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Characteristics of HPV16 transformed mice cells, transduced with genes for immunomodulating factors and endostatin
Lakatošová, Monika ; Vonka, Vladimír (advisor) ; Reiniš, Milan (referee) ; Forstová, Jitka (referee)
Genesfor two cytokines,one chemokineandgenecoding for oneangiostaticfactor were used in the presentwork for tansfection ofmouse IIPV-16- transformedtumor cells. Main characteristics oftransduced cells werc t€stedrnraTo and,in vivo and,cnrnparedwith theparentaltumor cells. In the first part I useda thymidine-kinase deficient (cTK) cell line designated123IA, which had beenderived from IIPVI6 transformedmouse(C57BL/6) cells MK16. To obain genetically modified cells, 1231Acells were transfectedwith bicistronic plasmids carrying the herpessimplex type 1thymidine kinase(HSV-TK) gen,eandeitherthe genefor the mouseB7-l (CD80) co-stimulatory molecule or the gene for the monocyte-chemoattractantprotsin 1 (MCP-|). For control putposes,a plasmid vector carrying only the HSV-TK genewas used.For comparativepurposesI also used89 cells, previously isolated in our laboratory, which expressthe mousegranulocyte-macrophagecolony stimulation factor (GM-CSF) andHSV-TK gene.All the cell lines testedwere found to be sensitiveto minute amountsof ganciclovir, revealing the production of functional HSV-TK. When inoculatedinto syngeneic mice, cells expressing either GM-CSF or B7-1 were non-oncogenic. Nearly all mice inoculatedwith MCP-l-producingcellsdevelopedtumors.Animalsinjectedwith GM-CSF or B7-l- producing cells were...
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Viruses and cytosketelon of the cell nucleus
Cibulka, Jakub ; Forstová, Jitka (advisor) ; Šťovíček, Vratislav (referee)
The nuclear cytoskeleton (the nucleoskeleton) provides a structural integrity to the nucleus and is involved in number of key processes including transcription, chromatin remodelling and mRNA transport. The nucleoskeleton consists of nuclear lamins, nuclear actin and other proteins. Some viruses, which replicate themselves in the nucleus, use nuclear cytoskeleton in their life-cycle. On the other hand the nucleosketon may also represent a barrier for viral infection. Herpesviruses need nuclear actin for capsid assembly and transport, but they have to desintegrate the nuclear lamina in order to escape the nucleus. Nuclear actin also participates in the morphogenesis and probably nuclear export of baculovirus capsids. Some retroviruses transport their unspliced RNAs from the nucleus using nuclear actin and there is also some evidence of retrovirus-induced nuclear lamina disruption. In this work, I focus on the interactions of above-mentioned viruses with the nuclear cytoskeleton (namely nuclear actin and lamins).
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Interference of selected DNA viruses with apoptotic processes
Sauerová, Pavla ; Forstová, Jitka (advisor) ; Štěpánek, Luděk (referee)
This work is focused on selected DNA viruses and some of their mechanisms used for inhibition or induction of the apoptotic processes. The selected DNA viruses are Hepatitis B virus, polyomaviruses, papillomaviruses and herpesviruses. Viruses developed different strategies for fighting the host defense mechanism during their evolution. One of the host defense mechanisms that reacts against virus infection is apoptosis. In case of viruses we can observe the phenomenon of inhibition or induction of apoptosis (which both depend on the life cycle phase of the virus). The purpose of these "fighting" strategies is to ensure successful replication, virus releasing from the cell and finally to let it spread in an organism or among them. Some "fighting" strategies are similar e.g. targeting and manipulation on p53 oncosupresor level or production of Bcl-2 homologs; other strategies are very specific. Certain viruses have mechanisms which allow them to survive in a host organism for a long time.
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Recombinant vaccinia virus for cancer therapy, the analysis of biological and biochemical features.
