Neurospora crassa transcriptomics reveals oxidative stress and plasma membrane homeostasis biology genes as key targets in response to chitosan

Chitosan is a natural polymer with antimicrobial activity. Chitosan causes plasma membrane permeabilization and induction of intracellular reactive oxygen species (ROS) in Neurospora crassa. We have determined the transcriptional profile of N. crassa to chitosan and identified the main gene targets involved in the cellular response to this compound. Global network analyses showed membrane, transport and oxidoreductase activity as key nodes affected by chitosan. Activation of oxidative metabolism indicates the importance of ROS and cell energy together with plasma membrane homeostasis in N. crassa response to chitosan. Deletion strain analysis of chitosan susceptibility pointed NCU03639 encoding a class 3 lipase, involved in plasma membrane repair by lipid replacement, and NCU04537 a MFS monosaccharide transporter related to assimilation of simple sugars, as main gene targets of chitosan. NCU10521, a glutathione S-transferase-4 involved in the generation of reducing power for scavenging intracellular ROS is also a determinant chitosan gene target. Ca2+ increased tolerance to chitosan in N. crassa. Growth of NCU10610 (fig 1 domain) and SYT1 (a synaptotagmin) deletion strains was significantly increased by Ca2+ in the presence of chitosan. Both genes play a determinant role in N. crassa membrane homeostasis. Our results are of paramount importance for developing chitosan as an antifungal.

Electrolyte transport in the mammalian colon: mechanisms and implications for disease Kunzelmann and Marcus Mall

Electrolyte Transport in the Mammalian Colon: Mechanisms and Implications for Disease. Physiol. Rev. 82: 245-289, 2002.The colonic epithelium has both absorptive and secretory functions. The transport is characterized by a net absorption of NaCl, short-chain fatty acids (SCFA), and water, allowing extrusion of a feces with very little water and salt content. In addition, the epithelium does secret mucus, bicarbonate, and KCl. Polarized distribution of transport proteins in both luminal and basolateral membranes enables efficient salt transport in both directions, probably even within an individual cell. Meanwhile, most of the participating transport proteins have been identified, and their function has been studied in detail. Absorption of NaCl is a rather steady process that is controlled by steroid hormones regulating the expression of epithelial Na+ channels (ENaC), the Na+-K+-ATPase, and additional modulating factors such as the serum- and glucocorticoid-regulated kinase SGK. Acute regulation of absorption may occur by a Na+ feedback mechanism and the cystic fibrosis transmembrane conductance regulator (CFTR). Cl- secretion in the adult colon relies on luminal CFTR, which is a cAMP-regulated Cl- channel and a regulator of other transport proteins. As a consequence, mutations in CFTR result in both impaired Cl- secretion and enhanced Na+ absorption in the colon of cystic fibrosis (CF) patients. Ca2+- and cAMP-activated basolateral K+ channels support both secretion and absorption of electrolytes and work in concert with additional regulatory proteins, which determine their functional and pharmacological profile. Knowledge of the mechanisms of electrolyte transport in the colon enables the development of new strategies for the treatment of CF and secretory diarrhea. It will also lead to a better understanding of the pathophysiological events during inflammatory bowel disease and development of colonic carcinoma.

Analytical electron microscopy of proton exchange membrane fuel cells

Microtome sections of proton exchange membrane cells produce a wide range of information ranging from macroscopic distribution of components through specimens in which the detailed distribution of catalyst particles can be observed. Using modern data management practices it is possible to combine information at different scales and correlate processing and performance data. Analytical electron microscopy reveals the compositional variations across used cells at the electrolyte/electrode interface. In particular analytical techniques indicate that sulphur concentrations are likely to diminish at the interface Nafion/anode interface. © 2006 Elsevier B.V. All rights reserved.

