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Study of interaction of charged catanionic vesicles with oppositely charged polyelectrolytes
Havlíková, Martina ; Lehocký, Marián (referee) ; Táborský, Petr (referee) ; Mravec, Filip (advisor)
The presented dissertation is focused on positively charged catanionic vesicles and their interactions with oppositely charged hyaluronan. The catanionic vesicles were composed of ion pair amphiphile of hexadecyltrimethylammonium-dodecyl sulfate (HTMA-DS), a positively charged double-chain surfactant that electrostatically stabilized the vesicles and cholesterol. Cholesterol may strongly affect the physico-chemical properties of the vesicles, therefore it was necessary to determine its optimal amount in the membrane. Especially, the size and stability of vesicles were studied by dynamic and electrophoretic light scattering, membrane microviscosity by fluorescence anisotropy and hydration of the outer part of the membrane by generalized polarization. It was found that vesicles show ideal properties at a cholesterol content about 40 mol.%. These catanionic vesicles interacted with hyaluronan regardless of its molecular weight. Visible aggregates were observed around their isoelectric point, but they disintegrated with increasing polymer concentration. However, this decay was different for different hyaluronan molecular weights. An increase in ionic strength, or more precisely concentration of sodium chloride above 25 mM had a destabilizing effect on both uncoated and hyaluronan coated vesicles. It was also interesting that increasing the ionic strength affected the microviscosity of the membrane, which became more fluid, but there were no significant changes in the hydration of its outer part. The presented work is also focused on the preparation and characterization of a new type of catanionic vesicles, where the cationic surfactants HTMAB was replaced by Septonex. The same methods were chosen for their characterization as in the case of the HTMA-DS vesicles. This exchange of surfactants had a major effect on the resulting properties of the vesicles, which are stable even at very low cholesterol content. In accordance with long-term stability, a suitable concentration of cholesterol was chosen from 15 mol.% and higher.
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Atomic force microscopy of soft materials
Šudáková, Anna ; Havlíková, Martina (referee) ; Smilek, Jiří (advisor)
This bachelor thesis focuses on atomic force microscopy (AFM), mainly on studying the measurement procedure and optimalization of measurement conditions with future perspective on imaging hydrogels and measuring mechanical properties (such as adhesion or stiffness) of hydrogels and semi-rigid materials at the microlevel. Atomic force microscopy is gaining significant importance in research due to its versatility, when it can provide topographical image of conductive and non-conductive samples while measuring mechanical properties of the samples, such as adhesion, elasticity or stiffness. Advantage of AFM method is that it can provide mechanical properties of the samples not only on macro levels as we are used to, for example, from rheology, but also on local level. Optimalization was performed on standard sample, one micrometre polystyrene nanoparticles , when the individual modes were measured, such as non-contact (AC Mode Imaging) mode and quantitative mode (QITM Advanced Imaging). This was followed by the measurement of a PVA foils, the measurement was performed because they are xerogels and will be studied more in the future. They were made with polyvinyl alcohol and chitosan. The internal environment of the hydrogels has been modified by adjusting the pH (NaOH) or by changing the ionic strength (NaCl). Furthermore, the effect of freezing on the PVA foils was observed, it is one of the possible ways of preparation of physically linked hydrogels. This work also examines the effect of porosity on concentration of physically linked thermoreversible agarose gels.
