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Pong, Mona Wrenn Steven Parker. "Ultrasound and model membrane interaction /." Philadelphia, Pa. : Drexel University, 2007. http://hdl.handle.net/1860/2520.
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Bories, Florent. "Interaction entre inclusions transmembranaires transmise par la membrane cellulaire." Sorbonne Paris Cité, 2015. http://www.theses.fr/2015USPCC224.
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L'objet de cette thèse est d'étudier les interactions entre inclusions transmembranaires imposant un excès d'épaisseur en utilisant un modèle élastique décrivant les membranes au niveau de leur épaisseur. Dans un premier temps, je montre que ce modèle généralise bien les précédents en prenant en compte toutes les constantes physiques possibles. J'ajoute ensuite une condition d'ancrage au bord de l'inclusion qui peut induire ou non une pente préférentielle. Je m'assure que les résultats trouvés dans le cadre de mon modèle rejoignent le précédent pour une seule inclusion dans deux cas limites. Dans un deuxième temps, je développe une méthode de calcul multipolaire me permettant d'envisager des calculs de la forme d'une membrane où plusieurs inclusions sont présentes. Je donne les solutions générales de ce modèle et donne une manière de déterminer la solution dans le cas où deux inclusions sont présentes dans une membrane de taille infinie. Enfin, je donne pour une bicouche lipidique typique certaines courbes de profil et d'énergie d'interaction attendues. Je compare mes résultats à des expériences de Constantin à l'aide d'un algorithme de traitement reposant sur l'équation d'Ornstein-Zernike et une relation de fermeture. Le premier système "C12E5 + gramicidine", dont la membrane est constituée de surfactants, me permet de faire concorder le modèle théorique avec les expériences et je donne ainsi pour celui-ci les premières mesures de paramètres physiques nouveaux. Le deuxième système "DLPC + gramicidine" donne un moins bon accord entre la théorie et les expériences mais je donne une nouvelle piste de traitement afin de donner des estimations pour ce systèmeThe present thesis is a study of interactions between transmembrane proteins inducing a hydrophobic mismatch with an elastic model describing the membranes at the scale of their thickness. I begin by showing that this model generalizes the precedent ones found in litterature by taking in account every possible physical constants. I add also an anchoring term at the edge of the inclusion that can induce a preferential slope. I verify that the results found with this addition is what was found previously with one inclusion in a membrane in two différent cases. Next, I develop a multipolar computation method that allows me to compute the shape of a membrane where several inclusions are presents. I give the general solutions of this model and gives an algorithm in the case where two inclusions are present in an infinite membrane. Then, I give the expected profile and the interaction energies for a typical lipidic bilayer. I compare my results to experiments performed by Constantin with an algorithm using Omstein-Zernike equation and closure relations. The first system "C12E5 + gramicidin", where the membrane is made of surfactant, gives good agreement between the theory and the experiments and allows me to give a first measurement for new physical parameters. The second system "DLPC + gramicidin" does not allow such an agreement between the theory and the experiments but I give a new lead which may give a measurement for this system
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Devaka, K. Weerakoon Cheung H. Tak. "Interaction of macrophages with the basement membrane." Normal, Ill. Illinois State University, 1995. http://wwwlib.umi.com/cr/ilstu/fullcit?p9603526.
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Thesis (Ph. D.)--Illinois State University, 1995.Title from title page screen, viewed May 8, 2006. Dissertation Committee: Hou Tak Cheung (chair), David W. Borst, Herman E. Brockman, Alan J. Katz, Anthony J. Otsuka. Includes bibliographical references (leaves 98-110) and abstract. Also available in print.
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Ma, Xin. "The interaction between amyloid beta peptide and phospholipids." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/29637.
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The aim of the thesis project was to examine what form(s) of Amyloid beta (Aβ) (25-‐35) peptide interact with phospholipids in vitro and the implications of this for the mechanism of Alzheimer’s Diseases (AD). The mechanism of AD is thought to involve protein folding and misfolding. An increasing amount of evidence has shown that protein misfolding plays an important role in the biological and pathological processes of AD. Although seen as the biomedical markers of those diseases, the roles of amyloid aggregates themselves are still not fully understood. Whether the aggregates, or the monomer, or some other intermediates of Aβ cause AD is still unknown. In order to investigate the membrane-‐interaction of Aβ and its implications for AD, two forms of Aβ, namely levorotary and dextrorotary (L-‐ and D-‐) Aβ isomers were used. Evidence has shown that L-‐ and D-‐ peptide can each form aggregates in a humid environment. However, when mixed together, L-‐ and D-‐ peptides tend not to form any aggregates. Using the mixtures of L-‐ and D-‐ peptides at different proportions and as well as using L-‐ and D-‐ alone can help us to determine the toxic form of Aβ. Phospholipids have been used to mimic membrane bilayers. Biological membranes in vivo are a complicated system. They contain three types of lipids, namely phospholipids, glycolipids, and steroids. Different types of cells and different membranes have different proportions of those lipids. Studying the interaction between Aβ and membranes in vivo can be extremely difficult. Artificial membranes, which only contains one kind of lipids, on the other hand, are a useful tool for the study of molecular interactions. Phospholipids are the most abundant type of membrane lipid and thus that can be seen as representative of cell membranes. The interactions of Aβ and different kinds of phospholipids have been investigated in this project. This thesis discusses the secondary structure of Aβ in different environment, the interaction between Aβ and phospholipids at the air-‐water surface, and the location of Aβ in membranes during the interaction. The study provides useful information of the mechanisms and the origin of AD. At the end of the thesis, a discussion chapter analyses the difficulties of studying Aβ and AD and the potentials and inadequacies of this research.
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Blixt, Ylva. "Early interaction between adenovirus type 2 and HeLa cells significance of the plasma membrane constitution /." Lund : Dept. of Microbiology, University of Lund, 1992. http://books.google.com/books?id=DzhrAAAAMAAJ.
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Carvalho, Kévin. "Interaction entre membrane plasmique et cytosquelette : approche biomimétique pour l’étude des interactions entre ezrine, PIP2 et actine." Montpellier 2, 2009. http://www.theses.fr/2009MON20175.
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La membrane plasmique de la cellule est composée de lipides et interagit notamment avec le squelette de la cellule (le cytosquelette), par l'intermédiaire de protéines d'ancrages et de lipides clefs qui jouent un rôle spécifique dans certains types d'interactions. Parmi les protéines intervenant dans l'ancrage direct de la membrane plasmique au cytosquelette, des protéines de la famille des ERM (Ezrine, Radixine, Moésine) peuvent interagir spécifiquement avec un lipide, le phosphatidylinositol (4,5) biphosphate (PiP2), d'une part et avec l'actine du cytosquelette d'autre part. Dans le but de comprendre les interactions entre membrane plasmique et cytosquelette, nous avons réalisé des expériences in vitro sur des systèmes comportant un nombre minimal de constituants : des vésicules géantes (GUV) contenant du PiP2, de l'ezrine recombinante et de l'actine purifiée. Nous avons mis en évidence que la liaison au PiP2 induit des changements conformationnels de l'ezrine. L'ezrine est alors capable d'interagir avec les filaments d'actine. Nous avons caractérisé quantitativement l'incorporation de PiP2 dans la membrane de vésicule géantes, et montré que l'interaction de l'ezrine avec les vésicule géante contenant du PiP2, induit un partitionnement du lipide dans la bicouche lipidique et conduit à la formation d'agrégats de PiP2 et d'ezrine sur la membrane. La connaissance des effets de l'ezrine sur les membranes contenant du PiP2 et la connaissance des différents mécanismes se produisant lors des interactions permettra de définir plus précisément le rôle de l'ezrine in celluloThe plasma membrane is composed of several different lipids and can interact directly with the actin cytoskeleton via specific lipids and linker proteins. Among these is the ERM (Ezrin, Radixin, Moesin) family of proteins, which is involved in the direct linkage of the membrane to the actin cytoskeleton via a phosphatidylinositol (4,5) biphosphate (PiP2) lipid binding site. Our aim is to understand the interactions between these proteins and PiP2 using in vitro simplifed biomimetic systems like giant unilamellar vesicles (GUV) containing PiP2. We showed that a conformational change of ezrin occur when the protein binds to PiP2, this conformational change allowing ezrin to bind to actin filaments. We have characterized quantitatively the incorporation of PIP2 in the membrane of giant vesicles, and showed that the interaction of ezrin with GUV induce a partitioning of the lipid within the membrane as well as ezrin aggregates on the membrane. Likewise, ezrin oligomers were observed only in the presence of PiP2. A better understanding of the interplay between ezrin, PIP2-containing membranes and actin will help to get a better view of the role of ezrin in cellulo
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Magzoub, Mazin. "Cell-penetrating peptides in model membrane systems : interaction, structure induction and membrane effects /." Stockholm : Institutionen för biokemi och biofysik, Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-247.
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Veiro, Jeffrey. "Multi-nuclear NMR studies of phospholipid membranes and their interaction with membrane active substances." Thesis, University of South Wales, 1985. https://pure.southwales.ac.uk/en/studentthesis/multinuclear-nmr-studies-of-phospholipid-membranes-and-their-interaction-with-membrane-active-substances(2650bad7-1bc8-463a-8d27-56d1347a7a8f).html.
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Multinuclear magnetic resonance spectroscopy in conjunction with lanthanide shift reagents was used to investigate the interaction of unilamellar phospholipid vesicles with membrane active substances. The regulation of ion channels by lipids such as the phosphatidic acids was investigated using 1H-NMR and small phospholipid vesicles. Transport across lipid vesicles in the presence of the ionophores alamethicin, melittin and nystatin was monitored using the lanthanide probe ion Pr3+. Channel characteristics were found to be dependent upon molecular interactions between the lipid and the individual ionophores. The results were discussed in terms of the phosphatidylinositol effect. The NMR technique provided methods whereby intervesicle ionophore exchange was studied. The results revealed that ionophore exchange between vesicles, the mechanism of exchange and the rate of ion conduction were dependent upon the initial environment of the ionophore and also the lipid composition of the vesicle. The modulation of a variety of mechanisms of channel-mediated transport across small phospholipid vesicles by a range of general anaesthetics were investigated using 1H-NMR. Membrane permeability was found to be inhibited by inhalation anaesthetics independently of the channel system or lipid composition used. The results indicated the importance of hydrogen bonding as an explanation of the observed inhibition. 19F-NMR was used to monitor signals from the fluorinated anaesthetics themselves, the results providing information on the disposition of the anaesthetics within the bilayer. 23Na + and 7Li transport across large vesicles was monitored using 23Na+ and 7Li+-NMR. The effect of the general anaesthetics on ion transport was found to be dependent upon the ionophore and the type of metal ions present in the vesicular solution, further suggesting the importance of hydrophilic interactions of the anaesthetics. Finally, 31P-NMR was used to show the inhibition by general anaesthetics of the hydrolysis of glucose-6-phosphate by the enzyme glucose-6-phosphatase which further supported the above conclusions on anaesthetic action.
