The complexity of cell membranes is far from being only a simple assembly of lipids and proteins separating cells from the surrounding environment. Each of the thousands of different membrane components performs its specific role in cellular functions, since a multitude of biological processes is mediated by membranes. The understanding of the molecular basis of these processes is one of the important aims of current biological research. Our research employing single- molecule fluorescence methods (e.g. FCS, FCCS, FLIM-FRET) has made a contribution to the knowledge of membrane lateral organization or mechanism of membrane fusion. Furthermore, we revealed the mechanism of membrane activity of a small natural compound. As native cell membranes are very complex structures, we performed the experiments on simplified model lipid membranes that allow studying lipid-lipid or lipid-protein interactions at the molecular level in a controlled way. The first part of this thesis deals with the mode of action of a membrane active secondary metabolite didehydroroflamycoin (DDHR). We demonstrated that DDHR is a pore-forming agent and that this activity is influenced by the presence of cholesterol. Direct visualization of intrinsic fluorescence of DDHR revealed its preferential partitioning into membrane areas...

The complexity of cell membranes is far from being only a simple assembly of lipids and proteins separating cells from the surrounding environment. Each of the thousands of different membrane components performs its specific role in cellular functions, since a multitude of biological processes is mediated by membranes. The understanding of the molecular basis of these processes is one of the important aims of current biological research. Our research employing single- molecule fluorescence methods (e.g. FCS, FCCS, FLIM-FRET) has made a contribution to the knowledge of membrane lateral organization or mechanism of membrane fusion. Furthermore, we revealed the mechanism of membrane activity of a small natural compound. As native cell membranes are very complex structures, we performed the experiments on simplified model lipid membranes that allow studying lipid-lipid or lipid-protein interactions at the molecular level in a controlled way. The first part of this thesis deals with the mode of action of a membrane active secondary metabolite didehydroroflamycoin (DDHR). We demonstrated that DDHR is a pore-forming agent and that this activity is influenced by the presence of cholesterol. Direct visualization of intrinsic fluorescence of DDHR revealed its preferential partitioning into membrane areas...

Euglenophyta are monophyletic group of euglenids defined by presence of green, three membrane- bound plastid which has been aquired via secondary endosymbiosis with chlorophyte alga. Mechanism of transport of nuclear-encoded proteins into this plastid is not yet completely understood. It was observed that the proteins are transported to the outermost plastid membrane in vesicles passing through ER and Golgi, but the mechanism of their recognition and fusion with the target membrane remains unclear. Translocation system of inner two membranes is still completely unknown, regarding the situation in other plastids, it has been proposed that homologues of TOC and TIC complexes are present. In this work we analyzed sequence data from proteome of isolated plastid membranes of model organism Euglena gracilis and transcriptome of E. gracilis and its distant relative Eutreptiella gymnastica. We studied whether they contain proteins potentially involved in transport and homologues of proteins of transport systems known from plastids in other organisms (TOC/TIC, ERAD-like transport, SNARE). However, all our results are negative. It is hard to determine whether these findings indicate the possible absence of TOC and TIC complexes in euglenid plastid, or rather the insufficiency of our data. Powered by TCPDF (www.tcpdf.org)

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...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis University of Porto Faculty of Pharmacy Department of Chemical Sciences Department of Biological Sciences Candidate: Kristína Morgošová Supervisor: assoc. prof. PharmDr. Radim Kučera, Ph.D. Consultants: prof. Maria da Conceição Branco da Silva, Ph.D. prof. Maria Alice dos Santos Silva Gomes Martins, Ph.D. Susana Maria Santos Rocha, Ph.D. Title of diploma thesis: Development and characterization of modified polysulfone hemodialysis membranes by means of immobilized neutrophil elastase inhibitors. Chronic kidney disease (CKD) is a major health and financial burden, mainly because of the costly renal replacement therapy and treatment associated. The last stage, end-stage renal disease, is associated with high morbidity and mortality rate, generally due to cardiovascular complications. Chronic inflammation is frequently present in CKD patients, which is enhanced by the long term intra-dialytic recurrent contact between blood and hemodialysis (HD) membrane and further contributes to development of atherosclerosis. Contact with the artificial material of HD membranes leads to oxidative stress and neutrophil activation with release of neutrophil serine proteases such as human...

