Dissertations / Theses on the topic 'Membrane transport systems'

In this article, we review our results on diffusion and phase separation in lipid membranes, as well as on interaction of membranes with colloidal particles, biomacromolecules, and supramolecular assemblies, which were obtained within the framework of the Saxon Research Unit FOR 877 “From Local Constraints to Macroscopic Transport”.

The work in this thesis shows that it is possible to design a diffusion cell which will allow access to the flux and lag time of a permeant without the need for invasive sampling and that this novel cell is both sensitive and reproducible. It was also shown that the cell could be used in conjunction with both simple model membranes and more complex biological membranes, namely the epidermis. From the data achieved from the cell it was possible to derive a series of equations which allowed access to thermodynamic parameters such as ?H, ?G and ?S. An extension of this calculational approach revealed that manipulation of the van’t Hoff isochore, under the condition where enthalpy is constant over the temperature range, it should be possible to calculate the partition coefficient. Ultimately these parameters can be used in the description of structure activity relationships. The systems described in this thesis are of a complex biological nature consequently the returned data reflect this complexity. In order to utilise the data to their full potential some method for dealing with this complexity was sought. One approach widely discussed in the literature is that of chemometric analysis or soft modelling. Initial studies into the use of chemometric analysis proved positive for the data presented in this thesis, and suggested that formulation contributions from components with close absorbance maxima could be separated.

In this article, we review our results on diffusion and phase separation in lipid membranes, as well as on interaction of membranes with colloidal particles, biomacromolecules, and supramolecular assemblies, which were obtained within the framework of the Saxon Research Unit FOR 877 “From Local Constraints to Macroscopic Transport”.

Amorphous silica membranes have molecular sieving properties for the separation of hydrogen from gas mixtures at high temperature. Consequently, they are considered to be applied in separation of a shifted syngas coming out of a water-gas-shift-reactor into the syngas and hydrogen. This separation is a key to an Integrated Gasification Combined Cycle (IGCC) plant, which would allow reducing the carbon footprint in power generation industry. The main objective of this thesis was to carry out a preliminary assessment of suitability of currently available amorphous silica membranes for this separation. However, the separation properties of amorphous silica membranes reported in the open literature vary by orders of magnitude. Therefore, in the first part of this thesis the separation properties of hypothetical silica membrane with different pore size distributions were predicted from first principles. Considering different possible gas transport mechanisms, it was concluded that gas transport in amorphous silica membranes is dominated by the activated and non-activated Knudsen diffusion. The activation energy for transport of different species was predicted using the concept of suction energy. Then, with arbitrary pore size distributions gas permeance of hypothetical silica membrane was predicted for different gas species. Since the pore size distribution of amorphous silica membrane cannot be known a priori, the developed model was used to determine the pore size distribution based on experimentally measured single gas permeances of three different species (kindly provided by Natural Resources Canada, CANMET Energy Technology Center (CETC) laboratory in Ottawa) by minimizing the error of the calculated permeance ratios with respect to the experimental values. The results indicate that, depending on how the objective function is defined, more than one pore size distribution can be found to satisfy the experimental permeance ratios. It is speculated that by increasing the number of experimentally determined permeances, a more unique pore size distribution for the tested silica membrane can be obtained. However, even at this early stage, the developed model provides a rational explanation for the effect of membrane densification on the properties of silica membranes. More specifically, a simultaneous decrease in membrane permeance and selectivity due to membrane densification, reported in the literature, is explained by shrinking the size of pores beyond a certain critical value, which depends on the kinetic diameter of gas molecules that are being separated. Comparing theoretically determined permeances, which match experimentally observed permeance ratios, revealed that the experimental permeances are considerably smaller than the theoretical values. The ratio of the two provided the basis for a scaling factor, a new concept that was introduced in this thesis. To simulate membrane module performance, a novel approach was introduced. More specifically, co- and counter-current flow configurations as well as cross-flow configuration were modeled by assuming no change in feed composition over an infinitesimally small element of membrane area. This led to a system of linear, rather than differential equations, which was readily solved numerically.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2011.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 187-192).
Oxy-fuel combustion, particularly using an integrated oxygen ion transport membrane (ITM), is a thermodynamically attractive concept that seeks to mitigate the penalties associated with CO 2 capture from power plants. Oxygen separation in an ITM system consists of many distinct physical processes, ranging from complex electrochemical and thermochemical reactions to conventional heat and mass transfer. The dependence of ITM performance on power cycle operating conditions and system integration schemes must be captured in order to conduct meaningful process flow and optimization analyses. An axially spatially-distributed, quasi two-dimensional ITM model is developed based on fundamental conservation equations, semi-empirical oxygen transport equations obtained from the literature, and simplified fuel oxidation kinetic mechanisms. Aspects of reactor engineering such as geometric structure, flow configuration and the relationship between oxygen transport, fuel conversion and pressure drop are explored. Emphasis is placed on model robustness, modularity, and low computational expense. The model seeks to bridge the gap between detailed CFD studies and overly-simplified black-box models and provides a tool for the analysis and design of ITM systems. The ITM model is used to explore the dependence of ITM performance on reactor geometric structure, flow configuration, operating conditions, membrane material properties, and uncertainty in key modeling assumptions. Many operational constraints are presented that are usually overlooked by black-box modeling strategies, and the implications of these constraints are explored. Further, a comparison is made between reactive and separation-only ITMs to assess the relative merits and disadvantages of each. The results show that although a reactive ITM significantly improves the partial pressure driving force, practical reactor engineering considerations indicate that this concept is not superior to counter-current separation-only ITMs, mainly due to stringent temperature limitations of the membrane material. A Second Law assessment of certain ITM configurations is performed to evaluate the potential of ITM technology to reduce the air separation penalty and to guide effective systems-level integration. Finally, simulations of various ITM-based zero-emissions power cycles using the intermediate fidelity ITM model are performed. The first objective is to analyze the prevalent ITM-based power cycle designs and develop novel design modifications. The second objective is to investigate the effect of reactive ITM improvement design strategies proposed herein, such as the multiple compartment reactive ITM (MCRI), the low activation energy (LAE) materials reactive ITM, and hybrid reactive and separation-only concepts. An assessment of the potential for these novel ITM designs to reduce both the penalty and size associated with ITM air separation technology is conducted. The power cycle simulation and analysis clearly demonstrates the various challenges associated with implementing reactive ITMs. The hybrid cycle displays the potential to reduce the size of the ITM compared to the best separation-only concept while maintaining a comparable First Law efficiency. Additionally, the MCRI simulation results indicate comparable ITM size and pressure drops to the best separation only-concepts, greatly improving the attractiveness of reactive ITMs. Overall, the work herein finally allows for detailed optimization analyses to determine the best possible ITM oxy-combustion power cycles.
by Nicholas D. Mancini.
S.M.

Nucleosides are transported across the inner membrane of E. coli by at least two separate energy-driven systems known as NupC and NupG. These systems are encoded by the genes nupC and nupG respectively. CTN1 (a mammalian transporter isolated form the jejunum) is a homologue of NupC. CNT1 is able to transport the nucleoside analogue AZT, used to combat HIV infection. CNT1 and NupC share substrate specificity and they have amino acid sequence identity of 27%, particularly in their C-terminal region. With the completion of the E. coli genome project paralogues of NupC and NupG have been found, designated as YeiM and YeiJ, and XapB and YegT respectively. To functionally characterise the putative nucleoside transporter-encoding genes xapB, yegT, yeiM and yeiJ, mutants were constructed which the coding sequence of these genes had been deleted from their chromosome. From this work it was established that all four putative nucleoside transporters XapB, YegT, YeiM and YeiJ were able to transport adenosine in the assays at similar levels as the positive control. Furthermore in kinetic analysis of the transporters it was possible to determine Km values for the four transporters with adenosine as substrate, which were in the same order of magnitude as mammalian nucleoside transporters and that shown for xanthosine uptake by XapB. The mutant strains were used to construct a bioassay, in which their ability to transport nucleoside analogues was assessed. AZT was used as model substrate. The use of multiple mutants in experiments involving nucleoside uptake as a sole carbon source further established the fact that the collection of genes studied in this project was indeed nucleoside permeases. Preliminary experiments gave an insight in developing a high throughput bioassay which can be developed further in order to produce a useful tool for nucleoside analogue drugs assessment.

