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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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Host-virus interactions of mammalian endogenous retroviruses
Farkašová, Helena ; Elleder, Daniel (advisor) ; Hirsch, Ivan (referee) ; Mělková, Zora (referee)
Endogenous retroviruses (ERVs) originate by germline infection and subsequent mendelian inheritance of their exogenous counterparts. With notable exceptions, all mammalian ERVs are evolutionarily old and fixed in the population of its host species. Some groups of retroviruses were believed not to be able to form endogenous copies. We discovered an additional endogenous Lentivirus and a first endogenous Deltaretrovirus. Both of these groups were previously considered unable to form endogenous copies. Endogenous lentiviruses were discovered only recently and are still quite rare. These are still just small pieces of evidence insufficient to give a broader picture about the history of virus endogenization. We described a novel endogenous Lentivirus in the genome of Malayan colugo (Galeopterus variegatus) denoted ELVgv (endogenous Lentivirus of G. variegatus). Based on several analyses we proved that this is the oldest Lentivirus discovered up to date and confirmed its presence in the only other extant species of Dermoptera - Cynocephalus volans. Endogenous deltaretroviruses were the last group without a single endogenous member. We detected the remnants of endogenous Deltaretrovirus in the genome of Natal Long-fingered bat (Miniopterus natalensis). However, this sequence was present in the genome only in one...
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Mechanismy kontroly endogenních retrovirů v hostitelské buňce.
Matoušková, Magda ; Hejnar, Jiří (advisor) ; Mělková, Zora (referee) ; Pačes, Jan (referee)
In my thesis I am dealing with human endogenous retroviruses (HERVs), which are involved in placenta development, and with porcine endogenous retroviruses (PERVs) in the context of the risk of their transmission to a patient xenotransplanted with a pig organ. We have shown DNA methylation to be an important silencing mechanism regulating HERV as well as PERV. (1) Whereas in placenta the demethylation of HERVs ERVWE1 and ERVFRDE1 is crucial for its correct function, in the testis it is connected with seminoma development. (2) It seems that methylation is partially responsible for low PERV expression in tissues and notably reduces the risk of zoonotic transmission during xenotransplantation. (3) However, the risk remains because in contrast with mouse and rat cells, some human cells are permissive to porcine retroviruses in vitro thanks to functional receptors and their inability to efficiently silence the integrated PERVs.
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Type of cell death and superinfection exclusion phenomenon in vaccinia virus-infected cells
Lišková, Jana ; Mělková, Zora (advisor) ; Anděra, Ladislav (referee) ; Němečková, Šárka (referee)
Vaccinia virus (VACV) was formerly used in the eradication campaign against smallpox. VACV infection causes lysis of most cell types, including epithelial ones, which is equivalent to necrosis. However in our laboratory, we have previously detected activation of caspases during infection of HeLa G and BSC-40 epithelial cell lines with VACV, a typical sign of apoptosis. In this thesis, the type of cell death in HeLa G and BSC-40 cell lines infected with VACV strain WR was further studied and the activation and activity of caspases in the infected cells was characterized. In HeLa G cells infected with VACV strain WR, apoptosis was identified, as demonstrated by condensed nuclei, activity of caspase-3 and cleavage of death substrates. Additionally, activation and activity of caspase-2 and -4 was detected in infected HeLa G cells. In infected BSC-40 cells, neither apoptosis nor caspase activity were observed, but activation of caspase-2 and 4 was detected in these cells also. Finally, cleavage of procaspase-3 and -12 was detected in infected cells of both cell lines. Vaccination strains Praha and Dryvax induced apoptosis in both HeLa G and BSC-40 cells, as was demonstrated by the apoptotic morphology of nuclei and by the cleavage of PARP, substrate of the executioner caspases. Our results suggest that...
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Mechanism of the inhibitory effect of ethacrynic acid on vaccinia virus growth
Váchová, Barbora ; Mělková, Zora (advisor) ; Španielová, Hana (referee)
Vaccinia virus (VACV) is globally known for its use in the eradication campaign against variola virus (VARV), the causative agent of smallpox. In 1980, WHO declared VARV as eradicated and the worldwide immunization has been stopped. After 2001, vaccination of selected groups of population was re-introduced because of concerns about variola misuse for bioterrorist purposes. Therefore, drugs effective against variola, post-vaccination complications and consequences of VACV transmission in the population, are intensively studied again. In 2009, laboratory of Dr. Mělková demonstrated that ethacrynic acid (EA) inhibited growth of VACV at the level of late gene expression. The goal of my diploma thesis was to perform a detailed study of the effects of EA on VACV late protein synthesis and on other stages of VACV replication cycle. To this end, the following methods were used: infection of tissue culture cells, plaque assay, northern blot, slot blot, western blot, direct and indirect immunofluorescence of fixed cells and fluorescence of EGFP expressed by VACV in infected live cells. First, we wanted to determine whether EA affects VACV late gene transcription or translation. Our results indicate that EA does not decrease levels of VACV late gene mRNA's and therefore, it probably affects late gene...
