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The role of APOBEC proteins in HPV-induced carcinogenesis
Frolíková, Daniela ; Šmahelová, Jana (advisor) ; Šroller, Vojtěch (referee)
Apolipoprotein B mRNA editing enzyme, catalytic polypeptide (APOBEC) are a family of evolutionarily conserved cytidine deaminases with the ability to bind and modify RNA and/or ssDNA. APOBEC1-4 have a number of functions in cells. Members of the APOBEC3 subfamily cause restriction of foreign nucleic acids, retrotransposons and viruses, including human papillomaviruses (HPV), and may contribute to the clearance of infection. Certain HPVs are referred to as oncogenic viruses because of their ability to induce immortalization and transformation of epithelial cells via E5, E6 and E7 oncoproteins. E6 and E7 can also induce transcription or inhibit degradation of some APOBEC3. This results in an increase in their levels in cells. APOBEC3 also act as cellular mutators, as they can catalyze deaminations on transiently produced ssDNA during replication or transcription. Deregulation of APOBEC3 caused by oncoproteins may contribute to mutagenesis. This bachelor thesis focuses on APOBEC proteins, their activation and function during HPV-induced carcinogenesis, and in particular the extent and consequences of APOBEC3 mutations. Keywords: APOBEC, mutagenesis, papillomavirus, oncoproteins, carcinogenesis
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Preparation of expression vectors and virus mutants for studies of the minor structural proteins of polyomaviruses.
Cibulka, Jakub ; Forstová, Jitka (advisor) ; Šroller, Vojtěch (referee)
Polyomaviruses are small non-enveloped DNA viruses infecting birds and mammals, including human. Their capsid consists of the major capsid protein, VP1, and two minor capsid proteins, VP2 and VP3. The VP2 and VP3 proteins are supposed to have an important function in the transport of viral genome into the cell nucleus, which is a key step to facilitate viral replication. VP2 and VP3 proteins of mouse polyomavirus and SV40 have an ability to bind and disrupt cellular membranes. This feature is believed to be involved in the transport of viral genome into the nucleus. Plasmids carrying genes of the minor capsid proteins of Merkel cell polyomavirus were prepared in order to produce and visualize these proteins in mammalian cells. These proteins are known to have very unusual sequences compared to other human polyomaviruses or related mouse polyomavirus. When produced alone, the minor capsid proteins of Merkel cell polyomavirus did not significantly interact with cellular membranes, unlike the minor proteins of the mouse polyomavirus. The second goal of this work was to prepare mouse polyomavirus mutants with deletion in hydrophobic domains of VP2 and VP3 proteins. These domains are likely responsible for the mentioned membrane interactions. Prepared mutants were non-infectious. The loss of infectivity was not...
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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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The role of PML nuclear bodies in herpesvirus infection
Bártová, Jana ; Šroller, Vojtěch (advisor) ; Saláková, Martina (referee)
PML nuclear bodies are protein structures in the cell nucleus that regulate many important cellular processes and are also implicated in antiviral defense. The permanent components of these nuclear bodies include PML, Sp100 and Daxx proteins, many other proteins are transiently associated with PML bodies. PML body components can be SUMOylated, this posttranslational modification is important for the formation of PML bodies and regulation of their function. Components of PML bodies such as PML, Sp100 and Daxx can act as restriction factors limiting the replication of many DNA and RNA viruses. Defense mechanisms mediated by PML bodies are suppressed by viral proteins that inactivate individual components or disrupt the structure of PML bodies. This thesis focuses on the role of PML bodies as restriction factors during infection by DNA viruses of the Herpesviridae family and describes the interactions of PML bodies and viral proteins, using herpes simplex virus 1, human cytomegalovirus and Epstein-Barr virus as examples. Keywords: PML nuclear bodies, restriction factor, antiviral defense, innate immunity, herpesviruses
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Role of helicases UAP56 and URH49 in viral infection
Nováková, Veronika ; Šroller, Vojtěch (advisor) ; Šmahel, Michal (referee)
Helicases are proteins with the catalytic ability to unwind double-stranded nucleic acids. An important group are helicases with a DEAD motif, which includes helicase UA56 and the more recently discovered helicase URH49. These helicases are orthologs and they share some functions. Both helicases are involved in the splicing of pre-mRNA and they take part in the transport of mRNA from the nucleus to the cytoplasm. Slight differences between the two helicases lead to different affinites for different mRNAs. Overproduction of the URH49 helicase has been reported in many cancer tissues and has therefore been suggested to function as a potential biomarker of adverse cancer prognosis. The association of helicases UAP56 and URH49 with nuclear export has led to research of their role in cells infected by viruses which replicate in the nucleus. The helicases UAP56 and URH49 have been shown to promote virus replication in several ways. They either participate in the transport of viral RNAs into the cytoplasm and thus help to translate important proteins for the virus or play a role in their encapsidation. They also help recruit the export complex, which is normally dependent on the formation of a splicing apparatus, to viral transcripts without introns. The URH49 helicase has also been described to suppress...
