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Molecular mechanisms of genome integrity maintenance under conditions of replication stress
Boleslavská, Barbora ; Dobrovolná, Jana (advisor) ; Hanzlíková, Hana (referee) ; Šebesta, Marek (referee)
Precise and timely duplication of chromosomal DNA is an essential process for all dividing cells. Failure to complete this process can lead to cell death or irreversible changes in genetic information ultimately leading to cell transformation and cancer. The sophisticated multiprotein complex ensuring DNA replication is often slowed down or stalled by numerous obstacles, including DNA lesions and secondary DNA structures, but also by another vitally important cellular process - by ongoing DNA transcription. Head-on transcription-replication collisions (TRCs) associated with the formation of R-loop structures are the major endogenous source of genomic instability. R-loops are three-stranded nucleic acid structures composed of an RNA:DNA hybrid and displaced DNA strand, with a strong potential to halt replication fork progression and to have a genotoxic effect. To ensure complete DNA replication, the eukaryotic cells have developed various mechanisms for removal and overcoming the obstacles to replication fork progression. However, despite the extensive research, the molecular mechanisms underlying the restart of R-loop-stalled replication forks remain unclear. Here, we provide evidence that in human cells the restart of DNA synthesis at the sites of TRCs is a multi-step process involving fork...
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Effects of natural compounds on viability of tumor cell lines
Boleslavská, Barbora ; Poljaková, Jitka (advisor) ; Bořek Dohalská, Lucie (referee)
Cancer is considered to be one of the most serious issues of medicine nowadays. Moreover, its incidence is still rising. Despite the huge progress in modern treatment methods, cancer therapy is still limited by many difficulties. This work focuses on the natural substances such as epigallocatechin gallate, caffeine, Cannabis sativa ethanol extract, Origanum acutidens water extract, Mentha piperita water extract and its effects on the human neuroblastoma cell line UKF-NB-4. The first part of the bachelor thesis deals with determining the viability of human neuroblastoma cell line UKF-NB-4 exposed to tested substances as well as it studies the effects of those substances on the cell cycle and caspase activity. Finally, it was tested, whether those substances are able to induce apoptosis in neuroblastoma cell lines. Tests were undertaken on the MUSETM cell analyzer and on the flow cytometer. The second part of the bachelor thesis focuses on the expression of protein p53 and retinoblastoma protein in neuroblastoma cell lines exposed to tested substances. Detection was carried out by Western blot analysis. Epigallocatechin gallate exhibited the most significant effect on human neuroblastoma cell line. It lowers the expression of retinoblastoma protein as well as it arrests cells between G0/G1 and S...
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Histone post-translational modifications and tumor cell lines resistence
Boleslavská, Barbora ; Stiborová, Marie (advisor) ; Bořek Dohalská, Lucie (referee)
Tumor cell resistance to anticancer drugs represents a major problem for the long- term effectiveness of chemotherapy. The explanation for the development of therapy resistance and tumor recurrence suggests genomic alterations and selection of mutant cells carrying a relevant random mutation resulting before therapy. However, dynamic non- genetic heterogeneity of cell population can produce different phenotypic variants, some of which can be chemoresistant. We analyzed a cell population of neuroblastoma cell line UKF-NB-4, that was able to survive a concentration of vincristine (VCR), which corresponded to 115 times higher concentration of the IC50 value (3 μM VCR). Only 3,7±0,02% of the cell population survives a 48 hour treatment with 3 μM VCR. Moreover, when transferred into a fresh media, these cells are able to proliferate and produce subcolonies. However, these subcolonies are VCR-sensitive. According to SNP array carried out in the study, cells surviving the 48 hour treatment with 3 μM VCR as well as the non-treated cells are not genetically different. Cells surviving the 48 hour treatment with 3 μM VCR exhibit distinctly lower histone acetylation levels together with increased histone methylation levels at specific lysine residues (H3ac, H4ac, H3K14ac, H3K4met3, H3K27met3, and H4K20met3)....
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Effects of natural compounds on viability of tumor cell lines
Boleslavská, Barbora ; Poljaková, Jitka (advisor) ; Bořek Dohalská, Lucie (referee)
Cancer is considered to be one of the most serious issues of medicine nowadays. Moreover, its incidence is still rising. Despite the huge progress in modern treatment methods, cancer therapy is still limited by many difficulties. This work focuses on the natural substances such as epigallocatechin gallate, caffeine, Cannabis sativa ethanol extract, Origanum acutidens water extract, Mentha piperita water extract and its effects on the human neuroblastoma cell line UKF-NB-4. The first part of the bachelor thesis deals with determining the viability of human neuroblastoma cell line UKF-NB-4 exposed to tested substances as well as it studies the effects of those substances on the cell cycle and caspase activity. Finally, it was tested, whether those substances are able to induce apoptosis in neuroblastoma cell lines. Tests were undertaken on the MUSETM cell analyzer and on the flow cytometer. The second part of the bachelor thesis focuses on the expression of protein p53 and retinoblastoma protein in neuroblastoma cell lines exposed to tested substances. Detection was carried out by Western blot analysis. Epigallocatechin gallate exhibited the most significant effect on human neuroblastoma cell line. It lowers the expression of retinoblastoma protein as well as it arrests cells between G0/G1 and S...
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