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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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Analysis of functional interaction between PKN3 kinase and CARMIL1 protein
Novotná, Petra ; Rösel, Daniel (advisor) ; Groušl, Tomáš (referee)
Cancer cell motility and cytoskeletal rearrangements are crucial for metastasis formation. These complex changes involve multiple cellular processes affected by many different proteins. One such protein is the Ser/Thr kinase PKN3. This kinase has been shown to be essential for metastasis formation in some aggressive types of breast and prostate cancer. Interestingly, the PKN3 kinase is not only important in malignant cancers but also in normal tissues. In endothelial cells, the PKN3 kinase can alter their adhesion, or in osteoclasts it helps to promote bone resorption. The effects of the PKN3 kinase on cancer malignancy and cell motility are well documented, but the mechanism behind these effects is still unclear. Therefore, our laboratory seeks to identify novel substrates and interaction partners of the PKN3 kinase. This work focuses on a novel potential substrate of the PKN3 kinase, CARMIL1. This protein is involved in actin cytoskeleton rearrangements by regulating actin polymerisation and thus cell motility. Here we provide evidence that the PKN3 kinase interacts with CARMIL1. Key words: PKN3, CARMIL1, actin cytoskeleton, cancer, invasion
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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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Biogenesis of plasma membrane Na+/H + antiporters of eukaryotic cells
Černá, Karolína ; Zimmermannová, Olga (advisor) ; Groušl, Tomáš (referee)
A secretory pathway is a process which serves for maturation of approximately one third of nascent polypeptides during proteosynthesis. Correct function of each membrane protein depends on their effective transport into its target membrane. An important group of proteins interacting with maturating proteins and ensuring their pass through the secretory pathway are cargo receptors. Cargo receptors help to insert nascent proteins into COPII vesicles via which they are exported from endoplasmic reticulum to Golgi apparatus. Erv14 belongs to characterized cargo receptors in yeast. Based on similarity with this cargo receptor several homologous cargo receptors were also identified in higher eukaryotes, including human. Na+ /H+ antiporters are among membrane proteins passing the secretory pathway and requiring a cargo receptor for their biogenesis. The aim of this work is to summarize knowledge of the role of Erv14 cargo receptor and its human homologs CNIH1-4 in biogenesis of monovalent cation transporters, including Na+ /H+ antiporters. Key words: Na+ /H+ antiporters, Erv14, CNIH, cargo receptors, secretory pathway
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Real-time monitoring of cellular processes - current approaches
Švecová, Iva ; Hodný, Zdeněk (advisor) ; Groušl, Tomáš (referee)
This thesis aims to provide an overview of real-time live-cell imaging methods with a focus on the signalling pathways. The first, most thorough section is about fluorescence methods and is followed by sections about bioluminescence and label-free methods. In the fluorescence section, we will at first introduce the types of fluorophores and respective labelling approaches. Subsequently, we will go through the individual techniques, starting with single-fluorophore and FRET biosensors, continuing with kinetic modelling approaches, a FLIM method used to detect changes in the cellular environment, and ending with two methods used to improve the resolution. With each technique, we will shortly explain the working principle and look at the examples at which this method was used. Finally, we will look at the example of live-cell imaging of one signalling cascade.
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Role of RACK1 in translation regulation during stress conditions
Chvalová, Věra ; Groušl, Tomáš (advisor) ; Převorovský, Martin (referee)
RACK1 (Receptor for activated C kinase 1) is an evolutionary conserved protein which has essential role in most studied eukaryotic organisms, except for yeast. Although RACK1 was originally described as a binding partner of protein kinase C, later studies re- vealed its significant role in other cellular signalizations such as MAPK, Src or FAK. Thanks to this, RACK1 participates in the regulation of key cellular processes including migration, apoptosis or translation. As a binding partner of a small ribosomal subunit, RACK1 contributes to transla- tion regulation by integrating signals from different cellular pathways and several transla- tional components such as PKC and eIF6. Moreover, RACK1 has a role in translation regu- lation during stress. Under stress conditions there is a global reduction of translation, in- creased expression of specific mRNAs important for cellular stress response and formation of cytosolic foci called stress granules (SGs). SGs play an important role in protection of mRNAs and translation components against degradation. SGs also function in prevention of apoptosis. RACK1 has been identified as one of many components of SGs and its localization into SGs leads to inhibition of RACK1-mediated pro-apoptotic pathways. Aim of this diploma thesis was to elucidate the role of...
