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Processing of different substrates by mammalian Dicer
Faltýnková, Jana ; Svoboda, Petr (advisor) ; Černý, Jan (referee)
Small RNA pathways represent sequence specific mechanisms regulating gene expression or mediating antiviral defence in eukaryotes. The common feature of these pathways are ~20-30-nucleotide small RNAs, which function as sequence specific guides. Small RNA pathways differ from each other in their roles, biogenesis of small RNAs and mechanism of regulation their targets in different organisms. In mammals, there are three recognized small RNA pathways: RNA interference (RNAi), microRNA (miRNA) and PIWI interacting RNA (piRNA) pathways. Biogenesis of small RNAs of RNAi and miRNA pathways is dependent on the Dicer protein, which generates small interfering RNAs (siRNAs) and miRNAs from long double stranded RNAs (dsRNAs) and small hairpins, respectively. This bachelor thesis provides an insight into structure and function of mammalian Dicer, particularly into differences in Dicer processing of pre-miRNA and siRNA precursors.
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The Persistence of Human Polyomaviruses
Blažková, Kristýna ; Drda Morávková, Alena (advisor) ; Kuthan, Martin (referee)
Despite years of research, even the most scrutinized Polyomaviruses - BK and JC - have not yet been thoroughly understood. With a number of new Polyomaviruses - KIV, WUV, MCV, HPyV6, HPyV7, TSV and HPyV9 described in the past few years, the need to understand how Polyomaviruses operate in their hosts has become even more urgent. The probable route of transmission appears to be either respiratory or faecal-oral. The initial infection occurs most likely in the early childhood or early-adolescence and is followed by a life-long persistence. The seroprevalence of Human Polyomaviruses among healthy adult population is high: BKV (81-97 %), JCV (35-69 %), KIV (55 %), WUV (69 %), MCV (25-46 %) and TSV (70-80 %). Human Polyomaviruses can cause fatal diseases in immunocompromised patients. The site of persistence in humans probably varies depending on the specific Polyomavirus. BK and JC are known to persist in kidneys and the urinary tract. Human Polyomaviruses have been detected in the lymphatic tissues, blood, respiratory, urinary, and gastrointestinal systems. It is not clear, however, if they persist in all of these sites. Mechanisms which Polyomaviruses use to establish and maintain persistent infection could include the viral miRNA and viral agnoprotein, which would result in a modulation of viral...
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The role of microRNAs in chronic lymphocytic leukemia
Moravec, Martin ; Macůrková, Marie (referee) ; Hájek, Miroslav (advisor)
2 Abstract MicroRNAs (miRs, miRNAs) are recently discovered molecules (19-25 nucleotides long) that regulate gene expression at post-transcriptional level by either blocking protein synthesis or mRNA degradation. As a part of gene silencing mechanism, miRNAs are involved in cellular processes, such as apoptosis, cell proliferation, development and viral defence. miRNAs have been intensely studied in connection to disease pathogenesis. Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries affecting mostly elderly people. In my work I focus on explanation of miRNA functions and their contributions to chronic lymphocytic leukemia (CLL). I describe previously published data about miRNA-15, miRNA-16, miRNA-143, miRNA-145 and miRNA-155 in connection to this disease. Based on recent reports, I also discuss the potential role of miRNA-326 in CLL pathogenesis.
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RNA interference in plants
Čermák, Vojtěch ; Kulich, Ivan (referee) ; Fischer, Lukáš (advisor)
The process of RNA interference allows cells to regulate functions of their genes. This process is usually initiated by the presence of double-stranded RNA within a cell. Such double-stranded RNA is diced by a specific protein called Dicer into duplexes of small RNAs, usually 20-25 nucleotides long. Single-stranded small RNAs, released from the duplexes, are the heart of RNA interference and they can be categorize into several groups according to their biogenesis. There are two groups of small RNAs in plants: miRNA and siRNA. Small RNAs can associate with a protein called Argonaut and guide it to the target molecule on the bases of sequence complementarity. The Argonaut-small RNA complex can act on itself or it can interact with other proteins in a wide spectrum of processes. The complex can slice the target mRNA (which can be handled by the sole Argonaut and small RNA), it can suppress translation or it can direct chromatin modifications. The phenomena of RNA interference can be found in almost all Eukaryotes where it can serve many functions, for example it can control cell differentiation, participate in stress responses, direct changes in chromatin and defend the organism against viruses. A diverse set of operating modes of RNA interference can be found in plants, which we are only at the...
