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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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Organelle proteomics of parasitic protists
Jedelský, Petr ; Tachezy, Jan (advisor) ; Kolářová, Libuše (referee) ; Půta, František (referee)
Advances in DNA sequencing led to a technological breakthrough, that allowed analyzis of complete genomes including those of parasitic protists Trichomonas vaginalis and Giardia intestinalis . These organisms are studied not only for their clinical importance, but also from the evolutionary point of view for their adaptation to anaerobic environment. Genome sequencing and annotations of predicted proteins alone did not bring detail view into functioning of their mitochondrion related organelles in G. intestinalis mitosomes, notparticipating in energetic metabolism, in T. vaginalis hydrogenosomes, producing molecular hydrogen and ATP by means of substrate phosphorylation. Traditional methods based on a fractionation by ultracentrifuging in density gradient and subsequent biochemical and enzymological analyzes were extended by one and twodimensional electrophoresis with subsequent identification of proteins by mass spectrometry. Methods of multidimensional separation of peptides produced by specific proteolysis of a complex mixture...
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CSL proteins of Schizosaccharomyces pombe
Převorovský, Martin ; Půta, František (advisor) ; Dvořák, Michal (referee) ; Kořínek, Vladimír (referee)
The CSL family of transcription factors is essential for metazoan development,mostly due to their involvement in the Notch signaling pathway. We identified two novel classes of CSL genes in several fungal species, organisms lacking the Notch pathway. We characterized experimentally cbfl I- and cbfl2*, the two CSL genesof Schizosaccharomyces pombe, in order to elucidate the CSL function in fungi. We provide evidence supportingtheir identiý as genuine CSL genes.Both cbfll- and cbfl2- are non-essential; they have distinct expression profiles and code for nuclear proteins with transcription activation potential. Significantly, we demonstratedthat Cbfl1 recognizesspecifically the canonical CSL response element GTGA/6GAA in vitro. The deletion of cbfLt_ is associated with growth phenoýpes and altered colony morphology. Furthermore, we found that Cbfl I and CbfIZ play opposite roles in cell adhesion, nuclear and cell division and their coordination. Disturbed balance of the two CSL proteins leads to cell separationdefects,cut phenotype, and high-frequency diploidization. Our data show that CSL proteins operate in an organism predating the Notch pathway, which should be of relevance to the understandingof (Notch- independent)CSL functions in metazoans.
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Regulation of S. cerevisiae TUB1 and TUB3 paralogous genes expression by Prp45
Cihlářová, Zuzana ; Půta, František (advisor) ; Schierová, Michaela (referee)
Prp45 is an essential splicing factor of budding yeast Saccharomyces cerevisiae. The human ortholog of Prp45 - protein SNW1/SKIP - is involved in splicing and probably influences transcription and histone modification. The genetic interacion of Prp45 with splicing factors is well described. We have additionally demonstrated that Prp45 genetically interacts also with factors involved in transcription regulation and histone modification enzymes. Our preliminary data therefore suggest that Prp45 might be a factor that connects processes of splicing, transcription, and chromatine modification and dynamics in S. cerevisiae. The first aim of this project was to investigate the role of introns in intra- and intergenic expression regulation of paralogous genes TUB1 and TUB3 and whether is this regulation influenced by aberrant splicing. Using quantitative PCR we found that expression of paralogous genes TUB1 and TUB3 is not dependent on the presence of their introns or correct splicing. The second aim of this project was to explore the potential role of Prp45 in the regulation of chromatin state. For this purpose, we used the system of β-estradiol-induced expression of myc-tagged histon H3 and determined its incorporation into nucleosomes by chromatin immunoprecipitation. Despite the lack of...
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Does the pre-mRNA splicing occur in S. cerevisiae co- or post-transcriptionally?
Cihlářová, Zuzana ; Půta, František (advisor) ; Kozáková, Eva (referee)
Until recently, the splicing and transcription were seen as almost independent processes. However, today a lot of studies provide plenty of evidence about their connection, even in the yeast Saccharomyces cerevisiae. The connection of these processes is particularly mediated by C-terminal domain of RNA polymerase II, which is consisted of tandemly repeated heptapeptide sequence - YSPTSPS. Amino acid residues of this heptapeptide sequence are specifically phosphorylated during transcription, which regulates transcription process and also the binding of specific factors. These factors are necessary for processing of the nascent transcript. Modifications of the primary transcript occur especially cotranscriptionally in higher eukaryotes, thus before the transcription is terminated and also before the functional mRNA is released. Opinion on cotranscriptional splicing in S. cerevisiae were significantly changed in the last years. However, nowadays the splicing of pre-mRNA of most genes in S. cerevisiae is seen as cotranscriptional process. RNA polymerase II pauses within the terminal exons and this pausing event provides sufficient time for each spliceosomal component to assemble on the pre-mRNA and also for catalysis of splicing before the transcription termination. Keywords: cotranscriptional...
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