The activity of pH membrane disruptive pseudopeptides and their subcellular fate in mammalian cells cultured in-vitro

This thesis describes investigations upon pseudopeptides which were conducted to improve our understanding of the fate of synthetic macromolecules in cells and to develop approaches to influence that fate. The low uptake of molecules across the external cellular membrane is the principal barrier against effective delivery of therapeutic products to within the cell structure. In nature, disruption of this membrane by amphiphilic peptides plays a central role in the pathogenesis by bacterial and toxin infections. These amphiphilic peptides contain both hydrophobic and weakly charged hydrophilic amino acid residues and upon activation they become integrated into the lipid bilayers of the extracellular or endosomal membranes. The architectures of the pseudopeptides described here were designed to display similar pH dependent membrane rupturing activity to that of peptides derived from the influenza virus hemagglutinin HA-2. This HA protein promotes fusion of the influenza virus envelope with the cell endosome membrane due to a change in conformation in response to the acidic pH of the endosome lumen (pH 5.0-6.0). The pseudopeptides were obtained by the copolymerisation of L-lysine and L-lysine ethyl-ester with various dicarboxylic acid moieties. In this way a linear polyamide comprising of alternating pendant carboxylic acids and pendant hydrophobic moieties was made. At physiological pH (pH 7.4), electrostatic repulsion of pendant anionic carboxyl groups along the polymer backbone is sufficient to overcome the intramolecular association of the hydrophobic groups resulting in an extended conformation. At low pH (typically pH 4.8) loss of charge results in increased intramolecular hydrophobic association and the polymer chain collapses to a compact conformation, leading to precipitation of the polymer. Consequently, a conformation dependent functional property could be made to respond to small changes in the environmental pH. Pseudopepides were investigated for their cytoxicity towards a well known cell line, namely C26 (colorectal adenocarcinoma) and were shown through the use of a cell viability assay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) to be well tolerated by C26 cells over a range of concentrations (2-500,μg/ml) at physiological pH (pH 7.4). A modified version of a shorter 30-minute coupled enzymatic assay, the LDH (lactate dehydrogenase) assay was used to evaluate the ability of the pseudopeptides to disrupt the membrane of two different cell lines (COS-1; African green monkey, kidney and A2780; human ovarian carcinoma) at low pH (pH 5.5). The cell membrane disruption property of the pseudopeptides was successfully demonstrated for COS-I and A2780 cell lines at this pH (pH 5.5). A variety of cell lines were chosen owing to limited availability and to compare the cytotoxic action of these pH responsive psudopeptides towards normal and tumorogenic cell lines. To investigate the intracellular delivery of one of the pseudopeptides, poly (L-lysine iso-phthalamide) and its subcellular location, a Cy3 bisamine fluorophore was conjugated into its backbone, at ratios of dye:lysine of 1:20, 1:30, 1:40, 1:60 and 1:80. Native polyacrylacrylamide gel electrophoresis (PAGE) and high voltage paper electrophoresis (HVPE) studies of the polydyes were conducted and provided evidence that that the Cy3 bisamine fluorophore was conjugated into the backbone of the polymer, poly (L-lysine iso-phthalamide). The subcellular fate of the fluorescentlylabelled "polydye" (hereafter PD20) was monitored by laser scanning confocal microscopy (LSCM) in CHO (Chinese hamster ovary) cells cultured in-vitro at various pH values (pH 7.4 and 5.0). LSCM images depicting time-dependent internalisation of PD20 indicated that PD20 traversed the extracellular membrane of CHO cells cultured in-vitro within ten minutes and migrated towards the endosomal regions where the pH is in the region of 5.0 to 6.0. Nuclear localisation of PD20 was demonstrated in a subpopulation of CHO cells. A further study was completed in CHO and HepG2 (hepatocellular carcinoma) cells cultured in-vitro using a lower molecular weight polymer to demonstrate that the molecular weight of "polydye" could be tailored to attain nuclear trafficking in cells. Prospective use of this technology encompasses a method of delivering a payload into a living cell based upon the hypercoiling nature of the pseudopeptides studied in this thesis and has led to a patent application (GB0228525.2; 20(2).