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Study of UV-generated fluorescent zinc complexes by fluorescence spectroscopy
Havlíková, Martina ; Vaculovičová, Markéta (referee) ; Mravec, Filip (advisor)
This thesis focuses on the study of UV light-generated zinc complexes with cadmium and organic molecules SAM, SAH, CYS, HCYS and GSSG, specifically at 375 nm. Furthemore, the aim of the work is to characterize the precursors spectrally and temporally before and after irradiation in the transilluminator at 250 nm. Study of genesis these complexes was performed by FLIM. Thanks to this method, it was found that the formation of complexes occurs only with Zn:SAH, Zn:GSSG and Zn:Cd. The formation of complexes is influenced by the method of preparation. The spectral characteristic was performed on a fluorimeter where the increase in fluorescence intensity of the irradiated solution with the precursors was expected. These were turbid solutions where sedimentation of the particles was observed and the intensity of fluorescence was changed. In the Zn:SAM and Zn:CYS sample, the sedimentation increased in intensity, while in Zn:SAH and Zn:HCYS decreased. The Zn:Cd precursor solution was clear and there was no change in intensity. Zn:Cd showed the best spectral properties, while the Zn:SAM sample, whose excitation and emission maxima are very close to each other, appeared to be the worst. A sample with Zn:CYS and Zn:HCYS showed almost the same spectra and respective peak results. Based on lifetime characteristics by TCSPC, the sample with Zn:CYS, Zn:HCYS and Zn:GSSG, which showed 3 lifetimes, was best treated. Lifetime could not be unambiguously determined for SAM and SAH samples. Zn:Cd had 4 lifetimes
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Releasing of the solubilized substances from the phase-separated hydrogels
Havlíková, Martina ; Krouská, Jitka (referee) ; Mravec, Filip (advisor)
This bachelor thesis is focused on releasing of the solubilized substances from the phase separeted hydrogels. The aim of this work is to determine the solubilization capacity of these hydrogels. Preparation of them were based on interaction between hyaluronan and cationic surfactant carbethopendecinium bromide. Hydrogels were prepared by two possible ways, „wet“ and „dry way“. For solubilization experiments were used fluorescein and acridine orange as fluorescent probes. Primarily UV-VIS spectrophotometry was used to determine if these probes are released form hydrogel or not. Hydrogels were monitored after 24, 48 and 72 hours from their preparation. But this method proved to be inappropriate and inaccurate, because of cloudy supernatants over the hydrogels and also because of very low concentrations of fluorescent probe in this solution. For these reasons instead of this metod was used method, which is based on fluorescence. The instrument is called MicroTime 200. It´s very accurate method and results of this are considered like relevant. It was proved that fluorescein as a negatively charged molecule is released less than acridine orange.
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Atomic force microscopy of soft materials
Šudáková, Anna ; Havlíková, Martina (referee) ; Smilek, Jiří (advisor)
This bachelor thesis focuses on atomic force microscopy (AFM), mainly on studying the measurement procedure and optimalization of measurement conditions with future perspective on imaging hydrogels and measuring mechanical properties (such as adhesion or stiffness) of hydrogels and semi-rigid materials at the microlevel. Atomic force microscopy is gaining significant importance in research due to its versatility, when it can provide topographical image of conductive and non-conductive samples while measuring mechanical properties of the samples, such as adhesion, elasticity or stiffness. Advantage of AFM method is that it can provide mechanical properties of the samples not only on macro levels as we are used to, for example, from rheology, but also on local level. Optimalization was performed on standard sample, one micrometre polystyrene nanoparticles , when the individual modes were measured, such as non-contact (AC Mode Imaging) mode and quantitative mode (QITM Advanced Imaging). This was followed by the measurement of a PVA foils, the measurement was performed because they are xerogels and will be studied more in the future. They were made with polyvinyl alcohol and chitosan. The internal environment of the hydrogels has been modified by adjusting the pH (NaOH) or by changing the ionic strength (NaCl). Furthermore, the effect of freezing on the PVA foils was observed, it is one of the possible ways of preparation of physically linked hydrogels. This work also examines the effect of porosity on concentration of physically linked thermoreversible agarose gels.
