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Roles of cytoskeleton in mouse polyomavirus trafficking
Klímová, Lucie ; Forstová, Jitka (advisor) ; Šmahel, Michal (referee)
6 Roles of cytoskeleton in mouse polyomavirus trafficking ABSTRACT: Mouse polyomavirus (mPyV) is small non-enveloped DNA virus. Its endocytic pathway is studied for a potential utilisation of polyomaviral virus-like particles in gene therapy and/or immunotherapy. mPyV enter cells by internalisation into smooth monopinocytic vesicles. During it's journey through the cell, it pass through early endosomes, and at the time 3 hours post infection, it is localised in endoplasmic reticulum and recycling endosomes. Many aspects of mPyV trafficking and nuclear entry are not clear yet. Time-lapse live imaging fluorescence confocal microscopy was used to describe the mouse polyomavirus intracellular movements. For these studies, we utilised mPyV fluorophore-labeled virions and cells expressing GFP-tagged g-actin or alpha-tubulin. Some virion-loaded vesicles were seen to move with actin organised into dynamic structures. Some of these structures resembled actin comets created by Listeria or vaccinia virus. At the same time post infection (40-60 min post infection), movement of the virion loaded vesicles along mirotubules was observed suggesting the simultaneous involvement of actin and tubulin during mPyV trafficking. Dynamitin, a dominant negative inhibitor of dynein-dynactin function reduced mPyV infection. Taken...
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The role of Hepatitis B virus capsid protein in the host ubiquitin proteasome pathway
Eliáš, Vratislav ; Weber, Jan (advisor) ; Šmahel, Michal (referee)
Hepatitis B virus (HBV) is a Hepadnaviridae virus infecting mammals. Its infection can result in an acute or chronic infection. Chronic infection can result in hepatocellular carcinoma and liver cirrhosis, potentially leading to death of the patient. HBV is a small 42 nm virus with a genome length of 3.2 kb encoding seven viral proteins. HBV Core protein (HBc) is a capsid forming protein which is pleiotropic in function. We have identified two ubiquitin ligases which could interact with this protein: F-box only protein 3 (FBXO3; E3 ubiquitin ligase) and Ubiquitin conjugating enzyme E2 O (UBE2O; E2/E3 ubiquitin ligase). By employing multiple methods we have confirmed these interactions. Co- immunoprecipitation and further western blot analysis unveiled multiple new insights into the ligases′ impact on HBc: FBXO3-mediated HBc polyubiquitination stimulation and UBE2O-mediated HBc monoubiquitination promotion. FBXO3's and UBE2O's role in HBV life cycle was investigated as well. By silencing the expression of FBXO3 and UBE2O respectively, we have observed changes in HBV replication levels: FBXO3 serves as an inhibitor of HBV replication, while UBE2O stimulates the course of HBV life cycle. Further investigation of these newly-discovered understandings may lead to a whole new HBV - host interplay...
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Characterisation of the cell line TRAMP-C2 side population, mouse model of prostate cancer
Žlabová, Anna ; Reiniš, Milan (advisor) ; Šmahel, Michal (referee)
Side population is a minor subpopulation (SP) of some cell lines, exporting staining dye Hoechst 33342 out of their cytoplasm. It is discussed as a possible source of "cancer stem cells", "tumour initiating cells" or "metastasis initiating cells". However, broad literature suggest, that stemness and other privileged properties of SP are very variable between different cell types, cell lines and stage of disease. Cell lines TRAMP are the only widely available murine models for testing of prostate cancer therapy. We noticed in literature a mention about existence of 1-2% of cells constituting side population, but detailed characteristic have not been described until now. In this diploma thesis, we worked on characterisation of SP of the TRAMP-C2 cell line in comparison to other cells (nonSP). In the first part, we compared stem properties of SP and nonSP. We started with checking the existence of SP by its verapamil sensitivity. Using mRNA analysis, we showed that neither SP nor nonSP have increased c-Kit expression and that there are no differences in Bmi-1 expression. We found that SP is heterogenic mixture of CD24-CD44-, CD24-CD44+ and CD24+CD44+ cells, while nonSP is almost solely CD24-CD44+. We documented that SP and nonSP returned back to original SP ratio during cultivation. Then we showed on...
