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Analysis of different forms of autophagy in differentiated cells of yeast colonies
Nejedlý, Adam ; Palková, Zdena (advisor) ; Groušl, Tomáš (referee)
The yeast Saccharomyces cerevisiae forms differentiated colonies on solid agar medium that resemble simple multicellular organisms. Similarities with the functioning of higher organisms can also be observed and the structures of some pathways may be analogous to the interaction of cancer cells with healthy cells in human tissues. Along with chronological aging and gradual depletion of nutrients in the medium, growing yeast colonies differentiate into several cell types, especially the so-called U (Upper) and L (Lower) cells. The two cell types differ in morphology and metabolism, U cells being characterized by the presence of several vacuoles, a lower level of respiration, synthesis of storage substances and greater resistance to stress. L cells, on the other hand, usually contain one large vacuole, respiring mitochondria and have increased activity of some degradative pathways. They are also less resistant to stress and have a lower life span. In an aging colony, the lower L cells provide material and nutrients to the upper U cells, which thus survive longer periods of starvation. One important process that occurs especially in the more viable U cells is autophagy. This is a pathway designed to degrade a number of damaged or unnecessary cellular components in the vacuole. The aim of this thesis...
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Antagonistic regulation by global transcription factors Tup1p, and Cyc8p of Flo11 and Flo11 -dependent phenotypes in wild yeast
Nguyen Van, Phu ; Palková, Zdena (advisor) ; Heidingsfeld, Olga (referee) ; Malcová, Ivana (referee)
Biofilms are a common mode of yeast growth in which cells adhere to each other and adhere to abiotic surfaces to form complex multicellular structures. Living together in biofilms provides cells with several beneficial features compared to planktonic cells. Undoubtedly, protection and resistance are advantages of life inside colony biofilms. Biofilms are found in many environments and play many important roles in commercial industries. However, biofilms can also be extremely dangerous in clinical settings. There is thus great interest in studying biofilms and how to eliminate them. In this study, we used wild yeast Saccharomyces cerevisiae colony biofilm as an ideal system to investigate potential functions of the yeast Cyc8-Tup1 transcriptional corepressor complex in the regulation of yeast adhesion, and biofilm formation on agar and at solid-liquid interfaces. Unexpectedly, we have found that Cyc8p and Tup1p antagonistically control the formation of structured biofilm colonies on agar and FLO11 expression. Cyc8p itself acts as a key repressor of FLO11, whereas Tup1p promotes the formation of biofilm colonies and induces FLO11 expression by inhibiting the repressive function of Cyc8p and preventing Flo11p degradation possibly by inhibiting an extracellular protease. In addition, other features...
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Factors influencing cell adhesion and the formation of yeast biofilms on solid surfaces
Světlíková, Daniela ; Palková, Zdena (advisor) ; Groušl, Tomáš (referee)
Yeasts are unicellular microorganisms that naturally aggregate to form multicellular structures. One of these formations is biofilm. The key properties of biofilm in S. cerevisiae are determined by the expression of the FLO11 gene. The large FLO11 gene promoter is the target of several pathways and factors, resulting in the complex regulation of this gene. Among the regulators of the FLO11 gene are the conserved Cyc8p and Tup1p proteins, which play a role in the regulation of many processes, especially as a corepressor complex. In previous years, our laboratory has demonstrated an antagonistic effect of the Cyc8p a Tup1p regulators on adhesion of BRF strain (Nguyen et al., 2018). In my work, I investigated whether this regulation is also valid in the selected clinical isolate S. cerevisiae YJM320 and whether, as in the BRF strain, positive regulation of FLO11 via Tup1 and negative regulation via Cyc8 exist here or whether their effect is different in this strain. The broader goal of my work was to identify the role of various factors on adhesion as the first step in biofilm formation. My work began by testing the effect of nutrients and temperature on the adhesion of strains BY, BRS and BRF and continued by testing the adhesion of selected clinical isolates of Saccharomyces cerevisiae and Candida...
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Expression of genes involved in chronogical ageing in yeast multicellular structures.
