No description available.

No description available.

Aeromonas hydrophila ML09-119 is an important fish pathogen that severely affects channel catfish aquaculture. To better understand this strain’s virulence factors, outer membrane vesicles (OMVs) were isolated, and their proteome was assessed. Using transmission electron microscopy and dynamic light scattering, OMVs were shown to be monodispersed particles with an average diameter of 120.33 nm. OMV proteins were identified using mass spectrometry, and analysis of the resulting proteome of 74 proteins revealed that many originated from the cytoplasm, but there was an enrichment of outer membrane, periplasmic, and extracellular proteins compared to the total proteome. The majority of the functional classifications were associated with bacterial metabolism. Of the predicted virulence factors, several had a putative function in adherence, and there were type III secretions system proteins as well as three secreted exotoxins. Overall, our data reveal new insights into A. hydrophila OMVs and their potential roles in physiology and virulence.

<p> The mitochondrial inner membrane is highly selective with regard to permeability to solutes and the movement of a large number of large or charged molecules across it therefore requires specific transport processes provided by specific membrane proteins. </p> <p> In order to study the spatial arrangement of one such protein the adenine nucleotide translocator protein which transports ADP and ATP across the mitochondrial inner membrane, a number of chemical labelling studies of the mitochondrial inner membrane were carried out. </p> <p> Mitochondrial inner membrane preparations of normal (mitoplasts) and inverted (submitochondrial particles) config~ration with respect to mitochondria have been isolated and the external phosphatidylethanolamine and proteins modified by 3H isethionyl acetimidate. An upper limit of 40-46% of the total PE in mitoplasts was found to be located in the external monolayer. </p> <p> Differences in protein labelling patterns of isethionyl acetimidate modified mitochondria and SMP was observed. JAI was found to penetrate the outer membrane but not the inner membrane of intact mitochondria. </p> <p> A tritiated photoreactive phospholipid, 1 palmitoyl-2-(mdiazirinophenoxynonanoyl) phosphatidylcholine (DAP-PC) was incorporated into mitoplasts and submitochondrial particles symmetrically into both monolayers by sonication and asymmetrically using phospholipid exchange protein isolated from beef heart. Photolysis yielded the translocator as a major crosslinked product in both types of particles and with both methods of incorporation. </p> <p> It was shown that the adenine nucleotide translocator can be asymmetrically labelled by modification of membrane.particles of opposite orientation by water soluble and membrane soluble probes. </p> / Thesis / Master of Science (MSc)

No description available.

No description available.

Microorganisms primarily acquire phosphorus (P) in the form of inorganic phosphate (PO4-3 or Pi) through expression of a suite of phosphate scavenging or phosphate transporter systems in response to limiting environmental phosphate. One such system is the Pit family of single protein Pi transport systems found in all domains. These vary in size from 300 to 800 amino acids (a.a.) in size. Previously, the pit gene of the soil bacterium Sinorhizobium meliloti, was found to encode a 334 a.a. Pi uptake system (KM 1-2µM) that is repressed in low Pi conditions. However, the S. meliloti pit gene is encoded in an operon and overlaps the coding sequence of a protein of unknown function, which was denoted as pap (pit-accessory protein). Using a conditional Pi-transport deficient mutant strain of S. meliloti, the effects of pap or pit mutations on Pit-mediated Pi uptake were studied by conducting growth experiments in minimal media (with Pi as the sole source of P) and Pi uptake experiments. Both pap and pit deletions resulted in a loss of growth and Pi uptake, which could be complemented by integration of the pap and pit genes into the deletion locus. Heterologous Pap-Pit systems from Bacteroides thetaiotaomicron and Shewanella oneidensis were found to have KM values (17 and 8.5 µM, respectively) similar to previously reported values of S. meliloti Pap-Pit. However, the Shewanella Pit protein was capable of transporting Pi in the absence of the cognate Pap protein, albeit with greatly reduced velocity at all measured concentrations. Pap-Pit orthologs were identified in ~2000 diverse prokaryotic proteomes using Pfam motifs of Pit (PHO4) and Pap (PhoU_div) protein domains. pap-pit operons were found in a third of all proteomes, and were predicted to be a co-transcribed operon in >95% of cases. This provided additional evidence that Pap is directly involved in Pit-mediated Pi uptake, and also that Pap-Pit systems have a significant role in microbial Pi uptake. Pap protein sequences and structures show striking similarities with that of PhoU, a protein of unknown function implicated as a modulator of the Pst uptake system. Pap and PhoU proteins share highly conserved putative metal-binding motifs (E/DXXXD) of which several Pap missense mutations were found to result in reduced Pi transport. This suggests that like PhoU, Pap may function as a modulator of Pi uptake by an interaction with its cognate transporter, Pit. However, the molecular mechanisms of PhoU and Pap proteins have yet to be defined. / Thesis / Master of Science (MSc) / Microbes acquire and assimilate phosphorus (P) in the form of inorganic phosphate (Pi) through a variety of mechanisms. Pit (Pi transporter) are a family of diverse transporters found in all kingdoms of life. Unlike other Pit systems, the Sinorhizobium meliloti pit gene is encoded in an operon with a protein of unknown function, denoted the pit-accessory protein (pap). Using S. meliloti Pap-Pit and orthologues from other bacteria as model systems, we demonstrate that Pap functions as a positive modulator of Pi uptake via Pit, as Pap is required for active uptake of Pi. Pap-Pit systems are found in 30% of all bacteria and archaea, and thus broadly distributed. Understanding the mechanism of Pap-Pit has biotechnological applications, as multiple Pap-Pit systems are present in phosphorus-accumulating bacteria utilized for waste-water treatment.

