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Polyomavirus minichromosomes: interactions with components of innate imunity
Satratzemis, Christos ; Forstová, Jitka (advisor) ; Trejbalová, Kateřina (referee)
The genome of polyomaviruses is a circular dsDNA (double-stranded DNA) which is associated with cellular histones within virions and during the entire viral replication cycle. Given the structural similarity to eukaryotic chromatin, the complex of polyomaviral DNA with histones is called minichromosome. The chromatin state of minichromosomes influences viral transcription and replication which could be exploited by the host innate immune response. One of the components of innate immunity, that affects viral chromatin, is the non-canonical histone H3.3, its chaperone DAXX- ATRX (death domain associated protein 6-alpha-thalassemia, mental retardation X-linked syndrome) and protein complexes called PML (promyelocytic leukemia protein). In order to trigger the innate immune response, foreign and/or stress molecules have to detected. During mouse polyomavirus (MPyV) infection, the innate immune response is initiated via the DNA sensor cGAS (cyclic GMP- AMP synthase). In this master's thesis, the distribution of histone H3.3, its chaperone DAXX-ATRX and the PML protein was analyzed during infection with MPyV. Using mass spectrometry, the histone was detected within viral chromatin. The data suggest that the chaperone complex and PML are involved in the regulation of H3.3 incorporation into the chromatin...
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Interplay between small DNA viruses and the PML Nuclear Bodies
Lovtsov, Alexey ; Huerfano Meneses, Sandra (advisor) ; Kadlečková, Dominika (referee)
This work focuses on interactions between small DNA viruses and PML NBs. PML NBs are membrane- free nuclear bodies that contain, permanently or transiently, more than 170 proteins. The permanent ones include, e.g., the PML, Daxx, and Sp100 proteins. Many PML NB proteins are subjected to SUMOylation. In addition to functions in regulating apoptosis and gene expression, chromatin remodelling, and DNA damage responses, they are also involved in antiviral cell defence. On the other hand, viruses have developed strategies to thwart the restrictive effects of PML NBs, or even to misuse some of their components to support the infection. Apparently, PML NBs play a significant role in some viral infections, but models of such processes are still incomplete and sometimes controversial. Some adenovirus proteins interact with PML NB components to prevent the negative effects of PML NBs on transcription and replication. In contrast, papillomavirus genomes can be protected by PML NBs from degradation upon entry into the nucleus and during transcription. For polyomaviruses, both promoting and restriction functions have been published. In Chicken anaemia virus infection, the interaction of the viral apoptin and PML led to the induction of cell apoptosis, but the results were questioned. In patients with chronic...
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Numerical simulations of optical response of nanostructures using FDTD method
Novák, Ondřej ; Veis, Martin (advisor) ; Vozda, Vojtěch (referee)
Title: Numerical simulations of optical response of nanostructures using FDTD method Author: Ondřej Novák Institute: Institute of Physics of Charles University Supervisor: RNDr. Martin Veis, Ph.D., Institute of Physics of Charles University Abstract: Abstract: The aim of this thesis is to develope an efficient algorythm to compute optical response of nanostructures and to equip it with usefull tools for further data processing. Considered problem is reduced to two dimensions and the method used is the Finite Difference Time Domain (FDTD). This method operates on finite grid called Yee grid and is often called Yee algorithm. En extra emphasis is given on optimalization of the algorithm and writing the computer code efficiently. Evolution equations are written in tensor form and the core algorithm is moved to graphic card using CUDA. Various boundary conditions are introduced to reduce reflections on the edge of the grid. Representation of a real object on the Yee-grid is discussed with introduction of several smoothing methods to improve the shape convergence of simulated object. Useful post- processing methods are introduced - discrete Fourier transform, from which the frequency response of simulated object can be computed and a way to compute the far field from the near field. Finaly, there is an attempt...
