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Use of transcriptomics to study mechanism of the action of complex mixtures of organic compounds occurring in the ambient air focusing on polycyclic aromatic hydrocarbons
Líbalová, Helena ; Topinka, Jan (advisor) ; Krásný, Libor (referee) ; Postlerová, Pavla (referee)
Polycyclic aromatic hydrocarbons (PAH) represent a large group of organic compounds occuring as pollutants in ambient air. Besides their genotoxic effect, some of them are known to be complete carcinogens and act via nongenotoxic and tumor promoting mechanism. Although effects of many individual compounds are well-documented, human exposure to polycyclic aromatic hydrocarbons in ambient air occurs through complex mixtures and only few studies describe the behavior of PAH in real complex mixtures. The first part of the thesis is dealing with the global gene expression changes in human embryonic lung fibroblasts (HEL) as a consequence of the effect of complex mixtures containing PAH extracted from the respirable airborne particles PM2.5. These particles were collected in 4 localities in the Czech republic (Ostrava - Bartovice, Ostrava - Poruba, Karviná, Třeboň) differing in the level of the air pollution. Gene expression changes induced by three subtoxic concentrations of organic extracts (EOM - extractable organic matter) from each locality after 24 hour incubation were examined by microarray analysis. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was applied to interpret gene expression data. In each locality we identified several deregulated signaling pathways...
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Regulation of alternative splicing via chromatin modifications
Hozeifi, Samira ; Staněk, David (advisor) ; Krásný, Libor (referee) ; Lanctôt, Christian (referee)
Alternative splicing (AS) is involved in expansion of transcriptome and proteome during cell growth, cell death, pluripotency, cell differentiation and development. There is increasing evidence to suggest that splicing decisions are made when the nascent RNA is still associated with chromatin. Here, I studied regulation of AS via chromatin modification with main focus on histone acetylation. First, we demonstrate that activity of histone deacetylases (HDACs) influences splice site selection in 700 genes. We provided evidence that HDAC inhibition induces histone H4 acetylation and increases RNA Polymerase II (RNA Pol II) processivity along an alternatively spliced element. In addition, HDAC inhibition reduces co-transcriptional association of the splicing regulator SRp40 with the target fibronectin exon. Further we showed that histone acetylation reader, Brd2 protein, affect transcription of 1450 genes. Besides, almost 290 genes change their AS pattern upon Brd2 depletion. We study distribution of Brd2 along the target and control genes and find that Brd2 is specifically localized at promoters of target genes only. Surprisingly, Brd2 interaction with chromatin cannot be explained solely by histone acetylation, which suggests that other protein-domains (in addition to bromodomains) are important for...
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Complex structural and functional analysis of individual subunits of yeast translation initiation factor 3.
Herrmannová, Anna ; Valášek, Leoš (advisor) ; Krásný, Libor (referee) ; Pospíšek, Martin (referee)
5 ABSTRACT Translation initiation in eukaryotes is a complex process promoted by numerous proteins or protein complexes called eukaryotic initiation factors (eIFs). The eukaryotic initiation factor 3 (eIF3) is a multisubunit complex that has been implicated in several steps of the translation initiation pathway. In yeast Saccharomyces cerevisiae, eIF3 is composed of five essential subunits (a/TIF32, b/PRT1, c/NIP1, g/TIF35, i/TIF34) and one nonessential subunit (j/HCR1). It is our long-term goal to understand how eIF3 promotes different stages of translation initiation and which subunits or their domains play a critical role in this process as well as to map the binding sites of eIF3 on 40S ribosomal subunit and to create a structural model of eIF3 complex. Here I present two structural studies showing interactions between the RNA recognition motif of eIF3b and a short peptide of eIF3j subunits of human eIF3 solved by NMR spectroscopy, and a crystal structure of the C-terminal fragment of yeast b/PRT1 in complex with the full length i/TIF34 subunit at 2.2 Å resolution. In the former study, me and my colleagues showed that the critical determinants mediating this eIF3b-eIF3j interaction are evolutionary conserved, since their mutations in yeast proteins reduced cellular growth rate, eliminated j/HCR1...
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Characterization of the HelD protein from Bacillus subtilis
Sudzinová, Petra ; Krásný, Libor (advisor) ; Lichá, Irena (referee)
BACKGROUND: Bacterial RNA polymerase (RNAP) is an extensively studied enzyme required for gene expression. In our Laboratory we found a new protein named HelD. HelD copurifies with B. subtilis RNAP. HelD is a ~90 kDa protein from the UvrD/Rep helicase family, which contains protein with the 3'-5' DNA unwinding activity. The molecular role(s) HelD in cell are still unknown and its potential role in transcription has not been studied so far. OBJECTIVE: The main aim of this Diploma project was to describe HelD. APPROACHES: The characterization was carried out on three levels: (i) bioinformatics analysis in silico was used to identify HelD homologs in other bacteria; (ii) growth tests in vivo were used to determine the phenotype(s) of the HelD-null mutant strain compared to wt; and (iii) biochemical experiments in vitro were utilized to describe the effects of HelD on transcription, and to test whether HelD has DNA binding and DNA unwinding activities. RESULTS: The in silico analysis revealed that HelD is present in Firmicutes, an industrially and medicinally important group of G+ bacteria. The phenotypic experiments showed that HelD is required for rapid adaptations to nutritional changes in the environment. The biochemical experiments showed that HelD stimulates transcription despite the fact that it...
