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Regulation of STING function during murine polyomavirus infection
Šnejdarová, Aneta ; Horníková, Lenka (advisor) ; Pimková Polidarová, Markéta (referee)
Stimulator of Interferon Genes (STING) is the adapter protein of an innate immunity signalling pathway, involved in detection of double-stranded DNA (dsDNA) in the cell cytoplasm, which leads to the expression of pro-inflammatory genes, including the production of type I interferon. Eventhough during the infection with a dsDNA virus, murine polyomavirus (MPyV), the STING protein is activated, the resulting interferon production is moderate. Therefore, it can be assumed that the function of the STING protein is regulated in MPyV-infected cells. The aim of this thesis was to investigate three mechanisms by which the regulation can occur, namely through protein interaction partners, post- translational modifications, or changes in the subcellular localization of the STING protein. A cell-line of mouse fibroblasts stably expressing the STING protein fused with the HA-tag was established to facilitate the research. Furthermore, two plasmids were prepared, that encode the STING protein fused with the green fluorescent protein, facilitating the monitoring of the localization of the protein in the cell, or with a composite tag containing an in vivo biotinylated BioEaseTM -tag enabling effective isolation of the STING protein. The results of colocalization observations and coimmunoprecipitation suggest that...
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Expression of ubiquitin ligases in gastrointestinal tract
Pícková, Markéta ; Sedláček, Radislav (advisor) ; Čermák, Lukáš (referee)
Ubiquitin (Ub) ligases are important regulatory and signalling molecules, which are involved in majority of cellular processes such as differentiation, DNA repair, and regulation of energetic metabolism or immune response. E3 Ubiquitin ligases are also responsible for pathophysiological changes in the organism and their activity is associated with many human diseases including cancers. This makes E3 Ubiquitin ligases to be new diagnostic markers and interesting pharmaceutical targets. Based on previous studies, these enzymes evince very specific expression in the level of tissues or cell populations. Determination of this specific expression is important for a better understanding of their biological function. In this diploma thesis we systematically screened presence of 370 genes of E3-Ub ligases in gastrointestinal tract under physiological conditions and during acute inflammatory damage of distal colon. Obtained data allowed us to select genes, which can play important role in homeostasis as well as pathophysiology and regeneration of gastrointestinal tract. The screening was based on the expression profiling using qPCR, followed by in situ hybridization to determine the exact localization of the gene expression within tissues. From qPCR analysis was predicted hundred thirty seven candidates for...
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Studium ubiquitinace proteinů DELLA
Breineková, Alžběta
Posttranslational modifications (PTMs) of proteins represent fascinating extensions of the dynamic complexity of living cells' proteomes, but also present a solid obstacle in the proteome analysis. Identification and mapping of PTMs in proteins have improved dramatically, but to comprehend complex mechanisms and biological functions, one must address also a very low abundant proteins. Here, in this thesis entitled 'Analysis of DELLA protein ubiquitination' we demonstrate the use of a recombinant protein standard for the determination of in vivo modified peptides of the DELLA family protein RGA. The candidate peptide sequences were targeted in an SRM-based analysis to detect the ubiquitination site and the results of this analysis and that of an MS/MS data processing indicate that the modification is localized in the conserved N-terminal region of RGA protein.
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Interakce rostlin s pathogeny: význam proteasomu a cytoskeletu
Zelená, Pavla
Modification of substrate proteins by ubiquitin is one of the major regulatory events in eukaryotic cells, and plants use this modification to react to exogenous and endogenous cues. Mounting evidence indicates that the ubiquitin-proteasome system is also a key component in response to biotic stressors. This thesis entitled "Plant-pathogen interactions: The role of proteasome and cytoskeleton" reviews the role of proteasome in plant signaling and briefly summarizes interactions between cytoskeleton and pathogen and outlines the role of cytokinin in plant-pathogen interactions. In the experimental part, proteasome-mediated signaling is probed using growth response assay, identification of proteasome substrates and confocal microscopy of cytoskeleton structures. The results present evidence that the employed methodology will be useful for plant-pathogen interaction analyses.
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