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ADP-ribosylation in ARH3-deficient cells and its impact on cellular functions
Kuttichová, Barbora ; Hanzlíková, Hana (advisor) ; Valihrach, Lukáš (referee)
ADP-ribosylation is a crucial post-translational modification that regulates various cellular processes, including DNA repair. It is catalysed by poly-ADP-ribose polymerases (PARPs) and involves the transfer of ADP-ribose moieties from the redox cofactor NAD+ to proteins, including histones. To maintain cellular homeostasis, ADP-ribose chains need to be rapidly degraded by ADP-ribosyl glycohydrolases. While poly-ADP-ribose glycohydrolase (PARG) is highly efficient, it cannot cleave the terminal ADP-ribose moiety. For the removal of the terminal mono-ADP-ribose, two glycohydrolases, TARG1 and ARH3, are involved. This removal process is necessary because it enables DNA repair factors to access the site of DNA damage. The primary goal of this thesis is to characterise cells derived from patients with homozygous ARH3 mutations and to develop appropriate tools to improve our understanding of the molecular mechanism by which ARH3 mutations affect ADP-ribosylation and how it contributes to the onset of the associated neurological disease. To achieve this, I measured the levels of ARH3 protein and detected increased mono-ADP-ribosylation in ARH3-mutated patient-derived fibroblasts. Furthermore, I assessed the sensitivity of these cells to different PARP inhibitors, which hold potential for the therapeutic...
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The use of induced pluripotent stem cells in the treatment of spinal cord injury and ALS.
Gajdoš, Roman ; Jendelová, Pavla (advisor) ; Vargová, Lýdia (referee)
Induced pluripotent stem cells (iPSCs) have become a new phenomenon of regenerative medicine. It is obvious that they share some common characteristics with embryonic stem cells (ESCs) such as stemness potential, self-renewal p., differentiation p. iPSCs retain their epigenetic memory, allowing becoming patient-specific and so it is not necessary to apply immunosuppressants. The use of ESCs is controversial, because their acquisition is associated with embryo destruction. As a cell source for iPSCs derivation we can use any somatic cells, however, fibroblasts are preferably used due to their easy availability. With transcriptional reprogramming cocktail (OCT4, SOX2, KLF4, c- MYC / OCT4, SOX2, NANOG, LIN28) we can obtain required iPSCs line, which is then further differentiated into neural precursors (NPCs). These cells can be grafted into lesion site, where they can facilitate regeneration by several mechanisms (cell replacement, protective effect, facilitation the expression of trophic factors). Nevertheless, here we are still dealing with the risk of tumorogenesis or low cell derivation efficiency that limits the use of iPSCs in clinical practice. In this thesis we will therefore mainly focus on the therapeutic potential of iPSCs in preclinical studies, their use in the treatment of...
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Generation and application of induced pluripotent stem cells in hematology
Berková, Linda ; Láníková, Lucie (advisor) ; Krulová, Magdaléna (referee)
Induced pluripotent stem cells (iPSC) are one of the key discoveries in cell biology of the last decade. These cells are pluripotent stem cells derived from differentiated somatic cells while having used only four exogenous transcription factors. Pluripotent cells, which can be derived from somatic cells carrying genetic mutation, have a great potential to be used in the testing of new drugs and in discovering molecular mechanisms of genetic disorders. iPSC derived from healthy cells can be used in regenerative medicine. Originally, retroviral vectors were used for delivering reprogramming transcription factors to cells. However such approach is not safe for medicinal use, because of the ability of retroviruses to integrate into the host genome. This fact initiated development of safer delivering methods of transcription factors into the cells. In this work I present the overview of methods which have been used for reprogramming including the most common techniques used to test pluripotency. In addition, I will describe iPSC application options for therapy of genetically determined hematological disorders (sickle cell anemia, β-thalassemia, X-linked chronic granulomatous disease) and for modelling of their molecular mechanism (polycythemia vera). Key words: iPSC, reprogramming, hematological...
