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Time-Resolved Fluorescence in Research of Hyaluronan-Colloidal Systems Interactions
Mondek, Jakub ; Táborský, Petr (referee) ; Peter, Kapusta (referee) ; Pekař, Miloslav (advisor)
The aim of the doctoral thesis was to study advanced fluorescence techniques and its use in colloids or hyaluronan-surfactant systems and hydrogels based on hyaluronan, respectively. Steady-state and time-resolved fluorescence were used to study excited state proton transfer fluroescen probes in hyaluronan-surfactant systems to asses the influence of hyaluronan hydration to its interactions with oppositely charged surfactants. Firstly, different excited state proton transfer fluorescence probes were discussed to choose the most suitable candidate for next research. The influence of hyaluronan on inner environment of micells was determined based on the sensitivity of excited state proton transfer of chosen fluorescence probe 1-naphtol and, based on above mentioned experiments, the structure of hyaluronan hydration shell was discussed. The next part of doctoral thesis was focused on fluorescence lifetime correlation spectroscopy and on the development of method of nanorheology. Measured correlation functions were transformed to mean square displacement with developed MATLAB script. Firstly, the fluorescence method was compared with well described methods such as videomicrorheology and dynamic light scattering to asses the reliability of fluorescence correlation spectroscopy in microrheology. Secondly, nanorheology method was developed and its use in passive nanorheology of hyaluronan hydrogels was discussed. Based on mentioned experiments, the fluorescence correlation spectroscopy microrheology and nanorheology methods were optimized to use the methods in hydrogel research.
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Advanced microrheological techniques in the research of hydrogels
Kábrtová, Petra ; Smilek, Jiří (referee) ; Mravec, Filip (advisor)
This diploma thesis deals with the use of fluorescence correlation spectroscopy technique for microrheological characterization of hydrogel in a system of hyaluronate-cetyltrimethylammonium bromide. Fluorescently labelled particles were used for microrheological FCS analysis. To optimize the method the most appropriate size of particles was chosen on the basis of Newtonian glycerol solutions analysis. Among other things, the discussion was focused on the influence of refractive index change of analysed solutions on analysis results. After hyaluronate solutions analysis it was possible to assess the biopolymer concentration and molecular weight impact on the FCS microrheology results, which could then be compared with analysis results of model hydrogels of hyaluronate and CTAB. Finally, usability and limitations of FCS microrheology have been discussed.
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Fluorescence correlation spectroscopy in hydrophilic colloids
Rýcová, Eva ; Mondek, Jakub (referee) ; Venerová, Tereza (advisor)
This work is focused on studying of hydrophilic colloids using fluorescence correlation spectroscopy (FCS). The gel systems were studied with several fluorescent probes, in 0,15 M sodium chloride (NaCl) consist of hyaluronan and surfactant cetyltrimethylammonium bromide (CTAB). Probe ATTO 655 was chosen for closer examination due to the suppression of triplet state. Every surrounding occuring in the gel systems was examine individually. Results showed that the measurement of gel systems by this method can be realized under certain conditions, the repeatability of the results was loaded with a small standard deviation. Extension of diffusion times caused the presence of micelles, triplet state was caused by a characteristic environment of the hydrogel. The probe also showed free diffusion motion in all environments. This diffusion motion was represented by the shortest diffusion time.
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Diffusion characterization of hydrogels at microscopic level
Uher, Tadeáš ; Smilková, Marcela (referee) ; Sedláček, Petr (advisor)
This Bachelor’s thesis deals with study of diffusion in gels on microscopic level. The method of fluorescence correlation spectroscopy is used for the study of diffusion and this method is based on monitoring the diffusion coefficients in a confocal volume of studied substance. As fluorescence probe is used organic pigment called Rhodamin 6G and diffusion experiments are realized in hydrogel matrix, whose medium forms thermoreversible linear polycarbohydrate – agarose, with addition of reactive component – biopolymer called sodium polystyrenesulphate in certain concentrations. The purpose of the thesis is to research the impacts of interaction between diffusing pigment and specific type of hydrogel. The change of final diffusion coefficient is observed. It is assumed, that sodium polystyrenesulphate influences the diffusion of fluorophore (Rhodamin 6G) in agarose hydrogel.
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Study of diffusion properties of vesicular systems using Fluorescence Correlation Spectroscopy
Rašticová, Barbora ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
This master's thesis deals with the study of the diffusion properties of vesicular systems composed of two oppositely charged tensides SDS and CTAB with the addition of the double-chain tenside DODAC and cholesterol. The aim of this work was to select suitable fluorescent probes and investigate their use in imaging these systems using dynamic light scattering and fluorescence correlation spectroscopy. Different concentrations of hyaluronan were also added to the prepared systems. Two representatives of carbocyanine (DiI, DiO) and two xanthene (F16, RBOE) dyes were selected as fluorescent probes. The suitability of their use was verified for all probes except RBOE. The measurement results from the two methods were also compared. It was found that the data do not correlate with each other, due to the considerable complexity of the analyzed systems and the available mathematical models to fit the FCS curves are therefore insufficient.
