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Role of nuclear lamins and Nup358 in BK polyomavirus infection
Išler, Lukáš ; Bruštíková, Kateřina (advisor) ; Němečková, Šárka (referee)
BK polyomavirus (BKPyV) has been causing serious health complication for several decades, especially in immunocompromised patients. The aim of this work was to investigate the progress of BKPyV replication during infection of human cells, as well as the influence of BKPyV infection on the nuclear lamina and nuclear pore proteins (NPC; Nuclear Pore Complex). During characterization and comparison of BKPyV infection in RPTEC/hTERT1 and MRC-5 cells we showed that BKPyV replicates better in RPTEC/hTERT1 cells as the productive infection results in six times higher viral titer. Using confocal microscopy we did not observe any nuclear lamina disruption nor VP1 accumulation under nuclear lamina that was previuosly observed in mouse polyomavirus infection. We verified previous observations of cytoplasmic deposits of NPC colocalizing with VP1 protein 24 hours post infection (hpi) with BKPyV and we showed that Nup358, protein of NPC, is a component of NPC deposits colocalizing with VP1. Neither transient expression from vectors encoding late region of BKPyV genome (pEF- BKV-late) or VP1 alone (pIaW), nor LT antigen expression analysis did not suggest any conection of observed phenomena to the productive infection. However, pseudoinfection of RPTEC/hTERT1 cells with VLPs derived from BKPyV induced VP1...
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Vazba paralogů EXO70 na ATG8 a funkční rozdělení rodiny EXO70 dle účasti v autofagii (Arabidopsis thaliana).
Semerádová, Hana ; Kulich, Ivan (advisor) ; Motyka, Václav (referee)
The exocyst, an octameric protein complex conserved among all eukaryotes, mediates tethering of the vesicle prior to its fusion with the target membrane. Apart from the function of exocyst in exocytosis, new studies from both mammalian and plant fields report its involvement in the cellular self-eating process called autophagy. In land plants the number of paralogs of some exocyst subunits is extraordinarily large. There are 23 paralogs of Exo70 subunit in Arabidopsis thaliana. It is supposed that these paralogs have acquired functional specialization during the evolution - including involvement in autophagy. Using yeast two- hybrid assay it is shown here that Exo70B1 and Exo70B2, but not other Arabidopsis Exo70 paralogs interact with Atg8, an autophagosomal marker. The proximity of these two paralogs and Atg8 in vivo was confirmed by independent Förster resonance energy transfer (FRET) method. Interestingly, interaction of Atg8f with Exo70B2 paralog appears to be stronger than with Exo70B1. Exo70B1-mRUBY expressed under the natural promoter shows punctate membrane structures that are mostly static. That changes after the tunicamycin treatment - movement of some of these dots was induced. Homology modeling of Exo70B1 and Exo70B2 proteins tertiary structure in combination with bioinformatic prediction based...
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Autophagy in the immune system
Vávra, Dan ; Černý, Jan (advisor) ; Janštová, Vanda (referee)
Autophagy is an essential, homeostatic process - survival mechanism that protects cells by various ways: cells break down their own components to recycle nutrients, remodel and dispose unwanted cytoplasmic constituents. Autophagy is involved in the degradation of long-lived proteins and entire organelles, but paradoxically, considering important prosurvival functions, autophagy may be deleterious. It plays an important role during development, tumor suppression, immunity and is required for the adaptation to environmental stresses such as starvation. Recent studies indicate, that autophagy is a central player in the immunological control of bacterial, parasitic and viral infections. The process of autophagy may degrade intracellulal pathogens. This work describes the mechanism of autophagy and highlights the role of autophagy in innate and adaptive imunity, summarizes some advances in understanding the functions of autophagy and its possible roles in the causation and prevention of human deseases.
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Vazba paralogů EXO70 na ATG8 a funkční rozdělení rodiny EXO70 dle účasti v autofagii (Arabidopsis thaliana).
Semerádová, Hana ; Kulich, Ivan (advisor) ; Motyka, Václav (referee)
The exocyst, an octameric protein complex conserved among all eukaryotes, mediates tethering of the vesicle prior to its fusion with the target membrane. Apart from the function of exocyst in exocytosis, new studies from both mammalian and plant fields report its involvement in the cellular self-eating process called autophagy. In land plants the number of paralogs of some exocyst subunits is extraordinarily large. There are 23 paralogs of Exo70 subunit in Arabidopsis thaliana. It is supposed that these paralogs have acquired functional specialization during the evolution - including involvement in autophagy. Using yeast two- hybrid assay it is shown here that Exo70B1 and Exo70B2, but not other Arabidopsis Exo70 paralogs interact with Atg8, an autophagosomal marker. The proximity of these two paralogs and Atg8 in vivo was confirmed by independent Förster resonance energy transfer (FRET) method. Interestingly, interaction of Atg8f with Exo70B2 paralog appears to be stronger than with Exo70B1. Exo70B1-mRUBY expressed under the natural promoter shows punctate membrane structures that are mostly static. That changes after the tunicamycin treatment - movement of some of these dots was induced. Homology modeling of Exo70B1 and Exo70B2 proteins tertiary structure in combination with bioinformatic prediction based...
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Autophagy in the immune system
Vávra, Dan ; Černý, Jan (advisor) ; Janštová, Vanda (referee)
Autophagy is an essential, homeostatic process - survival mechanism that protects cells by various ways: cells break down their own components to recycle nutrients, remodel and dispose unwanted cytoplasmic constituents. Autophagy is involved in the degradation of long-lived proteins and entire organelles, but paradoxically, considering important prosurvival functions, autophagy may be deleterious. It plays an important role during development, tumor suppression, immunity and is required for the adaptation to environmental stresses such as starvation. Recent studies indicate, that autophagy is a central player in the immunological control of bacterial, parasitic and viral infections. The process of autophagy may degrade intracellulal pathogens. This work describes the mechanism of autophagy and highlights the role of autophagy in innate and adaptive imunity, summarizes some advances in understanding the functions of autophagy and its possible roles in the causation and prevention of human deseases.
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