In mammalian cells, homeostasis and fate during development relies on the proper transport of membrane-bound cargoes to their designated cellular locations. The hetero-tetrameric adaptor protein complexes (APs) are required for sorting and concentration of cargo at donor membranes, a crucial step during targeted transport. AP2, which functions at the plasma membrane during clathrin-mediated endocytosis, is well characterized. In contrast, AP1 a clathrin adaptor mediating the delivery of lysosomal hydrolases via mannose 6-phosphate receptors (MPRs) and AP3 an adaptor ensuring the proper targeting of lysosomal membrane protein are difficult to study by classic cell biology tools. To gain new insights on these APs, our lab has previously designed an in vitro system. Reconstituted liposomes were modified with small peptides mimicking the cytosolic domains of bona fide cargoes for AP1 and AP3 respectively and thereby enabling the selective recruitment of these APs and the identification of the interacting protein network. In the study at hand we utilize above-described liposomes to generate supported lipid bilayers and Giant Unilamellar Vesicles (GUVs), large-scale membrane systems suited for analysis by fluorescence microscopy. By using cytosol containing fluorescently-tagged subunits, we visualized clathrin coats on artificial membranes under near physiological conditions for the first time. Moreover, we demonstrated clathrin-independent recruitment of AP3 coats on respective GUVs. Presence of active ARF1 was sufficient for the selective assembly of AP1-dependent clathrin coats and AP3 coats on GUVs. By using dye-conjugated ARF1, we show that ARF1 colocalized with AP3 coats on GUVs and that increased association of ARF1 with GUVs coincided with AP1-dependent clathrin coats. Our previous study identified members of the septin family together with AP3 coats on liposomes. Here we show on GUVs, that active ARF1 stimulated the assembly of septin7 filaments, which may constrain the size and mobility of AP3 coats on the surface. Subsequent cell biology studies in HeLa cells linked septins to actin fibers on which they may control mobility of AP3-coated endosomes and thus their maturation. An actin nucleation complex, based on CYFIP1 was identified together with AP1 on liposomes before. Here we show on GUVs, that CYFIP1 is recruited on the surface surrounding clathrin coats. Upon supply of ATP, sustained actin polymerization generated a thick shell of actin on the GUV surface. The force generated by actin assembly lead to formation of long tubular protrusions, which projected from the GUV surface and were decorated with clathrin coats. Thereby the GUV model illustrated a possible mechanism for tubular carriers formation. The importance of CYFIP1-reliant actin polymerization for the generation of MPR-positive tubules at the trans-Golgi network (TGN) of HeLa cells was subsequently demonstrated in our lab. The notion that tubulation of artificial membranes could be triggered by actin polymerization allowed us to perform a comparative mass spectrometry screen. By comparing the abundance of proteins on liposomes under conditions promoting or inhibiting actin polymerization, candidates possibly involved in stabilization, elongation or fission of membrane tubules could be identified. Among the proteins enriched under conditions promoting tubulation, we identified type I phosphatidylinositol-4-phosphate 5-kinases. Their presence suggested an involvement of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in tubule formation. By cell biology studies in HeLa we show, that down regulation of these enzymes altered the dynamics of fluorescently-tagged MPRs, illustrating the importance of locally confined PI(4,5)P2 synthesis during formation of coated carriers at the TGN. Bin–Amphiphysin–Rvs (BAR) domains are known to sense membrane curvature and induce membrane tubulation. Among various BAR domain proteins, Arfaptin2 was enriched under conditions allowing tubulation of liposomes. By microscopy studies on HeLa cells we show, that Arfaptin2 as well as its close paralog Arfaptin1 were present on AP1-coated MPR tubules emerging from the TGN. We further show, that tubule fission occurred at regions were Arfaptin1 is concentrated and that simultaneous down regulation of both Arfaptins lead to increased number and length of MPR tubules. Since fission of coated transport intermediates at the TGN is poorly understood, our findings contribute a valuable component towards a model describing the entire biogenesis of coated post-Golgi carriers. In conclusion, combining artificial membrane systems and cell biology studies allowed us to propose new models for formation as wall as for fission of AP1-coated transport intermediates at the TGN. Further we gained new insights on AP3 coats and the possible involvement of septin filaments in AP3-dependent endosomal maturation.

Ce projet de thèse étudie la synthèse et les propriétés des triarylamines pour le transport d'électrons, d'ions ou de molécules d'eau, en se basant sur les propriétés d'auto-assemblage et de conductivité de certaines triarylamines, récemment découvertes par notre équipe. La thèse débute par une introduction sur la chimie supramoléculaire et les polymères supramoléculaires, et aborde ensuite dans son deuxième chapitre la synthèse et les propriétés d'analogues macrocycliques de triarylamine présentant des propriétés électromagnétiques intéressantes. Dans le chapitre suivant, ce travail de thèse explore l'applicabilité des triarylamines pour le transport biomimétique des ions métalliques et des molécules d'eau à travers des membranes de bicouches lipidiques. Le dernier chapitre de cette thèse s'intéresse ensuite à la fabrication de surfaces conductrices organiques / inorganiques hybrides par dopage de surfaces non conductrices avec des assemblages de triarylamines
Based on the navel and highly interesting self-assembly properties found for certain triarylamines, together with the resulting conducting properties, this PhD project investigates the synthesis and properties of triarylamines towards the transport of electrons, ions or water molecules. The thesis starts with an introduction on supramolecular chemistry and supramolecular polymers, and then discuss in its second chapter the synthesis and properties of macrocyclic triarylamine analogues with interesting electromagnetic properties. ln the following third chapter this thesis work explores the applicability of triarylamines towards the biomimetic transport of metal ions and water molecules through lipid bilayer membranes. The last chapter of this thesis then deals with the fabrication of hybrid organic/inorganic conducting surfaces through doping of non-conducting surfaces with triarylamine assemblies

Glucose is transported in crustacean hepatopancreas and intestine by Na+-dependent co-transport, while Na+-dependent D-fructose influx has only been described for the hepatopancreas. It is still unclear if the two sugars are independently transported by two distinct cotransporter carrier systems. In this study lobster (Homarus americanus) hepatopancreas brush border membrane vesicles (BBMV) were used to characterize, in detail, the cation-dependency of both D-[3H] glucose and D-[3H] fructose influxes, while in vitro perfused intestines were employed to determine the nature of cation-dependent sugar transport in this organ. Over the sodium concentration range of 0-100 mM, both 3H-D-glucose and 3H-D-fructose influxes (0.1 mM; 1 min uptakes) by hepatopancreatic BBMV were hyperbolic functions of [Na+], exhibiting Km values of 2.30 ± 0.59 and 2.58 ± 0.95 mM, respectively. D-[3H] glucose and fructose influxes by hepatopancreatic BBMV over a potassium concentration range of 15-100 mM were hyperbolic functions of [K+], exhibiting Km values of 9.85 ± 0.41 and 12.6 ± 0.80 mM respectively. Both sugars displayed significant (p < 0.01) Na+/K+-dependent and Na+-independent uptake processes. Transepithelial 25 μM D-[3H] glucose and D-[3H] fructose fluxes across lobster intestine over a luminal sodium and potassium concentration range of 0 – 50 mM and 5-100 mM, respectively, were hyperbolic functions of luminal [Na+] and [K+]. As with hepatopancreatic sugar transport, transepithelial intestinal sugar transport exhibited both significant (p < 0.01) Na+/K+-dependent and Na+-independent processes. Results suggest that both D-glucose and D-fructose are transported by a single carrier process in each organ with sodium being the preferred cation for both sugars in the hepatopancreas, and potassium being the preferred cation for both sugars in the intestine.