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PIZZICHEMI, MARCO. "Interaction of pulsed electric fields with cell membrane." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2009. http://hdl.handle.net/10281/7790.
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Pulsed Electric Fields (PEF) allow non-thermal pasteurization and sterilization of liquids, involving the application of high intensity electric field pulses (20-80 kV/cm) of short duration (1-10 microseconds). The electric field interacts with microorganisms at the level of plasma membrane, through the mechanism of electroporation, but, although many theories have been proposed to describe this phenomenon, a satisfactory explanation has not been found yet. An extensive description of the state of the art of the knowledge on this field is given, along with the current research needs and the main obstacles to a wide diffusion of PEF applications.
The time course of cell transmembrane voltage is studied, developing an analytical expression for planar, spherical, cylindrical and prolate spheroidal membranes, with the aim to evaluate the charging time constants and the steady state intensity upon variation of treatment conditions. The results of this approach are used to investigate the impact of the electric field in rod-like bacteria. The electric field distribution in test chamber is studied by means of computer simulations for various electrode geometries, optimizing the shape for intensity and uniformity of electric field. The impact on PEF treatment of a normal distribution of cell dimensions in a microbial population and the rotational movement of bacteria inside treatment chambers are also investigated. Computer simulations are used to obtain a possible explanation to the deviations from first order inactivation kinetics, and to the intrinsic variability of microbial laboratory results.
PEF inactivation experiments of Escherichia Coli are carried out in a test system under different conditions of treatment duration and microbial concentration. Inactivation kinetics is compared to theories of electroporation and computer simulations previously carried out. Experimental evidence of a variation in the effectiveness of PEF as a function of bacterial concentration is discovered and a possible explanation proposed. The application of high permittivity ceramics materials to PEF is also studied, with the aim of increasing the volumes of treatment chambers, improving the duration of electrodes, and allowing the use of the most energy efficient square wave pulses to large volumes of liquid. These materials can also be used to test the possibility of a relation between energy deposited in treatment chambers and microbial inactivation, in order to acquire more insight on the interaction between Pulsed Electric Fields and cell membranes. A novel chamber is prepared with the use of a high permittivity non-toxic material (Sodium Potassium Niobate) and it is ready to be tested in PEF applications.
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Hernandez, Lopez Agustin. "Plasma membrane sterols and fatty acids : effects on membrane properties and H'+-ATPase of Ustilago maydis." Thesis, University of Bristol, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336825.
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Dannehl, Claudia. "Fragments of the human antimicrobial LL-37 and their interaction with model membranes." Phd thesis, Universität Potsdam, 2013. http://opus.kobv.de/ubp/volltexte/2013/6814/.
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A detailed description of the characteristics of antimicrobial peptides (AMPs) is highly demanded, since the resistance against traditional antibiotics is an emerging problem in medicine. They are part of the innate immune system in every organism, and they are very efficient in the protection against bacteria, viruses, fungi and even cancer cells. Their advantage is that their target is the cell membrane, in contrast to antibiotics which disturb the metabolism of the respective cell type. This allows AMPs to be more active and faster. The lack of an efficient therapy for some cancer types and the evolvement of resistance against existing antitumor agents make AMPs promising in cancer therapy besides being an alternative to traditional antibiotics.
The aim of this work was the physical-chemical characterization of two fragments of LL-37, a human antimicrobial peptide from the cathelicidin family. The fragments LL-32 and LL-20 exhibited contrary behavior in biological experiments concerning their activity against bacterial cells, human cells and human cancer cells. LL-32 had even a higher activity than LL-37, while LL-20 had almost no effect. The interaction of the two fragments with model membranes was systematically studied in this work to understand their mode of action. Planar lipid films were mainly applied as model systems in combination with IR-spectroscopy and X-ray scattering methods. Circular Dichroism spectroscopy in bulk systems completed the results.
In the first approach, the structure of the peptides was determined in aqueous solution and compared to the structure of the peptides at the air/water interface. In bulk, both peptides are in an unstructured conformation. Adsorbed and confined to at the air-water interface, the peptides differ drastically in their surface activity as well as in the secondary structure. While LL-32 transforms into an α-helix lying flat at the water surface, LL-20 stays partly unstructured. This is in good agreement with the high antimicrobial activity of LL-32.
In the second approach, experiments with lipid monolayers as biomimetic models for the cell membrane were performed. It could be shown that the peptides fluidize condensed monolayers of negatively charged DPPG which can be related to the thinning of a bacterial cell membrane. An interaction of the peptides with zwitterionic PCs, as models for mammalian cells, was not clearly observed, even though LL-32 is haemolytic.
In the third approach, the lipid monolayers were more adapted to the composition of human erythrocyte membranes by incorporating sphingomyelin (SM) into the PC monolayers. Physical-chemical properties of the lipid films were determined and the influence of the peptides on them was studied. It could be shown that the interaction of the more active LL-32 is strongly increased for heterogeneous lipid films containing both gel and fluid phases, while the interaction of LL-20 with the monolayers was unaffected. The results indicate an interaction of LL-32 with the membrane in a detergent-like way.
Additionally, the modelling of the peptide interaction with cancer cells was performed by incorporating some negatively charged lipids into the PC/SM monolayers, but the increased charge had no effect on the interaction of LL-32. It was concluded, that the high anti-cancer activity of the peptide originates from the changed fluidity of cell membrane rather than from the increased surface charge. Furthermore, similarities to the physical-chemical properties of melittin, an AMP from the bee venom, were demonstrated.Aufgrund der steigenden Resistenzen von Zellstämmen gegen traditionelle Therapeutika sind alternative medizinische Behandlungsmöglichkeiten für bakterielle Infektionen und Krebs stark gefragt. Antimikrobielle Peptide (AMPs) sind Bestandteil der unspezifischen Immunabwehr und kommen in jedem Organismus vor. AMPs lagern sich von außen an die Zellmembran an und zerstören ihre Integrität. Das macht sie effizient und vor allem schnell in der Wirkung gegen Bakterien, Viren, Pilzen und sogar Krebszellen. Das Ziel dieser Arbeit lag in der physikalisch-chemischen Charakterisierung zweier Peptidfragmente die unterschiedliche biologische Aktivität aufweisen. Die Peptide LL-32 und LL-20 waren Teile des humanen LL-37 aus der Kathelizidin-Familie. LL-32 wies eine stärke Aktivität als das Mutterpeptid auf, während LL-20 kaum aktiv gegen die verschiedenen Zelltypen war. In dieser Arbeit wurde die Wechselwirkung der Peptide mit Zellmembranen systematisch anhand von zweidimensionalen Modellmembranen in dieser Arbeit untersucht. Dafür wurden Filmwaagenmessungen mit IR-spektroskopischen und Röntgenstreumethoden gekoppelt. Circulardichroismus-Spektroskopie im Volumen komplementierte die Ergebnisse. In der ersten Näherung wurde die Struktur der Peptide in Lösung mit der Struktur an der Wasser/Luft-Grenzfläche verglichen. In wässriger Lösung sind beide Peptidfragmente unstrukturiert, nehmen jedoch eine α-helikale Sekundärstruktur an, wenn sie an die Wasser/Luft-Grenzfläche adsorbiert sind. Das biologisch unwirksamere LL-20 bleibt dabei teilweise ungeordnet. Das steht im Zusammenhang mit einer geringeren Grenzflächenaktivität des Peptids. In der Zweiten Näherung wurden Versuche mit Lipidmonoschichten als biomimetisches Modell für die Wechselwirkung mit der Zellmembran durchgeführt. Es konnte gezeigt werden, dass sich die Peptide fluidisierend auf negativ geladene Dipalmitylphosphatidylglycerol (DPPG) Monoschichten auswirken, was einer Membranverdünnung an Bakterienzellen entspricht. Eine Interaktion der Peptide mit zwitterionischem Phosphatidylcholin (PC), das als Modell für Säugetierzellen verwendet wurde, konnte nicht klar beobachtet werden, obwohl biologische Experimente das hämolytische Verhalten zumindest von LL-32 zeigten. In der dritten Näherung wurde das Membranmodell näher an die Membran von humanen Erythrozyten angepasst, indem gemischte Monoschichten aus Sphingomyelin (SM) und PC hergestellt wurden. Die physikalisch-chemischen Eigenschaften der Lipidfilme wurden zunächst ausgearbeitet und anschließend der Einfluss der Peptide untersucht. Es konnte anhand verschiedener Versuche gezeigt werden, dass die Wechselwirkung von LL-32 mit der Modellmembran verstärkt ist, wenn eine Koexistenz von fluiden und Gelphasen auftritt. Zusätzlich wurde die Wechselwirkung der Peptide mit der Membran von Krebszellen imitiert, indem ein geringer Anteil negativ geladener Lipide in die Monoschicht eingebaut wurde. Das hatte allerdings keinen nachweislichen Effekt, so dass geschlussfolgert werden konnte, dass die hohe Aktivität von LL-32 gegen Krebszellen ihren Grund in der veränderten Fluidität der Membran hat und nicht in der veränderten Oberflächenladung. Darüber hinaus wurden Ähnlichkeiten zu Melittin, einem AMP aus dem Bienengift, dargelegt. Die Ergebnisse dieser Arbeit sprechen für einen Detergenzien-artigen Wirkmechanismus des Peptids LL-32 an der Zellmembran.
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Hemming, Nicola Jane. "The interaction of protein 4.1 with the erythrocyte membrane." Thesis, University of Bristol, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240878.
