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The role of Interferon regulatory factors in virus infections
Suchý, Tomáš ; Forstová, Jitka (advisor) ; Matyska Lišková, Petra (referee)
Viruses are intracellular parasites, which expose their proteins and nucleic acids during their interaction with a cell. Thanks to co-evolution the host immune system developed mechanims how to recognize these components and subsequently activate defensive mechanisms. This work summarizes the knowledge about selected transcription factors with interferon regulatory function. Induction of the antiviral state is complicated and strictly regulated process. Primary function of selected transcription factors is to connect virus detection and synthesis of cellular molecules with antiviral potential. The work includes information about the direct interaction of viral proteins with interferon regulatory factors. These facts enable us insight into the molecular struggle between a host and a virus. As the name of factors suggests, the most important function is to influence the interferon production. Gathering of information about coordination of the immune system is beneficial for humane medicine and future therapeutic purposes. Key words: interferon regulatory factors; viral infection; regulation of innate immunity; interferon; pathogen recognition
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Vesicular trafficking from acidic compartments to the endoplasmic reticulum
Polidarová, Markéta ; Forstová, Jitka (advisor) ; Plocek, Vítězslav (referee)
The cell uses retrograde transport from endosomes to Golgi apparatus and further to the endoplasmic reticulum to recycle its receptors and other proteins. There are several pathways starting on different types of endosomes aimed to the trans-Golgi network and from it further to the endoplasmic reticulum. From the early and maturing endosomes the proteins are transported using the retromer complex. Rab9 GTPase is essential for transport from the late endosomes. Rab6 and Rab11 play major role in the transport form the recycling endosomes. There are two pathways going through the Golgi apparatus. The first one is mediated by COPI vesicles which are regulated by Arf1 GTPase and the pathway is sensitive to brefeldin A. The second pathway is regulated by Rab6 GTPase. Except for endogenous proteins the retrograde transport is used by protein toxins and small unenveloped DNA viruses as well. Rab6 pathway from the recycling endosomes and through the Golgi apparatus is characteristic for Shiga toxin. The retrograde transport of ricin starts on the early endosomes and is less clear. Scientists only started uncovering the transport of small unenveloped DNA viruses.
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Studies of the Mouse polyomavirus: properties of the minor structural proteins, requirements for virion productive trafficking to the nucleus and observed side effects of DNA transfection.
Huérfano- Meneses, Sandra ; Forstová, Jitka (advisor) ; Mělková, Zora (referee) ; Reiniš, Milan (referee)
In this thesis, we aimed to investigate the role of Mouse polyomavirus (MPyV) minor structural proteins, VP2 and VP3 in the viral life cycle as well as to study specific aspects of the productive trafficking of the virus. The thesis is divided into 3 chapters as follows: chapter 1 addresses questions about the interaction of the minor structural proteins, VP2 and VP3, of MPyV with host cells and their role during late stages of virus infection. In the chapter 2, aspects concerning the role of late endosomes, recycling endosomes and actin cytoskeleton in MPyV productive infection are addressed and in the chapter 3, results of studies of interferon (IFN) responses induced after DNA nucleofection are presented. The study in chapter 3 was preceded by a microarray analysis primary performed to reveal cell responses to the presence of the minor proteins within host cells. We showed that both MPyV minor structural proteins, VP2 and VP3, when produced individually (without VP1) are highly cytotoxic, inducing fast, caspase dependent apoptosis. Immuno-electron and confocal microscopy revealed affinity of both proteins to endocellular membranes. Both VP2 and VP3 were found to be bound to damaged ER and mitochondrial membranes. Interaction of the proteins with membranes causing physical damage of organelles...
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Studies of properties of gene products of the Merkel cell carcinoma polyomavirus: Antibody preparation and expression vector construction.
