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Study of cholesterol depletion in human skin barrier
Jančálková, Pavla ; Vávrová, Kateřina (advisor) ; Nováková, Veronika (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Organic and Bioorganic Chemistry Pavla Audrlická Supervisor: prof. PharmDr. Kateřina Vávrová, Ph.D. Title of diploma thesis: Study of cholesterol depletion in human skin barrier Barrier function of human skin barrier dwells in intercellular lipid membranes of the uppermost skin layer, the stratum corneum (SC), composed of equimolar mixture of ceramides (Cer), free fatty acids (FFA) and cholesterol (Chol).1 Chol is required for proper lipid organization of SC, however, it stays unclear, why is it present in an amount so high that it separates from other lipids.2 Experiments using synthetic model membranes with decreased Chol content suggested that molar ratio of Cer:FFA:Chol 1:1:0,4 is sufficient for lipid barrier formation and its complex functionality.3 The aim of this work was to manipulate Chol content directly in human SC and to study the effects of decreased Chol content on the SC permeability and microstructure. Ex vivo SC obtained from healthy donors was extracted by methyl-β-cyclodextrin (MCD) to reduce natural Chol content. The extracted SC did not show significant changes in Cer or FFA whilst the amount of Chol was lowered to 78 %. SC barrier properties were evaluated by measurements of transepidermal water loss...
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Synthesis and evaluation of human 6-hydroxyceramides
Majcher, Adam ; Opálka, Lukáš (advisor) ; Krátký, Martin (referee)
Title of the Master thesis: Synthesis and evaluation of human 6-hydroxyceramides Candidate: Adam Majcher Supervisor: PharmDr. Lukáš Opálka, Ph.D. Consultant: PharmDr. Andrej Kováčik, Ph.D. Charles University, Faculty of Pharmacy in Hradec Králové Skin Barrier Research Group, Department of Organic and Bioorganic Chemistry Ceramides (Cer), the members of sphingolipid family, occur in all human cells and play an important role in cell signaling. In high concentrations, Cer can also be found in the uppermost layer of epidermis called stratum corneum, along with free fatty acids and cholesterol (in equimolar ratio), where they form the intercellular multi-lamellar lipid matrix. The key function of stratum corneum is to ensure a permeability barrier, thus, to provide water and electrolyte homeostasis, and to prevent entry of harmful substances into the organism. Cer are composed of a sphingoid base and an acyl part derived from a long-chain fatty acid. Cer based on 6-hydroxysphingosine (H) are amongst the most unusual sphingolipids. In contrast to sphingosine-based Cer, 6-hydroxysphingosine-based Cer (H-Cer) are unique for the epidermis and, in addition, H-Cer are not typical for all mammals. Moreover, the function and biosynthesis of H-Cer in the skin is still not completely understood. Several...
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Study of cholesterol depletion in human skin barrier
Audrlická, Pavla ; Vávrová, Kateřina (advisor) ; Nováková, Veronika (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Organic and Bioorganic Chemistry Pavla Audrlická Supervisor: prof. PharmDr. Kateřina Vávrová, Ph.D. Title of diploma thesis: Study of cholesterol depletion in human skin barrier Barrier function of human skin barrier dwells in intercellular lipid membranes of the uppermost skin layer, the stratum corneum (SC), composed of equimolar mixture of ceramides (Cer), free fatty acids (FFA) and cholesterol (Chol).1 Chol is required for proper lipid organization of SC, however, it stays unclear, why is it present in an amount so high that it separates from other lipids.2 Experiments using synthetic model membranes with decreased Chol content suggested that molar ratio of Cer:FFA:Chol 1:1:0,4 is sufficient for lipid barrier formation and its complex functionality.3 The aim of this work was to manipulate Chol content directly in human SC and to study the effects of decreased Chol content on the SC permeability and microstructure. Ex vivo SC obtained from healthy donors was extracted by methyl-β-cyclodextrin (MCD) to reduce natural Chol content. The extracted SC did not show significant changes in Cer or FFA whilst the amount of Chol was lowered to 78 %. SC barrier properties were evaluated by measurements of transepidermal water loss...
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Evaluation of preparation of monolayer lipid skin barrier models
Růžičková, Karolína ; Švecová, Barbora (advisor) ; Vraníková, Barbora (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Author: Karolína Růžičková Supervisor: PharmDr. Barbora Švecová, Ph.D. Consultant: Mgr. Anna Nováčková Title of thesis: Evaluation of preparation of monolayer lipid skin barrier models Skin, the protective barrier of human body, consists of several layers. The uppermost one is the stratum corneum, part of epidermis, whose extracellular matrix is composed mainly of ceramides, cholesterol and free fatty acids. The composition and arrangement of skin lipids are essential for the proper skin barrier function. Various multilayer and monolayer models are used to study skin lipids at the molecular level. Some of the evaluation methods are Langmuir monolayers at the air interface. In this work I dealt with the behavior of monolayer lipid models at four different pH values of the liquid subphase. Lipids isolated from human skin, lipid mixture prepared from the individual components, and a mixture of fatty acids were compared as well. Langmuir isotherms and the Brewster angle microscopy at different compression rates were used for this purpose. The results showed that pH of the subphase has no major effect on lipids arrangement. Lipids were most likely to form a tight monolayer at neutral pH 7,0, at a...
