National Repository of Grey Literature 51 records found  1 - 10nextend  jump to record: Search took 0.01 seconds. 
Factors affecting gene expression in Bacillus subtilis
Sudzinová, Petra ; Krásný, Libor (advisor) ; Vopálenský, Václav (referee) ; Vohradský, Jiří (referee)
Bacterial DNA-dependent RNA polymerase (RNAP) is a key enzyme of bacterial transcription. Its activity must be tightly regulated. This could be done on the level of promoter DNA topology recognition, by changing the intracellular levels of metabolites, or by binding proteins, known as transcription factors. Even though the RNAP regulatory network has been intensively studied for decades, new regulators are still being described. The main focus of this Thesis is to characterize some of them: i) HelD, a novel RNAP interacting factor, with so far unknown protein 3D structure; ii) RNase J1, an enzyme with a unique mechanism of functioning; iii) Spx, a major regulator of gene expression in Bacillus subtilis, with still new roles to be defined and iv) the effect of the topological state of promoters on transcription. We identified HelD as an interacting protein of RNAP in Bacillus subtilis and described its biochemical properties. It stimulates transcription in an ATP-dependent manner, by enhancing recycling of RNAP molecules (Publication I). We published the first insight into the HelD structure by SAXS (small angle X-ray scattering) and deepened the understanding of HelD domain composition (Publication III). And finally, we were able to solve the cryo-EM structure of HelD:RNAP complexes from...
The role of elF3 a Rps3 in stop codon readthrough
Poncová, Kristýna ; Valášek, Leoš (advisor) ; Vopálenský, Václav (referee) ; Krásný, Libor (referee)
Translation represents a highly regulated, interconnected process of protein synthesis in the cell. It could be divided into 4 phases: initiation, elongation, termination, and ribosomal recycling. Our laboratory is involved in in-depth studies of a complex eukaryotic initiation factor 3 protein (eIF3). We are interested not only in revealing its molecular roles in the translational cycle in general but also in specific mechanisms that allow translational regulation according to specific cellular needs. In the budding yeast, the eIF3 is composed of five essential subunits (a/Tif32, b/Prt1, c/Nip1, g/Tif35 and i/Tif34). In mammals, the protein is even more complex, comprising of 12 subunits (a-i, k-m). eIF3 is a key player not only in translation initiation but also in ribosomal recycling and, surprisingly, in translation termination and stop codon readthrough as well. The latter process harbors important clinical potential, as approximately 1/3 of genetically inherited diseases is caused by the presence of a premature termination codon in the protein-coding region. Therefore, understanding the molecular mechanism underlying this phenomenon provides important tools for the targeted and less toxic drug development approaches needed for patient therapy. In this Ph.D. Thesis, I uncovered the role of...
Development of a technique for gene transfer into T-lymphocytes using polyomavirus structures and the LAH4 peptide
Schreiberová, Lucie ; Španielová, Hana (advisor) ; Vopálenský, Václav (referee)
Efficient delivery of genetic material to T-lymphocytes is key in gene therapy using T-lymphocytes with chimeric antigen receptors. Current procedures require the use of potentially dangerous viral vectors or large amount of input material. The diploma thesis therefore focuses on exploring new approaches for gene transfer into T-lymphocytes: use of safe virus-like particles (VLPs) derived from mouse polyomavirus in combination with the amphipathic cationic peptide LAH4. LAH4 has the potential to increase the efficiency of DNA and viral vector transport into cells. The system which combines VLPs and the LAH4 peptide was optimized for the delivery of reporter gene (encoding GFP and luciferase) to the model T-cell line Jurkat. It has been found that Jurkat cells cannot be efficiently transduced by DNA packed into VLPs. When cells were transfected only with DNA and LAH4, consistent results were not obtained, and the transfection efficiency ranged from 0.5 to 19%. The diploma thesis also analysed the effect of phosphorylation of viral structures on gene transfer. The impact of treatment of virus particles by alkaline phosphatase on the infectivity of the virus was studied and it was necessary to analyse the effect of the reaction components. Sublytic concentration of Triton-X100 in the reaction buffer...
