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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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Study of transport systems of microorganisms
Jančíková, Iva ; Gášková, Dana (advisor) ; Amler, Evžen (referee) ; Krůšek, Jan (referee)
Title: Study of transport systems of microorganisms Author: Mgr. Iva Jančíková Department: Institute of Physics of Charles University Supervisor: Assoc. Prof. RNDr. Dana Gášková, CSc., Institute of Physics Abstract: Overexpression of drug efflux pumps is responsible for a multidrug resistance (MDR). We used the potentiometric fluorescent probe diS-C3(3), which is a substrate of major MDR pumps in three yeast species, Saccharomyces cerevisiae (ScPdr5p, ScSnq2p), Kluyveromyces lactis (KlPdr5p) and Candida albicans (CaCdr1p, CaCdr2p), to monitor inhibition of selected membrane transporters caused by various chemical stressors (diS-C3(3) assay). The extent of inhibition of probe transport points to a tighter arrangement of the K1Pdr5p binding pocket compared to that of ScPdr5p. Furthermore, we discovered that while deletion of the KlPDR16 gene does not affect K1Pdr5p activity, it only caused cell hyperpolarization, deletion of the KlERG6 gene results in both change in membrane potential and in a suppression of the pump's activity. We developed an effective method to search for inhibitors of MDR proteins of. C. albicans, which is based on pre-screening their potential to block the probe efflux from S. cerevisiae cells. Using this method we identified the substance H, derivative of 1,4-dihydropyridine, which...
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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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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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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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Monitoring the effect of medically important drugs on activity of human P-glycoprotein using fluorescent probes
Veľas, Lukáš ; Gášková, Dana (advisor) ; Sigler, Karel (referee)
One of the main causes of failure in cancer treatment when using chemotherapy is the phenomenon of multidrug resistance - MDR. The most important protein mediating MDR in human cells is P-glycoprotein. The main goal of this thesis was the modification of fluorescent method - developed for studying yeast MDR pumps at the Department of Biophysics at the Institute of Physics of Charles University - to study the activity of P-glycoprotein in human tumor cells. The fluorescent method is based on the use of redistribution potentiometric probe diS-C3(3) which we found to be a substrate of P-glycoprotein. The optimalization of the method (experimental window) allowed sensitive monitoring of changes in the activity of P-glycoprotein caused by the effect of its several known inhibitors/substrates: oligomycin, amiodarone, verapamil, vinblastine, ketoconazole, itraconazole and FK506. New important results regarding the effect of these medically significant drugs on human cells were obtained. The developed method will undoubtedly be of great benefit in the search for new effective inhibitors and the study of their mechanisms in the future.
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Development of Scaffolds for Bone Defects Regeneration
Prosecká, Eva ; Amler, Evžen (advisor) ; Rosina, Jozef (referee) ; Gášková, Dana (referee)
A promising new smart scaffold with potential for clinical use was prepared during our experiments. The biocompatible, biodegradable, osteoconductive and also osteoinductive 3D scaffold contains 0.5% type I collagen and 50% of hydroxyapatite with pore size around 400 um suitable for cell ingrowth and vascularization. Subsequently added poly-ɛ-caprolactone nanofibers improved the mechanical properties of the scaffold. The scaffold was enriched with mesenchymal stem cells and thrombocyte rich solution. The functionalized scaffold promoted new bone tissue formation throughout the defects, with uniform distribution of the newly-formed bone in vivo in a rabbit model, while the scaffold gradually degraded and was replaced by newly-formed bone tissue. In addition, we have found a fabrication process and materials which meet the European medicines agency requirements and can be developed for human applications. Hydroxyapatite-coated coaxial poly-ɛ- caprolactone/polyvinylalcohol nanofibers have been developed as a promising novel drug-delivery system suitable for bone tissue engineering.
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Plasma modification of functionalized PVA nanofibers for the enhancement of mesenchymal stem cell adhesion, viability and proliferation.
Bezděková, Dagmar ; Amler, Evžen (advisor) ; Gášková, Dana (referee)
Electrospinning is widely used technique to produce nanoscale constructs for tissue engineering. This technique can be used to spin wide range of polymers. One of them is polyvinyl alcohol (PVA), which has very good properties for use in this field. PVA is nontoxic, has good mechanical strength and it's degradable and biocompatible. Electrospun PVA nanofibers have limitations because of their -OH side groups. These groups cause solubility of PVA in water. The solubility can be adjusted with crosslinking techniques, but PVA still remains very hydrophilic, which is causing low adhesion of cells. In recent research we decided to reduce the hydrophilicity of PVA using plasma modification. Polymer modification with cold plasma is an economic and quite simple process to change the surface chemistry without side effects that come with conventional chemical treatment. With radical, formed by discharge, we have deposited hydrocarbons on the PVA surface and we rapidly increased hydrophobicity of the polymer surface. The change of surface chemistry has only a little effect on the fiber morphology. The increase of hydrophobicity allowed better adhesion of mesenchymal stem cells on plasma modified PVA as compared to non-modified PVA and a huge change in cell morphology was observed. These changes suggest that we...
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