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Studies of polyomavirus trafficking from late endosomes towards the cell nucleus
Štach, Martin ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee)
Mouse polyomavirus (MPyV) is a model virus of the Polyomaviridae family. Polyomaviruses are small non-enveloped DNA viruses. They cause severe problems to immunocompromised patients. Their oncogenic potential is known in animals and humans. Trafficking of MPyV within the cell is not clear yet. The virus enters via smooth monopinocytic vesicles and continues to early and late endosomes. From there, the virus is transported to the ER by unknown mechanism. It bypasses Golgi aparatus (GA). One possible pathway is from late endosomes to trans-Golgi network (TGN) facilitated by Rab9 GTPase and then in COPI vesicles to the ER. In this thesis, the effect of inhibitors of retrograde transport (Brefeldin A, Golgicide A) on MPyV infection was evaluated. Brefeldin A is not completely specific; it has effect on whole endosomal system. Golgicide A causes specific disruption of transport via TGN and GA. Both inhibitors suppressed infection of MPyV. Confocal microscopy revealed colocalization of some MPyV virions with markers of TGN and COPI vesicles. MPyV didn't colocalize with cis-Golgi marker. Unfortunately, the effect of overexpression of Rab9 dominant negative mutant couldn't been evaluated due to its high cytotoxicity. However, overexpression of wild type Rab9 slightly increased infectivity. The results...
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Studies of polyomavirus trafficking from late endosomes towards the cell nucleus
Štach, Martin ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee)
Mouse polyomavirus (MPyV) is a model virus of the Polyomaviridae family. Polyomaviruses are small non-enveloped DNA viruses. They cause severe problems to immunocompromised patients. Their oncogenic potential is known in animals and humans. Trafficking of MPyV within the cell is not clear yet. The virus enters via smooth monopinocytic vesicles and continues to early and late endosomes. From there, the virus is transported to the ER by unknown mechanism. It bypasses Golgi aparatus (GA). One possible pathway is from late endosomes to trans-Golgi network (TGN) facilitated by Rab9 GTPase and then in COPI vesicles to the ER. In this thesis, the effect of inhibitors of retrograde transport (Brefeldin A, Golgicide A) on MPyV infection was evaluated. Brefeldin A is not completely specific; it has effect on whole endosomal system. Golgicide A causes specific disruption of transport via TGN and GA. Both inhibitors suppressed infection of MPyV. Confocal microscopy revealed colocalization of some MPyV virions with markers of TGN and COPI vesicles. MPyV didn't colocalize with cis-Golgi marker. Unfortunately, the effect of overexpression of Rab9 dominant negative mutant couldn't been evaluated due to its high cytotoxicity. However, overexpression of wild type Rab9 slightly increased infectivity. The results...
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Nuclear envelope interactions
Štach, Martin ; Forstová, Jitka (advisor) ; Forman, Martin (referee)
The nuclear envelope is composed of the inner membrane and outer membrane, which is a continuation of the rough ER. Lipids of the nuclear envelope compossess signaling functions. Ganglioside GM1 helps to regulate the concentration of Ca2+ in the nucleus. . Proteomic studies revealed about 100 proteins present in membranes nuclear envelope. Nevertheless, no functions for most of them were uncovered. LEM proteins (LAP2, emerin, MAN1) interact with BAF factor linking them to the lamina and chromatin. The main function of LAP1 and LAP2 is association with lamins. Emerin binds transcriptional repressors, is involved in signaling and RNA splicing. MAN1 binds lamins. LEM2 plays a role in nuclear morphology arrangement. LBR associates with lamin B, DNA-binding proteins and heterochromatin. LINC complex composed of nesprins and SUN proteins links the cytoskeleton with nucleoskeleton. Transport of proteins into the inner membrane is provided through the nuclear pore by several different mechanisms. Keywords: nuclear envelope, emerin, LBR, LAP, MAN1, LINC, transport
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