Caloxin2A 1 is a novel peptide that inhibits the activity of Plasma Membrane Calcium ATPase (PMCA). PMCA is known to play a role in homeostasis of cytosolic calcium and cell signaling. There are 4 genes (PMCA1-4) that code for the various isoforms of the calcium pump. Based on hydropathy plots, PMCA proteins have 5 putative extracellular domains. We screened combinatorial peptide phage display library for binding to specific extracellular targets. Caloxin 2A1 was obtained as a peptide sequence that would bind to the 2nd putative extracellular domain of PMCA 1 isoform. Caloxin2A1 selectively inhibited the Ca2+-Mg2+ ATPase activity in human erythrocyte leaky ghosts that express mainly PMCA 4 isoform. It produced 50% inhibition of the pump activity at 0.4 mM. Caloxin2A1 inhibited the formation of the acid stable 140 kDa acyl phosphate in the reaction cycle of the calcium pump in the human erythrocyte leaky ghosts. It also produced endothelium dependent relaxation in the pig coronary artery. The random peptide phage display library was screened again with higher stringency to obtain caloxin with higher affinity in order to be cost effective and with greater therapeutic potential. This time, the targets were the 2nd putative extracellular domain of PMCA 1 and 2nd and 3rd putative domains of PMCA 4. The peptides selected for binding to the 2nd putative extracellular domain of PMCA 4 selectively inhibited the Ca2^+-Mg^2+ ATPase activity in human erythrocyte leaky ghosts but with a similar affinity as Caloxin2A1. The peptide selected for binding to the 3rd putative extracellular domain of PMCA 4 was hydrophobic and water insoluble. Substitution of its C-terminus amino acid with lysine residue made the peptide water-soluble and it did inhibit the Ca^2 +-Mg^2 + ATPase with slightly higher affinity. However, the inhibition was due to hydrophobicity of the peptide as the randomized version of the peptide also produced inhibition. We have obtained the first selective inhibitor of PMCA and shown that perturbing extracellular targets can affect protein activity even though most of the functional groups of this protein are in the cytosol. / Thesis / Master of Science (MS)