Surfactin is an antibiotic produced by several strains of B. subtilis. Its broad range of biological activities is interesting from perspective of medicine, food industry and bioremediation and is based on its surface-active properties and interaction with biological membranes. The latter means mainly forming ion channels, conductive pores and with increasing concentration eventually disrupting membrane structure in detergent-like manner. Mechanism of resistance of producing strain against its own toxic product is not yet fully understood. This work shows that it could be based on surfactin target modification - which means altering membrane lipid composition. We were able to recognize surfactin-formed ion channels or pores with a broad range of conductivities spanning from 2 pS to 2 nS using BLM method. Liposome leakage assay with carboxyfluorescein revealed few distinct mechanisms of lysis, differing in amplitude, rate of lysis and cooperativity. Increased content of anionic lipids with conical shape, namely cardiolipin and phosphatidic acid led to substantial increased membrane resistance to surfactin-induced permeabilization. Key words: membrane, surfactin, Bacillus subtilis, cardiolipin, black lipid membranes, liposomes

Assessment of semen quality (sperm viability) is an essential for the efficient honeybee semen storage. The motility of spermatozoa is not the ultimate parameter of sperm viability. Instead, the viability of spermatozoa is assessed by testing their structural or functional plasma membrane integrity. These assays are based primarily on the technique of microscopy evaluation of either fluorescent DNA dyes or hypo-osmotic "tail-coiling" reaction of spermatozoa. However, the technique of microscopy evaluation is time-consuming, subjective and often is not accurate. On the other hand, the technique of flow cytometry is rapid, objective and precise. Nowadays, this technique is affordable for many institutions, as the price of "benchtop" flow cytometer is relatively low. To best of our knowledge, there is currently no flow cytometry assay available for evaluation of functional integrity of honeybee spermatozoa. Here we report the establishing of novel flow cytometry assay for simultaneous evaluation of structural and functional plasma membrane integrity. DRAQ7, the new fluorescent DNA dye was used to test the structural integrity of plasma membranes. The hypo-osmotic "tail-coiling" reaction was used for flow cytometric assessment of functional integrity of spermatozoa plasma membranes. The combination of both is a valuable tool for rapid and precise evaluation of sperm viability in honey bees.

Presented work focused on researching cold resistance in insects (Pyrrhocoris apterus), especially in qualitative and quantitative composition and seasonal changes in the content of tocopherols and sterols in membranes. Collection of samples, tissue extraction, separation, sample preparation and the final analysis was included. There were detected three different sterols (animal cholesterol, plant sterols: campesterol and sitosterol). Also were detected ?-tocopherol and ?-tocopherol. Volume of cholesterol was in the range of 0.2 to 0.8 mol%, for phytosterols was the range from 1.2 to 15.2 mol%, and for tocopherols was the range from 0.1 to 31.3 mol%. Seasonal fluctuations of sterols and tocopherols were evaluated with a minimum in summer and maximum in winter.

This diploma thesis deals with the general characteristics of membrane processes and separations. It includes a chapter on the history of development of the technology industry using membranes as a medium for separating chemicals based on their specific properties and also the importance of such processes at present. This work then introduces the basic principles of mass transport by membranes and describes various types of membranes and materials used for their production. It also deals with electromembrane separations, including electrodialysis. The practical part focuses on experimental separation of solutions of four sodium salts by means of electrodialysis. Based on the electrical current and conductivity measurements of these solutions in time during membrane separation are evaluated process parameters such as efficiency, energy consumption, transferred electrical charge, and mass of the sodium salt.

This diploma thesis deals with the preparation of the graphene membranes for depo-sition of gallium atoms by the molecular beam epitaxy. In the first part properties ofgraphene and methods of its production are described. Second part focuses on the gra-phene membranes, their specific properties, applications and methods of production. Thirdpart describes growth theory of the thin films. Practical part is focused on preparationof graphene membranes, which consists of covering the holes in the silicon substrate bygraphene layer. For that mechanical exfoliated and chemical vapor deposited graphenewere used. Subsequently, gallium atoms were deposited on these membranes by molecularbeam epitaxy and in situ observed by scanning electron microscopy.