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Hyaluronane interactions with hydrophobic solutes
Slezáková, Dagmar ; Knotková,, Kateřina (referee) ; Pekař, Miloslav (advisor)
The diploma thesis is based on the study of hydrophobic interactions of the native hyaluronan with selected solutes. On the basis of a literature search were chosen fluorescent probes and fluorescing biologically active substances, which are useful for investigation of colloids as 6-(p-toluidino)-2-naphthalenesulfonic acid (polarity probe), lipophilic vitamin (±)-alpha-tocopherol, pyrene (polarity probe) and finally hydrophilic vitamin riboflavin. In the experimental part of this thesis was studied the influence of solvents with different polarities, or more precisely dielectric constant, on the emission spectra, as well. There were investigated interactions of native hyaluronan with TNS and then interactions, which were influenced by the ionic strength. Such influenced interactions were not observed, that was probably due to the strong solvation´s wrapping of the hyaluronan. Interactions were observed after the process of lyophilisation followed-up by the rehydratation of the samples. For the next study of interactions the riboflavin was chosen and was investigated the REES effect in the native hyaluronan in different concentrations of its different molecular weights. In this case were not observed any shifts in the emission maximum with the excitation wavelenght shift and that is why the interactions of hyaluronan with riboflavin were not demonstrated in the field of chosen concentrations. By using another probe alpha-tocopherol was investigated the associative behaviour of hyaluronan and moreover was observed anisotropy of alpha-tocopherol in different concentrations of different molecular weights of native hyaluronan. The anisotropy reached high values in contrast to the reference solute that was the mixture of glycerol and ethanol. The anisotropy depended more on the molecular weight than on the concentration of hyaluronan. Interactions of hyaluronan were also studied by using the polarity probe pyrene in different concentrations of different molecular weights of the hyaluronan. The pyrene 1:3 ratio did not show the concentration dependence within the chosen concentrations except for the molecular weight 253.9 kg mol–1. Both probes alpha-tocopherol and pyrene were performed by the process of lyophilisation followed-up by the rehydratation, which improved interactions of these probes with hyaluronan.
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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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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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Hyaluronane interactions with hydrophobic solutes
Slezáková, Dagmar ; Knotková,, Kateřina (referee) ; Pekař, Miloslav (advisor)
The diploma thesis is based on the study of hydrophobic interactions of the native hyaluronan with selected solutes. On the basis of a literature search were chosen fluorescent probes and fluorescing biologically active substances, which are useful for investigation of colloids as 6-(p-toluidino)-2-naphthalenesulfonic acid (polarity probe), lipophilic vitamin (±)-alpha-tocopherol, pyrene (polarity probe) and finally hydrophilic vitamin riboflavin. In the experimental part of this thesis was studied the influence of solvents with different polarities, or more precisely dielectric constant, on the emission spectra, as well. There were investigated interactions of native hyaluronan with TNS and then interactions, which were influenced by the ionic strength. Such influenced interactions were not observed, that was probably due to the strong solvation´s wrapping of the hyaluronan. Interactions were observed after the process of lyophilisation followed-up by the rehydratation of the samples. For the next study of interactions the riboflavin was chosen and was investigated the REES effect in the native hyaluronan in different concentrations of its different molecular weights. In this case were not observed any shifts in the emission maximum with the excitation wavelenght shift and that is why the interactions of hyaluronan with riboflavin were not demonstrated in the field of chosen concentrations. By using another probe alpha-tocopherol was investigated the associative behaviour of hyaluronan and moreover was observed anisotropy of alpha-tocopherol in different concentrations of different molecular weights of native hyaluronan. The anisotropy reached high values in contrast to the reference solute that was the mixture of glycerol and ethanol. The anisotropy depended more on the molecular weight than on the concentration of hyaluronan. Interactions of hyaluronan were also studied by using the polarity probe pyrene in different concentrations of different molecular weights of the hyaluronan. The pyrene 1:3 ratio did not show the concentration dependence within the chosen concentrations except for the molecular weight 253.9 kg mol–1. Both probes alpha-tocopherol and pyrene were performed by the process of lyophilisation followed-up by the rehydratation, which improved interactions of these probes with hyaluronan.
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