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Study of the biogenesis of monovalent-cation transporters in eukaryotic cell
Černá, Karolína ; Zimmermannová, Olga (advisor) ; Schierová, Michaela (referee)
Regulation of intracellular levels of alkali metal cations (Na+ , K+ ) and protons depends on the proper localization and function of monovalent-cation transporters in cell membranes of most organisms (from bacteria to mammals). Cargo receptors, as a part of the endoplasmic reticulum and COPII vesicles, recognize membrane proteins and help them pass through the secretory pathway to reach their target membranes. The family of cargo receptors Erv14/Cornichon is conserved among eukaryotic organisms, and particular members participate in the biogenesis of several monovalent-cation transporters. In this diploma thesis, we analysed the structure and functions of selected cornichons from the yeast Saccharomyces cerevisiae (Erv14) and from humans (CNIH1-4) in more detail. Using a heterologous expression of human cargo receptors CNIH1, CNIH2, and CNIH4 in yeast S. cerevisiae, we studied their ability to fulfil the role of Erv14 cargo receptor in cell salt tolerance, maintenance of membrane potential and intracellular pH. Of the three human cornichons, the functions of Erv14 in maintaining monovalent cation homeostasis were best complemented by the CNIH1 isoform. Expression of CNIH proteins in S. cerevisiae also impacted the biogenesis and function of yeast Na+ , K+ /H+ antiporter Nha1. Moreover, we...
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pH sensitive fluorescence probes
Marková, Kateřina ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
The goal of this thesis was to suggest the suitable method for measuring bacterial cytosolic pH in bacteria strain Cupriavidus necator. Fluorescent microscopy was chosen to obtain scan of bacteria and time-resolved fluorescence was chosen to obtain a calibration curve. BCECF-AM was used as a pH-sensitive fluorescent probe. It was suggested that the external calibration is more suitable than internal one for the prokaryotic type of cells. Bacteria H16 shows a long fluorescence lifetime only on the granules containing PHB, while in PHB-4 the longest fluorescence lifetime occurs randomly throughout the cell.
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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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Changes of intracellular pH in yeast cells under stress conditions
Divín, Radek ; Plášek, Jaromír (advisor) ; Krůšek, Jan (referee)
Title: Changes of intracellular pH in yeast cells under stress conditions Author: Radek Divín Department: Institute of Physics of Charles University Supervisor: prof. RNDr. Jaromír Plášek, CSc. Abstract: Specific values of intracellular pH (pHi) can affect all biochemical processes in a cell and this phenomenon is closely connected with the degree of importance of changes in the intracellular pH under the stress conditions. In the Master Thesis, the yeast cells Saccharomyces cerevisiae were used as a model of organism eukaryotic cells. Monitoring of intracellular pH of the cells was performed by the method of synchronous fluorescence scan technique of genetically encoded fluorescent probes pHluorin which was located in the cytosol of the cells. The cells were exposed to stress conditions due to the chemical changes in the environment. Consequently, their ability to maintain a stable value of the intracellular pH in various acidic environments was studied in more detail. The attention was also focused on the impact on optimizing of glucose cytosolic pH. The work was centered on the changes in intracellular pH under the influence of the presence of KCl in suspension. Furthermore, the decrease of cytosolic pH of monitored cells by protonophore CCCP was investigated. The effect of stress environment on the...
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Response of plasma membrane potential and intracellular pH of yeast to changes in the concentration of extracellular K+
Babuka, David ; Plášek, Jaromír (advisor) ; Heřman, Petr (referee)
The bachelor thesis is focused on studying yeast cells and their response to various external conditions. Main focus was on the study of intracellular pH and membrane potential change under the condition of varying extracellular concentration of K+ ions. In particular we studied to what extent are the yeast cells able to compensate these changes. The ability of yeasts to resist the changes of external pH of the cell medium was studied in an experiment complementary to the measurements of intracellular pH. To measure the intracellular pH a genetically encoded fluorescent probe ratiometric pHluorin was used and to measure the changes of membrane potential a fluorescent probe diS-C3(3) was used. Also we successfully applied a method of synchronously scanned fluorescence to supress the cell autofluorescence.
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The analysis of membrane potential recovery in yeast under CCCP-induced stress
Babuka, David ; Plášek, Jaromír (advisor)
The master's thesis is focused on the study of response of the intracellular pH of the yeast cells on various external environments, primarily in a relation to the protonophore carbonyl cyanide m-chlorophenylhydrazone, CCCP. To measure the intracellular pH of the yeast cells we used a genetically coded fluorescent probe the ratiometric pHluorin. Using the method of synchronously scanned fluorescent spectra we were able to measure the intracellular pH of the cells with high precision. As a part of these experiments we also studied the influence of ionic strength of the cell suspensions buffers on the surface potential as well as the influence of the mineral salt KCl on the depolarization of the yeast membranes and cytosolic acidification induced by the protonophore CCCP. We examined the changes of cytosolic pH as such but we also used the measured pH as an indicator of the processes and the state of environment outside the cell. One of the most notable outcomes of this thesis is a new method of monitoring the value of the surface potential of the yeast cells by measuring the titration curves of cytosolic acidification induced by the protonophore CCCP.