The production of clinical dextran using membrane processes

Clinical dextran is used as a blood volume expander. The British Pharmacopeia (BP) specification for this product requires the amount of dextran below 12,000 MW and above 98,000 MW to be strictly controlled. Dextran is presently fractionated industrially using ethanol precipitation. The aim of this work was to develop an ultrafiltration system which could replace the present industrial process. Initially these molecular weight (MW) bands were removed using batch ultrafiltration. A large number of membranes were tested. The correct BP specification could be achieved using these membranes but there was a significant loss of saleable material. To overcome this problem a four stage ultrafiltration cascade (UFC) was used. This work is the first known example of a UFC being used to remove both the high and low MW dextran. To remove the high MW material it was necessary to remove 90% of the MW distribution and retain the remaining 10%. The UFC significantly reduced the amount of dialysate required. To achieve the correct specification below 12,000 MW, the UFC required only 2.5 - 3.0 diavolumes while the batch system required 6 - 7. The UFC also improved the efficiency of the fractionation process. The UFC could retain up to 96% of the high MW material while the batch system could only retain 82.5% using the same number of diavolumes. On average the UFC efficiency was approximately 10% better than the equivalent batch system. The UFC was found to be more predictable than the industrial process and the specification of the final product was easier to control. The UFC can be used to improve the fractionation of any polymer and also has several other potential uses including enzyme purification. A dextransucrase bioreactor was also developed. This preliminary investigation highlighted the problems involved with the development of a successful bioreactor for this enzyme system.

Transition metal ion coordination in hydrophilic polymer membranes

The research described within this thesis is concerned with the investigation of transition metal ion complexation within hydrophilic copolymer membranes. The membranes are copolymers of 4-methyl-4'-vinyl-2,2'-bipyridine, the 2-hydroxyethyl ester of 4,4'- dicarboxy-2,2'-bipyridine & bis-(5-vinylsalicylidene)ethylenediamine with 2-hydroxyethyl methacrylate. The effect of the polymer matrix on the formation and properties of transition metal iron complexes has been studied, specifically Cr(III) & Fe(II) salts for the bipyridyl- based copolymer membranes and Co(II), Ni(II) & Cu(II) salts for the salenH2- based copolymer membranes. The concomitant effect of complex formation on the properties of the polymer matrix have also been studied, e.g. on mechanical strength. A detailed body of work into the kinetics and thermodynamics for the formation of Cu(II) complexes in the salenH2- based copolymer membranes has been performed. The rate of complex formation is found to be very slow while the value of K for the equilibrium of complex formation is found to be unexpectedly small and shows a slight anion dependence. These phenomena are explained in terms of the effects of the heterogeneous phase provided by the polymer matrix. The transport of Cr(III) ions across uncomplexed and Cr(III)-pre-complexed bipyridyl-based membranes has been studied. In both cases, no Cr(III) coordination occurs within the time-scale of an experiment. Pre-complexation of the membrane does not lead to a change in the rate of permeation of Cr(III) ions. The transport of Co(II), Ni(II) & Cu(II) ions across salenH2- based membranes shows that there is no detectable lag-time in transport of the ions, despite independent evidence that complex formation within the membranes does occur. Finally, the synthesis of a number of functionalised ligands is described. Although they were found to be non-polymerisable by the methods employed in this research, they remain interesting ligands which provide a startmg pomt for further functionalisation.

