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Polyomavirus minichromosome structure
Satratzemis, Christos ; Forstová, Jitka (advisor) ; Mělková, Zora (referee)
The polyomavirus genome is present in the host cell as circular double-stranded DNA associated with nucleosomes. Consequently, the expression of polyomavirus genes is affected by the location of nucleosomes on DNA and histone modifications. This thesis reviews the current state of knowledge regarding the polyomavirus minichromosome structure and the effects of nucleosome phasing and histone modifications on polyomaviral replication cycle. In addition, factors conditioning these phenomena are discussed. Drawing on available literature, neither nucleosome phasing nor histone modifications appear to be random. However, not all viral DNA molecules are identical in these respects. Processes such as early and late transcription, replication and encapsidation thus occur only within certain fractions of the set of DNA molecules
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Interaction of transmembrane proteins ASCT1 and ASCT2 with retroviral envelope glycoproteins
Trávníček, Martin ; Trejbalová, Kateřina (advisor) ; Mělková, Zora (referee)
Transmembrane proteins ASCT1 and ASCT2 are ubiquitous neutral amino acid transporters. Apart from their transporter function in metabolically active cells, they also serve as receptors for a wide group of retroviruses. All retroviruses recognizing the transmembrane receptor ASCT2/ASCT1 share a similar env gene, encoding the envelope glycoprotein. Syncytin-1 is the envelope glycoprotein, encoded by human endogenous retrovirus type W, produced in placental cytotrophoblasts of primates, including human. Interaction of receptor binding domain of Syncytin-1 and specific extracellular region of ASCT2 is responsible for fusion of neighbouring cells and formation of multinucleated syncytiotrophoblast. The importance of syncytiotrophoblast lies in higher efficiency of feto-maternal exchange of nutrients and simultaneously in modulation of immune response of mother towards fetus. Defect in syncytiotrophoblast differentiation often leads to complications during pregnancy and impairs the proper development of embryo. Characterization of protein domains responsible for the interaction between Syncytin-1 and its receptors is important to uncover genetic causes of these pathologies. Furthermore, understanding the interaction helps us to clarify the mechanism of cell entry and explains the molecular basis of host...
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Changes of the intracellular redox state during virus infections
Kompas, Maroš ; Mělková, Zora (advisor) ; Trejbalová, Kateřina (referee)
Viruses are infectious agens, which cause disruption of a host cellular redox homeostasis. This effect is mediated by cellular defense machinery or via viral gene products. In order to restore cellular redox enviroment, activation of cellular adaptive response takes place. That is mediated by transcription factor Nrf2, which leads to upregulation of gene expression of antioxidant enzymes. Under suboptimal redox condition, or by detecting foreign nucleic acid, redox sensitive transcription factor Nf-κB is also being activated, what leads to expresion of proteins mediating cellular imunne responses. It is important to remember that these proteins might show malignant effects to surrounding tissues during long term inflammations. With respect to that, viruses have evolved mechanisms, through which they are able to overcome or hijack these pathways, in order to propagate the infection. Key words: intracellular redox state, ROS, RNS, oxidative stress, antioxidant enzymes, regulation of gene expression, virus infections
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Effects of the Interferon regulatory factor 3 on immune responses to vaccinia virus in the atopic organism
Pilná, Hana ; Mělková, Zora (advisor) ; Drbal, Karel (referee)
Vaccinia virus (VACV) is an enveloped DNA virus, member of the Orthopoxviridae genus. VACV genome size is about 200 kbp. This huge genome capacity allows VACV to encode a set of factors that are non-essential for virus replication and spread in vitro. While these factors are needed for interfering with host immune responses, VACV remains strongly immunogenic. Cell-mediated and humoral immune responses in atopic disorders are deregulated to a certain extent, leading to complications in case of infection or vaccination with vaccines based on replicating viruses, such as eczema vaccinatum caused by VACV. VACV effects on immune responses consist among others in the inhibition of expression of type I interferon (IFN) at various levels - for example in a specific inhibition of phosphorylation of the interferon regulatory factor-3 (IRF-3) via inhibition of the activity of TANK-binding kinase 1 (TBK 1) that normally phosphorylates IRF-3. Phosphorylation allows IRF-3 to translocate into the nucleus where it initiates transcription of IFNβ followed by induction of expression of IFN and interferon stimulated genes. Expression of these genes is shut down when IRF-3 activity is inhibited. To overcome this block, a recombinant VACV expressing murine IRF-3 under VACV p7.5 promotor (WR-IRF3) was generated....