Žůrková, Kamila ; Němečková, Šárka (advisor) ; Forstová, Jitka (referee) ; Trejbalová, Kateřina (referee)
151 8 SUMMARY Recombinant vaccinia virus has been used for elicitation of the immune response against expressed heterologous proteins which has led to protection of the host organisms against the agents producing that antigen (viruses, cancer cells). In our laboratory, we designed and evaluated several vaccines against cancer caused by human papillomavirus type 16 (HPV16). Vaccinia viruses derived from replication competent strain P13 or attenuated MVA were used for construction of recombinant viruses expressing HPV16-E7 in highly immunogenic fusion construct SigE7LAMP. Recombinant viruses were used both in prophylactic and therapeutic settings in mouse tumor models using TC-1 or TC-1/A9 cells. The genes encoding stimulatory cytokines GM-CSF or Flt3 ligand were inserted into the above viruses to support the immune system and to potentiate the anticancer response. Tumor microenvironment was modified using the recombinant viruses expressing both the E7 gene and soluble receptor for TGF-β which should decrease the inhibition of immune system caused by tumor TGF-β cytokine and elicit the response against tumor cells. Intratumoral or intraperitoneal administration of viruses enhanced anticancer response in mice, the viruses expressing Flt3 ligand induced the proliferation of E7- specific cytotoxic T lymphocytes....
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Fluorescence studies of bacterial membrane proteins and cell signalling.
Fišer, Radovan ; Konopásek, Ivo (advisor) ; Hof, Martin (referee) ; Forstová, Jitka (referee)
(English) This work is based on five publications studying mostly adenylate cyclase toxin (CyaA) from Bordetella pertussis and its interaction with biological membranes. CyaA permeabilizes cell membranes by forming small cationselective pores and subverts cellular signaling by delivering an adenylate cyclase (AC) enzyme that converts ATP to cAMP into host cells. First study clarifies the membrane disruption mechanisms of CyaA and another bacterial RTX toxin; αhemolysin (HlyA) from Escherichia coli. For this purpose, we employed a fluorescence requenching method using liposomes as target membranes. We showed that both toxins induced a graded leakage of liposome content with different ion selectivities (Fišer a Konopásek 2009). Both AC delivery and pore formation were previously shown to involve a predicted amphipathic αhelix(502522). In the second publication we investigated another predicted transmembrane αhelix(565591) that comprises a Glu(570) and Glu(581) pair. We examined the roles of these glutamates in the activity of CyaA, mostly on planar lipid membranes end erythrocytes. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu(570) in...
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Construction of various types of vaccines based on the structural proteins of mouse polyomavirus and analysis of immune response after their administration to mice
Hrušková, Veronika ; Forstová, Jitka (advisor) ; Roubalová, Kateřina (referee) ; Reiniš, Milan (referee)
Conclusion Humoral and cellular immune responses developed in mice after intranasal delivery of model mouse polyomavirus derived VLPs carrying epitope of enhanced green fluorescence protein (EGFP) Model chimeric EGFP-VLPs were purified and used for immunization of mice. Immune response of immunized animals was examined. No specific antibodies against EGFP protein, but high titers of specific antibodies against major structural protein VP1 were developed in the sera of immunized animals. Splenocytes derived from immunized animals secreted IL-2 and IFN-γ after their antigen (EGFP or VP1) restimulation. Proliferation of CD4+, but not CD8+ T cells from immunized mice after the stimulation with both EGFP and VP1 was observed. No EGFP specific cytotoxic activity of splenocytes from immunized mice was detected. The presentation of EGFP-VLPs in the context ofMHC class II was blocked by inhibitors of endo-lysosomal as well as proteasomal compartments. Changes in the numbers of CD25+Foxp3+ subpopulation of CD4+ T cells were observed in the spleens if immunized mice. Chimeric VLPs derived from mouse polyomavirus carrying epitopes of human Bcr-Abl fusion protein (Bcr-Abl VLPs) Chimeric Bcr-Abl VLPs carrying 171 amino acids sequence of Bcr-Abl protein (containing Bcr-Abl breakpoint region) were prepared. Chimeric VLPs...