This thesis analyses two strategies for decreasing anthropogenic carbon dioxide (CO2) emissions: to capture and store CO2, and to increase the use of biomass. First, two concepts for CO2 capture with low capture penalties are evaluated. The concepts are an integrated gasification combined cycle where the oxygen is supplied by a membrane reactor, and a hybrid cycle where the CO2 is captured at elevated pressure. Although the cycles have comparatively high efficiencies and low penalties, they illustrate the inevitable fact that capturing CO2 will always induce significant efficiency penalties. Other strategies are also needed if CO2 emissions are to be forcefully decreased. An alternative is increased use of biomass, which partially could be used for production of motor fuels (biofuels). This work examines arguments for directing biomass to the transport sector, analyses how biofuels (and also some other means) may be used to reduce CO2 emissions and increase security of motor fuel supply. The thesis also explores the possibility of reducing CO2 emissions by comparatively easy and cost-efficient CO2 capture from concentrated CO2 streams available in some types of biofuel plants. Many conclusions of the thesis could be associated with either of three meanings of the word sense: First, there is reason in biofuel production – since it e.g. reduces oil dependence. From a climate change mitigation perspective, however, motor fuel production is often a CO2-inefficient use of biomass, but the thesis explores how biofuels’ climate change mitigation effects may be increased by introducing low-cost CO2 capture. Second, the Swedish promotion of biofuels appears to have been governed more by a feeling for attaining other goals than striving for curbing climate change. Third, it seems to have been the prevalent opinion among politicians that the advantages of biofuels – among them their climate change mitigation benefits – are far greater than the disadvantages and that they should be promoted. Another conclusion of the thesis is that biofuels alone are not enough to drastically decrease transport CO2 emissions; a variety of measures are needed such as fuels from renewable electricity and improvements of vehicle fuel economy.
QC 20100823

Les membranes fluorées sont utilisées en particulier dans les dénommées piles à combustible à membrane électrolyte polymère. Grâce à sa grande mobilité en protons, le célèbre ionomer Nafion® (Dupont) est un matériau de référence pour les applications liées aux piles à combustible. En présence d’eau ou d’autres solvants hydrophiles la membrane se sépare en une matrice polymérique hydrophobe et une sous-phase aqueuse contenant des clusters d’eau et ions, dont les tailles et la connectivité augmente quand la quantité d’eau augmente [1]. Quelle est la morphologie du Nafion et la structure du solvant, dans de tels systèmes?Il a été récemment montré [2] sur des simulations de large systèmes que plusieurs modèles morphologiques reproduisent les données expérimentales de diffusion, évoquant l’incapacité des mesures de diffusion seules à élucider la véritable structure du Nafion.Néanmoins, un modèle ’aléatoire’ décrit dans [2], c’est à dire l’unique modèle étudié sans présumer d’une structure initiale particulière, n’a pas pu reproduire les données expérimentales.Générer en simulations moléculaires des configurations du système qui soient vraiment décorrélées de la configuration initiale reste un vrai défi statistique. Les échelles de temps réalisables ne permettent simplement pas d’obtenir des mouvements significatifs du polymère (comme des transitions de conformations, repliements de chaînes, etc.). Nous proposons ainsi dans cette étude un nouveau modèle de Nafion à morphologie aléatoire. Un algorithme récemment développé est utilisée pour générer des chaînes de Nafion avec des chemins et des points de départ aléatoires. Une différence majeure avec le modèle aléatoire dans [2] est que nous ne construisons pas nos systèmes à une densité proche de la densité finale. Pour ne pas démarrer avec des chaînes trop enchevêtrées, les systèmes sont initialement préparés à une densité en dessous de la référence expérimentale. La densité après équilibration est de nouveau proche de l’expérience. Bien qu’il soit facilement envisageable d’améliorer les nouveaux algorithmes, nous démontrons ici qu’avec la présente version plusieurs séries de configurations compatibles avec les données expérimentales de diffusion disponibles peuvent être générées et équilibrées. Douze large systèmes de Nafion à morphologie aléatoire sont construits avec des positions initiales des atomes ainsi que des quantités d’eau et des longueurs de chaînes (Nafion/Hyflon) différentes. Ils sont équilibrés puis simulés sur plusieurs dizaines de nanosecondes. Après équilibration, les structures sont, comme indiqué ci-dessus,compatibles avec les données expérimentales de diffusion. En plus nous étudions un modèle ressemblant à celui de Schmidt-Rohr and Chen [3], c’est à-dire le plus récent modèle morphologique. Avec ce modèle, les données expérimentales sont également reproduites de manière satisfaisante, d’où la prolongation du débat sur la structure du Nafion. La cohésion entre les valeurs calculées et celles mesurées expérimentalement incite à des analyses plus en détails de ces configurations obtenues. Nous caractérisons et analysons les structures locales, intermédiaires et à grande échelle avec divers paramètres structuraux et distributions des tailles de domaines. Nous calculons donc, par exemple, des fonctions de distribution radiale (rdf), des facteurs de structure (S(q)) totaux et partiels tout comme des nombres et des tailles de clusters hydrophiles (selon la définition d’un cluster). La dynamique de diverses espèces dans le système est également examinée,par exemple au travers des déplacements carrés moyens (msd) et des coefficients de diffusion. Ces simulations sont probablement à la limite de ce qui est réalisable aujourd’hui avec des simulations ’full-atom’ du type MD. Nous espérons que ce travail fera avancer le débat sur la structure et la dynamique de ces matériaux importants
Perfluorinated membranes are used in particular in polymer electrolyte fuel cells(PEFC). The well-known ionomer Nafion® (Dupont) is, due to its high proton mobility,a reference material for fuel cell applications. In water or other hydrophilic solvents themembrane segregates into a hydrophobic backbone matrix and a hydrophilic sub-phasecontaining clusters of both water and ions, where the cluster sizes and connectivity increasewith increasing water content [1].What is the Nafion morphology and the structure of the solvent in such systems? It hasbeen shown recently [2] on large simulated systems that several morphological modelsfit the experimental scattering data, suggesting the inability of scattering experimentsalone to elucidate the true structure of Nafion. However, a ’random’ model describedin [2], i.e. the only explored model that did not assume a particular initial structure,could not reproduce the experimental data.It remains a real computational challenge to generate in molecular simulations systemconfigurations which are really decorrelated from the initial one. The time scales thatcan be achieved simply do not allow to obtain significant motions of the polymer (e.g.conformational changes, folding, etc.). We thus propose in this work a new randommodel of Nafion. A newly developped algorithm is used to generate Nafion chains withrandom growth paths and random starting points. A significant difference with therandom model in [2] is that we do not build our systems at a density close to the finalone. In order not to start with too much entangled chains, the systems are initiallybuilt at a density below the experimental one. The density after equilibration is againclose to the experimental one.Even though further improvements of the new algorithms can easily be envisaged,we demonstrate here that with the present version several sets of configurations thatare compatible with the available scattering data can be generated and equilibrated.Twelve large random Nafion systems are built with different initial positions of theatoms as well as different water contents and side chain lengths (Nafion/Hyflon). Theyare equilibrated and then simulated for several ten nanoseconds. After equilibration,the structures are, as mentioned, compatible with the experimental scattering data. Inaddition we study a model similar to the one by Schmidt-Rohr and Chen [3], i.e. thenewest morphological model of Nafion. The experimental scattering data are also satisfactorilyreproduced with this model, hence, the prolonged debate over the structureof Nafion.This agreement gives confidence that a more detailed analysis of the so-obtained configurationsis scientifically warranted. We characterize and analyze the local, intermediateand large-scale structures by various structural parameters and domain size distributions.We therefore compute, for example, radial distribution functions (rdf), total andpartial structure factors (S(q)) as well as numbers and sizes of hydrophilic clusters (dependingon the definition of a cluster). The dynamics of various species in the systemis also investigated, e.g. via the computation of the mean square displacements (msd)and the self-diffusion coefficients. These simulations are probably at the limit of whatcan today be achieved with all-atom molecular simulations of the MD type. We hopethat this work will advance the ongoing debate on the structure and dynamics of theseimportant materials
Perfluorierte Membranen werden insbesondere in Polymerelectrolyt-Brennstoffzellen(PEFC) eingesetzt. Das wohlbekannte Ionomer Nafion® (Dupont) ist wegen seinerhohen Protonenbeweglichkeit ein Referenzmaterial für solche Anwendungen in Brennstoffzellen.Die Membran separiert in Wasser oder anderen hydrophilen Lösungsmittelin eine hydrophobe Polymermatrix und eine hydrophile Subphase, die Cluster mitWasser und Ionen enthält. Dabei vergroeßern sich die Ausdehnung der Cluster und ihreKonnektivität mit zunehmendem Wassergehalt [1].Welche ist die Morphologie des Nafions und die Struktur des Lösungsmittels in diesenSystemen? Es ist jüngst anhand großer simulierter Systeme gezeigt worden [2], dassmehrere morphologische Modelle die experimentellen Streudaten wiedergeben können,was nahelegt, dass solche Streudaten alleine nicht geeignet sind, die wahre Strukturdes Nafion aufzudecken. Ein in [2] beschriebenes ’Zufallsmodell’, d.h. das einzigeder untersuchten Modelle, das keine besondere Anfangsstruktur annahm, konnte dieexperimentellen Daten allerdings nicht wiedergeben.In molekularen Computersimulationen Konfigurationen zu erzeugen, die wirklich nichtmehr mit der angenommenen Anfangskonfiguration korreliert sind, bleibt eine echteHerausforderung. Die erreichbaren Zeitskalen sind zu kurz, um eine signifikante Bewegungdes Polymers (z.B Konformationsänderungen, Faltungen, usw.) zuzulassen. Indieser Arbeit wird daher ein neues Zufallsmodell für Nafion vorgestellt. Ein neuentwickelterAlgorithmus erzeugt Nafionketten mit zufälligem Wachstumspfad ausgehendvon zufälligen Anfangspunkten. Ein signifikanter Unterschied zu dem Zufallsmodellvon [2] ist, dass hier nicht versucht wird, die Systeme bei einer Dichte vergleichbarder experimentellen Dichte aufzubauen. Anstattdessen werden die Systeme, um alzustarkes Verknäuelung zu vermeiden, anfangs bei einer deutlich kleineren Dichte erzeugt.Nach äquilibrierung ist die Systemdichte wieder in etwa gleich der experimentellen.Wiewohl weitere Verbesserungen des neu Algorithmuses leicht ins Auge gefaßt werdenkönnen, so kann hier doch gezeigt werden, dass mit der gegenwärtigen VersionKonfigurationen erzeugt und äquilibriert werden können, die mit den verfügbarenStreudaten kompatibel sind. Zwölf große Nafion Zufallssysteme, mit verschiedenenAnfangspositionen der Atome, verschiedenem Wassergehalt und Längen der Seitenketten(Nafion/Hyflon) werden aufgebaut. Diese werden äquilibriert und mehrerezehn Nanosekunden lang simuliert. Nach der äquilibrierung sind die Strukturen, wieerwähnt, kompatibel mit den experimentellen Streudaten. Weiterhin wird ein Modellähnlich dem von Schmidt-Rohr und Chen [3], d.h. dem neuesten morphologischen Modellfür Nafion, studiert. Auch hier werden die experimentellen Streudaten zufriedenstellendwiedergegeben, daher die weiterhin bestehende Debatte über die Struktur desNafion.Die gefundenen übereinstimmungen lassen darauf vertrauen, dass eine detaillierte Analyseder simulierten Konfigurationen wissenschaftlich sinnvoll ist. So wird die Strukturder Systeme auf verschiedenen Längenskalen charakterisiert, zum Beispiel durch radialePaarverteilungsfunktionen (rdf), totale und partielle Strukturfaktoren (S(q)) sowieAnzahl- und Größenverteilungen hydrophiler Cluster (abhängig von der Definition einesClusters). Die Dynamik einzelner Spezies im System wird ebenfalls untersucht, zumBeispiel durch die Berechnung der mittleren quadratischen Verschiebungen (msd) undder Selbstdiffusionskoeffizienten. Diese Simulationen sind wahrscheinlich an der Grenzedessen, was heute mit ’all-atom’ molekularen MD-Simulationen möglich ist. Ich vertrauedarauf, dass diese Arbeit dennoch einen Fortschritt in der aktuellen Debatte überdie Struktur und Dynamik dieser wichtigen Materiale darstellt