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Contribution of gag region to overall HIV replicative fitness in patients with different disease progression
Suchý, Tomáš ; Weber, Jan (advisor) ; Šmahel, Michal (referee)
Human immunodeficiency virus (HIV) is globally spread virus without available cure. Since its life-long presence, virus is carefully monitored as well as patient's immunological status. Replicative fitness of the virus is one of important aspects which can be taken into account, when monitoring HIV. Here, we are measuring HIV replicative fitness of gag recombinant viruses and comparing the results with replicative fitness of primary isolates. Further, we are comparing our findings of replicative fitness change over time with disease progression in the patient. We found that gag can be major contributor to overall fitness, although not in all cases. Additionally, we observed a correlation of replicative fitness development and slope of patient's CD4+ T cells. Moreover, this relation was even more noticeable in patients with slow disease progression or in carriers of protective alleles. In summary, our results extend the understanding of replicative fitness and its role in disease progression; and pave the way to use the recombinant HIV for replicative fitness measurement in clinical practice. Keywords: HIV, replicative fitness, recombinant virus, HIV disease progression, gag
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Epigenetic regulation of expression of immunoactive genes on tumor cells
Hejhal, Tomáš ; Reiniš, Milan (advisor) ; Šmahel, Michal (referee)
The aim of this master thesis was to identify epigenetically silenced genes and to determine molecular effects of DNA methyltransferases inhibitor (5-aza-2'-deoxycytidine) and histon deacetylases inhibitor trichostatin A (TSA) on gene expression in cancer cell lines TC-1/A9 and RVP3. Epigenetic events play an important role in tumorigenesis and also in escape of cancer cells from immune surveillance. I analyzed global changes in gene expression profiles of two cell lines by microarray analysis with a special attention paid to immunoactive genes. The experimental model used for this purpose were murine tumor cell lines, MHC class I deficient. I identified epigenetic regulation of several genes that are involved in cancer and immune system interactions. These data demonstrate that epigenetic agents are capable to activate expression of genes that are important for antigen presentation, cell adhesion and apoptosis. Powered by TCPDF (www.tcpdf.org)
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Checkpoint blockade in cancer immunotherapy
Vacková, Julie ; Šmahel, Michal (advisor) ; Černý, Jan (referee) ; Říhová, Blanka (referee)
The immune checkpoint blockade is a novel approach of cancer therapy, which markedly enhanced treatment efficacy of several cancer types. However, the frequency of cancer patients non-responding to this treatment is high. Establishment of predictive markers to distinguish patients suitable for the immune checkpoint blockade would enhance the number of patients receiving benefit from the therapy. This dissertation thesis focuses on the enhancement of efficacy of immune checkpoint inhibitors (ICIs) and predictive markers in experimental models of mouse tumours induced by TC-1 and TC-1/A9 cell lines and its clones with deactivation of interferon (IFN)-γ signalling (TC-1/dIfngr1 and TC-1/A9/dIfngr1), or CD80 molecule (TC-1/dCD80-1). IFN-γ is presumed to be the main inducer of programmed death ligand 1 (PD-L1) and a major histocompatibility complex I (MHC-I). Moreover, PD-L1 expression may predict sensitivity to PD-1/PD-L1 blockade. Non-functional IFN-γ signalling or downregulated MHC-I expression has been associated with resistance to ICIs in some patients. We found that IFNs type I (IFN-α and IFN-β) induced the expression of PD-L1 and MHC-I on TC-1/A9/dIfngr1 tumour cells with reversible downregulation of both molecules. We also showed that deactivation of IFN-γ signalling in TC-1/A9 cells was not a...
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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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From adenoma to colorectal cancer: The study of DNA methylation profiles
Fabianová, Ivana ; Vymetálková, Veronika (advisor) ; Šmahel, Michal (referee)
Colorectal cancer (CRC) is a major public health problem worldwide and is one of the most common types of cancer in advanced countries. Recent statistics still present that the Czech Republic has a high incidence of CRC worldwide, especially in Czech men. CRC is known to be a disease that is caused not only by genetic and chromosomal abnormalities but also by epigenetic changes with the best-known DNA methylation. Changes in DNA methylation are the most prominent mechanisms that alter gene expression. Loss of gene function by epigenetic silencing of critical genes plays a key role in the development and progression of sporadic human tumors, including CRC. CRC usually develops from a harmless protrusion, known as an adenoma. However, little is known about the exact timing of DNA methylation changes in the transition from a healthy colon, through an adenoma to a malignant state. This bachelor thesis aims to summarize in detail the aberrant changes in DNA methylation in people with adenoma and in patients with CRC and at the same time to summarize the currently used methods of DNA methylation detection. Keywords: colorectal cancer, DNA methylation, adenoma, CpG island methylation phenotype, hypermethylation, hypomethylation
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