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Genome stability of human induced pluripotent stem cells
ŘEHÁKOVÁ, Daniela
Human induced pluripotent stem cells (hiPSCs) play roles in both disease modeling and regenerative medicine. It is critical that the genomic integrity of the cells remains intact and that the DNA repair systems are fully functional. I focused on the detection of DNA double-strand breaks (DSBs) by phosphorylated histone H2AX (known as H2AX) and p53-binding protein 1 (53BP1) in multiple lines of hiPSCs, their source cells, human embryonic stem cells (hESCs) and cells differentiated from hiPSCs. I measured spontaneously occurring DSBs throughout the process of reprogramming and during long-term in vitro culture and differentiation process. To assess the variations in the functionality of the DNA repair system among the samples, the number of DSBs induced by gamma irradiation and the decrease over time was analyzed. The foci number was detected by fluorescence microscopy separately for the G1 and S/G2 cell cycle phases. Source cells contained a low number of non-replication-related foci, while this number increased after reprogramming into hiPSCs and then decreased again after long-term in vitro passaging or differentiation. The artificial induction of DSBs revealed that the repair mechanisms function well in the source cells and hiPSCs at low passages but fail to recognize a substantial proportion of DSBs at high passages. Observations suggest that cellular reprogramming increases the DSB number but that the repair mechanism functions well. However, with long-term hipSCs culture reparation capacity decreases.
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Generation and application of induced pluripotent stem cells in hematology
Berková, Linda ; Láníková, Lucie (advisor) ; Krulová, Magdaléna (referee)
Induced pluripotent stem cells (iPSC) are one of the key discoveries in cell biology of the last decade. These cells are pluripotent stem cells derived from differentiated somatic cells while having used only four exogenous transcription factors. Pluripotent cells, which can be derived from somatic cells carrying genetic mutation, have a great potential to be used in the testing of new drugs and in discovering molecular mechanisms of genetic disorders. iPSC derived from healthy cells can be used in regenerative medicine. Originally, retroviral vectors were used for delivering reprogramming transcription factors to cells. However such approach is not safe for medicinal use, because of the ability of retroviruses to integrate into the host genome. This fact initiated development of safer delivering methods of transcription factors into the cells. In this work I present the overview of methods which have been used for reprogramming including the most common techniques used to test pluripotency. In addition, I will describe iPSC application options for therapy of genetically determined hematological disorders (sickle cell anemia, β-thalassemia, X-linked chronic granulomatous disease) and for modelling of their molecular mechanism (polycythemia vera). Key words: iPSC, reprogramming, hematological...
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The use of induced pluripotent stem cells in the treatment of spinal cord injury and ALS.
Gajdoš, Roman ; Jendelová, Pavla (advisor) ; Vargová, Lýdia (referee)
Induced pluripotent stem cells (iPSCs) have become a new phenomenon of regenerative medicine. It is obvious that they share some common characteristics with embryonic stem cells (ESCs) such as stemness potential, self-renewal p., differentiation p. iPSCs retain their epigenetic memory, allowing becoming patient-specific and so it is not necessary to apply immunosuppressants. The use of ESCs is controversial, because their acquisition is associated with embryo destruction. As a cell source for iPSCs derivation we can use any somatic cells, however, fibroblasts are preferably used due to their easy availability. With transcriptional reprogramming cocktail (OCT4, SOX2, KLF4, c- MYC / OCT4, SOX2, NANOG, LIN28) we can obtain required iPSCs line, which is then further differentiated into neural precursors (NPCs). These cells can be grafted into lesion site, where they can facilitate regeneration by several mechanisms (cell replacement, protective effect, facilitation the expression of trophic factors). Nevertheless, here we are still dealing with the risk of tumorogenesis or low cell derivation efficiency that limits the use of iPSCs in clinical practice. In this thesis we will therefore mainly focus on the therapeutic potential of iPSCs in preclinical studies, their use in the treatment of...
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