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Membrane microdomains in regulation of lipid metabolism
Veselá, Petra ; Malínský, Jan (advisor) ; Hašek, Jiří (referee) ; Zimmermannová, Olga (referee)
The fluid mosaic model described by Singer and Nicolson in 1972 was timeless and remains relevant to understanding the structure, function and dynamics of biological membranes more than fifty years later. From the outset, its authors acknowledged the existence of lateral membrane regions that differ in composition and biological function from their immediate surroundings. Therefore, even contemporary studies demonstrating the existence of many different membrane microdomains do not pose a fundamental challenge to the validity of this model. In particular, research over the last twenty years has shown that a number of cellular processes (nutrient transport, signaling, regulation of nucleic acid metabolism, lipophagy and many others) are linked to membrane microdomains. However, the molecular details of these links remain hidden in many cases. The aim of this work is to find specific links between membrane microdomains and the metabolism of selected lipids. Using the yeast model S. cerevisiae, we document the connection of a specialized plasma membrane microdomain, the membrane compartment of arginine permease Can1 (MCC), to the metabolism of sphingolipids and the mitochondrial anionic phospholipids, phosphatidylglycerol (PG) and cardiolipin (CL). The initial two chapters deal with the elucidation of...
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Yeasts as a tool for study of cation homeostasis in eukaryotic cells
Farbulová, Michaela ; Zimmermannová, Olga (advisor) ; Zahumenský, Jakub (referee)
One of the key abilities of cells is to maintain a balance in the internal environment when the external surroundings change. Among the cations that have an irreplaceable role in living organisms are K+ , Na+ and H+ . In yeasts, most proteins transporting monovalent cations across cell membranes have been identified, making them an ideal model organism for studying cellular physiological processes in eukaryotic cells, including the principles of maintaining monovalent cation homeostasis. Using a unicellular organism such as yeast, we can also characterise proteins from phylogenetically higher organisms and humans, thus revealing disorders of transport systems related to pathological diseases. This bachelor thesis aims to point out the importance of yeasts in the research of the structure and function of cation transporters from eukaryotic cells and, at the same time, summarise knowledge about diseases associated with the disruption of the functions of individual cation transporters.
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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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The role of stress granules and 4E-BP in heat-stressed cells of S. cerevisiae
Kolářová, Věra ; Hašek, Jiří (advisor) ; Zimmermannová, Olga (referee)
The cells are capable of very quick and specific reactions on stress conditions. Influence of translation, specifically initiation of translation by inhibition factors, is one of the main regulatory process. Two of eIF4E-binding proteins (4E-BP), Eap1p and Caf20p, are known as cap-dependent translation repressors in yeast Saccharomyces cerevisiae. We used in vivo fluorescent microscopy analysis to show different reaction of Caf20p and Eap1p to heat stress. Protein Caf20p does not react on heat shock and stays difused in cytoplasm. Contrary to Caf20p reaction, protein Eap1p accumulates in cytoplasm close to stress granules (SGs). This work shows that Eap1p is involved in stress granules assembly. In the absence of Eap1p, yeast cells react to the heat stress with small and less focused SGs. Dele- tion of CAF20 does not affect SG assembly. This points to specific function of SG in distribution of factors connected with stress reaction. Polysomal analysis shows that deletion of one of initiation translation repressors does not affect heat induced global repression of translation. In permisive condition deletion of EAP1 may cause defect in addition of 60S ribosomal subunits. Absence of protein Eap1p causes morphological defect. That point to a different reactions of Eap1p and Caf20p on heat stress and possible...
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Dynamics of yeast colony differentiation: New approaches
Jurečková, Gabriela ; Palková, Zdena (advisor) ; Zimmermannová, Olga (referee)
The yeast Saccharomyces cerevisiae when growing on solid medium forms structured colonies. During its development, two subpopulations of cells are formed, termed as U cells ("upper cells") and L cells ("lower cells"). This nomenclature derives from their position within the colony. These cells differ from each other considering their morphology, metabolism, physiology and are characterized by varying degrees of resistance to stress. This diploma thesis deals with new methodological approaches that can be used in further research of yeast differentiation. The first part deals with yeast differentiation and de-differentiation. In the experimental part, the development of mechanically mixed yeast colonies was monitored. The development was monitored in situ in the colony section using fluorescence and "wide-field" microscopy. The ability to de-differentiate of already specialized cells was evaluated mainly according to the nature of the expression of the fluorescently labeled protein Ato1p, which serves as a protein marker of U cells. The second part deals with the optogenetic system EL222 and its possible use in yeast. Many optogenetic tools have been described to control various cellular processes using light. One of these optogenetic tools is the EL222 system, which allows the induction of...