Sauerová, Pavla ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee)
Merkel cell polyomavirus (MCPyV) is a recently discovered human virus, having it's genome often integrated in a genome of Merkel carcinoma cells. Although this type of carcinoma is not so usual, it is very aggressive and it's incidence has been rising in last few years. It is not surprising that this virus is nowadays in the centre of scientific interest, as well as other pathogens and mechanisms affecting human life. Because the virus was discovered not so long ago, its research has been at the whole beginning. This diploma thesisaims to contribute to the study of this virus from the molecular-virology point of view. A neutralizing monoclonal antibody, type IgG2a, targeted against the main capsid protein of MCPyV, VP1, and recognizing its conformational epitote was prepared. This antibody was then used for a pilot study of VP1 VLPs MCPyV movement in mammalian cells. Results showed that the studied virus, at least particularly, utilizes caveolin-1-carrying vesicles for its movement in cells (colocalisation of VP1 VLPs and caveolin-1 was observedColocalisation with EEA1 marker of early endosomes, LamP2 marker of endolysosomal compartments or with BiP marker of endoplasmic reticulum was sporadic but significant. These preliminary results suggest that MCPyV might utilise an endocytic pathway leading...
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Gene Modified Cellular Vaccines against bcr-abl-transformed Cells
Petráčková, Martina ; Vonka, Vladimír (advisor) ; Forstová, Jitka (referee) ; Reiniš, Milan (referee)
of PhD. Thesis Gene Modified Cellular Vaccines against bcr-abl-Transformed Cells In our laboratory we are focused on the development of therapeutic vaccines against chronic myeloid leukaemia (CML), using as a model system mouse bcr-abl-transformed B210 and 12B1 cells. Both these cells are of early B-cell lineage, express BCR-ABL protein and induce leukaemia after i.v. inoculation; the 12B1 cells also induce solid lymphoma-like tumours after s.c. inoculation. Several types of experimental vaccines directed against these bcr-abl-transformed cells were developed in our laboratory. Since it has been recognized that BCR-ABL protein does not carry the immunodominant epitope, our attention switched to the development of cell-based vaccines that are capable of inducing immune responses against a whole complex of tumour-associated antigens. The present work describes studies on experimental cellular vaccines based on the B210 or 12B1 cells, gene-modified to express either IL-2 or GM-CSF or IL-12. For the transfection of these cells, an optimised electroporation method was used. The vaccines were tested in our mouse BALB/c model. All the cell lines derived from B210 cells secreting IL-2, GM-CSF or IL-12 were non-oncogenic. The oncogenicity of the IL-2 producing 12B1 sublines was reduced and most of the...
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Myosin-PIP2 interaction in the cell nucleus
Yildirim, Sükriye ; Hozák, Pavel (advisor) ; Černý, Jan (referee) ; Forstová, Jitka (referee)
Even though nuclear myosin 1 (NM1) and myosin 1C (Myo1C) are the products of the same gene, NM1 has additional 16 amino acids at the N-terminus due to alternative start of transcription. Studies claim that NM1 and Myo1C are nuclear and cytoplasmic proteins, respectively. Therefore, researchers thought that NM1 translocates into nucleus via nuclear localization signal (NLS) in its N-terminal extension. However, here we show that NLS is placed within second IQ domain where calmodulin (CaM) binds in a calcium- dependent manner. Since both NM1 and Myo1C have identical neck domains where NLS resides, we have confirmed that both myosin isoforms localize to nucleus. Based on findings indicating Myo1C binding to phosphatidylinositol 4,5-bisphosphate (PIP2) via its tail domain, we tested if NM1 and Myo1C can interact with PIP2 in the nucleus. We show that both isoforms can bind to PIP2 via their tail domains, and interactions with PIP2 can recruit other nuclear proteins into this lipo-protein complex. PIP2 makes complex with a subset of Pol I transcription and processing machinery proteins and modulate their functions in the nucleolus. Moreover, PIP2 depletion results in a dramatic loss of Pol I transcription activity. NM1 and actin were already shown to promote Pol I transcription. Here, we show that...
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Mechanisms of autophagy and their relation to virus infections
Slavková, Martina ; Forstová, Jitka (advisor) ; Schierová, Michaela (referee)
Autophagy is one of the defense mechanisms of the cells. It is highly conserved from yeast to man. Cytosolic proteins or whole organelles are degraded by this process. Autophagy is induced by various conditions. We distinguish several types of autophagy, e. g. macroautophagy, microautophagy and chaperone-mediated autophagy. Macroautophagy is carried out through the cellular structure called autophagosome. Degradation takes place in the lysosomal compartment of the cells. A large number of proteins and regulatory molecules is involved in the whole mechanism. Autophagy is connected with the other cellular mechanisms such as the cytoplasm-to-vacuole pathway which extends under optimal growth conditions of the cells. Autophagy also works as a defense mechanism during viral infection or it is misused by viruses for their benefit.
