Caratterizzazione di membrane metalliche e ionomeriche per applicazioni energetiche

The work of this thesis has been focused on the characterization of metallic membranes for the hydrogen purification from steam reforming process and also of perfluorosulphonic acid ionomeric (PFSI) membranes suitable as electrolytes in fuel cell applications. The experimental study of metallic membranes was divided in three sections: synthesis of palladium and silver palladium coatings on porous ceramic support via electroless deposition (ELD), solubility and diffusivity analysis of hydrogen in palladium based alloys (temperature range between 200 and 400 °C up to 12 bar of pressure) and permeation experiments of pure hydrogen and mixtures containing, besides hydrogen, also nitrogen and methane at high temperatures (up to 600 °C) and pressures (up to 10 bar). Sequential deposition of palladium and silver on to porous alumina tubes by ELD technique was carried out using two different procedures: a stirred batch and a continuous flux method. Pure palladium as well as Pd-Ag membranes were produced: the Pd-Ag membranes’ composition is calculated to be close to 77% Pd and 23% Ag by weight which was the target value that correspond to the best performance of the palladium-based alloys. One of the membranes produced showed an infinite selectivity through hydrogen and relatively high permeability value and is suitable for the potential use as a hydrogen separator. The hydrogen sorption in silver palladium alloys was carried out in a gravimetric system on films produced by ELD technique. In the temperature range inspected, up to 400°C, there is still a lack in literature. The experimental data were analyzed with rigorous equations allowing to calculate the enthalpy and entropy values of the Sieverts’ constant; the results were in very good agreement with the extrapolation made with literature data obtained a lower temperature (up to 150 °C). The information obtained in this study would be directly usable in the modeling of hydrogen permeation in Pd-based systems. Pure and mixed gas permeation tests were performed on Pd-based hydrogen selective membranes at operative conditions close to steam-reforming ones. Two membranes (one produced in this work and another produced by NGK Insulators Japan) showed a virtually infinite selectivity and good permeability. Mixture data revealed the existence of non negligible resistances to hydrogen transport in the gas phase. Even if the decrease of the driving force due to polarization concentration phenomena occurs, in principle, in all membrane-based separation systems endowed with high perm-selectivity, an extensive experimental analysis lack, at the moment, in the palladium-based membrane process in literature. Moreover a new procedure has been introduced for the proper comparison of the mass transport resistance in the gas phase and in the membrane. Another object of study was the water vapor sorption and permeation in PFSI membranes with short and long side chains was also studied; moreover the permeation of gases (i.e. He, N2 and O2) in dry and humid conditions was considered. The water vapor sorption showed strong interactions between the hydrophilic groups and the water as revealed from the hysteresis in the sorption-desorption isotherms and thermo gravimetric analysis. The data obtained were used in the modeling of water vapor permeation, that was described as diffusion-reaction of water molecules, and in the humid gases permeation experiments. In the dry gas experiments the permeability and diffusivity was found to increase with temperature and with the equivalent weight (EW) of the membrane. A linear correlation was drawn between the dry gas permeability and the opposite of the equivalent weight of PFSI membranes, based on which the permeability of pure PTFE is retrieved in the limit of high EW. In the other hand O2 ,N2 and He permeability values was found to increase significantly, and in a similar fashion, with water activity. A model that considers the PFSI membrane as a composite matrix with a hydrophilic and a hydrophobic phase was considered allowing to estimate the variation of gas permeability with relative humidity on the basis of the permeability in the dry PFSI membrane and in pure liquid water.

Statistical problems related to excitation threshold and reset value of membrane potentials