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Mechanism of uric acid transport in intestinal epithelium
Vrzáčková, Aneta ; Vávra, Jiří (advisor) ; Zimmermannová, Olga (referee)
Uric acid is main metabolite from purine degradation pathway in humans a higher primates. Serum uric acid level is controlled on one side by absorbtion and on the other side by excretion. This proceses run in kidney nephrone and in intestinal epithelium. This thesis will be summarize the newest knowledges about intestinal epithelium cells transport protein, which excreted or reabsorbed uric acid from lumen of intestine. The thesis will be contain informatin about the transport protein family, protein - substrate interaction, regulation and it's importance in other tissue. Possibilities of regulation the urate handling system in intestinal epithelial, physiological and pathological importance will be contained in this work too. Key words: Uric acid, enterocyte, transport protein, epithelial cell
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Changes in domain organization of the plasma membrane in the stress response
Vaškovičová, Katarína ; Malínský, Jan (advisor) ; Zimmermannová, Olga (referee) ; Cvačková, Zuzana (referee)
MCC/eisosomes are yeast plasma membrane microdomains that respond to changes in both extracellular and intracellular conditions and activate important stress-related signaling pathways. In this study, we investigated the function of MCC/eisosomes under the conditions of chronic glucose depletion. We found that MCC/eisosomes regulate mRNA decay under these conditions. Specifically, we demonstrated that the sequestration of the evolutionarily conserved Xrn1 exoribonuclease at MCC/eisosomes leads to the attenuation of its enzymatic activity. Modulation of activity by the enzyme localization may represent a novel and effective mechanism in regulation of biochemical pathways. Moreover, our results suggested that an MCC protein Nce102 might play a role in vacuolar fusion and lipid droplets degradation. We demonstrated that prolonged chronic glucose depletion induces the translocation of Nce102 from MCC to sterol-enriched microdomains in the vacuolar membrane. Deletion mutants lacking Nce102 and its functional homologue Fhn1 exhibited significant delay in vacuole maturation and in turnover of a lipid droplet marker Erg6. The function of MCC/eisosomes in the stress response have been demonstrated in many fungal species. Similar to the microdomain function, also individual protein components of...
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The role of stress granules and 4E-BP in heat-stressed cells of S. cerevisiae
Kolářová, Věra ; Hašek, Jiří (advisor) ; Zimmermannová, Olga (referee)
The cells are capable of very quick and specific reactions on stress conditions. Influence of translation, specifically initiation of translation by inhibition factors, is one of the main regulatory process. Two of eIF4E-binding proteins (4E-BP), Eap1p and Caf20p, are known as cap-dependent translation repressors in yeast Saccharomyces cerevisiae. We used in vivo fluorescent microscopy analysis to show different reaction of Caf20p and Eap1p to heat stress. Protein Caf20p does not react on heat shock and stays difused in cytoplasm. Contrary to Caf20p reaction, protein Eap1p accumulates in cytoplasm close to stress granules (SGs). This work shows that Eap1p is involved in stress granules assembly. In the absence of Eap1p, yeast cells react to the heat stress with small and less focused SGs. Dele- tion of CAF20 does not affect SG assembly. This points to specific function of SG in distribution of factors connected with stress reaction. Polysomal analysis shows that deletion of one of initiation translation repressors does not affect heat induced global repression of translation. In permisive condition deletion of EAP1 may cause defect in addition of 60S ribosomal subunits. Absence of protein Eap1p causes morphological defect. That point to a different reactions of Eap1p and Caf20p on heat stress and possible...
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The TUB3 intron splicing in PRP45 mutant cells
Konířová, Jana ; Půta, František (advisor) ; Zimmermannová, Olga (referee)
Protein Prp45, an essential factor of the yeast Saccharomyces cerevisiae, is implicated in pre-mRNA splicing. A truncated version of the PRP45 gene, prp45(1-169), which exhibits a temperature sensitivity, was previously prepared in our laboratory. The aim of this work is to contribute to better understanding of prp45(1-169) mutant phenotype. We tested the prp45(1-169) strain for its response to microtubule inhibitor benomyl and then we found that TUB3 overexpression from plasmid rescues discovered prp45(1-169) mutant cells hypersensitivity to benomyl. In addition, we studied the influence of TUB1, TUB3, and COF1 intron deletion on prp45(1-169) strain temperature sensitivity. Using RT-qPCR method we found that prp45(1-169) mutation results in the distinctive increase of pre-mRNA level for all tested genes, that could implicate that pre-mRNA splicing in these cells is affected before first transesterication.
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