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Walter, Vivien. "Lipid membrane interaction with self-assembling cell-penetrating peptides." Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAE032/document.
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Les peptides pénétrateurs de cellule (CPP) sont des oligopeptides cationiques faisant parti des vecteurs les plus étudiés dans le cadre du développement du transport ciblé de médicament à l’intérieur de l’organisme. Les applications principales sont par exemple le traitement des cancers ou la thérapie génique. Néanmoins, certaines caractéristiques des CPPs rendent leur utilisation médicale compliquée, tels que leur manque de spécificité à l’égard des cellules cibles ou la perte de leurs propriétés pénétrantes lorsqu’un cargo moléculaire leur est greffé. L’une des solutions envisagées pour résoudre ces problèmes est le greffage sur des polypeptides di-blocs auto-assemblés basés sur de l’élastine (ELPBC), des systèmes développés par l’équipe d’Ashutosh Chilkoti à l’Université de Duke (USA). Des travaux précédents ont montré que ces macromolécules, que l’on appelle CPP-ELPBC, retrouvaient les propriétés pénétrantes du CPP même en présence d’un cargo et permettaient également d’induire une spécificité à l’encontre des cellules cancéreuses. En revanche, le mécanisme de pénétration de ces systèmes restait inconnu.Dans cette thèse, je me suis concentré sur l’étude du mécanisme de pénétration des CPP et des CPP-ELPBC au travers de membranes lipidiques modèles, et en particulier sur l’adsorption de ces molécules à la surface de vésicules unilamellaires géantes (GUV). Le développement d’une nouvelle méthode de quantification de la fluorescence en microscopie confocale m’a permis de réaliser des mesures simples de comptage de peptides à la surface des vésicules, ce qui m’a permis par la suite de procéder à des mesures thermodynamiques de l’adsorption des peptidesCell-penetrating peptides (CPP) are cationic oligopeptides currently investigated as potential vectors for targeted drug delivery design, for applications in cancer treatment and/or gene therapy. Nevertheless, some drawbacks make the CPP complex for medical applications, such as their lack of specificity toward target cells or the loss of their penetrating properties once they have been grafted with a molecular cargo. One of the solutions studied to overcome these issues is the binding of the CPP unit on a self-assembling elastin-like diblock polypeptide (ELPBC), a macromolecular system designed by the team of Ashutosh Chilkoti from Duke University (USA). While it has already been proven that these molecules, named CPP-ELPBC, recover the penetrating properties of the CPP despite the presence of a cargo and also induce a selectivity toward tumorous cells, the exact mechanism of translocation is still under debate.In this PhD thesis, I focused on the investigation of the translocation mechanism of the CPP and CPP-ELPBC using model lipid membranes, and specifically the adsorption of these molecules at the surface of giant unilamellar vesicles (GUV). The development of a new quantification method of fluorescence in confocal microscopy allowed me to directly count the peptides adsorbed on the surface of the GUVs, which I used to perform thermodynamic measurements on the peptide adsorption
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Divet, François. "Fluctuations d'une membrane en interaction avec un champ diffusif." Université Joseph Fourier (Grenoble), 2001. http://www.theses.fr/2001GRE10194.
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Les bicouches de phospholipides sont des membranes modèles permettant de mimer les membranes des cellules et des organites cellulaires. Les membranes réelles contiennent en plus des protéines membranaires à la surface ou à l'intérieur de la bicouche. Ce travail de thèse constitue une contribution théorique à l'influence de l'adsorbtion de molécules (atomes, protéines. . . ) à la surface des membranes modèles. Les molécules absorbées agissent sur les propriétés élastiques des membranes de deux façons : (i) elles induisent une courbure spontanée proportionnelle à la concentration membranaire et/ou (ii) elles augmentent la constante de rigidité de la membrane proportionnellement à la concentration de molécules adsorbées. Les molécules adsorbées diffusent à la surface de la membrane et peuvent désorber dans le fluide environnant. Ce couplage entre molécules adsorbées et molécules en volume est pris en compte pour la première fois dans ce travail et s'applique au cas de membrane hors de l'équilibre (gradient de concentration volumique). Nous nous sommes cependant restreints, quant à l'exploitation de modèle, à la situation d'équilibre. Nous avons mis en évidence que les membrane planes et les vésicules quasi-sphériques sont déstabilisées par l'adsorbtion de molécules si le paramètre de couplage entre courbure et concentration membranaire est supérieur à une valeur critique. Lorsqu'elles sont stables (petits paramètres de couplage), l'étude des fluctuations thermiques donne des formations microscopiques sur les paramètres cinétiques comme le temps d'adsorbtion des molécules à la surface de la membrane. Nous avons également trouvé les formes d'équilibre bidimensionnelles pour des vésicules dégnoflées et montré que la connaissance de la concentration le long de la membrane permet de trancher entre les deux modèles (i) et (ii).
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See, Sarah Bihui. "Outer membrane protein immunity to Pasteurella pneumotropica and the interaction of allergy." University of Western Australia. School of Paediatrics and Child Health, 2010. http://theses.library.uwa.edu.au/adt-WU2010.0103.
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[Truncated abstract] Infectious and allergic diseases of the respiratory tract are major contributors to global mortality, morbidity and economic burden. Bacterial infections such as pneumonia and otitis media are important diseases, especially in children, while allergic diseases such as asthma and allergic rhinitis afflict up to 30% of the world's population. A confounding aspect of respiratory disease is the evidence of a complex relationship between respiratory allergy and respiratory infection, with infection suggested to both promote and prevent the pathogenesis of allergic disease. Additionally, allergy is a risk factor for bacterial infection such as otitis media, pneumonia and sinusitis, while respiratory infection can exacerbate allergic symptoms. Given the burden of bacterial respiratory disease and respiratory allergy, the development of preventative treatments for these diseases is needed and will benefit from clearer knowledge of the underlying immune mechanisms. This thesis aimed to to extend current knowledge by using Pasteurella pneumotropica, a similar bacteria to the human pathogen nontypeable Haemophilus influenzae (NTHi), to study respiratory infection and protective anti-outer membrane protein (OMP) immunity as well as the interaction of respiratory infection and allergic inflammation. Homologues of the important NTHi vaccine candidates P4, P6, P26 and D15 were found to be encoded by P. pneumotropica and a high level of amino acid sequence identity was noted between the different P. pneumotropica strains, as well as between other Pasteurellaceae members. ... In contrast, anti-P6his serum antibodies transferred to naïve mice did not confer protection. These results suggested that T-cellmediated mechanisms were involved in P6his-mediated protection, and showed that the P. pneumotropcia model was useful for elucidating protective mechansims. The interaction of P. pneumotropica infection and papain-induced allergy was studied to investigate immune mechanisms underlying respiratory infection and allergy. Mice with ongoing allergic inflammation were intranasally challenged with bacteria and exhibited reduced pulmonary bacterial numbers, prolonged eosinophilia in the lungs and the induction of Th2 cytokines in the BALF, compared to nonallergic, infected mice. This suggested a protective role for allergic inflammation in this model. The effect of papaininduced inflammation on mice colonised by P. pneumotropica was also examined and allergic inflammation appeared to worsen infection in colonised mice. This suggested that allergic inflammation may also have a role in promoting infection in this model. In conclusion, this thesis explored mechanisms involved in vaccine-mediated immunity and the interaction of respiratory infection and allergy using a P. pneumotropica infection in its natural host. It was shown that intranasally administered recombinant P6 and P4 protected mice from lung infection, which justifies the inclusion of these OMPs as NTHi vaccine candidates. Additionally, it was demonstrated that the interaction of allergy and respiratory infection modulated immune responses. Overall, these results emphasize that a clearer understanding of the complex mechanisms underlying these interactions is required, and may be aided by the development of suitable animal models.
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König, Ireen. "Investigation of the actin-membrane interaction at the leading edge and its influence on membrane diffusion." Thesis, University of Glasgow, 2009. http://theses.gla.ac.uk/1413/.
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Actin polymerisation is a highly dynamic process which drives many cellular events, including endocytosis and cell motility. It is known that actin monomers are added to the filaments at the leading edge of a migrating cell and that this polymerisation is the driving force of protrusion. Much is known about the activation and regulation of this dynamic actin remodelling, but many questions about the exact nature of the interaction between the actin filaments and the plasma membrane remain. Weisswange et al (Weisswange et al., 2005) found that the leading edge of protruding fish keratocytes functions as a diffusion barrier for lipid dyes. The aim of the here presented thesis was to continue this project and to study the actin-membrane interaction at the leading edge, using the diffusion barrier as an initial read-out method. First it was investigated whether this diffusion barrier is present in other cell types and could therefore be seen as a general feature of protrusion. Using Fluorescence Recovery After Photo-bleaching (FRAP)in B16 F1 cells, it could be shown that the diffusion of membrane anchored GFP (GFP-F) is significantly inhibited at the leading edge compared to lamellar regions. No reduction in diffusion could be observed after destruction of the actin meshwork or at non-protruding sites of the lamellipodial periphery, showing that the diffusion barrier depends on active protrusion. The diffusion of cytoplasmic GFP was not altered near the leading edge compared to in the lamellipodium, indicating that only membrane bound proteins are affected. After showing that the diffusion barrier is a general feature of protrusion, the exact nature of the actin-membrane interaction causing this phenomenon was investigated. No direct interaction between actin and the membrane could be observed using FLIM-FRET, but FRAP experiments on fixed cells and a correlation between the strength of the diffusion barrier with the speed of protrusion indicate that the reason for the reduction in diffusion around the leading edge is the force created by the actin filaments pushing against the membrane. Further FRAP experiments indicate that actin regulating proteins such as IRSp53 are influenced by the restricted diffusion zone at the leading edge. We propose that the lipid diffusion barrier traps regulatory proteins at the leading edge and can therefore be seen as a positive feedback mechanism for actin polymerisation.
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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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Saurabh, Rahul. "Interaction between the Alzheimer's peptide, beta-amyloid and lipid membrane." Thesis, University of Hull, 2015. http://hydra.hull.ac.uk/resources/hull:13635.