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Preparation of expression vectors and virus mutants for studies of the minor structural proteins of polyomaviruses.
Cibulka, Jakub ; Forstová, Jitka (advisor) ; Šroller, Vojtěch (referee)
Polyomaviruses are small non-enveloped DNA viruses infecting birds and mammals, including human. Their capsid consists of the major capsid protein, VP1, and two minor capsid proteins, VP2 and VP3. The VP2 and VP3 proteins are supposed to have an important function in the transport of viral genome into the cell nucleus, which is a key step to facilitate viral replication. VP2 and VP3 proteins of mouse polyomavirus and SV40 have an ability to bind and disrupt cellular membranes. This feature is believed to be involved in the transport of viral genome into the nucleus. Plasmids carrying genes of the minor capsid proteins of Merkel cell polyomavirus were prepared in order to produce and visualize these proteins in mammalian cells. These proteins are known to have very unusual sequences compared to other human polyomaviruses or related mouse polyomavirus. When produced alone, the minor capsid proteins of Merkel cell polyomavirus did not significantly interact with cellular membranes, unlike the minor proteins of the mouse polyomavirus. The second goal of this work was to prepare mouse polyomavirus mutants with deletion in hydrophobic domains of VP2 and VP3 proteins. These domains are likely responsible for the mentioned membrane interactions. Prepared mutants were non-infectious. The loss of infectivity was not...
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Properties and function of middle T antigen of the murine polyomavirus
Fabiánová, Anna ; Forstová, Jitka (advisor) ; Čáp, Michal (referee)
Polyomaviruses are small DNA viruses, which are able to induce a broad variety of tumors. The main oncoprotein of the mouse polyomavirus (MPyV) is middle T antigen (MT antigen) which is able to transform cells. MT antigen has not an enzymatic activity of its own. It is able to activate signal transduction of host cells through its interactions with certain cellular proteins. These proteins include protein phosphatase 2A (PP2A), Src kinase, phosphatidylinositol 3 kinase (PI3K), Shc protein, 14-3-3 protein and phospholipase Cγ1 (PLCγ1). This work is focused on interaction between MT antigen and cellular proteins and on the impact of this interaction on cell transformation. Since MT antigen is a potent oncogene, the work also deals with the character of transformed cells and tumor development in mouse mammary epithelium. Keywords: polyomaviruses, MT antigen, PP2A, PI3K, PLCγ1, Shc protein, 14-3-3 protein
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Preparation of monoclonal antibody against the major structural protein, VP1, of BK virus and construction of plasmids for protein interaction analysis by BiFC method.
Kozmanová, Alexandra ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee)
The aim of this study was to create a monoclonal antibody against VP1 protein BK virus for study of individual steps of its replication cycle. BK virus is human polyomavirus which causes serious disease - polyomavirus associated nephropathy in immunocompromised patients after renal transplantation or hemorrhagic cystitis after bone-marrow transplantation. Virions of BK virus have capsids with icosahedral symmetry consisting of 360 molecules of the major capsid protein VP1 and two minor structure proteins VP2 and VP3, which are not exposed on the capsid surface. VP1 is highly immunogenic what gives us possibility to produce antibodies capable of BK virus monitoring by capsid protein detection. Empty BK virus particles produced in insect cells from a recombinant baculovirus were used as antigen to immunize mice for preparing hybridomas. Cloning of hybridomas created by fusion of myeloma cells with B-lymphocytes from immunized mouse gave us series of primary hybridoma clones. One of them, 5/G10/A5/B2/8, was cloned to homogenity and monoclonal antibody against VP1 BK virus was purified from hybridomas medium by affinity chromatography. The analysis of antibody properties revealed that it recognizes a conformation epitope and can be used for VP1 protein and virion detection in cells by indirect...
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