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1
Gutlederer, Erwin Johann. "On the morphology of vesicles. - [überarb. Diss.]." Universität Potsdam, 2007. http://opus.kobv.de/ubp/volltexte/2007/1506/.
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This dissertation contains theoretical investigations on the morphology and statistical mechanics of vesicles. The shapes of homogeneous fluid vesicles and inhomogeneous vesicles with fluid and solid membrane domains are calculated. The influence of thermal fluctuations is investigated. The obtained results are valid on mesoscopic length scales and are based on a geometrical membrane model, where the vesicle membrane is described as either a static or a thermal fluctuating surface. The thesis consists of three parts.
In the first part, homogeneous vesicles are considered. The focus in this part is on the thermally induced morphological transition between vesicles with prolate and oblate shape. With the help of Monte Carlo simulations, the free energy profile of these vesicles is determined. It can be shown that the shape transformation between prolate and oblate vesicles proceeds continuously and is not hampered by a free energy barrier.
The second and third part deal with inhomogeneous vesicles which contain intramembrane domains. These investigations are motivated by experimental results on domain formation in single or multicomponent vesicles, where phase separation occurs and different membrane phases coexist. The resulting domains differ with regard to their membrane structure (solid, fluid). The membrane structure has a distinct effect on the form of the domain and the morphology of the vesicle. In the second part, vesicles with coexisting solid and fluid membrane domains are studied, while the third part addresses vesicles with coexisting fluid domains. The equilibrium morphology of vesicles with simple and complex domain forms, derived through minimisation of the membrane energy, is determined as a function of material parameters. The results are summarised in morphology diagrams. These diagrams show previously unknown morphological transitions between vesicles with different domain shapes. The impact of thermal fluctuations on the vesicle and the form of the domains is investigated by means of Monte Carlo simulations.Die vorliegende Arbeit enthält theoretische Untersuchungen zur Morphologie und statistischen Mechanik von Vesikeln. Es wird die Gestalt homogener fluider Vesikel und inhomogener Vesikel mit fluiden und festen Membrandomänen berechnet. Der Einfluss thermischer Fluktuationen wird untersucht. Die erzielten Ergebnisse beziehen sich auf mesoskopische Längenskalen und basieren auf einem geometrischen Membranmodell, in welchem die Vesikelmembran als statische, beziehungsweise thermisch fluktuierende Fläche beschrieben wird. Die Arbeit besteht aus drei Teilen. Im ersten Teil werden homogene fluide Vesikel betrachtet. Das Interesse gilt dem thermisch induzierten Morphologieübergang zwischen prolaten und oblaten Vesikelformen. Mit Hilfe von Monte-Carlo-Simulationen wird ein freies Energieprofil für diese Vesikel ermittelt. Es kann gezeigt werden, dass die Formumwandlung zwischen prolaten und oblaten Formen kontinuierlich verläuft und mit keiner freien Energiebarriere verbunden ist. Der zweite und dritte Teil beschäftigt sich mit inhomogenen Vesikeln, die intramembrane Domänen enthalten. Ausgangspunkt und Motivation der Berechnungen sind experimentelle Studien über Domänbildung in ein- oder mehrkomponentigen Vesikelmembranen, bei denen Phasentrennung stattfindet und unterschiedliche Membranphasen koexistieren. Die dabei auftretenden Domänen unterscheiden sich hinsichtlich ihrer Membranstruktur (fest, fluid). Diese beeinflusst die Form der Domäne und des gesamten Vesikels auf entscheidende Weise. Im zweiten Teil werden Vesikel untersucht, bei denen feste und fluide Membrandomänen koexistieren, Teil drei widmet sich Vesikeln mit zwei koexistierenden fluiden Membranphasen. In Abhängigkeit von Materialparametern werden durch Minimierung der Membranenergie die Grundzustandsformen von Vesikeln mit einfachen und komplexen Domänenformen bestimmt. Die Ergebnisse werden in Morphologiediagrammen zusammengefasst. Dabei werden bisher unbekannte Morphologieübergänge zwischen Vesikeln mit unterschiedlichen Domänformen beobachtet. Die Auswirkungen thermischer Fluktuationen auf die Vesikel und die Form ihrer Domänen werden mittels Monte-Carlo-Simulationen untersucht.
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Oldham, Alexis Jean. "Modulation of lipid domain formation in mixed model systems by proteins and peptides." View electronic thesis, 2008. http://dl.uncw.edu/etd/2008-1/r1/oldhama/alexisoldham.pdf.
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Wilkinson, Debbie Isabelle. "Visualisation of osteoclast membrane domains." Thesis, University of Aberdeen, 2010. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=158808.
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Osteoclasts polarise upon activation and form four distinct membrane domains; the basolateral domain, the sealing zone, the functional secretory domain and the ruffled border. The ruffled border is the resorptive organelle of the cell and provides a large surface area for the release of protons and enzymes into the space beneath the osteoclast. Defects in osteoclast formation or function can lead to diseases such as osteopetrosis. Ruffled border formation is a critical event in osteoclast function but the process by which it and other membrane domains form is only partially understood. Vesicular trafficking is essential for the tight regulation of the osteoclast membrane domains and it has been shown previously that treatment with pharmacological inhibitors causes disruption of trafficking. The aims of this PhD were to increase our understanding of vesicular trafficking in osteoclasts and to optimise ways of visualising osteoclast membrane domains. My studies of patients with osteoclast-poor osteopetrosis identified defects in RANKL as a cause of the defect. This in turn has identified a potential therapy of recombinant RANKL for patients with this form of the disease. Although purification of wild type or mutant RANKL was not completely successful, it did suggest that the mutant forms of RANKL were not functional. I have used pharmacological inhibitors to study osteoclast membrane domains, and found that transmission electron microscopy is an essential tool for studying membrane changes following pharmacological inhibition at the ultrastructural level. I also established that the study of vesicular trafficking to analyse formation of membrane domains can make excellent use of immuno-electron methods. Furthermore, genetic diseases associated with defective ruffled border formation such as XLA and osteopetrosis provide useful tools to further analyse the dynamics involved in the formation and maintenance of the ruffled border, as well as revealing more about the diseases themselves.
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Daste, Frédéric. "Function and regulation of coiled‐coil domains in intracellular membrane fusion." Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015PA05T001.
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Les mécanismes moléculaires impliqués dans la fusion membranaire ont été amplement étudiés au cours des trente dernières années. Notre compréhension actuelle de ce phénomène est principalement basée sur des résultats obtenus par (1) le développement de modèles physiques décrivant la fusion des membranes biologiques, (2) l’étude mécanistique et structurale des protéines de fusion membranaire des virus à enveloppe et (3) l’étude des évènements de fusion intracellulaire médiés par les protéines SNARES dans les cellules eucaryotes. La découverte du complexe SNARE fut l’aboutissement de travaux interdisciplinaires qui ont exigés un large éventail de techniques tel que la génétique de la levure, l’électrophysiologie, la biologie moléculaire, la biochimie cellulaire, la biophysique expérimentale et l’imagerie. Tirant parti des paradigmes et techniques biophysiques qui ont émergés de ces études, nous avons examiné les fonctions et mécanismes de régulation des domaines « coiled-coil » dans les processus de fusion intracellulaire impliquant des protéines de la famille des Longin-SNAREs ou des Mitofusines, deux machineries protéiques de fusion dont le mode d’action exact reste encore peu clair. La conception exacte des mécanismes moléculaires de la fusion membranaire requiert la reconstitution in vitro des protéines de fusion dans un large spectre d’environnement membranaire avec des propriétés biophysiques définies et facilement modulables. Idéalement, ces systèmes membranaires devraient permettre à l’expérimentateur de contrôler la composition lipidique et protéique, ainsi que la topologie membranaire, afin de rendre compte de l’importante variabilité observée entre les différents compartiments de fusion cellulaire. La reconstitution dans des liposomes offre une incroyable flexibilité avec la possibilité de faire varier la plupart des paramètres clefs et de créer un environnement minimal dans lequel les facteurs solubles et/ou membranaires peuvent être ajoutés, seuls ou en combinaison, pour dévoiler leur rôle avec clarté. Nous avons mis au point des systèmes in vitro de reconstitution de protéines dans des plateformes membranaires artificielles pour nos deux systèmes d’études (les deux protéines Longin-SNAREs TI-VAMP et Sec 22b, ainsi que les domaines « coiled-coil » des Mitofusines) et nous avons réalisé des expériences biochimiques pour caractériser le mode d’action de ces protéines. L’objectif à long-terme de ce projet est de comparer les mécanismes moléculaires des machineries de fusion associés aux protéines SNAREs et Mitofusines, et ainsi de dévoiler des similitudes structurelles et fonctionnelles entre (1) leur protéines de fusion principales et (2) leur facteurs régulateursThe molecular mechanisms involved in membrane fusion have been extensively studied for the past thirty years. Our current understanding of this phenomenon is mainly based on results obtained by (i) the development of physical models describing the fusion of membranes, (ii) structural and mechanistic investigations on fusion proteins of enveloped viruses and (iii) studies of SNARE protein-mediated intracellular fusion events of eukaryotic cells. Discovery of the SNARE complex was the outcome of interdisciplinary works which involved a wide range of techniques including yeast genetics, electrophysiology, molecular biology, cell-free biochemistry, adhesion/fusion biophysics and imaging. Taking advantage of the paradigms and biophysical techniques that emerged from these studies, we investigated the function and regulation of coiled-coil domains in intracellular fusion processes involving Longin-SNAREs or Mitofusins, two fusion protein machineries whose exact mode of action still remains unclear. A comprehensive understanding of the molecular mechanisms of membrane fusion requires the in vitro reconstitution of fusion proteins into a wide variety of membrane environments with defined and tunable biophysical properties. Ideally, these membrane systems should allow the experimentalists to control the lipid and protein composition as well as the membrane topology, to account for the variability observed across cellular fusing compartments. Reconstitution into liposomes offers amazing flexibility with the capacity to vary most of these relevant parameters, and to create a minimal environment in which membrane and/or soluble factors can be added, one at a time or in combination, to reveal their role with clarity. We have set up the in vitro reconstitution of proteins into various artificial membrane platforms for both systems (the Longin-SNAREs TI-VAMP and Sec22b and the coiled-coil domains of Mitofusins) and performed biochemical assays to gain insight into how these proteins execute their functions. The long-term goal of this project is to compare the molecular mechanisms of SNARE and Mitofusin fusion machineries and thus reveal structural and functional similitudes between (i) their core fusion proteins, and (ii) their regulatory factors
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Jean-François, Frantz. "Vers un nouveau mode d’action de peptides antimicrobiens structurés en feuillets ß : formation de domaines membranaires par la cateslytine." Thesis, Bordeaux 1, 2008. http://www.theses.fr/2008BOR13638/document.