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Alzheimer’s disease (AD) is a neurodegenerative disorder. The patho-physiological effects of amyloid beta (Aβ) may be mediated by Aβ-membrane interaction. However, the molecular mechanism of interaction between Aβ and neuronal membrane remains unknown. The aim of the study was to investigate the interaction of a toxic fragment of amyloid beta, Aβ₂₅-₃₅ with the lipid membrane model using model lipid bilayers and beta breaker peptide, KLVFF. Liquid ¹H NMR assay was used to investigate the aggregation properties of Aβ₂₅-₃₅. The sharp NMR peaks of Aβ₂₅-₃₅ appeared immediately after sample preparation and these peaks were lost after 24 hrs incubation. However, on addition of KLVFF to Aβ₂₅-₃₅, the amyloid peptide peaks remain unchanged even after long period of incubation. The data suggest that KLVFF has ability to inhibit the aggregation of Aβ₂₅-₃₅. Magic angle spinning solid state NMR were used to investigate the location and interaction of Aβ₂₅-₃₅ to lipid bilayers. NOESY cross-relaxation rates suggest that the soluble form of Aβ₂₅-₃₅ may interact predominantly with the lipid chains near glycerol region. The cholesterol molecules did not exhibit direct interaction with soluble Aβ₂₅-₃₅. However, cross relaxation rate data suggest that cholesterol may push the soluble Aβ₂₅-₃₅ towards the head region of lipid bilayers. The data indicates that soluble form of Aβ₂₅-₃₅ may enter into lipid bilayers and interact with phospholipids. ²H NMR was used to analyse the effect of Aβ₂₅-₃₅ on lipid phase behaviour. M₁ analysis and methyl splitting data were used to observe the phase transitions. Aβ₂₅-₃₅ lowers the lipid phase transitions temperature in presence and absence of cholesterol. The data suggest that the insoluble form of Aβ₂₅-₃₅ may develop the lipid order (stiffness) and thus lowers the phase transition temperature. The Aβ₂₅-₃₅ plus KLVFF with cholesterol may also significantly raise the phase transition temperature and also elongate the phase transition boundaries, indicating that cholesterol molecules may enhance the lipid order parameter. In conclusion, KLVFF may stop the amyloid beta aggregation either in solution or in the lipid bilayers. Cholesterol molecules may not interact directly with amyloid beta and it may also affect the location of amyloid beta in the lipid bilayers. The results of the study may be important to understand the interactions between Aβ and lipid bilayers which may act as new therapeutic strategies for the development of new drugs for amyloid diseases.
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Bettio, Martina [Verfasser]. "Membrane-model systems to study EGFR-ARNO interaction / Martina Bettio." Bonn : Universitäts- und Landesbibliothek Bonn, 2017. http://d-nb.info/1150777745/34.
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Olofsson, Annelie. "Helicobacter pylori outer membrane vesicles and the host-pathogen interaction." Doctoral thesis, Umeå universitet, Institutionen för medicinsk kemi och biofysik, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-68744.
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Sheridan, Norman P. "The interaction of plant growth regulators with cell membrane constituents." Thesis, Kingston University, 1986. http://eprints.kingston.ac.uk/20346/.
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The thesis describes the interaction of auxins with membrane fractions prepared from etiolated epicotyl tissue of ‘Pisum Sativum’ seedling. The interaction of auxins with phospholipids was also examined. Two classes of high affinity binding tissue sites were found in the growing region of the epicotyl tissue. Kinetic analysis of the data resulted in dissociation constant values of: K[sub]1=2.2x 10[sup]-7 M, n[sub]1=1.8x10[sup]-10 moles/g fresh wt; K[sub]2=11x10[sup]-7M, n[sub]2=3x10[sup]-10 moles/g fresh wt. These sites were not found in the non-growing region of the pea epicotyl suggesting that they may be involved in the growth process. From the competition studies reported here, it would appear that site 2 showed greater auxin specificity than site 1 and this could be considered a candidate as an auxin receptor. Sucrose gradient fractionation techniques were employed to further separate the two binding sites and it was shown that site 2 binding was associated with fractions rich in plasma membrane while site 1 was associated with the endoplasmic reticulum. Separation of the solubilized sites by gel permeation methods indicated an apparent molecular weight of 42,000 daltons. IAA was shown to complex with the polar head group region of phospholipids, in CDCl[sub]3, although the strength of the complex was rather low (Kd=1.9x10[sup]-2 Molal). The strength of binding was influenced by the polar head groups of the phospholipids, but did not appear to be affected by the fatty acyl chain length. The physiological significances of such interactions are discussed.
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Serrano, Galiano Sonia. "Fluid-structure interaction of membrane aerofoils at low Reynolds numbers." Thesis, University of Southampton, 2016. https://eprints.soton.ac.uk/414110/.
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This thesis investigates the fluid-structure interaction (FSI) problem of elastic membrane aerofoils at low Reynolds numbers. The dynamics of the fluid and membrane coupled system is studied via direct numerical simulation (DNS) using a newly developed computational framework whose characteristics and validation are included in this report. A set of two-dimensional DNS were performed for varying Reynolds number, membrane elasticity and aerofoil geometry in order to investigate the effect of these relevant fluid and structural parameters on the behaviour of fluid-structure coupled system. Static and dynamic features of the system, and their effect in aerodynamic properties, are described and compared for the different parameter combinations. The case with highest Reynolds number, Re = 10; 000, and intermediate elasticity was chosen as a base case to further study the fluid-structure coupling mechanism, particularly at low angle of attack conditions. The dynamic behaviour was characterised via spectral analysis in the frequency and wavenumber-frequency domains, which allowed the propagating wave nature of the membrane vibrations and their effect on the surrounding pressure field fluctuations to be clarified. The membrane vibrations are found to introduce upstream-propagating pressure waves that seem to be responsible for a loss in aerodynamic efficiency compared to a rigid aerofoil. Stability aspects of the FSI problem are also investigated by performing numerical experiments to analyse the response of the system to initial flow perturbations. The solutions of the 2D DNS are used as initial conditions for three-dimensional simulations, upon which initial perturbations in spanwise velocity are added. As the simulation is advanced in time the evolution of the perturbations is studied to determine the stability characteristics of the flow. Amplifications of the perturbations are found for Re > 10; 000. The coupling of the fully three-dimensional developed flow and the elastic aerofoil is also analysed with spectral techniques. Comparison of two- and three-dimensional results reveals that the three-dimensional flow development causes a decrease in the amplitude of the system fluctuations, but the same coupling mechanism found in the two-dimensional approach is also present in the three-dimensional case.
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Kalapos, Thomas Lawrence. "Interaction of Water with the Proton Exchange Fuel Cell Membrane." Case Western Reserve University School of Graduate Studies / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=case1175891061.
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Largueze, Christophe. "Raffinage des huiles végétales par microfiltration : interaction milieu hydrophobe/membrane." Montpellier 2, 1997. http://www.theses.fr/1997MON20062.
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Yuerueker, Ayse Banu. "Organization, membrane interaction and functional regulation of the neutrophil cytoskeleton /." [S.l.] : [s.n.], 1992. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Dessolin, Samuel. "Membrane models for a controllable surface." Thesis, Georgia Institute of Technology, 2003. http://hdl.handle.net/1853/17527.
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Carvalho, Kevin. "Interaction entre membrane plasmique et cytosquelette : Approche biomimétique pour l'étude des interactions entre ezrine, PIP2 et actine." Phd thesis, Université Montpellier II - Sciences et Techniques du Languedoc, 2009. http://tel.archives-ouvertes.fr/tel-00514808.
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La membrane plasmique de la cellule est composée de lipides et interagit notamment avec le squelette de la cellule (le cytosquelette), par l'intermédiaire de protéines d'ancrages et de lipides clefs qui jouent un rôle spécifique dans certains types d'interactions. Parmi les protéines intervenant dans l'ancrage direct de la membrane plasmique au cytosquelette, des protéines de la famille des ERM (Ezrine, Radixine, Moésine) peuvent interagir spécifiquement avec un lipide, le phosphatidylinositol (4,5) biphosphate (PIP2), d'une part et avec l'actine du cytosquelette d'autre part. Dans le but de comprendre les interactions entre membrane plasmique et cytosquelette, nous avons réalisé des expériences in vitro sur des systèmes comportant un nombre minimal de constituants : des vésicules géantes (GUV) contenant du PIP2, de l'ezrine recombinante et de l'actine purifiée. Nous avons mis en évidence que la liaison au PIP2 induit des changements conformationnels de l'ezrine. L'ezrine est alors capable d'interagir avec les filaments d'actine. Nous avons caractérisé quantitativement l'incorporation de PIP2 dans la membrane de vésicule géantes, et montré que l'interaction de l'ezrine avec les vésicule géante contenant du PIP2, induit un partitionnement du lipide dans la bicouche lipidique et conduit à la formation d'agrégats de PIP2 et d'ezrine sur la membrane. La connaissance des effets de l'ezrine sur les membranes contenant du PIP2 et la connaissance des différents mécanismes se produisant lors des interactions permettra de définir plus précisément le rôle de l'ezrine in cellulo.
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Akabane, Hiroto. "Protein kinase C activity in mouse eggs regulates gamete membrane interaction." Huntington, WV : [Marshall University Libraries], 2006. http://www.marshall.edu/etd/descript.asp?ref=655.
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Hall, Esther. "Calpastatin isoforms and interaction with membrane triads in porcine skeletal muscle." Thesis, University of Nottingham, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312238.
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Zhou, Lianqun. "Study of the membrane-fluid interaction in micro lamb wave sensor." Besançon, 2010. http://www.theses.fr/2010BESA2041.
