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...

Charles University Faculty of Pharmacy in Hradec Králové Department of pharmeceutical technology Candidate: Oksana Yanok Supervisor: Pharm.Dr. Andrej Kováčik, Ph.D. Title of Diploma Thesis: The effects of glucosyl sphingosine on barrier function of skin and complex skin model. The skin barrier, which provides protection from water loss and harmful environmental influences is located in the stratum corneum. The dominant group of lipids within the stratum corneum are ceramides (Cer), which also have the most important role in ensuring the barrier properties of the skin. The enzymes sphingomyelin deacylase and glucosylceramide deacylase hydrolyze the amide bond of Cer precursors, which leads to highly polar metabolites, called lysolipids. The increased activity of these enzymes is considered to be one of the major factors leading to the development of a number of skin diseases characterized by a skin barrier disorder (for example atopic dermatitis). In this study we prepared model membranes mimicking a healthy skin barrier as an equimolar mixtures of human Cer, cholesterol, free fatty acids with the addition of 5 % cholesterol sulfate. We also prepared models in which the amount of Cer was gradually reduced and replaced by the hydrophilic lysolipid glucosylsphingosine. The permeability was measured...

1. Abstract (EN) This thesis is collection of work focused mainly on the understanding of mast cell activation and its regulation by Orm1-like (ORMDL) proteins. ORMDL family is a group of endoplasmic reticulum (ER) membrane resident proteins that are highly conserved amongst mammalian species. ORMDL proteins can be found in diverse range of organisms from plants through fungi to animals. ORMDL proteins were first discovered in yeasts and the interest in these proteins skyrocketed after the discovery that ORMDL3 is associated with childhood onset asthma in genome wide association studies. Following research connected ORMDL3 also with allergic inflammation and inflammatory bowel disease. Since mast cells are mainly known for their role in allergy and allergen induced inflammation, we decided to investigate the role of ORMDL proteins in regulation of mast cell activation and signaling. In our first study we focused on the role of ORMDL3 in mast cell activation via the high affinity IgE receptor 1 (FcεRI). We prepared bone marrow-derived mast cells with decreased (ORMDL3-KD) or increased (ORMDL3-OE) ORMDL3 expression. We showed that ORMDL3 is a negative regulator of mast cell activation events like degranulation, cytokine release and migration, without any effect on calcium mobilization. ORMDL3 was previously...

Adaptation to chronic hypoxia provides myocardial protection against ischemia - reperfusion injury (IR). Cardioprotective effect of adaptation depends on the degree and duration of hypoxic exposure and daily regime of adaptation. Certain protective regimes of adaptations to hypoxia have been reported to activate proapoptotic signaling pathways and bioactive sphingolipids were recently shown to play important role in the regulation of apoptosis in the heart. We aimed to determine the mRNA level of selected genes related to apoptotic pathways and to sphingolipid metabolism in two models of hypoxic adaptation, continous normobaric hypoxia (CNH 10% O2) with different exposures (4h, 48h, 120h, 21days) and intermitent hypobaric hypoxia (IHH 7000 m, 8h/day). Both ventricles, LV and RV, were analysed after adaptation to CNH and only LV was analysed after IHH adaptation. Our results show that both types of adaptation increased mRNA of proapoptotic genes, CNH mainly in RV and IHH in LV. Furthermore, increased expressions of proapoptotic genes were accompanied by the increase of expression of enzymes producing predominantly protective kinds of sphingolipids. The exact role of apoptosis and sphingolipid signaling molecules in endogenous myocardial protection requires further research. Key words: Apoptosis,...