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Le peptide antimicrobien Cateslytine (bCGA RSMRLSFRARGYGFR ) inhibe la libération des catécholamines des cellules chromaffines. Des études biologiques ont montré que ce peptide est capable d’inhiber aussi la croissance de nombreux microorganismes notamment des bactéries, des levures ainsi que le parasite Plasmodium falciparum responsable de la malaria. Cependant, le mode d’action moléculaire demeurait inconnu. Afin de mieux comprendre le ciblage et la sélectivité de ce peptide sur les membranes de mammifères ou de microorganismes, nous avons donc envisagé la reconstitution du système biologique composé initialement de peptides en contact avec des cellules, en le substituant par des modèles de membrane, de composition mimant celle des différents microorganismes. Des études structurales ont été menées en utilisant la technique d’ATR-FTIR polarisé, le dichroïsme circulaire et la RMN à haute résolution. La dynamique membranaire a été étudiée en utilisant la RMN des solides du phosphore et du deutérium. Des expériences de patch-clamp ont été effectuées afin de mesurer des flux d’ions au travers de la membrane. Enfin, de la simulation par ordinateur a permis de comprendre cette interaction au niveau moléculaire. Trois résultats principaux sont ressortis de cette approche pluridisciplinaire : i) Des flux ioniques au travers de la membrane attestent de la présence de cannaux. ii) La formation de domaines membranaires rigides constitués de lipides chargés négativement est démontrée. iii) Une structuration des peptides en feuillets ß antiparallèles est observée sur des membranes chargées négativement mimant les microorganismes. L’ensemble de ces résultats conduit à la proposition d’un mode d’action dans lequel la déstabilisation membranaire est induite par les domaines rigides stabilisés par les agrégats de peptides structurés en feuillets ßThe antimicrobial peptide Cateslytin (bCGA RSMRLSFRARGYGFR ) is a five positively charged arginin rich peptide known to inhibit the release of catecholamine in chromaffin granules. Although biological data showed that it is able to inhibit the growth of several microorganisms such as bacteria, yeast and Plasmodium falciparum parasite involved in malaria, the mechanism of action has not been yet studied. In order to better understand both targeting and selectivity of this peptide towards microorganisms, model membranes of variable compositions have been chosen to respectively mimic microorganisms or mammalian membranes. Structural studies have been performed using polarised ATR-FTIR, circular dichroïsm and high resolution NMR Membrane dynamics has been followed using deuterium labelled lipids and solid state NMR Patch clamp experiments were also performed on lipid vesicles to measure channe conductivity. All-atom molecular dynamics on hydrated peptide-lipid membrane systems was also used to assess the interaction from the atomic level. Main results from this interdisciplinary approach are three-fold. i) Electric current passages through membranes demonstrate permeation akin to pore formation. ii) Peptide-induced formation of rigid domains mainly made of negatively charged lipids is found. iii) Peptide antiparallel ß-sheets are observed preferentially with negatively charged lipids mimicking microorganism membranes. The general picture leads to the proposal that membrane destabilization/permeation is promoted by rigid domains stabilised by peptide ß-sheets
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Goulding, Rebecca Ellen. "Membrane localization of RasGRPs by C1 domains." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/24211.
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Ras and Rap GTPases are membrane-bound activators of signal transduction pathways that regulate several cell processes including proliferation, apoptosis and adhesion. Guanine nucleotide exchange factors (GEF5) positively regulate Ras and Rap GTPases by exchanging guanosine diphosphate (GDP) for guanosine triphosphate (GTP). In order to activate Ras and Rap GTPases, GEFs must be at the same membrane compartments where their target GTPases are located. The Ras guanine nucleotide releasing protein (RasGRP)
family of four GEFs regulate both Ras and Rap GTPases, with differential specificities. All
RasGRPs contain Cl domains, which have the potential to bind the lipid second messenger
diacylglycerol (DAG) that is generated at membranes in response to the ligation of many cell
surface receptors. Binding of their Cl domains to DAG could serve to co-localize RasGRPs
with membrane bound Ras and Rap GTPases. While some evidence exists for each member of the RasGRP family being potentially regulated by their Cl domains binding to DAG, there is contradictory evidence for RasGRP2. My thesis research focused on Cl domain-mediated mechanisms of RasGRP membrane localization, with special focus on RasGRP2. I found that the Cl domains of RasGRP2 and the β splice variant of RasGRP4 do not bind either DAG or phorbol ester, a DAG analog. However, all RasGRP Cl domains were shown to
bind anionic phospholipids. I determined that the Cl domain of RasGRP2 is required for
constitutive plasma membrane localization in NIH 3T3 fibroblasts and T-cells, and also for
translocation to the plasma membrane in SDF-1α-stimulated T-cells. I also identified a putative PDZ protein binding site which is required for RasGRP2 localization at the Golgi. My experiments showed that while RasGRP2 localization can occur at the plasma membrane and Golgi of NIH 3T3s, RasGRP2 mediated Rapi activation at the plasma membrane via its Cl domain is required for changes in cell morphology that are induced by RasGRP2
expression. My thesis research has demonstrated that all four members of the RasGRP family utilize their Cl domains to localize to membranes, although in the case of RasGRP2 this occurs via a DAG-independent mechanism, which targets RasGRP2 to the plasma membrane.
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Headlam, Madeleine Joyce. "Cytochrome P450scc (CYP11A1) : effects of glycerol and identification of the membrane binding domain." University of Western Australia. School of Biomedical and Chemical Sciences, 2004. http://theses.library.uwa.edu.au/adt-WU2004.0065.
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The first step in the synthesis of steroid hormones occurs in the mitochondria where cholesterol is converted to pregnenolone by cytochrome P450scc (CYP11A1). Cholesterol is insoluble in water and is supplied to the CYP11A1 directly from the inner mitochondrial membrane to which the enzyme is bound. The aim of this study was to characterise the interaction of bovine CYP11A1 with the phospholipid membrane. The effect of osmotic stress provided by glycerol on the spin-state, activity and degree of hydration of CYP11A1 was also investigated. Multiple sequence alignment of mitochondrial P450s revealed that there are 46 absolutely conserved residues, with the highest conservation in the heme-binding region at the C-terminal. The greatest variablility between subfamilies is in the regions believed to be involved in substrate binding (SRSs), as defined for the CYP2B family. The secondary structure prediction for CYP11A1 suggests that there is strong similarity in secondary structure to P450s of known structure. A model structure of CYP11A1 was built from primary sequence alignment to template P450 structures using the SwissModel automated server. From the model and other bioinformatic analyses, the distal face of the P450 which includes the A’ helix, F-G loop and beta sheet 1 regions, were predicted to interact with the membrane. Tryptic digests of CYP11A1 were performed with the aim of identifying membrane bound peptides that may be protected from protease activity. HPLC tryptic maps were similar in profile between soluble and vesicle-bound P450 which suggests that there is not a large region of CYP11A1 protected from protease digestion when the enzyme is attached to a membrane. Mass spectrometric analysis of peptides resulting from tryptic digestion revealed a number of peptides in the soluble digest that were not present in the digest of vesicle-bound P450. These peptides were located at the N-terminal and the J to J’ helix and interestingly, there was an absence of C-terminal peptides for both digests. This C-terminal peptide could be detected in digests of vesicle-bound P450 but not in digests of soluble P450 by tricine SDS polyacrylamide gel electrophoresis, Western transfer and N-terminal sequence analysis. Based upon the bioinfomatic and tryptic digestion data, a set of N- and C-terminal deletion mutants of CYP11A1 were expressed in E. coli and fractionated based on their association with the soluble or membrane fraction of the cells. The N-terminal deletion of the A’ helix resulted in an increase in the proportion of CYP11A1 in the soluble fraction while the C-terminal deletion did not alter membrane localisation. There are eight tryptophan residues in mature CYP11A1. The accessibility of these tryptophans to a water-soluble fluorescence quencher was determined for soluble and vesicle-bound enzyme. When CYP11A1 was associated with the vesicle membrane an average of 2 tryptophan residues were protected from quenching compared to soluble CYP11A1. This suggests that these tryptophan residues become buried within the membrane following association of CYP11A1 with the vesicles and are no longer accessible to quencher. The only free cysteine (C265S) of bovine CYP11A1 was removed by site directed mutagenesis and new cysteine residues introduced at selected sites based upon earlier results and the modelled CYP11A1 structure. The cysteine mutants were expressed, purified and labelled with the environmentally sensitive fluorescent probe, N-(7-nitrobenz-2-oxal-3-diazol-4-yl)ethylenediamine (NBD). There was an increase in the hydrophobicity of the NBD environment following the association of CYP11A1 with vesicles for the labeled mutants V212C and L219C. This indicates that these residues which are in the F-G loop, become localized to a more hydrophobic environment following membrane binding. Labeled cysteine residues introduced into the A’, B’ and G helices and β4-2 did not show major changes in hydrophobicity following membrane integration of CYP11A1. Osmotic stress of CYP11A1 induced by glycerol resulted in a low-spin spectral response and inhibition of activity. The change to low spin correlated with the dissociation of five or six water molecules from CYP11A1 and the inhibition of activity with cholesterol as substrate correlated with the dissociation of two molecules of water. In conclusion, this study shows that CYP11A1 is held to the membrane, at least in part, by the F-G loop region, and that the removal of water from the active site of CYP11A1 by osmotic stress causes a low spin spectral response and inhibition of activity.
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Salkhordeh, Mahmoud. "Localization of membrane binding domains in synapsin I." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ57790.pdf.
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Salkhordeh, Mahmoud Carleton University Dissertation Biology. "Localization of membrane binding domains in synapsin I." Ottawa, 2000.
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Brechin, C. "14-3-3 proteins and cholesterol-dependent membrane domains." Thesis, University of Edinburgh, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.641914.
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In this thesis I set out to characterise the interaction of 14-3-3 with lipid raft-like membrane domains. 14-3-3 associated with DRMs isolated from rate brain extract in a 14-3-3 isoform specific manner. Inhibition of 14-3-3 target protein interactions suggested that this association was reliant on binding of 14-3-3 to a membrane protein. The interaction of 14-3-3 with DRMs was further investigated in N2a and PC12 cells and also be detergent-free method. There are, however, some concerns over whether DRMs represent physiological membrane domains. Therefore I also investigated quantitatively the colocalisation of 14-3-3 with membrane domains in intact cells by confocal microscopy, using the lipid raft marker cholera toxin B subunit (CTXB). For comparison, other DRM-resident proteins, Thy-1, syntaxin-1a and SNAP-25, were also examined. SNAP-25 and Thy-1 showed a high degree of coincidence with CTXB in PC12 cells, 14-3-3 also colocalised with CTXB but to a much lower extent. As lipid rafts have been implicated in the control of regulated exocytosis the high coincidence of SNAP-25 and lack of coincidence of syntaxin-1 with CTXB is of interest. Cholesterol depletion, which affects the integrity of lipid raft-like domains, revealed some discrepancies between the association of 14-3-3., Thy-1, SNAP-25 and syntaxin-1a with DRMs and CTXB domains imaged in intact PC12 and N2a cells. CTXB clusters were partially disrupted in N2a cells and the coincidence of SNAP-25, but not 14-3-3, with CTXB was reduced. These findings indicate a role for lipid raft-like domains in controlling the spatial distribution of SNAP-25 on the plasma membrane. However, the lack of cholesterol dependent 14-3-3 localisation indicates that other membrane compartmentalisation mechanism may affect 14-3-3 distribution.
