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Study of sphingosine, dihydrosphingosine and phytosphingosine in skin barrier models
Kubátová, Denisa ; Kováčik, Andrej (advisor) ; Svačinová, Petra (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Author: Denisa Kubátová Supervisor: PharmDr. Andrej Kováčik, Ph.D. Consultant: PharmDr. Lukáš Opálka, Ph.D. Title of diploma thesis: Study of sphingosine, dihydrosphingosine and phytosphingosine in skin barrier models The stratum corneum (SC), the uppermost layer of the skin, localized in the uppermost part of the epidermis, represents the skin barrier of the organism. SC is composed of corneocytes and an intercellular lipid matrix, which is formed by ceramides (Cer), free fatty acids (FFA), and cholesterol (Chol) in an equimolar ratio. Substances from the group of sphingolipids - Cer, are sphingoid bases (for example, sphingosine (S), dihydrosphingosine (dS), phytosphingosine (P)) acylated with a fatty acid (for example, lignoceric acid (LIG)). In the lipid matrix, the metabolic products of Cer (free sphingoid bases) are also present, but their role in SC barrier functions is not clear. Some studies show that Cer with different sphingoid bases, and increased presence of free sphingoid bases, can lead to a change in the permeability of the skin barrier. This work aimed to study the effect of permeability of sphingoid bases on the model membrane permeability. Nine types of membranes were prepared; they...
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Study of free sphingoid bases in skin barrier
Jarešová, Zuzana ; Kováčik, Andrej (advisor) ; Vraníková, Barbora (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Author: Zuzana Jarešová Supervisor: PharmDr. Andrej Kováčik, Ph.D. Consultant: PharmDr. Lukáš Opálka, Ph.D. Title of diploma thesis: STUDY OF FREE SPHINGOID BASES IN SKIN BARRIER The skin barrier, localized in the stratum corneum (SC), consists of corneocytes and an intercellular matrix formed from three types of lipids - ceramides, free fatty acids, and cholesterol, represented in an equimolar ratio. The overall arrangement of lipids is organized and highly specialized. Ceramides are structurally formed from the fatty acid acyl attached to a sphingoid base. In minor but not insignificant amounts, free sphingoid bases can also be found in the skin barrier. Several studies show that there is an increased concentration of free sphingoid bases in skin barrier disorders, such as atopic dermatitis. Although it is assumed that the presence of free sphingoid bases affects the skin barrier, it is not elucidated the way of their participation till today. The lack of studies or their diverse results leads us to the main goal of this thesis - to clarify how free sphingoid bases influence the skin barrier. In this work, the model membranes were prepared by the isolation of human SC ex vivo. Sphingosine (S),...
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In vitro models of skin barrier
Šimek, Matěj ; Zbytovská, Jarmila (advisor) ; Pullmannová, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Candidate: Matěj Šimek Consultant: doc. Dr. rer. nat. Mgr. Jarmila Zbytovská Title of thesis: In vitro models of skin barrier The aim of this work is to summarize information about various types of skin models which are used for testing of permeability, toxicity, irritability and other aspects of drugs, through professional, verified and reviewed literature. These characteristics are necessary to know in order to grant optimal safety, effectiveness nad quality of transdermally administered drugs. Transdermal administration of drugs has got lots of benefits in contrast with classic peroral administration. An administration of drugs through this way is quite simple and it can be interrupted quite easily. We can also easily change a place of administration in which a drug is released and the risk of overdosing is very low. Furthermore, transdermal administration makes possible to maintain constant plasmatic concentration of drug in a blood stream and also to prolong the duration of effect of drugs with small halftime thanks to constant releasing of drug. And primarily, transdermally administered drug normally avoids the "first-pass" effect of liver, so the dose of drug can be lowered. The risk of drug...
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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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