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Localisation of selected proteins during yeast colony development
Plocek, Vítězslav ; Palková, Zdena (advisor) ; Heidingsfeld, Olga (referee)
Yeasts is unicellular organisms which can create remarkably complex colonies. By studying multicellular structures of Saccharomyces cerevisiae yeast it was found that cells within the yeast colony behave differently. (Kamath and Bungay, 1988; Mináriková et al., 2001; Scherz et al., 2001; Palková and Váchová, 2006; Váchová et al., 2009; Piccirillo et al., 2010; Váchová et al., 2011). Through microarray analysis of the developing yeast colony (Palková et al., 2002; Váchová et al., 2009) were described genes whose expression changes basically during the development of the colony. Of those genes, I chose four - PD5, STL1, PHO89, FET3 - that , as I thought, could affect the growth and differentiation of the yeast colony. I created their fusion variants with GFP and, using techniques of yeast colony cuts, yeast colony differentiation in sucrose gradient and measuring by flow cytometry I identified places in the colony, where the gene expression occurs. I found out that in the yeast colony differentiation and different expression take place in early phases of the development.
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Molecular mechanisms of formation and development of colonies of wild Saccharomyces cerevisiae strains
Šťovíček, Vratislav ; Palková, Zdena (advisor) ; Hašek, Jiří (referee) ; Heidingsfeld, Olga (referee)
Yeasts are capable of forming a wide range of multicellular communities, which enable the survival in harmful and changing environment. Surface associated biofilms, often connected with infections in human body, and colonies can serve as an example of such populations. This work investigates formation and development of complex structured colonies of Saccharomyces cerevisiae, which can be considered as a distinctive feature of yeast strains isolated from the wild. Architecture and properties of such colonies are fundamentally different from the spatially undifferentiated colonies of most of laboratory strains and resemble in many ways rather natural biofilms of pathogenic yeasts. Yeast populations use specific developmental processes induced by communication mechanisms to synchronize the early stages of their development. Formation of specific three-dimensional colony architecture is enabled by the presence of extracellular matrix and adhesive protein Flo11p which provide stability and integrity of the whole structure. Protection of the colonies is accomplished by spatially differentiated cell subpopulations using various mechanisms such as expression of efflux pumps capable of removing toxic substances or production of extracellular matrix functioning also as selectively permeable barrier. Phenotypic...
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Participation of Selected Carbonyl Reductases in Deactivation of Anticancer Drugs
Odiana, Romana ; Wsól, Vladimír (advisor) ; Szotáková, Barbora (referee) ; Heidingsfeld, Olga (referee)
Reduction is the reverse of oxidation and therefore it can involve loss of oxygen atom or the addition of two hydrogen atoms. The reduction of carbonyl groups in xenobiotics was the main topic of this thesis. We tried to identify and characterize human carbonyl reductases responsible for anticancer drugs deactivation. When cancer is among the most common death causes in the developed world, it is necessary to look for new and efficient ways of its treatment. Inhibition of enzymes, which may contribute to disease development or relapses and/or treatment efficacy decrease by drug inactivation, could be a possible way of treatment improvement and might also lead to decrease of drug doses and side effects of cytostatics. In the first part of our project, we focused on a soluble cytosolic reductase AKR1C3. This enzyme is involved in sex hormone metabolism and might play an important role in breast and prostate cancer development. We tested its ability to metabolize anticancer drugs by its incubation with oracin and doxorubicin with subsequent metabolite determination with use of HPLC. Our experiment proved that it can deactivate these two drugs with Km 355 μM for doxorubicin and 110 μM for oracin, respectively. AKR1C3 can therefore influence the anticancer therapy, expecially when overexpressed. The...
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Plasma-membrane alkali-metal-cation transporters involved in salt tolerance of pathogenic Candida species
Krauke, Yannick ; Sychrová, Hana (advisor) ; Malcová, Ivana (referee) ; Heidingsfeld, Olga (referee)
of Ph.D.Thesis Conclusions All the aims of the thesis were achieved.The toleranceto alkali metalcationsof four pathogenic Candida species was studied in detail and revealed differencesamong the yeasts. These differences in sall tolerance remained the same under various growth conditions.For the first time, the internalsodium and potassiumconcentrationsof several Candidaspecieswere estimatedunder highsalt-stressgrolvth.The internalK./Na* ratiowas not in relationwith the salt tolerancerevealingdifferentadaptationmechanismsto salt stress in Candida species. A first study on combinatoryuse of fluconazoleand NaCl revealed severe synergisticeffects of both compounds, leading to grovvthinhibitionand increased internalNa* concentrationsin C. albicans.The molecularbasis of this synergismremainsto be established. Ihe C. dubliniensis, C. glabrata and C. parapsilosis Cnhl NalH- antiporters were cloned and functionallycharacterizedupon heterologousexpression in S. cerevlslae to understandthe mechanismsinvolvedin the differentsalttolerancesof Candidaspecies.The three antiportersdifferedin theiractivityfor alkalimetalcations,which roughlycorrelatedwith the observed differences in salt tolerance among the species. Additionally,during the characterizationof heteroiogouslyexpressed antiporters,two antiporter chimeras...