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A potential role of DAXX in cell cycle arrest and cellular senescence
Valášek, Ján ; Hanzlíková, Hana (advisor) ; Vopálenský, Václav (referee)
Death domain-associated protein 6 (DAXX) is a multifunctional protein involved in diverse cellular processes. It acts as a histone chaperone or regulator of transcription and apoptosis, in which is its role quite controversial. DAXX also participates in regulation of cell DNA damage response (DDR). DAXX co-creates and stabilizes complex with Mdm2, which negatively regulates the stability of p53, an important tumor suppressor, which is a part of signalling node in the DDR. If DNA damage is not lethal for the cell and unables it to proliferate, the irreversible state of cell cycle called cellular senescence takes place. Under physiological conditions, induction of senescence can prevent the development of tumorigenesis. Therefore, the description of mechanisms involved in the induction of senescence has potential clinical significance. In my thesis, I aimed to determine changes in the level of DAXX protein in senescent cells and to characterize the manner of its regulation. In tumor cells MCF-7 and primary BJ fibroblasts, I observed decrease in DAXX protein level and its regulation. I tested the hypothesis according to which an increase in DAXX level before DNA damage canprevent induction of cellular senescence, or increase in its expression during senescence can cause recovery of cell proliferation....
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Immunity and polyomaviruses
Janovec, Václav ; Drda Morávková, Alena (advisor) ; Belovičová, Martina (referee)
Human polyomaviruses JC and BK belongs to the group of small non-enveloped DNA viruses, and are widespread in the human population. After usually asymptomatic primary infection subsequently persist throughout life in the body in a state of persistence. Thus, this thesis aims to summarize present knowledge concerning the involvement of immune mechanisms involved in immune surveillance against persistent JC and BK viruses. The published findings show that the immune surveillance against human polyomaviruses is a very complex process where an important factor is the involvement and cooperation of non-specific and specific immune defense. Long-term immune surveillance against persistent viruses is mainly mediated by specific T lymphocytes. If this immune surveillance disrupted, it can lead to reactivation of the virus. The change in cytokine environment and the genetic makeup of an individual are another important factors in cases of reactivation. The two human polyomaviruses were developed mechanisms that allow them seemed to partially escape immune surveillance. It also raises the question whether this immune escape contribute to induce tumorigenesis.
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Role of the tumour suppressor PML in DNA damage response and cellular senescence after genotoxic stress
Knoblochová, Lucie ; Hodný, Zdeněk (advisor) ; Horníková, Lenka (referee)
The promyelocytic leukemia protein (PML) is a tumour suppressor. It has been reported that PML interaction with the p53 protein is involved in the activation of cell cycle checkpoints and, when persistent, may lead to the premature onset of cellular senescence. Cellular senescence is a state of permanent cell growth arrest that is associated with characteristic morphological and metabolic changes and persistent DNA damage signalling. Importantly, PML nuclear bodies coassociate with persistent DNA damage foci in senescent cells; however, the role of this interaction is still obscure. My goal was to characterize the role of PML in DNA damage response (DDR) and the induction of premature cellular senescence after genotoxic stress, namely X-radiation, using both siRNA-mediated PML knock down (PML KD) and complete PML knock out (PML KO) in human cells. The dynamics of DNA damage foci, levels of various proteins involved in DDR, and proliferation rate were measured in both PML KD and KO cells. No significant changes in the formation of DNA damage foci, activated DDR (p53 and Chk2), activated p21CIP1/WAF1 cyclin-dependent kinase inhibitor, senescent morphology, and SA-β-galactosidase activity in PML KO cells were observed. However, PML KO cells displayed higher levels of retinoblastoma protein (Rb) and...
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Role of PML in nucleolar functions
Kučerová, Alena ; Hodný, Zdeněk (advisor) ; Skalníková, Magdalena (referee)
Promyelocytic leukemia protein (PML) is a tumour suppressor which is frequently downregulated in human tumours. PML plays a role in many cellular processes including DNA damage response, senescence and apoptosis and is mainly localized in special structures called PML nuclear bodies (PML NBs). The nucleolus is a key nuclear compartment, where transcription of ribosomal DNA and biogenesis of ribosomes take place. The nucleolus is also called a stress sensor because of its role, for instance, in stabilization of tumour suppressor p53. Localization of PML to the nucleolar periphery appears to be prominent after disturbance of nucleolar functions - for example inhibition of rRNA transcription or processing. Thus the relationship between the nucleolus and PML nuclear bodies may be important for cellular response to stress. However, the role of PML nucleolar associations in nucleolar function including mechanism of formation of these structures remain unclear. Here we characterised PML nucleolar structures and mechanism of their formation. We showed that formation of PML nucleolar structures is not caused by replication stress, is not dependent on any specific phase of cell cycle and is not caused by DNA damage response but is induced by topological stress due to inhibition of toposiomerase function....
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