Cette thèse s’applique à améliorer la compréhension de l’absorption du cadmium (Cd) par le maïs et le tabouret calaminaire, dans l’objectif de mieux modéliser son transfert vers les plantes comestibles ou hyperaccumulatrices. Le modèle utilisé étant sensible aux caractéristiques d’absorption racinaire, le premier objectif était de développer une méthode rigoureuse de mesure de ces paramètres. Deux protocoles ont été mis au point pour décrire précisément, en fonction de la concentration de Cd en solution, l’influx net de Cd dans les parois et dans le milieu intracellulaire des racines. Les résultats ont mis en évidence, pour la première fois, l’existence d’un système de transport à faible affinité (LATS) qui agit en même temps que le système de transport à forte affinité pour le Cd (HATS). Les nouveaux paramètres cinétiques mesurés n’ont cependant pas amélioré significativement le modèle : le prélèvement de Cd par le maïs est surestimé de 100%, et son prélèvement par le tabouret calaminaire est sous-estimé de 66%. Plusieurs facteurs ont alors été étudiés pour comprendre les raisons de ce décalage. Nous avons montré que les conditions dans lesquelles ont été mesurés les paramètres cinétiques présentaient des caractéristiques capables de modifier radicalement leurs valeurs : la composition ionique de la solution d’exposition au Cd, la concentration de Cd durant la croissance et la structure racinaire engendrée par l’hydroponie. Cette thèse suggère de cultiver les plantes en aéroponie et de mesurer les paramètres cinétiques dans une composition ionique représentative du voisinage des parois et des membranes racinaires en sol, composition qu’il reste encore à déterminer
This thesis aimed to improve the comprehension of cadmium (Cd) absorption by maize and alpine pennycress plants in order to model better its transfer into edible and hyperaccumulating plants. Since the model used is sensitive to the characteristics of root absorption, the first objective was to develop a rigorous method for measuring those parameters. Two protocols were finalized to describe precisely Cd net influx in both root compartments (apoplast and symplast) according to Cd concentration in solution. The results highlighted, for the first time, the existence of a low-affinity transport system (LATS) that works at the same time with the high-affinity transport system (HATS). However, the kinetics parameters measured through these experimentations did not succeed in improving significantly the model: Cd uptake by maize is overestimated by 100%, and Cd uptake by alpine pennycress is under-estimated by 66%. Several factors were investigated in order to understand the reasons of this difference. We showed that the experimental conditions used to measure the kinetics parameters present characteristics that are able to modify their values significantly: the ionic composition of the solution of exposition to Cd, Cd concentration during growth, and the root structure that forms in hydroponics. This thesis suggests to choose aeroponics as controlled culture condition, and to measure the kinetics parameters in an ionic composition that is representative of the close vicinity of the root cell walls and membranes in soil. This composition remains to be investigated

Les peptides de la famille du LAH4 sont des peptides cationiques capables de se replier en hélice α amphiphile. Ces peptides sont riches en histidines ce qui permet de moduler les interactions de ces peptides de manière pH dépendante et dans une gamme physiologique. Leurs capacités à interagir et perturber les membranes ont été utilisées pour divers applications biologique, et notamment pour l'amélioration de systèmes de transport de gènes.Le travail de cette thèse a été divisé en trois parties dans le but de caractériser de manière biophysique les différentes interactions ayant lieu lors de la livraison du système de transport de gènes à l’intérieur d’une cellule. L’interaction peptide-peptide : avec l’étude de l’agrégation en fibrilles de la VF1 ; l’interaction peptide-membrane : avec l’effet du LAH4L1 en présence de membranes ; et l’interaction peptide-ADN : avec le suivit de l’interaction entre le LAH4L1 et de l'ADN
The LAH4 family consists of cationic amphiphilic peptides with propensity to fold in α-helical secondary structures. They contain histidines allowing the modulation of their interactions in a pH dependent manner in the physiological range. In membranes, at neutral or acidic pH the peptide assumes a transmembrane or an in planar configuration, respectively.In the field of gene delivery systems, peptides like LAH4 are used. They are able to firstly interact with different cargoes in order to form stable complexes, then interact with the cell membrane, and finally, promote to escape from the endosome.This PhD has been divided into three parts in order to characterize, with biophysical methods, the interactions occurring during the delivery of these gene systems: peptide-peptide interactions with a focus on the study of VF1 fibre formation; peptide-membrane interactions: with the investigation of the effect of LAH4L1 in different membranes; and peptide-DNA interactions, where the interactions of LAH4L1 with a small DNA fragment were measured