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Bdiri, Myriam. "Etude du décolmatage, par procédés chimiques et biologiques, des membranes échangeuses d'ions utilisées en électrodialyse dans le domaine agroalimentaire." Thesis, Paris Est, 2018. http://www.theses.fr/2018PESC1070.
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L’électrodialyse (ED) est principalement basée sur l’action spécifique des membranes échangeuses d’ions (MEIs) et est largement répandue en industrie agroalimentaire pour la stabilisation tartrique des vins, la désacidification et le traitement des jus de fruits, la déminéralisation du lactosérum ou l’élimination et le fractionnement des protéines du lait. Le colmatage organique, accentué par la complexité de composition des effluents alimentaires et leur richesse en composés phénoliques, représente un facteur majeur de limitation de l’efficacité des procédés et des performances des MEIs. Ce phénomène provoque une diminution de la sélectivité de membranes, une augmentation de leur résistance électrique et réduit le rendement énergétique du procédé conduisant à des pertes économiques en industrie. Cette étude consiste principalement à étudier le décolmatage de MEIs par procédés chimiques et biologiques. Des lots de membranes échangeuses de cations (MECs) et d’anions (MEAs) neuves (1 lot de MEC et 1 lot de MEA) et usées (3 lots de MECs et 2 lots de MEAs) à différentes durées d’utilisation en ED dans l’industrie agroalimentaire –application confidentielle- ont été étudiés. L’ensemble des échantillons ont préalablement été caractérisés pour détermination des paramètres physicochimiques (capacité d’échange (CE), épaisseur (Tm), conductivité électrique (km), angle de contact (θ), teneur en eau (WC) ainsi que la fraction volumique de la solution inter-gel (f2) résultant de l’exploitation du modèle microhétérogène), de structure et morphologiques par spectroscopie IR-TF, microscopie optique, microscopie électronique à balayage et mécaniques par essais de traction. Les effets directs et indirects (causés par les opérations de lavage régulières en industrie) du colmatage ainsi que l’anisotropie des propriétés mécaniques de membrane ont été mis en évidence. Des méthodes de nettoyage non agressives et respectueuses de l’environnement ont été expérimentées en mode statique en ex-situ : Solutions salines (NaCl à 35 g.L-1 et eau de mer reconstituée), solution hydro-alcoolique (mélange eau-éthanol 12%, pH=3,5) et solutions biologique utilisant 3 catégories d’agents enzymatiques (Rohalase BX-BXL, β-glucanase / Corolase 7089, endo-peptidase / Tyrosinase, polyphenol-oxydase) dont les conditions opératoires d’activité enzymatiques optimale ont été déterminées. L’évolution de CE, km, θ et f2 ont été suivis en fonction de la durée de nettoyage. Les solutions salines ont un effet négligeable sur le nettoyage en profondeur mais restent efficaces pour le nettoyage de surface. Cependant, l’application de la solution hydro-alcoolique et des solutions d’enzymes se sont avérées être efficaces pour le décolmatage interne et externe et parviennent à rétablir significativement les paramètres suivis. Il a été démontré que les composés phénoliques, principaux constituants des effluents traités, sont en majeure partie responsables du colmatage des MEIs. Ceux-ci forment des nanoparticules colloïdales denses, non perméables aux ions dans les méso- et macropores des MEIs et ne pénètrent pas dans ses micropores. Une modification du modèle microhétérogène selon cette hypothèse a permis de fournir une interprétation adéquate du km et de modéliser la modification structurale de la phase inter-gel engendrée par les mécanismes de colmatages de polyphénols et expliquer les raisons de diminution du facteur f2app. Une méthode d’extraction utilisant un mélange de solvants (25%V/V, acétone/méthanol/isopropanol/eau) a été mise au point et a permis d’extraire certains composés phénoliques de différents lots de MECs et MEAs usées et ont été identifiés par chromatographie liquide à haute performance. Il a été démontré que les interactions entre les composés phénoliques et la matrice polymère étaient principalement régies par l’empilement des cycles aromatiques et des interactions électrostatiques du type CH-pi et pi-pi ainsi que les liaisons hydrogènesConventional electrodialysis (ED) is mainly based on the specific action of ion exchange membranes (IEMs) and is widely used in food industry for tartaric stabilization of wines, deacidification and treatment of fruit juices, demineralization of whey or elimination and fractionation of milk proteins. The organic fouling, accentuated by the complex composition of the food effluents and their richness in phenolic compounds, represents a major limitative factor of the process efficiency and the IEMs performance. This phenomenon causes a decrease in the selectivity of membranes, an increase in their electrical resistance and reduces the energy efficiency of the process leading to economic losses in industry. This study mainly consists in studying the IEMs cleaning by chemical and biological methods. Two batches of new membranes (cation- (CEMs) and anion-exchange membranes (AEMs)) and five batches of used ones (3 CEMs and 2 AEM) with different durations of use in ED units in food industry -confidential application- have been studied. All the samples have been previously characterized to determine their physicochemical parameters (ion-exchange capacity (IEC), thickness (Tm), electrical conductivity (km), contact angle (θ), water content (WC) and the volume fraction of the inter-gel solution (f2) resulting from the study of the micro heterogeneous model), structure and morphology by FTIR spectroscopy, optical microscopy, scanning electron microscopy and mechanical by tensile strength tests. The direct and indirect effects (caused by the regular cleaning operations in industry) of fouling as well as the anisotropy of the membranes mechanical properties have been highlighted. Non-aggressive and environmentally friendly cleaning methods have been experimentally tested in ex-situ static mode: Saline solutions (35 g.L-1 NaCl and reconstituted seawater), hydro-alcoholic solution (12% water-ethanol mixture, pH = 3,5) and biological solutions using 3 categories of enzymatic agents (Rohalase BX-BXL, β-glucanase / Corolase 7089, endo-peptidase / Tyrosinase, polyphenol oxidase) whose operating conditions of optimal enzymatic activity have been determined. The evolution of IEC, km, θ and f2 were followed in function of the cleaning duration. Saline solutions have a negligible effect on intern cleaning but remain efficient for extern cleaning. However, the application of the hydro-alcoholic solution and enzyme solutions have been found to be efficient for both intern and extern cleaning and led to significant recoveries of the studied parameters. It has been shown that phenolic compounds, the principal constituents of treated effluents, are mainly responsible for MEIs fouling. Apparently, they form dense colloidal nanoparticles not permeable for ions within membrane meso- and macropores, not penetrating into micropores. A modification of the micro heterogeneous model under this assumption allowed an adequate interpretation of km and the modelization of structural modifications of the inter-gel phase generated by the fouling mechanisms by polyphenols and explained the reasons why the f2app decreases. An extraction method using a mixture of solvents (25% V/V, acetone/methanol/ isopropanol/water) was developed and made it possible to extract certain phenolic compounds from different batches of used CEMs and AEMs that were identified by high performance liquid chromatography. It has also been demonstrated that the interactions between the phenolic compounds and the polymer matrix are mainly governed by the stacking of aromatic rings and electrostatic interactions of the CH-pi and pi-pi type as well as the hydrogen bonds
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Regula, Camille. "Etude du vieillissement des membranes dans le domaine de l’agroalimentaire : production d’eau potable et filtration du lait." Thesis, Aix-Marseille, 2013. http://www.theses.fr/2013AIXM4313.
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Quel que soit le domaine d'application, les membranes doivent fréquemment subir des procédures de nettoyage et/ou de désinfection afin d'éliminer le colmatage qui découle de la filtration. Ces nettoyages sont responsables d'une accélération du vieillissement des matériaux qui impacte directement les performances techniques et économiques des procédés membranaires. La pérennité de ces derniers passe par l'identification des principaux facteurs responsables du vieillissement et par la compréhension des mécanismes qui le régissent. Le vieillissement statique de membranes d'ultrafiltration spiralée en polyethersulfone et fibres creuses en polysulfone respectivement utilisées pour la filtration du lait et pour la production d'eau potable a été étudié. Ce vieillissement a été mis en place selon la méthodologie des plans d'expériences qui a permis l'étude simultanée de la concentration, de la température et du temps de contact membrane/détergents et ce, pour différents détergents formulés couramment utilisés sur les sites de production (alcalin, acide, enzymatiques, oxydant chloré et oxydant non chloré). Les résultats ont permis de modéliser statistiquement les modifications membranaires induites par chaque détergent. Ces modifications ont été suivies aussi bien à l'échelle microscopique que macroscopique afin d'appréhender le phénomène de vieillissement dans sa globalité : propriétés de transport, propriétés mécaniques, propriétés de surface, ainsi que les modifications de la composition chimique du polymère constitutif de la membrane. Des préconisations d'utilisation des différents produits sont proposées : véritable carnet de bord les industrielsIn Food Industries, using membrane processes, contact with cleaning chemicals is believed to play an important role in membrane ageing. In this thesis, polysulfone hollow fibers and polyethersulfone spiral wound membranes were used to simulate the industrial cleaning in static conditions. Ageing of the membrane was mimicked by immersing samples in solutions containing commercial detergents with various concentrations, temperatures and soaking times defined by experimental designs. In an innovative way in the chemical membranes ageing research, an approach based on methodological tools has been realized for different formulated detergents (alkaline, acidic, enzymatic, oxidant and biocide). The main interest is to achieve a relevant ageing pattern without using an accelerated ageing protocol which has been proved to be not relevant. Results allow modeling macroscopic ageing involved by each detergent. Those modifications have been studied as well at the macroscopic scale as at the microscopic scale in order to put membrane ageing in a global perspective: flux, mechanical properties, surface properties, polymer modifications. Specifications of using can be advised according to membranes and products which represent a real oversight handbook about membrane cleaning for industrial users
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Kelm, Sebastian. "Structural modelling of transmembrane domains." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:b4c9fba9-ee25-469b-8baf-b7c1d70c9d05.
