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Extrachromosomal hereditary elements such as organelles, viruses, and plasmids are important for the cell fitness and survival. Their transcription is dependent on host cellular RNA polymerase (RNAP) or intrinsic RNAP encoded by these elements. The yeast Kluyveromyces lactis contains linear cytoplasmic DNA virus-like elements (VLEs, also known as linear plasmids) that bear genes encoding putative non-canonical two-subunit RNAP. Here, we describe the architecture and identify the evolutionary origin of this transcription machinery. We show that the two RNAP subunits interact in vivo, and this complex interacts with another two VLE-encoded proteins, namely the mRNA capping enzyme and a putative helicase. RNAP, mRNA capping enzyme and the helicase also interact with VLE-specific DNA in vivo. Further, we identify a promoter sequence element that causes 5′ mRNA polyadenylation of VLE-specific transcripts via RNAP slippage at the transcription initiation site, and structural elements that precede the termination sites. As a result, we present a first model of the yeast virus-like element transcription initiation and intrinsic termination. Finally, we demonstrate that VLE RNAP and its promoters display high similarity to poxviral RNAP and promoters of early poxviral genes, respectively, thereby pointing to their evolutionary origin., Author summary Yeast cytoplasmic double-stranded DNA virus-like elements (VLEs, also known as linear plasmids) were widely investigated in the past but the topic was almost entirely abandoned, partly due to an inability to express VLE-encoded proteins using conventional expression systems. In this study, we re-opened investigation of these elements focused on considerably underexplored nucleus-independent transcription of K. lactis VLEs. Using systematic in vivo study, we were able to characterize composition of the previously hypothesized VLE-encoded transcription complex. Further, we identified new DNA and RNA elements that were directly connected with formation of VLE-specific mRNA ends, as demonstrated by mutagenesis of these elements and its effect on VLE transcription in vivo. Finally, our phylogenetic and sequence analysis of VLE-encoded non-canonical RNA polymerase and its promoters, respectively, suggested evolutionary relationship of VLE transcription machinery to much better explored transcription machinery of cytoplasmic double-stranded DNA viruses of the Poxviridae family.

IRESite is an exhaustive, manually annotated non-redundant relational database focused on the IRES elements (Internal Ribosome Entry Site) and containing information not available in the primary public databases. IRES elements were originally found in eukaryotic viruses hijacking initiation of translation of their host. Later on, they were also discovered in 5'-untranslated regions of some eukaryotic mRNA molecules. Currently, IRESite presents up to 92 biologically relevant aspects of every experiment, e.g. the nature of an IRES element, its functionality/defectivity, origin, size, sequence, structure, its relative position with respect to surrounding protein coding regions, positive/negative controls used in the experiment, the reporter genes used to monitor IRES activity, the measured reporter protein yields/activities, and references to original publications as well as cross-references to other databases, and also comments from submitters and our curators. Furthermore, the site presents the known similarities to rRNA sequences as well as RNA-protein interactions. Special care is given to the annotation of promoter-like regions. The annotated data in IRESite are bound to mostly complete, full-length mRNA, and whenever possible, accompanied by original plasmid vector sequences. New data can be submitted through the publicly available web-based interface at http://www.iresite.org and are curated by a team of lab-experienced biologists.

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...

Background: Sequence variability in the hepatitis C virus (HCV) genome has led to the development and classification of six genotypes and a number of subtypes. The HCV 5′ untranslated region mainly comprises an internal ribosomal entry site (IRES) responsible for cap-independent synthesis of the viral polyprotein and is conserved among all HCV genotypes. Description: Considering the possible high impact of variations in HCV IRES on viral protein production and thus virus replication, we decided to collect the available data on known nucleotide variants in the HCV IRES and their impact on IRES function in translation initiation. The HCV IRES variation database (HCVIVdb) is a collection of naturally occurring and engineered mutation entries for the HCV IRES. Each entry contains contextual information pertaining to the entry such as the HCV genotypic background and links to the original publication. Where available, quantitative data on the IRES efficiency in translation have been collated along with details on the reporter system used to generate the data. Data are displayed both in a tabular and graphical formats and allow direct comparison of results from different experiments. Together the data provide a central resource for researchers in the IRES and hepatitis C-oriented fields. Conclusion: The collation of over 1900 mutations enables systematic analysis of the HCV IRES. The database is mainly dedicated to detailed comparative and functional analysis of all the HCV IRES domains, which can further lead to the development of site-specific drug designs and provide a guide for future experiments. HCVIVdb is available at http://www.hcvivdb.org.

