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Hydrophilic polymers-based delivery systems for the transport and controlled release of siRNA
Blažková, Jana ; Laga, Richard (advisor) ; Vopálenský, Václav (referee)
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...
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Preparation and characterization of diamond-based nanocarriers for transfection of siRNA
Majer, Jan ; Cígler, Petr (advisor) ; Fišer, Radovan (referee)
Although nanodiamonds were discovered and produced tens of years ago, they have been utilized in medical and biological fields just recently, particularly in drug and gene delivery into a cell and in bioimaging methods. Nanodiamonds can be modified with specific positively charged moieties for complexation with negatively charged nucleic acids. These complexes afterwards overcome extracellular and intracellular barriers and transport the nucleic acid either into cytosol or into the nucleus. Owing to fluorescence centres nitrogen- vacancy, which can be formed in the nanodiamonds, nanodiamonds exhibit excelling optical properties, as they emit stable fluorescence without "photoblinking" or "photobleaching". This thesis reviews properties, synthesis and modifications of nanodiamonds and other selected nanoparticles and their in vitro applications. This thesis also compares their cytotoxicity and gene knockdown efficiency.
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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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Polymer systems for siRNA delivery
Blažková, Jana ; Pechar, Michal (advisor) ; Šťovíček, Vratislav (referee)
The process of RNA interference (RNAi) is a natural phenomenon posttranscriptionally controlling gene expression by means of small double-stranded RNA molecules (dsRNA). Small interfering RNA (siRNA) is a small dsRNA that can be used for targeted gene silencing as an alternative therapeutic treatment of genetic diseases. For in vivo administration, siRNA must be protected against degradation to ensure its efficient delivery to target cells using sophisticated vectors. This work is focused on description of non-viral vectors based on cationic polymers, forming polyelectrolyte complexes with siRNA (polyplexes), and surface-modifying hydrophilic polymers enabling protection of the vector during its transport in the bloodstream.
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RNA directed DNA methylation in Arabidopsis thaliana
Motylová, Šárka ; Fischer, Lukáš (advisor) ; Moravec, Tomáš (referee)
The differential transcriptional activity of the genome is provided by epigenetic modifications, which include DNA methylation, alteration of histone N-terminal amino acids and changes in histone variants. RNA interference is a regulatory process, in which transcriptional or post-transcriptional silencing of exogenous or endogenous sequences is mediated by the action of small RNAs derived from these sequences. The 24-nucleotide siRNAs, forming a fraction of small RNAs, direct de novo DNA methylation and participate in the maintenance of DNA methylation (RNA-directed DNA methylation; RdDM), which facilitates transcriptional silencing of heterochromatin and transposable elements representing a large part of plant genomes. The presence of two RNA polymerases involved in this pathway is characteristic for flowering plants, which were discovered for the first time in the genome of Arabidopsis thaliana, which has also become the main plant model for the study of RdDM. Polymerase IV transcribes siRNA precursors; siRNAs are subsequently associated with AGO4 proteins and guide methylation enzymes to the target sequences via complementarity with polymerase V transcripts.
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Size matters - siRNAs biogenesis and function in Arabidopsis
Přibylová, Adéla ; Fischer, Lukáš (advisor) ; Honys, David (referee)
RNA interference (RNAi) play a key role in various biological processes including regulation of gens and transposons, phylogenetic of part plant body, stress response, chromatin remodeling and antiviral mechanism. The ground of RNAi is short RNA molecules (small RNA, sRNA). In plants they are produced in range from 21 to 24 nucleotides (nt) and on the basis of being complementary they recognize target molecule of RNAi. It is possible to divide small RNA in two basic classes: microRNAs (miRNA) and small interfering RNAs (siRNA). To product and put small RNA into activate needs proteins from several gene family. DICER-LIKE (DCL) proteins create small RNAs from double-strand RNA precursors, which are often created by RNA dependent RNA polymerase (RDR) activity. With these small RNAs interact ARGONAUTE (AGO) proteins and together create RNA-Induced Silencing Complex (RISC). Those complexes play a key role in recognizing target molecule in active phase of RNAi. Structure and biogenesis of sRNAs has decisive influence on RISC complex and its next way in biogenesis. RNAi cause effect on post-transcriptional level (PTGS), as degradation of target molecule or repression of translation. And on transcriptional level (TGS) as sRNA intermediate histone and DNA methylation.
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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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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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