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Membrane proteins represent about one third of all known vertebrate proteins and over half of the current drug targets. Knowledge of their three-dimensional (3D) structure is worth millions of pounds to the pharmaceutical industry. Yet experimental structure elucidation of membrane proteins is a slow and expensive process. In the absence of experimental data, computational modelling tools can be used to close the gap between the numbers of known protein sequences and structures. However, currently available structure prediction tools were developed with globular soluble proteins in mind and perform poorly on membrane proteins. This thesis describes the development of a modelling approach able to predict accurately the structure of transmembrane domains of proteins. In this thesis we build a template-based modelling framework especially for membrane proteins, which uses membrane protein-specific information to inform the modelling process.Firstly, we develop a tool to accurately determine a given membrane protein structure's orientation within the membrane. We offer an analysis of the preferred substitution patterns within the membrane, as opposed to non-membrane environments, and how these differences influence the structures observed. This information is then used to build a set of tools that produce better sequence alignments of membrane proteins, compared to previously available methods, as well as more accurate predictions of their 3D structures. Each chapter describes one new piece of software or information and uses the tools and knowledge described in previous chapters to build up to a complete accurate model of a transmembrane domain.
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Leidy, Chad. "Thermotropic behavior of lipid domains in model membranes /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2002. http://uclibs.org/PID/11984.
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Rink, Jochen C. "Rab-domain dynamics in endocytic membrane trafficking." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2005. http://nbn-resolving.de/urn:nbn:de:swb:14-1117095871452-66763.
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Eukaryotic cells depend on cargo uptake into the endocytic membrane system, which comprises a functionally interconnected network of endosomal compartments. The establishment and maintenance of such diverse compartments in face of the high rates of exchange between them, poses a major challenge for obtaining a molecular understanding of the endocytic system. Rab-GTPases have emerged as architectural key element thereof: Individual family members localize selectively to endosomal compartments, where they recruit a multitude of cytoplasmic effector proteins and coordinate them into membrane sub-domains. Such "Rab-domains" constitute modules of molecular membrane identity, which pattern the endocytic membrane system into a mosaic of Rab-domains. The main objective of this thesis research was to link such "static" mosaic-view with the highly dynamic nature of the endosomal system. The following questions were addressed: How are neighbouring Rab-domains coordinated? Are Rab-domains stable or can they undergo assembly and disassembly? Are the dynamics of Rab-domains utilized in cargo transport? The first part of this thesis research focused on the organization of Rab-domains in the recycling pathway. Utilizing Total Internal Reflection (TIRF) microscopy, Rab11-, but neither Rab4- nor Rab5-positive vesicles were observed to fuse with the plasma membrane. Rab4-positive membranes, however, could be induced to fuse in presence of Brefeldin A. Thus, these experiments complete the view of the recycling pathway by the following steps: a) Rab11-carriers likely mediate the return of recycling cargo to the surface; b) such carriers are presumably generated in an Arf-dependent fission reaction from Rab4-positive compartments. Rab11-chromatography was subsequently carried out in the hope of identifying Rab11-effectors functioning at the Rab4-Rab11 domain interface. An as yet uncharacterized ubiquitin ligase was identified, which selectively interacts with both Rab4 and Rab11. Contrary to expectations, however, the protein (termed RUL for *R*ab interacting *U*biquitin *L*igase) does not function in recycling,but appears to mediate trafficking between Golgi/TGN and endosomes instead.In order to address the dynamics of Rab-domains, fluorescently tagged Rab-GTPases were imaged during cargo transport reactions in living cells. Herefore high-speed/long-term imaging procedures and novel computational image analysis tools were developed. The application of such methodology to the analysis of Rab5-positive early endosomes showed that a) The amount of Rab5 associated with individual endosomes fluctuates strongly over time; b) such fluctuations can lead to the "catastrophic" loss of the Rab5-machinery from membranes; c) Rab5 catastrophe is part of a functional cycle of early endosomes, involving net centripetal motility, continuous growth and increase in Rab5 density. Next, the relevance of Rab5 catastrophe with respect to cargo transfer into either the recycling- or degradative pathway was examined. Recycling cargo (transferrin) could be observed to exit Rab5-positive early endosomes via the frequent budding of tubular exit carriers. Exit of degradative cargo (LDL) from Rab5-positive endosomes did not involve budding, but the rapid loss of Rab5 from the limiting membrane.Rab5-loss was further coordinated with the concomitant acquisition of Rab7, suggesting "Rab conversion" as mechanism of transport between early- and late endosomes.Altogether, this thesis research has shown that first, Rab-machineries can be acquired and lost from membranes. Second, such dynamics provide a molecular mechanism for cargo exchange between endosomal compartments. Jointly, these findings lead to the concept of Rab-domain dynamics modulation in /trans/ between neighbouring domains as mechanistic principle behind the dynamic organization of membrane trafficking pathways.
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Kreder, Rémy. "Sondes moléculaires multifonctionnelles pour l'imagerie de fluorecence de membranes cellulaires." Thesis, Strasbourg, 2015. http://www.theses.fr/2015STRAJ006/document.
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Conçues à partir d’une approche rationnelle, nous avons créé de nouvelles sondes membranaires permettant l’imagerie de l’organisation de la membrane plasmique cellulaire. Dans ce travail, nous avons d’abord développé un groupe d’outils, à partir du fluorophore solvatochrome Nile Red et de Black Hole Quencher-2, capable de marquer spécifiquement les domaines ordonnés et désordonnés (radeaux) en les identifiant par leur couleur d’émission. Les études cellulaires, à l’aide de ces sondes, suggèrent que la membrane plasmique est composée de deux phases distinctes. Puis dans le but de créer de nouvelles sondes basées sur Nile Red compatibles avec le sérum et fixables par formaldéhyde/glutaraldéhyde, nous avons modifié la sonde, préalablement développée, NR12S avec un groupement PEG ou amino, respectivement. Etonnamment, la sonde PEGylée est rapidement internalisée dans la cellule et le dérivé animo agrège avec l’agent fixant. D’un autre côté,basée sur Nile Red, nous avons conçu une sonde capable de détecter un récepteur donné et de visualiser son environnement lipidique. Initialement, nous avons obtenu des sondes capables d’allumer leur fluorescence en se liant sur le RCPG à l’ocytocine. Puis, nous avons conjugué NR12Spar l’intermédiaire d’un espaceur PEG(12) au ligand de l’intégrine, RGD. Les résultats préliminaires montrent que la molécule peut se lier à la membrane et détecter l’ordre lipidique, cependant les études cellulaires nécessitent un achèvement. Nous avons aussi travaillé sur des sondes membranaires fluorogéniques (turn-on) pour de l’imagerie multi-couleurs. Basées sur le fluorophore3-méthoxychromone, nous avons obtenu des sondes plus brillantes et plus photostables que la sonde développée originellement à partir de 3-hydroxychromone (F2N12S). Grâce à l’important déplacement de Stokes, elles permettent une imagerie de la membrane cellulaire avec une autofluorescence minimale dans la région spectrale bleue, compatible avec les marqueurs communs verts et rouges. Pour finir, basées sur le fluorophore squaraine, nous avons développé trois nouvelles sondes opérant dans la région rouge lointain, qui est particulièrement intéressante pour l’imagerie in vitro et in vivo. Ces sondes montrent une orientation parallèle avec la membrane lipidique, alors que les expériences cellulaires indiquent que seule la sonde avec deux ancres lipidiques est capable de marquer de façon stable la membrane plasmique. Ces sondes développées ici sont prévues pour être utilisées dans la recherche des radeaux lipidiques aussi bien que pour l’imagerie super-résolution et multi-couleurs de cellules vivantesBased on rational molecular design, we design new membrane probes that enable fluorescence imaging of cell plasma membrane organization. In this work, we first synthesized a toolkit, based on solvatochromic Nile Red dye and Black Hole Quencher-2, that can stain specifically ordered and disordered lipid domains (rafts) and identify them by the emission color. Cellular studies with these probes suggested that the plasma membrane is composed of two distinct phases. Then,with the idea to make Nile Red-based probes compatible with serum medium and fixable by formaldehyde/glutaraldehyde, we modified previously developed probe NR12S with PEG and aminogroups, respectively. Surprisingly, the PEGylated probe is quickly internalized inside the cell and the amino-derivative aggregates with the fixing agent. On the other hand, based on Nile Red we designed probes capable to detect a given receptor and visualize its lipid environment. Initially, we obtained probes that can turn-on fluorescence on binding to the oxytocin GPCR receptor. Then, we conjugated NR12S through a PEG(12) spacer to the ligand of intergrin, RGD. The first data show that the molecule can bind to the membrane and detect the lipid order, though cellular studies have to be completed. We also worked on fluorogenic (turn-on) membrane probes for multi-color imaging. Based on blue 3-methoxychromone dyes, we obtained probes that are brighter and more photostable than the originally developed probe based on 3-hydroxychromone (F2N12S). Due to large Stocks shift, they enabled cell membrane imaging with minimal auto-fluorescence in the blue spectral region, compatible with common green and red probes. At the end, based on squaraine fluorophore, we developed three new probes operating in the far red region, which is also very interesting for in vitro and in vivo imaging. These dyes show a parallel orientation with the lipid membrane, while the cellular experiments point out that only the probe with two anchor groups is able to stain stably the plasma membrane. The probes developed here are expected to be used for lipid rafts research as well as for super-resolution and multi-color imaging of living cells
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Karathanassis, Dimitrios. "Structural insights into membrane binding by phox homology (PX) domains." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.619724.
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Brechin, Carolyn. "SNAREs, 14-3-3 proteins and cholesterol dependent membrane domains." Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/29276.
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Lipid rafts are suggested to be sphingolipid and cholesterol rich domains that segregate out from the bulk plasma membrane by forming a more ordered lipid phase. Lipid raft-like domains have been described as cell signalling platforms and have been implicated in the regulation of an array of signal transduction events. This study investigates the association of two classes of protein, 14-3-3 proteins and plasma membrane SNAREs, with lipid raft-like domains. The 14-3-3 family of proteins are important regulators of numerous cell signalling pathways and are essential for cell survival; recently there has been some interest in the roles of these soluble proteins at the membrane, though this area remains poorly characterised. 14-3-3 has also been linked to CJD progression, which is directed by the lipid raft associated prion protein. SNAREs are essential mediators of exocytosis, a process that is also reported to depend on cholesterol, implying lipid raft involvement. SNAREs have also been isolated in detergent resistant membranes (DRMs) that are believed to represent clustered lipid rafts. To examine the association of 14-3-3 and SNAREs with lipid raft-like domains in N2a and PC 12 cells two approaches were taken. Initially, detergent resistant membranes were isolated and analysed for protein association. The second approach involved quantitative analysis of the colocalisation of 14-3-3 and SNAREs with membrane domains in intact cells by confocal microscopy, using the lipid raft marker cholera toxin B subunit (CTXB). Discrepancies between results from these two methods add to evidence implying that DRMs do not necessarily represent pre-existing membrane domains. Cholesterol depletion, which affects the integrity of lipid raft-like domains, caused a rearrangement CTXB labelled clusters in N2a and PC 12 cells. The colocalisation of 14-3-3 with CTXB was unaffected by cholesterol depletion, a result which does not support the localisation of 14-3-3 to lipid raft-like domains. Interestingly however, the membrane distribution of the lipid raft marker Thy-1, a GPI-anchored protein, was also unaltered when cholesterol was depleted. In contrast to previous reports, disruption of SNAP-25 or syntaxinla (SNARE) clusters was not observed following cholesterol depletion. However, in N2a cells, the colocalisation of SNAP-25 with CTXB was reduced, though this was not the case in PC 12 cells. Taken together these results suggest that cholesterol depletion may affect various raft-associated proteins and cell types in different ways. The findings from N2a cells indicate a role for lipid raft-like domains in controlling the spatial distribution of SNAP-25 on the plasma membrane. The membrane distribution of syntaxinla appears to be differently regulated from that of SNAP-25, which may have implications for the regulation of exocytosis.