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Micro lamb wave sensor is one of useful tools to study the membrane-fluid interaction, especially in interdisciplinary and not yet explored areas, such as gas sensing, aerodynamics, the multi-parameter decoupling and so on. This is because micro Lamb wave sensor holds high sensitivity, low losses and multi-modes. This thesis deals with the membrane-fluid interaction with micro lamb wave sensor is investigated, and the potential function method is used to analyze the dispersion curves, the displacements, the stress curves, etc. A model is built to analyze the modes distribution in micro Lamb wave sensor. The gase effects on lamb wave propagations are investigated. The applications of Lamb wave in aerodynamics and multi-parameters decoupling are discussed. The details are described as follows. In theory, combinations of the potential function method and the boundary conditions at all interfaces are used to analyze the membrane-fluid interaction. Compared with traditional plate shell theory, the particle movements in the membrane and fluid are taken into account in this method. Several aspects of the problem can be addressed, including the displacement, the stress, velocity dispersion curve, pointing vector, group velocity, energy velocity and so on. All of these are available to investigate the interaction at the membrane-fluid interface. In the case of micro Lamb wave sensor, the resonant modes can be excited and work simultaneously with the travelling modes, as the membrane is width limited and not large enough comparing with the wavelength of Lamb waves. The established model car reveal the mode distributions in micro Lamb wave device clearly and accurately. These works provide insight into the understandings of the modes in micro Lamb wave device, which is useful for further experiments. In literatures, little attention has been paid on the Lamb wavesۥ propagation near the membrane-gas interface, as both the density and sound velocity of gas are low. We aim to provide more studies of gases effects on the evanescent wave the leaky wave near the membrane-gas interface. It is shown that the relative frequency shifts in the low frequency range of the A0 mode (evanescent wave, EW) is rather important and the shape of the curve looks like ۥUۥ shape; in the high frequency range of this mode (leaky Lamb wave, LLW), the quality factor decreases rapidly when the Lamb wave phase velocity approaches the gas sound velocity. The Sº mode shows immune to gas loading, which is a good choice for a reference mode. This provides theoretical and experimental work for related fields in gas sensing. The application of membrane-gas interaction in aerodynamics in investigated theoretically and experimentally. The interaction between the gas flow boundary layer and the acoustic sound field (EW and LLW) at the membrane-gas interface can give out the parameter in the gas flow. The parameters in gas flow can be obtained from the measuring the interaction, which happens between the gas flow boundary layer and the acoustic sound field (EW and LLW) at the membrane gas interface. In the EW case, the thicknesss of the gas flow boundary layer and the penetration depth of the evanescent wave combined determine this interaction. When the Lamb wave phase velocity approaches the gas sound velocity, this effect is clearly observed. In the LLW case, it is shown experimentally that the gas flow has not evident effects on Lamb wave’s propagations. It suggests that Lamb wave is promising for applications in wind tunnel experiments, micro channels characterization, and can lead to multi-parameters measurements. The effects of the different physical parameters (density, sound velocity, viscosity, etc) on the modesۥ propagations at he membrane-liquid interface are studied. Combination of the relative frequency shifts of the A01 mode (he fundamental mode of low frequency A0 mode) and the A03 mode (the third harmonic wave of the A01 mode), the density and the sound velocity of liquid, but is amplitude changes with the viscosity. This work makes Lamb wave have promising applications in the investigation of the molecular thermodynamics, molecular labels free detection,etcCette thèse traite, théoriquement et expérimentalement, de l’interaction fluide-membrane dans un capteur a onde de Lamb. Un modèle est utilisé pour calculer les courbes de dispersion, le déplacement, les contraintes. Un autre modèle est utilisé pour analyser la distribution des modes. L’effet des gaz est étudié théoriquement et expérimentalement. Les applications des ondes de Lamb à l’aérodynamique et aux mesures multiparamétriques sont présentées. Voici quelques détails. Le premier modèle utilise les fonctions potentielles et recherche les fonctions solution des équations de propagation qui remplissent les conditions aux limites avec ou sans la présence d’un liquide. Ce modèle permet d’obtenir de nombreux paramètres, le déplacement des particules, les contraintes, le vecteur de Poynting, les vitesses de groupe et d’énergie etc. La membrane étant limitée dans le sens latéral il y a coexistence dans la membrane de modes stationnaires et d’ondes progressives. Un modèle donne la position et l’intensité relatives des modes. Le but est d’apporter des connaissances complémentaires sur l’action des gaz sur la propagation des ondes de Lamb. On montre que pour les basses fréquences de A0 (ondes évanescentes dans le gaz) l’action est principalement un changement de fréquence , tandis aux plus hautes fréquences de A0 (Ondes «fuyantes» l’action est principalement une atténuation. Le S0 mode étant très peu modifié par la présence de gaz. L’application de l’interaction gaz-membrane en aérodynamique est étudiée théoriquement et expérimentalement. Le principal effet ce produit quand la vitesse de phase de l’onde de Lamb est proche de la vitesse du son dans le gaz. Les résultats suggèrent que les applications dans ce domaine seront très prometteuses. Les effets sur l’onde de Lamb de différents paramètres (densité, vitesse du son viscosité) d’une solution liquide sont étudiés. On montre que l’utilisation conjointe de A01 mode (fondamental du A0 mode) et du A03 mode (harmonique 3 DU A0 mode) permet de mesurer la densité et la vitesse du son. La densité étant connue, le S0 mode permet d’obtenir la viscosité
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Nilsson, Martin. "GIANT UNILAMELLAR VESICLES FOR PEPTIDE-MEMBRANE INTERACTION STUDIES USING FLUORESCENCE MICROSCOPY." Thesis, Linköpings universitet, Biofysik och bioteknik, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-167467.
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Vesicles are a type of biological or biomimetic particle consisting of one or more often spherical bilayers made up of amphipathic molecules, creating a closed system. They can function as an encapsulating device, holding hydrophilic molecules on the inside of the bilayer membrane(s) or hydrophobic molecules in the non-polar interstitial space in the middle of the bilayers. Because of this capacity to carry molecules, vesicles are a premier system for drug delivery and even theranostics in vivo. A peptide-based approach to release of encapsulated molecules has previously been developed but since drug delivery vesicles are in the size range of nanometers, the mechanisms have not been visualized. This project aims to produce giant unilamellar vesicles as a model system used to visualize membrane interactions vital to the understanding and further development of smaller vesicle-based systems for drug delivery. Giant unilamellar vesicles were produced successfully and a preparation protocol was established. Additionally, some membrane interactions were investigated using fluorescence microscopy.
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Pauletti, Miguel Sebastian. "Parametric AFEM for geometric evolution equations and coupled fluid-membrane interaction." College Park, Md.: University of Maryland, 2008. http://hdl.handle.net/1903/8603.
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Thesis (Ph. D.) -- University of Maryland, College Park, 2008.Thesis research directed by: Dept. of Mathematics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Betaneli, Viktoria. "Voltage dependent anion channel: Interaction with lipid membranes." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2012. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-85742.
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Evidence has accumulated that the voltage dependent anion channel (VDAC), located on the outer membrane of mitochondria, plays a central role in apoptosis. The involvement of VDAC oligomerization in apoptosis has been suggested in various studies. However, it still remains unknown how exactly VDAC supra-molecular assembly can be regulated in the membrane. Previous studies suggested the possible influence of various proteins on the formation of VDAC oligomers, but the important issue of the VDAC oligomeric state regulation by lipids has not been studied so far. Nevertheless, the effect of lipids on the oligomerization of several membrane proteins has been mentioned in the literature and in general, protein-lipid interactions are under extensive investigation.
In the present work, I addressed the influence of lipids on VDAC oligomerization experimentally by reconstituting the fluorescently labelled VDAC in giant unilamellar vesicles (GUVs)—a chemically well defined, cell-free minimal model system. Fluorescence cross-correlation spectroscopy was performed to determine the oligomeric state of VDAC. I investigated the effect of important for apoptosis anionic lipids, phosphatidylglycerol and cardiolipin, on VDAC oligomerization. I demonstrated that phosphatidylglycerol significantly enhances VDAC oligomerization in the membrane, whereas cardiolipin disrupts VDAC oligomers. These results suggest that up- or down- regulation of these lipids in mitochondria during apoptosis can tune VDAC oligomerization in the membrane.
Thus, this study sheds light on the role played by the above-mentioned lipids in the regulation of VDAC oligomerization during apoptosis and provides additional information on the molecular mechanisms of the programmed cell death.
Another objective of this work was to investigate the partitioning of VDAC into liquid disordered or liquid ordered lipid phases. The existence of lipid domains or the lipid rafts in mitochondria and VDAC enrichment in these rafts is still under debate. Additionally, mitochondrial VDAC was recently found in the plasma membrane. The role of this VDAC is not known, however, it was shown to be located in caveolae (specialized lipid rafts) and play an important role in neuronal apotosis and Alzheimer’s disease. Therefore, VDAC partitioning to the lipid rafts is an interesting question for investigation.
The possibility to reconstitute VDAC into minimal model systems–GUVs with phase separation, allowed to reveal the preferential partitioning of VDAC into liquid disordered lipid domain, which suggests either non-raft localization of VDAC or the requirement of the other factors for the recruitment of VDAC into lipid rafts.
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Kemayo, Koumkoua Patricia. "Structural characterisation of highly specific membrane protein-lipid interactions involved in cellular function." Thesis, Strasbourg, 2015. http://www.theses.fr/2015STRAF055/document.
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Les membranes cellulaires sont des systèmes complexes composés de lipides variés qui interagissent avec les protéines pour accomplir une fonction. Leur adressage spécifique dans la cellule est crucial pour le fonctionnement cellulaire. Les vésicules COP (coat protein) sont impliquées dans leur transport dans les premières étapes de la voie de sécrétion. Récemment, une interaction très spécifique a été identifiée entre le domaine transmembranaire de la protéine p24 (p24TMD) abondante dans la membrane des vésicules COP et la sphingomyéline C18:0. Cette spécificité a été identifiée dans le cas d’interaction protéine-protéine et protéine-acide nucléique comme impliquée dans la régulation de fonctions cellulaires, C’est pourquoi nous avons décidé d'étudier sur cette interaction. A cet effet, le p24TMD a été obtenu par synthèse chimique et sa structure étudiée par RMN du solide en présence de la sphingomyèline avec pour but ultime de comprendre la fonctionCell membranes are complex systems composed of variety of lipids that interacts with proteins to trigger cellular function. The delivery of these lipids to the right compartment is crucial for cells to work efficiently. The coat protein (COP) complex vesicles are involved in lipids traffic in the early stages of the secretory pathway. Recently, a highly specific interaction has been found between the transmembrane domain of p24 protein (p24TMD) abundant in COPI membrane and sphingomyelin C18:0. As such highly specific interaction have been reported for protein-protein and protein-nucleic acid interactions to be involved in regulation of cell functions, we decide to investigate this specific interaction. The p24TMD was obtained chemically and investigated by solid state NMR in presence of sphingomyelin with the ultimately goal to understand the function behind
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Zhou, Xiongtu. "Investigation of cell-material interaction using topographical patterns and cell imprinting techniques." Paris 6, 2010. http://www.theses.fr/2010PA066599.