Non-templated addition of adenosine on the 3' end of the product of several DNA polymerases commonly used in molecular biology has been a problematic yet unexplained phenomenon for a few decades now. The only other instance where adenosine is added to the 3' end of an information molecule in a cell is polyadenylation. At the same time, several RNA viruses appear to perform similar action to either edit or polyadenylate their mRNAs. This work focuses on the details of these three highlighted processes and relationship between them and has found out that all of them appear to stem from a common conserved property of the ancestral polymerase. Key words: DNA polymerase, RNA polymerase, non-templated addition, Taq polymerase, polyadenylation

After the whole genome duplication event, Saccharomyces cerevisiae lost 90 % of its paralogs. 59 ribosomal protein genes (RPG) retained a duplicated form. The cell has to balance expression of RPGs as a part of adaptation to changing conditions, thus ensuring the production of the right ratio of ribosomal proteins (RP). In addition, RPGs contain 1/3 of all introns found in the S. cerevisiae genome. Rpl22A/B are part of the large ribosomal subunit where they contact 25S rRNA. Deletion of these RPGs results in 2X slower growth in comparison with WT cells. Within their extraribosomal function Rpl22A/B are able to intragenically and intergenically regulate their expression. Binding of Rpl22A/B to the intronic part of pre-mRNA of RPL22A or RPL22B results in splicing inhibition, which is stronger in the case of the RPL22B intron (RPL22Bi). However, the exact mechanism is not known. We know that Rpl22s from various organisms bind a hairpin structure that can be found in 25S rRNA also. Since the predicted structure of RPL22Bi does not contain this binding motif, it rises a question of whether the RNA binding surface of Rpl22A/B is different or more extensive than the one with which Rpl22 contacts 25S rRNA. We compared the ability of Rpl22 from different organisms to complement the functions of Rpl22A/B. These...

Hepatitis C virus (HCV) is a virus of the family Flaviviridae whose genome consists of ,,+RNA" molecule. It causes the disease hepatitis Cepatitida typu C, which affects tens of millions of people worldwide. Although there is an effective treatment for this type of hepatitis, a preventive vaccine against the HCV virus has not yet been developed. Several ambigious works focused on the relationship between HCV and the enzyme adenosine deaminase acting on double-stranded RNA 1 (ADAR1) were published in the past. This dimeric double-stranded RNA binding enzyme is a part of innate immunity and causes catalytic conversion of adenosine to inosine, which is recognized by cellular mechanisms as guanine, which ultimately leads to mutations in the affected dsRNA molecule. The works published so far attribute an antiviral function to the ADAR1 enzyme in the context of HCV infection. However, vectors containing the entire HCV genome were not used in these works, and a cell line with deletion od the ADAR1 gene has never been used so far. The actual relationship between the ADAR1 enzyme and the HCV virus has not yet been reliably verified. The aim of this thesis was to gain a deeper understanding of the relationship between the HCV virus and the ADAR1 enzyme. For this task, HCV permissive Huh7.5 cell lines with...

There is currently very few information about the transaltion of linear cytoplasmatic plasmids occured in yeast cells Kluyveromyces lactis. However, there is a relatively well developer information about their transcription apparatus. A study of transkript linear plasmids revealed an atypical organization at the 5ʼ end. Those ends contain nontemplate polyadenylation and they are missing the N7 methylguanosine hat. Because of the presence of this structure, which is localized at 5ʼend of plasmids specific mRNA, raised a question regarding the iniciation of the translation. The present thesis is focused on the preparation of reporter systém suitable for studying the influence of a number of the nontemplate adenosins, which were added at the 5ʼ ends of mRNA linear plasmids. The frist step was making a construction of dual yeast cell plasmids carring two reporters genes, which are under the controle of two different promoters. After a successfull construction, the aktivity of promoters TEF1 and PGK1 was measured, whereby the promoter TEF1 proved twice stronger. The transcription start site of both promotor was determined. The second step was the construction of a reporter system directly in yeast cell plasmid pGKL. Reporter genes were under the controle of two promoters originating from the pGKL...

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...

0 Abstract: Non-canonical amino acids allow the introduction of new chemical properties into proteins, which is useful both for studying proteins, and designing proteins. However, the synthesis of proteins containing non-canonical amino acids faces problems with decreased effectivity of translation. This thesis examines the role of genetic code for coding non-canonical amino acids, proteosynthesis and aminoacilation of tRNA and finally the non-canonical amino acids and their application in proteins while including examples.

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 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...

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.

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...

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

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...

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...

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...

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...

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...

