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Fish Herpesviruses
Čečetka, Petr ; Forstová, Jitka (advisor) ; Kuthan, Martin (referee)
Herpetic viruses that attack fish are ones of the most widespread virus pathogens, threatening modern day fish population not only in the wild but also those that are farmed. There are many types of the pathogen, from the ones those do not cause severe diseases to those that are extremly dangerous, spread without any control and caused vast losses in commercial farming and in wild populations. Most of the viruses exhibit increased sensitivity on heat and stress factors, which are the most common reasons of the repeating outbreak of disease. To the best described fis herpesvirus viruses attacking fresh water fishes belong. Nowadays more and more herpetic viruses which attack mainly salt water fis are beeing discovered. Powered by TCPDF (www.tcpdf.org)
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Analysis of cell signaling mediated by the adapter protein Daxx
Švadlenka, Jan ; Anděra, Ladislav (advisor) ; Forstová, Jitka (referee) ; Stopka, Tomáš (referee)
2 Abstract Multifunctional adapter protein and histone chaperone Daxx has been described in nu- merous cellular processes, including the regulation of apoptotic and stress signalling, antiviral response and processes connected to chromatin (e. g. transcription). Its influ- ence on chromatin-related processes is mainly carried out by several associated en- zymes, such as DNA-methyltransferase-1, histone deacetylases and chromatin- remodelling ATPase ATRX. In the complex with ATRX Daxx functions as a chaperone of histone-3.3, maintaining the constitutive heterochromatin e. g. at centromeric and telomeric regions. The main aim of this Thesis was a better understanding of the Daxx cellular functions through identification and functional characterization of its novel interacting proteins. Using the yeast two-hybrid screen, several such new Daxx-interacting proteins were identified. These proteins were mainly nuclear, connected to the regulation of chroma- tin-related processes. More detailed analysis focused on the interaction of Daxx with chromatin-remodelling ATPase Brg1. This interaction was confirmed both in vitro and in the cells, where Daxx and Brg1 associated mainly in high molecular weight pro- tein complexes. These likely chromatin-remodelling complexes contain, in addition to Brg1, several...
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Histone modifications and methylation of polyomaviral genomes during the infection
Mrkáček, Michal ; Forstová, Jitka (advisor) ; Šmahelová, Jana (referee)
Similarly to other viruses, polyomaviruses require for their successful replication enzymes and other proteins encoded by their host cells. Additionally, because of their relatively small genome with only a few genes, polyomaviruses utilize for their efficient replication cellular regulation mechanisms. One of these regulations are posttranslational modifications of histones, which form nucleosomes together with viral DNA. The spectrum of these modifications is very wide, but in case of polyomaviruses, almost only ones studied are histone acetylations and methylations. Second possible regulation is a methylation of cytosine in CpG dinucleotides, which is associated with repression of gene expression. Current knowledge however suggest that polyomaviruses do not utilise this kind of modification. Moreover, because of a relatively small amount of CpG dinucleotides present in their genomes, they seem to avoid it. The goal of this work is to describe the individual types of these modifications and show their possible importance in the infectious cycle of polyomaviruses. Key words: polyomavirus, epigenetics, histone modification, DNA methylation, CpG dinucleotides
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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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Endoplasmic reticulum-associated degradation and its role in virus infection
Svobodová, Terezie ; Forstová, Jitka (advisor) ; Mašek, Tomáš (referee)
The endoplasmic reticulum-associated degradation pathway, ERAD, is an important mechanism for maintaining cellular homeostasis. The function of ERAD is degradation of accumulated unfolded proteins in the endoplasmic reticulum. ERAD is carefully regulated by pathway called "Unfold protein response" and by "ERAD tuning" mechanism. Some viruses have adopted the ways how to exploit this system or its factors for their own benefit. These utilizations include targeting of specific host proteins for degradation, transfer of viral products or virions from the endoplasmic reticulum into the cytoplasm, or the use of a membrane platform arising from the cooperation with "ERAD tuning" for viral replication. Role of ERAD in viral infection can manifest itself in different ways, it can contribute to degradation not only of host proteins but also of viral products. In this work I summarize mechanisms of ERAD pathway and their regulatory pathways. Meanwhile, in the specific examples, I present roles of ERAD pathway and associated systems in viral infections.
