiting energy resources, and plays a crucial role in the adaptation of cells to stress conditions. Localization of many proteins in the cell varies depending on its physiology. Therefore the specific localization of such proteins may be considered as a "marker" of a specific physiological condition. There are proteins in each type of granule that can be...
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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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Alakali-metal-cation homeostasis in pathogenic Candida species
Elicharová, Hana ; Sychrová, Hana (advisor) ; Heidingsfeld, Olga (referee) ; Půta, František (referee)
Several tens of Candida species belong to the opportunistic human pathogens capable of inducing life-threatening infections in immunocompromised patients. Virulence of single Candida species depends among others on their resistance to the variable external conditions. The maintenance of alkali-metal-cation homeostasis, which means the ability to accumulate sufficient amount of potassium cations and on the other hand to survive under high extracellular concentrations of alkali-metal cations, is essential for growth and virulence of Candida cells. We observed the negative effect of fluconazole (FLC) on salt-tolerance of six Candida species and found that it is independent of the species level of FLC- resistance. FLC hyperpolarizes plasma membrane of Candida cells and therefore increases non-specific uptake of alkali-metal cations which results in strongly increased salt-sensitivity of Candida cells. The FLC-induced hyperpolarization also results in an increased sensitivity of Candida cells to the antifungals which are positively charged and are driven into the cells by the membrane potential. The effect of fluconazole on membrane potential and thus on the uptake of alkali- metal cations into the cell turned our attention to the homeostasis of potassium cations whose high intracellular concentration is...
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Participation of Selected Carbonyl Reductases in Deactivation of Anticancer Drugs
Odiana, Romana ; Wsól, Vladimír (advisor) ; Szotáková, Barbora (referee) ; Heidingsfeld, Olga (referee)
Reduction is the reverse of oxidation and therefore it can involve loss of oxygen atom or the addition of two hydrogen atoms. The reduction of carbonyl groups in xenobiotics was the main topic of this thesis. We tried to identify and characterize human carbonyl reductases responsible for anticancer drugs deactivation. When cancer is among the most common death causes in the developed world, it is necessary to look for new and efficient ways of its treatment. Inhibition of enzymes, which may contribute to disease development or relapses and/or treatment efficacy decrease by drug inactivation, could be a possible way of treatment improvement and might also lead to decrease of drug doses and side effects of cytostatics. In the first part of our project, we focused on a soluble cytosolic reductase AKR1C3. This enzyme is involved in sex hormone metabolism and might play an important role in breast and prostate cancer development. We tested its ability to metabolize anticancer drugs by its incubation with oracin and doxorubicin with subsequent metabolite determination with use of HPLC. Our experiment proved that it can deactivate these two drugs with Km 355 μM for doxorubicin and 110 μM for oracin, respectively. AKR1C3 can therefore influence the anticancer therapy, expecially when overexpressed. The...
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