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Hagelbäck, Johan, and Kenny Svensson. "Locating transmembrane domains in protein sequences." Thesis, Blekinge Tekniska Högskola, Institutionen för programvaruteknik och datavetenskap, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:bth-2752.
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We have developed a new approach for locating transmembrane domains in protein sequences based on hydrophobicity analysis and backpropagation neural network or k-nearest-neighbor as classifiers. Our system was able to locate over 98% of the transmembrane domains and the total accuracy including overpredictions was above 95%.
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Yandrapalli, Naresh. "Role of HIV-1 Gag protein multimerization in the generation of nanodomains in lipid membranes." Thesis, Montpellier, 2016. http://www.theses.fr/2016MONTT097/document.
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La polyprotéine Gag du VIH-1 qui contient quatre principaux domaines (Matrix (MA), capside (CA), nucléocapside (NC), et P6) est l’orchestrateur privilégié de l'assemblage du virus HIV-1, assemblage qui a lieu pendant la phase tardive de la réplication. Il est bien connu que Gag interagit avec les lipides de la membrane plasmique de la cellule hôte et s’auto-assemble sur le feuillet interne de cette dernière afin de générer de nouvelles particules virales. Le bourgeonnement de ces particules virales hors de la cellule hôte est décrit comme étant dépendant de la machinerie cellulaire ESCRT. Différentes études structurales, fonctionnelles ainsi que des simulations de dynamique gros grain ont montré que la liaison de Gag à la membrane est médiée par une interaction duale. Une spécifique de nature éléctrostatique, qui associe une région hautement basique (HBR) du domaine MA de Gag au lipide acide,phosphatidyl inositol biphosphate (PI(4,5)P2) du feuillet interne de la membrane plasmique. Une de type hydrophobe, qui consiste en l’insertion du myristate de Gag dans la membrane plasmique. Savoir si Gag reconnait spécifiquement des domaines lipidiques pré-existants de type « rafts » ou si, au contraire, Gag tri ses lipides et les réorganise latéralement afin d’optimiser sa multimérisation et son bourgeonnement est une question à la fois fondamentale et d’actualité en virologie.Durant ma thèse, j’ai vérifié l’existence de la seconde hypothèse en utilisant des membranes modèles contenant du PI (4,5) P2 marqué de façon fluorescente et différent mutants et produits de la protéine Gag non-myristoylée. Ces expériences ont montré de fortes affinités de ces protéines pour les membranes contenant du PI (4,5) P2. S’appuyant sur les propriétés d’auto-extinction de fluorescence du marqueur choisit et à l’aide des différents variants de la protéine Gag, j'ai pu montré que la multimérisation de Gag génère l’existence de nanodomaines contenant du PI (4, 5) P2 et du cholestérol, la sphingomyéline étant au contraire exclue de ces domaines. En marquant la protéine Gag par un autre fluorophore, j’ai pu montrer par microscopie optique sur des vésicules lipidiques géantes (GUVs) que la protéine Gag partitionnait préférablement dans des microdomaines lipidiques de type liquide désordonnés (Ld). Par la suite, j’ai testé la capacité de la protéine Gag d’induire la formation de vésicules sur des membranes modèles (Bicouches supportés et GUVs) contenant du PI(4,5) P2 et de la phosphatidyl sérine (PS). En utilisant une microbalance à cristal de quartz (QCM-D) et des techniques de microscopie de fluorescence, j’ai suivi l'auto-assemblage de Gag dans le temps et ai montré que la protéine Gag était suffisante pour générer une courbure de la membrane et libérer des vésicules lipidiques. Grâce à différents produits de maturation de cette protéine, j’ai montré que la présence des domaines MA et CA est suffisante pour produire ces vésicules.L’ensemble de ces résultats suggèrent que la liaison et la multimérisation de la protéine Gag ne se produit pas dans des domaines lipidiques préexistants de type « raft », mais, au contraire, que la liaison et multimérisation de la protéine Gag génère l’existence de domaines lipidiques enrichis en PI (4,5) P2 et en cholestérol. La générescence de ces domaines lipidiques pourrait participer à la courbure de la membrane plasmique nécessaire au bourgeonnement du virusGag polyprotein of HIV-1 is made of four main domains Matrix (MA), Capsid (CA), Nucleocapsid (NC), and P6 and is the prime orchestrator of virus assembly that occurs during the late phase of replication. It is well known that Gag interacts with host cell lipids and self-assemble along the inner-leaflet of the plasma membrane in order to generate virus like particles (VLPs). Budding of these VLPs out of the living cell is described to be ESCRT dependent. Structural, functional and simulation based studies has shown that Gag membrane binding is mediated by a bipartite interaction. One specific electrostatic interaction, between the highly basic region (HBR) of its MA domain and the host cell acidic lipid phosphatidyl inositol bisphophate (PI(4,5)P2), plus a hydrophobic interaction through Gag’s myristate insertion in the plasma membrane. It is still an opened question whether Gag would specifically recognize pre-existing lipid domains such as rafts to optimize its multimerization or, on the contrary, would reorganize lipids during its multimerization. During my Ph.D. I explored the second hypothesis using purified myr(-) Gag protein and model membranes containing fluorescently labelled PI(4,5)P2.Bonding experiments have shown strong affinities of these purified proteins towards PI(4,5)P2 containing lipid bilayers. Using PI(4,5)P2 fluorescence self-quenching properties, I found that multimerization Gag generates PI(4,5)P2/Cholesterol enriched nanoclusters. On the opposite, sphingomyelin was excluded from these nanoclusters. In addition to this, using a fluorescently labelled myr(-) Gag, I have observed its preferable partitioning into lipid disordered (Ld) phases of giant unilamellar vesicles (GUVs). Further, possibility of whether HIV-1 Gag alone, as a minimal system, can induce the formation of vesicles on PI(4,5)P2/PS containing supported lipid bilayers (SLBs) & GUVs was tested. Using quartz crystal microbalance (QCM-D) and fluorescence microscopy techniques, I monitored the self-assembly of HIV-1 Gag with time and found that Gag was sufficient to generate membrane curvature and vesicle release. Moreover, using mutants of this protein, I found that having MA and CA domain is enough for Gag to produce vesicle like structures. Taken together, these results suggest that binding and multimerization of Gag protein does not occur in pre-existing lipid domains (such as “rafts”) but this multimerization is more likely to induce PI(4,5)P2/Cholesterol nanoclusters. This nanophase separation could locally play a role in the membrane curvature needed for the budding of the virus
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Smith, Karen Louise. "Structure and function of neuronal GPI domains." Thesis, King's College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313392.
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King, Gavin W. "Investigating helix-helix interactions in the transmembrane domains of membrane proteins." Thesis, University of Warwick, 2009. http://wrap.warwick.ac.uk/3157/.
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Helix-helix interactions between membrane-spanning transmembrane (TM) domains have been shown to drive the assembly of α-helical membrane proteins within biological membranes. However, the rules that determine these interactions are not yet fully understood, despite such interactions being found in an increasing number of proteins. Recent work has implicated TM domain interactions in the formation of the protein complex Ii-MHC, formed from the association of Major Histocompatibility Complex Class II (MHC) and the MHC-associated-Invariant Chain (Ii) proteins. Following biosynthesis, three MHC α/βheterodimers bind to the Ii homotrimer to form a nonameric Ii-MHC complex within the endoplasmic reticulum. This is a critical step in the export of MHC molecules to the antigen presentation system and hence the activation of an immune response to a pathogen. In this study we have explored the TM domain interactions within the Ii-MHC complex. Results from in vivo and in vitro experiments revealed the TM domains of the α- and β-chains of MHC have a propensity to self-associate into homo-dimers and to associate with one another to form hetero-dimers. Highly conserved GxxxG motifs (known to drive dimerization) were implicated in these interactions. The TM domain of Ii was confirmed to self-associate to form trimers by in vivo and in vitro methods, but surprisingly also displayed additional oligomeric states suggesting the interaction is not as specific as was previously thought. Furthermore, we show that in vivo, the TM domain of Ii can associate with those of the α- and β-chains of MHC, whilst in vitro methods suggested Ii preferentially binds to α-chains. Collectively, these findings strongly suggest that the TM domains of Ii and MHC have a role to play in the assembly of the Ii-MHC complex, and hence the very important process of antigen presentation. Additionally, in this study we have undertaken development of NMR spectroscopy methods that have the potential to increase our understanding of not only the Ii-MHC complex, but protein-protein interactions in general.
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Kucherak, Oleksandr. "Development of new fluorescent membrane probes for apoptosis and raft domains." Strasbourg, 2011. http://www.theses.fr/2011STRA6056.
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La première sonde ratiométrique capable de détecter l’apoptose (F2N12S) a récemment été développée au laboratoire. Le but du présent travail est de développer des sondes fluorescentes pour l’apoptose présentant des caractéristiques améliorées permettant de palier certains défauts de F2N12S. Nous avons découvert que l’apoptose induit non seulement une modification de la charge surfacique du feuillet externe, mais également de son hydratation et de son état de phase, expliquant ainsi la réponse de la sonde. Ensuite, nous avons synthétisé une vingtaine de nouvelles sondes membranaires, qui ont été classées en trois catégories en fonction de leur réponse obtenue sur membranes modèles. Une première catégorie montre une sensibilité presque nulle vis-à-vis de l’état de phase, mais nettement améliorée vis-à-vis de la charge surfacique (>3 fois), comparée à F2N12S. Une seconde catégorie montre une meilleure sensibilité à l’état de phase comme à la charge surfacique. Une troisième catégorie, dérivée du Rouge Nil, est sensible seulement à l’état de phase. En se basant sur ces résultats, nous avons proposé des principes généraux pour la conception de sondes membranaires fluorescentes. Enfin, nous avons décrit deux nouvelles classes de fluorophores sensibles à l’environnement basés sur les résidus fluoréne et 3-methoxychromone afin d’élaborer de nouvelles sondes membranaires. Ces nouveaux fluorophores ont montré des propriétés spectroscopiques intéressantes, telles qu’une forte luminosité, une excellente photostabilité et un solvatochromisme exceptionnel. Les premières tentatives pour convertir ces fluorophores en sondes membranaires ont été décritesFluorescent dyes are of great importance for investigation of biological processes. Recently, the first ratiometric fluorescent probe for apoptosis detection (F2N12S) was developed in our laboratory. The aim of the present work was to develop improved fluorescent apoptosis probes that will overcome the drawbacks of F2N12S. Primarily, we performed systematic studies of F2N12S in model membranes and cells to better understand the mechanism of its response to apoptosis. We found that in addition to surface charge, the membrane hydration and phase state are changed on apoptosis, thus explaining the spectroscopic response of the probe. On the second step, we have synthesized about 20 new membrane probes, which were classified into three types according to the experiments in model membranes. The first type of dyes showed nearly no sensitivity to the phase state while their sensitivity to surface charge was much higher (>3-fold) as compared to F2N12S. The second type showed improved sensitivity both to the surface charge and to the phase state of the lipid bilayers. The third type, which was based on Nile Red, was sensitive only to the phase state. Basing on these results we proposed general principles for the design of fluorescent membrane probes. Finally we described two new classes of environment-sensitive fluorophores on the base of fluorene and 3- methoxychromone units for further construction of new advanced membrane probes. These new fluorophores showed attractive spectroscopic properties, such as high brightness and photostability as well as an exceptional fluorescence solvatochromism. The first attempts to convert these fluorophores into membrane probes were described
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Björkholm, Patrik. "Protein Interactions from the Molecular to the Domain Level." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-101795.