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Effects of heme arginate in HIV-1 acute infection and in latency reversal
Prakash, Shankaran ; Mělková, Zora (advisor) ; Hirsch, Ivan (referee) ; Hejnar, Jiří (referee)
The available antiretroviral compounds can effectively suppress the replication of HIV-1 and block the disease progression. However it is impossible to eradicate the virus from the organism as the HIV-1 integrated in the genome is not affected by the existing anti-HIV-1 drugs. Therefore, new latency reversing agents are being actively developed as part of "shock and kill" therapy to reactivate the provirus and clear the reservoir. Normosang (heme arginate; HA) is a human hemin- containing compound used to treat acute porphyria. Heme is physiologically catabolised by heme oxygenases to form iron (Fe2+ ), carbon monoxide (CO) and biliverdin that is further converted to bilirubin by biliverdin reductase. In this study, we have demonstrated that HA inhibited HIV-1 replication during the acute infection, which was accompanied by the inhibition of reverse transcription. On the other hand, HA synergised with phorbol myristyl acetate (PMA) and reactivated the HIV-1 provirus in ACH-2 cells and the HIV-1 "mini-virus" in Jurkat cell clones A2 and H12. HIV-1 ''mini-virus'' was reactivated also by HA-alone. Further, we have studied the effects of heme degradation products on latent HIV-1 reactivation when added individually. We employed addition of ascorbate to generate Fe2+ , resulting in an increased...
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Experimental and clinically used vaccines based on vaccinia virus
Pilná, Hana ; Mělková, Zora (advisor) ; Šroller, Vojtěch (referee)
Vaccinia virus (VACV) is an enveloped DNA virus belonging in the Orthopoxviridae genus. It is a laboratory virus in which the natural host and exact origin remain unclear. However it is of great significance for human kind. First of all, different VACV strains were used for preparation of vaccines used in the smallpox eradication campaign. Even today a significant effort is made to prepare more efficient and safer vaccines against smallpox, namely because of still remaining concerns that variola virus - causative agent of smallpox - could be misused as a biological weapon. Advances in genetic engineering allowed use of VACV for additional purposes, namely as a vaccination and expression vector. VACV enables insertion of large pieces of foreign DNA into its genome and expression of this DNA in a host. Furthermore VACV replicates exclusively in a cytoplasm, decreasing a risk of incorporation of the viral DNA into the host genome. These and other features make VACV an ideal candidate as a vector for preparation of recombinant vaccines against various infectious and oncological diseases. This thesis provides a summary of both clinically used and experimental vaccines derived from VACV. Powered by TCPDF (www.tcpdf.org)
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Study of the effect of immunological sdjuvants on experimental treatment of HPV-induced tumors by recombinant VACV and DNA vaccines
Gabriel, Pavel ; Němečková, Šárka (advisor) ; Mělková, Zora (referee) ; Reiniš, Milan (referee)
1 ABSTRACT The success of cancer vaccines depends on factors associated with the vaccine, which define the main parameters of effective immune responses such as its size and quality, as well as on factors related with the host, represented by the immunosuppressive mechanisms that allow the tumor to escape recognition by the immune system or negatively influence the function of effector T-cells. Attenuated, non-replicating viruses are at present preferred as VACV for safety reasons. A problem may arise concerning their lack of immunogenicity. Through the deletions of non-essential genes, vaccination vectors are therefore developed based on attenuated rVACV capable of replication, which induce a strong immune response. Genes of various immunological adjuvants (e.g., genes for cytokines and costimulatory molecules) are inserted into the vectors for the purpose of eliminating the influence of the immunosuppressive mechanisms of tumors. The first part of the work describes our study of the influence of vCCI on biological properties of rVACV derived from the Prague strain. Testing of vCCI deletion and insertion mutants expressing tumor associated protein HPV16 E7 has shown that secreted vCCI attenuated the virus in vivo, which correlated with reduced levels of the corresponding CC chemokines in the blood compared...
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