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Améliorer la connaissance des interactions cellules-matériau est un enjeu crucial pour la biologie cellulaire et l’ingénierie tissulaire ainsi que pour la fabrication de dispositifs biomédicaux élaborés. Ce travail de thèse s’est basé sur le développement de technologies de micro- et nanofabrications avancées et de l’imagerie cellulaire de haute résolution, afin d’obtenir une vue plus claire sur les phénomènes d’élongation, alignement, extention, et déformation locale de membrane cellulaire sur des motifs topographiques. La culture cellulaire sur des substrats comportant des trous et des sillons à dimensions micro et nanométriques a été étudiée, ce qui a permis de démontrer une forte dépendance de la géométrie des motifs et de la décoration de surface. En général, les membranes cellulaires sont localement déformées à cause de la présence des trous ou des sillons micrométriques, qui aident, entre autre, l’attachement et l’adhésion cellulaire. Nous avons aussi noté que les cellules cancereuses sont plus agressives pour exploiter les motifs topographiques. Enfin, nos études ont été élargies à des motifs de géometrie plus complexe tels que des réseaux de microsillons croisés avec des différentes périodicités et profondeurs de gravure.
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Nipper, Matthew Edward. "Characterization of membrane viscosity changes with the novel molecular rotor FCVJ." Diss., Columbia, Mo. : University of Missouri-Columbia, 2007. http://hdl.handle.net/10355/4973.
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Thesis (M.S.)--University of Missouri-Columbia, 2007.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on May 12, 2009) Includes bibliographical references.
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Valdés, Vázquez Jesús Gerardo. "Nonlinear Analysis of Orthotropic Membrane and Shell Structures Including Fluid-Structure Interaction." Doctoral thesis, Universitat Politècnica de Catalunya, 2007. http://hdl.handle.net/10803/6866.
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Problemas de interacciónn fluido-estructura representan hoy en día un gran desafío en diferentes áreas de ingeniería y ciencias aplicadas. Dentro de las aplicaciones en ingeniería civil, el flujo del viento y los movimientos estructurales pueden ocasionar inestabilidades aeroelásticas en construcciones tales como puentes de gran luz, rascacielos y cubiertas estructurales ligeras. Por otro lado, aplicaciones en biomecánica están interesadas en el estudio de hemodinámica, por ejemplo: flujo sanguíneo en arterias, donde grandes deformaciones de las venas interactúan con fluidos.En la parte estructural de este trabajo, una nueva metodología para el análisis geométricamente no-lineal ortótropo de membranas y láminas sin grados de libertad de rotación es desarrollada basándose en la orientación de la fibra principal del material. Una consecuencia directa de la estrategia de orientación de fibras es la posibilidad de analizar membranas y láminas pretensadas cuya configuración inicial está fuera del plano. Por otra parte, ya que la teoría convencional de membranas permite que existan tensiones de compresión, un modelo de arrugado basado en la modificación de la ecuación constitutiva se presenta. El desarrollo estructural es modelado con elementos finitos provenientes de las ecuaciones de la elastodinámica.La parte de fluidos de este trabajo está gobernada por las ecuaciones de Navier-
Stokes para flujos incompresibles, las cuales son modeladas por interpolaciones estabilizadas de elementos finitos. Ya que la solución monolítica de dichas ecuaciones tiene la desventaja que consumen mucho tiempo en la solución de grandes sistemas de ecuaciones, el método de pasos fraccionados se usa para aprovechar las ventajas computacionales que brinda gracias al desacoplamiento de la presión del campo de las velocidades. Además, el esquema α-generalizado para integración en el tiempo para fluidos es adaptado para que se use con la t´ecnica de los pasos fraccionados.
El problema de interacción fluido-estructura es formulado como un sistema de tres campos: la estructura, el fluido y el movimiento de la malla. El movimiento del dominio del fluido es tomado en cuenta mediante la formulación arbitraria Lagrangiana-Euleriana, para la cual se usan dos estrategias de movimiento de malla.
Para el acoplamiento entre el fluido y la estructura se usa un acoplamiento fuerte por bloques usando la técnica de Gauss-Seidel. Debido a que la interacción entre el fluido y la estructura es altamente no-lineal, se implementa el método de relajación basado en la técnica de Aitken, la cual acelera la convergencia del problema.
Finalmente varios problemas se presentan en los diferentes campos, los cuales verifican la eficiencia de los algoritmos implementados.
Nowadays, fluid-structure interaction problems are a great challenge of different fields in engineering and applied sciences. In civil engineering applications, wind flow and structural motion may lead to aeroelastic instabilities on constructions such as long-span bridges, high-rise buildings and light-weight roof structures. On the other hand, biomechanical applications are interested in the study of hemodynamics, i.e. blood flow through large arteries, where large structural membrane deformations interact with incompressible fluids.
In the structural part of this work, a new methodology for the analysis of geometrically nonlinear orthotropic membrane and rotation-free shell elements is developed based on the principal fiber orientation of the material. A direct consequence of the fiber orientation strategy is the possibility to analyze initially out-ofplane prestressed membrane and shell structures. Additionally, since conventional membrane theory allows compression stresses, a wrinkling algorithm based on modifying the constitutive equation is presented. The structure is modeled with finite elements emerging from the governing equations of elastodynamics.
The fluid portion of this work is governed by the incompressible Navier-Stokes equations, which are modeled by stabilized equal-order interpolation finite elements.
Since the monolithic solution for these equations has the disadvantage that take great computer effort to solve large algebraic system of equations, the fractional step methodology is used to take advantage of the computational efficiency given by the uncoupling of the pressure from the velocity field. In addition, the generalized-α time integration scheme for fluids is adapted to be used with the fractional step technique.
The fluid-structure interaction problem is formulated as a three-field system: the structure, the fluid and the moving fluid mesh solver. Motion of the fluid domain is accounted for with the arbitrary Lagrangian-Eulerian formulation with two different mesh update strategies. The coupling between the fluid and the structure is performed with the strong coupling block Gauss-Seidel partitioned technique.
Since the fluid-structure interaction problem is highly nonlinear, a relaxation technique based on Aitken's method is implemented in the strong coupling formulation to accelerate the convergence.
Finally several example problems are presented in each field to verify the robustness and efficiency of the overall algorithm, many of them validated with reference solutions.
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Sawyer, Janet Gail. "Interaction of macrophage cationic proteins with the outer membrane of Pseudomonas Aeruginosa." Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/26531.
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Purified macrophage cationic proteins were used in functional assays to determine their interactions with the outer membrane and lipopolysaccharide of Pseudomonas aeruginosa. A fluorescent derivative of polymyxin B (dansyl-polymyxin) was found to bind to saturation to purified lipopolysaocharide, with similar affinity for the aminoglycoside supersensitive strain H215 and wild type strain H103 lipopolysaocharide. MCP-1 could displace more dansyl-polymyxin bound to the lipopolysaocharide of both strains, and bound with greater affinity than MCP-2. When whole cells were used, MCPs also displaced bound dansyl-polymyxin. Effects on the outer membrane of whole cells were examined by determining the initial rate of uptake of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine. Uptake was enhanced in the presence of MCPs, indicating permeabilization of the outer membrane. MCP-1 caused maximal uptake of the probe at 40 µg/ml, MCP-2 at 70 µg/ml, and crude extract at only 20 µg/ml. Uptake of the probe was found to be enhanced at add pH, with maximal uptake occurring with only 7.5 µg/ml MCP-1 at pH 6.5. The data suggested that MCPs act to permeabilize the outer membranes of P. aeruginosa in a manner analagous to that defined for other polycationic agents.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Swan, Ruth. "Investigation into the membrane interaction of E. coli penicillin binding protein 4." Thesis, University of Central Lancashire, 2005. http://clok.uclan.ac.uk/20841/.
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The low molecular mass penicillin-binding proteins (PBPs) of E. coli play a regulatory role in the terminal stages of peptidoglycan biosynthesis. PBPs 4, 5 and 6 have been shown to possess DD-carboxypeptidase activity with PBP4 also having an endopeptidase activity. PBP5 and 6 are proposed to anchor to the periplasmic face of the inner membrane via C-terminal ahelices whereas the association of PBP4 with the membrane is more ambiguous and may involve a specific binding site and/or C-terminal amphiphilic a-helical interaction. PBP4 has also been tentatively proposed to function as part of an enzyme complex that serves to insert new peptidoglycan strands into the pre-existing layer surrounding the bacterial cell. The effect that P4, a C-terminal homologue of PBP4, had on an artificial bilayer has been investigated using 2H NMR. This has been compared with the interactions of P5, a PBP5 C-terminal homologue, and melittin, a well documented membrane interactive protein of similar molecular mass. In the NMR experiments it was found that splitting reduced with increasing P4 concentration (0 - 8.27 mg.g5, P < 0.088. A similar, though less convincing, effect was observed with P5 (0 - 6.94 mg.gj, P < 0.86, and melittin (0-3.06 mg.g 1 ), Pc0.39. This data implies that P4 interferes with the lamellar structure to a greater degree than either PS or melittin, possibly causing disruption, and would support a role for the C-terminus of PBP4 in membrane binding. In monolayer experiments, P4 produced an increase in surface pressure of between 1 and 7.5 mN m 1 , with monolayers formed with either dioleoylphosphatodylglycerol (DOPO), dioleoylphosphatidylethanolamine (DOPE), a mixed lipid system (75% DOPE, 20% DOPG, 5% cardiolipin) or E. coli lipid extract. Similar pressure increases were observed with the addition of NaC1 (1 and 10 gM) to the sub phase of DOPG and lipid extract monolayers. This data confirms that the C-terminus of the protein would be able to interact with the bilayer interface and implies that the driving force behind the association is hydrophobic in nature rather than electrostatic. These pressure changes were higher than those caused by the whole protein (PBP4.his) which was found to interact better with ionic DOPO monolayers, producing an increase in pressure of 3 mN m 4 , compared with no increase in pressure for the zwitterionic DOPE monolayers. PBP4.his also interacted with the E. coli lipid extract (2 mN rn4 ) better than with the mixed lipid monolayers (0.5 mN m 1 ). The addition of NaC1 (1 .tM) to the sub phase of DOPO and lipid extract monolayers prevented any interaction, indicating an electrostatic component to the membrane interaction mechanism of PBP4. The combination of data from the monolayer and NMR experiments shows that whilst the C-terminus could contribute to membrane association there may be stabilising electrostatic interactions between the ectornembraneous domain and the lipid. Cross-linking studies were also performed to investigate the possibility that PBP4 can interact with other membrane proteins. PBP4 was found to cross-link to another membrane protein implying that there is potential for PBP4 to bind to the membrane via a specific site as well as the potential to form an enzyme complex. In conclusion, these results supports the theory of a specific binding site involving protein-protein interaction for PBP4 with a requirement for ionic lipids to stabilise the membrane interaction of the whole protein. The monolayer and NMR data would support a role for the C-terminus possibly in stabilising initial interactions at the periplasmic face of the membrane.
