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The role of microRNAs in chronic lymphocytic leukemia
Moravec, Martin ; Hájek, Miroslav (advisor) ; Macůrková, Marie (referee)
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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Preparation of nanoparticles for hepatitis B viral therapy
Kružíková, Zuzana ; Grantz Šašková, Klára (advisor) ; Žáčková Suchanová, Jiřina (referee)
Hepatitis B virus (HBV) represents one of the hot topics of current basic and pharmaceutical research. Although an effective vaccine against HBV exists since 1982, the world prevalence of chronic infection is still alarming. The infection can lead to significant liver damage, often resulting in hepatocellular carcinoma. Chronic HBV infection cannot be cured due to the fact that the viral genome persists in the infected hepatocyte hidden from the host immune response as well as from the antiviral treatment. One of the novel approaches aiming for HBV cure suggests that this cccDNA pool could be destroyed using gene editing tools such as CRISPR/Cas9 system. In order to shift this gene editing system to possible medicinal application, CRISPR/Cas9 has to be specifically delivered into the target cell in order to minimize its putative off-target activity. This thesis focuses at first on the design and efficacy testing of new sgRNAs targeting HBV cccDNA and secondly, it describes modular lipid nanoparticles developed specially for delivery of the CRISPR/Cas9 system in the form of RNA. Keywords: hepatitis B virus, CRISPR/Cas9, gene editing, lipid nanoparticles, mRNA delivery, targeted delivery
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The m6A pathway at the molecular level and its role in neurological diseases
Švendová, Aneta ; Černá, Barbora (advisor) ; Čočková, Zuzana (referee)
N6-methyladenosine is the most abundant modification in eukaryotic messenger RNA. This modification is reversible, thanks to a complex of methyltransferases and demethylases. The biological effects of m6 A are mediated through reader proteins. This complex mechanism of proteins contributes to many molecular processes such splicing, translation and transport. It also plays a role in many serious neurological diseases, such as Alzeheimer's disease, Parkinson's disease, major depressive disorder and attention deficit hyperactivity disorder. The purpose of this thesis is to describe the m6 A pathway, its regulation at the molecular level and to put it into context with neurological diseases of today. Key words: mRNA, mRNA metabolism, N6-methyladenosine, m6 A regulation, FTO, METTL, ALKBH5, neurodegenerative disorders, Alzheimer's disease, Parkinson's disease, major depressive disorder, ADHD
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The Role of oncogenic microRNA - 155 and proto - oncogen MYB in chronic lymphocytic leukemia
Vargová, Karina
(EN) Chronic lymphocytic leukemia (B-CLL) represents a disease of mature-like B-cells. Due to failed apoptosis but also due to enhanced proliferative signals, the leukemic B-cells accumulate in the peripheral blood, bone marrow, lymph nodes and spleen. The clinical course of B-CLL is very heterogeneous; in some patients B-CLL progresses very rapidly into an aggressive form. Such patients need therapy sooner while in other patients with indolent B-CLL the onset of therapy takes years. Several standard prognostic and disease progression markers are used for disease staging and monitoring, however a reliable marker that will suggest when to start therapy is unknown. Expression of small, non-coding microRNAs is often deregulated and represent important prognostic markers in variety of cancers including leukemia. Hence in our study we concentrated to miR-155, an important molecule regulating differentiation of hematopoietic cells, inflammation process and antibody production. Its aberrant expression was described in Hodgkin`s as well as in non-Hodgkin`s lymphoma, including indolent lymphoproliferations like B-CLL. Our results confirmed elevated levels of both, primary miR-155 transcript and mature form of miR-155 in our B-CLL patient samples (N=239). The aberrant expression of miR-155 in B-CLL samples...
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Messenger RNA stability and microRNA activity in mouse oocytes
Flemr, Matyáš ; Svoboda, Petr (advisor) ; Motlík, Jan (referee) ; Hampl, Aleš (referee)
The oocyte-to-zygote transition represents the only physiological event in mammalian life cycle, during which a differentiated cell is reprogrammed to become pluripotent. For its most part, the reprogramming relies on the accurate post-transcriptional control of maternally deposited mRNAs. Therefore, understanding the mechanisms of post-transcriptional regulation in the oocyte will help improve our knowledge of cell reprogramming. Short non- coding microRNAs have recently emerged as an important class of post-transcriptional regulators in a wide range of cellular and developmental processes. MicroRNAs repress their mRNA targets via recruitment of deadenylation and decapping complexes, which typically accumulate in cytoplasmic Processing bodies (P-bodies). The presented work uncovers an unexpected feature of the microRNA pathway which is found to be suppressed in fully-grown mouse oocytes and through the entire process of oocyte-to-zygote transition. This finding is consistent with the observation that microRNA-related P-bodies disassemble early during oocyte growth and are absent in fully-grown oocytes. Some of the proteins normally associated with P-bodies localize to the oocyte cortex. At the final stage of oocyte growth, these proteins, together with other RNA-binding factors, form subcortical...