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IGFBP3 expressing rekombinant vaccinia virus used for tumor therapy
Musil, Jan ; Němečková, Šárka (advisor) ; Forstová, Jitka (referee)
IGFBP-3 expressing rekombinant vaccinia viruses used for tumor therapy Insulin-like growth factor-binding protein-3 (IGFBP-3) is a major regulator of endocrine effects of IGF and is capable to suppress the growth of variety of cancer. Several studies have shown that IGFBP-3 can induce the apoptosis of cancer cells via IGF-dependent and IGF-independent mechanisms. In our study, we have constructed recombinant vaccinia viruses (VACV) expressing IGFBP-3 under the control of the early H5 and synthetic early/late (E/L) promoter to investigate the potential effect on cancer growth in our cervical cancer model. We have shown that the expression of IGFBP-3 alone had no effect on tumor growth. On the other hand, the co-expression of IGFBP-3 enhanced the anti-cancer effect of immunization with the fusion protein SigE7LAMP, which gave rise to the anti-cancer immunity directed against HPV16 induced tumors. We have shown that the double-recombinant P13-SigE7LAMP-H5-IGFBP-3 can enhance the protective immune responses against MK16/ABC induced tumors. Furthermore, we have show that both double-recombinant viruses P13-SigE7LAMP-H5- IGFBP-3 and P13-SigE7LAMP-E/L-IGFBP-3 can increase the anti-cancer effect of SigE7LAMP expression in the therapy of TC-1 induced tumors. Key words: IGFBP-3, IGF, VACV, HPV16, E7 oncoprotein,...
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HIV - 1 Protease: Insights into Drug Resistance Development
Grantz Šašková, Klára ; Konvalinka, Jan (advisor) ; Forstová, Jitka (referee) ; Dohnálek, Jan (referee) ; Schiffer, Celia (referee)
Amino acid changes within HIV protease or its substrate that decrease the susceptibility to protease inhibitors represent a highly complex issue still not yet fully understood. Various mechanisms by which this often complicated pattern of mutations influence drug binding needs to be analyzed on a molecular level by a series of methods including experiments with recombinant viruses, biochemical enzyme analysis, structural and thermodynamical studies or molecular dynamics. Each result may help to complete the overall picture of protease inhibitor resistance evolution and therefore contribute to the design of more powerful 3rd generation HIV/AIDS drugs. This thesis presents several analyses of HIV resistance development on molecular level. We have focused on the nelfinavir resistance pathway, lopinavir mutation score, emergence of amino acid insertions in HIV protease gene and their contribution to protease inhibitor resistance and finally we analyzed a highly mutated protease species isolated from patients failing darunavir therapy. Since we are able to accomplish a wide combination of techniques, we could explain and put together some pieces of viral evolution considering the final steps of HIV life cycle and also provide knowledge necessary for novel inhibitor design. Aims of the Project There were...
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Roles of cytoskeleton in mouse polyomavirus trafficking
Klímová, Lucie ; Forstová, Jitka (advisor) ; Šmahel, Michal (referee)
6 Roles of cytoskeleton in mouse polyomavirus trafficking ABSTRACT: Mouse polyomavirus (mPyV) is small non-enveloped DNA virus. Its endocytic pathway is studied for a potential utilisation of polyomaviral virus-like particles in gene therapy and/or immunotherapy. mPyV enter cells by internalisation into smooth monopinocytic vesicles. During it's journey through the cell, it pass through early endosomes, and at the time 3 hours post infection, it is localised in endoplasmic reticulum and recycling endosomes. Many aspects of mPyV trafficking and nuclear entry are not clear yet. Time-lapse live imaging fluorescence confocal microscopy was used to describe the mouse polyomavirus intracellular movements. For these studies, we utilised mPyV fluorophore-labeled virions and cells expressing GFP-tagged g-actin or alpha-tubulin. Some virion-loaded vesicles were seen to move with actin organised into dynamic structures. Some of these structures resembled actin comets created by Listeria or vaccinia virus. At the same time post infection (40-60 min post infection), movement of the virion loaded vesicles along mirotubules was observed suggesting the simultaneous involvement of actin and tubulin during mPyV trafficking. Dynamitin, a dominant negative inhibitor of dynein-dynactin function reduced mPyV infection. Taken...
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