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Anisotropy techniques in study of cytoplasm
Sýkorová, Kateřina ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
The main goal of this thesis was to compare experiments using time-resolved anisotropy and steady-state anisotropy for measuring in bacteria strain Cupriavidus necator. Fluorescent probe for anisotropy imaging was chosen BCECF_AM, which is derivate of fluorescein. Using experiment in system glycerol/water with fluorescein, anisotropy has been verified and calculated molecular hydrodynamic volume of a single fluorescein molecule, which approximately corresponded with real value. By using fluorescence imaging anisotropy microscopy, images and values of average anisotropy in cells were taken. Images of living cells (bacteria) of CN H16 and mutant CN PHB-4 showed differences, mainly in the uniformity of the inside environment.
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Advanced Fluorescence Techniques in Research on Micellar Systems and Their Interactions with Biopolymers
Holínková, Petra ; Burgert, Ladislav (referee) ; Táborský, Petr (referee) ; Pekař, Miloslav (advisor)
The dissertation thesis deals with study of advanced steady-state and time-resolved fluorescence techniques, which can be used for study of micellar systems properties. Selected fluorescence techniques were used for characterization of Septonex and CTAB cationic micellar systems and theirs interactions with hyaluronan. Fluorescent probe pyrene was used for determination of critical micelle concentration (CMC) and micellar aggregation number of these surfactants. The changes of fluorescence behaviour of fluorescein and prodan were studied in wide concentration range of Septonex. Next chapter of thesis deals with study of Förster resonance energy transfer between perylene and fluorescein in Septonex and CTAB micellar solutions and the effect of hyaluronan addition to these systems. Also steady-state and time-resolved fluorescence anisotropy studies were used for research of the effect of hyaluronan addition to micellar solutions. The last chapter of this thesis is focused on photophysical behaviour of Prodan in different solutions (water, Septonex solutions below CMC, hyaluronan solution, Septonex micellar solution and Septonex micellar solution with hyaluronan), which was discussed on the basis of time-resolved emission spectra.
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Study of microviscosity of membrane systems based on ionic amphiphilic pairs
Moslerová, Lenka ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
In this master ‘s thesis, catanionic vesicles formed by the pseudo-double-chain complex CTA – DS were investigated from the point of view of microviscosity. Samplesand of cationic vesicles contained 23, 43 and 53 mol. % of cholesterol and the double-chain surfactant DODAC. Cationic vesicles were prepared for visual observation, their stability was determined by DLS and the prepared system was further investigated. Microviscosity was determined from fluorescence anisotropy. To study the outer part of the membrane, laurdan fluorescent probes were used whereas diphenylhexatriene was used for the inner part of the membrane. This method has been proven to be suitable because it reflects the conditions of the membrane. Moreover, a 1,3-bispyrenylpropane probe forming intramolecular excimers was used to study the microviscosity in the vesicle bilayer. The dicyanovinyljulolidine (DCVJ) probe was applied in the case of the molecular rotor technique. It has been shown that in the case of the DCVJ probe, the molecular rotor technique is practically unusable, due to the fact that the probe has a low quantum yield at low temperatures. Also, the excimer formation of P3P probes does not lead to the expected results. The cationic vesicles do not seem to support this formation, as they are too closely related. This type of probe can be used for the selected system with some restrictions.
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Correlation between steady-state fluorescence anisotropy measurement on fluorimeter and fluorescence microscope
Moslerová, Lenka ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
This Work concentrates on detection fluorescent anisotropy on a model system of fluorescent probe and glycerol. In this thesis, the ATTO 488 was used as a fluorescent substance and glycerol solutions of different concentrations were used to simulate different viscous environments. A fluorescence spectrofluorometer and confocal fluorescence microscope were used for the measurements. A linear encrease anisotropy with increasing viscosity of the environment was observed on fluorometer. The same trend was detected on fluorescence microscope. The values were compared and correlation factors were determined. The accuracy of the measurement was verified by calculations using the Perrin´s equation.
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Study of membrane properties of liposomal systems using fluorescence spectroscopy
Zbořilová, Hana ; Pekař, Miloslav (referee) ; Mravec, Filip (advisor)
The presented diploma thesis is focused on the preparation, characterization and study of membrane properties of liposomal systems which were composed of the neutral phosphatidylcholine (DPPC), cholesterol, negatively charged phosphatidylglycerol (DPPG), polyethylenglycol bounded to phosphatidylethanolamine (PEG5000–PE) and polycation N,N,N-trimethylchitosan (TMC). The influence of individual components and their concentrations on the average particle size, zeta potential and changes in the outer and inner part of the bilayer was investigated. In this matter, methods of dynamic and electrophoretic light scattering and fluorescence spectroscopy with the application of laurdan and DPH probes were used. Based on the above-mentioned parameters, concentrations of components that most suitably influence properties of liposomes in terms of the intended application were selected for the definite complex. It was managed to prepare a liposomal complex stealth liposome–N,N,N-trimethylchitosan, which, due to the optimized composition, could have suitable attributes as a drug delivery system for inhalation administration of biologically active substances.
