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The study of tumour DNA virus integrations
Frčková, Tereza ; Saláková, Martina (advisor) ; Šroller, Vojtěch (referee)
Most people perceive viruses primarily as a cause of diseases such as cold, flu or COVID-19. But there are also viruses with oncogenic potential. There are several ways in which viruses contribute to the development of tumors. In the case of human papillomavirus and Merkel cell virus, it is the expression of oncogenes encoded in viral genomes. The virus may be integrated into the host genome, which can also promote the development of carcinogenesis. In this work, the method of detection of integrated papillomavirus sequence by polymerase chain reaction (DIPS PCR) was used to detect the integration breakpoints in human papillomavirus (HPV) in positive cell lines originated from cervical cancer, clinical head and neck cancer samples associated with HPV, and clinical samples of Merkel cell carcinoma. In the case of Merkel cell carcinoma, DIPS PCR method was performed on samples isolated from fresh frozen tissues and from formalin fixed paraffin embedded (FFPE) sections of tumor samples. In the case of tonsillar tumors associated with HPV, only fresh frozen tissues were used. Integration breakpoints were detected in samples from both fresh frozen and FFPE tissues using DIPS PCR method. For the detection of HPV genome integration breakpoints, a new set of primers was tested and optimized on the SiHa...
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The study of tumour DNA virus intergrations
Frčková, Tereza ; Saláková, Martina (advisor) ; Šroller, Vojtěch (referee)
Most people perceive viruses primarily as the cause of diseases such as cold, flu or COVID-19. But there are also viruses with oncogenic potencial. There are several ways in which viruses provide to the development of tumors. In the case of human papillomavirus and Merkel cell virus, this is the expression of oncogens encoded in viral genomes. The virus may be integrated into the host genome, which can also promote to the development of carcinogenesis. In this work, the method of detection of integrated papillomavirus sequence by polymerase chain reaction (DIPS PCR) was used to detect the integration breakpoints in human papillomavirus (HPV) of positive cell lines originated from cervix cancer, clinical head and neck cancer samples associated with HPV, and clinical samples of cancer from Merkel cells. In the case of Merkel cell carcinoma, the DIPS PCR method was performed on samples isolated from freshly frozen tissue and from cuts of tumor samples stored in paraffin. In the case of tonsil tumors associated with HPV, it was only freshly frozen tissue. Using the DIPS PCR method, integration breakpoints were detected in samples from both freshly frozen tissue and tissue stored in paraffin. For the detection of HPV genome integration breakpoints, a new set of primers was tested, which was optimized on...
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Extracellular vesicles and middle T antigen of mouse polyomavirus
Kropáček, Václav ; Šroller, Vojtěch (advisor) ; Brázdová, Andrea (referee)
This study is focused on middle tumor antigen (MT Ag) of mouse polyomavirus (MPyV), potential consequences of it's secretion via extracellular vesicles (EVs) and it's effect on cellular signaling. MT Ag is membrane bound protein able to induce cellular transformation thanks to it's ability to interfere with cellular signal transduction. Mainly due to aberrant activation of MAP kinase pathway. Firstly we followed up previous observations of our group concerning ability of MT Ag to be secreted from cells via extracellular vesicles. We were interested if MT Ag could contribute to malignant transformation in recipient cells. We performed 2 types of EVs isolation from cell lines stably expressing middle T antigen (3T6MT). We confirmed presence of MT Ag in isolated EVs. Then we characterized isolated EVs by detection of exosomal markers and cryo-electron microscopy. In next step we exposed recipient cell line (3T6) to isolated EVs and with use of flow cytometry tried to detect internalization of MT Ag. Simultaneously we tried asses levels of Erk phosphorylation in 3T6 cells exposed to EVs. Secondly we tried to confirm and analyse previous unpublished observations of elevated levels of NF-kB phosphorylation in cells stably expressing MT Ag. We used western blot and detection of NF-kB dependent secreted...
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The role of IFI16 protein in viral infection
Karchňák, Jan ; Šroller, Vojtěch (advisor) ; Poláková, Ingrid (referee)
6 Abstract Viruses are obligate intracellular parasites causing a large variety of diseases. Most of their hosts, including humans, have developed particular mechanisms, which are meant to tackle such diseases. First line of defense against viruses are pattern recognition receptors. These receptors are responsible for detection of pathogen associated molecular patterns (PAMPs) and of damage associated molecular patterns (DAMPs). Inteferon γ inducible protein 16 (IFI16) is one of these receptors and is responsible for detecting alien and damaged DNA both in the nucleus and cytoplasm. Its structure contains two sequence independent DNA binding HIN domains and one PYD domain, which mediates its protein-protein interactions. In the cell it functions as a regulator of cell cycle, differentiation and plays a role in cell aging. IFI16 also triggers activation of non-specific immune response and it directly acts as a restrictive factor for many viruses. During evolution these viruses have evolved mechanism which they us to evade its imunne activity or even use it to their advantage. Keywords: IFI16, STING, DNA sensing, interferons, restriction factor, pattern recogniton receptors
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The role of cellular sensors in viral infection
Karchňák, Jan ; Šroller, Vojtěch (advisor) ; Poláková, Ingrid (referee)
Viruses are obligate intracellular parasites causing a large variety of diseases. Most of their hosts, including humans, have developed particular mechanisms, which are meant to tackle such diseases. First line of defense against viruses are germline encoded Pattern recognition receptors. These receptors are responsible for detection of pathogen associated molecular patterns (PAMPs) and of damage associated molecular patterns (DAMPs). Inteferon γ inducible protein 16 (IFI16) is one of these receptors and is responsible for detecting alien and damaged DNA both in the nucleus and cytoplasm. Its structure contains two sequence independent DNA binding HIN domains and one PYD domain, which mediates its protein-protein interactions. In the cell it functions as a regulator of cell cycle and differentiation. It also has telomerase activity and regulates cell senescence. IFI16 also triggers activation of non-specific immune response and it directly acts as a restrictive factor for many viruses. During evolution these viruses have evolved mechanism which they us to evade its imunne activity or even use it to their advantage. Keywords: IFI16, STING, DNA sensing, interferons, restriction factor, pattern recogniton receptors
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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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