The separation of hydrogen and carbon monoxide using polymer membranes

This work studies the development of polymer membranes for the separation of hydrogen and carbon monoxide from a syngas produced by the partial oxidation of natural gas. The CO product is then used for the large scale manufacture of acetic acid by reaction with methanol. A method of economic evaluation has been developed for the process as a whole and a comparison is made between separation of the H2/CO mixture by a membrane system and the conventional method of cryogenic distillation. Costs are based on bids obtained from suppliers for several different specifications for the purity of the CO fed to the acetic acid reactor. When the purity of the CO is set at that obtained by cryogenic distillation it is shown that the membrane separator offers only a marginal cost advantage. Cost parameters for the membrane separation systems have been defined in terms of effective selectivity and cost permeability. These new parameters, obtained from an analysis of the bids, are then used in a procedure which defines the optimum degree of separation and recovery of carbon monoxide for a minimum cost of manufacture of acetic acid. It is shown that a significant cost reduction is achieved with a membrane separator at the optimum process conditions. A method of "targeting" the properties of new membranes has been developed. This involves defining the properties for new (hypothetical -yet to be developed) membranes such that their use for the hydrogen/carbon monoxide separation will produce a reduced cost of acetic acid manufacture. The use of the targeting method is illustrated in the development of new membranes for the separation of hydrogen and carbon monoxide. The selection of polymeric materials for new membranes is based on molecular design methods which predict the polymer properties from the molecular groups making up the polymer molecule. Two approaches have been used. One method develops the analogy between gas solubility in liquids and that in polymers. The UNIFAC group contribution method is then used to predict gas solubility in liquids. In the second method the polymer Permachor number, developed by Salame, has been correlated with hydrogen and carbon monoxide permeabilities. These correlations are used to predict the permeabilities of gases through polymers. Materials have been tested for hydrogen and carbon monoxide permeabilities and improvements in expected economic performance have been achieved.

The fractionation of dextran polymer by ultrafiltration to yield clinical products

A review of ultrafiltration (UF) theory and equipment has been made. Dextran is fractionated industrially by ethanol precipitation, which is a high energy intensive process. The aims of this work were to investigate the fractionation of dextran using UF and to compare the efficiency and costs of UF fractionation with ethanol fractionation. This work is the continuation of research conducted at Aston, which was concerned with the fractionation of dextran using gel permeation chromatography (GPC) and hollow fibre UF membranes supplied by Amicon Ltd. Initial laboratory work centred on determining the most efficient make and configuration of membrane. UF membranes of the Millipore cassette configuration, and the DDS flat-sheet configuration, were examined for the fracationation of low molecular weight (MW) dextran. When compared to Amicon membranes, these membranes were found to be inferior. DDS membranes of 25 000 and 50 000 MW cut-offs were shown to be capable of fractionating high MW dextran with the same efficiency as GPC. The Amicon membranes had an efficiency comparable to that of ethanol fractionation. To increase this efficiency a theoretical UF membrane cascade was adopted to utilize favourable characteristics encountered in batch mode membrane experiments. The four stage cascade used recycled permeates in a counter- current direction to retentate flow, and was operated 24 hours per day controlled by a computer. Using 5 000 MW cut-off membranes the cascade improved the batch efficiency by at least 10% for a fractionation at 6 000 MW. Economic comparisons of ethanol fractionation, combined GPC and UF fractionation, and UF fractionation of dextran were undertaken. On an economic basis GPC was the best method for high MW dextran fractionation. When compared with a plant producing 100 tonnes pa of clinical dextran, by ethanol fractionation, a combined GPC and UF cascade fractionation could produce savings on operating costs and an increased dextran yield of 5%.

Surfactant-free purification of membrane proteins with intact native membrane environment

In order to study the structure and function of a protein, it is generally required that the protein in question is purified away from all others. For soluble proteins, this process is greatly aided by the lack of any restriction on the free and independent diffusion of individual protein particles in three dimensions. This is not the case for membrane proteins, as the membrane itself forms a continuum that joins the proteins within the membrane with one another. It is therefore essential that the membrane is disrupted in order to allow separation and hence purification of membrane proteins. In the present review, we examine recent advances in the methods employed to separate membrane proteins before purification. These approaches move away from solubilization methods based on the use of small surfactants, which have been shown to suffer from significant practical problems. Instead, the present review focuses on methods that stem from the field of nanotechnology and use a range of reagents that fragment the membrane into nanometre-scale particles containing the protein complete with the local membrane environment. In particular, we examine a method employing the amphipathic polymer poly(styrene-co-maleic acid), which is able to reversibly encapsulate the membrane protein in a 10 nm disc-like structure ideally suited to purification and further biochemical study.