Electron transport systems exist in the plasma membranes of all cells. Although not well characterised they play roles in cell growth and proliferation, hormone responses and other cell signalling events, but perhaps their most important role, especially in erythrocytes, is enabling the cell to respond to changes in both intra- and extracellular redox environments. Human erythrocytes possess a transmembrane electron transport capability that mediates the transfer of reducing equivalents from reduced intracellular species to oxidised extracellular species and is concomitant with proton extrusion. In the work for this thesis I showed that erythrocyte membranes contain a transmembrane WST-1 (water soluble tetrazolium-1) reductase activity that uses reducing equivalents from intracellular NADH to reduce extracellular WST-1. The rate of WST-1 reduction was increased by the presence of phenazine methosulfate and, although of low activity, it showed similar properties to a previously reported transmembrane NADH-oxidase activity. 1H NMR experiments showed that WST-1 was reversibly bound to the membrane and/or proteins in the membrane within the timeframe of the NMR experiment, confirming the location of the WST-1 reduction. Preliminary attempts to purify NADH:WST-1 reductase and NADH:ferricyanide reductase activities from the erythrocyte plasma membrane were inconclusive. The protein(s) responsible for the reduction of these oxidants appear to be of low abundance in the plasma membrane and may be part of a larger protein complex. Further work on the isolation of these redox activities is required before the protein(s) involved can be identified with any confidence. The ability of cells to export electrons suggests that an electron import mechanism might also exist to re-establish the cell�s redox-buffering equilibrium under conditions of oxidative stress. This hypothesis was tested in glucose-deprived erythrocytes using reduced glutathione and NADH as extracellular electron donors. It was shown that neither reduced glutathione nor NADH donated reducing equivalents through a transmembrane redox system. Extracellular NADH was, however, able to produce profound changes in starvation metabolism and methaemoglobin reduction rates. The addition of extracellular NADH caused a six-fold increase in the rate of lactate production above that observed in glucose-starved controls, together with a concomitant decrease in pyruvate production. In erythrocytes containing high levels of methaemoglobin, extracellular NADH increased the rate of methaemoglobin reduction in both the presence and absence of glucose. These results were explained by the leakage of lactate dehydrogenase from erythrocytes due to an admittedly low level of haemolysis. This caused the displacement of the intracellular pseudo-equilibrium of the lactate dehydrogenase reaction via transmembrane exchange of lactate, allowing the conversion of extracellular pyruvate to lactate and resulted in an increase in intracellular NADH concentrations. The latter increased the rate of methaemoglobin reduction. In conclusion, the work described in this thesis showed that erythrocyte membranes do not contain mechanisms for importing electrons or reducing equivalents from extracellular reduced glutathione or NADH. Erythrocytes do, however, contain an electron export system which can reduce extracellular oxidants such as WST-1 and the activity of this system depends on an intricate balance between intracellular antioxidants and enzyme activities. There is much still to be learnt about plasma membrane redox systems, little is known, for example, about the protein composition, mechanism of action, and the in vivo conditions under which these systems are most active.

Ce travail rapporte l’étude de systèmes membranaires à valves sélectives (SMV), qui combinent les avantages des techniques de séparation par membrane et par absorption. La 1ère partie du mémoire présente l'étude du transport de matière en régime permanent et transitoire dans les SMV élaborés à partir de membranes polymères denses et de solution aqueuses. Dans la 2ème partie le développement d'un modèle mathématique original pour la simulation des SMV est proposé ainsi que son évaluation expérimentale pour les séparations CO2/CH4/H2, avec des solutions aqueuses confinées de K2CO3. Des améliorations simultanées de la perméabilité (˜ 4 fois) et de la sélectivité (>20 fois) ont été obtenues pour CO2 avec le mélange CO2/H2. Un logiciel basé sur un nouveau modèle de transport de gaz dans ces contacteurs membranaires prenant en compte la sorption physique et chimique dans la phase liquide a été développé pour le calcul de la perméabilité en régime transitoire ou permanent
Combined gas-liquid membrane systems gather the advantages of membrane and absorption separation techniques. Specifically, selective membrane valves (SMV called flowing liquid membranes) were studied, i.e. creation, gas transport experiments, liquid nature and temperature dependences, and modelling. SMV use a flowing layer of liquid supported between two membranes. The 1st part of the dissertation reports the study of steady and non-steady state gas transports in SMV built out dense polymeric membranes. In the 2nd part is given the development of an original mathematical model for SMV simulation and its experimental assessment for CO2/CH4/H2 mixtures with aqueous solutions of K2CO3. From systematic studies of steady and non-steady state processes, both in flow-through and circulating modes, it was found that increasing of temperature and liquid carrier concentration (K2CO3) leads to the simultaneous rise of permeability and selectivity during CO2 recovery from CO2/H2 mixture

Oligopeptide transport in rat small intestine has been studied in intact tissue, using the luminally and vascularly perfused isolated jejunum in situ technique, and a hydrolysis-resistant dipeptide (D-Phe-L-Gln). The data in this thesis can be divided into two main sections: (l) identification of the transporter proteins, and (2) short-term regulation of transport. The basolateral peptide transporter protein has not, to date, been identified. A candidate protein was identified from membrane vesicles by a photo affinity labelling technique using a dipeptide derivative ([4-azido-3,5-3H-D-Phe]-L-Ala), previously shown to be an efficient substrate for the basolateral transporter. The labelled candidate protein was successfully isolated by 2-DE, which revealed an apparent Mr of 112 ± 2 kDa and a pI of approximately 6.5. Initial sequence analysis, tryptic digestion followed by MALDI-TOF analysis and Q-TOF fragmentation of a tryptic peptide, produced a peptide fingerprint and a sequence tag of 9 amino acids, respectively, which, together, did not completely and conclusively match to any known protein sequence contained within databases, therefore suggesting that the 112 kDa protein may be novel. Short-term regulation of peptide transport was also investigated using the vascular perfusion method. An amino acid-sensing pathway was discovered, using L-Leucine as the regulator, involving protein kinase cacades leading to p70S6k activation and subsequent stimulation ofbasolateral membrane peptide transport. A major conclusion arising from the data was the distinction between PepTl and the basolateral transporter, i.e. the sequence data obtained from the candidate protein did not match to the PepTl sequence; in addition there appeared to be distinct mechanisms of regulatory control at the two membranes. Efficient delivery of peptidomimetic drugs when adminsitered by the oral route requires knowledge of short-term regulation of intestinal peptide transport, in addition to the sequence and structure of the basolateral transporter. This thesis provides essential information, which may eventually contribute to the unequivocal identification and sequencing of the intestinal basolateral peptide transporter, ultimately leading to the future development of compounds with high bioavailability.

El objetivo principal de este trabajo es desarrollar un modelo sencillo que permita la caracterización hidrodinámica de membranas selectivas integradas en sistemas bi-iónicos, mediante la determinación de coeficientes de difusión y de espesores de las capas límite alrededor de la membrana. A tal fin, se empleó una célula de difusión rotatoria (CDR), que permite el establecimiento de condiciones hidrodinámicas bien definidas para el sistema de membrana, dado que la variación de la frecuencia de giro del cilindro interior (ω), permite disminuir el espesor de la capa límite sobre la membrana, lo que favorece el intercambio iónico a su través. Se puede comprobar éste comportamiento, mediante consideraciones en torno al coeficiente de difusión de los cationes en el sistema de membrana y del cálculo del propio espesor de la capa límite. El mencionado coeficiente se obtendrá a partir del flujo iónico en la membrana, determinado a partir de medidas de pH, junto a medidas de conductividad, en la fase externa (receptora), a diferentes temperaturas y a distintas valores de ω.La medida de los flujos, una vez establecida su dependencia con ω, permite obtener los coeficientes de difusión catiónicos en el sistema de membrana, en función de la temperatura y de ω. Las medidas de la conductividad permiten testar el modelo propuesto, mediante su correlación con los valores de pH obtenidos, proporcionando información adicional acerca de los coeficientes de difusión de los cationes.
From the experimental study of the ionic transport through selective membranes in biionic systems, a simple model which allows the characterising hydrodynamic of the membrane systems through the determination of diffusion coefficients and the thickness of the limit layer has been developed. With this purpose, a rotating diffusion cell that allows the setting of hydrodynamic conditions clearly for the membrane system has been used, studying the variation of the conductivity and the pH in the external phase (receiving) at different temperatures from 20ºC to 50ºC and at different rotating velocities ω. The measurement of the fluxes, once set its dependence with ω, allows obtained the diffusion coefficients cationics in the membrane system in accordance with the temperature and ω. The measurements of the conductivity allow the testing of this model, through its correlation with the values of the pH measured, obtaining additional data about the diffusion coefficient of the cations in the receiving phase.