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Holmes, Robert John. "The role of membrane proteins in the Erisiphe cichoracearum/Arabidopsis thaliana interaction." Thesis, University of Southampton, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.444953.
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Evans, Philip John. "Membrane-solute-cleaning agent interaction during the ultrafiltration of black tea liquor." Thesis, University of Bath, 2008. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.528172.
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Eriksson, Sylvia. "Molecular properties of disordered plant dehydrins : Membrane interaction and function in stress." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-136033.
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Dehydrins are intrinsically disordered plant stress-proteins. Repetitively in their sequence are some highly conserved stretches of 7-17 residues, the so called K-, S-, Y- and lysine rich segments. This thesis aims to give insight into the possible role dehydrins have in the stressed plant cell with main focus on membrane interaction and protection. The work includes four recombinant dehydrins from the plant Arabidopsis thaliana: Cor47 (SK3), Lti29 (SK3), Lti30 (K6) and Rab18 (Y2SK2). Initially, we mimicked crowded cellular environment in vitro to verify that dehydrins are truly disordered proteins. Thereafter, the proposal that the compulsory K-segment determines membrane binding was tested. Experiments show that only Lti30 and Rab18 bind, whereas Cor47 and Lti29 does not. As Lti30 and Rab18 binds they assembles vesicles into clusters in vitro, a feature used to characterize the interaction. From this it was shown that membrane binding of Lti30 is electrostatic and determined by global as well as local charges. Protonation of histidine pairs flanking the K-segments works as an on/off-binding switch. By NMR studies it was shown that the K-segments form a dynamic α-helix upon binding, so called disorder-to-order behaviour. Also, dehydrins electrostatic interaction with lipids can be further tuned by posttranslational phosphorylation or coordination of calcium and zinc ions. Finally, specific binding of Rab18 to inositol lipids, mainly PI(4,5)P2, is reported. The interaction is mainly coordinated by two arginines neighboring one of the K-segments. In conclusion, the K-segments are indeed involved in the binding of dehydrins to membrane but only in combination with extensions (Lti30) or modified (Rab18).At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.
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Sabapathy, Thiruvarutchelvan. "The Interaction of Insulin with the Insulin Receptor in a Membrane Environment." Thesis, Curtin University, 2018. http://hdl.handle.net/20.500.11937/73527.
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The first event in insulin action is the binding of insulin to insulin-receptors embedded in the cell membrane. This study investigated how plasma membrane cholesterol influences this interaction, as examined in several experimental models including cultured cells, virus-like-particles and liver cell plasma membranes. The study is of relevance to a better understanding of diabetes at a molecular level. Novel results include the demonstration that alterations in membrane cholesterol content significantly reduce insulin binding and signalling.
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Adrien, Vladimir. "Diffusion des lipides et interaction protéine-protéine dans des membranes modèles." Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCB033.
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Les membranes biologiques, qui compartimentent les différents éléments du vivant, jouent un rôle essentiel dans les processus biologiques comme la signalisation, le transport, la transmission du message nerveux, etc. Envisagées comme des fluides à deux dimensions, l’étude de leurs propriétés physiques peut nous aider à comprendre certains mécanismes biologiques. Ce travail de thèse s’est intéressé à la mobilité des molécules au sein des membranes, et notamment à deux paramètres essentiels, la viscosité membranaire, et la diffusion latérale. Après avoir optimisé la technique de recouvrement de fluorescence après photoblanchiment (FRAP) au microscope confocal, nous avons étudié la mobilité des molécules au sein de deux types de membranes modèles in vitro : la phase éponge d’un surfactant non-ionique (C12E5) et les vésicules géantes unilamellaires (GUVs) lipidiques. 1) La phase éponge (ou L3) : après avoir déterminé son diagramme de phase et montré que les protéines membranaires restent actives dans cette phase, nous avons mesuré la mobilité de protéines par recouvrement de fluorescence après photoblanchiment sur un motif à franges (FRAPP). Cela nous a permis d’obtenir les constantes d’association de protéines de la pompe d’efflux OprM-MexAB, impliquée dans la résistance aux antibiotiques de la bactérie Pseudomonas aeruginosa. Ces interactions dépendent très fortement du degré de confinement de chacune des protéines. 2) Les GUVs : après avoir développé une méthode simple de formation des GUVs, au sein desquelles les protéines membranaires restent actives, nous avons mesuré la diffusion des lipides par FRAP, et montré que dans certaines conditions, ils se déplacent en groupe, ce qui permet d’expliquer la diversité des résultats de la littérature. En mesurant la viscosité membranaire par imagerie microscopique du temps de vie de fluorescence (FLIM), nous avons également montré qu’elle ne se déduit pas nécessairement des modèles hydrodynamiques de diffusionBiological membranes, which divide the elements of life, are a key factor in biological processes such as signaling, transport, transmission of an nerve impulse, etc. Seen as two-dimensional fluids, the study of their physical properties could help us understand some unsolved biological mechanisms. This work focused on molecule mobility within membranes, and specifically on two essential parameters: membrane viscosity and lateral diffusion. After optimizing the Fluorescence Recovery After Photobleaching (FRAP) technique on confocal microscopes, we studied the mobility of molecules within two types of in vitro model membranes: the sponge phase made of a non-ionic surfactant (C12E5) and the giant unilamellar lipidic vesicles (GUVs). 1) Sponge phase (or L3) : after having established its phase diagram and shown that membrane proteins stay active in this phase, we measured protein mobility by Fluorescence Recovery After fringe Pattern Photobleaching (FRAPP). This allowed us to obtain the association constants of the proteins of the efflux pump OprM-MexAB involved in the resistance to antibiotics of the bacteria Pseudomonas aeruginosa. These interactions heavily depend on the degree of confinement of each protein. 2) GUVs : after having developed a simple method for the formation of GUVs, in which membrane proteins stay active, we measured the lipid diffusion by FRAP. We showed that, under certain conditions, they can move together, which explains the diversity of results in the literature. By measuring membrane viscosity by Fluorescence Lifetime Imaging Microscopy (FLIM), we also showed that viscosity should not be necessarily deduced from hydrodynamic diffusion models
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Wilkins, Simon. "Lectin-carbohydrate mediated interaction between Plasmodium ookinetes and the mosquito midgut." Thesis, Imperial College London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367836.
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Gires, Pierre-Yves. "Interaction hydrodynamique entre deux vésicules dans un cisaillement simple." Phd thesis, Université de Grenoble, 2012. http://tel.archives-ouvertes.fr/tel-00982554.
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Les vésicules sont des bicouches fermées de molécules tensioactives, remplies de liquide, à l'intérieur d'un autre liquide. Leur taille peut être comprise entre dix et 100 microns : elles sont alors dites géantes. Nous nous intéressons à la dynamique de deux de ces objets dans un cisaillement simple, c'est à dire l'écoulement d'un liquide situé entre deux plaques planes se translatant l'une par rapport à l'autre à vitesse et distance constante. Nous commençons par une étude asymptotique, pour des vésicules quasi-sphériques en interaction lointaine. Nous utilisons ensuite un code de calcul basé sur la méthode des éléments de frontière pour étudier le cas de vésicules moins sphériques et plus proches, et comparons les résultats obtenus avec des expériences. Nous présentons enfin comment cette étude peut être utilisée pour prédire certaines propriétés de diffusion d'une suspension de vésicules, dans le régime semi-dilué, où seul le détail des interactions à deux corps est considéré.
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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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Huang, Wenxin, and 黃聞馨. "Sperm fucosyltransferase-5 mediates the sperm-oviductal epithelial cell interaction to protect human sperm from oxidative damage." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/196485.
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Oxidative damage by reactive oxygen species (ROS) is a major cause of sperm dysfunction. Excessive ROS generation reduces fertilization and enhances DNA damage of spermatozoa. In mammals, including humans, oviduct functions as a sperm reservoir which is created by the binding of spermatozoa to the epithelial lining in the oviduct. Interaction between sperm and oviductal epithelial cells improves the fertilizing ability of and reduces chromatin damage in spermatozoa. However, the mechanism(s) by which spermatozoa-oviduct interaction producing these beneficial effects is unknown. One possibility is that oviduct protects spermatozoa from oxidative stress. The hypothesis of this project was that oviductal cell membrane proteins interact with spermatozoa to protect them from oxidative damage. Due to the limited availability of human oviductal tissue for research, an immortalized human oviductal epithelial cell line, OE-E6/E7, was used as a study model.
The first objective examined the effect of OE-E6/E7 membrane proteins on human spermatozoa. The extracted OE-E6/E7 membrane proteins bound to sperm head and preferentially to uncapacitated sperm. Pretreatment with OE-E6/E7 membrane proteins significantly suppressed ROS-induced adverse effects in sperm motility, membrane integrity, DNA integrity, and intracellular ROS level. OE-E6/E7 membrane proteins also increased the endogenous enzyme activities of sperm superoxide dismutase (SOD) and glutathione peroxidase (GPx).