Die vorliegende Arbeit ist motiviert durch biologische Fragestellungen bezüglich des Verhaltens von Membranpotentialen in Neuronen. Ein vielfach betrachtetes Modell für spikende Neuronen ist das Folgende. Zwischen den Spikes verhält sich das Membranpotential wie ein Diffusionsprozess X der durch die SDGL dX_t= beta(X_t) dt+ sigma(X_t) dB_t gegeben ist, wobei (B_t) eine Standard-Brown'sche Bewegung bezeichnet. Spikes erklärt man wie folgt. Sobald das Potential X eine gewisse Exzitationsschwelle S überschreitet entsteht ein Spike. Danach wird das Potential wieder auf einen bestimmten Wert x_0 zurückgesetzt. In Anwendungen ist es manchmal möglich, einen Diffusionsprozess X zwischen den Spikes zu beobachten und die Koeffizienten der SDGL beta() und sigma() zu schätzen. Dennoch ist es nötig, die Schwellen x_0 und S zu bestimmen um das Modell festzulegen. Eine Möglichkeit, dieses Problem anzugehen, ist x_0 und S als Parameter eines statistischen Modells aufzufassen und diese zu schätzen. In der vorliegenden Arbeit werden vier verschiedene Fälle diskutiert, in denen wir jeweils annehmen, dass das Membranpotential X zwischen den Spikes eine Brown'sche Bewegung mit Drift, eine geometrische Brown'sche Bewegung, ein Ornstein-Uhlenbeck Prozess oder ein Cox-Ingersoll-Ross Prozess ist. Darüber hinaus beobachten wir die Zeiten zwischen aufeinander folgenden Spikes, die wir als iid Treffzeiten der Schwelle S von X gestartet in x_0 auffassen. Die ersten beiden Fälle ähneln sich sehr und man kann jeweils den Maximum-Likelihood-Schätzer explizit angeben. Darüber hinaus wird, unter Verwendung der LAN-Theorie, die Optimalität dieser Schätzer gezeigt. In den Fällen OU- und CIR-Prozess wählen wir eine Minimum-Distanz-Methode, die auf dem Vergleich von empirischer und wahrer Laplace-Transformation bezüglich einer Hilbertraumnorm beruht. Wir werden beweisen, dass alle Schätzer stark konsistent und asymptotisch normalverteilt sind. Im letzten Kapitel werden wir die Effizienz der Minimum-Distanz-Schätzer anhand simulierter Daten überprüfen. Ferner, werden Anwendungen auf reale Datensätze und deren Resultate ausführlich diskutiert.

Membrane disrupting antimicrobial peptide dendrimers with multiple amino termini

Antimicrobial peptide dendrimer H1 Leu8(Lys-Leu)4(Lys-Phe)2Lys-LysNH2 (Lys = branching lysine) was identified by screening a 6750-membered combinatorial library by the bead-diffusion assay. Sequence variations also revealed dendrimer bH1 Leu8(Dap-Leu)4(Dap-Phe)2Dap-LysNH2 (Dap = branching 2,3-diaminopropanoic acid) as a more potent analog. H1 and bH1 showed good antimicrobial activities mediated by membrane disruption (MIC = 2–4 μg mL−1 on Bacillus subtilis and Escherichia coli) but low hemolytic activity (MHC = 310 μg mL−1 respectively >2000 μg mL−1).

Why is 11beta-hydroxysteroid dehydrogenase type 1 facing the endoplasmic reticulum lumen? Physiological relevance of the membrane topology of 11beta-HSD1

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is essential for the local activation of glucocorticoid receptors (GR). Unlike unliganded cytoplasmic GR, 11beta-HSD1 is an endoplasmic reticulum (ER)-membrane protein with lumenal orientation. Cortisone might gain direct access to 11beta-HSD1 by free diffusion across membranes, indirectly via intracellular binding proteins or, alternatively, by insertion into membranes. Membranous cortisol, formed by 11beta-HSD1 at the ER-lumenal side, might then activate cytoplasmic GR or bind to ER-lumenal secretory proteins. Compartmentalization of 11beta-HSD1 is important for its regulation by hexose-6-phosphate dehydrogenase (H6PDH), which regenerates cofactor NADPH in the ER lumen and stimulates oxoreductase activity. ER-lumenal orientation of 11beta-HSD1 is also essential for the metabolism of the alternative substrate 7-ketocholesterol (7KC), a major cholesterol oxidation product found in atherosclerotic plaques and taken up from processed cholesterol-rich food. An 11beta-HSD1 mutant adopting cytoplasmic orientation efficiently catalyzed the oxoreduction of cortisone but not 7KC, indicating access to cortisone from both sides of the ER-membrane but to 7KC only from the lumenal side. These aspects may be relevant for understanding the physiological role of 11beta-HSD1 and for developing therapeutic interventions to control glucocorticoid reactivation.