Comparison of hydrophilic and hydrophobic active molecules release from polymer-phospholipid complexes

Hypercoiling polymers can be suited for application to living systems because they are similar in structure to the protein-based lipid assemblies found at fluid interfaces within the body. This leads to a range of exciting possibilities, not only in membrane transport applications but also in biosensors, drug delivery and mechanistic studies of biological membrane function. This study is focused in the study of the stability and suitability of nanostructures made of a hypercoiling polymer for drug delivery applications. The polymer poly (styrene-maleic acid) (PSMA) was combined with the phospholipid dimyristoylphosphatidylcholine (DMPC) to form amphiphilic nanostructures. The stability and suitability of these polymer-phospholipid nanocarriers for hydrophobic and hydrophilic molecules load and release was analyzed by several techniques. It was found that several of the studied molecules had a substantial effect on the surface charge and stability of the nanocarrier. It was also demonstrated that two types of nanocarriers, chemically modified and unmodified, were able to control the release of the molecules, especially in the case of hydrophobic compounds. In addition, as the hydrophobicity increased the release slowed down. These clear nanocarriers have the potential to behave very favorably at interfaces such as the tear lipid film were transparency is a requirement, giving a new way of controlled drug release in the eye.

Basalt Fibre Reinforced Polymer Strengthening of Normal Strength Reinforced Concrete Floor Slabs Subject to Arching Effects

With ever increasing demands to strengthen existing reinforced concrete structures to facilitate higher loading due to change of use and to extend service lifetime, the use of fibre reinforced polymers (FRPs) in structural retrofitting offers an opportunity to achieve these aims. To date, most research in this area has focussed on the use of glass fibre reinforced polymer (GFRP) and carbon fibre reinforced polymer (CFRP), with relatively little on the use of basalt fibre reinforced polymer (BFRP) as a suitable strengthening material. In addition, most previous research has been carried out using simply supported elements, which have not considered the beneficial influence of in-plane lateral restraint, as experienced within a framed building structure. Furthermore, by installing FRPs using the near surface mounted (NSM) technique, disturbance to the existing structure can be minimised.
This paper outlines BFRP NSM strengthening of one third scale laterally restrained floor slabs which reflect the inherent insitu compressive membrane action (CMA) in such slabs. The span-to-depth ratios of the test slabs were 20 and 15 and all were constructed with normal strength concrete (~40N/mm2) and 0.15% steel reinforcement. 0.10% BFRP was used in the retrofitted samples, which were compared with unretrofitted control samples. In addition, the bond strength of BFRP bars bonded into concrete was investigated over a range of bond lengths with two different adhesive thicknesses. This involved using an articulated beam arrangement in order to establish optimum bond characteristics for use in strengthening slab samples.

Quantitative characterization of physico-chemical properties of the active layers of reverse osmosis and nanofiltration membranes, and their relation to membrane performance