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Study of UV-generated fluorescent zinc complexes by fluorescence spectroscopy
Havlíková, Martina ; Vaculovičová, Markéta (referee) ; Mravec, Filip (advisor)
This thesis focuses on the study of UV light-generated zinc complexes with cadmium and organic molecules SAM, SAH, CYS, HCYS and GSSG, specifically at 375 nm. Furthemore, the aim of the work is to characterize the precursors spectrally and temporally before and after irradiation in the transilluminator at 250 nm. Study of genesis these complexes was performed by FLIM. Thanks to this method, it was found that the formation of complexes occurs only with Zn:SAH, Zn:GSSG and Zn:Cd. The formation of complexes is influenced by the method of preparation. The spectral characteristic was performed on a fluorimeter where the increase in fluorescence intensity of the irradiated solution with the precursors was expected. These were turbid solutions where sedimentation of the particles was observed and the intensity of fluorescence was changed. In the Zn:SAM and Zn:CYS sample, the sedimentation increased in intensity, while in Zn:SAH and Zn:HCYS decreased. The Zn:Cd precursor solution was clear and there was no change in intensity. Zn:Cd showed the best spectral properties, while the Zn:SAM sample, whose excitation and emission maxima are very close to each other, appeared to be the worst. A sample with Zn:CYS and Zn:HCYS showed almost the same spectra and respective peak results. Based on lifetime characteristics by TCSPC, the sample with Zn:CYS, Zn:HCYS and Zn:GSSG, which showed 3 lifetimes, was best treated. Lifetime could not be unambiguously determined for SAM and SAH samples. Zn:Cd had 4 lifetimes
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Newly synthesized permanently positively charged monosubstituted beta-cyclodextrin derivatives as chiral selectors in capillary electrophoresis.
Havlíková, Martina ; Bosáková, Zuzana (advisor) ; Čabala, Radomír (referee)
This diploma thesis deals with the application of two permanently positively charged monosubstituted β-cyclodextrin derivatives (PEMEDA-β-CD, PEMPDA-β-CD) as chiral selectors in capillary electrophoresis. Use of PEMPDA-β-cyclodextrin in capillary electrophoresis has not been reported in literature. Properties of PEMEDA-β- cyclodextrin are already known, but its application for separation of amino acid enantiomers has not been published yet. Cyclodextrin derivatives were tested as additives in different buffers of different pH and with eventual addition of organic modifier. As suitable background electrolyte 15 mmol·l-1 borate buffer, pH = 9.5 without organic modifier was chosen. Furthermore the influence of chiral selector on separation and eventual enantioseparation of chosen analytes was evaluated. Addition of cyclodextrin derivatives in concentration range 0.0 - 5.0 was tested. Fourteen anionogenic analytes, including native amino acids, N- blocked amino acids and profens, were detected with UV-VIS detector at optimal wavelength 214, 254 or 280 nm. Both chiral selectors were suitable for enantioseparation of N-boc-D,L-tryptophan, which was baseline separated at concentration of selector as low as 0.5 mmol·l-1 . Tested amino acids blocked with terc-butoxycarbonyl and D,L-ketoprofen were partially...
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Releasing of the solubilized substances from the phase-separated hydrogels
Havlíková, Martina ; Krouská, Jitka (referee) ; Mravec, Filip (advisor)
This bachelor thesis is focused on releasing of the solubilized substances from the phase separeted hydrogels. The aim of this work is to determine the solubilization capacity of these hydrogels. Preparation of them were based on interaction between hyaluronan and cationic surfactant carbethopendecinium bromide. Hydrogels were prepared by two possible ways, „wet“ and „dry way“. For solubilization experiments were used fluorescein and acridine orange as fluorescent probes. Primarily UV-VIS spectrophotometry was used to determine if these probes are released form hydrogel or not. Hydrogels were monitored after 24, 48 and 72 hours from their preparation. But this method proved to be inappropriate and inaccurate, because of cloudy supernatants over the hydrogels and also because of very low concentrations of fluorescent probe in this solution. For these reasons instead of this metod was used method, which is based on fluorescence. The instrument is called MicroTime 200. It´s very accurate method and results of this are considered like relevant. It was proved that fluorescein as a negatively charged molecule is released less than acridine orange.
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