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Study of the function of selected genes in the colonies of wild yeast strains
Tarabová, Eva ; Kuthan, Martin (advisor) ; Heidingsfeld, Olga (referee)
Saccharomyces cerevisiae strains isolated from the wild are able to exhibit multicellular social behaviour and to form complex structured colonies resembling in many properties highly resistant biofilms of pathogenic yeasts. The capability of phenotypic variability, i.e. high frequency transition between two or more different phenotypes, is another feature typical for the wild yeast strains. Such phenotypic changes are in case of pathogenic yeast often connected with changes in virulence and resistance to stress and antifungal treatment. Long-term cultivation of the wild yeast strains under laboratory conditions leads to their domestication, i.e. transition to smooth colonies and loss of some features typical for structured colonies. This process is, similarly to phenotypic switching, accompanied by significant changes in gene expression and global change of colony lifestyle. Mechanisms underlying yeast phenotypic transitions are ascribed to epigenetic regulation of gene expression via transcriptional silencing conferred by histone deacetylases. This work deals with the study of such mechanisms using knock-outs of selected genes with putative function in formation of structured colonies in wild and domesticated strains. The achieved results show, that NAD+-dependent histone deacetylase Sir2p influences...
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Dynamics of selected DNA damage response proteins
Benada, Jan ; Hodný, Zdeněk (advisor) ; Kuthan, Martin (referee)
DNA damage response (DDR) represents a vital signaling network that protects genome integrity and prevents development of cancer. Therefore the study of DDR is of a crucial clinical importance and DDR proteins are promising therapeu- tic targets. Although the great advances have been made mapping out interac- tions between individual DDR proteins, better understanding of complex behav- ior of this network is still needed. One approach, which might help us in this task, is to describe the dynamics of key proteins under different conditions. The first objective of this study was to investigate whether the temporal dynamics of selected DDR proteins differ upon different genotoxic insults, particularly upon γ- irradiation and UV-C irradiation. We showed that under certain insult some DDR proteins exhibit a monotone continuous activation pulse, while the activation of others triggers a series of pulses. We observed a previously described pulsative dynamics of p53 after γ-irradiation in MCF7 cells. Interestingly, we detected a monotone increase of p53 in U2OS after γ-irradiation and similar dynamics upon UV-C irradiation. We suggest that p53 dynamics depends on the presence or ab- sence of effective negative feedback loops between the upstream p53-activating kinases and Wip1 phosphatase. In the second...
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The role of protein kinase StkP in regulation of the cell division in Streptococcus pneumoniae
Malíková, Eliška ; Doubravová, Linda (advisor) ; Kuthan, Martin (referee)
Protein phosphorylation by protein kinases is a key mechanizm that enables both eukaryotic and prokaryotic organizm sense and read environmental signals and convert these signals into changes in gene expression and thus proper biological response. One of the main phosphorylation systems in bacteria consists of eukaryotic-like Ser/ Thr protein kinases. The genome of human pathogen Streptococcus pneumoniae contains single Ser/ Thr protein kinase StkP. StkP regulates virulence, competence, stress resistance, gene expression and plays an important role in the regulation of cell division cycle. Analysis of phosphoproteome maps of both wild type and ΔstkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including the cell division protein DivIVA (NOVÁKOVÁ et al., 2010). DivIVA in S. pneumoniae is localized at midcell and at the cell poles. It was proposed to be primarily involved in the formation and maturation of the cell poles (FADDA et al., 2007). The aim of this thesis was to investigate phosphorylation of the cell division protein DivIVA in S. pneumoniae. Gene divIVA was cloned, expressed in E. coli and protein was purified via affinity chromatography. Phosphorylation of DivIVA by StkP was examined in a kinase assay. We confirmed that DivIVA is a direct...