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Immune reactions induced by SARS-CoV-2 infection
Krausová, Kateřina ; Šmahel, Michal (advisor) ; Šroller, Vojtěch (referee)
Coronavirus disease 2019 (COVID-19) pandemic caused by newly discovered Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes severe health and economic problems all over the world. The disease severity depends mainly on the host's immune response to SARS-CoV-2. This virus uses many mechanisms for escape from the host's immune system. The major evasion mechanisms include suppression of interferon production at the early phase of infection, exhaustion of natural killer cells and induction of a cytokine storm. After the innate immune response, mechanisms of adaptive immunity join the defense against the virus. Patients with severe cases have a significant reduction in the amount of both helper CD4+ T cells and cytotoxic CD8+ T cells. On the contrary, these patients have an increased level of antibodies. Even though there have been many findings about immune reactions to SARS-CoV-2 in the year after its discovery, there are still many unknowns. Vaccines, which are successful at preventing COVID-19, have been developed in a short time. However, an important remaining question for further research is the longevity of immune memory after vaccination or after suffering from COVID-19.
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Major capsid protein of polyomaviruses and its interactions with nuclear lamins
Žáčková, Sandra ; Horníková, Lenka (advisor) ; Šroller, Vojtěch (referee)
In this study, we focused on interactions of structural proteins of mouse polyomavirus (MPyV) and BK virus (BKV) with the nuclear lamina. Our goal was to examine whether and how can the virus, hence viral structural proteins, interact with the nuclear lamina and how would these interactions affect its properties. We supposed, that the expression of viral proteins would induce disintegration of the structure of nuclear lamina, thus enabling nuclear egress of virions in the late phase of infection. Viral structural proteins were expressed transiently in cells transfected with an expression vector pMPyV LATE. In these cells, VP1 was localized in a likewise manner as it shows in infected cells - mostly in a perinuclear area. Concurrently, defects in staining of nuclear lamina were observed in these cells, similarly to infected cells. Also, another expression vector was used in our experiments, the pMPyV mut3 VP1 encoding for a mutated protein VP1. When transiently expressed in cells, the mutated VP1 protein showed mostly diffuse nuclear localization. However, we observed significant morphological deformations and defective staining of the nuclear lamina. These observations imply an important role of VP1 in mechanical and biochemical properties alterations of the nuclear lamina in transfected and...
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Therapeutic use of bacteriophages and their modifications
Vaško, Michal ; Fraiberk, Martin (advisor) ; Šroller, Vojtěch (referee)
Bacteriophages, which infect the bacteria, form a vast group of viruses, are one of the most wide-spread organisms. Since their discovery they have been used to treat various diseases caused by the bacteria, but the development of antibiotics hindered their usage significantly. Since various bacteria strains acquire multirezistance nowadays more attention is brought to the usage of bacteriophages once again. But since the application of native bacteriophages has its limitations, e.g. too broad or too narrow host range, low efficiency, they can be overcome using molecular modifications. The goal of this thesis is to provide a brief outline of the therapeutic utilization of phages in bacteriophage therapy. The majority of this thesis focuses on potential methods of acquiring modified phages and their subsequent usage. So prepared bacteriophages characterized by their specific modified properties are not only used as bactericidal reagents but also as a tool to enhance the efficiency of antibiotics, drug delivery, diagnostics and vaccine development. Various studies describe the improvement or alteration of properties gained by phage modifications. Their clinical application, however, is still limited due to small number of in vivo trials.
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Indoleamine 2,3-dioxygenase 1 inhibitors in cancer immunotherapy
Muffová, Barbora ; Poláková, Ingrid (advisor) ; Šroller, Vojtěch (referee)
The indoleamine 2,3-dioxygenase (IDO 1) enzyme is expressed in small amounts in most of the mammalian tissues, and its production is detected also in various types of tumours. IDO 1 catalyses the very first step of the kynurenine pathway, the tryptophan conversion to N-formylkynurenine, which is further metabolized to kynurenine (Kyn). The kynurenine/ tryptophan ratio (kyn/trp) may be used as a prognostic marker in research and treatment of IDO 1+ tumours. The kyn/trp demonstrates the activity of IDO 1 in tumours. The goal of cancer immunotherapy based on IDO 1 inhibition is to reverse or reduce the protumour effects of IDO 1, such as avoiding NK and T cells inhibition and activation of regulatory T cells or association with tumour-associated macrophages (TAM). IDO 1 inhibitors have been examined alone in monotherapy or together with cytotoxic T-lymphocytes antigen 4 (CTLA-4) inhibitors and programmed cell death protein 1 (PD-1) inhibitors in combined therapy. Recently, several studies are dedicated to invent inhibitors, which are able to inhibit the activity of other trp-catalysing enzymes, the indoleamine 2,3-dioxygenase 2 (IDO 2) and tryptophan 2,3-dioxygenase (TDO), together with the IDO 1 activity. Cancer immunotherapy based on IDO 1 inhibition may be combined also with chemotherapy.
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