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Characterization of transcription apparatus encoded by the linear plasmids of the yeast Kluyveromyces lactis
Sýkora, Michal ; Vopálenský, Václav (advisor) ; Krásný, Libor (referee)
Transcription is an essential step in the expression of genetic information. This process depends on protein complex of multisubunit RNA polymerases that are exceptionally conserved among all cellular organisms. These enzymes together with eukaryotic RNA-dependent RNA polymerases involved in gene silencing form a monophyletic protein family whose members contain two double-ψ β-barrel structural motifs in their active center. This family also includes a group of mainly in silico predicted non-canonical DNA-dependent RNA polymerases which differ from multisubunit RNA polymerases in reduced composition. Putative non-canonical RNA polymerase consisting of two subunits is also encoded by cytoplasmic linear plasmids of the yeast Kluyveromyces lactis and highly likely transcribes genes of these plasmids. Characterization of a unique transcription machinery of Kluyveromyces lactis plasmids with major emphasis on non-canonical RNA polymerase has become the aim of this work. Bioinformatic analysis in silico was used to examine the evidence leading to an assumption of existence of specific RNA polymerase. Subsequent genetic and biochemical methods were used for: 1) production of putative RNA polymerase subunits in several expression systems; 2) testing interaction between several components of transcription...
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Effect of knock out of yxkO gene on environmental stress adaptation in genus Bacillus
Tkadlec, Jan ; Lichá, Irena (advisor) ; Krásný, Libor (referee)
We have previously characterized a Bacillus subtilis mutant defective in growth and osmoadaptation under limited K+ concentrations. In this mutant, the yxkO gene encoding a putative ribokinase is disrupted. This gene is supposed to belong to the sigma B operon and its expression is induced after osmotic, heat and ethanol shock. In comparison to the wild type, this mutation causes pleiotropic changes in host phenotype. In addition to its osmosensitivity, the mutant differs in cell shape, motility and ability to produce endospores. Our goal was to focus on manifestations of the mutation in the yxkO gene in other bacteria of the genus Bacillus. Using plasmid pMUTIN4 we have prepared mutants with disruptions of this gene derived from Bacillus amyloliquefaciens and Bacillus subtilis subsp. spizizenii strains differing in the yxkO surroundings and in the level of laboratory domestication. As in the previous study (with laboratory strain Bacillus subtilis 168) we demonstrate impaired ability of the mutant strain derived from Bacillus amyloliquefaciens to grow in potassium limitation and osmotic shock. We have studied this phenomenon at the level of the growth dynamics of the bacterial culture. We have also detected an increased sensitivity of the strain derived from Bacillus amyloliquefaciens to...
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Mapping of regulatory elements within 5' region of the Disp3 locus
Oltová, Jana ; Bartůněk, Petr (advisor) ; Krásný, Libor (referee)
Dispatched 3 (Disp3), a thyroid hormone-regulated gene, is studied extensively in our laboratory. Phenotype of cells with overexpressed Disp3 and its expression pattern make it a perfect candidate for a molecular link between thyroid hormone action and cholesterol homeostasis in the brain. Moreover, we hypothesize that it might play a role in certain neurodegenerative disorders and brain tumours. This thesis is aimed at the process of regulation of this gene via thyroid hormone receptor (TR), specifically identification of responsive elements of the thyroid hormone receptor that are necessary for the regulation. Also, we searched for elements recognized by liver X receptor (LXR), as LXR binds to the same arrangement of repeats as TR and there are a number of genes regulated by both of them. We combined in silico analysis of the Disp3 locus with reporter luciferase assays. A cluster of six elements identified around the first exon with two of them being conserved among human and mice draw our attention. In order to analyze this sequence in more detail, reporter vectors of various truncations of 3 kb region around exon 1 were constructed and tested in reporter assays. Reporter assays did not reveal any substantial element activated by TR or LXR; on the other hand, region containing repressor element(s)...
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Factors interacting with bacterial RNA polymerase
Sudzinová, Petra ; Krásný, Libor (advisor) ; Fišer, Radovan (referee)
The bacterial cell must be able to rapidly change its gene expression to survive unstable external conditions. Transcription is the key level that affects gene expression. The pivotal enzyme of transcription is RNA polymerase (RNAP). Activity of RNAP is tightly regulated by transcription factors (TFs). These factors affect RNAP in different ways. This work presents an overview of various proteins and others factors, description of their effects on transcription and also mechanisms of their actions. TFs could be divided according to various criteria. In this work, TFs are divided according to how they interact with RNAP: TFs interacting only with RNAP; TFs binding simultaneously DNA and RNAP; TFs interacting with RNA and RNAP. This work presents a comprehensive overview of various TFs that are involved in the bacterial cell's reprogramming of gene expression that is required to withstand the changes in the environment.
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Stress proteins in the cytoplasmic membrane fraction of Bacillus subtilis
Šemberová, Lenka ; Svobodová, Jaroslava (advisor) ; Krásný, Libor (referee)
Stress proteins in the cytoplasmic membrane fraction of Bacillus subtilis A primary habitat of the Gram-positive bacterium Bacillus subtilis is the upper layer of the soil. Within this ecosystem, B. subtilis experiences a wide variety of environmental challenges and nutrient limitations that induce several mechanisms to help the cells to survive. A process known as a general stress response belongs among them. Expression of about 150 genes is enhanced and their products called general stress proteins (GSP) minimize the cell damage. Although the size and structure, as well as the regulation, of many stress proteins have been fairly well elucidated the information about stress membrane proteins is very limited. We characterized the membrane proteome of Bacillus subtilis 168 trp2- exposed to acidic pH and ethanol. Cells were 1) grown under optimum conditions in complex medium (pH 7.0) at 40řC with aeration; 2) grown in complex medium at pH 5.0 for 20 hours or 3) challenged with 3% v/v ethanol for 30 minutes during exponential growth. The cultures were harvested in the mid-exponential phase by rapid filtration. Isolated membrane fractions were analysed by an optimized two-dimensional gel electrophoresis. Two alternative methods of protein detection were compared: silver staining and radioactive labeling with...
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