The effect of selected endogenous and exogenous factors on bacterial growth
Šiková, Michaela ; Krásný, Libor (advisor) ; Valášek, Leoš (referee) ; Vopálenský, Václav (referee)
The growth of bacteria by binary division is a key characteristic of these organisms. This growth depends on two types of factors: endogenous and exogenous. Endogenous factors make up the molecular apparatus of cells. Among important endogenous factors belong also those involved in gene expression and its regulation. Exogenous factors are external conditions such as nutrient availability, temperature, pH, various stresses or the presence of antibacterial agents. The main aim of my Thesis was to study the effects of selected endogenous and exogenous factors on bacterial growth. As endogenous factors I studied RNase J1 in Bacillus subtilis and a small RNA called Ms1 in Mycobacterium smegmatis, which are involved in regulation of gene expression at the transcriptional level. I showed that RNase J1 can, besides its role in RNA degradation, play a role in genome integrity by removing stalled RNA polymerase (RNAP) complexes from DNA. I further showed that Ms1 binds to the RNAP core and affects the level of RNAP in the cell. The results revealed new mechanistic aspects of the transcription apparatus and show how individual components or their combinations affect bacterial growth. As exogenous factors I studied the recently discovered antibacterial compounds, called lipophosphonoxins, their interaction...
Effect of adenosine deaminase acting on RNA on viral infection of eukaryotic cells
Kubů, Martin ; Vopálenský, Václav (advisor) ; Fraiberk, Martin (referee)
Double-stranded RNA is a molecule rarely found in a cell, but it is specific for viral infection. It is also a substrate of ADAR enzymes. These enzymes convert adenosin to inosine, which is recognized as guanosine by cellular machinery. Apart from editing activity, ADAR enzymes interact with cellular proteins, such as Dicer and protein kinase R, which together with editing affects viral replication. In this work, the information about antiviral activity of ADAR enzymes and their impact on infection of selected primarily human viruses is reviewed.
Analysis of miRNAs in HPV-associated carcinomas
Pagáčová, Lucie ; Tachezy, Ruth (advisor) ; Vopálenský, Václav (referee)
Papillomaviruses are small DNA viruses that are associated with the induction of epithelial tumors. HPV is an important infectious agent causing almost 100 % of cervical tumors but it can also cause tumors in other anogenital and head and neck locations in both men and women. Active HPV infection induces changes in miRNA expression that contribute to the tumor formation and progression. It is already known that papillomaviruses do not encode their own viral miRNAs but they affect the expression of cellular miRNAs. In my thesis I have in selected epithelial tumors (vulva, cervix, anus and tonsils) determined their etiology and analyzed the presence of miRNAs in tissues by next generation sequencing. From these data I determined the expression profiles of deregulated miRNAs in tumors relation to healthy tissues of corresponding location. Even though, sufficient number of samples was analyzed, it was not possible to detect HPV-core miRNA common to all analyzed HPV-induced tumors due to the absence of statistically relevant differentially expressed miRNAs in HPV positive vulvar tumors. Among the tumors of the other sites I found an overlap in three miRNAs. One of these miRNAs (miR-139-5p) and another one (miR-9-5p) which I have selected based on the study of other published data, were used for...
Characterization of non-canonical RNA polymerase encoded by the yeast linear plasmids
Sýkora, Michal ; Vopálenský, Václav (advisor) ; Macíčková Cahová, Hana (referee) ; Valášek, Leoš (referee)
Transcription is the control point of gene expression. This process relies on protein complex of multisubunit RNA polymerases, which are extremely conserved among all cellular organisms. Transciption of extrachromosomal hereditary elements such as organelles, viruses and plasmids is dependent on host cellular RNA polymerases or intrinsic RNA polymerase is contained within these elements. Putative non-canonical two-subunit RNA polymerase is also encoded by linear cytoplasmic plasmids of the yeast Kluyveromyces lactis and most likely transcribes genes of these plasmids. Besides the two subunits of RNA polymerase encoded by linear plasmids of Kluyveromyces lactis there are another two estimated components of the transcription apparatus, namely capping enzyme that adds the cap to 5' mRNA ends and putative DExD/H box helicase. Characterization of the unique and underexplored transcription machinery of Kluyveromyces lactis plasmids was the principal objective of this work. The main goal was to: 1) clarify evolutionary origin of the linear plasmid transcription apparatus; 2) describe architecture of the linear plasmid transcription complex in vivo focused on putative RNA polymerase binding partners; 3) reveal mechanisms of transcription initiation and termination of the yeast linear plasmids. The main...