Local degradation of structural, mechanical, electrical and chemical properties of membrane electrode assembly in polymer electrolyte fuel cell

Polymer electrolyte fuel cell (PEMFC) is promising source of clean power in many applications ranging from portable electronics to automotive and land-based power generation. However, widespread commercialization of PEMFC is primarily challenged by degradation. The mechanisms of fuel cell degradation are not well understood. Even though the numbers of installed units around the world continue to increase and dominate the pre-markets, the present lifetime requirements for fuel cells cannot be guarantee, creating the need for a more comprehensive knowledge of material’s ageing mechanism. The objective of this project is to conduct experiments on membrane electrode assembly (MEA) components of PEMFC to study structural, mechanical, electrical and chemical changes during ageing and understanding failure/degradation mechanism. The first part of this project was devoted to surface roughness analysis on catalyst layer (CL) and gas diffusion layer (GDL) using surface mapping microscopy. This study was motivated by the need to have a quantitative understanding of the GDL and CL surface morphology at the submicron level to predict interfacial contact resistance. Nanoindentation studies using atomic force microscope (AFM) were introduced to investigate the effect of degradation on mechanical properties of CL. The elastic modulus was decreased by 45 % in end of life (EOL) CL as compare to beginning of life (BOL) CL. In another set of experiment, conductive AFM (cAFM) was used to probe the local electric current in CL. The conductivity drops by 62 % in EOL CL. The future task will include characterization of MEA degradation using Raman and Fourier transform infrared (FTIR) spectroscopy. Raman spectroscopy will help to detect degree of structural disorder in CL during degradation. FTIR will help to study the effect of CO in CL. XRD will be used to determine Pt particle size and its crystallinity. In-situ conductive AFM studies using electrochemical cell on CL to correlate its structure with oxygen reduction reaction (ORR) reactivity

Dendritic spine morphology determines membrane-associated protein exchange between dendritic shafts and spine heads.

The purpose of this study was to examine whether variability in the shape of dendritic spines affects protein movement within the plasma membrane. Using a combination of confocal microscopy and the fluorescence loss in photobleaching technique in living hippocampal CA1 pyramidal neurons expressing membrane-linked GFP, we observed a clear correlation between spine shape parameters and the diffusion and compartmentalization of membrane-associated proteins. The kinetics of membrane-linked GFP exchange between the dendritic shaft and the spine head compartment were slower in dendritic spines with long necks and/or large heads than in those with short necks and/or small heads. Furthermore, when the spine area was reduced by eliciting epileptiform activity, the kinetics of protein exchange between the spine compartments exhibited a concomitant decrease. As synaptic plasticity is considered to involve the dynamic flux by lateral diffusion of membrane-bound proteins into and out of the synapse, our data suggest that spine shape represents an important parameter in the susceptibility of synapses to undergo plastic change.

Tracking individual membrane proteins and their biochemistry: The power of direct observation

The advent of single molecule fluorescence microscopy has allowed experimental molecular biophysics and biochemistry to transcend traditional ensemble measurements, where the behavior of individual proteins could not be precisely sampled. The recent explosion in popularity of new super-resolution and super-localization techniques coupled with technical advances in optical designs and fast highly sensitive cameras with single photon sensitivity and millisecond time resolution have made it possible to track key motions, reactions, and interactions of individual proteins with high temporal resolution and spatial resolution well beyond the diffraction limit. Within the purview of membrane proteins and ligand gated ion channels (LGICs), these outstanding advances in single molecule microscopy allow for the direct observation of discrete biochemical states and their fluctuation dynamics. Such observations are fundamentally important for understanding molecular-level mechanisms governing these systems. Examples reviewed here include the effects of allostery on the stoichiometry of ligand binding in the presence of fluorescent ligands; the observation of subdomain partitioning of membrane proteins due to microenvironment effects; and the use of single particle tracking experiments to elucidate characteristics of membrane protein diffusion and the direct measurement of thermodynamic properties, which govern the free energy landscape of protein dimerization. The review of such characteristic topics represents a snapshot of efforts to push the boundaries of fluorescence microscopy of membrane proteins to the absolute limit.

