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Non-resonant Raman Spectroscopic Study of Guanine Quadruplex Structures
Golan, Martin ; Mojzeš, Peter (advisor) ; Mašek, Vlastimil (referee)
Parts of human telomere sequences containing at least 4 guanine subsequences show the ability to form intrastrand quadruplexes of remarkable conformational diversity. Former studies using conventional Raman spectroscopy have revealed that the sequence G3(TTAG3)3 at milimolar concentrations in phosphate buffer solution doped with Na+ ions (ionic strength 150 mM) adopts antiparallel conformation regardless of the length of standing at room temperature or annealing, whereas K+ ions cause gradual transition to "3+1" or even parallel conformation. On the other hand, measurements carried out upon sequence AG3(TTAG3)3 at similar concentrations using Photonic Crystal Fibre-enhanced Raman Spectroscopy (PCFRS) suggest that in the respective presence of both Na+ and K+ (ionic strength 100 mM), a parallel structure is adopted. The hereby presented work employs conventional Raman spectroscopy and Drop Coating Deposition Raman spectroscopy to examine the sequence AG3(TTAG3)3 at concentrations ranging from units to hundreds of milimoles in strands. It concludes that the structure adopted in the presence of Na+, resp. K+ ions is antiparallel, resp. "3+1", and doesn't change over time despite both long standing and annealing. Two hypotheses about the cause of the differences between the results obtained by PCFRS and...
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Complexes of Cationic Porphyrins with Nucleic Acids Studied by Surface Enhanced Resonance Raman Spectroscopy
Lásková, Barbora ; Mojzeš, Peter (advisor) ; Michl, Martin (referee)
Complexes of cationic porphyrins with nucleic acids are interesting from the point of view of delivery of therapeutic oligonucleotides into cells as well as for photodynamic therapy of cancer. Diploma thesis deals with study of the complexes of cationic metalloporphyrin CuTMPyP4 with poly(dG-dC)2, where intercalation of porphyrin within polynucleotide's base pairs is supposed and with poly(dA-dT)2, poly(dA)·poly(dT), in which external groove-binding of CuTMPyP4 on the helix is assumed. The measurements were made by SERRS, RRS and absorption spectroscopies. From the time evolution of the SERRS spectra for each complex it was found, that intensity of SERRS spectra and rate of SERRS kinetics for complexes fall short of intuitive supposition about low SERRS signal and its slow increasing in the case of intercalation of porphyrin, because for complex of CuTMPyP4 with poly(dG-dC)2, the SERRS kinetic are very fast and final SERRS signal is more intense than for poly(dA-dT)2, poly(dA)·poly(dT). Comparing the SERRS and RRS spectra of the complexes it was found that SERRS spectra correspond to the porphyrin molecules released from the complexes rather that to the entire complexes. Study of various colloidal systems used for SERRS measurements revealed that SERRS signal evolves with time of exposition, namely for...
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In situ Luminescence Detection of Singlet Oxygen
Vyklický, Vojtěch ; Dědic, Roman (advisor) ; Mojzeš, Peter (referee)
The thesis summarises already published results of singlet oxygen luminiscence detection in relation to photodynamic therapy treatment. The spectroscopic and photosensitising properties of the photosensitizer TMPyP and their comparison with already published properties of TPPS4 are presented. Also the inuence of phosphatidylcholine on photosensitising properties of TPP and singlet oxygen lifetime was investigated. The thesis is mainly focused on adaptation and optimisation of experimental setup for singlet oxygen luminescence deteciton from the surface of solid samples with time and spectral resolution using optical fibres with luminescence probe. The usability was tested on polymer matrix (PMMA or polystyrene) containing protoporphyrine IX. Finally, the kinetics of singlet oxygen phosphorescence measured from surface of thin layer of 3T3 murine fibroblasts containing TPPS4 or TMPyP are presented. Thereby the feasibility of singlet oxygen detection in situ was demonstrated using this setup.
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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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Biologically important non-canonical structures of nucleic acids in complexes with cationic porphyrins
Palacký, Jan ; Mojzeš, Peter (advisor) ; Setnička, Vladimír (referee) ; Víglaský, Viktor (referee)
Guanine quadruplexes are a class of unusual nucleic acids conformations based on stacked planar guanine tetrads stabilized via Hoogsteen pairing and cation coordination. They are implicated in numerous cellular processes including replication, recombination or transcription. Guanine quadruplexes are widespread within the human genome but their occurrence is highest in the single stranded guanine-rich regions at telomeres. Telomeric guanine quadruplexes are gaining growing interest due to their ability to inhibit the activity of the telomerase enzyme, which is responsible for the proliferation of tumor cells. Specifically, we investigated the conformational polymorphism of the human core telomeric sequence G3(TTAG3)3 conditioned by the concentration of DNA, metal cations (K+ , Na+ ) and/or annealing. Raman spectroscopy was employed as the primary method for this study because, unlike common spectroscopic methods, it allowed us to monitor the quadruplex structure at very high DNA concentrations mimicking molecular crowding conditions in the cell. We demonstrate that the G3(TTAG3)3 quadruplex switch between the antiparallel and parallel strand alignment as a function of nucleoside and potassium concentration. In addition, we demonstrate that cationic porphyrins can be used as sensitive probes of the quadruplex...