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The basic unit of life is the cell, from single-cell bacteria to the largest creatures on the planet. All cells have DNA, which contains the blueprint for proteins. This information is transported in the form of messenger RNA from the genome to ribosomes where proteins are produced. Proteins are the main functional constituents of the cell, they usually have one or several functions and are the main actors in almost all essential biological processes. Proteins are what make the cell alive. Proteins are found as solitary units or as part of large complexes. Proteins can be found in all parts of the cell, the most common place being the cytoplasm, a central space in all cells. They are also commonly found integrated into or attached to various membranes. Membranes define the cell architecture. Proteins integrated into the membrane have a wide number of responsibilities: they are the gatekeepers of the cell, they secrete cellular waste products, and many of them are receptors and enzymes. The main focus of this thesis is the study of protein interactions, from the molecular level up to the protein domain level. In paper I use reoccurring local protein structures to try and predict what sections of a protein interacts with another part using only sequence information. In papers II and III we use a randomization approach on a membrane protein motif that we know interacts with a sphingomyelin lipid to find other candidate proteins that interact with sphingolipids. These are then experimentally verified as sphingolipid-binding. In the last paper, paper IV, we look at how protein domain interaction networks overlap and can be evaluated.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.
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Fuhrer, Andrew B. "The Role of Lipid Domains and Sterol Chemistry in Nanoparticle-Cell Membrane Interactions." Ohio University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1596569401131742.
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Georgiev, Alexander. "Membrane Stress and the Role of GYF Domain Proteins." Doctoral thesis, Stockholm : Department of Biochemistry and Biophysics, Stockholm university, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-7764.
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Rubin, Darrell. "ACUTE REGULATION OF GLUT1 FUNCTION: THE ROLE OF DETERGENT-RESISTANT MEMBRANE DOMAINS." Connect to text online, 2004. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1087996732.
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Laurent, Nelson. "Caractérisation des mécanismes régulant les modifications de la structuration membranaire induites dans la signalisation des réponses de défense des plantes." Thesis, Bourgogne Franche-Comté, 2020. http://www.theses.fr/2020UBFCK026.
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Afin d’assurer un état physiologique optimal des cellules, l’organisation de tous les composants de leur membrane plasmique (MP) est finement régulée. L’un des paramètres permettant de rendre compte de l’agencement des composants entre eux et du niveau d’organisation de la MP est le degré d’ordre. Ici, nous avons utilisé, la di-4-ANEPPDHQ, une sonde fluorescente sensible à son environnement, pour suivre le degré d’ordre de la MP de cellules de tabac BY-2 dans deux contextes différents ; lors de la régénération de cellules à partir de protoplastes et dans le cadre d’une élicitation par la cryptogéine.Nous avons ainsi mesuré que le degré d’ordre de la MP de cellules de tabac BY-2 est modulé au cours du lent processus de leur régénération. Cette régulation est découplée de la néosynthèse de la paroi et indépendante de la forme des cellules, mais est corrélée au niveau de différenciation cellulaire. A cette dimension temporelle, s’ajoute une dimension locale de la régulation de l’organisation de la MP. De fait, la cartographie des périmètres de protoplastes et de cellules en suspension de tabac a montré une organisation sous forme de mosaïque de petits domaines (288X288 nm) aux niveaux d’ordre très variés. Les deux modèles étudiés diffèrent par l’organisation de cette mosaïque. En effet, les cellules en suspension présentent une zone, à dimension micrométrique, d’enrichissement en domaines de degré d’ordre plus élevé, située entre deux cellules adjacentes. Nous discutons l’implication du trafic membranaire dans la modulation du RGM des cellules, via un adressage de lipides/protéines à la MP, qui mènerait à l’acquisition d’une identité cellulaire.Une approche pharmacologique a permis d’identifier les formes actives d’oxygènes responsables de la réorganisation rapide de la MP chez les cellules de tabac BY-2 observée en réponse à la cryptogéine. Ainsi, l’effet du H2O2 sur le degré d’ordre présente des similarités avec celui de la cryptogéine, dont un effet dose dépendant. De plus, une inhibition partielle de l’accumulation de H2O2 en réponse à la cryptogéine est associée à une inhibition partielle de la hausse du degré d’ordre membranaire normalement induite chez les cellules de tabac BY-2. Ces résultats suggérant que le H2O2 soit capable de réguler finement le degré d’ordre membranaire des cellules lors de la réponse à la cryptogéine. Le mécanisme d’action du H2O2 a été étudié, et deux hypothèses classiques ont pu être éliminées. La première implique une modification de la composition de la MP via une arrivée de domaines ordonnés à la MP et la seconde une formation de novo de ces domaines à la surface de la MP via une modification directe des composants membranaires. Nous discutons l’existence d’un nouveau rôle pour le H2O2 qui agirait directement sur le degré d’ordre de la MPSpatial distribution of pasma membranes (PM) components is tightly regulated to provide the cell an optimal physiological state. The level of order degree is a suitable parameter to study PM organization, reflecting the intensity of interactions taking place between PM components and so the level of their packing. During our work, we used the environment sensitive probe di-4-ANEPPDHQ to assess the level of order degree of tobacco BY-2 cells PM in different situations: during the time-course of cell regeneration and in the particular case of an elicitation by cryptogein.We measured that the level of order degree of PM is modulated during the slow process of cell regeneration. This regulation isn’t related to the cell wall neo-synthesis neither the cell morphology, but is correlated to the differentiation state. The mapping of the level of order degree along the PM perimeter of protoplasts and suspension cells revealed a global common organization with a mosaic like distribution of domains (288X288 nm) exhibiting high diversity of level of order degree. The two models differed by the organization of this mosaic, the cell PM exhibiting enrichment in highest ordered domains in a specific area between two adjacent cells. Then, we discus about a possible involvement of membrane trafficking in the regulation of the level of order degree allowing specific targeting of lipids/proteins, and subsequent cellular identity acquisition.A pharmacological approach allowed the identification of the reactive oxygen species involved in the PM reorganization observed at the surface of tobacco BY-2 cells in response to cryptogein. We report that H2O2 has similar effects on the PM level of order degree compared to cryptogein’s one, both acting doses dependently. Furthermore, a partial inhibition of the H2O2 accumulation occurring in response to cryptogein is linked to a partial inhibition in the increase of the level of order degree usually induced by cryptogein. Those results suggest that H2O2 could finely regulates The PM level of order degree during the response of tobacco cells to cryptogein. The potential mechanisms involved has been studied. Two current hypotheses were studied in parallel, and both were rejected. The first one involves a modification of PM composition by an arrival of ordered domain to the PM. In the second one, a de novo formation of PM component structure. So, we discuss about a new role for H2O2, which would act directly on the PM level of order degree
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Laudon, Hanna. "Functional domains in the Alzheimer's disease-associated presenilin 1 protein /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-085-0/.
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Stie, Jamal Talal. "Modulation of the Plasma Membrane Domain Structure of Human Neutrophils." Thesis, Montana State University, 2006. http://etd.lib.montana.edu/etd/2006/stie/StieJ0806.pdf.
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Eukaryotic cell plasma membranes form an interface between cells and their environment and function to detect and interpret environmental cues. The work described in this dissertation examines the changes that occur in membrane structure during plasma membrane function in human neutrophils and a fungal opportunist. The body of this work examines how circulating neutrophils can remain functionally inactive in the presence of perturbing influences inherent in the blood circulation, and yet rapidly activate upon exposure to proinflammatory agents. It is hypothesized that the regulated modulation of plasma membrane domain structure determines the activation of blood-leukocytes, in vivo. Experimentation is based the isolation of blood-neutrophils in either nonactivated or activated (primed) cellular states using dextran- or gelatin-based preparative methods, respectively. Analysis of plasma membrane cortical components actin, fodrin, ezrin, CD45 and CD43 by sucrose density sedimentation, flow cytometry and indirect immunofluorescence microscopy indicated significant differences in the plasma membrane structure of both neutrophil populations. In nonactivated neutrophils, cortical actin and fodrin were cytosolic, thus indicating the absence of cortical structure in this population. However, cortical actin and fodrin were membrane-associated in activated neutrophils showing the existence of a cortex. Fodrin, actin, ezrin and their respective anchors, CD45 and CD43 did not codistribute with the plasma membrane marker, alkaline phosphatase, in sucrose density gradients made with primed neutrophils. These latter results suggested the lateral compartmentalization of the plasma membrane cortex into compositionally distinct surface domains. Additional studies were performed to examine the surface-association of hCap, a soluble microbicidal component of neutrophil specific granules. Results indicated association of hCap with primed and degranulated but not nonactivated plasma membranes. This interaction was resistant to 1M salt but labile to 10 mM NaOH, indicating a high affinity association. In support of this, hCap also co-partitioned with detergent in Triton X-114 phase experiments. In separate studies, marked alterations in the plasma membrane lipid metabolism of isolates from Candida glabrata are correlated with an ability to survive and grow in vivo. Altogether, this work provides insight into structure-function relationships at the plasma membrane level.
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Baranova, Ekaterina. "Electron crystallography of the membrane domain of respiratory complex I." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612931.
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Xu, Yuanda. "Thermodynamic and Hydrodynamic Coupling Effects on Compositional Lipid Domains in Membrane Stack Systems." Thesis, Princeton University, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10642189.
