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Competition of cells within the population of yeast colony
Očková, Veronika ; Váchová, Libuše (advisor) ; Gášková, Dana (referee)
Competition is a very important natural phenomenon, which causes the rivalry of organisms, in cases such as space limitation or lack of nutrients. It occurs mainly in situations where organisms, including microorganisms live in large populations. Multicellular yeast colonies represent an example of such a population. After the population of yeast cells spends nutrients from the environment, the cells in colonies are able to respond to these changes by production of ammonia functioning as a signaling molecule. Subsequently, the cells are able to change their morphology and metabolism and, dependently on their location within the colony, to create a subpopulation of cells with specific characteristics and functions. It is likely that in the case of mixed colonies formed by the two different strains, a competition between the cells of these two strains could exist. Such rivalry can result in changes in the ratio of cells of the two strains within the colony population, so that the cells of one strain outweigh the other. In this diploma thesis, I compared the growth and development of giant colonies and competition between the cells of selected pairs of strains forming mixed colonies. I focused on the parental strain Saccharomyces cerevisiae BY and its variants labeled with fluorescent proteins. For...
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Study of membrane transport processes in yeast using potentiometric fluorescent porbe diS-C3(3)
Bartl, Tomáš ; Gášková, Dana (advisor) ; Krůšek, Jan (referee)
1 Title: Study of membrane transport processes in yeast using potentiometric fluorescent probe diS-C3(3) Author: Tomáš Bartl Department: Institute of Physics of Charles University Supervisor: doc. RNDr. Dana Gášková, CSc., Institute of Physics of Charles University Abstract: Yeast membranes contain a number of transporters. Some are responsible for flow of nutrients to the inside of the cell, others for disposing of waste and foreign substances and some for transport of small ions or protons across the membrane. The focus of this work is on the activity of specific transport membrane proteins, so-called MDR pumps, which are responsible for transport of foreign substances or drugs, out of the cell. Using the series of mutant strains of the yeast Saccharomyces cerevisiae (AD1-3, AD1-8 and AD12) differentiated in the presence of specific MDR pumps in their membrane, an influence of various chemical substances on the intracellular concentration of the potentiometric fluorescent probe diS-C3(3), which is actively being transported out of the cell by some of the MDR pumps, was observed. By the examination of the effect of 2-deoxyglucose we proved the active contribution of not only the main MDR pump, Pdr5p, but also of some other pumps, in lowering the intracellular probe concentration. It was observed that...
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Functionalized nanofiber system for accelerated regeneration of internal tissues
Kráľovič, Martin ; Amler, Evžen (advisor) ; Gášková, Dana (referee) ; Chvojka, Jiří (referee)
Functionalized nanofiber system for accelerated regeneration of internal tissues Abstract Functionalization can adjust the properties of nanofibers prepared by electrospinning to best replace the missing extracellular matrix in the healing wound. In the rabbit intestinal anastomoses, functionalization of nanofibers by cryogenic fractionation has proven to be crucial. While the use of functionalized PVA and chitosan nanofiber membranes led to intestinal strictures and severe inflammation, further functionalization of PVA and chitosan nanofibers by their cryogenic fractionation led to the possibility of reducing the polymer dose, which significantly reduced the inflammatory response and significantly increased the biocompatibility of the material used. The application of fractionated nanofibers increased the fraction of microvessels, fibroblasts and collagen, which resulted in an increase in the biomechanical strength of healed anastomoses. The functionalization of the polycaprolactone nanofibers by special adhesion to the polypropylene mesh made it possible to use them as a support for the healing of the fascia in the abdominal wall of pigs. Functionalization of PVA nanofibers with glyoxal led to their increased stability in the aqueous environment. By applying a membrane of PVA functionalized nanofibers to...
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Analysis of the arrangement of the binding pocket of the MDR pump Cdr1p of the pathogenic yeast Candida albicans - a major contributor to clinical drug resistance.
