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Study of RNAi mechanisms in tobacco BY-2 cell line and potato plants
Tyč, Dimitrij ; Fischer, Lukáš (advisor) ; Kovařík, Aleš (referee) ; Moravec, Tomáš (referee)
Knowledge of the processes of RNA interference, the regulation of gene expression by small RNAs (sRNAs), has grown at an unprecedented rate over the last 30 years. Some of the findings were literally revolutionary, as they revealed events that overturned many long-held notions. Many phenomena have been shown to be highly conserved and common to organisms of different species, but others are specific to certain lineages or have not yet been fully explored. There is also a lack of knowledge about the interconnection of numerous pathways - for example between silencing at the transcriptional (TGS, leading to the promoter methylation) and post-transcriptional levels (PTGS, affecting mRNA stability or translation). The present work summarizes the findings of two published and two unpublished works and attempts to describe some of the less known sites of RNA interference using various plant model organisms. Research on Solanum tuberosum transgenic lines has revealed the ability of 5-azacytidine to restore the expression of transcriptionally silenced transgenes at the whole plant level. De novo regeneration from leaves of such plants can lead to re-silencing of reactivated transgenes and thus serves as a selection method to exclude lines prone to spontaneous silencing. The nature of changes in the...
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Role of 5-azacytidine in therapy of myelodysplastic syndrome
Machalová, Veronika ; Hodný, Zdeněk (advisor) ; Indrová, Marie (referee)
The myelodysplastic syndrome (MDS) is a group of hematopoietic clonal disorders resulting in the inefficient production of myeloid lineage blood cells, with the prevalence of patients older than 65 years. One of the possible treatment options for MDS is 5- azacytidine and 5-aza-2'-deoxycytidine therapy. These compounds have been shown to cause the induction of cell-cycle arrest, cell differentiation and/or apoptosis. The in vitro experiments with 5-aza-2'-deoxycytidine indicated that this compound causes the premature cellular senescence, a state of the irreversible cell-cycle arrest. We have asked, whether 5-azacytidine, as a molecule with similar structure, is capable of causing the same effect. This treatment strategy could be beneficial in case that the negative pro- inflammatory effect of senescent cells on their surroundings can be nullified. In this thesis we have shown that 5-azacytidine induces DNA damage response, which is described as a fundamental event for the onset of the cell senescence. We tested 5- azacytidine treated HeLa cells for several markers of the cell senescence - the increase of the β-galactosidase activity, the PML and PML nuclear bodies and the formation of persistent DNA damage signaling lesions - albeit all these markers were positive, it was the very low increase in...
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Immunotherapy of HPV16 - associated cancers and regulation of antitumour immune response
Štěpánek, Ivan ; Reiniš, Milan (advisor) ; Lipoldová, Marie (referee) ; Němečková, Šárka (referee)
The MHC class I status of tumour cells during immunotherapy is often underestimated. It represents one of important tumour escape mechanisms and thus can contribute to the failure of most of the cancer clinical trials that are usually based on the induction of cytotoxic T cell responses. Epigenetic changes in the promoters of genes involved in the MHC class I Ag presentation can result in decreased expression of the cell surface MHC molecules on tumour cells. Thus, epigenetic modifiers can restore an expression of the MHC class I molecules and make tumours visible to the CD8+ effector cells. Besides the epigenetic changes on the tumour cells, epigenetic modulators affect cells of the immune system such as dendritic cells (DC). Tumour cells can escape from the immune response not only by changes in the cancer cells, but also by influencing, expanding and/or activating immunoregulatory cell populations, such as regulatory T cells (Treg). This thesis focuses on the potential of the DC-based vaccines against HPV-16-associated tumours with a different MHC class I expression, on the combination of cancer immunotherapy with the treatment using epigenetic modifiers, with special attention paid to their effects on DC, and, finally, on the impacts of the anti-CD25 antibody (used for Treg elimination) on Treg and NKT...
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Impacts of chemotherapy on imunoregulatory gene expression in the tumor microenvironment
Paračková, Zuzana ; Reiniš, Milan (advisor) ; Zajícová, Alena (referee)
Tumor microenvironment is an area, where the local immunosuppressive effects dominate and prevents the immune system to perform its physiological functions. The cells infiltrating the microenvironment have an important function among many cell types since they produce a large quantity of factors suppressing the immune response. In our work, we monitored the immune changes in the microenvironment during tumor growth and chemotherapy. For these purposes, we utilized the methods for analysis of the proportion and phenotype of the distinct populations of immunocytes and for analysis of the total level of expressions of selected genes associated with immunosuppression or with distinct populations of immunocytes. The aim of our work was to discover, using two types of mouse tumors (TRAMP-C2 and TC-1/A9), how 5-azacytidine (5AC), a cytostatic drug with epigenetic activity, affects the proportion of leukocytes infiltrating the tumor microenvironment and, further, whether these changes are accompanied by decreased expression of immunosuppressing genes. In addition, we have also focused on the changes of relative expression of genes encoding markers of lymphoid lines and, on other immunoregulating genes, encoding IL-6, IL-10, IL-12, IL-4 and IFNγ cytokines, in the microenvironment of these tumors....
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