Fedorová, Viktória ; Palková, Zdena (advisor) ; Groušl, Tomáš (referee)
The yeast Saccharomyces cerevisiae forms structured multicellular populations that undergo several growth phases during growth depending on the amount of nutrients present in the medium. The last growth phase is called chronological ageing, during which cell division is minimal and physiologically and metabolically distinct subpopulations are formed. The aim of this work is to describe the expression and cellular localization of selected genes that may serve as marker genes specifically expressed during the different growth phases of yeast multicellular populations in fermentative and respiratory media. The selected genes were tagged with the GFP gene using the transformation method and stran fluorescence subsequently analyzed during growth in liquid media. Strains with specific expression, localization, or significant differences in expression between the 2 carbon sources were analyzed by Western blots and during growth on solid media as microcolonies. Based on the amount of protein expressed in the cells, 5 genes were selected that were specifically expressed during the exponential and stationary growth phases, and their expression or localization differed significantly during growth on fermentative and respiratory media. Key words: yeast, growth phases, gene expression, fermentation, respiration
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Analysis of metabolites produced by yeast colonies and biofilms
Karásek, Filip ; Palková, Zdena (advisor) ; Dostál, Jiří (referee)
Some time ago it was shown (proved, described) that bacteria can communicate and regulate a gene expression by using small molecules. This phenomenon is called quorum sensing. The same phenomenon has been recently described in yeast. In yeast there are still many questions about the mechanism of sensing via small molecules. Few works and publications have described the impact of some small molecules, such as phenylethylalcohol or tryptophol, on morphology on Saccharomyces cerevisiae cell. The knowledge about the impact of the small molecules on the yeast multicellular community, like colonies, is still very limited these days. The aims of this work are analysis of exometabolites produced by colonies of different yeast strains cultivated on various solid complex media, by an untarged metabolomic approach. In accordance with the result of the exometabolites analyses we are going to test the impact of the selected substance on Saccharomyces cerevisiae colony morphology. The third part of this work is about the construction of Saccharomyces cerevisiae strains with deletion or lowering the expression of genes which encode proteins that are important in decarboxylation step of the Ehrlich pathway. Key word: Saccharomyces cerevisiae, microcolonies, tryptophol, isoamyl alcohol, isovaleric acid, colony...
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Development and differentiation of different types of yeast colonies: Regulation of metabolic diversification and development of cells with novel properties
Maršíková, Jana ; Palková, Zdena (advisor) ; Heidingsfeld, Olga (referee) ; Demnerová, Kateřina (referee)
Yeasts are unicellular organisms, but on a solid substrate they are capable of forming complex organized structures that behave like primitive multicellular organisms. Examples of these structures include colonies and biofilms, whose cells interact with each other, coordinate their growth and development, differentiate spatially and form specialized cell subpopulations in which specific processes and regulatory pathways occur. The basis of cellular differentiation and specialization is the formation of gradients of nutrients, metabolites and signaling molecules. Thus, multicellular yeast communities differ significantly from planktonic populations in their characteristics. The aim of this work is to increase knowledge related to the development and differentiation of both smooth and structured colonies of the yeast Saccharomyces cerevisiae. The literature introduction of the thesis provides an overview of the current knowledge on the development of yeast colonies and biofilms, especially of S. cerevisiae species, and also includes selected regulations important for the formation of multicellular populations. The thesis provides insights into the antagonistic function of the transcriptional regulators Cyc8p and Tup1p in the development of structured biofilm colonies. Genome-wide transcriptomic...
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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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Ammmonium transport in yeast
Faltýnková, Kateřina ; Palková, Zdena (advisor) ; Princová, Jarmila (referee)
Bachelor thesis - Kateřina Faltýnková - Ammmonium transport in yeast Ammonium and ammonia are an essential nutrient for every yeast cells, not only in metabolism, for example in amino acid synthesis, but also as signalling molecules that serve for communication between colonies or for the regulation of pseudohyphal growth. Transport of ammonia and ammonium ions requires active transport, which is provided by MEP permeases inside the cell likely by exporters ATO proteins out of the cell. In this work there are described families of genes MEP and ATO with main focus on their importance for uptake and export of ammonium ions by yeast and also the regulation of these two gene families in the yeast Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans a Yarrowia lipolytica.
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Comparison of specific expression in bacterial and yeast biofilms
Kicko, Peter ; Palková, Zdena (advisor) ; Lichá, Irena (referee)
The development and maintenance of biofilm is a complex process that is based on a change in genetic expression. The biofilm formation is observed in some prokaryotic and eukaryotic cells. During its formation, cell aggregation occurs, extracellular matrix is created and we observe the formation of metabolically differentiable cells, often with increased resistance to antimicrobial drugs. This work focuses on important steps leading to biofilm formation associated with specific gene expression and highlights the similar and different processes between bacterial and yeast cells. The work begins by comparison of cell signalling, it continues by comparing the expression of the adhesive proteins and extracellular enzymes, synthesis of exopolysaccharides, formation of extracellular nucleic acid, and in the last chapter we focused on the formation of persistors. The aim of this work is to connect the acquired information and to contribute to the understanding the complexity of this process. Key words: biofilm, signalling, adhesins, exopolysaccharides, extracellular nucleic acid, persistor
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