Bartl, Tomáš ; Gášková, Dana (advisor) ; Krůšek, Jan (referee)
Title: Analysis of the arrangement of the binding pocket of the MDR pump Cdr1p of the pathogenic yeast Candida albicans - a major contributor to clinical drug resistance. Author: Tomáš Bartl Department: Institute of Physics of Charles University Supervisor: doc. RNDr. Dana Gášková, CSc., Institute of Physics of Charles University Abstract: Candida infections are becoming an increasing cause of death in hospitalized patients. The main reason for drug resistance in the most common yeast pathogen Candida albicans is an increased production of transport proteins, which are removing the drug from the cell cytosol and thus producing the phenomenon called multidrug resistance - MDR. The goal of this thesis was to verify the suitability of the yeast strain Saccharomyces cerevisiae with heterologously expressed MDR pumps from the pathogenic yeast Candida albicans and comparison to the results from C. albicans with homologous expression. The results that the azole drugs miconazole, bifonazole, and ketoconazole, together with potentiometric fluorescent probe diS-C3(3), are substrates of the CaCdr1p and CaCdr2p pumps, but not, or minimally, of the pump CaMdr1p, are consistent with the previously published work. The binding pocket of CaCdr1p was explored using the disc diffusion assay and the diS-C3(3) fluorescent probe...
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Functional characterization of MDR pump Pdr5p responsible for multiple drug resistance in yeast Saccharomyces cerevisiae
Sayedová, Shirin ; Gášková, Dana (advisor) ; Krůšek, Jan (referee)
One of the main reasons for the treatment failure of infections caused by pathogenic microorganisms is the overexpressing of efflux membrane proteins, which actively remove drugs from cells, leading to a phenomenon called multidrug resistance MDR. In this work, we focused on the functional characterization of the MDR pump Pdr5p in the yeast Saccharomyces cerevisiae. We have verified that diS-C3(3) fluorescence method can be used to determine the binding sites where the substrates bind in the binding pocket of the pump ScPdr5p. We focused on the study of the ScPdr5p binding pocket using triazole derivatives: ravuconazole, voriconazole and fluconazole. Using disc diffusion assay, we showed that all three studied triazoles are substrates of the pump ScPdr5p. We have found that these structural analogs have a significantly different effect on the inhibition of the potentiometric fluorescent probe diS-C3(3) transport by the pump ScPdr5p, and also that ravuconazole and voriconazole compete with each other for transport by the pump ScPdr5p. We have used a fluorescent approach to study the binding of azoles to the binding pocket of pump ScPdr5p using benchmark substrates, that bind selectively to only one binding site in the binding pocket of the pump ScPdr5p, and we have supported the hypothesis that ravuconazole...
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Study of the performance of yeast MDR pumps by fluorescent probes: effect of potential inhibitors
Drietomská, Andrea ; Gášková, Dana (advisor)
Using a set of five isogenic mutant strains, we tested the effects of a set of twelve aminoesters of fatty acids belonging to two structural groups, which have been previously classified among lysosomotropic antifungals (LA), on membrane potential and the activity of MDR pumps Pdr5p, Snq2p and Yor1p in S. cerevisiae by a newly developed assay - a combination of the diS-C3(3) method and biological tests [1]. Depending on their chemical structure and concentration, the LA displayed several effects: (a) membrane depolarization, (b) interaction with MDR pumps, and (c) membrane damage leading to cell permeabilization. Membrane depolarization was observed with nearly all LA while only three of the tested compounds interacted with the MDR pumps - a competitive inhibition was detected. The above diagnostic fluorescence method using the cationic redistribution probe diS-C3(3) can readily be employed to exclude from the group of potential inhibitors those compounds (even though they can be substrates of Pdr5p and Snq2p) that do not satisfy the necessary condition of inhibiting MDR pumps Pdr5p and Snq2p already at low concentration.
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Study of the differences in the architecture of the binding pockets of two major MDR pumps of yeast Saccharomyces cerevisiae, Pdr5p and Snq2p, using their common substrates
Backová, Lenka ; Gášková, Dana (advisor) ; Krůšek, Jan (referee)
Multidrug resistance (MDR) is responsible for the decrease in drug effectiveness on pathogenic microorganisms or tumours. One of the mechanisms of multidrug resistance is drug transport out of the cell (efflux) by membrane transporters - pumps. Main MDR pumps of a yeast species Saccharomyces cerevisiae are Pdr5p and Snq2p, who share high amino acid sequence identity. This thesis focuses on the differences of these pumps, their binding pockets and their arrangement. The binding pocket of Pdr5p is better researched and comparing the results with those of pump Snq2p leads to broader knowledge about the binding pocket of Snq2p. We use disc diffusion assay to determine common substrates of both pumps, ketoconazole and bifonazole. These substrates are used in potentiometric fluorescent probe diS-C3(3) assay. Results of these experiments lead us to the findings that the binding pocket of Snq2p has multiple binding sites. Binding pockets of pump Pdr5p and Snq2p differ in binding sites and their conformation. However, the conformation of both pumps is dynamic, which has been shown after the addition of glucose to supply the pumps with energy. 1
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