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The effect of antimicrobial peptides on Saccharomyces cerevisiae and other yeast species
Makarova, Anna-Marie ; Hodek, Petr (advisor) ; Malcová, Ivana (referee)
The increased use of antibiotics, antifungal agents and disinfectants in the last decades has resulted in development of microbial resistance to these drugs. Candida species are the fourth most common cause of hospital-acquired bloodstream infection and kill 40% of those patients. Natural antimicrobial peptides are promising candidates for the development of new agents to treat yeast and bacterial infections, as their presumed mechanism of action differs significantly from the mechanism of action of current drugs. This work is focused on several peptides isolated from the venom of wild bees and their synthetic analogues and the identification of the most effective ones against non-pathogenic Saccharomyces cerevisiae and several pathogenic Candida species. Antifungal activity of eight cationic antimicrobial peptides was tested and compared under various conditions. The overall susceptibility of pathogenic yeast species to currently used antifungal drugs and the antimicrobial peptides was screened with the aim to identify potential synergistic and species-specific effects. The effect of antimicrobial peptides on membrane potential was measured by a fluorescent probe (diS-C3(3)), and the relative hyperpolarization of plasma membrane was shown for each peptide. The effect of antimicrobial peptides on...
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Monitoring of mitochondria in living cells using fluorescence methods
Taliánová, Eliška ; Čejková, Darina (referee) ; Čmiel, Vratislav (advisor)
This bachelor thesis deals with the topic of monitoring mitochondria and evaluating their activity. The structure and function of cellular mitochondria are briefly discussed. Contains a list of commonly available fluorescent dyes. It investigates the possibilities of influencing mitochondria in vitro. It deals with the influence of mitochondrial membrane potential by means of various concentrations of glucose and subsequently evaluates mitochondrial activity using the dye JC-10.
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Monitoring of mitochondria in living cells using fluorescence methods
Taliánová, Eliška ; Čejková, Darina (referee) ; Čmiel, Vratislav (advisor)
This bachelor thesis deals with the topic of monitoring mitochondria and evaluating their activity. The structure and function of cellular mitochondria are briefly discussed. Contains a list of commonly available fluorescent dyes. It investigates the possibilities of influencing mitochondria in vitro. It deals with the influence of mitochondrial membrane potential by means of various concentrations of glucose and subsequently evaluates mitochondrial activity using the dye JC-10.
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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 effect of antimicrobial peptides on Saccharomyces cerevisiae and other yeast species
Makarova, Anna-Marie ; Hodek, Petr (advisor) ; Malcová, Ivana (referee)
The increased use of antibiotics, antifungal agents and disinfectants in the last decades has resulted in development of microbial resistance to these drugs. Candida species are the fourth most common cause of hospital-acquired bloodstream infection and kill 40% of those patients. Natural antimicrobial peptides are promising candidates for the development of new agents to treat yeast and bacterial infections, as their presumed mechanism of action differs significantly from the mechanism of action of current drugs. This work is focused on several peptides isolated from the venom of wild bees and their synthetic analogues and the identification of the most effective ones against non-pathogenic Saccharomyces cerevisiae and several pathogenic Candida species. Antifungal activity of eight cationic antimicrobial peptides was tested and compared under various conditions. The overall susceptibility of pathogenic yeast species to currently used antifungal drugs and the antimicrobial peptides was screened with the aim to identify potential synergistic and species-specific effects. The effect of antimicrobial peptides on membrane potential was measured by a fluorescent probe (diS-C3(3)), and the relative hyperpolarization of plasma membrane was shown for each peptide. The effect of antimicrobial peptides on...
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Mode of daptomycin action and mechanism of resistance
Helusová, Michaela ; Mikušová, Gabriela (advisor) ; Lichá, Irena (referee)
Daptomycin is one of antimicrobial peptides. These molecules are part of immune system of all organisms. Daptomycin consists of a cyclic peptide core and a lipophilic tail. Daptomycin is produced by Streptomyces roseosporus and is used to treat serious gram-positive infections. Daptomycin is active also against methicillin-resistant Staphylococcus aureus. Its antimicrobial effect depends on the presence of calcium ions and phosphatidylglycerol. Daptomycin targets bacterial cytoplasmic membrane, where it forms oligomers. Mode of action of daptomycin probably includes pore-forming effect leading to membrane damage. This disturbance causes ion efflux from cytoplasm which leads to membrane potential disruption, which results in inhibition of macromolecular synthesis and cell death. Daptomycin also causes changes in cell morphology. Despite its unusual mode of action, several mechanisms of resistance have emerged in some pathogenic strains. These are for example decrease in the amount of phosphatidylglycerol in the membrane, increase in the amount of lysylphosphatidylglycerol, release of membrane phospholipids or mutations in genes which control peptidoglycan synthesis.
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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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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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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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