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DNA damage response in Huntington disease
Vachová, Veronika ; Šolc, Petr (advisor) ; Roth, Jan (referee)
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, which leads to loss of striatal neurons in basal ganglias. It is characterized by involuntary movements and progressive cognitive impairment. HD is a relatively rare disease and the prevalence is approximately 0,01 % of the population of Western European. HD is caused by a CAG repeat expansion in the huntingtin gene (HTT). This mutation results in an elongated stretch of glutamin. Mutant huntingtin (mHTT) expression leads to accumulation of DNA double-strand breaks (DSB) due to reduced ability of effective reparation, which contributes to the pathogenesis of HD, however this mechanism is not fully understood. There are several angles of view how mHTT impaires DNA damage response (DDR). Some studies say that the expression of the mHTT initiates excessive activation of the DDR including p53 signaling pathway leading to apoptosis. Other studies represent results for dysfunction of non-homologous end joining after recognition of DSB or that the cell is not able to recognize DSB. All theories would explain cell death as a consequence of high level of unrepaired DNA damage. The understanding of these mechanisms is important for the development of therapeutical strategies. Key words: Huntington's disease, huntingtin, DNA...
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Cellular senescence and tumour immuno-surveillance
Včelková, Terézia ; Hodný, Zdeněk (advisor) ; Štěpánek, Ivan (referee)
Cellular senescence represents the antitumor mechanism that has been considered to be irreversible. However, it appears that under certain circumstances the cell is able to escape from senescent state, that led to increased risk of tumor transformation. Senescent cells secrete a plethora of substances including cytokines that modulate their surrounding environment. It turns out that they are able to induce senescence in neighboring cells and, paradoxically, they are the reason of tumor promoting effects of cellular senescence. According to the latest findings, senescent cells are subject to surveillance of the immune system, which is named as senescent surveillance. This event provide the ablation of these non- proliferating, damaged cells and protects the body from pathologies that are associated specifically with the phenomenon of cellular senescence. The aim of this bachelor thesis is to compile the current knowledge about the interactions of senescent cells with the immune system and to show their relevance in the war against cancer. Powered by TCPDF (www.tcpdf.org)
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Novel Approaches in Electrochemical Determination of Xenobiotic Compounds and in Study of Their Interaction with DNA
Hájková, Andrea
Presented Ph.D. Thesis is focused on the development of analytical methods applicable for determination of selected xenobiotic compounds and for monitoring DNA damage they can induce. The main attention has been paid to the development and testing of non-toxic electrode materials for preparation of miniaturized electrochemical devices and novel electrochemical DNA biosensors. 2-Aminofluoren-9-one (2-AFN) was selected as a model environmental pollutant, which belongs to the group of hazardous genotoxic substances. Its carcinogenic and mutagenic effects may represent a risk to living and working environment. 2-AFN has one oxo group, where the cathodic reduction occurs, and one amino group, where the anodic oxidation occurs. The voltammetric behavior of 2-AFN in the negative potential region was investigated at a mercury meniscus modified silver solid amalgam electrode (m-AgSAE) representing a non-toxic and more mechanically robust alternative to mercury electrodes. This working electrode was subsequently used for the development of a newly designed miniaturized electrode system (MES), which has many benefits as the possibility of simple field measurements, easy portability, and the measurement in sample volume 100 µL. Moreover, a glassy carbon electrode (GCE) was used for further investigation of...
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Tolerance of DNA damage by novel biologically active platinum complexes
Vystrčilová, Jana ; Vrána, Oldřich (referee) ; Nováková,, Olga (advisor)
The anti-tumor activity of platinum based drugs is mediated by their ability to attack DNA. Platinum complexes can alter the structure of DNA by modifying the bases, mainly guanines. The biological consequnces of such interactions are compromising replication and transcription. RNA polymerase complex can stall at a damaged site in DNA and mark the lesion for repair by proteins that are utilized to execute nucleotide excision repair, a pathway commonly associated with the removal of bulky DNA damage from the genome. This RNA polymerase-induced repair pathway is called transcription-coupled nucleotide excision repair. Main goal of this thesis was to study RNA polymerases tolerance of DNA damage by novel, biologically active platinum (II) complexes involving derivatives of aromatic cytokinines as the ligands; cis-[Pt(2-chloro-6-(4-methoxybenzylamino)-9-isopropylpurin)2Cl2](PR-001), cis-[Pt(2-chloro-6-(benzylamino)-9-isopropylpurin)2Cl2](PR-002 )and cis-[Pt(2-(3-hydroxypropylamino)-6-(benzylamino)-9-isopropylpurin)2Cl2](PR-005). DNA templates (constructs) that contain a single, site-specific DNA lesion and support transcription by human RNA polymerase II and bacteriophage T7 RNA polymerase were prepared. The method is making use of polymerase chain reaction (PCR) and biotin-streptavidin interactions and paramagnetic particles to purify the final product. Synthetic oligomers duplexes (75-mer, 56-mer and 15-mer) are ligated to 5´-biotin pCI-neo-G-lessT7 PCR fragment, the 15-mer is either unmodified or modified with a site-specific lesion of PR-005 and cisplatin. We also studied the inhibition of RNA polymerases activity on globally modified plasmid pCI-neo and pUC 19 by novel platinum complexes and cisplatin. We found that bifunctional adducts of complex PR-005 contrary to adducts of PR-001 and PR-002 effectively decrease amount of full lenght transcripts produced by both, human and bacterial RNA polymerases. This result can be explained by a sterical block, induced to DNA by intrastrand cross-link of PR-005 with bulky aromatic ligands.
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DNA damage response in green algae Chlamydomonas reinhardtii and Scenedesmus quadricauda
HLAVOVÁ, Monika
The effect of FdUrd, zeocin, caffeine and their combination on the cell cycle of green algae Chlamydomonas reinhardtii and Scenedesmus quadricauda and response of these model organisms to DNA damage were studied. Both, FdUrd and zeocin, caused DNA damage that led to cell cycle arrest in these algae. In contrast, caffeine partially abolished G2 phase block imposed by zeocin. Protein levels of three crucial cell cycle regulators - CdkA, CdkB and Wee1 kinases were measured to identify mechanisms controlling reaction to DNA damage.
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