|
|
Molekulárně-biologická diagnostika lidských polyomavirů
Ryšavá, Markéta
Most of the human population encounters human polyomaviruses during childhood, when the first infection is asymptomatic or with mild symptoms. However, these viruses persist in the human body and most often, they reactivate during immunosuppression. Together with JC virus reactivation, the progressive multifocal encephalopathy is associated. Hemorrhagic cystitis is associated with BK virus, resulting in the loss of allograft during kidney transplantation. Accurate diagnostics can detect viruses in a timely manner and mitigate tissue damage. This diploma thesis deals with biotechnologies, which can be used in virus detection with a focus on real time PCR, which is the gold standard in virus diagnostics. The literary research summarizes basic information about polyomaviruses with a focus on human BKV and JCV polyomaviruses. It summarizes the biotechnological methods used for detection of polyomaviruses, both in routine diagnosis and in alternative approaches involving biosensors and CRISPR. The experimental part of the work includes the design of a detection system for polyomaviruses from in silico genome analysis, through the design of potential primers, to theoretical specificity analysis. The practical part of the work compares the efficiency and sensitivity of amplification of two reaction mixtures, where one is intended for simple systems and the other for multiplexes. It also includes testing of selected additives of PCR and determining the sensitivity and validity parameters of the reaction mixture test selected for PCR system development. The results showed that detection using a multiplex reaction mixture is sufficiently sensitive, as it meets the conditions and requi-rements of clinical recommendations and at the same time shows very good values of sensitivity and specificity of the test comparable to published technologies. This reaction mixture appears to be relevant to the use of the development and optimization of a PCR system for the detection of polyomaviruses.
|
|
|
MicroRNAs encoded by polyomaviruses.
Zachovalová, Veronika ; Bruštíková, Kateřina (advisor) ; Malík, Radek (referee)
MicroRNAs are small regulating molecules of RNA that are encoded by orgamism's genome. Biogenesis of microRNA takes place partly in the nucleus and partly in the cytoplasm. Result of this biogenesis is a 22 nt long microRNA molecule. They are able to silence the genes thanks to sequence- specific degradation of a target mRNA or thanks to the repression of translation of target, complementary mRNA. In mammalian cells the mechanism of translational repression is more common. During this mechanism the microRNA molecule is not entirely complementary to 3'UTR of its target mRNA. Polyomaviruses are small, non-enveloped dsDNA viruses with a circular genome and icosahedral capsid composed of VP1 protein pentamers. These viruses belong in a group called onkoviruses, which can transform infected cells and contribute to development of serious illnesses such as Merkell cell carcinoma. Their genome encodes regulating proteins called T antigens, structural capsid proteins and also microRNAs. My main focus in this thesis will be SV40, MPyV, MCPyV, BKPyV and JCPyV encoded microRNA molecules. Key words: polyomaviruses, small interfering RNA, microRNA, siRNA, RNA interference, mouse polyomavirus, BK virus, JC virus, SV40
|
|
|
The role of JC virus in etiology and therapy of multiple sclerosis.
Musil, Dominik ; Španielová, Hana (advisor) ; Šmahelová, Jana (referee)
JCPyV is a human polyomavirus. Infections caused by this type of virus is often without any clinical symptoms and JCPyV mostly persists in kidneys of human for the rest of the life. The most serious disease caused by the JCPyV is progressive multifocal leukoencephalopathy, which causes severe demyelination of neurons in the brain. Multiple sclerosis, as well as progressive multifocal leukoencephalopathy, is characterised by the demyelinization of neurons. In the past few years, it was shown that the development of PML occurs in some pacients who suffered from multiple sclerosis and who were treated with the monoclonal antibody called natalizumab (Tysabri). This text describes the analysis of the conncetion between viral infection of JCPyV and the etiology of multiple sclerosis and progressive multifocal leukoencephalopathy. As the risk factors for the progression of progressive multifocal leukoencephalopathy in natalizumab treated multiple sclerosis patients the duration of the use, previous immunosuppressant therapy and the presence of the anti-JCPyV antibodies have been demonstrated. In addition to these risk factors, it has also been demonstrated that the people with progressive multifocal leukoencephalopathy have mutations in the genome of virus in the regulatory region, as well as in the...
|
guest ::
