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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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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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Characterization of ligand binding to M1 muscarinic acetylcholine receptor using fluorescence anisotropy method
Danková, Hana ; Vokřál, Ivan (advisor) ; Červený, Lukáš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Hana Danková Supervisors: Prof. Ago Rinken, PhD. MSc. Tõnis Laasfeld PharmDr. Ivan Vokřál, PhD. Title of diploma thesis: Characetrization of ligand binding to M1 muscarinic receptor using fluorescence anisotropy method Muscarinic acetylcholine receptors (mAChRs), members of the superfamily of G-protein coupled receptors (GPCRs), regulate vital physiological processes and are important targets in drug research. Five different subtypes (M1 - M5) have been identified. M1 mAChR is mainly distributed in the central nervous system and is linked to pathophysiology of neurodegenerative diseases. In recent years, fluorescent methods have been frequently used in studies of ligand binding to receptors. The fluorescence anisotropy (FA) is a homogenous assay to characterize ligand binding to receptors. In this work, we have evaluated the FA method with fluorescent ligand MK342 binding to M1 mAChRs expressed on budded baculovirus (BBV) particles. The fluorescence ligand was binding with the high affinity (4,4 nM) to M1 receptor in constructed BBV preparation. The apparent binding affinities (pKi) of eleven classical and three bitopic muscarinic ligands were screened and compared to previously published...
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Characterization of ligand binding to M1 muscarinic acetylcholine receptor using fluorescence anisotropy method
Danková, Hana ; Vokřál, Ivan (advisor) ; Červený, Lukáš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Hana Danková Supervisors: Prof. Ago Rinken, PhD. MSc. Tõnis Laasfeld PharmDr. Ivan Vokřál, PhD. Title of diploma thesis: Characetrization of ligand binding to M1 muscarinic receptor using fluorescence anisotropy method Muscarinic acetylcholine receptors (mAChRs), members of the superfamily of G-protein coupled receptors (GPCRs), regulate vital physiological processes and are important targets in drug research. Five different subtypes (M1 - M5) have been identified. M1 mAChR is mainly distributed in the central nervous system and is linked to pathophysiology of neurodegenerative diseases. In recent years, fluorescent methods have been frequently used in studies of ligand binding to receptors. The fluorescence anisotropy (FA) is a homogenous assay to characterize ligand binding to receptors. In this work, we have evaluated the FA method with fluorescent ligand MK342 binding to M1 mAChRs expressed on budded baculovirus (BBV) particles. The fluorescence ligand was binding with the high affinity (4,4 nM) to M1 receptor in constructed BBV preparation. The apparent binding affinities (pKi) of eleven classical and three bitopic muscarinic ligands were screened and compared to previously published...
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