Thesis (Ph.D.)--Indiana University, 2009.
Title from screen (viewed on December 9, 2009). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Jeffrey S. Elmendorf, Peter J. Roach, Joseph T. Brozinick, Michael S. Sturek, Robert V. Considine. Includes vitae. Includes bibliographical references (leaves 97-124).

Aspects of the molecular basis of potassium transport via the KefC potassium efflux system in E. coli were examined. Separate cloning of the proposed membrane-spanning and cytoplasmic domains of KefC was carried out. A KefC construct was created that contained a amber stop codon at residue 382, between the two domains. When cloned into a prokaryotic expression vector, transformed strains grew poorly. No activity was detected in either an amber suppressor strain, or a strain lacking an amber suppressor, in the presence or absence of IPTG. A series of strains possessing mutant kefC alleles that cause spontaneous potassium loss via KefC were studied. The mutations in the mutant kefC strains were identified as missense mutations, which mapped to two regions of KefC; a sequence of amino acids in the N-terminal domain, (HALESDIE), and the Rossman fold within the C-terminal, cytoplasmic, domain. Recently, it has been discovered that an upstream gene, yabF, which overlaps kefC by eight base pairs, is involved in KefC activity. The sequence of yabF, from Frag5 and the mutant strains was examined and was not mutated in any of the strains, but the sequence of yabF was different from that published by the E. coli genome sequencing project. Studies on kefB mutant strains were initiated. In vitro site-directed mutagenesis of cloned kefC was used to analyse the importance of two residues in the Rossman fold, R416 and S420. The KefCR416S mutations results in spontaneous potassium loss, whereas the KefCS420A mutation does not. The interaction of KefC subunits was confirmed. The R416S mutation, (in the Rossman fold), and the D264A mutation, (in the HALESDIE sequence), were co-dominant. Spontaneous potassium loss via KefCR416S was found to require YabF and glutathione.

The twin-arginine protein transport (Tat) system is one of two general protein transport systems in the cytoplasmic membranes of bacteria, and is conserved in the thylakoid membranes of plants. The Tat system transports fully folded proteins of varying sizes across the membrane. This feat is achieved by the use of just three conserved integral membrane proteins, TatA, TatB and TatC. Passenger proteins are designated for transport by the system by the use of an N-terminal extension, termed a signal peptide which contains an invariant twin arginine motif. The TatC component of the transport system is responsible for recognising the signal peptide, as well as binding to TatB and the TatA components. TatC therefore emerges as the functional and organisational ‘core’ of the Tat system. There is a wealth of functional data relating to the TatC protein, but no high resolution structural information on TatC was available to interpret this data. In order to gain an understanding of the Tat system at the molecular level, a structure was required of TatC. Preliminary expression screening using green fluorescent protein had previously identified the TatC protein from the thermophilic bacterium Aquifex aeolicus as being amenable for structural work. This study purified the A. aeolicus TatC construct in a variety of detergents, used native mass spectrometry and light scattering techniques to assay the sample homogeneity and set up extensive crystallisation trials. Following optimisation of a selenomethionine minimal media growth protocol, crystals grown from selenomethionine-substituted TatC purified in lauryl maltose neopentyl glycol diffracted to 3.5Å resolution, and allowed the structure of TatC to be solved by single wavelength anomalous dispersion. Analysis of the novel structure of TatC, in combination with an isothermal titration calorimetric binding assay, gave information on how TatC binds to signal peptides. Further structural analysis combined with additional experimental data from collaborators allowed a model to be proposed for how TatC binds to the two additional integral membrane components of the system, TatB and TatA, as part of the transport mechanism. Subsequent work was undertaken in this study to attempt to gain a co-crystal structure of TatC with the signal peptide. Construct and detergent screening was carried out in order to aim towards high resolution structural characterisation of the TatBC complex.

The Tat protein export system transports folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. In Escherichia coli, the Tat system is composed of the TatA, TatB and TatC proteins. TatB and TatC assemble into a multimeric receptor complex that recognises and binds the substrate, before the TatA protomers cluster at the TatBC complex to facilitate substrate transport. A genetic screen was devised to explore the oligomeric state of TatC, reasoning that the isolation of dominant negative TatC variants that inactivate the Tat system in the presence of a functional copy of wild type TatC would provide strong evidence TatC is an obligate oligomer. Single dominant negative TatC substitutions were isolated that were located in the first and second periplasmic loops of TatC. These substitutions did not prevent TatC from interacting with TatB, TatA, itself or with a Tat substrate. Blue Native PAGE analysis showed that the TatC variants were unable to form the 440 kDa TatBC complex. Surprisingly, the substitutions did not prevent TatC:TatC self-interactions in the periplasmic regions, detected by disulphide cross-linking, but they did abolish a substrate-induced interaction at the fifth transmembrane helix of TatC. Fluorescence microscopy experiments revealed that the dominant negative TatC variants prevented the polymerisation of TatA-YFP in vivo. These results show that TatC possesses at least two interaction interfaces and imply that the periplasmic loops are critical for the transition between substrate binding and TatA polymerisation. Accessibility of single cysteine substitutions in TatC was probed by PEG-Mal labelling in intact cells. TatB was shown to be important for the proper insertion of TatC into the membrane. The absence of TatA led to accessibility changes in the vicinity of the fifth transmembrane domain of TatC, where both TatA and TatB are known to dock. This suggests that TatA and TatB may share an overlapping binding site.

Le travail presenté dans ce mémoire a été entrepris dans le but de purifier et de caractériser le transporteur des dicarboxylates des mitochondries, qui catalyse l'échange un contre un des dicarboxylates entre eux (malate, malonate, succinate) ou contre le phosphate. Cette étude a été effectuée simultanément sur les mitochondries de foie de rat et sur les mitochondries d'une souche de levure: Saccharomyces cerevisiae. Les mitochondries de levure ont été solubilisées par le Triton X-100 et l'extrait a été chromatographié sur hydroxyapatite. Le filtrat, traité par électrophorèse en milieu dénaturant, a révélé par coloration à l'argent la présence d'environ 5 protéines de Mr compris entre 28000 et 35000. Après reconstitution de l'activité de transport dans des liposomes, un accroissement de 10 fois, de l'activité spécifique d'échange a été observé. Le passage des protéines du filtrat d'hydroxyapatite sur une colonne de malate deshydrogénase mitochondriale (EC 1. 1. 1. 37) immobilisée sur Sépharose, a conduit à la purification complète du transporteur. Cette protéine, purifiée à partir des deux types de mitochondries, possède toutes les propriétés caractéristiques du transporteur in situ et présente, en milieu dénaturant, un poids moléculaire de 28000. L'activité du transporteur purifié à partir des mitochondries de levure, reconstituée dans des liposomes, montre un Km pour le succinate de 2 mM et un Vmax de 1,5 lmol. Min-1. Mg-1 protéines tandis que l'activité spécifique augmente de 300 fois par rapport à l'extrait de départ. L'activité d'échange du transporteur reconstituée des deux types de mitochondries est inhibée par un réactif des groupements SH, le p-chloromercuriphénylsulfonate et par un réactif des acides aminés, le phosphate de pyridoxal, suggérant l'implication d'un ou plusieurs groupements SH et d'un ou plusieurs résidus lysine dans le mécanisme catalytique du transporteur. L'analyse de la composition en acides aminés de la protéine de transport des mitochondries de foie de rat indique qu'il s'agit d'une protéine hydrophobe, légèrement acide, avec la partie N-terminale bloquée