The main objectives of this dissertation were: (i) to develop experimental and analytical procedures to quantify different physico-chemical properties of the ultra-thin (~ 100 nm) active layers of reverse osmosis (RO) and nanofiltration (NF) membranes and their interactions with contaminants; (ii) to use such procedures to evaluate the similarities and differences between the active layers of different RO/NF membranes; and (iii) to relate characterization results to membrane performance. Such objectives were motivated by the current limited understanding of the physico-chemical properties of active layers as a result of traditional characterization techniques having limitations associated with the nanometer-scale spatial resolution required to study these ultra-thin films. Functional groups were chosen as the main active layer property of interest. Specific accomplishments of this study include the development of procedures to quantify in active layers as a function of pH: (1) the concentration of both negatively and positively ionized functional groups; (2) the stoichiometry of association between ions (i.e., barium) and ionized functional groups (i.e., carboxylate and sulfonate); and (3) the steric effects experienced by ions (i.e., barium). Conceptual and mathematical models were developed to describe experimental results. The depth heterogeneity of the active layer physico-chemical properties and interactions with contaminants studied in this dissertation was also characterized. Additionally, measured concentrations of ionized functional groups in the polyamide active layers of several commercial RO/NF membranes were used as input in a simplified RO/NF transport model to predict the rejection of a strong electrolyte (i.e., potassium iodide) and a weak acid (i.e., arsenious acid) at different pH values based on rejection results at one pH condition. The good agreement between predicted and experimental results showed that the characterization procedures developed in this study serve as useful tools in the advancement of the understanding of the properties and structure of the active layers of RO/NF membranes, and the mechanisms of contaminant transport through them.

Development and Detailed Characterization of Innovative, High-Performance Membrane Materials for CO2 Capture Processes

The scope of this dissertation is to study the transport phenomena of small molecules in polymers and membranes for gas separation applications, with particular attention to energy efficiency and environmental sustainability. This work seeks to contribute to the development of new competitive selective materials through the characterization of novel organic polymers such as CANALs and ROMPs, as well as through the combination of selective materials obtaining mixed matrix membranes (MMMs), to make membrane technologies competitive with the traditional ones. Kinetic and thermodynamic aspects of the transport properties were investigated in ideal and non-ideal scenarios, such as mixed-gas experiments. The information we gathered contributed to the development of the fundamental understanding related to phenomenon like CO2-induced plasticization and physical aging. Among the most significant results, ZIF-8/PPO MMMs provided materials whose permeability and selectivity were higher than those of the pure materials for He/CO2 separation. The CANALs featured norbornyl benzocyclobutene backbone and thereby introduced a third typology of ladder polymers in the gas separation field, expanding the structural diversity of microporous materials. CANALs have a completely hydrocarbon-based and non-polar rigid backbone, which makes them an ideal model system to investigate structure-property correlations. ROMPs were synthesized by means of the ring opening metathesis living polymerization, which allowed the formation of bottlebrush polymers. CF3-ROMP reveled to be ultrapermeable to CO2, with unprecedented plasticization resistance properties. Mixed-gas experiments in glassy polymer showed that solubility-selectivity controls the separation efficiency of materials in multicomponent conditions. Finally, it was determined that plasticization pressure in not an intrinsic property of a material and does not represent a state of the system, but rather comes from the contribution of solubility coefficient and diffusivity coefficient in the framework of the solution-diffusion model.

Influence of additives on the performance of a PVDF membrane

The field of use of membranes is wide and ranges from the automotive industry to biomedical uses. Many formulations and compositions find a niche where they are able to improve efficiency, running cost and quality of the product. The aim of this research is to expand GVS’s product portfolio introducing a new membrane formulation. A series of additives were researched and evaluated, adding them to the membrane solutions, which were then cast and characterised using techniques like Scanning Electron Microscopy (SEM), poroscopy, FT-IT ATR and measurements like Water Break Through (WBT), Air Flow (AF), thickness. This study ultimately focused on one additive, which effect on the membranes was studied in various compositions. Interesting insights were also collected on the stability of the polymer solutions over time, which was found to change the membrane properties significantly, mainly affecting airflow and water breakthrough. Properties of the membranes were studied to find possible correlations to the amount of additive. The additive seems however to change the membrane porometry considerably depending on the time of immersion in the water bath. A new procedure to yield uniform unsupported polymeric membranes for tensile tests was developed. The additive was found to reduce elongation at break and decrease tensile strength of the membranes, possibly hinting toward plasticization of the product.