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Analysis of IFI16 protein binding to DNA
Kratochvilová, Libuše ; Smetana, Jan (referee) ; Brázda, Václav (advisor)
This diploma thesis deals with the binding of interferon gamma-inducible protein 16 (IFI16) to DNA with the potential of G-quadruplex formation. The IFI16 protein contains two tandemly located DNA-binding HIN domains showing differential binding to DNA structures. IFI16 protein has been shown to preferentially bind G-quadruplex structures over other nucleic acid secondary structures. G-quadruplexes are secondary local structures of DNA (or RNA) that are easily formed under physiological conditions in a number of important regulatory regions of the genome, or are part of the genomes of a number of viruses and pathogens. The ability to recognize, specifically bind and stabilize G-quadruplex structures explains the involvement of the IFI16 protein in the cellular processes of replication, transcription and translation and the establishment of innate immune responses. In the first part of the thesis, the sequences of synthetic oligonucleotides with the potential for G-quadruplex formation were characterized by selected biophysical methods and the full-length IFI16 protein was isolated, which was subsequently used for in vitro binding and competitive binding experiments with characterized oligonucleotides. In the last part of the work, isogenic yeast strains differing in the sequences of the responsive element were transformed with plasmid vectors for the expression of p53 and IFI16 proteins with constitutive and GAL inducible promoters, and the one-hybrid yeast system model was optimized for the study of IFI16 protein interactions in vivo. The results show that most of the analyzed sequences are able to form G-quadruplex structures in vitro, even in the presence of only one run of three or more G-bases. While the presence of several G-runs separated by a single nucleotide spacer led to the formation of intermolecular G-quadruplex structures, mutation in the original G-quadruplex sequence induced the formation of intramolecular structures with different conformations. In vitro binding and competitive binding experiments demonstrated specific binding of the IFI16 protein to G-quadruplex structures without differences in protein binding preference to a particular G-quadruplex conformation. Stabilization of G-quadruplex structures in vivo behind the transcription factor responsive element (p53) in the gene promoter induced repression of the transcription of the given gene. In the absence of any binding site of the IFI16 protein, a protein-protein interaction between the IFI16 and p53 proteins occurred, which led to an increase in the transactivation potential of the p53 protein, while the binding of the p53 protein and initiation of reporter gene transcription was influenced not only by the presence of the G-quadruplex motif and its stabilization, but and the DNA sequence adjacent to the p53 responsive element.
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Studies of Lanthanide Complexes by a Combination of Spectroscopic Methods
Krupová, Monika ; Bouř, Petr (advisor) ; Kapitán, Josef (referee)
Studies of Lanthanide Complexes by a Combination of Spectroscopic Methods Monika Krupová (Department of Physical and Macromoecular Chemistry, Faculty of Science, Charles University in Prague) Since conventional structural analysis offers rather limited means for the chirality detection, a series of lanthanide tris-(β-diketonates) are investigated as effective receptors for a better chirality sensing in biomolecular substrates. These lanthanide complexes containing β-diketonate ligands are electrically neutral; they can further coordinate with various small organic molecules such as chiral alcohols, amino alcohols or amino acids in organic solvents and produce a strong chiral signal. Previously, a resonance in Raman scattering was observed in the studied systems due to the correspondence of europium electronic transition energy to the laser excitation wavelength, about a 100-fold signal enhancement if compared to non-resonant vibrational ROA was observed. This enabled shorter detection times as well as lower sample concentrations. In the current work, interaction of the Eu(FOD) complex with (R)- and (S)- enantiomer of 1-phenylethanol in n-hexane was studied using IR spectroscopy, Raman spectroscopy and Raman optical activity (ROA), UV-Vis spectroscopy and ultraviolet circular dichroism (UVCD). Only...
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Conformational transitions and stability of quadruplex nucleic acids
Johanovská, Zuzana ; Mojzeš, Peter (advisor) ; Profant, Václav (referee)
The work deals with guanine quadruplexes, i.e., non-canonical four-stranded structures of nucleic acids rich in repetitive guanine sequences with potential biological and nanotechnological significance. The first part summarizes basic information about the structure and topology of quadruplexes, in the second part, the present knowledge about the quadruplexes in vivo are overviewed. The third part is devoted to experimental methods used in the present study, namely Raman spectroscopy, circular dichroism, and absorption spectroscopy. The next sections are devoted to the acquisition and treatment of your own experimental results. Five sequentially related oligonucleotides differing in the bases in a single-member loop were investigated. The effect of concentration of oligonucleotide and potassium ions K+ on the stability of these quadruplexes was investigated by Raman spectroscopy, and an unusually high thermal stability was observed. Other experiments included measurements of CDs and absorption spectra at lower oligonucleotide concentrations, two different potassium ion concentrations, and a comparison of the stability of individual modifications with the previously published results. Furthermore, the possible interaction with the cationic porphyrin CuTMPyP4, similar to the interaction with the antiparallel...