Revers elektrodialyse (RED) er en fornybar, ikke-forurensende måte å generereelektrisitet på ved å blande vann med forskjellige saltkonsentrasjoner, e.g.elvevann og sjøvann. RED er avhengig av ioneselektive membraner. Dereseffektivitet kan bli beskrevet med deres effektive transporttall, som er ratioenmellom den målbare elektromotoriske kraften (Emf) over membranen og detideelle potensialet, som er sett på som det teoretiske maksimum. Her er detvist at den ikke-ideelle oppførselen til en ioneselektiv membran, det at denhar et effektivt transporttall under 1 og at det effektive transportallet går nedmed økende saltkonsentrasjon, kan bli forklart med elektroosmose. Dette er ikontrast til den utbredte tolkningen at det effektive transporttallet er et målpå selektiviteten til membranen. Vanntransporttallet beskriver retningen oggraden av elektroosmose.Denne tolkningen er testet eksperimentelt med Emf målinger av ioneselektivemembraner ved forskjellige konsentrasjoner. Fra resultatene er det vistat den anionselektive membranen Fumasep FAD oppfører seg som om den erhelt selektiv og har et vanntransporttall på -4.8±0.8 med saltvannsløsningeri konsentrasjonsområdet 2-30 gram natriumklorid (NaCl) per liter. Derfor,hvis produsenter som Fumatech vil produsere mer effektive membranertrenger de ikke å gjøre membranene mer selektive ovenfor ioner, men istedenformå de gjøre dem mer selektive ovenfor vann. Det er også vist teoretiskat vanntransporten i spesielle tilfeller kan gi transporttall over 1. Dette kanbidra til å forklare resultatene som nylig ble publisert av Nature.Effekten av denne modellen av det effektive transporttallet på predikertytelse av RED systemer ble undersøkt ved hjelp av Matlab simulasjoner.Det ble sammenliknet med hvordan det effektive transporttallet vanligvis erbehandlet, det vil si som en konstant som beskriver selektiviteten av membranen.Det effektive transporttallet er regnet ut fra Emf målinger og avhengerav konsentrasjonen som blir brukt under denne målingen. Det ble funnet atden gamle metoden er en god approksimasjon ved høye strømningshastigheter,med under 1 % forskjell ved strømningshastigheter over 1 mm/s, hvis deteffektive transporttallet er målt i konsentrasjonene ved innløpet. Hvis transporttalleter målt ved andre konsentrasjoner vil forskjellen være større. Denpredikerte kraftproduksjonen vil være 7 % mindre hvis konsentrasjonene 20og 30 g/l NaCl er brukt istedenfor 3 og 30 g/l NaCl. I disse simulasjonene etvanntransporttall på 10 var brukt. Et større vanntransporttall vil gi størreiiieffekt. Vanntransporttallene til ioneselektive membraner har blitt m°alt til °avære 4-50, men de fleste kommersielle membranene har ikke vanntransporttalls°a høye som 20-50.

Shigella flexneri attacks cells of the intestinal tract, causing over 1 million deaths annually from bacterial dysentery. A type III secretion system (T3SS) initiates the host-pathogen interaction and transports virulence factors directly into host cells via a needle complex (NC) comprising an extracellular needle and membrane-spanning basal body. Rings formed by the single-pass membrane proteins MxiG and MxiJ are arranged concentrically within the inner membrane ring (IMR) of the NC. The Neterminal domain of MxiG (MxiG-N) is the predominant IMR cytoplasmic structure, however it was structurally and functionally uncharacterised. Determination of the solution structure of MxiG-N in this study revealed it to be a forkhead associated (FHA) domain, although subsequent analyses of conserved residues suggested it does not have the canonical role in cell-signalling via phospho-threonine recognition. Subsequent positioning of the structure in the electron microscopy (EM) density for the S. flexneri NC supported models with 24-fold symmetry in the IMR. Both MxiG and MxiJ also have significant periplasmic domains, which were purified to homogeneity in this study, facilitating preliminary characterisation of their structures and intermolecular interactions. In addition, the entire IMR within the context of intact basal bodies was isolated and visualised in vitro by EM. The essential function of MxiG-N could be to localise the putative cytoplasmic ring (Cering) at the base of the T3SS. Although absolutely required for secretion, the Csring component, Spa33, was structurally uncharacterised. The crystal structure of the Cvterminal domain of Spa33 (Spa33-C) was determined in this study, showing an intertwined dimer that aligned with homologous structures and exhibited a novel interaction with the N-terminus of the ATPase regulator, MxiN. Subsequently, Spa33-C was identified as an altemative translation product of spa33 that formed a 2: 1 complex with Spa33 in vitro. This complex oligomerised further, demonstrating for the first time that Spa33 has the propensity to form the ordered, high molecular weight assemblies that would be required for C-ring formation in S. flexneri.

Les transporteurs a atp binding cassette sont caracterises par une organisation en quatre domaines : deux domaines hydrophobes qui forment un site de reconnaissance de substrat, et deux domaines hydrophiles conserves qui hydrolysent l'atp, source d'energie du transport. Ces transporteurs servent a l'import ou l'export de substrats varies en taille et nature chimique et jouent un role important en sante humaine. Mais la comprehension du mecanisme moleculaire du transport est encore imparfaite. Nous nous sommes interesses au role des interactions proteine-proteine dans le systeme modele du complexe de membrane interne du systeme de transport du maltose d'escherichia coli. Ce transporteur est constitue de quatre polypeptides : malf et malg, les domaines hydrophobes, qui servent a l'accrochage de deux copies de malk, les domaines hydrophiles. Nous avons etudie par mutagenese dirigee le role fonctionnel d'une boucle cytoplasmique conservee de malf et malg, la region eaa. Nous avons montre que cette region sert a l'interaction avec un domaine dit helical de malk. En incubant la proteine malk purifiee avec des vesicules a la topologie inversee contennant malf et malg, nous avons reconstitue un transporteur fonctionnel. En utilisant cette approche nous avons montre que la fixation de l'atp module l'interaction entre malk et malf-malg. La fixation de l'atp induit un changement de conformation de malk, et un site de son domaine helical devient accessible a la proteolyse. Ce site est protege de l'attaque d'une protease par la presence de malf-malg, suggerant de nouveau que ce domaine est implique dans les interactions avec malf-malg. Enfin, nous avons montre que l'insertion dans la membrane de malf surproduite sans malg induit une reponse de stress extracytoplasmique. Une grande boucle periplasmique de malf en est la cause et la presence de malg abolit cette induction, suggerant que l'interaction entre malf et malg joue un role important dans le repliement correct de malf.

