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DNA-binding properties of the CSL proteins of Schizosaccharomyces pombe
Ptáčková, Martina ; Půta, František (advisor) ; Doležal, Pavel (referee)
As the effector component of the Notch signaling pathway the transcription factors of the CSL family (CBF1/RBP-Jκ/Suppressor of Hairless/Lag-1) are essential for many developmental processes in metazoan organisms, but they can function also independently of Notch. Recently, their presence was proved in fungal organisms lacking the Notch pathway as well as most of the known metazoan interacting partners. Cbf11 and Cbf12, the CSL proteins of the unicellular yeast Schizosaccharomyces pombe, were determined experimentally as non-essential nuclear transcription factors, which regulate cell adhesion, extracellular material production, colony morphology, septation and daughter cell separation, coordination of nuclear and cell division, and ploidy maintenance in an antagonistic way. The responsive genes of these factors are not known yet. In this study, genes of S. pombe, whose promoter regions represent potential direct targets for the Cbf proteins binding, were predicted. The binding of the Cbf11 and Cbf12 proteins, and of a truncated version Cbf12∆N to CSL response elements contained in the regulatory regions of selected S. pombe genes was tested in vitro by EMSA, and consequently, in the case of the Cbf11 protein, also in vivo by ChIP. Cbf11 and Cbf12∆N recognize specifically the response elements in...
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Význam rostlinných proteinů z podrodiny ABCB pro transport auxinu
Kubeš, Martin ; Zažímalová, Eva (advisor) ; Hejátko, Jan (referee) ; Reinöhl, Vilém (referee)
Polar auxin transport provides essential directional and positional information for many developmental processes in plants. At the cellular level, it is realized by both passive diffusion and the active transport through the membrane proteins - AUX1/LAXes, PINs and ABCBs. The aim of this thesis was to characterize the role of ABCB1, ABCB4 and ABCB19 proteins in polar auxin transport using transformed tobacco BY-2 cell lines. It was shown that the plasma membrane (PM) localization of the ABCB1, 4 and 19 is not polar. The ABCB4 was also more stable on PM after the treatment with auxin influx inhibitors; making use of ABCB4-cell line helped to uncover new characteristics of markers of endocytosis - the FM- dyes. The induction of ABCB19 has led to a decrease in 3 H-NAA accumulation with characteristic auxin starvation phenotype, similar to PIN7 overexpressing cell line, that could be rescued in case of PIN7 cell line by application of the auxin efflux inhibitor NPA; however, the accumulation of auxin in ABCB19-overexpressing cell line was less sensitive to NPA and the rescue of the auxin starvation phenotype was ineffective. Importantly, unique property of the ABCB4 was demonstrated: It displayed dual, auxin-concentration-dependent auxin transport activity in Arabidopsis roots, tobacco BY-2 and yeast cells. The...
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Bordetella Adenylate Cyclase: Molecular Mechanism of Action and Its Use for Antigen Delivery
Kamanová, Jana
Dissertation title: Bordetella Adenylate Cyclase: Molecular Mechanism of Action and Its Use for Antigen Delivery Author: Jana Kamanová The first part of this PhD. thesis deals with molecular mechanism of action of the adenylate cyclase toxin (CyaA), a key virulence factor of the whooping cough agent Bordetella pertussis. CyaA belongs to the family of RTX (Repeat-in-ToXin) proteins secreted by Gram-negative bacteria and primarily targets myeloid phagocytes, expressing the CD11b/CD18 integrin receptor (also known as αMβ2, CR3 or Mac-1). Upon binding, CyaA permeabilizes cell membranes by forming small cation-selective pores, and subverts cellular signaling by delivering into host cells an adenylate cyclase (AC) enzyme that converts ATP to cAMP. Elevation of the cytosolic cAMP levels by CyaA then knocks down bactericidal functions of host innate immunity. CyaA is unique among other enzymatically active toxins in its capacity to penetrate cells directly from cell surface across the cytoplasmic membrane, without the need for endocytosis. Penetrating activity of CyaA depends on plasma membrane potential and on an intact, acylated and calcium-loaded RTX cytolysin moiety. By examining a set of 18 CyaA constructs that bear overlapping deletions within AC domain and a CD8+ OVA T-cell epitope tag, we showed that the...
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The TUB3 intron splicing in PRP45 mutant cells
Konířová, Jana ; Půta, František (advisor) ; Zimmermannová, Olga (referee)
Protein Prp45, an essential factor of the yeast Saccharomyces cerevisiae, is implicated in pre-mRNA splicing. A truncated version of the PRP45 gene, prp45(1-169), which exhibits a temperature sensitivity, was previously prepared in our laboratory. The aim of this work is to contribute to better understanding of prp45(1-169) mutant phenotype. We tested the prp45(1-169) strain for its response to microtubule inhibitor benomyl and then we found that TUB3 overexpression from plasmid rescues discovered prp45(1-169) mutant cells hypersensitivity to benomyl. In addition, we studied the influence of TUB1, TUB3, and COF1 intron deletion on prp45(1-169) strain temperature sensitivity. Using RT-qPCR method we found that prp45(1-169) mutation results in the distinctive increase of pre-mRNA level for all tested genes, that could implicate that pre-mRNA splicing in these cells is affected before first transesterication.
