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Study of nucleic-acid structure and interactions by resonance Raman scattering
Klener, Jakub ; Štěpánek, Josef (advisor)
Despite the decades of intensive research, nucleic acids represent still a permanent object of structural studies. Within the framework of the doctoral work, the apparatus for measurement of UV excited resonance Raman spectra (UV RRS) was built up and optimized. A realistic and complex interpretation table was prepared based on analysis of published data and extensive series of UV RRS measurements on NA model structures, mononucleotides, and polynucleotides. The established methodology was verified when applied in several structural studies of nucleic acids, mainly the study of the influence of magnesium ions on the equilibrium between duplexes and triplexes formed by PolyA and PolyU homopolynucleotides, a study of temperature-induced structural changes in DNA double helix and DNA hairpin, and investigation of slow structural transitions of guanine quadruplexes induced by the presence of potassium ions. The results of the test measurements and the above-mentioned studies have shown that the created methodology for studying UV RRS of nucleic acids brings most of the expected benefits of the resonance excitation: the possibility of Raman scattering measurements at the same concentrations as in the case of UV absorption, high sensitivity to fine temperature-induced structural changes and good interpretability...
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Study of nucleic-acid structure and interactions by resonance Raman scattering
Klener, Jakub ; Štěpánek, Josef (advisor) ; Michl, Martin (referee) ; Vlčková, Blanka (referee)
Despite the decades of intensive research, nucleic acids represent still a permanent object of structural studies. Within the framework of the doctoral work, the apparatus for measurement of UV excited resonance Raman spectra (UV RRS) was built up and optimized. A realistic and complex interpretation table was prepared based on analysis of published data and extensive series of UV RRS measurements on NA model structures, mononucleotides, and polynucleotides. The established methodology was verified when applied in several structural studies of nucleic acids, mainly the study of the influence of magnesium ions on the equilibrium between duplexes and triplexes formed by PolyA and PolyU homopolynucleotides, a study of temperature-induced structural changes in DNA double helix and DNA hairpin, and investigation of slow structural transitions of guanine quadruplexes induced by the presence of potassium ions. The results of the test measurements and the above-mentioned studies have shown that the created methodology for studying UV RRS of nucleic acids brings most of the expected benefits of the resonance excitation: the possibility of Raman scattering measurements at the same concentrations as in the case of UV absorption, high sensitivity to fine temperature-induced structural changes and good interpretability...
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Diagnostics of neurodegenerative diseases by means of Raman spectroscopy
Klener, Jakub ; Kopecký, Vladimír (advisor) ; Matějka, Pavel (referee)
Therapies of neurodegenerative diseases are often very difficult and their success depend on an early diagnose. From that reason we have been developing new diagnostic method for multiple sclerosis and Alzheimer disease by drop coating deposition Raman (DCDR) spectroscopy of cerebrospinal fluid (CSF) in this work. We found out conditions of measurements, where spectra were reproducible and accepted for standard diagnostic practices. We discovered that CSF has fast degradation at a room temperature, which was detectable in spectra after 5 hours, and degradation due to refreezing. DCDR spectra of CSF from individual patients were analyzed by factor and cluster analysis. Multiple sclerosis was manifested by lower intensity of a Raman band at 1080 cm−1 , which is probably connected with more general pathologic state. Spectral changes caused by Alzeheimer disease were more complex and beside changes mentioned above also changes connected with composition and conformation of proteins were identified in regions 1200-1800 cm−1 and 2870-2950 cm−1 . Additionally, we succeeded in distinguishing of young healthy patients from older patients in DCDR spectra. In this work were checked up, that DCDR is good diagnostic method for clinical practices for determining neurodegenerative diseases through the complex...
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