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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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Replication of Merkel cell polyomavirus (MCPyV) in human cell lines
Bučková, Alžbeta ; Saláková, Martina (advisor) ; Huerfano Meneses, Sandra (referee)
The Merkel cell polyomavirus (MCPyV) is the only human polyomavirus classified as probably carcinogenic to humans and is the causal factor of a rare aggressive skin malignancy the Merkel cell carcinoma (MCC) in around 80% of cases. Nevertheless, in addition to skin the virus was detected in various body tissues. In spite of that an ideal in vitro model system is still lacking. Three MCPyV isolates from healthy human skin are studied in this diploma thesis. The isolates harbour mutations, one has mutations is its LT coding gene, one has a rearranged non-coding control region (NCCR), and one is identical to the reference MPCyV isolate R17b. In this diploma thesis the replication capacities of the isolates were studied with the emphasis on the impact of a duplication within the NCCR and mutations on MCPyV genome replication in vitro after transfection. First, the results show that the NCCR rearrangement and the mutations in the viral protein coding genes impact the MCPyV replication in the 293 cell line. Preliminary data suggest the NCCR rearrangement negatively influences the MCPyV replication but certain mutations in protein coding genes could increase the replication. Moreover, none of the isolates replicated in the 293T cell line. The next part of the thesis focused on the replication of the MCPyV...
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