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Conformational transitions and stability of quadruplex nucleic acids
Johanovská, Zuzana ; Mojzeš, Peter (advisor) ; Profant, Václav (referee)
The work deals with guanine quadruplexes, i.e., non-canonical four-stranded structures of nucleic acids rich in repetitive guanine sequences with potential biological and nanotechnological significance. The first part summarizes basic information about the structure and topology of quadruplexes, in the second part, the present knowledge about the quadruplexes in vivo are overviewed. The third part is devoted to experimental methods used in the present study, namely Raman spectroscopy, circular dichroism, and absorption spectroscopy. The next sections are devoted to the acquisition and treatment of your own experimental results. Five sequentially related oligonucleotides differing in the bases in a single-member loop were investigated. The effect of concentration of oligonucleotide and potassium ions K+ on the stability of these quadruplexes was investigated by Raman spectroscopy, and an unusually high thermal stability was observed. Other experiments included measurements of CDs and absorption spectra at lower oligonucleotide concentrations, two different potassium ion concentrations, and a comparison of the stability of individual modifications with the previously published results. Furthermore, the possible interaction with the cationic porphyrin CuTMPyP4, similar to the interaction with the antiparallel...
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Study of single stranded DNA by biophysical techniques
Svoboda, Jakub ; Schneider, Bohdan (advisor) ; Novák, Petr (referee)
DNA is the fundamental functional molecule of all domains of life. One of its characteristics is the ability to self-associate to form a double stranded helix. Nevertheless, even single stranded DNA can form non-canonical structures such as hairpins, triplexes or tetraplexes. One of the interesting single stranded sequences are REP elements. These sequences form a part of bacterial transposable elements, so-called insertion sequences, which can be found in a great number of copies in wide range of bacterial species. This thesis studies structure and properties of selected REP sequences. It presents the results of spectral measurements by technique of circular dichroism, melting curves from differential scanning calorimetry, and monocrystal diffraction data. Key words Single-stranded DNA, crystallography, circular dichroism, calorimetry, transpozone, REP elements, insertion sequences.
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Studies of Lanthanide Complexes by a Combination of Spectroscopic Methods
Krupová, Monika ; Bouř, Petr (advisor) ; Kapitán, Josef (referee)
Studies of Lanthanide Complexes by a Combination of Spectroscopic Methods Monika Krupová (Department of Physical and Macromoecular Chemistry, Faculty of Science, Charles University in Prague) Since conventional structural analysis offers rather limited means for the chirality detection, a series of lanthanide tris-(β-diketonates) are investigated as effective receptors for a better chirality sensing in biomolecular substrates. These lanthanide complexes containing β-diketonate ligands are electrically neutral; they can further coordinate with various small organic molecules such as chiral alcohols, amino alcohols or amino acids in organic solvents and produce a strong chiral signal. Previously, a resonance in Raman scattering was observed in the studied systems due to the correspondence of europium electronic transition energy to the laser excitation wavelength, about a 100-fold signal enhancement if compared to non-resonant vibrational ROA was observed. This enabled shorter detection times as well as lower sample concentrations. In the current work, interaction of the Eu(FOD) complex with (R)- and (S)- enantiomer of 1-phenylethanol in n-hexane was studied using IR spectroscopy, Raman spectroscopy and Raman optical activity (ROA), UV-Vis spectroscopy and ultraviolet circular dichroism (UVCD). Only...
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Polarizace vakua v Coulombickém poli
Šimsa, Daniel ; Zamastil, Jaroslav (advisor) ; Skála, Lubomír (referee)
In the present work the vacuum polarization and the circular dichroism of hydrogen-like atoms are studied. We derive equations for the Fourier transforma- tion of the vacuum expectation value of the charge density. We use it to derive Uehling potential and calculate energy shifts caused by it. Then we discuss effects of vacuum polarization in higher orders of α. In second part we define circular dichroism and we express it in terms of reduced matrix elements. Then we derive the formula for parity vilating potential which is generated by weak interaction and together with other results we use it to find the expression for circular dichro- ism in terms of hydrogen radial functions. 1
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