|
|
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...
|
|
|
Role of phosphorylation in nuclear import of viral proteins and complexes
Pokorná, Karolína ; Forstová, Jitka (advisor) ; Roučová, Kristina (referee)
Replication of many different viruses occurs in the nucleus of the host cell. These viruses discovered ways how to overcome the nuclear membrane and often use cell transport machinery to transport their proteins and genome into the nucleus. For many viral proteins the timing of their nuclear import in order to secure correct viral replication is important. Regulated nuclear import also allows these proteins to perform several functions depending on their localization. Nuclear import of viral proteins and complexes can be regulated by phosphorylation. Phosphorylation can, for example, modulate affinity of proteins for importins or other cellular proteins. Phosphorylation can also cause conformational change, which can lead to unmasking of localization sequence.
|
|
|
Extraribosomal function of ribosomal proteins
Gredová, Alexandra ; Folk, Petr (advisor) ; Roučová, Kristina (referee)
Ribosomal proteins are important not only because they enable proteosynthesis, but also because of their functions outside of ribosome - in their extraribosomal functions. This thesis is mainly focused on the extraribosomal functions of higher eukaryotic ribosomal proteins. Specifically, it analyzes three ribosomal proteins, L13a, L22, and S3, which illustrate several aspects of extraribosomal functions. It discusses the mechanisms of action of these proteins and how they affect various levels of gene expression. Key words: extraribosomal function, L13a, L22, S3, p53, NF - κB, splicing
|
|
|
Regulatory network controlled by sigma factors of RNA polymerase in Corynebacterium glutamicum
Kučera, Tomáš ; Pátek, Miroslav (advisor) ; Roučová, Kristina (referee)
An important feature of bacteria is an ability to adapt to changing environment by regulating gene expression. Level of gene expression and its right timing depends mainly on activation or repression of the gene by transcriptional regulators and recognition of the respective promoter by the sigma factor which is a subunit of RNA polymerase. Transcription regulators with sigma factors and other control elements, form a complex regulatory network. The regulatory network in Corynebacterium glutamicum is one of the best studied networks among gram-positive bacteria owing to genome sequencing and application of a number of techniques at the genome level. There has been a lot of success in understanding the roles of individual regulators and interactions between regulators in response to changes in environment. This work summarizes currently known knowledge of mutual relationships between sigma factors, the influence of sigma factors on transcriptional regulators and their cooperative effect on the initiation of transcription. In the thesis, a regulatory network of sigma factors in C. glutamicum and a regulatory cascade in response to the stress situation is schematically created. Key words: sigma factor (FS), Corynebacterium glutamicum, transription regulator (TR), transcription, regulation
|
|
|
Domain structure and function of primary bacterial sigma factors
Kálalová, Debora ; Krásný, Libor (advisor) ; Roučová, Kristina (referee)
Transcription initiation is one of the crucial steps of gene expression. A multisubunit enzyme RNA polymerase (RNAP) transcribes the genetic information from DNA to RNA. However, RNAP itself is unable to recognize a specific promoter and initiate transcription. For this purpose, bacteria have a protein called σ factor, which binds to RNAP and together form the RNAP holoenzyme. In this thesis I describe the mechanism of bacterial transcription and the structure, function and regulation of σ factors. I focus mainly on the primary σ factors of two important model species, namely gramnegative Escherichia coli and grampositive Bacillus subtilis. I describe them in the context of alternative σ factors, and I point out their differences in structure, function and regulation. Key words: RNA polymerase, primary σ factors, transcription, bacteria, Bacillus subtilis, Escherichia coli
|
|
|
Effect of epigenetic modifications on replication and transcription of hepatitis B virus DNA
Hofman, Tomáš ; Hirsch, Ivan (advisor) ; Roučová, Kristina (referee)
The hepatitis B virus forms in a nuclei of infected hepatocytes a stable cccDNA resistant to degradation and capable to persist for many years. The main role in the persistence of cccDNA play the epigenetic mechanisms that regulates viral transcription and replication in various stages of the infection. Major epigenetic mechanisms involved in the regulation of HBV include CpG islands methylation, cccDNA-bound histone acetylation and methylation and RNA interference. CpG methylation is associated with the activity of capsid protein, which binds to the cccDNA and promotes transcription and structurally distinguishes viral and the host genome. HBx protein besides CpG methylation mainly affects histone modifications via its interaction with host proteins and thus eliminates the inhibitory effects and promotes the active modifications favoring virus transcription. This work summarizes current knowledges concerning the cccDNA epigenetic regulation. Keywords: HBV, cccDNA, replication and transcription of HBV DNA, histone modifications, methylation of CpG, CpG islands, HBx, HBc, IFNα, IL-6
|
|
|
Role of phosphorylation in nuclear import of viral proteins and complexes
Pokorná, Karolína ; Forstová, Jitka (advisor) ; Roučová, Kristina (referee)
Replication of many different viruses occurs in the nucleus of the host cell. These viruses discovered ways how to overcome the nuclear membrane and often use cell transport machinery to transport their proteins and genome into the nucleus. For many viral proteins the timing of their nuclear import in order to secure correct viral replication is important. Regulated nuclear import also allows these proteins to perform several functions depending on their localization. Nuclear import of viral proteins and complexes can be regulated by phosphorylation. Phosphorylation can, for example, modulate affinity of proteins for importins or other cellular proteins. Phosphorylation can also cause conformational change, which can lead to unmasking of localization sequence.
|
|
|
Regulation of bacterial transcription by alternative sigma factors.
Benda, Martin ; Krásný, Libor (advisor) ; Roučová, Kristina (referee)
An important feature of bacteria is their ability to respond to various environmental conditions by regulation of transcription. This thesis is focused on regulation of transcription initiation by different sigma factors. Sigma factor is a specific subunit of RNA polymerase, which ensures correct recognition of promotor sequences. During exponential growth phase under ideal conditions, the cell transcribes most of the genes under the control of the so-called housekeeping sigma factor. In the transition to stationary phase, during sporulation or upon exposure to different stresses, many genes are activated and transcribed under the control of alternative sigma factors. This work presents a list of these alternative sigma factors of Bacillus subtilis, focusing on conditions under which the individual sigma factor is used, methods of activation and repression of these factors and on regulons controled by these sigma factors.
|
|
|
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...
|
|
|
Internal ribosome entry site of the hepatitis C virus as a possible target for therapy
Roučová, Kristina ; Čáp, Michal (referee) ; Pospíšek, Martin (advisor)
Hepatitis C virus infects about 3 % of world's population. Progress in molecular biology and better knowledge of hepatitis C virus life cycle contribute to the development of specifically targeted antiviral therapies for HCV. This new treatment also targets the internal ribosome entry site (IRES), which is highly conserved and therefore an attractive target for intervention. In addition, inhibition of IRES function could disable the propagation of the virus early in the HCV life cycle. Novel therapeutics are aimed both at the HCV IRES structure and its nucleotide sequence. Small molecules and synthetic nucleic acids, e.g. antisense oligonucleotides and ribozymes, have been tested as potential therapeutic substances. In this paper particular attention is paid to small molecules interacting with HCV IRES.
|
guest ::
RSS feed.