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Role of phosphorylation in nuclear import of viral proteins and complexes
Pokorná, Karolína ; Forstová, Jitka (advisor) ; Roučová, Kristina (referee)
Replication of many different viruses occurs in the nucleus of the host cell. These viruses discovered ways how to overcome the nuclear membrane and often use cell transport machinery to transport their proteins and genome into the nucleus. For many viral proteins the timing of their nuclear import in order to secure correct viral replication is important. Regulated nuclear import also allows these proteins to perform several functions depending on their localization. Nuclear import of viral proteins and complexes can be regulated by phosphorylation. Phosphorylation can, for example, modulate affinity of proteins for importins or other cellular proteins. Phosphorylation can also cause conformational change, which can lead to unmasking of localization sequence.
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Mouse polyomavirus: The role of cell cytoskeleton in virus endosomal trafficking and properties of the minor capsid proteins
Žíla, Vojtěch ; Forstová, Jitka (advisor) ; Hozák, Pavel (referee) ; Rumlová, Michaela (referee)
Mouse polyomavirus (MPyV) is a non-enveloped DNA tumor virus, which replicates in the host cell nucleus. MPyV enters cells by receptor-mediated endocytosis and its subsequent transport towards the nucleus requires acidic environment of endosomes and intact microtubules, which are important for virus delivery to endoplasmic reticulum (ER). In ER, capsid disassembly and uncoating of viral genome take place. The mechanism of subsequent translocation of viral genome from ER into nucleoplasm is still only poorly understood process with predicted involvement of cellular factors and viral minor capsid proteins VP2 and VP3. Once the genome appears in the nucleus, early viral antigens are produced and mediate suitable environment for replication of viral genomes. After replication of viral DNA and morphogenesis of virions, virus progeny is released from the cells during its lysis. The research presented in the first part of thesis focused on intracellular transport of MPyV and involvement of cytoskeletal networks during virus delivery to the ER. In particular, we investigated still unclear role of microtubules during virus trafficking in endosomes, and involvement of microtubular motors. We found that MPyV trafficking leading to productive infection does not require the function of kinesin-1 and kinesin-2,...
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Functions of actin and myosin 1c in the cell nucleus and in the cytoplasm
Kalendová, Alžběta ; Hozák, Pavel (advisor) ; Binarová, Pavla (referee) ; Forstová, Jitka (referee)
Human MYO1C gene encodes three myosin 1c (Myo1c) isoforms which differ only at their N-ends. Interestingly, all three isoforms localize to the nucleus and also to the cytoplasm, where they are anchored to the plasma membrane by the interaction with phosphatidyl inositol-4,5-bisphosphate (PIP2). However, studies reporting functional involvement of these isoforms are inconsistent. While the shortest isoform C (Myo1c-isoC) has been implicated exclusively in the cytoplasmic processes, the longer isoform B (termed the nuclear myosin 1, NM1) has been employed in the nuclear and processes, such as DNA transcription and rRNA maturation. Similarly, the longest isoform A (Myo1c-isoA) exerts its functions in the nucleus solely. To complete the information on the cellular functions of Myo1c isoforms, we searched for the cytoplasmic functions of NM1 and nuclear functions of Myo1c-isoC. In mouse, only two isoforms (NM1 and Myo1c-isoC) are expressed. We prepared the knock-out mouse (KO) which lacks specifically NM1 while retaining Myo1c-isoC unchanged. Surprisingly, this manifested in no phenotype observed. Since we demonstrated that even Myo1c-isoC acts in the transcription in the similar manner as NM1, it suggests that Myo1c- isoC functionally overlap with NM1 in the nuclear functions. Besides its localization...
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Studies of properties of the minor structural proteins of the Murine polyomavirus
Bílková, Eva ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee)
Murine polyomavirus (MPyV) is a member of the Polyomaviridae family. Its capsid is composed of the major capsid protein, VP1, and the minor proteins, VP2 and VP3. The minor capsid proteins probably assure delivery of the viral genome through the endoplasmic reticulum membrane to the nucleus during early phase of infection. However, precise mechanism is not known. Expression plasmids encoding mutated VP2 or VP3 fused with EGFP have been constructed to study the interaction of VP2 and VP3 with membranes. The mutated proteins have deletions in the predicted hydrophobic domains. In this thesis, cell localisation of mutated proteins was followed. The study revealed that the hydrophobic domain 2 is the most important for association of VP2 and VP3 with membranes, while domains 1 and 3 are rather expendable. Further, nature of VP2 and VP3 isoforms has been studied. Isoforms with different electrophoretic mobility were separated on SDS-PAGE. Consequent mass spectrometry analysis showed that they differ in deamidation of asparagine, present at both minor proteins (position 253 of VP2 and 137 of VP3). Previously, acetylation of VP3 N-terminal alanine has been identified. To elucidate the function of these modifications, mutated viruses were constructed with substitution of these amino acids. Pilot...
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