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Major capsid protein of mouse polyomavirus: interaction with cellular structures
Horníková, Lenka ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee) ; Mělková, Zora (referee)
Mouse polyomavirus (MPyV) is small non-enveloped DNA virus. Although this virus has been studied for almost 60 years, it still remains unclear, how can virus transport its genetic information to the cell nucleus. Also, the mechanism of virion morphogenesis is not well understood. First part of this work is focused on endocytic pathway which is used by MPyV for trafficking toward the cell nucleus. Using dominant negative mutant of caveolin-1 we showed that caveolin-1dependent endocytic pathway, described for SV40, is not used by MPyV for productive infection. MPyV is transported to early endosomes. Acidic milieu of endosomes is indispensable for productive infection. Preventing virus localisation into early endosomes (dominant negative mutant of Rab 5 GTPase) or endosomes alkalisation (by ammonium chloride or bafilomycin A1) led to dramatic decrease of virus infectivity. Alkalisation of endosomes entailed retention of MPyV in early endosomes. It indicates that virus is further transported to late endosomes. Finally, we confirmed by FRET that MPyV is in perinuclear space localized into recycling endosomes. Another poor characterized process is virion morphogenesis. To characterize the participation of cellular proteins in virion precursor complexes, nuclear as well as whole-cell lysates of infected cells or...
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Pathogenesis of eczema vaccinatum
Elsterová, Jana ; Mělková, Zora (advisor) ; Forstová, Jitka (referee)
Vaccinia virus (VACV) is primarily known as a vaccine against its relative variola virus, the causative agent of smallpox. In the seventies of the 20th century, the vaccination campaign with VACV led to eradication of smallpox. Consequently, vaccination of the general population was stopped. Currently, the vaccination was reintroduced, namely among army and healthcare professionals. However, vaccination with VACV is accompanied with a high incidence of vaccination-related complications, namely among immunocompromised individuals. One of the complications is eczema vaccinatum, occuring in patients with atopic dermatitis. The laboratory of Dr. Melkova has focused on development of a model of eczema vaccinatum in mice Nc/Nga and on studies of pathogenesis of this complication. The goal of my diploma thesis is to contribute to characterization of imunopathogenesis of eczema vaccinatum in mice Nc/Nga infected either with VACV strain Western Reserve (WR) or with a recombinant VACV with the integrated cDNA for IRF-3 (Interferon Regulatory Factor 3; WR-IRF3). IRF-3 regulates the expression of interferon type I in response to viral infection. This recombinant virus has been constructed in the laboratory of Dr. Melková. The objective of my work was to verify the expression of the integrated cDNA for IRF-3 and to...
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Molecular mechanisms of apoptosis regulation by fatty acids in pancreatic β-cells
Němcová, Vlasta ; Kovář, Jan (advisor) ; Anděra, Ladislav (referee) ; Mělková, Zora (referee)
The incidence of type 2 diabetes is growing rapidly and represents a big threat for the human health care and economy system as well in the 21st century. The association of type 2 diabetes with obesity is apparent and dysfunction and apoptosis of pancreatic β-cells caused by elevated levels of fatty acids in circulation are considered as an important factor contributing to the development of this disease. However, molecular mechanisms that underlie these detrimental effects of fatty acids are only partially understood. The aim of this research project was to contribute to elucidation of mechanisms by which saturated and unsaturated fatty acids regulate viability and apoptosis induction in human pancreatic β-cells in vitro. Employing human pancreatic β-cell line NES2Y, we showed that increased levels of relevant dietary saturated fatty acids (palmitic and stearic acid) induce apoptosis of pancreatic β-cells, in contrast to relevant dietary unsaturated fatty acids (e.g. palmitoleic and oleic acid). We found that stearic acid-induced apoptosis is accompanied by significant activation of caspase-2, -6, -7, -8 and -9, but not by significant activation of caspase-3. Nevertheless, it was not associated with significant cytochrome c release, alteration in PIDD, Fas receptor and Fas ligand expression and...
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Charakter of the host cell iduced by vaccinia virus and inhibitory effects of the lipoic acid on vaccinia virus growth
Spišáková, Martina ; Mělková, Zora (advisor) ; Němečková, Šárka (referee) ; Pospíšek, Martin (referee)
Vaccinia virus is a typical member of the poxvirus family. It had been successfully used during the worldwide smallpox eradication campaign. Currently, it is used as vector for prophylactic, as well as experimental purposes. This PhD thesis stems from the findings previously published in our lab. It is partially focused on a further characterization of vaccinia virus effects on the type of host cell death. The results point to the activation of apoptosis during vaccinia virus infection, but it cannot be completed and the cell dies by necrosis. Our attempts to shift the necrotic type of cell death induced by vaccinia virus infection towards apoptosis using a pharmacological inhibition of activity of a key enzyme PARP (Poly-(ADP-ribose) polymerase) remained unsuccessful. The effects of vaccinia virus-encoded anti-apoptotic factors appear superior to the inhibition of this single enzyme. The second part of the thesis is focused on inhibitory effects of a redox-modulating compound, lipoic acid, on vaccinia virus infection. Our results demonstrated its inhibitory effects in cell lines of different embryonic origin. It appears that the lipoic acid inhibits vaccinia virus growth at the stage of late gene expression or possibly later, during virus morphogenesis. Lipoic acid could be potentially used as a supportive...
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