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Analysis of gene regulatory regions in the genome of oxymonad Monocercomonoides
Brzoň, Ondřej ; Hampl, Vladimír (advisor) ; Vopálenský, Václav (referee)
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)...
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Elucidation of ERK1 and ERK2 protein kinases effect on cap-independent translation initiation
Přibyl, Miroslav ; Vopálenský, Václav (advisor) ; Vomastek, Tomáš (referee)
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...
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The Design of siRNAs and the Influence of Modifications on Their Stability and Efficiency
Kuldanová, Kateřina ; Vopálenský, Václav (advisor) ; Čáp, Michal (referee)
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,...
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DNA methylation in haematological malignancies
Šeborová, Karolína ; Beličková, Monika (advisor) ; Vopálenský, Václav (referee)
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
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A potential role of DAXX in cell cycle arrest and cellular senescence
Valášek, Ján ; Hanzlíková, Hana (advisor) ; Vopálenský, Václav (referee)
Death domain-associated protein 6 (DAXX) is a multifunctional protein involved in diverse cellular processes. It acts as a histone chaperone or regulator of transcription and apoptosis, in which is its role quite controversial. DAXX also participates in regulation of cell DNA damage response (DDR). DAXX co-creates and stabilizes complex with Mdm2, which negatively regulates the stability of p53, an important tumor suppressor, which is a part of signalling node in the DDR. If DNA damage is not lethal for the cell and unables it to proliferate, the irreversible state of cell cycle called cellular senescence takes place. Under physiological conditions, induction of senescence can prevent the development of tumorigenesis. Therefore, the description of mechanisms involved in the induction of senescence has potential clinical significance. In my thesis, I aimed to determine changes in the level of DAXX protein in senescent cells and to characterize the manner of its regulation. In tumor cells MCF-7 and primary BJ fibroblasts, I observed decrease in DAXX protein level and its regulation. I tested the hypothesis according to which an increase in DAXX level before DNA damage canprevent induction of cellular senescence, or increase in its expression during senescence can cause recovery of cell proliferation....
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Characterization of the molecular mechanism of translation reinitiation in yeast.
Pondělíčková, Vanda ; Valášek, Leoš (advisor) ; Hašek, Jiří (referee) ; Vopálenský, Václav (referee)
Translation initiation is a multi-step process culminating in formation of the elongation- competent 80S ribosome. It requires accurate assembly of small and large ribosomal subunits, mRNA, initiation Met-tRNAi Met and at least 12 eukaryotic initiation factors (eIFs). This phase of protein synthesis is also one of the key points of regulation of gene expression. One of the main aims of our laboratory is a complex characterization of the multiprotein eIF3 complex that has been implicated in most of the steps of translation initiation. For example, we revealed and described its novel role in translation reinitiation (REI), a gene-specific translational control mechanism that among others governs expression of an important yeast transcriptional activator GCN4. Here I present a detailed characterization of the multi-functional N-terminal domain of Tif32 (subunit eIF3a). We demonstrated that the Tif32-NTD functionally interacts with the 5' sequences of short upstream ORF (uORF1) in the GCN4 mRNA leader and thus allows efficient reinitiation downstream of this critical reinitiation-permissive uORF. Four REI- promoting elements (RPEs) were identified in the 5' sequences of uORF1, two of which were shown to work in the Tif32-NTD-dependent manner. The structure of the 5' sequences was determined...
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ERK1/2 MAP kinase - Structure and Interaction Partners
Přibyl, Miroslav ; Vopálenský, Václav (advisor) ; Žíla, Vojtěch (referee)
Extracellular signal molecules are recognized by membrane receptors on the surface of eukaryotic cells. Receptors transmit the signal into the intracellular space where activation of the concrete enzymes occurs. Activated enzymes may be protein kinases that phosphorylate the substrate proteins corresponding to the requirements for specific recognition by a protein kinase. Substrate proteins may be structural proteins and enzymes, which in turn transmit the signal or directly affect the physiological processes of the cell. The protein kinase family accounts for ERK1 and ERK2 (Extracellular signal-regulated kinase 1 and 2), enzymes which affect cell growth, cell cycle and numerous other physiological processes of the cell. Protein kinases are dynamic molecules, which undergo a series of conformational changes during their catalytic cycle and whose stability and function are affected by conformational changes. Conserved amino acid residues carry out the function of protein kinases. These factors are also involved in interactions with protein substrates and regulatory proteins, and are responsible for specific function of protein kinase.
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Gene expression study of oxysterol signal pathway in breast cancer patients
Kloudová, Alžběta ; Souček, Pavel (advisor) ; Vopálenský, Václav (referee)
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)
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Single cell gene expression profiling and quality control
Švec, David ; Kubista, Mikael (advisor) ; Beneš, Vladimír (referee) ; Vopálenský, Václav (referee)
Single cell gene expression profiling and quality control David Švec Institute of Biotechnology AS CR, Laboratory of Gene Expression, Prague, Czech Republic TATAA Biocenter, Research & Development, Gothenburg, Sweden Abstract: Gene expression profiling has become an exceedingly important tool for describing occurence of mRNA in tissue samples and even single cells. Most often we use it for characterization of cell types, degree of differentiation and pathology on a molecular level. In our newly established laboratory, we developed high resolution qPCR tomography to show distribution of tens of maternal mRNAs within a single oocyte. We demonstrated that distribution of mRNAs has an important role in further development of the organism. For high resolution qPCR tomography, where one oocyte is divided in tens of samples and about fifty genes are studied in each sample, we optimized dye based protocol for microfluidic high-throughput platform BioMark. Next step was complementing the molecular profile of tens most important genes with information about histology of each selected tissue section using laser microdissection. As a model we used embryonic development of mouse molar. Our goal was to describe interaction of up to one hundred genes in different stages of development and on the single cell level. This...
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Single cells gene expression profiling and analysis
Novosadová, Vendula ; Kubista, Mikael (advisor) ; Beneš, Vladimír (referee) ; Vopálenský, Václav (referee)
Cells are the basic units of life. Studying complex tissues and whole organs requires an understanding of cell heterogeneity and responses to stimuli at the single-cell level. Even the cells, which belong to the same cell type, behave differently at a specific moment and contain different amount of mRNA. Quantitative polymerase chain reaction (qPCR) is one the most sensitive methods for the detection of mRNA, however, gene expression profiling in single cells leads to a large amount of missing data due to the fact that the transcript is missing, or is below the level of detection. Therefore, it is necessary to establish a new statistical approach for analysis of single cells. In this thesis the potential of single-cell gene expression profiling using the high throughput instrument Biomark, focusing on data analysis and biological interpretation, is discussed. Data normalization and handling of missing data are two important steps in data analysis that are performed differently at the single-cell level. Single cells are not normalized by reference genes but the number of cells as a normalizer is applied. Missing data are replaced by value, which is equaled one quarter of transcript amount in the cell. Furthermore it is shown how single-cell gene expression data can be viewed and how subpopulations...
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