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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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Role of RAD18 in ubiquitin signaling at DNA double-strand breaks
Palek, Matouš ; Macůrek, Libor (advisor) ; Čermák, Lukáš (referee)
RAD18 is an E3 ubiquitin ligase that prevents the replication forks from collapsing caused by damaged DNA. As an important factor controlling replication, its dysregulation was shown to be associated with some human tumours. However, the clinical relevance of this finding is unknown. The aim of the thesis was evaluation of selected RAD18 variants that had been identified in breast and ovarian cancer patients. This work revealed functional defects of RAD18 variants not only in replication fork protection but also in repair of DNA double-strand breaks. This unconventional role of RAD18 is known to be dependent on upstream ubiquitination events, however, its contribution to the repair per se is not understood. This work aimed to elucidate the function of RAD18 in DNA double-strand break repair by homologous recombination focusing especially on its relationship with 53BP1. Data presented here show that RAD18 effectively disrupts 53BP1 accumulation in the repair foci by competition for the same binding partner and thus promotes resection of DNA ends. This antagonistic function of RAD18 is restricted both spatially (to the vicinity of the repair centre) and temporarily (to S phase). Moreover, it seems to be regulated by existence of RAD18 in two distinct complexes. Potential models for this regulation...
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Cancerogenic and metastatic potential of cancer cells with non-functional CRL4 ubiquitination complex
Slámová, Monika ; Procházka, Jan (advisor) ; Grantz Šašková, Klára (referee)
Ubiquitination complex CRL4 (Cullin Ring Ligase) attracts a lot of attention due to its involvement in physiological and pathological processes, especially in the development of cancer. Cullin4 a/b proteins are reported to serve as oncoproteins in various malignancies. Due to their role in the regulation of cancer drugs targeting CRL4 have been identified, including thalidomide and its derivatives inhibiting one of the substrate receptors of the complex, the Cereblon protein. The adapter protein within the CRL4 complex - DDB1, which is involved i.a. in DNA repair, also has a role in cancer. However, the mechanism of this function has not yet been fully elucidated. The subject of this master thesis was to study the effects of elimination and suppression of CRL4 complex functions in the prostate cancer cell line LNCaP. Significantly variable changes in cell proliferation and migration have been observed if the complex functions were affected by thalidomide. The creation of the LNCaP cell line with conditionally suppressed DDB1 function was used to study tumor dynamics in a mouse model. Results show that suppression of DDB1 function has an inhibitory effect on tumor cell proliferation but increases their ability to invade adjacent tissues. Complete deletion of the DDB1 gene in the LNCaP cell line...
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Dysregulation of E3 ubiquitin ligases in inflammatory bowel diseases
Armerová, Eliška ; Petrezsélyová, Silvia (advisor) ; Červená, Klára (referee)
E3 ubiquitin ligases are a large family of enzymes involved in many cellular processes such as DNA damage repair, transport of membrane proteins, chromatin modification, cell cycle and apoptosis. E3 ubiquitin ligases have been shown to play a significant role in the maintenance of intestinal homeostasis, and their abnormal function associated with their deregulation contributes to inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. In recent years, GWAS has identified approximately 200 risk loci that are susceptible to these diseases. Including those encoding E3 ubiquitin ligases.The aim of this work is to compare the already identified E3 ubiquitin ligases associated with these diseases and to give an overview of them with a focus on the regulation of intestinal homeostasis. Key words: E3, ubiquitination, inflammatory bowel diseases, intestinal homeostasis, CD, UC
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Dysregulation of E3 ubiquitin ligases in inflammatory bowel diseases
Armerová, Eliška ; Petrezsélyová, Silvia (advisor) ; Červená, Klára (referee)
E3 ubiquitin ligases are a large family of enzymes involved in many cellular processes such as repair of damaged DNA, transport of membrane proteins, chromatin modification, cell cycle and apoptosis. E3 ubiquitin ligase has been shown to play a significant role in the maintenance of intestinal homeostasis, and their abnormal function associated with their deregulation contributes to inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. In recent years, approximately 200 risk loci have been identified that are susceptible to these diseases, including those encoding E3 ubiquitin ligase. 10 of them have been identified. The aim of this work is to compare the already identified E3 ubiquitin ligases associated with these diseases and to give an overview of them with a focus on the regulation of intestinal homeostasis. Key words: E3, ubiquitination, inflammatory bowel diseases, intestinal homeostasis, CD, UC
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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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