Mobility of cytochrome P450 in the endoplasmic reticulum membrane

Cytochrome P450 2C2 is a resident endoplasmic reticulum (ER) membrane protein that is excluded from the recycling pathway and contains redundant retention functions in its N-terminal transmembrane signal/anchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retention/retrieval signals. One hypothesis to explain exclusion of resident ER proteins from the transport pathway is the formation of networks by interaction with other proteins that immobilize the proteins and are incompatible with packaging into the transport vesicles. To determine the mobility of cytochrome P450 in the ER membrane, chimeric proteins of either cytochrome P450 2C2, its catalytic domain, or the cytochrome P450 2C1 N-terminal signal/anchor sequence fused to green fluorescent protein (GFP) were expressed in transiently transfected COS1 cells. The laurate hydroxylase activities of cytochrome P450 2C2 or the catalytic domain with GFP fused to the C terminus were similar to the native enzyme. The mobilities of the proteins in the membrane were determined by recovery of fluorescence after photobleaching. Diffusion coefficients for all P450 chimeras were similar, ranging from 2.6 to 6.2 × 10−10 cm2/s. A coefficient only slightly larger (7.1 × 10−10 cm2/s) was determined for a GFP chimera that contained a C-terminal dilysine ER retention signal and entered the recycling pathway. These data indicate that exclusion of cytochrome P450 from the recycling pathway is not mediated by immobilization in large protein complexes.

Lipidic cubic phases: A novel concept for the crystallization of membrane proteins

Understanding the mechanisms of action of membrane proteins requires the elucidation of their structures to high resolution. The critical step in accomplishing this by x-ray crystallography is the routine availability of well-ordered three-dimensional crystals. We have devised a novel, rational approach to meet this goal using quasisolid lipidic cubic phases. This membrane system, consisting of lipid, water, and protein in appropriate proportions, forms a structured, transparent, and complex three-dimensional lipidic array, which is pervaded by an intercommunicating aqueous channel system. Such matrices provide nucleation sites (“seeding”) and support growth by lateral diffusion of protein molecules in the membrane (“feeding”). Bacteriorhodopsin crystals were obtained from bicontinuous cubic phases, but not from micellar systems, implying a critical role of the continuity of the diffusion space (the bilayer) on crystal growth. Hexagonal bacteriorhodopsin crystals diffracted to 3.7 Å resolution, with a space group P63, and unit cell dimensions of a = b = 62 Å, c = 108 Å; α = β = 90° and γ = 120°.

Facilitated diffusion of fructose via the phosphoenolpyruvate/glucose phosphotransferase system of Escherichia coli

From mutants of Escherichia coli unable to utilize fructose via the phosphoenolpyruvate/glycose phosphotransferase system (PTS), further mutants were selected that grow on fructose as the sole carbon source, albeit with relatively low affinity for that hexose (Km for growth ≈8 mM but with Vmax for generation time ≈1 h 10 min); the fructose thus taken into the cells is phosphorylated to fructose 6-phosphate by ATP and a cytosolic fructo(manno)kinase (Mak). The gene effecting the translocation of fructose was identified by Hfr-mediated conjugations and by phage-mediated transduction as specifying an isoform of the membrane-spanning enzyme IIGlc of the PTS, which we designate ptsG-F. Exconjugants that had acquired ptsG+ from Hfr strains used for mapping (designated ptsG-I) grew very poorly on fructose (Vmax ≈7 h 20 min), even though they were rich in Mak activity. A mutant of E. coli also rich in Mak but unable to grow on glucose by virtue of transposon-mediated inactivations both of ptsG and of the genes specifying enzyme IIMan (manXYZ) was restored to growth on glucose by plasmids containing either ptsG-F or ptsG-I, but only the former restored growth on fructose. Sequence analysis showed that the difference between these two forms of ptsG, which was reflected also by differences in the rates at which they translocated mannose and glucose analogs such as methyl α-glucoside and 2-deoxyglucose, resided in a substitution of G in ptsG-I by T in ptsG-F in the first position of codon 12, with consequent replacement of valine by phenylalanine in the deduced amino acid sequence.

Single-molecule spectroscopy of the β2 adrenergic receptor: Observation of conformational substates in a membrane protein

Single-molecule studies of the conformations of the intact β2 adrenergic receptor were performed in solution. Photon bursts from the fluorescently tagged adrenergic receptor in a micelle were recorded. A photon-burst algorithm and a Poisson time filter were implemented to characterize single molecules diffusing across the probe volume of a confocal microscope. The effects of molecular diffusion and photon number fluctuations were deconvoluted by assuming that Poisson distributions characterize the molecular occupation and photon numbers. Photon-burst size histograms were constructed, from which the source intensity distributions were extracted. Different conformations of the β2 adrenergic receptor cause quenching of the bound fluorophore to different extents and hence produce different photon-burst sizes. An analysis of the photon-burst histograms shows that there are at least two distinct substates for the native adrenergic membrane receptor. This behavior is in contrast to one peak observed for the dye molecule, rhodamine 6G. We test the reliability and robustness of the substate number determination by investigating the application of different binning criteria. Conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes. These studies provide insight into the conformational heterogeneity of G protein-coupled receptors in the presence and absence of a bound agonist.

