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Analyses of inverted repeats in human patogen genomes
Dobrovolná, Michaela ; Kouřilová, Xenie (referee) ; Brázda, Václav (advisor)
Helminth parasites are highly prevalent in humans in developing countries. According to WHO, approximately 2 billion people are infected worldwide. The etiological agents of parasitic infections are mainly Nematodes (roundworms) and Platyhelminths (flatworms), causing inflammatory responses, malnutrition, and anemia that are the primary cause of mortality. Drug resistance is accelerated by the overuse of human anthelmintics, as well as poor infection prevention and control. The therapeutic potential of small molecule ligands binding G-quadruplexes (G4s) has been demonstrated. For instance, that it can be used to stabilize the quadruplex structures and eliminate drug-resistant pathogens. G4s are secondary structures formed in guanine-rich nucleic acid sequences, which can regulate the process of gene expression, DNA damage repair, transcription, and translation of oncogenes. Here we used the G4Hunter Web Tool to identify and compare G-quadruplex sequences (PQS) in the nuclear and mitochondrial genomes of six Platyhelminth and four Nematode species to identify targets for G4 ligands to predict new drug targets and more effective drugs. We found that PQS are nonrandomly distributed in these genomes. Most of the G-quadruplexes are in the proximity of genes, suggesting their role in genetic regulation. Interestingly, less infective Platyhelminthes were found enriched with PQS, compared to highly infective species with a lower PQS frequency. In contrast, a Nematoda, Ascaris lumbricoides, was found to be highly enriched in stable PQS. This highly infective species can tolerate high-stability G4 structures, which are not counter-selected at all in contrast to Caenorhabditis elegans.
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The effect of indole alkaloids on selected cell lines and DNA structures
Dobrovolná, Michaela ; Hoová, Julie (referee) ; Brázda, Václav (advisor)
G-quadruplexes (G4) are secondary DNA structures formed by a cluster of guanines that have been shown to play a role in many biological functions, including regulation of replication timing and repression of oncogene expression. In this work, four plant alkaloids (harmine, harmane, harmaline, and brucine) were tested as potential G4 ligands that could regulate, induce, or convert different G4 topologies. Furthermore, their effect on fibroblast cell line HDF164 and breast cancer cell line MCF-7 was studied, in which case a dose-dependent growth inhibition mechanism was demonstrated. Interaction with the parallel G4 promoter of the proto-oncogene c-Myc was confirmed for harmine, harmaline, and brucine, and with the hybrid G4 of telomeric DNA for harmine, harman, and harmaline. In addition, the ability of the tested compounds to interact with calf thymus double-stranded DNA (ctDNA) was verified by UV-Vis absorption spectroscopy. The methylation assay showed that the DNA of MCF-7 cells treated with harmine and brucine was hypermethylated.
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Analyses of inverted repeats in human patogen genomes
Dobrovolná, Michaela ; Kouřilová, Xenie (referee) ; Brázda, Václav (advisor)
Helminth parasites are highly prevalent in humans in developing countries. According to WHO, approximately 2 billion people are infected worldwide. The etiological agents of parasitic infections are mainly Nematodes (roundworms) and Platyhelminths (flatworms), causing inflammatory responses, malnutrition, and anemia that are the primary cause of mortality. Drug resistance is accelerated by the overuse of human anthelmintics, as well as poor infection prevention and control. The therapeutic potential of small molecule ligands binding G-quadruplexes (G4s) has been demonstrated. For instance, that it can be used to stabilize the quadruplex structures and eliminate drug-resistant pathogens. G4s are secondary structures formed in guanine-rich nucleic acid sequences, which can regulate the process of gene expression, DNA damage repair, transcription, and translation of oncogenes. Here we used the G4Hunter Web Tool to identify and compare G-quadruplex sequences (PQS) in the nuclear and mitochondrial genomes of six Platyhelminth and four Nematode species to identify targets for G4 ligands to predict new drug targets and more effective drugs. We found that PQS are nonrandomly distributed in these genomes. Most of the G-quadruplexes are in the proximity of genes, suggesting their role in genetic regulation. Interestingly, less infective Platyhelminthes were found enriched with PQS, compared to highly infective species with a lower PQS frequency. In contrast, a Nematoda, Ascaris lumbricoides, was found to be highly enriched in stable PQS. This highly infective species can tolerate high-stability G4 structures, which are not counter-selected at all in contrast to Caenorhabditis elegans.
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