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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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Extracellular vesicles and middle T antigen of mouse polyomavirus
Kropáček, Václav ; Šroller, Vojtěch (advisor) ; Brázdová, Andrea (referee)
This study is focused on middle tumor antigen (MT Ag) of mouse polyomavirus (MPyV), potential consequences of it's secretion via extracellular vesicles (EVs) and it's effect on cellular signaling. MT Ag is membrane bound protein able to induce cellular transformation thanks to it's ability to interfere with cellular signal transduction. Mainly due to aberrant activation of MAP kinase pathway. Firstly we followed up previous observations of our group concerning ability of MT Ag to be secreted from cells via extracellular vesicles. We were interested if MT Ag could contribute to malignant transformation in recipient cells. We performed 2 types of EVs isolation from cell lines stably expressing middle T antigen (3T6MT). We confirmed presence of MT Ag in isolated EVs. Then we characterized isolated EVs by detection of exosomal markers and cryo-electron microscopy. In next step we exposed recipient cell line (3T6) to isolated EVs and with use of flow cytometry tried to detect internalization of MT Ag. Simultaneously we tried asses levels of Erk phosphorylation in 3T6 cells exposed to EVs. Secondly we tried to confirm and analyse previous unpublished observations of elevated levels of NF-kB phosphorylation in cells stably expressing MT Ag. We used western blot and detection of NF-kB dependent secreted...
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Study of exosomes in polyomavirus infection
Hyka, Lukáš ; Šroller, Vojtěch (advisor) ; Saláková, Martina (referee)
Exosomes are extracellular vesicles of endosomal origin. It was thought, that exosomes are used by cells only as carriers for cellular waste, but it was found out, that exosomes serve in the cellular communication and have a role in viral infections. Exosomes are exploited by viruses for example for the transport of viral protein or viral RNA/DNA. One of the viruses, where the mechanism of exploitation is unknown (if any exists) is murine polyomavirus. Murine polyomavirus belongs to the family Polyomaviridae, to which other human viruses belong for example, JC virus or virus of Merkel cell carcinoma. Murine polyomavirus codes for small, large and middle T antigen and three capsid proteins. Middle T antigen is known to bind to cellular membranes. Exosomes are membrane derived structures, so we investigated a possible transfer of middle T antigen. To this goal the successful isolation of exosomes and their characterization was necessary. Exosomes were isolated by ultracentrifugation and further purified by the density gradient OptiPrep. Exosomes were characterized by electron microscopy, NanoSight and by protein exosomal markers. These markers are for example Alix and flotillin-1. The cells were transfected in order to produce middle T antigen. It was shown, that exosomes isolated from these cells...
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