Imaging of single molecule diffusion.

In recent years observations at the level of individual atoms and molecules became possible by microscopy and spectroscopy. Imaging of single fluorescence molecules has been achieved but has so far been restricted to molecules in the immobile state. Here we provide methodology for visualization of the motion of individual fluorescent molecules. It is applied to imaging of the diffusional path of single molecules in a phospholipid membrane by using phospholipids carrying one rhodamine dye molecule. For this methodology, fluorescence microscopy was carried to a sensitivity so that single fluorescent molecules illuminated for only 5 ms were resolvable at a signal/noise ratio of 28. Repeated illuminations permitted direct observation of the diffusional motion of individual molecules with a positional accuracy of 30 nm. Such capability has fascinating potentials in bioscience--for example, to correlate biological functions of cell membranes with movements, spatial organization, and stoichiometries of individual components.

Chronopotentiometric study of a Nafion membrane in presence of glucose

Chronopotentiometric and swelling experiments have been conducted to characterize the behavior of a Nafion membrane in NaCl and KCl aqueous solutions without and with glucose. A mixture solution with similar composition to the cerebrospinal fluid and blood plasma has also been studied. From the chronotentiograms, current-voltage curves have been obtained, and the values of the limiting current density, diffusion boundary layer thickness, difference between counter-ion transport number in membrane and free solution, and transition times have been determined for the investigated membrane systems. The obtained results indicate that the presence of glucose affects the ion transport through the membrane depending on the electrolyte and glucose concentrations. At low electrolyte concentration, experimental transition times are found to be smaller in presence of glucose, which has been related to an effective membrane area reduction in presence of glucose. The membrane system corresponding to the mixture solution shows a behavior similar to the single high concentration NaCl membrane system, indicating that the observed behavior is mainly associated to the Na^+ ions transport in higher proportion. In this case, the glucose presence does not affect significantly the investigated properties of the membrane, which is interesting for its utilization in a glucose fuel cell.

Electrocatalytic hydrogenation of acetophenone using a Polymer Electrolyte Membrane Electrochemical Reactor

The use of a solid polymeric electrolyte, spe, is not commonly found in organic electrosynthesis despite its inherent advantages such as the possible elimination of the electrolyte entailing simpler purification processes, a smaller sized reactor and lower energetic costs. In order to test if it were possible to use a spe in industrial organic electrosynthesis, we studied the synthesis of 1-phenylethanol through the electrochemical hydrogenation of acetophenone using Pd/C 30 wt% with different loadings as cathode and a hydrogen gas diffusion anode. A Polymer Electrolyte Membrane Electrochemical Reactor, PEMER, with a fuel cell structure was chosen to carry out electrochemical reduction with a view to simplifying an industrial scale-up of the electrochemical process. We studied the influence of current density and cathode catalyst loading on this electroorganic synthesis. Selectivity for 1-phenylethanol was around 90% with only ethylbenzene and hydrogen detected as by-products.

Sub-Cellular Localization and Complex Formation by Aminoacyl-tRNA Synthetases in Cyanobacteria: Evidence for Interaction of Membrane-Anchored ValRS with ATP Synthase

tRNAs are charged with cognate amino acids by aminoacyl-tRNA synthetases (aaRSs) and subsequently delivered to the ribosome to be used as substrates for gene translation. Whether aminoacyl-tRNAs are channeled to the ribosome by transit within translational complexes that avoid their diffusion in the cytoplasm is a matter of intense investigation in organisms of the three domains of life. In the cyanobacterium Anabaena sp. PCC 7120, the valyl-tRNA synthetase (ValRS) is anchored to thylakoid membranes by means of the CAAD domain. We have investigated whether in this organism ValRS could act as a hub for the nucleation of a translational complex by attracting other aaRSs to the membranes. Out of the 20 aaRSs, only ValRS was found to localize in thylakoid membranes whereas the other enzymes occupied the soluble portion of the cytoplasm. To investigate the basis for this asymmetric distribution of aaRSs, a global search for proteins interacting with the 20 aaRSs was conducted. The interaction between ValRS and the FoF1 ATP synthase complex here reported is of utmost interest and suggests a functional link between elements of the gene translation and energy production machineries.