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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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The role of ABC and MFS transporters in drug resistance of pathogenic Candida yeasts
Chaun, Martin ; Sychrová, Hana (advisor) ; Půta, František (referee)
Pathogens and their hosts lead an ancient battle of survival among themselves. New strategies are constantly being developed to defeat an opponent and counter-strategy to resist to a rival. Recently, due to increased drug use, pathogens have adapted to the prevalence of these substances in the environment, and are increasingly unresponsive to drug treatment, resulting in drug resistance status. Progressively increasing drug resistance also affects the most common fungal pathogens, Candida genus. One of the mechanisms by which organisms are able to withstand the effects of toxic substances is their transport out of the cell by membrane transport proteins. These transporters are members of the ABC and MFS protein superfamily in the Candida genus. This thesis presents ABC and MFS proteins involved in drug efflux in four Candida species, namely Candida albicans, Candida glabrata, Candida parapsilosis and Candida auris.
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Production of biologically active substances by yeasts and algae cultivated on different nutrition sources
Kohútová, Darina ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
Carotenoids are lipophilic pigments and the most widespread antioxidants occurring in nature with great application potencial in the cosmetics, pharmaceuticals and food industry. Lipids are one of the essential organic substances of living organisms as a nutrition source and energy supply. This work deals with the optimalization of nutritional characteristics of cultivation medium for cultivation of carotenogenic yeasts and algae. Two waste substrates were used and different nitrogen sources, NaCl concentrations, and various C/N ratios were applied. The biomass obtained was analysed by chromatography, and the production of carotenoids, ergosterol, coenzyme Q and lipids was evaluated. Of the tested nitrogen sources urea was the most favourable for yeasts, and sodium nitrate for algae. Higher salt concentration had a negative effect on yeast growth but increased degree of fatty acid unsaturation. Tested C/N led different effects: while the C/N ratio 16 most suitable for growth, the increasing C/N ratio caused the increase of the polyunsaturated fatty acids content. Of the used yeast strains, Rhodotorula mucilaginosa and Sporidiobolus salmonicolor exhibited the best production characteristics, of the used algae strains, it was Chlorella.
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The antimicrobial effects of plant extracts against yeast skin mycoses
Posztósová, Gyöngyi ; Rapta, Marek (referee) ; Němcová, Andrea (advisor)
Superficial cutaneous mycoses are relatively widespread and affect more and more people. Currently are used drugs as ketoconazole, fluconazole and others to treat mycoses. Pathogens occasionally become resistant to these drugs. Medicinal plants have a long history in alternative medicine and they could be a good solution. This bachelor thesis deals with the study of antifungal activity of selected plants. Were prepared ethanol extracts of basil, betony, eucalyptus, turmeric, geranium, oregano and ginseng. The theoretical part of thesis contains a review of the most frequently occurring dermatomycoses. It describes plants as sources of natural substances with antimicrobial effect and the content of selected active substances. The experimental part is focused on the analysis of plant extracts. Was determined the content of total polyphenols, flavonoids and antioxidants. The antimicrobial activity was monitored against yeast Candida glabrata and against G- and G+ bacteria, specifically Escherichia coli and Micrococcus luteus. Finally, the cosmetic products with the addition of plant extract were prepared and tested against mycoses.
