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Studies of properties of viral capsid proteins and development of recombinant vaccines and diagnostic components based on artificial viral structures
Fraiberk, Martin ; Forstová, Jitka (advisor) ; Hubálek Kalbáčová, Marie (referee) ; Hejnar, Jiří (referee)
The aim of this study was to develop a system for easy production of different veterinary chimeric vaccines based on stable mouse polyomavirus (MPyV) structures. The system is designed for antigens that are problematic in production or stability. First, universal vectors for baculovirus-directed production of chimeric MPyV VLPs or pentamers based on the major capsid protein VP1 were designed to be exploited as vaccines against other pathogens. The different strategies used in this study are based on: A) exposure of selected immunogenic epitopes on the surface of MPyV VLPs by inserting them into a surface loop of the VP1 protein, B) insertion of foreign protein molecules inside the VLPs, or C) fusion of a foreign protein or its part with the C-terminus of VP1 protein, thus forming giant pentamers of a chimeric protein. Candidate vaccine antigens against porcine circovirus 2 (PCV2), the causative agent of porcine circovirus 2 systemic diseases (PCV2-SD) which causes significant economic losses in swine breeding, were prepared using the constructed vectors. All candidate vaccines induced the production of antibodies against the capsid protein of PCV2 after immunization of mice. The candidate vaccine Var C based on fusion of MPyV and PCV2 capsid proteins, is able to induce production of antibodies with...
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Targeting DNA and DNA synthesis in situ
Strunin, Dmytro ; Koberna, Karel (advisor) ; Hocek, Michal (referee) ; Forstová, Jitka (referee)
The thesis is focused on the development and application of the new methods for targeting DNA and DNA synthesis in situ. Three methodical approaches were developed, which significantly improve detection of cellular DNA. Concurrently, a new insights in the mech- anism of 2'-deoxy-5-ethynyluridine cytotoxicity were described. The approaches developed in this work provide an important tool for study of DNA replication and repair mechanisms, the study of mito- chondrial DNA, as well as the analysis of cell proliferation. The first part of the thesis presents the new method for the detection of 5-bromo-2'-deoxyuridine incorporated into the newly synthesized DNA using monovalent copper ions in the presence of oxygen. This method is applicable for the detection of nuclear DNA synthesis as well as for the mitochondrial DNA synthesis. Next part of the work describes the approach enabling highly efficient detection of over- all nuclear and mitochondrial DNA. The third approach concerned the elimination of the unspecific binding of anti 5-bromo-2'-deoxyuridine antibodies to another modified nucleoside - 2'-deoxy-5-ethynyluridine. This method is useful in the studies where the necessity of two con- secutive labeling pulses is needed. The approach is based on the use of non-fluorescent azido-molecules. The...
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Exosomes, their biogenesis, composition and role
Hyka, Lukáš ; Forstová, Jitka (advisor) ; Motlová, Lucia (referee)
Exosomes are a subtype of extracellular vesicles. Exosomes are distinguishable from other extracellular vesicles by their endosomal origin and their typical cup-shaped morphology. The biogenesis of exosomes begins in the early endosomes by inward budding. The endosomal sorting complex required for transport sorts ubiquitinylated proteins into the vesicles. The small volume of cytosol is also encapsulated during budding. These vesicles are called intraluminal vesicles and the whole body is called multivesicular body. Multivesicular body fuses with the plasma membrane and vesicles are released as exosomes into the extracellular space. Exosomes are present in all bodily fluids and are secreted by a high number of cells. Exosomes present antigens on their surface to trigger immunity or serve in the cellular communication by the transfer of small RNAs.
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Interactions of mouse polyomavirus with Toll-like receptors
Pokorná, Karolína ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee)
Toll-like receptors (TLRs) are important receptor family of innate immunity. They enable fast recognition of infection through so called pathogen associated molecular patterns (PAMPs). In this thesis, we studied interaction of mouse polyomavirus (MPyV) with TLRs of mouse embryonic fibroblasts (MEF cells). We observed that inhibition of TLR4 signaling abolished response of MEF cells to MPyV. This suggested that TLR4 plays a role in MEF cells recognition of MPyV. To detect response of MEF cell to MPyV, we measured IL-6 production by ELISA. Next, we investigated effect of TLR4 signalization on MPyV infection. Inhibition of TLR4 signaling with CLI-095 inhibitor did not affect number of infected cells. Presence of TLR4 antagonist, LPS-RS, led to significant decrease in quantity of infected cells 20 hours post infection. Decrease in number of infected cells was also observed in presence of LPS. Viral infection was also inhibited by TLR9 antagonist ODN 2088. We also investigated role of MAP kinases in MPyV infection. We tested, whether inhibition of selected MAP kinases would affect number of infected cells. Inhibition of kinase p38 did not affect infection. On the other hand, inhibition of MEK kinase or JNK resulted in decrease of number of cells infected by MPyV.
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