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This dissertation will focus on my work in biophysics, and my work in mean field games and glucose predictive analysis will not be presented. Several problems relating to the effects of thermodynamic coupling and hydrodynamic coupling within the membrane stack system are discussed. Three theoretical approaches are employed and proposed to study the membrane stack system: a diffuse-interface approach is utilized for numerical simulations; a coarse-grained sharp-interface approach is utilized to provide physical understanding of various kinetics; a hybrid intermediate sharp-interface approach is adopted to study the domain coalescence in the absence of diffusion.
In the first part of the thesis, we discuss the thermodynamic coupling in membrane stack systems. Comprehensive analyses are presented to understand the accelerated coarsening kinetics with respect to single layer and long-range alignment. Numerical simulations are conducted for three systems, namely a diffusion dominated system, an advective interlayer friction dominated system, and an advective membrane viscosity dominated system. Experimental results regarding the advective interlayer friction dominated system are supported by simulations. We investigate the mechanism of the enhanced coarsening kinetics in membrane stack systems and the relationship between the coarsening process and vertical alignment. An intuitive understanding along with analytical explanations are further presented. Moreover, numerical results regarding the critical mixture are also discussed.
We then investigate the interfacial fluctuation behavior within membrane stack systems. The hydrodynamic coupling is found to play a significant role and several physical length scales are found to be crucial. Both a sharp-interface approach and a diffuse-interface approach are employed to numerically simulate decay of interface fluctuations in representative two-membrane systems.
To measure the thermodynamic coupling in experiments, the hydrodynamic force needs to be quantified, especially for the non-circular domains. In the last part of this thesis, the drag coefficient relating domain velocity and force acting on the domain is calculated using perturbation theory within two limits: the first limit refers to a domain much larger than the hydrodynamic screening length; the second limit refers to a domain that is much smaller than the hydrodynamic screening length.
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Lefevre, Rémy. "Conception et réalisation d'une micropompe intelligente : applications dans le domaine biomédical." Phd thesis, Université de Grenoble, 2013. http://tel.archives-ouvertes.fr/tel-00935192.
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Cette thèse s'inscrit dans le développement d'un Dispositif Médical d'Injection (DMI) automatisé dans lequel est intégrée une micropompe en technologie silicium. Le cœur de cette micropompe est constitué d'une membrane actionnée permettant de déplacer un volume de liquide à travers des canaux micro-fluidiques. Deux types de membranes actionnées ont été étudiés : une membrane à actionnement bimétallique intégré et une membrane à actionnement piézoélectrique externe. Des simulations FEM ont permis d'affiner les modèles théoriques existants et de mieux rendre compte des effets non linéaires qui régissent le fonctionnement de ces membranes. Une méthode d'optimisation spécialement mise en place a permis de calculer des configurations géométriques optimales en fonction des plages de fonctionnement visées. Des membranes ont ensuite été fabriquées en salle blanche. Leurs caractéristiques mécaniques ont été mesurées et comparées aux prédictions des simulations FEM.
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Thevenin, Damien. "Roles of transmembrane domains in the folding and assembly of the adenosine A2A receptor." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 170 p, 2007. http://proquest.umi.com/pqdweb?did=1260822171&sid=2&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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Polonovski, Véronique. "Etude du mécanisme d'action de l'homéoprotéine Engrailed 2 : interaction protéine - protéine avec la protéine à domaine forkhead Foxa2 et interaction protéine - membrane." Paris 6, 2007. http://www.theses.fr/2007PA066490.
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L’identification et la compréhension des bases d’interactions entre protéines ou entre protéines et membranes sont fondamentales pour mieux comprendre les mécanismes impliqués et leurs applications. Les travaux présentés ici s’intéressent à l’homéoprotéine Engrailed 2 (En2HD) et à son interaction avec deux partenaires différents : le facteur de transcription à domaine forkhead Foxa2 et les bicouches lipidiques. La première partie de cette étude a concerné la détermination structurale partielle de la protéine Foxa2 par résonance magnétique nucléaire, puis l’analyse de résultats préliminaires de l’interaction entre les deux protéines obtenus par des tests de stabilité ou par une étude par résonance plasmonique de surface. La deuxième partie a permis d’identifier le milieu membranaire le plus proche des bicouches lipidiques natives et de réaliser une réattribution partielle de la structure de l’homéodomaine dans ce milieu, puis de réaliser une étude comparative des interactions entre l’homéodomaine ou l’hélice III d’Engrailed 2 et différents milieux membranaires (micelles, bicelles) par résonance magnétique nucléaire 1H-15N ou par résonance magnétique nucléaire du phosphore.
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Petrov, Eugene P., Rafayel Petrosyan, and Petra Schwille. "Translational and rotational diffusion of micrometer-sized solid domains in lipid membranes." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-139339.
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We use simultaneous observation of translational and rotational Brownian motion of domains in lipid membranes to test the hydrodynamics-based theory for the viscous drag on the membrane inclusion. We find that translational and rotational diffusion coefficients of micrometer-sized solid (gel-phase) domains in giant unilamellar vesicles showing fluid–gel phase coexistence are in excellent agreement with the theoretical predictionsDieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich
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Petrov, Eugene P., Rafayel Petrosyan, and Petra Schwille. "Translational and rotational diffusion of micrometer-sized solid domains in lipid membranes." Royal Society of Chemistry, 2012. https://tud.qucosa.de/id/qucosa%3A27824.
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We use simultaneous observation of translational and rotational Brownian motion of domains in lipid membranes to test the hydrodynamics-based theory for the viscous drag on the membrane inclusion. We find that translational and rotational diffusion coefficients of micrometer-sized solid (gel-phase) domains in giant unilamellar vesicles showing fluid–gel phase coexistence are in excellent agreement with the theoretical predictions.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
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Liang, Ying. "Characterization of Sad1/UNC-84 domain protein 2 (SUN2)." Click to view the E-thesis via HKUTO, 2006. http://sunzi.lib.hku.hk/hkuto/record/B36616515.
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Liang, Ying, and 梁瑛. "Characterization of Sad1/UNC-84 domain protein 2 (SUN2)." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B36616515.
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Kongmanas, Kessiri. "Roles of Seminolipid and Its Associated Membrane Domain in Male Fertility." Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/32509.
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Our research aims at understanding the roles of seminolipid (sulfogalactosylglycerolipid or SGG) and its associated membrane domains in male reproduction. SGG is a sulfoglycolipid present selectively and abundantly in mammalian male germ cells. Therefore, information on its properties would be relevant towards the development of male fertility biomarkers and spermicide-based contraceptives. We have shown that SGG has direct affinity for zona pellucida (ZP, egg extracellular matrix) and plays a role in the formation of sperm lipid rafts, the ZP-binding platforms on the sperm anterior head plasma membrane (APM), the initial ZP binding site. For a better understanding of mechanisms underlying sperm-ZP interaction, I performed proteomic characterization of APM vesicles (SGG-associated membrane domains with ZP affinity) isolated from sperm before and after capacitation, a process through which sperm gain maximal ZP affinity. Proteomic results revealed that capacitated APM vesicles contained high-molecular-weight protein complexes, with higher ZP affinity and levels of ZP-binding proteins as compared with those of the non-capacitated samples. ZP-binding proteins known to exist in the acrosome (i.e., zonadhesin, proacrosin/acrosin) were found in these APM protein complexes. Immunofluorescence suggested that a fraction of these proteins trafficked from the acrosome to APM during capacitation. These findings provided a new mechanism on how sperm gain full ZP-binding ability during capacitation. Since SGG is a major component of APM, proper SGG levels at this site would be important for male fertility. Levels of sperm SGG are regulated through the synthesis and degradation. In fact, lack of SGG-synthesis enzymes causes a spermatogenesis disruption, resulting in male infertility. However, significance of SGG degradation remains unknown. SGG can be desulfated in vitro by arylsulfatase A (ARSA), an enzyme existing in the acrosomes of sperm/spermatids and lysosomes of Sertoli cells, testicular somatic cells that nurture developing germ cells. Sertoli cells also phagocytose ~50% of germ cells that become apoptotic during spermatogenesis. To understand physiological importance of SGG degradation, the fertility status and SGG levels of Arsa-/- male mice were determined. We found that Arsa-/- males became subfertile when they were older than 5 months, and when they were 8-month-old (~40-year-old men) they produced sperm at 50% wild type rate. Arsa-/- sperm had minimal in vitro fertilizing ability and a number of them showed abnormal morphology. Quantitative mass spectrometry revealed that SGG levels in Sertoli cells of 8-month-old Arsa-/- mice were increased to ~250% of the wild type level; this SGG accumulation may lead to a decrease in Sertoli cell ability to support spermatogenesis. However, SGG levels in sperm of 8-month-old Arsa-/- mice were ~50% of the wild type value, a result that partly explained the decreased fertilizing ability of these sperm. The reduced SGG level of Arsa-/- sperm was likely due to a lack of SGG’s building-block lipid (palmitylpalmitoylglycerol) putatively generated in Arsa-/- Sertoli cells and recycled to the next generation of primary spermatocytes for SGG synthesis. Hence, levels of sperm SGG are a promising bioindex for male fertility. Since Sertoli cells also regulate SGG homeostasis, their functionality should be now included in male fertility/subfertility diagnosis.
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Scott, Angela Michelle. "Mechanism of membrane binding of the C2 domains of conventional protein kinase C isoforms." Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2006. http://wwwlib.umi.com/cr/ucsd/fullcit?p3222056.
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Thesis (Ph. D.)--University of California, San Diego, 2006.Title from first page of PDF file (viewed September 20, 2006). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 127-132).
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Saleh, Mazen T. "Identifying domains of Shiga-like toxin I that are responsible for its membrane translocation." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0002/NQ27715.pdf.
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Collinson, Ian Richard. "Studies of the membrane and stalk domains of ATP synthase from bovine heart mitochondria." Thesis, University of Cambridge, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360698.
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Lu, Ruifeng, and Jean M. Wilson. "Rab14 specifies the apical membrane through Arf6-mediated regulation of lipid domains and Cdc42." NATURE PUBLISHING GROUP, 2016. http://hdl.handle.net/10150/622499.
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The generation of cell polarity is essential for the development of multi-cellular organisms as well as for the function of epithelial organs in the mature animal. Small GTPases regulate the establishment and maintenance of polarity through effects on cytoskeleton, membrane trafficking, and signaling. Using short-term 3-dimensional culture of MDCK cells, we find that the small GTPase Rab14 is required for apical membrane specification. Rab14 knockdown results in disruption of polarized lipid domains and failure of the Par/aPKC/Cdc42 polarity complex to localize to the apical membrane. These effects are mediated through tight control of lipid localization, as overexpression of the phosphatidylinositol 4-phosphate 5-kinase a [PtdIns(4) P5K] activator Arf6 or PtdIns(4) P5K alone, or treatment with the phosphatidylinositol 3-kinase (PtdInsI3K) inhibitor wortmannin, rescued the multiple-apical domain phenotype observed after Rab14 knockdown. Rab14 also co-immunoprecipitates and colocalizes with the small GTPase Cdc42, and Rab14 knockdown results in increased Cdc42 activity. Furthermore, Rab14 regulates trafficking of vesicles to the apical domain, mitotic spindle orientation, and midbody position, consistent with Rab14' s reported localization to the midbody as well as its effects upon Cdc42. These results position Rab14 at the top of a molecular cascade that regulates the establishment of cell polarity.