The thesis research was designed to study the characteristics of two important physiological processes in smooth muscle: Ca2+ transport mediated by the plasmalemmal Ca2+-ATPase and muscarinic receptor-G protein interactions. In resting smooth muscle, several Ca2+ extrusion or sequestration processes offset the passive inward leak of Ca2+. Although biochemical evidence suggests that the plasmalemmal Ca2+ pump plays a key role in this process, the precise role of this enzyme could not be proven until a reliable estimate of the inward Ca2+ leak was measured. Recent studies using dispersed smooth muscle cells from the toad stomach provided an estimate of the basal transmembrane Ca2+ flux rate; thus, we examined the transport capacity of the plasmalemmal Ca2+pump in this tissue. Gastric smooth muscle tissue was disrupted by homogenization and nitrogen cavitation. Membranes enriched 20 fold for plasma membrane markers were obtained using differential centrifugation and purification by flotation on discontinuous sucrose gradients. The membrane vesicles exhibited an ATP-dependent 45Ca uptake that was insensitive to azide or oxalate but sensitive to stimulation by calmodulin or inhibition by orthovanadate and the calmodulin antagonists trifluoperazine (TFP) or calmidazolium (CMZ). 45Ca accumulated in the presence of ATP was rapidly released by Ca2+ ionophore but not by agents that stimulate Ca2+ release from the sarcoplasmic rettculum (caffeine, inositol trisphosphate, GTP). However, both CMZ and TFP evoked a Ca2+ release that was comparable to that observed in the presence of Ca2+ ionophore, suggesting that these compounds have profound effects on membrane Ca2+permeability. 45Ca transport exhibited a high affinity for Ca2+ (KD 0.2 μM) and a high transport capacity, producing a > 12,000-fold gradient for Ca2+and a transmembrane flux rate at least 3-fold greater than that observed in resting smooth muscle cells. As a first step toward understanding the biochemical basis for the diversity of muscarinic cholinergic actions on smooth muscle, we examined the distribution of muscarinic receptor subtypes and coupling to guantne nucleotide-binding (G) proteins in airway and gastric smooth muscle. Receptor subtypes were classified in membranes prepared from bovine trachea and toad stomach based on the relative abilities of the selective antagonists pirenzepine (M1), AF-DX 116 (M2) and 4-DAMP (M3) to displace the binding of nonselective antagonist [3H]QNB (quinuclidinyl benzilate). Based on the binding profiles for these antagonists, it was concluded that both smooth muscle types contain a mixture of M2 and M3 subtypes. In trachea the majority of receptors (86%) were M2, whereas in stomach the majority of receptors (88%) were M3. The displacement of [3H]QNB binding by the agonist oxotremorine indicated a mixed population of high affinity (KD = 4 nM) and low affinity (KD = 2-4 μM) binding sites. The addition of GTPγS abolished all high affinity agonist binding, suggesting that coupling of the receptors to G proteins may confer high affinity. Reaction of membranes with pertussis toxin in the presence of [32P]NAD caused the [32P]-labelling of a ~ 41 kD protein in both gastric and tracheal smooth musc1e. Pretreatment of the membranes with pertussis toxin and NAD completely abolished high affinity agonist binding in gastric smooth muscle, but produced little if any decrease in high affinity agonist binding in trachea. We conclude that, although muscarinic receptor activation leads to the elevation of intracellular Ca2+ and to contraction of both airway and gastric smooth muscle, the dissimilar distributions of receptor subtypes and distinct patterns of coupling to G proteins may indicate that each smooth muscle type uses different receptor-G protein interactions to regulate intracellular signalling pathways.

Etude de la recuperation selective de agno#3 en presence d'un exces de cu(no#3)#2 par le dicyclohexano-18-crown-6. L'approche proposee pour rationaliser l'effet du diluant sur le coefficient de transfert indique l'importance de la viscosite sur le coefficient de diffusion et sur l'epaisseur des couches de nernst

The topic of this thesis is the degradation of fuel cell electrodes in proton exchange membrane fuel cells (PEMFCs). In particular, the degradation associated with localized fuel starvation, which is often encountered during start-ups and shut-downs (SUs/SDs) of PEMFCs. At SU/SD, O2 and H2 usually coexist in the anode compartment. This situation forces the opposite electrode, i.e. the cathode, to very high potentials, resulting in the corrosion of the carbon supporting the catalyst, referred to as carbon corrosion. The aim of this thesis has been to develop methods, materials and strategies to address the issues associated to carbon corrosion in PEMFC.The extent of catalyst degradation is commonly evaluated determining the electrochemically active surface area (ECSA) of fuel cell electrode. Therefore, it was considered important to study the effect of RH, temperature and type of accelerated degradation test (ADT) on the ECSA. Low RH decreases the ECSA of the electrode, attributed to re-structuring the ionomer and loss of contact with the catalyst.In the search for more durable supports, we evaluated different accelerated degradation tests (ADTs) for carbon corrosion. Potentiostatic holds at 1.2 V vs. RHE were found to be too mild. Potentiostatic holds at 1.4 V vs. RHE were found to induce a large degree of reversibility, also attributed to ionomer re-structuring. Triangle-wave potential cycling was found to irreversibly degrade the electrode within a reasonable amount of time, closely simulating SU/SD conditions.Corrosion of carbon-based supports not only degrades the catalyst by lowering the ECSA, but also has a profound effect on the electrode morphology. Decreased electrode porosity, increased agglomerate size and ionomer enrichment all contribute to the degradation of the mass-transport properties of the cathode. Graphitized carbon fibers were found to be 5 times more corrosion resistant than conventional carbons, primarily attributed to their lower surface area. Furthermore, fibers were found to better maintain the integrity of the electrode morphology, generally showing less degradation of the mass-transport losses. Different system strategies for shut-down were evaluated. Not doing anything to the fuel cell during shut-downs is detrimental for the fuel cell. O2 consumption with a load and H2 purge of the cathode were found to give around 100 times lower degradation rates compared to not doing anything and almost 10 times lower degradation rate than a simple air purge of the anode. Finally, in-situ measurements of contact resistance showed that the contact resistance between GDL and BPP is highly dynamic and changes with operating conditions.
Denna doktorsavhandling behandlar degraderingen av polymerelektrolytbränslecellselektroder. polymerelektrolytbränslecellselektroder. Den handlar särskilt om nedbrytningen av elektroden kopplad till en degraderingsmekanism som heter ”localized fuel starvation” oftast närvarande vid uppstart och nedstängning av bränslecellen. Vid start och stopp kan syrgas och vätgas förekomma samtidigt i anoden. Detta leder till väldigt höga elektrodpotentialer i katoden. Resultatet av detta är att kolbaserade katalysatorbärare korroderar och att bränslecellens livslängd förkortas. Målet med avhandlingen har varit att utveckla metoder, material och strategier för att både öka förståelsen av denna degraderingsmekanism och för att maximera katalysatorbärarens livslängd.Ett vanligt tillvägagångsätt för att bestämma graden av katalysatorns degradering är genom mätning av den elektrokemiskt aktiva ytan hos bränslecellselektroderna. I denna avhandling har dessutom effekten av temperatur och relativ fukthalt studerats. Låga fukthalter minskar den aktiva ytan hos elektroden, vilket sannolikt orsakas av en omstrukturering av jonomeren och av kontaktförlust mellan jonomer och katalysator.Olika accelererade degraderingstester för kolkorrosion har använts. Potentiostatiska tester vid 1.2 V mot RHE visade sig vara för milda. Potentiostatiska tester vid 1.4 V mot RHE visade sig däremot medföra en hög grad av reversibilitet, som också den tros vara orsakad av en omstrukturering av jonomeren. Cykling av elektrodpotentialen degraderade istället elektroden irreversibelt, inom rimlig tid och kunde väldigt nära simulera förhållandena vid uppstart och nedstängning.Korrosionen av katalysatorbäraren medför degradering av katalysatorn och har också en stor inverkan på elektrodens morfologi. En minskad elektrodporositet, en ökad agglomeratstorlek och en anrikning av jonomeren gör att elektrodens masstransportegenskaper försämras. Grafitiska kolfibrer visade sig vara mer resistenta mot kolkorrosion än konventionella kol, främst p.g.a. deras låga ytarea. Grafitiska kolfibrer visade också en förmåga att bättre bibehålla elektrodens morfologi efter accelererade tester, vilket resulterade i lägre masstransportförluster.Olika systemstrategier för nedstängning jämfördes. Att inte göra något under nedstängning är mycket skadligt för bränslecellen. Förbrukning av syre med en last och spolning av katoden med vätgas visade 100 gånger lägre degraderingshastighet av bränslecellsprestanda jämfört med att inte göra något alls och 10 gånger lägre degraderingshastighet jämfört med spolning av anoden med luft. In-situ kontaktresistansmätningar visade att kontaktresistansen mellan bipolära plattor och GDL är dynamisk och kan ändras beroende på driftförhållandena.

QC 20131104

La pyoverdine excretee par pseudomonas aeruginosa a un role de siderophore. Complexe a la pyoverdine, le fer est transporte activement a l'interieur de la cellule par l'intermediaire d'une proteine de la membrane externe qui est un recepteur specifique de la pyoverdine excretee par la souche. La complementation de mutants de biosynthese par une banque genomique de la souche etudiee permet d'isoler des transconjugants qui ont le phenotype fluorescent caracteristique de la pyoverdine