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Role of autophagy in yeast cell adaptation
Brádlerová, Michaela ; Kuthan, Martin (advisor) ; Zikánová, Blanka (referee)
Autophagy is an evolutionarily conserved degradative pathway. Autophagy occurs constitutively at a basal level and it is involved in the recycling and turnover of damaged or superfluous organelles and proteins. It has a critical role in cellular homeostasis. Autophagy can be induced in response to starvation or other types of stress. Induction of autophagy during these conditions has a major role in protection and adaptation of the cell. Autophagy needs to be properly regulated. A wide range of diseases is associated with dysregulation of autophagy. Better understanding of autophagy mechanisms can help to develop strategies designed to modulate autophagic responses occuring in a number of diseases. This work is focused on current knowledge of main types of autophagy and how autophagy helps yeast cells to adapt. Key words: autophagy, yeast, degradative pathway, adaptation, TORC1
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Regulation of yeast chronological ageing
Némethová, Ema ; Palková, Zdena (advisor) ; Motlová, Lucia (referee)
Yeast cells are able to differentiate during chronological aging and form different cell- types which acquire different viability. Chronological aging cells do not divide and thus mimic the aging of cells within multicellular organisms, such as mammals. Understanding of mechanisms of chronological aging of yeasts thus could help to understand similar processes of more complex organisms. Two types of cells, called "quiescent" and "non- quiescent" cells, are formed when yeast are grown in liquid medium. These cells have different morphology and gene expression and also differ in their metabolism. Yeast cells within agar-plate-grown colonies can sense different gradients of nutrients and signaling compounds and diversify to different cell types such as U and L cells, located in different colony regions. Both the "quiescent" and U cells are more vital and able to proliferate, whereas "non-quiescent" and L cells are less viable and exhibit stress features. Chronological aging involves many cellular processes including accumulation of storage compounds, mitochondrial activity, functions of specific genes and can be affected by calorie restriction and mild stress. Key words: chronological aging, yeast, differenciation of cell populations, metabolic change
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Inducible promoters and their use in yeast cell manipulation
Přibáňová, Gabriela ; Palková, Zdena (advisor) ; Vopálenský, Václav (referee)
Promoters which can be regulated by different chemical or physical factors are often used in cell manipulations. This thesis focuses predominantly on promoter systems which use light as an inductor. There are two main approaches to controlling a promoter by light. The first one uses so-called "caged molecules", chemical inducers whose inducing activity is "masked" by a photolabile protecting group. The second approach includes optogenetic systems, which can regulate transcription in cells. These systems are encoded in the DNA of the organism, and light is the only external regulatory stimulus. Photoreceptors that need a specific cofactor (chromophore) are the main components of optogenetic systems. There are several groups of photoreceptors classified by the type of chromophore and photoactivation mechanism. This thesis gives an overview of optogenetic systems used for transcription regulation and focuses on different photoreceptors and induction mechanism used. The systems using photocaged molecules are described as well. Furthermore, the thesis deals with light- systems in yeast as a model organism as well as organism used for biotechnological purposes. Finally, some limitations of light inducible promoters are discussed, including the chromophore type, the wavelength of the light, and the...
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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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Production of carotenoids grown on waste fat
Holub, Jiří ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
Carotenoids are lipid-soluble pigments which are contained in plants and microorganisms. Carotenoids are known for their antioxidant effects. Lipids are predominantly non-polar substances, which are essential and present in all types of organisms. The research was made by theoretical and practical form. It was dealing with production of carotenoids pigments and different lipophilic substances by yeasts of Rhodotorula glutinis, Rhodosporidium toruloides and Sporidiobolus metaroseus, cultivated in media containing waste fat or glycerol. Further, methods for obtaining lipid substances from microorganisms and with further analysis of samples by gas or liquid chromatography were optimized. The main task of the research was to find out which types of media and yeasts are the most suitable for production of lipid substances. For production of lipid substances in glycerol media, as the best producing strain Rhodosporidium toruloides was found, which simultaneously reported the highest concentrations of carotenoids on fatty and fatty hydrolyzed media.
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Production of beta-glucans by some yeasts and algae
Veselá, Markéta ; Kočí, Radka (referee) ; Márová, Ivana (advisor)
Several yeast strains and microalgae were selected for this diploma thesis. -glucans, lipids, carotenoids, ergosterol and coenzyme Q were determined in selected producers, and the cultivation conditions for yeast strains were optimized to gain enhanced production of -glucans. Microalgae cultivations were carried out according to the instructions of the Collection of Autotrophic organisms (CCALA). Selected microalge strains include Desmodesmus acutus, Dunaliella salina, Arthrospira maxima and Cyanothece sp. Selected yeast species include Rhodotorula glutinis, Cystofilobasidium macerans and Sporidiobolus metaroseus. Edible yeast Saccharomyces cerevisiae was cultivated to compare with other yeast strains because of it's verified production of -glucans. -glucans were then determined by the enzymatic kit K-YBGL Megazyme, carotenoids, ergosterol and coenzyme Q were analyzed by HPLC/PDA and fatty acids were analyzed by GC/FID. The best producer of yeast -glucans was R. glutinis and S. metaroseus, and the best conditions for the production of -glucans and other metabolites was the C/N ratio of 70. Within the microalgae species, only -glucan production was observed, the best producer was D. acutus.
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