In this dissertation the gas permeation process within four hybrid inorganic-organic membranes is modeled at the micro level using molecular dynamics (MD) and at the meso scale level using a diffusion mechanism. The predicted permeances and relative selectivity of CO₂ and CH₄ are compared with the experimental results. In the MD simulation a single-pore silica crystal framework model with and without inserted phenyl groups are used to define two membrane structures. We designate the two cases as PSPM and SPM respectively. To mimic the diffusion of gas across the membrane, a three-region system with a repulsive wall potential on the edge is employed. Results from the SPM model indicate that the pore size affects the permeance but not the selectivity. In the PSPM model the permeance decreases significantly when the pore size is below a critical value. The extent of decrease varies with the type of gas and this is reflected in the large selectivity in the PSPM model. When the initial diameter is 0.4 nm the model shows a selectivity of 17.3, which is very close to experimental results. At this selectivity the CO₂ permeance is 2.87 Ã 10^-4 mol m⁻²s⁻¹Pa⁻¹ and the CH₄ permeance is 1.66 Ã 10⁻⁵ mol m⁻²s⁻¹Pa⁻¹. For different gases we also studied the motions of the phenyl groups in the pore during the permeation process. The results show that in CO₂ diffusion the phenyl groups moves in a larger range than in CH₄ diffusion. The density profile of gas molecules that the phenyl groups see is analyzed using double layer phenyl groups . The results show that the number of phenyl groups cannot affect the permeation. In the meso scale study a mixed mechanism model with a grid framework is developed to model the permeation process. In the model the membrane is assumed to consist of various grids which follow three major diffusion mechanisms. Models with different grid sizes are employed for the four membranes. Parameters in each model are estimated from the permeance results of the two gases. By comparing the estimated parameters in the surface diffusion mechanism with the reported values, the acceptable grid models are determined and the models with the minimum number of grids are studied. The diffusion is dominated by the activated Knudsen diffusion mechanism at lower temperatures and follows the surface diffusion mechanism when the temperature is above a critical value. In the diffusion of both gases within the four membranes the surface diffusion portion is very close but the activated Knudsen diffusion portion is not. This explains why the permeation with high selectivity occurs at lower temperatures. By comparing the results it shows the two studies can validate each other. On the other hand the two methods can be complementary as the diffusion model is able to predict the permeance within the right range and the MD model is able to predict the selectivity more accurately. / Ph. D.

Sulfonated poly(arylene ether sulfone)s are a class of engineering thermoplastics well-known for their mechanical properties and chemical/oxidative stability. The research in this dissertation focuses on modifying the structure of sulfonated poly(arylene ether sulfone)s to improve membrane performance. Blends of a 20% disulfonated poly(arylene ether sulfone) (BPS20) with poly(ethylene glycol) (PEG) were investigated with the objective of promoting water flux across a reverse osmosis membrane. It was considered desirable to investigate poly(arylene ether sulfone)s with a hydroquinone unit that could be controllably post-sulfonated without degradation, providing a polymer with controlled sulfonation through controlling hydroquinone content. It also avoided the disadvantages noted previously in polymers with post-sulfonated biphenol units. Initial experiments focused on determining sulfonation conditions to confirm quantitative sulfonation of the hydroquinone without side reactions or degradation. A polymer with 29 mole % hydroquinone-containing units was used to study the rate of sulfonation. Successful post-sulfonation was confirmed and reaction conditions were applied to a series of polymers with varying hydroquinone comonomer contents. These polymers were sulfonated, characterized and evaluated for transport properties. Of interest was the high sodium rejection in the presence of calcium, which in the directly copolymerized disulfonated materials is compromised. The post-sulfonated poly(arylene ether sulfone)s showed no compromise in sodium rejection in a mixed-feed of sodium chloride and calcium chloride. In the membrane electrolysis of water, Nafion's high permeability to hydrogen, particularly above about 80 C, results in back-diffusion of hydrogen across the membrane. This reduces efficiency, product purity, and long-term electrode stability. Hydrophilic-hydrophobic multiblock copolymers based on disulfonated and non-sulfonated poly(arylene ether sulfone) oligomers feature a lower gas permeability. Various multiblock compositions and casting conditions were investigated and transport properties were characterized. A multiblock poly(arylene ether sulfone) showed a significant improvement in performance over Nafion at 95°C. Multiblock hydrophilic-hydrophobic poly(arylene ether sulfone)s have been extensively investigated as alternatives for proton exchange membrane fuel cells. One concern with these materials is the complicated multi-step synthesis and processing of oligomers, followed by coupling to produce a multiblock copolymer. An streamlined synthetic process was successful for synthesizing membranes with comparable morphologies and performance to a multiblock synthesized via the traditional method. / Ph. D.