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Analysis of short Argonaute isoforms from mouse oocytes
Jankele, Radek ; Svoboda, Petr (advisor) ; Petr, Jaroslav (referee)
AnalysisofshortArgonauteisoformsfrommouseoocytes Abstract: Argonaute proteins carrying small RNAs form the conserved core of RNA silencing mechanisms, which repress viruses, mobile genetic elements, and genes in a sequence specific manner. The microRNA (miRNA) pathway is a dominant mammalian RNA silencing mechanism in somatic cells, which post-transcriptionally regulates large fraction of genes and thereby adjusts protein levels. miRNA-guided Argonautes inhibit translation and induce deadenylation of complementary mRNAs, ultimately resulting in their decay. In contrast to RNA interference (RNAi), which employs Argonaute slicer activity to directly cleave perfectly complementary RNAs, an effective miRNA-mediated mRNA repression requires multiple Argonaute-associated protein factors and enzymes. The miRNA pathway has been implicated in many complex biological processes ranging from organogenesis, stress-response to haematopoiesis or cancer. Surprisingly, canonical miRNAs are not essential for oocytes and early embryonic development in mice. Even the most abundant miRNAs present in mouse oocytes are unable to effectively repress target genes. However, RNAi, which shares key enzymes with the miRNA pathway, is highly active in oocytes and early embryos. The cause of miRNA inactivity in mouse oocytes remains...
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Formation of spatio-temporal molecular gradients in early embryonic development of Xenopus laevis.
Šídová, Monika ; Tlapáková, Tereza (advisor) ; Pěknicová, Jana (referee) ; Petr, Jaroslav (referee)
Clarifying the underlying spatio-temporal mechanisms that determine body pattern is important for detailed understanding of embryonic development. A crucial question of vertebrate embryogenesis remains: when and how are single blastomeres determined for differentiation that subsequently leads to body axes specification and the formation of different tissues and organs? The answer to this question will be beneficial for primary research as well as in the field of applied medicine. The main aim of the presented thesis was to study spatio-temporal molecular gradients of cell fate determinants during early embryonic development. The African clawed frog Xenopus laevis was used as a model organism because of their large size of oocytes and external embryonic development. Due to late activation of embryonic transcription, a crucial mechanism of early blastomeres determination is dependent on asymmetric localization of maternal factors within oocyte and their uneven distribution into single blastomeres during early cell division. Two main localization patterns were identified along the animal-vegetal axis of the mature Xenopus oocyte using qPCR tomography. The localization gradient with preference in either animal or vegetal hemisphere was found for maternal mRNA as well as miRNAs. Moreover, two vegetal...
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DNA čipy a jejich využití pro molekulární diagnostiku a predikci u onkologických onemocnění
VORÁČKOVÁ, Lucie
Many visible factors produced during carcinogenesis refer to oncological disease, e.g. modified gene's expression or increased number of miRNA. These factors can be diagnosed by analyzing of gene's expression. For this diagnostics, there are used many methods, of whom DNA microarray analysis is the most significant progress in genetic research. A large number of DNA sequences can be detected DNA with microarrays on a single microarray in a relatively short time. This analysis means a positive turnover for the diagnosis and prediction of disease.
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Biofyzikální studium malých RNA
Šmerková, Kristýna
Thanks to the prove of connection between the aberrant occurrence of small RNA and various diseases and their potential in diagnostics and treatment led to discovery of new methods and materials facilitating their detection and targeted transport during gene therapy. This work summarizes present knowledge about chosen groups of small RNA, their significance in medical science and the possibilities of their detection. This work primarily concentrates on combination of magnetic separation with electrochemical detection. Magnetic particles (MPs) with different surface modifications were used for isolation. Non-specific isolation was carried out using silanol-coated MPs; streptavidin-coated MPs modified with specific biotinylated probe were used for specific separation. Square wave voltammetry (SWV) was used as a very sensitive electrochemical detection method. Optimized method based on specific magnetic separation with SWV was able to reach nanomolar detection limit (4 nM) with microRNA. The method was applied on human embryonic cells for specific isolation and detection of miR-124. The CdTe quantum dots (QDs) were studied as a nanomaterial tool for nucleic acid detection. The QDs were modified with streptavidin for their bioconjugation with biotinylated molecules were used. Interaction of QDs with nucleic acids was studied using capillary electrophoresis.
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Classification of Small Noncoding RNAs
Žigárdi, Tomáš ; Martínek, Tomáš (referee) ; Vogel, Ivan (advisor)
This masters's thesis contains description of designed and implemented tool for classification of plant microRNA without genome. Properties of mature and star sequences in microRNA duplexes are used. Implemented method is based on clustering of RNA sequences (with CD-HIT) to mainly reduce their count. Selected representants from each clusters are classified using support vector machine. Performance of classification is more than 96% (based on cross-validation method using the training data).
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