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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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The purinosome - a multi-protein complex involved in the de novo purine synthesis
Kráčmarová, Markéta ; Zikánová, Marie (advisor) ; Macůrková, Marie (referee)
The purinosome is a multiprotein complex involved in the de novo purine synthesis (DNPS). Through a several steps of this metabolic pathway 5-phosphoribosyl-1-pyrophosphate is converted to inosine monophosphate, the precursor of purine nucleotides. Purine nucleotides are also synthesized from inosine monophosphate via a salvage pathway that utilizes hypoxanthine. The purinosome is a dynamic multienzyme complex which is assembled and diassembled by actual need and availability of purines. The purinosome assembly is disrupted in the inherited disorders of the DNPS enzymes - AICA- ribosiduria and adenylosuccinate lyase deficiency. Detailed studies of assembly and dynamics of purinosome and identification of molecular changes associated with the formation of purinosome under physiological and pathological conditions are object of research. Besides better understanding of purine metabolism, in the future, it can open up new possibilities of drug development especially of new use of chemotherapeutics that block DNPS. Key words: purinosome, de novo purine synthesis, defects of enzymes, metabolism, purinosome interactions, cell control
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Mechanisms of retromer - dependent protein recycling from endosomes
Horázná, Monika ; Macůrková, Marie (advisor) ; Černý, Jan (referee)
Most processes in nature are very effective concerning saving energy and minimizing waste. A good example of saving on cellular level is receptor recycling. Whether it concerns receptors for lysosomal enzymes or for proteins destined for secretion, after releasing their cargo protein the fate of the receptor would be sealed in lysosomes. Nevertheless, some transmembrane receptors contain a signal motif through which they are recognized by specific proteins or protein complexes and they escape the degradation in lysosomes. One such complex is the retromer. Its first discovered function was the recycling of receptors for lysosomal hydrolases in yeast. Later it was proved that it has a similar role in transport of many other proteins in other eukaryotes. The task for retromer is to sort the cargo proteins on the endosomal membrane and together with others auxiliary proteins create a transport vesicle which is then transported to the Golgi. This makes the cell able to recycle proteins that would otherwise be transported from endosomes to lysosomes for degradation.
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Gene expression regulation by nuclear receptors in a specific metabolic context - evolutionary perspective
Kaššák, Filip ; Kostrouch, Zdeněk (advisor) ; Macůrková, Marie (referee) ; Leontovyč, Ivan (referee)
In animals, some of the most critical regulators of gene expression are nuclear hormone receptors (NRs) and their coregulators, specifically the Mediator complex. Of particular interest are the NRs implicated in metabolic and developmental regulation and in carcinogenesis: thyroid hormone receptors (TRs) and retinoid X receptors (RXRs). In this work, I venture to elucidate some aspects of gene expression regulation by these NRs: the degree of evolutionary conservation of signalling based on NRs and their coregulators; the mechanisms of negative regulation by NRs; and possible implications of these findings for clinical medicine. State-of-the-art bioinformatical, genome editing and microscopic techniques are applied at three levels of animal evolution to study NRs and Mediator. Reverse genomics in human patients suffering from the syndrome of resistance to thyroid hormones β are used to infer the structure and function of TRβ subdomains. Alignments, binding studies and in vivo experiments in Trichoplax adhaerens allow identification of a close orthologue of human RXR at the basis of metazoan evolution. Employing database queries, genome editing and microscopy, we describe a correct orthologue of the Mediator subunit 28 in Caenorhabditis elegans, indicating a complete homology of the Mediator complex...
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The role of phosphatidylinositol-4-phosphate 5-kinase PPK-1 in Wnt signalling regulation in Caenorhabditis elegans
Zavadilová, Kristýna ; Macůrková, Marie (advisor) ; Kostrouch, Zdeněk (referee)
The Wnt signalling pathway is highly conserved signalling among Eukaryotes that regulates many cellular processes. In particular, it plays a role during invidual's development, but it is also important at later stage of life, when it is involved in maintaining homeostasis in the body. Recent studies have shown that phosphatidylinositol-4-phospahte 5-kinase (PIP5K), which is key to the production of phosphatidylinositol (4,5)-bisphosphate in the cell, is also involved in regulation of the canonical Wnt signalling cascade. In mammalian cells, a model has been described in which PIP5K is involved in signal transduction in a Wnt receiving cell. We decided to verify the involvement of PIP5K in the regulation of Wnt signalling also in Caenorhabditis elegans, an excellent model organism for study of signalling pathways, and thus contribute to a better understanding of this evolutionarily conserved pathway. In this work, we found that decreased expression of PPK-1/PIP5K in wild type animals does not result in Wnt signalling disruption. Nevertheless, in conditions, where the activity of Wnt signalling is already reduced, decrease in PPK-1 levels leads to defective migration of the QL neuroblast daughter cell. By analyzing the migration of QL progeny, which is controlled by EGL-20/Wnt dependent Wnt...