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The Persistence of Human Polyomaviruses
Blažková, Kristýna ; Drda Morávková, Alena (advisor) ; Kuthan, Martin (referee)
Despite years of research, even the most scrutinized Polyomaviruses - BK and JC - have not yet been thoroughly understood. With a number of new Polyomaviruses - KIV, WUV, MCV, HPyV6, HPyV7, TSV and HPyV9 described in the past few years, the need to understand how Polyomaviruses operate in their hosts has become even more urgent. The probable route of transmission appears to be either respiratory or faecal-oral. The initial infection occurs most likely in the early childhood or early-adolescence and is followed by a life-long persistence. The seroprevalence of Human Polyomaviruses among healthy adult population is high: BKV (81-97 %), JCV (35-69 %), KIV (55 %), WUV (69 %), MCV (25-46 %) and TSV (70-80 %). Human Polyomaviruses can cause fatal diseases in immunocompromised patients. The site of persistence in humans probably varies depending on the specific Polyomavirus. BK and JC are known to persist in kidneys and the urinary tract. Human Polyomaviruses have been detected in the lymphatic tissues, blood, respiratory, urinary, and gastrointestinal systems. It is not clear, however, if they persist in all of these sites. Mechanisms which Polyomaviruses use to establish and maintain persistent infection could include the viral miRNA and viral agnoprotein, which would result in a modulation of viral...
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Screening for the HCV IRES interacting proteins
Roučová, Kristina ; Pospíšek, Martin (advisor) ; Kuthan, Martin (referee)
Hepatitis C virus (HCV) is a worldwide spread pathogen infecting up to 3 % of the human population. Nowadays, research of new drugs against this virus is focused on the individual steps in its life cycle, including the translation initiation. In the case of HCV translation initiation is dependent on the internal ribosome entry site (IRES). Besides of components of the translational machinery also other components of the cell, so called IRES trans-acting factors (ITAF), contribute to its proper progress. This work continues in previous research of our laboratory focused on searching for new ITAF. In order to search for potential ITAF increasing HCV IRES activity new recombinant plasmid vectors and reference strains were prepared and selection conditions of the selection system were optimized. The differences in the growth characteristics of the reference strains were analyzed and quantified under selective and non-selective conditions. A set of pilot high efficiency transformations of the yeast strain pJ69-4A carrying bicistronic construct with HCV IRES were conducted using human expression cDNA library in order to optimize the efficiency of transformation and selection conditions and to attempt to identify new ITAF. Several dozens of randomly selected clones from these transformations obtained under...
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Seroprevalence of polyomaviruses in human
Blagoevová, Kateřina ; Drda Morávková, Alena (advisor) ; Kuthan, Martin (referee)
Human BK polyomavirus, also known as Polyomavirus hominis type 1, is a small animal tumorigenic virus. It penetrates into the host cell by caveolin-mediated endocytosis and then through the ER pathway to get into the nucleus where the virus replicates and expresses viral proteins. BKV primary infection typically occurs during childhood and id mostly asymptomatic, it is only occasionally accompanied by mild respiratory or urinary tract illnesses associated with viruria. After primary infection the virus occurs mainly in the kidney and urinary tract and in immunocompetent individuals remains in nonreplicative state. Healthy individuals have no health problems and it persists as a lifelong infection. In immunosuppressed individuals, particularly renal and bone marrow transplant patients, causing virurie, viremia, ureteral stenosis and serious nephropathy, this can lead to graft failure. The prevalence of this virus in the world is significant, scientific publications indicate that up to 80% of the human population has specific antibodies. Any small amounts of the virus prevalence reported in some publications are probably caused by using various sensitive detection methods and different types of samples.
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Yeast gene silencing.
Tarabová, Eva ; Kuthan, Martin (advisor) ; Schierová, Michaela (referee)
Each cell contains a complete copy of the entire genetic equipment of the organism. However not all genes are expresed, cells are differentiated in higher eukaryots and only certain proteins are transcribed in each cell. This is possible thanks to a gene silencing, that is stable throughout the whole cell cycle and epigeneticaly inherited from one generation to another. Gene silencing serves also in the maintainance of the chromosomal integrity, it is connected with the right progression of the cell division. It even enables mating type switching and ensures right cells' identity in yeasts. The basis is compact and a higher-ordered structure of chromatin called heterochromatin. The mechanism is common to many various organisms, although the proteins, which ensure silencing, are different.
