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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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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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Elucidating the interactions of interleukin-1alpha with components of the eukaryotic transcription machinery
Zámostná, Blanka ; Pospíšek, Martin (advisor) ; Černý, Jan (referee) ; Mělková, Zora (referee)
4 ABSTRACT Interleukin-1α (IL-1α) is a pleiotropic cytokine and a key mediator of host immune response. It is synthesised as a 31-kDa precursor, that is cleaved by the cysteine protease calpain into the 17-kDa mature IL-1α and the 16-kDa N- terminal peptide of IL-1α (IL-1αNTP). Although IL-1α can be secreted, act on target cells through the surface receptor IL-1RI and trigger the signal transduction pathway, increasing evidence points toward the involvement of IL-1α in certain nuclear processes. IL-1αNTP is highly conserved among higher eukaryotes and contains a nuclear localisation sequence; indeed, both the precursor and IL-1αNTP are found in the cell nucleus. Previously, a genetic interaction of IL-1α with nuclear histone acetyltransferase (HAT) complexes has been reported from mammalian cells and, interestingly, also from the heterologous yeast model. This thesis extends the research of the nuclear function of IL-1α and demonstrates that IL-1α physically associates with the HAT/Core module of yeast SAGA and ADA HAT complexes. Results of the HAT subunit gene knock-out experiments followed by a set of co-immunoprecipitations also suggest a novel model of the yeast SAGA complex assembly, in which ADA appears to represent only a partly functional HAT complex. In its natural milieu of mammalian cells, IL-1α...
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