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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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Fluorescence in hydrophilic polymers research
Kotouček, Jan ; Pilgrová, Tereza (referee) ; Venerová, Tereza (advisor)
This bachelor thesis deals with the use of fluorescence in the study of hydrophilic polymers such as polyvinylpyrrolidone, carboxymethylcellulose, bovine serum albumin, and hyaluronan. The first measurement was a fluorescence intensity of the anionic form of 3-hydroxy naphtholic acid which undergoes an intramolecular proton transfer in the excited state then the steady-state fluorescence anisotropy of 3-hydroxy naphtholic acid and also the lifetime of the fluorescent probe. The measured data were evaluated in terms of interaction of the fluorescence probe with hydration shell of polymer in an aqueous environment by determination of a binding constant of the system, polymer - fluorescent probe. The interaction between anionic form of the probe with polymer was found in BSA, PVP and CMC systems. There was no interaction of fluorescent probe with hydration shell of hylauronan due to charge repulsion between the probe and hyaluronan.
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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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Influence of ionic strength on the properties of ionic amphiphilic pairs
Filipová, Lenka ; Smilek, Jiří (referee) ; Mravec, Filip (advisor)
The Bachelor thesis studies catanionic vesicle systems consisting of ion pair amphiphile (IPA) and their properties in the presence of ionic strength. Ion pair amphiphile was prepared from single-chained surfactants: cationic surfactant HTMAB (hexadecyltrimethylammonium bromide) and anionic surfactant SDS (sodium lauryl sulfate). Cationic double-chained surfactant DDAC (dimethyldioctadecylammonium chloride) was added in order to stabilize the vesicle system and make it positively charged. Cholesterol was also added to further stabilize the vesicle system. The system is considered to be relatively stable when consisting of 90 % IPA and 10 % DDAC with 43 mol.% cholesterol in a membrane. The stability of the system can be disrupted by external factors such as ionic strength. The Ionic strength was induced by NaCl solutions of varying concentrations (0.0, 0.5, 1.0, 2.0, 50.0, 100.0, 150.0 and 300.0 mM). The change of properties of a vesicle system, which was induced by ionic strength, was analyzed by electrophoretic and dynamic light scattering, fluorescence anisotropy, and generalized polarization. –potential was obtained by electrophoretic light scattering analysis which determines the stability of the system. Dynamic light scattering measurement resulted in the determination of the size of vesicles. The fluidity of vesicles’ membranes was examined by fluorescence anisotropy with DPH (1,6-diphenyl-1,3,5-hexatriene) as a probe. The hydration shell of vesicles was observed by generalized polarization with Laurdan (2-(dimethylamino)-6-dodecanoylnaphthalene) as a probe. As a result, it was found out that low ionic strength (0.5 to 2.0 mM) causes a decrease in the size of vesicles and high ionic strength (50.0 to 300.0 mM) causes vesicles to grow in size. –potential showed a consistent trend for the whole concentration series – its value rapidly decreasing with increasing ionic strength. The samples with the concentrations of salt 50.0 to 300.0 mM were considered unstable according to the –potential data. Fluorescence anisotropy decreases with increasing temperature and increasing ionic strength. The value of generalized polarization decreases with increasing temperature; hence there is more solvation of a vesicle membrane. The highest value of generalized polarization was measured when the concentration of sodium chloride was relatively high (from 50.0 to 300.0 mM), as a result of which the membrane was less hydrated and therefore, more organized.
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Study of membrane properties of catanionic vesicles from new SEPT-DS amphiphilic ion pairs
Vajcíková, Katarína ; Krouská, Jitka (referee) ; Mravec, Filip (advisor)
This work describes the possibility of determining the membrane properties of vesicular systems formed by ion pair amphiphile (IPAs), which were prepared by mixing positively and negatively charged surfactants, namely the positively charged surfactant carbethopendecinium bromide (Septonex) and negatively charged surfactant sodium dodecyl sulphate with the addition of dioctadecyldimethylammonium chloride and cholesterol for stability enhancement. Such systems are potential drug carriers and hence their careful characterization is very important for further research. In this work, the possibilities of studying the membrane properties of such systems using spectrometric methods, namely fluorescence anisotropy and generalized polarization, by which the fluidity and solvation of the bilayer membrane were detected as a function of the amount of added cholesterol and the change in temperature, are described. The size and stability of the prepared vesicular systems formed from the new amphiphilic pairs were investigated by dynamic and electrophoretic light scattering, and finally, the phase transition temperature was detected using differential scanning calorimetry method.
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Study of the interaction between the C-terminus of DNA-binding domain of FOXO4 and DNA
Zusková, Iva ; Obšil, Tomáš (advisor) ; Teisinger, Jan (referee)
Forkhead transcription factors are structurally similar molecules containing approximately 110-amino-acid-long DNA-binding domain known as a forkhead domain. Protein FOXO4 is a member of subgroup "O" of forkhead transcription factors. Members of this subgroup play a key role in many biologically important processes. For example, FOXO factors participate in metabolism control, cell-cycle control, apoptosis and oxidative stress resistance. The forkhead domain (DNA-binding domain) consists of three α-helices (H1, H2 and H3), three β-strands (S1, S2 and S3) and two flexible loops (called wings W1 and W2). The role of the wing W2 in FOXO binding to the target DNA is still elusive. Wing W2 probably interacts with the DNA in the region upstream of the core motif. It has been speculated that the FOXO DNA-binding affinity depends on A-T content (number of A-T pairs) in the region upstream of the core motif. In order to investigate this hypothesis, DNA- binding domain of the FOXO4 protein was expressed and purified and it was determined its binding affinity for three molecules of double stranded DNA containing different number of A-T pairs in the region upstream of the core motif using steady-state fluorescence anisotropy- based method. Our results show no significant differences between obtained FOXO4...
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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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