CTCF is a ubiquitously expressed nuclear protein that binds to DNA through its central zinc finger domain. Thousands of CTCF binding sites have been identified throughout the human genome at gene promoters, in intergenic regions or in non-coding sequences. CTCF can function either as a positive or as a negative regulator of gene expression and is also involved in creating and maintaining long-range chromosomal interactions. Various developmentally important genes have been shown to be regulated by CTCF and its malfunction is frequently associated with developmental defects or diseases. CTCF undergoes various posttranslational modifications such as phosphorylation or SUMOylation which also affect its function in the regulation of gene expression. Keywords: CTCF, three dimensional genome, cohesin, regulation of gene expression, insulation, HOX genes

The progression of replication forks can be slowed down or paused by various external and internal factors during DNA replication. This phenomenon is referred to as replication stress and substantially contributes to genomic instability that is a hallmark of cancer. Transcription complex belongs to the internal replication-interfering factors and represents a barrier for progression of the replication complex. The replication forks are slowed down or paused while passing through the transcriptionally active regions of the genome that can lead to subsequent collapse of stalled forks and formation of DNA double-strand breaks, especially under conditions of increased replication stress. DNA helicase RECQ5 is significantly involved in maintenance of genomic stability during replication stress, but the mechanisms of its action are not clear. In this diploma theses, we have shown that RECQ5 helicase, in collaboration with BRCA1 protein, participates in the resolution of collisions between replication and transcription complexes. BRCA1 protein is a key factor in the homologous recombination process, which is essential for the restart of stalled replication forks. Furthermore, we have shown that RECQ5 helicase is involved in ubiquitination of PCNA protein at stalled replication forks. Key words DNA...

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...

Protein kinases ERK1 and ERK2 are one of the most studied proteins in cell signalling. Both proteins are involved in a plethora of processes, such as phosphorylation and activation of kinases as part of signalling pathways. Enzymes ERK1 and ERK2 are part of MAPK/ERK signalling cascade, connected to many cellular including cell proliferation, cell growth or differentiation. The MAPK/ERK signalling cascade is often activated in different types of tumors, making it a candidate for developing new chemical inhibitors. One of the important questions in fundamental research of ERK1 and ERK2 protein kinases is the search for difference between these proteins. Current knowledge points to redundancy of both proteins, howver several examples suggest otherwise. Recently, the work presented in Casanova et al. 2012 indirectly suggests divergent effect of ERK1 and ERK2 on cap-independent translation initiation. In the Laboratory of RNA biochemistry we focus on HCV IRES (Hepatitis C Virus Internal Ribosome Entry Site) dependent translation initiation. This diploma thesis lead to establish RNA interference method in our laboratory and to establish reporter system to study ERK1 and ERK2 effect on HCV IRES dependent translation initiation. Based on our data acquired during our research, we present in this work...

Gene regulatory networks, underlying the molecular regulation of eye development are conserved across many animal phyla. Genes from the Pax family of transcription factors are one of the most conserved members through the evolution, regulating the development of crucial parts of eye, including the photoreceptor cells. Pax transcription factors are considered to be regulators of opsins, molecules providing the conversion of the light stimulus into the electrochemical signalisation in the photoreceptors cells. In this thesis, pax6 and pax2/5/8 transcription factors are investigated as potential regulators of eye development in Platynereis dumerilii. pax6 and pax2/5/8 transcription factors are tested as potential regulators of the r-opsin in Platynereis, based on the observed early expression onsets of these genes. Wild-type expression analysis of pax6 and pax2/5/8 using the whole mount RNA in-situ hybridization is provided, accompanied by the initial analysis of the Platynereis pax6 knockout line. pax6 heterozygote mutants are shown to be viable and able to reproduce, however, homozygote mutation of pax6 in Platynereis is lethal. Our data suggest that transcription factors pax2/5/8, otx and six3 are not regulated by the pax6 in Platynereis. Concerning the r-opsin present in the Platynereis eyes, pax6...

iv Abstract Regulation of gene expression is a key ability of every single cell in its development, differentiation and homeostasis. On the other hand, rather sparse amount of information is available for protists and our understanding of regulation of gene expression in eukaryotes is limited to a few model organisms. Our research is aimed at oxymonads, poorly studied group of anaerobic protists, which inhabit digestive tract of some animals. In this study we focus on the genus Monocercomonoides. Gene expression is modulated at multiple levels by many mechanisms. This thesis is focused on structure of promoter regions, 5' untranslated regions and basal transcription and translation initiation factors. Our results are compared to the closest studied relatives of Monocercomonoides - Trichomonas vaginalis and Giardia intestinalis. We have identified several conserved motifs in promoter regions of Monocercomonoides, including TATA box and TATA-like motif. These motifs potentially play a role in the transcription regulation. 5' untranslated regions are relatively short (typically 20 - 30 nucleotides) and GC content in these regions is low compared to model organisms. In selected genes, the quality of the automatic prediction of UTR was verified by RACE. We have annotated sets of basic transcription (23 proteins)...