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Alakali-metal-cation homeostasis in pathogenic Candida species
Elicharová, Hana ; Sychrová, Hana (advisor) ; Heidingsfeld, Olga (referee) ; Půta, František (referee)
Several tens of Candida species belong to the opportunistic human pathogens capable of inducing life-threatening infections in immunocompromised patients. Virulence of single Candida species depends among others on their resistance to the variable external conditions. The maintenance of alkali-metal-cation homeostasis, which means the ability to accumulate sufficient amount of potassium cations and on the other hand to survive under high extracellular concentrations of alkali-metal cations, is essential for growth and virulence of Candida cells. We observed the negative effect of fluconazole (FLC) on salt-tolerance of six Candida species and found that it is independent of the species level of FLC- resistance. FLC hyperpolarizes plasma membrane of Candida cells and therefore increases non-specific uptake of alkali-metal cations which results in strongly increased salt-sensitivity of Candida cells. The FLC-induced hyperpolarization also results in an increased sensitivity of Candida cells to the antifungals which are positively charged and are driven into the cells by the membrane potential. The effect of fluconazole on membrane potential and thus on the uptake of alkali- metal cations into the cell turned our attention to the homeostasis of potassium cations whose high intracellular concentration is...
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CUG Codon in Pathogenic Yeasts of the Genus Candida
Marečková, Lucie ; Půta, František (referee) ; Heidingsfeld, Olga (advisor)
2. Abstract In many Candida species the standard leucine CUG codon is translated as a serine, although not in 100% cases. This dual specifity of the CUG codon has evolved through a mechanism that required codon ambiguity mediated by a unique tRNACAG, which is in vitro aminoacylated more often by serine than by leucine. This codon ambiguity has been tolerated for more than 170 million years. The explanation at least for now is that the CUG codon reassignment could have generated genetic diversity that facilitated occurrence of new phenotypes resistant to stress. Beside this, an important step was to reduce negative impact of the codon ambiguity by crucial mutations in the structure of the ser-tRNACAG. Candida species became a valuable experimental model for elucidation of the genetic code changes. While consequences of the CUG codon reassignment have been extensively studied in Candida albicans, this topic has not yet been addressed in Candida parapsilosis. Solving the structure of C. parapsilosis secreted proteinase Sapp1p provided a tool to carry out a "case study" of possible effects of the CUG codon ambiguity. The SAPP1 gene contains one CUG codon, and the respective serine is located on the loop in the close proximity of the active site of the proteinase.
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Ammonia production by colonies of mutants and aging of wrinkled colonies of Saccharomyces cerevisiae
Nedbálková, Jana ; Heidingsfeld, Olga (referee) ; Janderová, Blanka (advisor)
Production of ammonia by the colonies of mutants and aging of wrinkled colonies of Saccharomyces cerevisiae The aim of this diploma thesis is to observe the development, respectively the aging of cells in yeast colonies Saccharomyces cerevisiae. Yeast cells S. cerevisiea form multicellular organized structures on a solid substrate, i.e. colonies, which the intercellular interactions occur in. These interactions influence forming, morphology and aging of yeast colonies. This diploma thesis is focused partly on the changes in ammonia production by giant colonies of deletion mutants and partly on the aging of colonies with the wrinkled morphology. I characterized mutant strains of S. cerevisiae with the deletion in RTG1, RTG2, RTG3, FIS1, CIT2 genes. Their products play an important role in the colony development. The transcription of these genes changes during the transition from the acidic to alkali phase during developmental process of the colonies. I have found out that the ammonium production rate was in accordance with the results of the alkalization in giant colonies surroundings and mentioned mutants derived from the BY strain has been producing ammonia since the 15th day. The rate of the ammonia production by rtg3∆ strain was comparable to the parental strain. Compared to parental strain, lower...
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Antifungal Drug Discovery: Focus on Incrustoporin Derivatives
Silva, Luis André Vale ; Buchta, Vladimír (advisor) ; Heidingsfeld, Olga (referee) ; Hamal, Petr (referee)
1 CONCLUSIONS In the context of Medical Mycology today, the development of new more effective antifungal agents is a priority. In fact, there are still only about six drugs in use to treat invasive fungal infections, concretely the polyene amphotericin B (including its new lipidic formulations), the antimetabolite flucytosine, the triazoles fluconazole, itraconazole, and voriconazole, and the echinocandin caspofungin. Simultaneously, the incidence of invasive opportunistic mycoses has been increasing steadily with the increasing number of immunocompromised patients, caused both by HIV (human immunodeficiency virus) infection and AIDS and the development of medical techniques, particularly referring to oncology or transplant patients. In this setting, the relevance of the development of new antifungal agents and, hence, our work presented here, is easily understandable. The group of the acyloxymethylated incrustoporin derivatives is now in a higher stage of development, after being previously studied concerning structure-antifungal activity relationship and tuning of antifungal activity. Our work has shown the best derivatives from the group, compounds LNO6-22, LNO15-22, and LNO18-22, to have broad spectrum in vitro antifungal activity and high potency, inhibiting growth of a variety of pathogenic yeasts and...
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