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Advanced fluorescence techniques applied on biomolecules (lipid membranes and DNA)
Beranová, Lenka ; Hof, Martin (advisor) ; Svoboda, Petr (referee) ; Večeř, Jaroslav (referee)
The thesis describes time dependent fluorescence shift method and fluorescence correlation spectroscopy method (FCS) with its extensions FLCS, Z-scan FCS and dual-focus FCS applied on specific problems in DNA and lipid research. Compaction mechanism of a DNA molecule smaller than a resolution of a confocal microscope was elucidated. The process was revealed to be "all or non" for a polycation spermine as a condenser in contrast with the gradual compaction caused by a cationic surfactant. Biophysical properties of a phospholipid bilayer influenced by presence of oxidized phospholipids with truncated sn-2 chain were explored. The dynamics of hydrated functional groups in the headgroup region was proved to get faster while the hydration of the headgroup region increased. These effects are in relation with the reorientation of the short sn-2 chains observed in molecular dynamics simulations. Presence of oxidized species may also influence the lateral diffusion of the lipids - a slight increase of the diffusion coefficient was observed. Decrease of hydration and mobility in the headgroup region was found as an influence of heavy water on the phospholipid membrane. These finding are in line with molecular dynamics simulations which show longer lifetimes of hydrogen bonds between water and lipid molecules in...
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Fluorescence Spectroscopy: Advanced methods and their defined applications in protein science
Pospíšil, Petr ; Hof, Martin (advisor) ; Heřman, Petr (referee) ; Polívka, Tomáš (referee)
The hydration and dynamics of the biomolecules appear to be vital for their proper biological functioning. In the presented thesis, various fluorescence techniques were developed and applied to access these properties and their changes upon the mutual interactions of the biomolecules. Initially, the solvent relaxation method based on recording time-dependent fluorescence shift (TDFS) was used to map DNA interactions with proteins and lipids by the newly synthesised fluorene dye covalently bound to the DNA. Secondly, copper-transporting ATPase was probed by Badan attached to the copper-binding cysteine-proline-cysteine motif. The variations in hydration were found to be crucial for the proper ATPase function. Third, a detailed study on quenching of Badan/Prodan fluorescence by tryptophan revealed the limitations of the TDFS method for protein studies, which is essential finding for further applications of TDFS. Fourth application involves investigations of heavy atom effects on the excited state relaxation processes by up-conversion approach in iodinated metallocorroles, which are promising dyes for biological imaging. The obtained findings shall help in further tuning of the optical properties of the corroles desired for the variety of applications. Finally, fluorescence correlation spectroscopy...
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Supported Phospholipid Bilayers and their Interactions with Proteins Studied by Ellipsometry, Atomic Force Microscopy and Confocal Fluorescence Microscopy
Macháň, Radek ; Hof, Martin (advisor) ; Fidler, Vlastimil (referee) ; Konopásek, Ivo (referee)
Supported lipid bilayers have been used as an artificial model of biological membranes and their interaction with 5 selected antimicrobial peptides was studied by several experimental techniques, mainly ellipsometry, laser scanning microscopy and fluorescence correlation spectroscopy. The thesis explains basic principles of the applied techniques focusing on their aspects relevant to characterization of lipid bilayers. The biological significance of antimicrobial peptides, their modes of interaction with membranes and the basic characteristics of the selected peptides are briefly discussed. The following text describes the main types of experimental studies performed and the interpretation of their results. Peptide-induced changes in lipid bilayer morphology were characterized by ellipsometry and laser scanning microscopy. Most interesting effects were observed in the case of melittin, which induced formation of long lipid tubules protruding from the bilayer. Lipid lateral diffusion measured by fluorescence correlation spectroscopy can provide information on bilayer organization on length-scales below resolution of optical microscopy.
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Supported Phospholipid Bilayers and their Interactions with Proteins Studied by Ellipsometry, Atomic Force Microscopy and Confocal Fluorescence Microscopy
Macháň, Radek
Supported lipid bilayers have been used as an artificial model of biological membranes and their interaction with 5 selected antimicrobial peptides was studied by several experimental techniques, mainly ellipsometry, laser scanning microscopy and fluorescence correlation spectroscopy. The thesis explains basic principles of the applied techniques focusing on their aspects relevant to characterization of lipid bilayers. The biological significance of antimicrobial peptides, their modes of interaction with membranes and the basic characteristics of the selected peptides are briefly discussed. The following text describes the main types of experimental studies performed and the interpretation of their results. Peptide-induced changes in lipid bilayer morphology were characterized by ellipsometry and laser scanning microscopy. Most interesting effects were observed in the case of melittin, which induced formation of long lipid tubules protruding from the bilayer. Lipid lateral diffusion measured by fluorescence correlation spectroscopy can provide information on bilayer organization on length-scales below resolution of optical microscopy.
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Preparation, characterization and evaluation of application potential of semiIPN hydrogels
Papežíková, Hana ; Pekař, Miloslav (referee) ; Sedláček, Petr (advisor)
Aim of this thesis os to optimize set up of hydrogel based on semi-interpenetrated polymer networks. Polyhydroxyethylmethyl acrylate was chosen as an model example of hydrogel due to its common use in biomedicine. At first, thermoiniciation was used for preparation and changed to photoiniciation afterwards. Following the optimisation of the hydrogel set up with proper qualities, the polyelectrolyte sodium polystyrene sulfonate was implanted. Object of research was its impact on structure, flowing and transport features of hydrogels. All qualities were tested by drying, rheology, and fluorescence correlation spectroscopy. Results clearly validate influence of hydrogel features by polyelectrolytes.
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