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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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Study of the performance of microbial MDR pumps by fluorescent probes: effect of potential inhibitors
Kodedová, Marie ; Gášková, Dana (advisor) ; Höfer, Milan (referee) ; Sychrová, Hana (referee)
The current increased use of antifungal agents has resulted in the development of resistance to these drugs. Search for new antifungals with different mechanisms of action overcoming the multidrug resistance is thus underway. Surface-active antifungals have the advantages of minimizing host toxicity and the emergence of drug resistance. We have developed a fluorescence method based on the use of the potentiometric fluorescent probe diS-C3(3), substrate of two major S. cerevisiae MDR pumps, Pdr5p and Snq2p. It allows us to monitor with high sensitivity and in real time changes in the activities of both pumps and also in membrane potential. We present here an efficient strategy for identifying pump inhibitors with minimal side effects on membrane integrity, and compare the potencies of different inhibitors towards MDR pumps. New efficient inhibitors of MDR pumps could potentially be used in conjunction with current antimicrobials that are MDR pump substrates. The method can be also used to determine the mechanism of action of surface-active drugs and their lowest effective concentrations.
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Nanofibrous scaffolds in controlled delivery of autologous growth factors
Buzgo, Matej ; Amler, Evžen (advisor) ; Gášková, Dana (referee)
Platelet preparations are a source of various autologous growth factors and have numerous applications in tissues engineering. The aim of this work was to development electrospun nanofiber scaffolds with platelet preparations. Scaffolds based on the adhesion of platelets on nanofiber meshes were developed. The scaffolds were able to enhance chondrocyte proliferation in vitro. The main disadvantage of this system is the burst release of growth factors immediately after adhesion. To overcome this, we developed coaxially electrospun scaffolds with incorporated alpha granules. Alpha granules are novel platelet preparations with high amounts of growth factors. This system was able to stimulate chondrocyte proliferation and maintain TGF- 1 concentrations for 7 days. Additionally, a novel drug delivery system with coaxially incorporated liposomes was developed. Liposomes incorporated into nanofibers remain intact and can be used for the delivery of various molecules. The ability to maintain HRP activity was compared to systems based on coaxial electrospinning with liposomes, coaxial electrospinning without liposomes and blend electrospinning. When compared to other systems, coaxial electrospinning with liposomes preserves enzyme activity twice as long. These results clearly indicate the potential of...
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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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Měření membránového potenciálu u mutantních kmenů Saccharomyces cerevisiae deficientních v různých membránových transportérech
Džamba, Dávid ; Gášková, Dana (advisor) ; Krůšek, Jan (referee)
Potassium transport across the cell membrane is supposed to contribute to the maintenance of membrane potential. In this work, the diS-C3(3) assay was used to determine the contribution of three main potassium transporters of S. cerevisiae cells (Trk1p, Trk2p and Tok1p) to the membrane potential changes in three different growth phases. It was shown that deletions of these transporters cause hyperpolarisation of the cell membrane in exponential and early diauxic growth phases; no difference was detected in post-diauxic cells. Another contribution of this work is in a deeper study of the activity of ABC pumps which are crucial in multidrug resistance. It was proved that apart from Pdr5p and Snq2p pumps, there are also other active extrusion pumps (most likely Pdr10p and Pdr15p). It was also showed that pdr1, pdr3 mutant strain is not an equivalent of strain lacking the multidrug pumps Pdr5p, Snq2p and Yor1p. The results in this work indicate that dis-C3(3) probe is probably a substrate of other pumps such as Pdr15p. Study of these transporters with our method is thus very suitable and could provide detailed information about their kinetics and help in finding their inhibitors.
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Interakce xenobiotik s MDR pumpami u S. cerevisiae
Hendrych, T. ; Zlámalová, Marta ; Gášková, Dana ; Pichová, Alena ; Sigler, Karel
The interactions of a number of xenobiotics with two main S. cerevisiae MDR pumps, Pdr5p and Snq2p were studied. The results showed that some compounds do not interact with MDR pumps whereas others affect one or another of the pumps, causing a loss of their ability to transport an indicator substrate out of the cells. This apparent inactivation of the pumps can be due to competition for transport with the indicator substrate or to actual (noncompetitive) inhibition of the pumps
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