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Grosjean, Kevin. "Microdomaines ordonnés de la membrane plasmique végétale : caractérisation et rôle dans la signalisation associée à la défense." Thesis, Dijon, 2015. http://www.theses.fr/2015DIJOS089/document.
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Au cours de ces dernières années, des études ont montré l’existence d’une compartimentation latéraledes composants de la membrane plasmique végétale, de manière analogue à ce qui avait été montréchez les animaux et les levures. L’objectif de cette thèse était d’apporter de nouveaux éléments decaractérisation de cette compartimentation (propriétés physiques de domaines particuliers, mécanismesde mise en place de ces domaines, de contrôle de leur taille, etc…) et d’étudier son rôle dans laphysiologie de la cellule végétale.Le développement d’une méthodologie de microscopie confocale spectrale couplée à l’utilisationd’une sonde environnementale a permis d’apporter la première description à l'échellesubmicrométrique de l’organisation du plasmalemme en territoires aux propriétés physiquesdifférentiées. Ces domaines coexistent au sein de la membrane plasmique de cellules en suspension,comme à celle de membranes artificielles composées de lipides modèles ou de lipides de membranescellulaires, de vésicules géantes constituées de membrane plasmique purifiée, ou de protoplastes.Cependant, les différences de l’organisation latérale observées chez ces différentes membranes ontpermis de montrer l’importance des phytostérols qui seraient, par le biais d'interactions spécifiquesavec d’autres lipides végétaux tels que les GIPCs, des composés essentiels pour la formation locale dedomaines lipidiques ordonnés. La grande diversité des lipides végétaux organiserait ainsi lacompartimentation de la membrane plasmique permettant la ségrégation dynamique des composantsmembranaires. Si les stérols augmentent de manière importante le degré de compaction de la bicouche,les protéines le diminuent. Le cytosquelette et la paroi ne semblent, quant à eux, modifier ni laprésence, ni l’organisation des domaines ordonnés de la membrane plasmique. Nous avons égalementmontré que l’organisation de ces domaines évolue transitoirement lors des étapes précoces de lacascade de signalisation induite par des réactions de défense. De fait, nous avons identifié desmodifications des propriétés physiques globales et de l’organisation fine de la membrane provoquéespar différents éliciteurs de réactions de défense, dont la cryptogéine, une protéine sécrétée parl’oomycète Phytophthora cryptogea. Nous avons montré que ces modifications sont un élémentgénérique de la signalisation de défense, sous la dépendance de phénomènes de phosphorylation, leburst oxydatif étant également une étape clé de l’augmentation du degré d’ordre observé dans lesphases précoces de cette signalisation. La cryptogéine, qui présente une aptitude singulière pour piégerles stérols, a également montré une capacité spécifique à augmenter la fluidité membranaire, ceparamètre pouvant contrôler l’intensité de la cascade de signalisation, mesurée par la production deformes actives d’oxygène.Ces résultats ouvrent de nouvelles perspectives dans la compréhension des interactions cellule-élicitineet apportent un nouvel éclairage sur le rôle des lipides végétaux dans l’organisation latérale de lamembrane plasmique végétale et positionne la dynamique membranaire comme un élément designalisation de défense des plantesRecent studies have shown the existence of lateral sub-compartmentalization of plant plasmamembrane similar to that of animal cells and yeasts. The aim of this thesis was to provide newelements to characterize this compartmentalization (physical properties of specific domains,mechanisms of their formation, determination of their size, etc...) and to study its role in thephysiology of plant cells.The development spectral confocal microscopy coupled with the use of an environment-sensitiveprobe enabled to obtain the first description at the submicron scale of plasma membrane organizationinto domains exhibiting various physical properties. These domains coexist at the plasma membranesurface of tobacco suspension cells as well as the membrane of vesicles composed of models lipids orcell plasma membrane lipids, purified plasma membrane vesicles, and protoplasts. However,differences in the lateral organization observed in these membranes have shown the importance ofphytosterols which are, through specific interactions with neighboring plant lipids such as GIPCs,essential for local formation of ordered domains. The huge diversity of plant lipids drives thecompartmentalization of the plasma membrane allowing the dynamic segregation of membranecomponents. Sterols greatly increase membrane order, whereas proteins tends to decrease it.Cytoskeleton and cell wall do alter neither presence nor organization of ordered domains of the plasmamembrane. We have also shown that the organization of these domains is transiently modified duringthe early stages of defense signaling cascade. In fact, we have identified changes in overall physicalproperties and fine lateral organization of the membrane caused by various elicitors of defensereactions, including cryptogein, a protein secreted by the oomycete Phytophthora cryptogea. We haveshown that these changes are a generic element of defense signaling cascade and depend onphosphorylation processes; oxidative burst being also a major actor of the control of the increase ofmembrane order observed during the very early stages of the signalling process. Cryptogein, whichexhibits the particular ability to trap sterols, also showed a specific capacity to increase membranefluidity and amplify the intensity of the signalling cascade, as measured by the production of reactiveoxygen species.These results open new perspectives in the understanding of cell-elicitin interactions and provide anew view on the central role of sterol composition in the lateral organization of plant plasmamembrane. They also identify membrane dynamics as a new player in the signalling cascade occurringduring plant defense
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Chojnacki, Jakub. "Envelope protein domains of duck hepatitis B virus : role in assembly and infectivity /." Connect to thesis, 2005. http://eprints.unimelb.edu.au/archive/00001738.
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Thesis (Ph.D.)--University of Melbourne, Dept. of Microbiology and Immunology, The Macfarlane Burnet Institute for Medical Research and Public Health, 2006.Typescript. Includes bibliographical references (leaves 155-177).
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Pinto, Belinda Sophia Geyer Pamela Kent. "Understanding the role of LEM domain proteins in Drosophila development." [Iowa City, Iowa] : University of Iowa, 2009. http://ir.uiowa.edu/etd/421.
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Nikolaus, Jörg. "Hemifusion and lateral lipid domain partition in lipid membranes of different complexity." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16437.
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Die Fusion von Membranen erfordert die Verschmelzung von zwei Phospholipiddoppel-schichten, wobei dies über dieselben Zwischenschritte abzulaufen scheint. Eine lokale Störung (‚Stalk’) stellt eine erste Verbindung der äußeren Membranhälften dar, die anschließend lateral expandiert und ein Hemifusionsdiaphragma (HD) bildet. Das Öffnen einer Fusionspore im HD führt zur vollständigen Fusion. Mittels konfokaler Mikroskopie wurde die Fusion von Giant unilamellar vesicles (GUVs) mit negativ geladenen Lipiden und transmembranen (TM) Peptiden in Anwesenheit von zweiwertigen Kationen beobachtet, wobei die Peptide bei der HD Entstehung völlig verdrängt wurden. Eine detaillierte Analyse zeigte, dass es sich bei diesem Mikrometer-großen Bereich um ein HD handelt, dessen Größe von der Lipidzusammensetzung und Peptidkonzentration in den GUVs abhängt. Laterale Lipiddomänen gelten als entscheidend für Signal- und Sortierungsprozesse in der Zelle. Liquid ordered (Lo) Domänen in Modellsystemen wie GUVs ähneln den mit Sphingo-lipiden und Cholesterol angereicherten biologischen Raft-Domänen, allerdings scheinen Membraneigenschaften wie die Lipidpackung sich von biologischen Membranen zu unterscheiden. In diesem Zusammenhang wird die Sortierung des TM-verankerten Hemag-glutinin (HA) des Influenzavirus und von lipidverankerten Ras-Proteinen in GUVs wie auch in abgelösten Plasmamembran-Ausstülpungen (GPMVs) untersucht. HA Protein und TM-Pepitde von HA wurden ausschließlich (GUVs) bzw. vorwiegend (GPMVs) in der liquid disordered (Ld) Domäne gefunden. K-Ras wurde inmitten der Ld detektiert, während N-Ras zur Lo/Ld Grenzlinie diffundierte. Diese Ergebnisse werden im Zusammenhang mit den Unterschieden der Lipidpackung innerhalb der verschiedenen membranverankerten Systeme diskutiert. Es ist wahrscheinlich, dass die Bildung, Größe und Stabilität sowie die physikalischen Eigenschaften der Lipiddomänen in biologischen Membranen stark von Protein-Lipid-Wechsel-wirkungen beeinflusst werden.Membrane fusion is ubiquitous in life and requires remodelling of two phospholipid bilayers. Fusion likely proceeds through similar sequential intermediates. A stalk between the contacting leaflets forms and radially expands into a hemifusion diaphragm (HD) wherein finally a fusion pore opens up. Direct experimental verification of this key structure is difficult due to its transient nature. Confocal microscopy was used to visualize the fusion of giant unilamellar vesicles (GUVs) comprising negatively charged phosphatidylserine and fluorescent transmembrane (TM) entities in the presence of divalent cations. A complete displacement of TM peptides preceded full fusion. This is consistent with HD formation. Detailed analysis provided proof that the micrometer sized structures are in fact HDs. HD size is dependent on lipid composition and peptide concentration. Lateral lipid domain formation is believed to be essential for sorting and signalling processes in the cell. Liquid ordered (Lo) domains in model systems like GUVs resemble biological rafts enriched in sphingolipids and cholesterol, but their physical properties seem distinct from biological membranes as judged by e.g. lipid order and packing. In this context the sorting of TM anchored influenza virus hemagglutinin (HA) and different lipid anchored Ras proteins is studied in GUVs and giant plasma membrane derived vesicles (GPMVs). Authentic HA or the TM domain peptides were sorted exclusively (GUVs) or predominantly (GPMVs) to the liquid disordered (Ld) domains. Whereas K-Ras was found in the bulk Ld domains, N-Ras diffuses to the Lo/Ld interface. These results are discussed with respect to differences in lipid packing in the different membrane systems and regarding the membrane anchors and their hydrophobic matching. The results suggest that the formation, size and stability as well as the physical properties of lipid domains in biological membranes are tightly regulated by protein-lipid interactions.
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Holmlund, Camilla. "Identification and investigations of leucine-rich repeats and immunoglobulin-like domains protein 2 (LRIG2)." Doctoral thesis, Umeå : Department of Radiation Sciences, Oncology, Umeå university, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-33784.
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Young, Mark. "Studies of the transmembrane domain of the human erythrocyte anion exchanger (band 3)." Thesis, University of Bristol, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340324.
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