|
|
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...
|
|
|
Molecular interactions of Staphylococcus aureus with the host
Cabrnochová, Marie ; Melter, Oto (advisor) ; Vopálenský, Václav (referee)
The aim of this thesis is to summarize S. aureus interactions with selected mechanisms of innate host immunity especially interactions with neutrophils and processes on the cell level which lead to host colonization. S. aureus surface proteins MSCRAMM interact with host cell surface proteins such as fibrinogen, keratin and thereby mediate adhesion to the host cell, which is an essential point for colonization of the host cell. The central mechanism of innate immunity against any S. aureus infection is the interaction of the pathogen with neutrophils, which produce neutrophil extracellular traps and phagocytes S. aureus cells. A crucial role in the elimination of bacterial cells in the phagosome of neutrophils is lysis by the antimicrobial peptides and degradation of bacterial biomolecules by the oxygen radicals. S. aureus defence mechanisms against action of immune system are considered to be virulence factors, due to its contribution to the establishment of the infection. These mechanisms are based on cell wall modification, inhibition of neutrophil chemotaxis, and production of enzymes that inhibit the effect of antimicrobial peptides, lysozyme, oxygen and nitrogen radicals. Expression of virulence factors of a particular S. aureus strain and host-specific risk factors can lead through successful...
|
|
|
Inducible promoters and their use in yeast cell manipulation
Přibáňová, Gabriela ; Palková, Zdena (advisor) ; Vopálenský, Václav (referee)
Promoters which can be regulated by different chemical or physical factors are often used in cell manipulations. This thesis focuses predominantly on promoter systems which use light as an inductor. There are two main approaches to controlling a promoter by light. The first one uses so-called "caged molecules", chemical inducers whose inducing activity is "masked" by a photolabile protecting group. The second approach includes optogenetic systems, which can regulate transcription in cells. These systems are encoded in the DNA of the organism, and light is the only external regulatory stimulus. Photoreceptors that need a specific cofactor (chromophore) are the main components of optogenetic systems. There are several groups of photoreceptors classified by the type of chromophore and photoactivation mechanism. This thesis gives an overview of optogenetic systems used for transcription regulation and focuses on different photoreceptors and induction mechanism used. The systems using photocaged molecules are described as well. Furthermore, the thesis deals with light- systems in yeast as a model organism as well as organism used for biotechnological purposes. Finally, some limitations of light inducible promoters are discussed, including the chromophore type, the wavelength of the light, and the...
|
|
| |
|
|
Importance of 5'-end structures of eukaryotic mRNA molecules
Vopálenský, Václav ; Pospíšek, Martin (advisor) ; Lukeš, Julius (referee) ; Staněk, David (referee)
73 V. SHRNUTÍ VÝSLEDKŮ IRESITE: THE DATABASE OF EXPERIMENTALLY VERIFIED IRES STRUCTURES (WWW.IRESITE.ORG) IRESite je kurátorovaná relační databáze typu MySQL-4.1 využívající InnoDB tabulky. Do databáze je možné vložit položky dvojího typu: o natural položky obsahují veškerá experimentální data týkající se určité IRES struktury (kompletní sekvence mRNA s vymezením pozic IRES sekvence a kódovaného proteinu, veškeré proteiny interagující s IRES, případná sekundární struktura) o engineered položky popisují uměle vytvořené plazmidy. Většinou se jedná o bicistronní plazmidy sloužící k testování funkčnosti sekvence, o níž se předpokládá, že obsahuje IRES element, a případné mutantní deriváty této sekvence. Do IRESite se v tomto případě vkládají údaje o sekvenci mRNA s vymezením IRES oblasti, informace o reportérových proteinech, informace o translačních experimentech včetně použitého promotoru a informace o pozitivních a negativních kontrolách. V databázi je nyní uloženo 288 položek; 67 natural (20 virových a 47 buněčných) a 221 engineered. IRESite slouží nejen k ukládání experimentálních dat, ale umožňuje také jejich prohledávání podle klíčových slov, případně následné porovnávání vybraných experimentů. A BIOINFORMATICAL APPROACH TO THE ANALYSIS OF VIRAL AND CELLULAR INTERNAL RIBOSOME ENTRY Publikace...
|
|
| |
|
| |
|
| |
|
| |
|
|
Induction of endogenous RNAi in mammalian cells
Demeter, Tomáš ; Svoboda, Petr (advisor) ; Vopálenský, Václav (referee)
Double-stranded RNA (dsRNA), a double helix formed by two antiparallel complementary RNA strands, is a unique structure with a variety of biological effects. dsRNA can be introduced into the cell from exogenous sources or it can be produced endogenously. There are four basic mechanisms producing dsRNA: inverted repeat transcription, convergent transcription, pairing of sense and antisense RNAs produced in trans, and RNA dependent RNA polymerase-mediated synthesis dsRNA. Different mechanisms of production determine additional structural features of dsRNA, such as dsRNA termini, mismatches etc. These features may affect cellular response to dsRNA. Recognition of dsRNA can trigger several responses that act in sequence-specific or sequence-independent manners. The main sequence- specific response triggered by dsRNA is RNA interference (RNAi) is. Our laboratory has been studying mechanism of induction of RNAi in mammalian cells using one specific type of long dsRNA expression system. The dsRNA used in these experiments formed hairpin structure with long 5' and 3' single-strand RNA overhangs. We hypothesized that other dsRNA substrates might be more efficient than the one used in mammalian RNAi experiments since 2002. Accordingly, the main aim of my thesis was to compare efficiency of different dsRNA...
|
guest ::