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Notch-independent functions of CSL transcription factors
Teska, Mikoláš ; Folk, Petr (advisor) ; Převorovský, Martin (referee) ; Kuthan, Martin (referee)
Notch pathway plays a critical role during development and life of Metazoan organisms. CBF1 is a component of the Notch pathway that mediates the regulation of target genes. The discovery of CBF1-like proteins in yeast raised the question of their function in unicellular organisms - before the origin of canonical Notch pathway. CBF1-homologs in yeast are conserved in parts that are important for DNA binding and bind to CBF1-binding elements in vitro. CBF1 and related transcription factors in Metazoa (CSL) interact with many proteins in Notch-dependent as well as Notch-independent complexes. The Notch receptor has likewise some CSL-independent functions. This assay reports about interacting partners of CSL in Metazoa along with homologous proteins in yeast with the aim to highlight potential interactions of CBF1-homologs in evolutionary ancestral context.
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Hybrid sterility genes in mice: mapping of epistatic interactions
John, Václav ; Forejt, Jiří (advisor) ; Kuthan, Martin (referee)
MOUSE HYBRID STERILITY GENES: A MAPPING OF EPISTATIC INTERACTIONS Hybrid sterility is an important postmeiotic reproductive isolation barrier. The phenotype of mouse hybrid sterility includes decreased testes weight and an absence of sperm. It occurs only in F1 males from PWD/Ph (PWD; Mus musculus domesticus) x C57BL/6J (B6, M. m. musculus) cross but not reciprocal B6 x PWD F1 males (FOREJT & IVÁNYI, 1974; FOREJT et al., 1991). Nowadays we know that the phenotype of hybrid sterility is controlled by the PWD/B6 allelic combination on chromosome 17 at Prdm9 locus is needed and by the PWD allele on chromosome X at Hstx2 locus. This combination of two loci is necessary but not sufficient obtain full sterility (MIHOLA et al., 2008; FOREJT, JIŘÍ, pers. comm.), so another genes must play role. In this experimental work I prepared the F2 cross from B6 and PWD mouse strains and I genotyped the males of F2 crosses and I carried out the QTL analysis. Extended QTL analysis was carried out with all animals carrying Prdm9PWD/B6 - Hstx2PWD allelic combination. The results showed unexpectedly low number of sterile males in F2 generation (1.90 %), and significant QTLs on chromosomes 17 (in Prdm9 loci) and X (different from Hstx2) in sperm, no other significant peak on any chromosome was found. These results indicate a more...
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Mammalian circadian clock in peripheral organs, molecular mechanism and entrainment
Polidarová, Lenka ; Kuthan, Martin (referee) ; Sumová, Alena (advisor)
Mammalian circadian clock in peripheral organs, molecular mechanism and entrainment The circadian system controls timing of behavioral and physiological processes in most organisms. In mammals, central oscillator is located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. Apart from the SCN, peripheral oscillators are located in numerous organs like liver, heart, lung, muscle, intestine etc. The central and peripheral oscillators need to be synchronized by external cues (Zeitgeber). The SCN coordinates and entrains the phase of the clocks in numerous peripheral tissues via neuronal and humoral signals. For the SCN, dominant synchronizer is external light-dark cycle. Peripheral oscillators are cell-autonomous, they could work also independently of the SCN as a consequence of a feeding cycle. The basic molecular core clock mechanism responsible for generating circadian rhythms in the central and peripheral clocks is composed of transcriptional/translational feedback loops between the clock genes and their protein products. The aim of the present thesis was to ascertain whether the clock gene and protein expressions exhibit circadian rhythms in the rat intestine and whether the core clock mechanism drives expression of a cell cycle regulator rWee1. Next aim was to reveal how the circadian...
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