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Studium exprese MDR pump u kvasinek Saccharomyces cerevisiae za různých růstových podmínek: metoda s fluorescenční sondou diS-C3(3)
Zahumenský, Jakub ; Gášková, Dana (advisor) ; Krůšek, Jan (referee)
In this work, we studied two yeast ABC transporters, Pdr10p and Pdr15p. At the time of assignment of this thesis, it was believed that these proteins contribute to the yeast MDR phenotype (PDR) on the grounds of their high homology to another yeast MDR protein, Pdr5p. In order to study these pumps, two sets of isogenic null-mutant strains were prepared with all possible combinations of gene deletions. We report that both of the studied proteins are very important in sus- taining the normal plasma membrane microenvironment for the most abundant, and essential, yeast plasma membrane protein, H+ -ATPase and so influence the membrane potential. Pdr10p and Pdr15p thus play an as yet unknown role in reg- ulation of the activity of this enzyme. Furthermore, we report that deletion of the genes coding for these proteins severely reduces the ability of the H+ -ATPase to be activated by the protonophore CCCP which is a weak acid. Studies performed with immunosuppressant FK506 further show that this compound reduces the viability of S. cerevisiae mutant strain PLY643 lacking genes coding for Pdr5p, Snq2p and Yor1p. Further deletion of Pdr10p and Pdr15p does not increase the lethality of this compound. Neither CCCP nor FK506 are substrates of the stud- ied pumps. 1
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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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Dynamics of yeast plasma membrane domains
Oláhová, Dominika ; Zahumenský, Jakub (advisor) ; Vopálenská, Irena (referee)
Plasmatic cellular mebrane consists of specialized domains which differ in shape, function and size. This compartmentalization allows cells to coordinate and separate a large number of diverse processes. Disorders of cell organization at the level of membrane domains are increasingly associated with the expansion of pathological disorders. Microdomains also play a role in the virulence of pathogenic organisms, e.g. some types of yeast. One of the best described microdomains of the yeast plasma membrane is the MCC (membrane compartment of arginine permease Can1), which is stabilized by a protein complex called the eisosome. The composition of the MCC/eisosome is described in detail, the dynamics of the individual components in the basal state (i.e. without stress) has not yet been described. The aim of this thesis is to summarize current knowledge about the dynamics of the proteins of this microdomain and its relationship with other domains of cell membranes through a detailed review of the available literature. And at the same time by using the advanced fluorescence method FRAP (fluorescence recovery after photobleaching) to show in my own experiment that in the basal state there is an exchange of individual Nce102 molecules in the MCC/eisosome microdomain. Key words: yeast, plasma membrane,...
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Characterization of native and heterologously expressed membrane transporters in yeast using fluorescent probes
Zahumenský, Jakub ; Gášková, Dana (advisor) ; Cebecauer, Marek (referee) ; Krůšek, Jan (referee)
Yeast plasma membrane transporters play crucial roles in many cellular processes, including detoxification and build-up and maintenance of the plasma membrane potential (ΔΨ). The former development of the diS-C3(3) fluorescence assay by the Biophysics Group of the Institute of Physics, Charles University, enabled us to conveniently study both, including their changes, using a simple fluorescent probe diS-C3(3). Many studies carried out on both animal and yeast cells have revealed that ethanol and other alcohols inhibit the functions of various membrane channels, receptors and solute transport proteins, and a direct interaction of alcohols with these membrane proteins has been proposed. Using the diS- C3(3) assay for multidrug-resistance pump inhibitors in a set of isogenic yeast pdr5 and snq2 deletion mutants we found that n-alcohols (from ethanol to hexanol) exhibit an inhibitory effect on both pumps, increasing with the length of the alcohol carbon chain. The inhibition is not connected with loss of plasma membrane structural or functional integrity and is fully reversible. This supports a notion that the inhibitory action does not necessarily involve only changes in the lipid matrix of the membrane but may entail a direct interaction of the alcohols with the pump proteins. Tok1p is a highly specific...
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Finding the answer in the answers
Záhumenský, Jakub ; Vidová Hladká, Barbora (advisor) ; Bojar, Ondřej (referee)
Title : Searching for the answer in answers Author : Jakub Záhumenský Contact : zahumensky.jakub@gmail.com Department : Institute of Formal and Applied Linguistics Supervisor : Mgr. Barbora Vidová Hladká, Ph.D. Contact on supervisor : hladka@ufal.mff.cuni.cz Abstract : We design a question-answering system Interviewer that enables users to fictionally (virtually) interview this person by asking questions as similar as possible to questions that journalists have already asked. The interviews with a given person posted on the web are being collected as a corpus of (question, answer) pairs. The user asks his/her question and the Interviewer system searches questions in the corpus to provide the answer that belongs to the most similar question. Matching questions is based on the frequency analysis and on the applications coming from natural language processing, namely tagging and parsing. We work with the interviews with Vaclav Havel posted on his personal page.
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Studium exprese MDR pump u kvasinek Saccharomyces cerevisiae za různých růstových podmínek: metoda s fluorescenční sondou diS-C3(3)
Zahumenský, Jakub ; Gášková, Dana (advisor) ; Krůšek, Jan (referee)
In this work, we studied two yeast ABC transporters, Pdr10p and Pdr15p. At the time of assignment of this thesis, it was believed that these proteins contribute to the yeast MDR phenotype (PDR) on the grounds of their high homology to another yeast MDR protein, Pdr5p. In order to study these pumps, two sets of isogenic null-mutant strains were prepared with all possible combinations of gene deletions. We report that both of the studied proteins are very important in sus- taining the normal plasma membrane microenvironment for the most abundant, and essential, yeast plasma membrane protein, H+ -ATPase and so influence the membrane potential. Pdr10p and Pdr15p thus play an as yet unknown role in reg- ulation of the activity of this enzyme. Furthermore, we report that deletion of the genes coding for these proteins severely reduces the ability of the H+ -ATPase to be activated by the protonophore CCCP which is a weak acid. Studies performed with immunosuppressant FK506 further show that this compound reduces the viability of S. cerevisiae mutant strain PLY643 lacking genes coding for Pdr5p, Snq2p and Yor1p. Further deletion of Pdr10p and Pdr15p does not increase the lethality of this compound. Neither CCCP nor FK506 are substrates of the stud- ied pumps. 1
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