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Anisotropy and time-resolved anisotropy techniques in colloidal systems research
Holínková, Petra ; Táborský,, Petr (referee) ; Mravec, Filip (advisor)
In this diploma thesis were investigated in terms of microviscosity liquid and condensed systems composed of hyaluronan (Hya) and cationic surfactant cetyltrimethylammonium bromide (CTAB). The excitation and emission spectra, lifetime, steady-state fluorescence anisotropy and time-resolved fluorescence anisotropy of the samples were measured. First, was studied the formation of hydrophobic domains in the system Hya-CTAB at concentration of CTAB lower than its critical micelle concentration in an aqueous solution and 0.15M NaCl. It was found that in an aqueous solution small hydrophobic domains linked to chains Hya are formed. Then an increasing concentration of CTAB leads to phase separation and formation of gel. Due to the addition of NaCl then leads to the reorganization of this system and probably the formation of free micelles in the solution. Were also studied condensed phase of system Hya-CTAB-NaCl at high concentrations of surfactant during fourteen days of ageing. It was found that the microviscosity of hydrophobic domains is constant, but the microviscosity of hydrophilic parts gradually decreases.
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Study of microviscosity of membrane systems based on ionic amphiphilic pairs
Moslerová, Lenka ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
In this master ‘s thesis, catanionic vesicles formed by the pseudo-double-chain complex CTA – DS were investigated from the point of view of microviscosity. Samplesand of cationic vesicles contained 23, 43 and 53 mol. % of cholesterol and the double-chain surfactant DODAC. Cationic vesicles were prepared for visual observation, their stability was determined by DLS and the prepared system was further investigated. Microviscosity was determined from fluorescence anisotropy. To study the outer part of the membrane, laurdan fluorescent probes were used whereas diphenylhexatriene was used for the inner part of the membrane. This method has been proven to be suitable because it reflects the conditions of the membrane. Moreover, a 1,3-bispyrenylpropane probe forming intramolecular excimers was used to study the microviscosity in the vesicle bilayer. The dicyanovinyljulolidine (DCVJ) probe was applied in the case of the molecular rotor technique. It has been shown that in the case of the DCVJ probe, the molecular rotor technique is practically unusable, due to the fact that the probe has a low quantum yield at low temperatures. Also, the excimer formation of P3P probes does not lead to the expected results. The cationic vesicles do not seem to support this formation, as they are too closely related. This type of probe can be used for the selected system with some restrictions.
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Study of membrane properties of liposomal systems using fluorescence spectroscopy
Zbořilová, Hana ; Pekař, Miloslav (referee) ; Mravec, Filip (advisor)
The presented diploma thesis is focused on the preparation, characterization and study of membrane properties of liposomal systems which were composed of the neutral phosphatidylcholine (DPPC), cholesterol, negatively charged phosphatidylglycerol (DPPG), polyethylenglycol bounded to phosphatidylethanolamine (PEG5000–PE) and polycation N,N,N-trimethylchitosan (TMC). The influence of individual components and their concentrations on the average particle size, zeta potential and changes in the outer and inner part of the bilayer was investigated. In this matter, methods of dynamic and electrophoretic light scattering and fluorescence spectroscopy with the application of laurdan and DPH probes were used. Based on the above-mentioned parameters, concentrations of components that most suitably influence properties of liposomes in terms of the intended application were selected for the definite complex. It was managed to prepare a liposomal complex stealth liposome–N,N,N-trimethylchitosan, which, due to the optimized composition, could have suitable attributes as a drug delivery system for inhalation administration of biologically active substances.
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Advanced Fluorescence Techniques in Research on Micellar Systems and Their Interactions with Biopolymers
Holínková, Petra ; Burgert, Ladislav (referee) ; Táborský, Petr (referee) ; Pekař, Miloslav (advisor)
The dissertation thesis deals with study of advanced steady-state and time-resolved fluorescence techniques, which can be used for study of micellar systems properties. Selected fluorescence techniques were used for characterization of Septonex and CTAB cationic micellar systems and theirs interactions with hyaluronan. Fluorescent probe pyrene was used for determination of critical micelle concentration (CMC) and micellar aggregation number of these surfactants. The changes of fluorescence behaviour of fluorescein and prodan were studied in wide concentration range of Septonex. Next chapter of thesis deals with study of Förster resonance energy transfer between perylene and fluorescein in Septonex and CTAB micellar solutions and the effect of hyaluronan addition to these systems. Also steady-state and time-resolved fluorescence anisotropy studies were used for research of the effect of hyaluronan addition to micellar solutions. The last chapter of this thesis is focused on photophysical behaviour of Prodan in different solutions (water, Septonex solutions below CMC, hyaluronan solution, Septonex micellar solution and Septonex micellar solution with hyaluronan), which was discussed on the basis of time-resolved emission spectra.
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Correlation between steady-state fluorescence anisotropy measurement on fluorimeter and fluorescence microscope
Moslerová, Lenka ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
This Work concentrates on detection fluorescent anisotropy on a model system of fluorescent probe and glycerol. In this thesis, the ATTO 488 was used as a fluorescent substance and glycerol solutions of different concentrations were used to simulate different viscous environments. A fluorescence spectrofluorometer and confocal fluorescence microscope were used for the measurements. A linear encrease anisotropy with increasing viscosity of the environment was observed on fluorometer. The same trend was detected on fluorescence microscope. The values were compared and correlation factors were determined. The accuracy of the measurement was verified by calculations using the Perrin´s equation.
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