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Evaluation of preparation of monolayer lipid skin barrier models
Růžičková, Karolína ; Švecová, Barbora (advisor) ; Vraníková, Barbora (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Author: Karolína Růžičková Supervisor: PharmDr. Barbora Švecová, Ph.D. Consultant: Mgr. Anna Nováčková Title of thesis: Evaluation of preparation of monolayer lipid skin barrier models Skin, the protective barrier of human body, consists of several layers. The uppermost one is the stratum corneum, part of epidermis, whose extracellular matrix is composed mainly of ceramides, cholesterol and free fatty acids. The composition and arrangement of skin lipids are essential for the proper skin barrier function. Various multilayer and monolayer models are used to study skin lipids at the molecular level. Some of the evaluation methods are Langmuir monolayers at the air interface. In this work I dealt with the behavior of monolayer lipid models at four different pH values of the liquid subphase. Lipids isolated from human skin, lipid mixture prepared from the individual components, and a mixture of fatty acids were compared as well. Langmuir isotherms and the Brewster angle microscopy at different compression rates were used for this purpose. The results showed that pH of the subphase has no major effect on lipids arrangement. Lipids were most likely to form a tight monolayer at neutral pH 7,0, at a...
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Influence of cholesterol concentration on permeability and microstructure of model skin barrier lipid membranes
Červená, Martina ; Vávrová, Kateřina (advisor) ; Zbytovská, Jarmila (referee)
Cholesterol is a substance of a steroidal nature that has a number of functions in the human body. One of them is also an irreplaceable role in the proper functioning of the skin barrier. Cholesterol is an integral part of the lipid matrix, together with ceramides and free fatty acids in an equimolar ratio (1: 1: 1), and 5% cholesterol sulfate, which fills the intercellular space between stratum corneum cells and is responsible for the barrier properties of the skin. Cholesterol is therefore necessary for epidermal homeostasis, but its role in SC permeability is unknown. The aim of this work was to study the influence of cholesterol concentration on the permeability and microstructure of model lipid membranes of the skin barrier. Eight sets of model membranes with decreasing cholesterol concentrations (100%, 80%, 70%, 60%, 40%, 20%, 0%) and cholesterol alone were studied for which permeability and microstructure were monitored.The study of permeability of membranes was carried out in the Franz diffusion cells by measuring four permeation parameters: water loss, electrical impedance measurement, and the cumulative amount of two model drugs (theophylline and indomethacin). The microstructure of these model membranes was verified by X-ray powder diffraction. X-ray powder diffraction measurements...
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Optimization of the synthesis of 32-hydroxydotriacontanoic acid
Sommerová, Veronika ; Opálka, Lukáš (advisor) ; Roh, Jaroslav (referee)
1 Abstract Acylceramides belong to the subgroup of ultralong chain ceramides. They are essential components of the extracellular lipid matrix of stratum corneum, where they play a crucial role in proper function of skin barrier (they help preventing the excessive water loss and penetration of exogenous substances and pathogens to the organism). The 32-hydroxydotriacontanoic acid is one of the fatty acids forming the backbone of all the acylceramides. In the molecule of acylceramide, the carboxyl group of this acid is bound to a primary amino group of the sphingoid base and the ω-hydroxy group is esterified with linoleic acid. In the stratum corneum, 32-hydroxydotriacontanoic acid may remain as a part of free acylceramides or it can be covalently linked to the surface of corneocytes and form the "first lamela", which then serves as a basis for the orientation of other lipids in the matrix. The recent literature describes the synthesis of 32-hydroxydotriacontanoic acid but only with relatively small overall yields. The most problematic part of the synthesis seems to be the connection of two shorter fragments leading to the ultralong chain. The main aim of this research project was to optimalise the reaction conditions to increase the yield of formation of the utralong acid, focusing on the most complicated...
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Study of penetration of selected substances from elderberry extracts through biological membrane
Škubalová, Zuzana ; Němcová, Andrea (referee) ; Vespalcová, Milena (advisor)
This Diploma thesis focuses on the study of penetration of selected phenolic substances contained in extract from black elder (Sambucus nigra) via modelling systems represented by two types of synthetic membranes and a real system represented by a biological membrane. As a biological membrane, we have chosen skin from pig´s earlobe. Experiments of transdermal absorption and absorption through synthetic membranes were conducted through vertical (Franz´s) cells. Selected substances contained in the extract from black elder were rutin and chlorogenic acid. The main focus of this work was to determine how readily, or if at all, these two selected phenolic substances penetrate the skin and selected synthetic membranes. The percentage of absorbed amount of the substance was determined by HPLC analysis.
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Experimental study and modelling of the transdermal penetration of active species from gels
Palanová, Veronika ; Opatřilová, Radka (referee) ; Sedláček, Petr (advisor)
This diploma thesis deals with design and experimental study of transdermal transport of pharmaceutically active agents from gel matrices, which contain humic substance in its structure. A model absorption membrane was represented by the skin of pig´s earlobes. The study of the release of active substances and Lignohumte was performed due to the vertical diffusion cells. The amount of released humic substance was characterized by UV-VIS method and the amount of released active agent from gel matrix was determined by HPLC-DAD. The most interesting finding of this diploma thesis was that Lignohumate enhances transdermal transport of active agents and supports their release from gel samples to the particular environment.
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Laboratory Measurements of Skin Conductance
Slouka, Petr ; Harabiš, Vratislav (referee) ; Sekora, Jiří (advisor)
This thesis contains an introduction to anatomy and physiology of the skin highlighting its barrier function. The barrier function of the skin enables it to separate internal organism from external environment. thermore, the electrical properties of the skin are described and possibilities of skin conductance measurement are discussed. The thesis presents a design of a device for the skin conductance measurement as the method of evaluation for barrier function. Alternating current is used for measurement during iontophoresis for evaluation of skin conductance. Circuit design and circuit board are included with a list of parts. The designed device was realized and in the end it was tested and the results were discussed.
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