Saccharomyces cerevisiae cells produce 2000 ribosomes per minute under normal conditions. The expression of ribosomal proteins is massive - it takes 50% of RNA polymerase II transcription and 90% of pre-mRNA splicing in rapidly growing cells. Since cells need an equimolar amount of individual ribosomal proteins, the tight coregulation of gene expression is required. The transcription is a main target of regulation, however, it is inherently unable to set a stoichiometric balance of ribosomal proteins. Various types of post-transcriptional regulation deal with fluctuations of individual ribosomal proteins and fine-tune their expression. Intron-dependent regulation appears to by predominant among ribosomal protein genes. Besides balancing their expression, presence of introns provides a rapid global regulation (repression) of ribosomal protein genes in response to environmental stress. KEY WORDS ribosomal protein genes, RPG, ribosomal protein, gene expression regulation, coregulation, Saccharomyces cerevisiae

Therapeutics based on siRNA represent a promising hope for the treatment of many congenital and acquired disorders. This method is based on posttranscriptional silencing of pathological gene or set of genes (RNAi process), which are responsible for the actual cause of the disease. Access is therefore based on the assumption of treatment options for the disease at the point of origin of the defect intervention at the molecular level, which is different from the conventional, so-called symptomatic therapy, which focuses only on the treatment or suppression of symptoms. Despite rapidly increasing understanding of gene function and cause a number of genetic diseases, the expansion of siRNA therapeutics limited the development of efficient and safe transport systems (vectors). In order to ensure efficient transport of siRNA in vivo conditions, the vectors must sufficiently reduce the size of the siRNA, protect it against degradation during transport, and release in the cytoplasm of the target cell. For this purpose they were developed sophisticated transport systems based on viral and non-viral origin. This diploma thesis is focused on the preparation of new transport systems, siRNA-based synthetic hydrophilic polymers, such as non-viral vectors. For in vitro testing the effectiveness during transport of siRNA...

Design of small interfering RNA (siRNA) is a basic step to successful RNA interference (RNAi). However, it is not trivial to design a suitable siRNA duplex at all, because there are some problematic areas like stability of siRNA duplexes in the presence of nucleases, efficiency of target mRNA elimination, sequence-specific elimination of non-target mRNAs, cytotoxicity and immunogenicity for example, which can influence the aliveness of siRNA duplexes and consequently also their utility in medicine and other fields. The understanding of the principles of siRNA duplexes function during RNAi is a necessary step to solution of the obstacles on the way to efficient drugs. The mean of optimisation of described troublesome properties is an alteration of siRNA duplexes design by use of different modifications. Phosphodiester backbone, bases and also ribose can be modified. Some modifications beneficial for siRNA duplexes and consequently RNA interference are known in every one of these groups. This work presents existing pieces of knowledge about some of the most known modifications, just like phosphorothioate substitution, 2′-deoxy-2′-fluoronucleotides, 2′-O-methyl ribonucleotides and LNA nucleotides are, as well as about a higher number of less known modifications like boranophosphate substitution,...

Hormonal therapy is a common part of breast carcinoma treatment in patients whose tumors express estrogen and progesterone receptors. The aim of hormonal therapy is to prevent proliferative effect of hormones througt their receptor proteins in order to inhibit tumor growth. However, certain number of tumors is resistant to hormonal therapy despite expression of hormonal receptors. Presently, the reasons of this resistance are not fully understood. Oxysterols are hydroxylated cholesterol derivates, which may play some role in development of the resistance. They may interfere with hormonal therapy effect and influence some signal pathways leading to cancer progression. This study comes with results of gene expression of proteins influenced by oxysterol action, metabolic and transport proteins, transcription factors and members of signaling pathways that may be related to oxysterol effect. This thesis identifies some candidate genes for future analysis on the basis of comparison of gene expression between estrogen receptor positive and negative tumors and correlation with clinopathological data. The final goal should lead to discovery of new diagnostic markers for breast cancer therapy. Powered by TCPDF (www.tcpdf.org)

DNA methylation is one of the most common epigenetics modifications, during which a methyl group from the donor molecule S-adenosyl-L-methionine is transferred to the 5'position of the cytosine ring to create 5-methylcytosine. This reaction is catalyzed by DNA methyltransferases. Epigenetics modification plays an important role in the regulation of the transcription, genomic imprinting, X-chromosome inactivation and the development of the organism. This role in the regulation of transcription is important for the cancer. Especially the aberrant forms, like hypermethylation, which leads to transcriptional silencing of the tumor suppressing genes leading to the tumor progression, or hypomethylation causing genomic instability. Key words: DNA methylation, demethylating drugs, haematological malignancies, methods of detection, myelodysplastic syndrome