Sperm fucosyltransferase-5 (sFUT5) is a membrane carbohydrate-binding protein on human sperm. The second objective investigated the involvement of sFUT5 in sperm-oviduct interaction. Purified sFUT5 bound to OE-E6/E7 cells and anti-FUT5 antibody inhibited this interaction. Pre-absorption of OE-E6/E7 membrane proteins with purified sFUT5 or blocking of sFUT5 on sperm with anti-FUT5 antibody significantly inhibited the protective effects of OE-E6/E7 membrane proteins against ROS-induced damages in spermatozoa. Asialofetuin, a reported sFUT5 substrate, can partly mimic the protective effect of OE-E6/E7 membrane proteins.
Sperm processing in assisted reproductive technology (ART) treatment, including centrifugation and cryopreservation, has shown to induce ROS production and oxidative damage in sperm. The third objective investigated the possible use of OE-E6/E7 membrane proteins or asialofetuin as an antioxidant supplement during centrifugation and cryopreservation. No adverse effect on sperm functions was detected by centrifugation using our centrifugation protocols. OE-E6/E7 membrane proteins or asialofetuin pretreatment suppressed the cryopreservation-induced damage on sperm in terms of motility and DNA fragmentation.
The fourth objective aimed to identify the sFUT5-interacting proteins from OE-E6/E7 membrane extracts. By using immuno-affinity chromatography and mass spectrometry analysis, cell adhesion molecule 4 (CADM4) was identified as a potential sFUT5-interacting protein. This result was further supported by co-immunoprecipitation, immunofluorescent staining and immunohistochemistry. CADM4 expression level was shown to be higher at follicular phase when compared to luteal phase of the menstrual cycle.
In conclusion, this thesis demonstrated that oviductal epithelial cell membrane proteins bind to the human spermatozoa and protect them from ROS-induced damages in terms of motility, membrane integrity, and DNA integrity. sFUT5 mediates the spermatozoon-oviductal epithelial cell interaction and the beneficial effects of such interaction on the fertilizing ability of spermatozoa. Results from this study provide the potential use of sFUT5-interacting proteins to enhance the fertilization ability of human spermatozoa by protecting them from oxidative stress.published_or_final_version
Obstetrics and Gynaecology
Doctoral
Doctor of Philosophy
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Jin, Sha. "Membrane interaction of amyloid–beta (1–42) peptide induces membrane remodeling and benefits the conversion of non–toxic Aβ species into cytotoxic aggregate." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2016. http://dx.doi.org/10.18452/17632.
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Das Amyloid-beta Peptid (Ab) ist der Hauptbestandteil der extrazellulären Plaques bei der Alzheimerschen Krankheit. Das Ziel der vorliegenden Arbeit ist es, die Mechanismen der Wechselwirkungen des Ab mit der Plasmamembran und der nachfolgenden zellulären Aufnahme aufzuklären. Die Aggregation, die zelluläre Aufnahme und die Zytotoxizität von Ab42 wurden durch Verwendung von fluoreszenzmarkierten Ab42 in einem Neuroblastomzellkulturmodell untersucht. Sowohl bei Inkubation mit Monomeren als auch mit Aggregaten wurde in den Zellen Ab42 detektiert. Dabei binden Ab42 Monomere und kleine Aggregate zunächst an die Zellmembran. Allerdings erfolgt keine direkte Aufnahme von Monomeren in die Zelle. Erst nach Ausbildung von Aggregaten mit geordneter Sekundärstruktur wurde Ab42 in den endozytotischen Vesikel detektiert. Voraussetzung für den an der Membran ablaufenden Aggregationsprozess ist, dass die Monomere oberhalb einer kritischen Konzentration anwesend sind, um eine Bildung von beta-Faltblatt-Strukturen (bF) und entsprechenden Aggregaten zu ermöglichen. Ab42 Aggregate, die sich durch eine bF auszeichneten, benötigten keine kritische Schwellenkonzentration für die endozytotische Aufnahme. Eng mit der Aufnahme von Ab42 Aggregaten war die Veränderung des zellulären Metabolismus verbunden. Um die Wechselwirkung zwischen Ab und der Membrannäher zu charakterisieren, wurden Modellmembransystemen einschl. riesigen Membranvesikeln genutzt. Dabei wurde beobachtet, dass sowohl Ab42 als auch Ab40 Einstülpungen in der Membran induzieren können. Kleine Aggregate beider Isoformen, die noch keine bF aufweisen, interagierten bevorzugt mit der ungeordneten Lipidphase und induzierten dabei eine negative Membrankrümmung. Diese Beobachtungen legen den Schluss nahe, dass möglicherweise das Ab selbst den endozytotischen Prozess unterstützt oder diesen sogar einleiten könnte. Dies könnte auch auf eine mögliche physiologische Funktion von Ab Aggregaten, die nicht toxisch sind, hindeuten.The accumulation of Amyloid beta peptide 1-42 (Ab42) in extracellular plaques is one of the pathological hallmarks of Alzheimer’s disease. Several studies have suggested that a cellular reuptake of Ab42 may be a crucial step in its cytotoxicity, but mechanisms of Ab-membrane interaction and subsequent cellular uptake are not yet understood. The first aim of the present study is to answer the question whether aggregate formation is a prerequisite or a consequence of Ab-membrane interaction and of Ab endocytosis. We visualized aggregate formation of fluorescently labeled Ab42 by Förster resonance energy transfer and tracked its internalization by human neuroblastoma cells. Both monomeric and aggregated Ab42 entered the cells, however, monomer uptake faced a concentration threshold and occurred only at concentrations and time scales that allowed beta-sheet-rich (bS) aggregates to form. By uncoupling membrane binding from internalization, we found that Ab42 monomers as well as small aggregate species bound rapidly to the plasma membrane and formed bS aggregates. These structures were subsequently taken up and accumulated in endocytic vesicles. This process correlated with inhibition of cellular metabolism activities. Our data therefore imply that the formation of bS aggregates at the cell membrane is a prerequisite for Ab42 uptake and cytotoxicity. The second aim of the study is to investigate the Ab-membrane interaction in vitro by using giant unilamellar vesicles and giant plasma membrane vesicles as model membrane systems. We found that both Ab isoforms, Ab42 and Ab40, interacted with the liquid disordered phase of model membranes. Early aggregation intermediates, which did not yet bind to the amyloiddophilic dye Thioflavin T, induced negative membrane curvature. The ability of Ab to induce membrane deformation suggests that Ab may facilitate its own endocytosis. It also hints at a possible physiological function of non-toxic Ab aggregate species.
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Tabdanov, Erdem. "Aspects biophysiques de l'adhérence intercellulaire : rôle des cadhérines dans les interactions membrane-cytosquelette." Paris 11, 2008. http://www.theses.fr/2008PA112105.
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Le cytosquelette cortical est impliqué dans tous processus impliquant la surface cellulaire. Il est associé à la membrane soit de manière transitoire ou durable. La E-cadhérine est une molécule d’adhérence qui stimule le développement de contact intercellulaire et augmente l’énergie d’adhérence intercellulaire en induisant le remodelage du cytosquelette d’actine. J’ai utilisé la technique d’étirement hydrodynamique de nano-tubes membranaires à partir de cellules pour analyser les interactions membrane-cytosquelette. J’ai réalisé des étirements de tubes soit à partir de billes recouvertes de polylysine (extrusion aspécifique) ou d’anticorps spécifiques de la E-cadhérine et à partir de cellules exprimant ou non des cadhérines. Les résultats oobtenus montre que l’extrusion « aspécifique » de tube de membrane est gouvernée par deux modes principaux d’écoulement de composants membranaires selon leur relation avec le cytosquelette cortical sous-jacent : (1) le détachement du cortex sous la membranes à proximité du tube, et (2) la dissipation visqueuse dans la zone distale. Les résultats provenant des extrusions « spécifiques » de tubes de membranes montrent que l’engagement des E-cadhérines augmente fortement l’énergie d’adhésion membrane-cytosquelette cortical. Cependant, cela se produit dans une zone strictement localisée au site adhésif. Après détachement des interactions membrane-cytosquelette, une récupération complète de leur adhésion est observée en 30 secondes montrant le rôle crucial des cadhérines dans la dynamique du remodelage cortical. Parfois lors de l’extrusion « spécifique » il y a formation de protubérance cylindrique d’un diamètre bien supérieur à celui d’un tube de membrane classique et d’environ 10-20 µm de long, appelée « tube cortical géant ». Il permet de mesurer le module de courbure du cortex cellulaire kc=2. 4. 10-16J. Ces résultats montrent le rôle crucial de la e-cadhérine dans le contrôle de l’interaction membrane-cytosqueletteCytoskeleton cortex is the basic cellular structure, determining cell viscoelastic properties and membrane dynamics. It is involved in every cell-surface associated process. Cadherins are transmembrane adhesion receptors spatially associated with actin cytoskeleton remodelling and promoting cell-cell adhesion and increasing adhesion energy. I used the tether extrusion technique, which consists of a hydrodynamic pulling of membrane tube from the cell surface, to directly measure the membrane-cytoskeleton interaction and adhesion energy. I performed specific or unspecific tether extrusions to compare the effect of E-cadherin-induced modification of cortex-membrane interactions. The results show that membrane tether unspecific extrusion is ruled by two main modes of the flow of membrane components through cytoskeleton related membrane-supporting network : (1) the detachment of the flowing membrane apart the cortex in the proximal zone of the tether’s neck and (2) the viscous permeation of molecules in the flowing membrane at the distal zone. For specific extrusion, the initial stage of tube elongation is extremely resistant to the hydrodynamic force, followed by the regular extrusion resistance, comparable to unspecific case. Together, these data show that E-cadherin engagement largely increases the energy of the plasma membrane-cortex adhesion, but restricted at the cell-bead interface zone. In some cases of specific extrusion, a giant cortical tube is formed corresponding to a cylindrical protrusion of 4-5µm in diameter and 10-20µm in length. It reveals the presence of the membrane-supporting cytoskeleton inside the tube and its contractile activity. This phenomenon allows measuring the cell cortex curvature modulus Kc =2. 4. 10-16J and also displays the strong anchoring of the cortex to the cell surface through E-cadherins. The results show the E-cadherin-promoted cortex reorganization and reveal its restriction to the adhesive zone