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Metal chip management of production machine
Bílek, Vít ; Zbožínek, Adam (referee) ; Holub, Michal (advisor)
This thesis is concerned with cutting farm machine and it consists of three parts. Theoretical part describes mechanism of chip formation, shape of the chips, temperature of the chips, technological variables describing chips, chip conveyor, chip crusher, briquetting press and coolant filter. In the next part there is proposal of design of cutting farm machine for production cell consists of six machining center. At last part there is design proposal and calculation of chip conveyor.
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Synchronization of circadian system during prenatal and early postnatal development
Houdek, Pavel ; Sumová, Alena (advisor) ; Novotná, Růžena (referee)
One of the few attributes common to almost all living organisms is an ability to generate and maintain endogenous rhythms, which are controlled by a biological clock. The processes, which recur with a period of about 24 hours, are known as the circadian rhythms. The circadian clock controls rhythms of molecular, physiological as well as behavioral processes and adapts their activity to regularly appearing changes in day and night or season. In case of mammals, central oscillator is located in the hypothalamic suprachiasmatic nuclei (SCN). The SCN clock entrains rhythms of peripheral oscillators located in cells of other tissues. The central oscillator itself is synchronized with external environment mainly by a light-dark cycle, however, other cues can entrain the SCN clock as well. For example, during prenatal development, entrainment of a fetal clock is entirely dependent on non-photic cues derived from maternal organism. This study aimed to investigate a mechanism of the communication between the maternal and fetal central oscillators. A hypothesis was tested whether maternal melatonin may play a role in entrainment of the circadian clock in the fetal SCN. Furthermore, a mechanism, how melatonin may entrain the fetal clock was investigated at molecular level. The results provided evidence, that...
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Influence of some foods on growth and viability of probotic bacteria
Vajglová, Klára ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
The goal of this work was a study the influence of food and beverages on the viability and growth of probiotic bacteria. The influence of food and beverages was tested on monocultures of Lactobacillus acidophilus, Bifidobacterium breve and mixed culture of probiotic microorganisms. In the experimental part, probiotic cultures were incubated in selected foods and beverages. After that they were tested in a model conditions of digestive tract. In some probiotic cultures, growth of viable cells during incubation in the digestive tract was observed. The increase of probiotic cells was showed predominantly in foods that contained higher levels of sugars and fats or a suitable combination. Their increase was up to four times in some cases. Based on the results, mixed probiotic cultures aren’t surprisingly exhibited better survival and maintain sufficient amount of viable cells even during the digestive process. Moreover, probiotic microorganisms could be recommended to consumption during meals better than just with a beverage.
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Biotyping of ascomycetous yeasts
Jurnečková, Alena ; Dudášová,, Hana (referee) ; Stratilová, Eva (advisor)
In total, 84 yeast strains (originated from water, plants, fruits and soil) were selected for MALDI-TOF biotyping. All strains were cultivated on malt agar and YPD medium. Samples for biotyping were processed according to methods of Bruker Daltonik GmbH company, Institute of Chemistry of SAS and combination of these methods. Single strains were identified based on the analysis of intracellular ribozomal proteins using MALDI-TOF mass spectrometry. In case of ambiguous results, the DNA was isolated and the D1/D2 26S rRNA domain sequencing was performed. The strain identification was carried out by comparing its mass spectra with spectra of sequenced strains using MALDI Biotyper 3.0 software. The mutual similarity of strains was considered by score value, which was the result of the analysis. In total, 18 strains from 84 were previously sequenced and used as model strains for comparison with unknown isolates. Altogether 51 strains were definitely taxonomically categorized into 18 phylogenetic groups at the species level. The MALDI-TOF biotyping was repeated for overall 6 strains because of ambiguity of results. The taxonomic classification of 15 strains was not clearly determined and, therefore, these strains were suggested for D1/D2 26S rRNA domain sequencing. It was not possible to identify one strain, based on the results of sequencing, therefore, the DNA isolation was repeated. In the case of 8 strains, the results were identical with originally designed taxonomic classification. Conversely, the remaining 6 strains were identified as species. For 20 selected strains the basic characteristics were determined using microbiological methods. The shape of colonies growing on solid medium and appearance of cultures in liquid medium was assessed. Furthermore, the radial growth constant and the presence of urease were determined. Finally the microscopic observation of cells and the fermentation test for carbohydrate substrates were performed.
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The role of Prp45p in mRNA expression and maturation
Abrhámová, Kateřina ; Půta, František (advisor) ; Pichová, Alena (referee) ; Janderová, Blanka (referee)
Prp45p of Saccharomyces cerevisiae and Snw1p of Schizosaccharomyces pombe are essential proteins, which share extensive homology with the mammalian transcription regulator and splicing factor SNW/SKIP. We have analyzed the essential function of these proteins in both yeasts and found a mutation (prp45(1-169)) that exhibited temperature sensitivity. The mutant strain harboring the corresponding chromosomal deletion shows temperature sensitive phenotype and hypersensitivity to cycloheximide, hydroxyurea, calcofluor white, and to microtubule inhibitors. At 30řC, the cells are often elongated, deformed, and larger than wt. After synchronization prp45(1-169) cells stop their growth with 2N DNA content at 37řC. We found that the temperature sensitivity is not overcome and the hypersensitivity to microtubule destabilizing drugs is only partially suppressed by the excision of intron from TUB1 gene. This distinguishes prp45(1-169) from those splicing factor's mutants that cause tubulin-dependent G2/M arrest, which can be relieved by the expression of intronless tub1. We performed analysis of splicing in vitro and found that splicing of optimal substrates is not impaired. We also compared the content and stability of RNA in wt-cells and in prp45(1-169) cells at variol temperatures using microarrays. The...
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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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