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Counterbalances: antagonistic regulation of fission yeast growth and proliferation under favourable conditions and stress
Hohoš, Patrik ; Převorovský, Martin (advisor) ; Groušl, Tomáš (referee)
Microorganisms come across dramatically changing conditions in the environment. It is important for them to be agile for a quick and effective response. Signal transduction pathways are essential for this ability. They can sense a broad spectrum of extracellular and intracellular stimuli and regulate a great number of processes in the cell. For unicellular microorganisms, the most essential ability is to sense environmental conditions for proliferation or abnormal stress conditions. One of the most popular model microorganisms, the fission yeast Schizosaccharomyces pombe, is used for the signal transduction pathways research. Findings obtained by research on the fission yeast are applicable to other eukaryotic organisms, thanks to the high conservation of the signal transduction pathways between the fission yeast and other eukaryotic organisms. Proliferation-promoting signal transduction pathways promote cell proliferation, growth and mitotic cell cycle in fission yeast. The stress-response signal transduction pathways play an opposite role. They promote cellular defence against stress stimuli and promote the sexual differentiation process alongside meiotic cell cycle. At first sight, the whole machinery may look like a switch mechanism. There is, however, a more complex crosstalk mechanism...
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Heat-induced stress granules of Saccharomyces cerevisiae.
Groušl, Tomáš ; Hašek, Jiří (advisor) ; Janderová, Blanka (referee) ; Sychrová, Hana (referee)
(English) In response to environmental stresses, cells try to adapt to changed living conditions. Regulation of translation process provides fast-responding and versatile system enabling execution of stress-induced expression program. Messenger ribonucleoprotein complexes (mRNPs) engaged in translation and mRNA turnover are remodelled and may accumulate into higher-order assemblies, in connection to stress-induced translational changes. Stress granules (SGs) and processing bodies (PBs) are examples of such assemblies. Through them, further fate of mRNA molecules and certain translation machinery components is determined. In an effort to better understand the entire role of SGs in cellular metabolism, we performed the analysis of heat-induced SGs in model organism Saccharomyces cerevisiae. We contributed to the finding that SGs phenomenon is evolutionary conserved in eukaryotic kingdom proving that SGs are formed also in unicellular yeast S. cerevisiae under robust heat stress. The SGs reassemble their counterparts from higher eukaryotes in core composition and proposed functions. However, they possess also unique nature, which seems to be specific to the yeast. We further extended the data about heat-induced SGs, with a focus on additional composition, dynamics, associated proteins and a relation...
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Autophagy and other processes in colonies of natural yeast strains
Novosadová, Zuzana ; Palková, Zdena (advisor) ; Groušl, Tomáš (referee)
Abstract The yeast Saccharomyces cerevisiae on solid media forms multicellular colonies. Cells within colonies undergo differentiation and metabolic diversification, including formation of two layers of cells called Upper and Lower cells. The metabolic activity of U and L cells is different. For instance a higher level of autophagy was observed in U cells. This thesis includes a literature review of molecular mechanisms of autophagy in yeasts. Yeast colonies, under starvation conditions, produce volatile ammonia signal. This signal allows them communicate over a distance. Studies, revealing cell differentiation within colonies and ammonia signalling among colonies, were performed in colonies of laboratory strains. Strains isolated from nature, so-called wild strains, form distinct structured colonies, termed fluffy. Yeast within fluffy colonies also form different cell types. However the situation seems to be more complex that within smooth colonies of laboratory strains. Strains were constructed during this study, which express marker proteins Icl2p, Pox1p, Mae1p, Pma1p, Pma2p, Ino1p, Met17p and Atg8p fused with fluorescent labels in order to study cell differentiation and other processes within fluffy colonies. Furthermore, a new system for ...
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