Role of RNA helicases in antiviral defense
Krbušek, David ; Vopálenský, Václav (advisor) ; Janovec, Václav (referee)
Hepatitis C virus is an important human pathogen against which there is no immunization yet. This virus is detected by the immune system of the eukaryotic host cell by pattern recognition receptors of the RLR receptor family, which is part of the innate immune system. These RLR receptors detect the presence of hepatitis C virus and initiate a signaling cascade triggering an antiviral immune response. In this thesis, the role of cytoplasmic PRRs involved in antiviral defense during hepatitis C virus infection of eukaryotic cells has been described and determined. Key words Helicase, RIG-I, MDA5, pattern-recognition receptors, HCV, virus
Interaction of nucleic acids with RNA polymerase
Janoušková, Martina ; Krásný, Libor (advisor) ; Vopálenský, Václav (referee) ; Knejzlík, Zdeněk (referee)
Regulation of gene expression by RNA polymerase (RNAP) is an essential ability of living organisms, required for their adaption to a changing environment and ultimately enabling their survival. Interaction of RNAP with ribonucleic acids (DNA or RNA) is crucial for transcription and its regulation. This Doctoral Thesis contains two projects addressing interactions of RNAP with nucleic acids: (i) Transcription of modified DNA templates and (ii) Ms1, a small RNA (sRNA) from M. smegmatis. (i) We investigated the influence of modifications in the major groove of DNA on bacterial transcription in vitro. We found out that transcription of modified DNA templates is influenced on the transcription initiation level and that the promoter sequence is important for the effect of the modifications. Furthermore, we successfully performed transcription switch ON and OFF in vitro by bioorthogonal reactions. This regulation of transcription by artificial DNA modifications has a future in biotechnologies and/or medical therapy. (ii) Regulators of transcription are also small non-coding RNAs. These molecules have an important role in gene expression regulation among prokaryotes and eukaryotes. Ms1 is an sRNA found in mycobacteria. It makes a complex with the RNAP core and it is abundant in stationary phase (in amounts...
Bacterial RNA polymerase and molecules affecting its function
Jirát Matějčková, Jitka ; Krásný, Libor (advisor) ; Vopálenský, Václav (referee) ; Staněk, David (referee)
RNA polymerase (RNAP) transcribes DNA into RNA and is the only transcriptional enzyme in bacteria. This key enzyme responds to external and internal signals from the cell, resolves the intensity of transcription of individual genes and thus regulates gene expression. RNAP is not only affected by its own subunits, but also protein factors, small molecules or small RNAs (sRNAs). The aim of this Thesis was to contribute to the understanding of the regulation of the RNAP and to add missing fragments to this broad topic. The first part of this Thesis is focused on the influence of selected proteins (δ, YdeB, GreA) on the sensitivity of RNAP to the concentration of the initiating nucleoside triphosphate ([iNTP]) during transcription initiation in Bacillus subtilis. We showed that δ affects the sensitivity of RNAP to [iNTP] at [iNTP]-sensitive promoters, but not at [iNTP]-insensitive promoters neither in vitro nor in vivo. The δ subunit is essential for cell survival during competition with other strains, because it enables the cell to react immediately to changing conditions. Further we showed that YdeB protein does not bind to RNAP in B. subtilis, and has not shown any effect on transcription in vitro. We found that both, GreA and YdeB proteins (unlike δ subunit) were unable to affect RNAP by [iNTP] at...

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