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The analysis of membrane potential recovery in yeast under CCCP-induced stress
Babuka, David ; Plášek, Jaromír (advisor)
The master's thesis is focused on the study of response of the intracellular pH of the yeast cells on various external environments, primarily in a relation to the protonophore carbonyl cyanide m-chlorophenylhydrazone, CCCP. To measure the intracellular pH of the yeast cells we used a genetically coded fluorescent probe the ratiometric pHluorin. Using the method of synchronously scanned fluorescent spectra we were able to measure the intracellular pH of the cells with high precision. As a part of these experiments we also studied the influence of ionic strength of the cell suspensions buffers on the surface potential as well as the influence of the mineral salt KCl on the depolarization of the yeast membranes and cytosolic acidification induced by the protonophore CCCP. We examined the changes of cytosolic pH as such but we also used the measured pH as an indicator of the processes and the state of environment outside the cell. One of the most notable outcomes of this thesis is a new method of monitoring the value of the surface potential of the yeast cells by measuring the titration curves of cytosolic acidification induced by the protonophore CCCP.
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The analysis of membrane potential recovery in yeast under CCCP-induced stress
Babuka, David ; Plášek, Jaromír (advisor) ; Sigler, Karel (referee)
The master's thesis is focused on the study of response of the intracellular pH of the yeast cells on various external environments, primarily in a relation to the protonophore carbonyl cyanide m-chlorophenylhydrazone, CCCP. To measure the intracellular pH of the yeast cells we used a genetically coded fluorescent probe the ratiometric pHluorin. Using the method of synchronously scanned fluorescent spectra we were able to measure the intracellular pH of the cells with high precision. As a part of these experiments we also studied the influence of ionic strength of the cell suspensions buffers on the surface potential as well as the influence of the mineral salt KCl on the depolarization of the yeast membranes and cytosolic acidification induced by the protonophore CCCP. We examined the changes of cytosolic pH as such but we also used the measured pH as an indicator of the processes and the state of environment outside the cell. One of the most notable outcomes of this thesis is a new method of monitoring the value of the surface potential of the yeast cells by measuring the titration curves of cytosolic acidification induced by the protonophore CCCP.
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Response of plasma membrane potential and intracellular pH of yeast to changes in the concentration of extracellular K+
Babuka, David ; Plášek, Jaromír (advisor) ; Heřman, Petr (referee)
The bachelor thesis is focused on studying yeast cells and their response to various external conditions. Main focus was on the study of intracellular pH and membrane potential change under the condition of varying extracellular concentration of K+ ions. In particular we studied to what extent are the yeast cells able to compensate these changes. The ability of yeasts to resist the changes of external pH of the cell medium was studied in an experiment complementary to the measurements of intracellular pH. To measure the intracellular pH a genetically encoded fluorescent probe ratiometric pHluorin was used and to measure the changes of membrane potential a fluorescent probe diS-C3(3) was used. Also we successfully applied a method of synchronously scanned fluorescence to supress the cell autofluorescence.
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Changes of intracellular pH in yeast cells under stress conditions
Divín, Radek ; Plášek, Jaromír (advisor) ; Krůšek, Jan (referee)
Title: Changes of intracellular pH in yeast cells under stress conditions Author: Radek Divín Department: Institute of Physics of Charles University Supervisor: prof. RNDr. Jaromír Plášek, CSc. Abstract: Specific values of intracellular pH (pHi) can affect all biochemical processes in a cell and this phenomenon is closely connected with the degree of importance of changes in the intracellular pH under the stress conditions. In the Master Thesis, the yeast cells Saccharomyces cerevisiae were used as a model of organism eukaryotic cells. Monitoring of intracellular pH of the cells was performed by the method of synchronous fluorescence scan technique of genetically encoded fluorescent probes pHluorin which was located in the cytosol of the cells. The cells were exposed to stress conditions due to the chemical changes in the environment. Consequently, their ability to maintain a stable value of the intracellular pH in various acidic environments was studied in more detail. The attention was also focused on the impact on optimizing of glucose cytosolic pH. The work was centered on the changes in intracellular pH under the influence of the presence of KCl in suspension. Furthermore, the decrease of cytosolic pH of monitored cells by protonophore CCCP was investigated. The effect of stress environment on the...
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