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Eucaryotic cells systems and their biotechnologycal applications
Porubiaková, Otília ; Obruča, Stanislav (referee) ; Vorlíčková, Michaela (referee) ; Brázda, Václav (advisor)
The submitted dissertation is divided into several parts. The first part deals with the interactions of the p53 protein and its isoforms with different potential DNA substrates under different experimental conditions. These are predominantly DNA and its non-canonical structural motifs, such as G-quadruplexes or cruciform structures, whose interactions have been studied in yeast isogenic systems or by in vitro methods. The seconds part deals with the bioinformatic analysis of the mentioned secondary DNA structures in different organismal groups, and the result is a set of publications that show their non-random distribution in the genome and their relationship with regulation. The last part of the work contains unpublished results, including the results of testing the effect of natural and synthetic substances on aging in model human cells.
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Eucaryotic cells systems and their biotechnologycal applications
Porubiaková, Otília ; Obruča, Stanislav (referee) ; Vorlíčková, Michaela (referee) ; Brázda, Václav (advisor)
The submitted dissertation is divided into several parts. The first part deals with the interactions of the p53 protein and its isoforms with different potential DNA substrates under different experimental conditions. These are predominantly DNA and its non-canonical structural motifs, such as G-quadruplexes or cruciform structures, whose interactions have been studied in yeast isogenic systems or by in vitro methods. The seconds part deals with the bioinformatic analysis of the mentioned secondary DNA structures in different organismal groups, and the result is a set of publications that show their non-random distribution in the genome and their relationship with regulation. The last part of the work contains unpublished results, including the results of testing the effect of natural and synthetic substances on aging in model human cells.
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DNA POLYMERASE STOP ASSAY FOR DETECTION OF G-QUADRUPLEXES
Petr, Marek ; Bažantová, Pavla ; Adámik, Matěj ; Kejnovská, Iva ; Dvořáková, Zuzana ; Vorlíčková, Michaela ; Pečinka, Petr ; Brázdová, Marie
In this work we performed DNA polymerase stop assays while using template DNA derived from promoter regions of VEGF and c-Myc proto-oncogenes under variety of experimental conditions. Partial stopping of DNA synthesis along the template strand was observed in the presence of K+ ions due to formation of stable G-quadruplex structures. In contrast, Na+ ions alone were unable to stabilize G-quadruplexes to stop the reaction at their site. These data suggest that K+ and Na+ ions, which are both known to stabilize G-quadruplexes, do this in different manner or extent.
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In situ study of nuclear acids interactions key for gene expression and therapy based on its silencing
Špringer, Tomáš ; Štěpánek, Josef (advisor) ; Brynda, Eduard (referee) ; Vorlíčková, Michaela (referee)
In this doctoral thesis we study novel analogues based on R06 aptamers and targeting TAR hairpins of the HIV virus by means of surface plasmon resonance biosensor, which allows for sensitive and real-time monitoring of molecular interactions. We investigate seven different modifications placed at nine different positions on the R06 aptamer in order to find out their applicability in the construction of efficient and stable anti-TAR oligonucleotides. We also determine which positions are suitable for substitutions with a modification and interpret the results in the context of the local nucleotide geometries and interactions in the TAR/anti-TAR complex. In this doctoral thesis we further develop a new fluidic system. This fluidic system eliminates sample dispersion and intermixing effects and thus enables accurate monitoring of molecular interactions on the surface of an SPR chip. We also characterize experimental conditions on the surface of an oligonucleotide chip and their relations towards bio-molecular assays. Specifically, we study the shielding effect of monovalent and divalent cations, which are crucial for the interaction of negatively charged oligonucleotides.
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