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Study of nucleic acids by means of drop coating deposition Raman microspectroscopy (DCDR)
Souček, Pavel ; Kopecký, Vladimír (advisor) ; Mojzeš, Peter (referee)
The work was focused on possibilities of the new technique of nonresonance Raman spectroscopy - drop coating deposition Raman (DCDR) spectroscopy upon study of nucleic acids. DCDR spectroscopy is based on deposition of a small droplet of the studied sample on a hydrophobic surface, where after evaporation of the solvent, ring of the studied material in glass phase is formed. That way the material is concentrated and upon measurements by means of Raman microspectrometry the increase of signal of several orders of magnitude is observed with respect to the standard technique of sample measurements in solvent. In this work we studied behavior of DNA 12-mer and DNA with 3000 base pairs. Our research showed that DCDR spectra of DNA dissolved in deionized water can be measured up to concentration of 30 M per base. The DCDR spectra are similar to those measured from solvents. Nevertheless, the deposited sample never forms a ring. It was shown that the size of nucleic acids didn't play dominant role upon ring formation but their charge which lead to repulsion of the molecules. After adding of sodium ions and subsequently also magnesium ions the ring formation was observed. Factor analysis of spectral maps demonstrated that formed rings are not fully homogenous and that upon their origin condensation of DNA could...
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Raman microspectroscopy of living cells and biological tissues
Moudříková, Šárka ; Mojzeš, Peter (advisor) ; Matthäus, Christian (referee) ; Samek, Ota (referee)
Title: Raman microspectroscopy of living cells and biological tissues Author: Šárka Moudříková Department / Institute: Institute of Physics of Charles University Supervisor of the doctoral thesis: doc. RNDr. Peter Mojzeš, CSc., Institute of Physics of Charles University Abstract: Raman microscopy combines Raman spectroscopy with optical confocal microscopy and thus provides information on chemical composition of a sample with a µm3 resolution. In this thesis, Raman microscopy has been used to study microalgae-unicellular photosynthetic organisms that are greatly relevant for the Earth's environment as well as for biotechnological applications. Raman microscopy of photosynthetic organisms struggles with a highly intensive background of the spectra, which is formed by fluorescence of cellular photosynthetic apparatus. In this thesis, we have developed a fast and reliable photobleaching method that suppresses the unwanted background; this method has enabled us to study intracellular distribution of algal biomolecules such as proteins, starch, lipids and polyphosphate. We have investigated an evolution of these structures during a cell cycle of a model microalga Desmodesmus quadricauda. Next, we have developed a method for quantitative analysis of polyphosphate in a cellular culture of a microalga Chlorella...
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Raman Microspectroscopy in Microfluidic Devices
Peksa, Vlastimil ; Mojzeš, Peter (advisor) ; Šloufová, Ivana (referee)
Miniaturization of devices to study chemical interactions and processes in liquid samples has led to the emergence of microfluidics and construction of lab-on-a-chip systems. Present work was devoted to implementation, development and testing of microfluidic systems with detection by confocal Raman microscopy and surface enhanced Raman scattering under the conditions of training department. Several options of performing standard macroscopic measurements in microscopic scales were explored. A method for measuring thermal stability of biopolymers in microsystems with contactless detection of temperature has been designed and tested. Furthermore, possibilites for studying the SERS effect within microfluidic channels were explored. It was demonstrated that the microfluidic chips provide promising opportunity to study hydrodynamics of liquids at microscopic level and chemical reactions and kinetics.
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Intensity Normalization of Raman Spectra by Means of Various Internal Standards
Sláma, Vladislav ; Mojzeš, Peter (advisor) ; Procházka, Marek (referee)
The work deals with the testing of selected inorganic anions as internal standards for intensity normalization of Raman spectra. Using the concentration and temperature series of aqueous solutions of PO4 3- , (CH3)2AsO2 - , NO3 - a SO4 2- , impact of the spectral changes of the standard bands for reliability and accuracy of the intensity calibration was studied. Temperature and concentration series of the anion Raman spectra are then used to test normalization by means of the bands of stretching and deformation vibrations of water molecules. In addition, reliability and robustness of the calibration procedures exploiting different spectral parameters present in the Raman spectra of standards was tested. Results of the study reveal possible sources of artifacts and misinterpretations arising from insufficient understanding of the standard behavior due to changes of the physical and chemical parameters, or due to improper calibration procedure applied.
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