|
|
Optical Micromanipulation Techniques Combined with Microspectroscopic Methods
Pilát, Zdeněk ; Prášil,, Ondřej (referee) ; Mojzeš, Peter (referee) ; Zemánek, Pavel (advisor)
Předložená dizertační práce se zabývá kombinací optických mikromanipulací s mikrospektroskopickými metodami. Využili jsme laserovou pinzetu pro transport a třídění živých mikroorganismů, například jednobuněčných řas, či kvasinek. Ramanovskou spektroskopií jsme analyzovali chemické složení jednotlivých buněk a tyto informace jsme využili k automatické selekci buněk s vybranými vlastnostmi. Zkombinovali jsme pulsní amplitudově modulovanou fluorescenční mikrospektroskopii, optické mikromanipulace a jiné techniky ke zmapování stresové odpovědi opticky zachycených buněk při různých časech působení, vlnových délkách a intenzitách chytacího laseru. Vyrobili jsme různé typy mikrofluidních čipů a zkonstruovali jsme Ramanovu pinzetu pro třídění mikro-objektů, především živých buněk, v mikrofluidním prostředí.
|
|
|
Spectroscopic study of verteporphin in liposomes
Laubrová, Veronika ; Dědic, Roman (advisor) ; Mojzeš, Peter (referee)
The diploma thesis examined the photochemical properties of verteporphin in liposomes. Absorption and fluorescence spectroscopy and time-resolved detection of singlet oxygen generated by verteporphin were used. The kinetics of singlet oxygen emission were studied under conditions of varying concentrations of dissolved oxygen, the presence of heavy water, and a specific quencher - sodium azide. It was found that verteporphin in liposomes is present in two distinct groups. It was also found that verteporphin in liposomes undergoes a photochemical change, and the formation of a photoproduct was observed in absorption and fluorescence spectra. The formation of the photoproduct is reflected in the times of increase and decrease of the kinetics of singlet oxygen luminescence. It has been demonstrated that the photoproduct is formed in the presence of oxygen in the ground state by an electron transfer mechanism from the triplet state of verteporphin and the formation and subsequent reaction of a superoxide radical.
|
|
|
Supramolecular complexes of oxoporphyrinogens with organic molecules
Březina, Václav ; Hanyková, Lenka (advisor) ; Mojzeš, Peter (referee) ; Tošner, Zdeněk (referee)
Title: Supramolecular complexes of oxoporphyrinogens with organic molecules Author: Václav Březina Department: Department of Macromolecular Physics Supervisor: doc. RNDr. Lenka Hanyková, Dr., Department of Macromolecular Physics Abstract: Oxoporphyrinogens are flat macrocyclic molecules possessing binding and protonation sites, and capable of light absorption in the visible region. These properties are prerequisites for a colori- metric molecular sensor, i.e. a specific detector of other molecules in the sample. In this work, we studied chromic properties of three oxoporphyrinogens, OxP and its partially (Bz2OxP) and fully (Bz4OxP) N-benzylated derivatives. Their colorimetric response to organic acids is caused by protonation and subsequent formation of supramolecular host-guest complex. We have shown that colorimetric sensitivity is highest for OxP and gradually weakens for Bz2OxP and Bz4OxP since the N-benzylation blocks the central binding sites, decreasing binding affinity of the ox- oporphyrinogens. Furthermore, solvatochromic response of the oxoporphyrinogens to varying solvent polarity showed similar sensitivity decrease in Bz2OxP and Bz4OxP. The chromic and binding properties were studied by UV/vis and NMR spectroscopy, host-guest binding models were applied to describe the formation of...
|
|
| |
|
|
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...
|
|
|
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...
|
|
|
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.
|
|
|
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...
|
|
|
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...
|
|
|
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...
|
guest ::
