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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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Adaptive immune response against BK polyomavirus infection
Rezlerová, Adéla ; Saláková, Martina (advisor) ; Roubalová, Kateřina (referee)
BK polyomavirus is a small non-enveloped virus that is found in a large proportion of the human population. BKPyV infection commonly occurs in early childhood. The virus establishes persistent infection in renal tubular cells and uroepithelial cells. In immunosuppressed individuals, especially after renal or haematopoietic stem cell transplantation, BKPyV reactivation can lead to serious complications such as BKPyV-associated nephropathy (BKVAN), urethral stenosis or haemorrhagic cystitis. Adaptive immunity plays a crucial role in controlling the replication and progression of BKPyV infection. The T cell response is particularly important, with the production of CD4+ and CD8+ T-lymphocytes. B-lymphocytes, which produce virus neutralising antibodies, are also important. Currently, there are no effective antiviral agents against BKPyV infection and reducing immunosuppression remains the main strategy to suppress reactivation. Exploration of immune- based therapies offers promising possibilities for effective treatment of complications associated with polyomavirus infection. Key words: BK polyomavirus, T cell response, antibodies, BK polyomavirus-associated nephropathy, hemorrhagic cystitis
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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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The importance of cell-free HPV DNA detection
Milt, Petr ; Saláková, Martina (advisor) ; Horníková, Lenka (referee)
Human papillomaviruses (HPV) are small, nonenveloped DNA viruses that are abundant in the population. They are sexually transmitted or spread by close contact with mucosa and skin. Papillomaviruses can cause lesions and warts on the skin and mucosa. In addition, high-risk HPV types, especially HPV 16 and 18, are associated with squamous cell carcinomas such as cervical cancer, oropharyngeal cancer and carcinomas of the vulva, anus, penis and vagina. Early detection and the right evaluation of the risk of recurrence are crucial for effective treatment. Cell-free DNA released from cells into body fluids has potential in cancer diagnosis. Cell-free circulating HPV DNA, in the blood of patients with HPV-associated cancers is a promising and highly sensitive biomarker, useful for monitoring treatment efficiency, early detection of the disease and estimation of recurrence risk. Key words: HPV, carcinogenesis, cfDNA, cfHPV DNA, significance of detection, cervical cancer, oropharyngeal cancer
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Virom of lower urinary tracts
Cirbusová, Adéla ; Saláková, Martina (advisor) ; Španielová, Hana (referee)
The human urinary tract was considered to be a sterile environment for many years. However, studies over the past decade have shown that urine harbours rich microbial community which includes also viruses. Nevertheless, there is only very little known about urinary virome so far. Optimised Next Generation Sequencing (NGS) protocol was used to describe the urinary virome of three individuals. However, characterization of the virome from urine samples using NGS proved to be quite challenging, mainly due to observed viral genomes fragmentation. Despite this problem, it was possible to identify human endogenous retroviruses in all individuals and also JC polyomavirus in two of them. Quantitative PCR was further used to characterize part of the urinary virome represented by human DNA viruses. Possible differences in prevalence and viral load of human DNA viruses were observed in individuals with and without bladder carcinoma (bc). Urine of these patients was obtained from different sites of the urinary tract to further establish, if there is a difference in these samples. Torque Teno virus and JC polyomavirus were found as the most common viruses. Torque Teno virus was detected in 75 % patients with and 60 % patients without bc, JC polyomavirus in 43,8 % patients with and 50 % patients without bc. BK...
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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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Functional analysis of differentially expressed miRNAs in HPV positive and HPV negative cell lines derived various anatomical localization
Krbušek, David ; Saláková, Martina (advisor) ; Dostálová Merkerová, Michaela (referee)
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. MiRNA expression profiles showed several differentially expressed miRNAs specific to human papillomavirus (HPV)-associated tumors. These miRNAs include miR-139-5p, which has reduced expression in these tumors. In this thesis, the role of miR-139-5p was studied in vitro on cell lines that were HPV positive (CRL-3240, SiHa) and HPV negative (FaDu, C-33A). Cell lines were transfected with mimic miRNA, the ability of cells to proliferate and migrate was then studied. Cell proliferation was studied using MTT assay, while Scratch wound healing assay and transwell assay were used to evaluate the migratory abilities of the cells. Mediator RNAs (mRNAs) of target genes of miR-139-5p were predicted using TargetScan and miRDB databases. The change in gene expression of target mRNAs, as well as the verification of the successful increase in miRNA expression in the cell lines, was verified using RT-qPCR. Increase of miR-139-5p expression in all used cell lines did not lead to statistically significant changes (p≤0.05) in proliferative or migratory abilities. The mRNAs of FOS, JUN, KIF13A and GDF10 genes were selected as targets of miR-139-5p. Transfected cell lines did not show a noticable reduction in the expression of the target mRNAs....
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Minor Structural Proteins of Polyomaviruses: Attributes and Interactions with Cellular Structures
Vinšová, Barbora ; Horníková, Lenka (advisor) ; Saláková, Martina (referee)
Even though polyomaviruses have been intensively studied for more than 60 years, the role of minor structural proteins VP2 and VP3 in some important steps of viral life cycle has still not been fully elucidated, explicitly their role in viral genome delivery to the cell nucleus and their involvement in late phases of viral life cycle. This diploma thesis focuses on the study of minor proteins of Mouse polyomavirus (MPyV) and Human polyomavirus BK (BKV). Four rabbit polyclonal antibodies against minor proteins of polyomaviruses MPyV or BKV have been prepared within this diploma thesis. Two of these prepared antibodies target minor proteins of MPyV (α-MPyV VP2/3) or BKV virus (α-BKV VP2/3), other two prepared antibodies recognize C-terminal sequence common to minor proteins VP2 and VP3 of MPyV (α-MPyV C-termVP2/3) or BKV virus (α-BKV C-termVP2/3). In the second part of this diploma thesis we aimed to study toxicity of BKV virus minor proteins during individual production in mammalian cells. Obtained results suggest that minor proteins of BKV virus might not exhibit as high levels of cytotoxicity as minor proteins of MPyV virus. Third part of this diploma thesis is devoted to investigation of interactions of BKV and MPyV minor proteins with cellular proteins and within one another respectively....
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The noncoding control region of human polyomaviruses
Pešek, David ; Saláková, Martina (advisor) ; Váňová, Jana (referee)
Genome of human polyomavirus consists of circular dsDNA around 5000 base and can be divided into three functional regions - the early viral gene region (EVGR), that encodes the regulatory T antigen and miRNAs, noncoding control region (NCCR) harboring the minimal cis- acting elements involved in viral replication and the late viral gene region (LVGR), that encodes the structural capsid proteins. Noncoding control region contains the origin of viral replication that overlaps the promoters that control expresion of early and late gene region. Noncoding control region sequences include a large number of various binding sites for cellular transcription factors involved in regulation expression from LVGR and EVGR. This thesis describes the organization of the most variable region of the PyV genome, NCCR, in chosen polyomaviruses SV40, BKPyV and JCPyV. This region often undergoes rearrangements, deletion and point mutations that affects exression of human polyomavirus. Key words: polyomavirus, noncoding control region, BKPyV virus, JCPyV virus, SV40, large T antigen, transcriptional factor
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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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