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Lipid Membranes at the Nanoscale: Single-Molecule Fluorescence Approach
Koukalová, Alena ; Černý, Jan (advisor) ; Malínský, Jan (referee) ; Benda, Aleš (referee)
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...
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Novel materials for membrane gas separation
Giel, Verena ; Pientka, Zbyněk (advisor) ; Izák, Pavel (referee) ; Vopička, Ondřej (referee)
The implementation of polymer membranes in gas separation applications has been investigated to a great extent. Nevertheless, only a few types of polymers are used in commercial applications, disclosing the need for new materials with superior membrane performance to make membrane processes a more competitive technology over the conventional ones. Based on this context, this work focuses on the development of new polymeric membranes. Polyaniline (PANI), a multifaceted polymer that can change its structural properties upon various modification procedures, was chosen as membrane material. PANI membranes possess attractive O2/N2 selectivities, wherefore it is an interesting candidate for the use in gas separation applications, such as generation of oxygen-enriched air or inert gas generation. However, membranes made from neat PANI are suffering from brittleness and thus create leak paths through the membrane. Therefore PANI was blended with polybenzimidazole (PBI), a temperature stable polymer with good film-forming properties facilitating the preparation of thin, stable polymer films. Furthermore, several techniques were investigated including acid-doping, thermal treatment, and addition of titanate nanotubes (TiNTs) to enhance the separation properties. The materials that have been prepared are: 1)...
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Modeling interactions of proteins with ions and membranes
Kadlec, Jan ; Jungwirth, Pavel (advisor) ; Kolafa, Jiří (referee)
The protein recoverin, localized in the eye in the rod outer segment of the retina, is a neuronal calcium sensor involved in vision adaptation. Recoverin reversibly associates with cellular membranes via its calcium-activated myristoyl switch. This reversible interaction is vastly dependent on the concentration of calcium ions in the cytosol and on conformation of recoverin. By using methods of molecular dynamics simulations and free energy calculations this work presents a detailed analysis of the energetics of myristoyl insertion into a lipid bilayer and interactions of non-myristoylated recoverin with the membrane. These results are in a perfect agreement with experimental data. The thesis provides a piece of puzzle to the so far unexplored mechanism of myristamide insertion into the membrane and also to recoverin conformational change. It gives an important insight into binding of recoverin to a membrane, which has a significant biological role.
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Membrane separation
Babíková, Barbora ; Másilko, Jiří (referee) ; Svěrák, Tomáš (advisor)
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.
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Biocompatible Amphiphilic Compounds and their Interactions with Polymers
Burdíková, Jana ; Sedlařík, Vladimír (referee) ; Bakoš, Dušan (referee) ; Pekař, Miloslav (advisor)
This work is focused on the study of interaction between hyaluronan and high-biocompatible amphiphilic molecules. Using fluorescent probe method, screening of the interaction of cationic lipid 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), mixture of this cationic lipid with zwitterionic lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphochloline (DPPC), with hyaluronan, both native and hydrophobically modified was carried out. Results showed the self-aggregation of DPPC and DPTAP independently on lipids ratio in the mixture and the interaction of DPTAP and DPPC/DPTAP aggregates with hyaluronan at specific ratio of DPTAP and hyaluronan concentration. Physical properties of formed membranes and the influence of cholesterol were also investigated at different DPPC and DPTAP concentration ratio. Last but not least, the non-ionic surfactant-DPPC systems were studied, namely, the size of the formed aggregates, the thermodynamics of solubilisation and the interaction with native hyaluronan.
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Antimicrobial peptides isolated from the venom of hymenopterous insect
Monincová, Lenka ; Čeřovský, Václav (advisor) ; Macek, Tomáš (referee) ; Fusek, Martin (referee)
Rapid development of bacterial resistance and multiresitance to conventional antibiotics has resulted in an intensive search for alternative antimicrobial agents. Antimicrobial peptides (AMPs) belong to promising anti-infective candidates since they do not development bacterial resistance. They kill microbes by disturbing or permeabilizing the cytoplasmic membrane, or may target putative key intracellular compartments. Their advantages include fast action and selectivity between prokaryotic and eukaryotic cells. We have isolated several novel AMPs from the venom of wild bees: halictines (HAL-1 and HAL-2) from Halictus sexcinctus, lasiocepsin (Las) from Lasioglossum laticeps and macropin (MAC-1) from Macropis fulvipes. They are active against Gram-positive and Gram- negative bacteria and against yeast Candida albicans. While halictines and macropin have moderate hemolytic activity, Las shows no hemolytic activity. A novel AMP was isolated also from the mucus of Xiphydria camelus. This AMP belongs to the category of insect defensins. It contains 55 amino acid residues, three disulphide bridges and its C-terminus is amidated. CD and NMR studies of HAL-1, HAL-2 and MAC-1 revealed propensity to form amphipathic α-helical structure in the presence of sodium dodecyl sulfate or trifluoroethanol. For the...
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Membrane proteome of euglenid plastid
Vanclová, Anna ; Hampl, Vladimír (advisor) ; Füssy, Zoltán (referee)
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)
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Transport of charged and neutral particles across the model biomembranes
Parisová, Martina ; Stiborová, Marie (advisor) ; Moserová, Michaela (referee)
This work was focused on the preparation of model stabilized phospholipid membranes formed on porous polycarbonate carrier. 1,2-dipalmitoyl-sn-glycero-3-phosphocholin was used for their formation in hydrophilic pores of polycarbonate carrier. For characterization of the formation of phospholipid layers, their changes and a study of transport processes, electrochemical impedance spectroscopy and voltammetry were used. Transport of cadmium and copper ions was studied in the presence and in the absence of ionophore calcimycin which was incorporated into the formed of phospholipid membrane. Because these ions are often bound in complexes with various substances, such as low molecular weight organic acids (LMWOAs), this work was also focused on the transport of copper and cadmium ions across the model phospholipid membranes in the presence of malic acid, citric acid and oxalic acid at different pH. Besides the use of ionophore, some pilot experiments were performed to realize the transfer of copper ions using two peptides, nisin and transportan 10. Formation of phospholipid membranes and the transport processes were characterized by two proposed electric equivalent circuits which correspond to the covered and to the uncovered polycarbobate carrier. Keywords: Phospholipids, Membranes, Ionophore, Peptid....
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Spectroscopic study of interaction of antimicrobial peptides with model membranes
Tesař, Adam ; Kopecký, Vladimír (advisor) ; Kočišová, Eva (referee)
Antimicrobial peptides (AMP) have a great potential in medicine and pharmacy. Mechanism of their impact is an interaction with a cell membrane leading to the penetration of the membrane. The way of disruption of the cell membrane is not completely understand, therefore we focused on the interaction of AMP HAL-1, isolated from the venom of the bee Halictus sexctinctus, with a model membrane of 100 nm liposomes consisting of phosphatidylcholin and phosphatidylglycerol. Circular dichroism and infrared spectroscopy (FTIR) proved the change of the secondary structure from the random coil of free HAL-1 to -helix in an interaction with the membrane. The next step was preparation of the lipid bilayer on the surface of ATR prism, which will enable usage of the polarized FTIR spectroscopy to study the interaction of AMP with model membranes in future. Therefore, the ATR-FTIR spectroscopy and factor analyses were applied to study dynamics of drying of the liposomes and their subsequent hydration also with an addition of HAL-1. We focused on the stabilization of the system. Hydratation of the lipid bilayer by 2 µl sample showed stability for minutes, nevertheless after dilution the stabilization decay in minutes. The protective influence of the peptide on the lipid bilayer and slowing down of the drying out of the...
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