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Analysis and mapping of binding sites of gene expression regulators in the genus of Streptomyces.
Šmídová, Klára ; Bobek, Jan (advisor) ; Krásný, Libor (referee) ; Kopecký, Jan (referee)
Streptomyces are medically important soil-living bacteria that undergo morphological changes from spores to aerial hyphae and are important producers of bioactive compounds including antibiotics. Their gene expression is tightly regulated at the early level of transcription and translation. In the transcriptional control, sigma factors play a central role; the model organism Streptomyces coelicolor possesses astonishing 65 sigma factors. The expression of sigma factors themselves is controlled on the post-transcriptional level through the action of sRNAs that modify their mRNA level. However, only several sigma factors in Streptomyces have known regulons and also their sRNAs-mediated regulation has not been studied so far. According to previously measured gene expression data, we selected several highly expressed sigma factors. Using mutant strains with HA-tagged sigma factors, regulons of two important sigma factors, SigQ and HrdB, were analyzed by ChIP-seq procedure. Other sigma factors were further studied to see if they possess asRNAs, using 5' and 3' RACE method and northern blotting. Our data confirm the essentiality of HrdB sigma factor during the vegetative phase of growth. The other sigma factor, SigQ, has been revealed to be an important regulator of nitrogen metabolism and osmotic...
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Effect of SR-like proteins in maturation and transfer of mRNA to the cytoplasm
Hájková, Karolína ; Abrhámová, Kateřina (advisor) ; Staněk, David (referee)
Metazoan SR proteins is a group of proteins involved in splicing. They are RNA-biding proteins characterized by the presence of serine and arginine-rich domain. The yeast, Saccharomyces cerevisiae, encodes genes for proteins that are similar in amino acid composition and structure to the group of SR proteins and are therefore called SR-like proteins. There are three proteins with RS domain in yeast: Npl3, Gbp2 and Hrb1. These proteins have been shown to have many functions, including, in addition to splicing, quality control of spliced mRNAs and their export from the nucleus to the cytoplasm. This Bachelor's thesis will deal with these three yeast proteins and their role in splicing, export and degradation.
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The effect of synthetic modified mRNAs induced proliferation on pancreatic beta cells
Veľasová, Adriana ; Koblas, Tomáš (advisor) ; Bořek Dohalská, Lucie (referee)
Diabetes mellitus is a chronic disease caused by the loss of pancreatic beta cells due to autoimmune destruction or increased apoptosis. Beta-cell deficiency results in reduced insulin production, which plays an important role in glucose metabolism. The number of beta-cells in the body is one of the main factors that influence the development of this chronic disease. Therefore, it is necessary to find a way by which the number of beta-cells of the organism can be increased and thus the insulin production can be restored in a natural way without any need for the use of insulin infusions. However, the ability of beta-cells to divide decreases with age and is virtually nil in adulthood. The study of the cell cycle, especially the early and late cyclins and cyclin-dependent kinases, which act as cell cycle regulators, thus appears to be a promising way to restore natural insulin-producing tissues. In order to increase the number of beta cells entering the cell cycle, we focused on studying the effect of in vitro transcribed (IVT) mRNAs, encoding cyclins type D and cyclin dependent kinases 4 and 6 on stimulating cell division of isolated beta-cells. We found that transfection IVT mRNAs for type D cyclins in combination with cyclin-dependent kinases 4 and 6 significantly increased the proliferation of beta-cells...
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Generation of synthetic modified mRNAs encoding cell cycle regulators
Petroušek, Filip ; Koblas, Tomáš (advisor) ; Vašková, Michaela (referee)
One of the modern therapeutic approaches is the application of in vitro transcribed mRNA into the target cells to produce the required target proteins. This method has prooved to be more effective than therapeutic application of proteins prepared in advance, because their insertion into the organism lead to denaturation. The main task of this work was to prepare mRNA in vitro and mRNA transfection into the cells and monitoring the expression of target proteins with the method of Western blotting. The target proteins were from families of cyclins and cyclin-dependent kinases, which play an important role in cell cycle regulation. The mRNAs for 8 types of molecules have been prepared, including non-mutated forms of cyclins D1, D2, and D3, and cyclin-dependent kinase 6, as well were synthesized mutated versions of cyclins D1 T286AT288A, D2 T280A, D3 T283A and cyclin-dependent kinase 4 R24C. As for cyclins, there was a substitution of aminoacid threonine for alanine. This substitution could cause slowdown degradation of proteins in comparision with those existing in natural form. Regarding the cyclin- dependent kinase 4 the exchange of aminoacid arginine for cysteine causes its bigger stability in vivo. The prepared mRNA which encodes mentioned regulators of cell cycle was transfected into isolated...
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