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Characterization of the Caenorhabditis elegans pop-1 gene
Jakšová, Soňa ; Vacík, Tomáš (advisor) ; Macůrková, Marie (referee)
The TCF/LEF transcriptional factors regulate the target genes of the Wnt signalling pathway - one of the key signalling mechanisms involved in development of multicellular organisms. The TCF/LEF genes produce a number of various protein isoforms, which consequently leads to a great functional diversity of the TCF/LEF proteins. In this diploma project we focused on the Caenorhabditis elegans gene pop-1, the ortholog of the TCF/LEF genes, whose isoforms have not been studied yet. Using the Northern blot analysis we tried to identify alternative isoforms of the pop-1 mRNA in C. elegans. Using quantitative RT-PCR we also analyzed the pop-1 mRNA levels during seven developmental stages of C. elegans. Further, we also determined the expression profile of two important partners of pop-1, the bar-1 and sys-1 genes, whose protein products function as transcriptional co-activators. Key words: canonical Wnt signaling pathway, TCF/LEF transcription factors, Caenorhabditis elegans, pop-1
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Mechanisms regulating the function of adaptor protein 2 complex during endocytosis
Zounarová, Apolena ; Macůrková, Marie (advisor) ; Pleskot, Roman (referee)
Adaptor protein complex 2 (AP2) mediates the interaction of clathrin with the plasma membrane and thus enables the clathrin-coated vesicle formation. AP2 is also responsible for cargo recognition and it recognizes cargo either directly using endocytic motifs YxxΦ or [DE]xxxL[LI] in the cytosolic domains of cargo or indirectly via additional adaptor proteins from which β-arrestin and ARH are the best-known. The binding sites for endocytic motifs are located in the core of AP2 complex and, similarly to the clathrin-binding site, they are blocked by autoinhibitory mechanism in the inactive cytosolic form of AP2. Therefore, binding of endocytic motifs and clathrin must be preceded by conformational change of AP2 complex which is triggered by membrane-bound phosphatidylinositol-4,5-bisphosphates and greatly facilitated by phosphorylation at Thr156 by AAK1 kinase. AP2 is also important for later stages of endocytosis during which it recruits proteins responsible for membrane curvature, fission, and eventual disassembly of clathrin coat. Repeated association of AP2 with the plasma membrane is prevented by the protein NECAP, but the mechanism of inactivation is still poorly understood.
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Molecular basis of interactions between Dishevelled 3 (Dvl3) and Protein Regulator Of Cytokinesis 1 (PRC1)
Kropáčková, Veronika ; Bařinka, Cyril (advisor) ; Macůrková, Marie (referee)
Scaffolding protein Disheveled (Dvl) is a key component of Wnt signaling cascades. Dvl participates in a number of biological processes, such as cell proliferation, differentiation and migration, determination of cell polarity, and also stem cell self-renewal. It is therefore indispensable for the correct embryo development and tissue homeostasis in adulthood. The protein regulator of cytokinesis (PRC1) is a microtubule-associated protein. PRC1 is involved in spindle midzone formation during cell division. Spindle midzone precedes the contractile ring assembly and is essential for normal cell cleavage. In our laboratory, PRC1 was identified as a putative interaction partner of DVL3. This master thesis is focused on delineation of the interaction interface between DVL3 and PRC1 using TIRF microscopy (Total Internal Reflection Fluorescence microscopy). To this end, full-length DVL and PRC1 proteins together with their truncated variants were designed, expressed and purified. It was discovered that PRC1 interacts with all three DVL isoforms and the N-terminal part of PRC1 is required for the interaction between PRC1 and DVL3. Furthermore, the DEP domain of DVL3 is likely involved in PRC1interactions. Key words: Dishevelled 3, DVL3, Protein regulator of cytokinesis 1, PRC1, interaction interface, TIRF...
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Analysis of casein kinase γ function in the model organism Caenorhabditis elegans
Hávová, Daniela ; Macůrková, Marie (advisor) ; Obšilová, Veronika (referee)
Casein kinase 1 (CK1) protein family is an important part of signalling apparatus in eukaryotic organisms. These serin-threonine kinases play roles in several important developmental and cell-maintaining signalling pathways including Wnt signalling. CK1 kinases influence this signalling pathway in various manners; some of them positively - leading to expression of Wnt regulated genes and some of them negatively - contributing to degradation of the transcription activator β-catenin. In this thesis, we were focusing on casein kinase 1 gamma (CK1γ), which in mammalian cells in the presence of Wnt signal phosphorylates Wnt co-receptor LRP5/6. This phosphorylation leads to binding and inhibition of a β-catenin degradation complex. In C. elegans LRP5/6 receptor is probably missing from the genome, yet we can still observe influence of CSNK-1/CK1γ on Wnt signalling. Using reduction-of-function experiments we have found out that loss of csnk-1 expression enhanced a canonical Wnt signalling defect in mutants with reduced Wnt production. Using overexpression approach, we were not able to uncover the tissue specificity of CSNK-1 action. In order to obtain a better tool to study the function of CSNK-1, we began to optimize conditions for the use of auxin-inducible degron system for conditional CSNK-1 depletion.
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The role of inactive MTMR phosphatases in mammalian cells
Sixtová, Nikola ; Macůrková, Marie (advisor) ; Klíma, Martin (referee)
Variable composition of the cellular membranes influences many cellular events such as endosomal transport, autophagy or cellular signalling. The membrane identity is significantly determined by the specific distribution of phosphoinositide derivatives. These derivatives are specifically distributed among cellular membranes and they are tightly regulated by the interplay of corresponding lipid kinases and phosphatases. Myotubularins (MTMRs) form a family of phosphatases dephosphorylating phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate at the 3rd position of the inositol ring. Similarly to their substrates, MTMRs are involved in various cellular events such as endosomal transport or autophagy. Mutations in MTMR proteins lead to dysregulation of the cellular events and manifestation of severe pathologies. Among the most studied are two hereditary diseases, X- linked myotubular myopathy and Charcot-Marie-Tooth syndrome, caused by mutations in MTM1 and MTMR2 genes, respectively. One of the specific features of the MTMR family is the presence of catalytically inactive members. These members were found to regulate protein stability, activity and localization of their active partners. MTMR10 and MTMR12 are two inactive members of the MTMR family, directly interacting with the...
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