Clean water is critical to the safety, security and survivability of humankind. Nearly 41% of the Earth's population lives in water-stressed areas, and water scarcity will be exacerbated by an increasing population. Over 96% of the total water is saline and only 0.8% is accessible fresh water. Thus, saltwater desalination has emerged as the key to tackle the problem of water scarcity. Our current work deals with the membrane process of reverse osmosis. Sulfonated polysulfones are a potential alternative to state-of-the-art thin film polyamides. Synthesized by step growth polymerization, polysulfone membranes have smooth surfaces and they are more chemically resistant relative to polyamides. Previously studied sulfonated polysulfone membranes were synthesized by direct copolymerization of pre-disulfonated comonomer and the sulfonate ions were placed on adjacent rings of bisphenol moiety. This study focuses on placing the sulfonate ions differently along the polysulfone backbone on isolated rings of hydroquinone moiety, and on adjacent rings of biphenol moiety- and its effect on the transport and hydrated mechanical properties of the membranes. Selective post sulfonation of poly(arylene ether sulfone) in mild conditions was also found to be an effective way to strategically place the sulfonate ions along the backbone of the polymer chain without the need to synthesize a new monomer. Hydroquinone based, amine terminated oligomers were synthesized with block molecular weights of 5000 and 10,000 g/mol. They were post-sulfonated and crosslinked at their termini with epoxy reagents. Such crosslinked and linear membranes had sulfonate ions on isolated rings of hydroquinone moiety. Synthesis and kinetics of controlled post-sulfonation of poly(arylene ether sulfones) that contained biphenol units were also reported. The sulfonation reaction proceeded only on the biphenol rings. The linear membranes had sulfonate ions on adjacent rings of biphenol moieties. The tensile measurements were performed on the membranes under fully hydrated conditions. All membranes remained glassy at values of water uptake. It was found that elastic moduli and yield strengths in the hydroquinone- based linear and crosslinked membranes increased with decrease in water uptakes in the membranes. The effect of plasticization of water superseded the effect of block length and degree of sulfonation in the membranes. The highest elastic modulus of 1420 MPa at lowest water uptake of 18% was observed in cross linked membrane with 50% repeat units being sulfonated (50% repeat units contain hydroquinone)and target molecular weight of 5000 g/mol. However, the hydroquinone membranes broke at low strains of < 20%. The hydrated mechanical properties could be improved by replacing the hydroquinone with biphenol moieties. The biphenol based post-sulfonated membrane showed high elastic modulus that was comparable to the hydroquinone-based counterparts at similar values of water uptake. The biphenol based membrane broke at higher strains of >80%. The post-sulfonated membranes- hydroquinone-based linear and crosslinked membranes and biphenol-based linear membranes had better transport properties than the previously studied sulfonated polysulfones that were synthesized by disulfonated comonomers.The post sulfonated hydroquinone-based membranes did not show a compromise in the rejection of monovalent ions in the presence of divalent ions in mixed feed water. The superior properties of the post-sulfonated membranes can potentially be attributed to the kinked backbone that potentially increased the free volume in the membranes and the sulfonate ions were spaced apart to potentially reduce their chelation with calcium (divalent) ions in mixed feed water. Interestingly, the biphenol based post-sulfonated membranes also did not have any compromise in the rejection of monovalent ions in the presence of divalent ions. This was potentially because the sulfonate ions were spaced far apart on the non-planar biphenol rings. / PHD / According to the World Economic Forum, the water crisis has remained one of the top five global risks that has had a huge impact on the society. The world population has tripled in the twentieth century. Close to 2 billion people live in water scarce regions, 1.2 billion people lack access to safe drinking water, 2.6 billion have little or no access to sanitation and countless die due to diseases transmitted through unsafe water. Industrialization and climate change have worsened the water crisis. Furthermore, in today’s economies food, energy and water are inherently linked. Thus, a water crisis can have a cascading effect on availability of food and energy. To obtain an adequate and sustainable supply of water, it is important to improve already existing methods and develop new and inexpensive technologies for water purification. According to the U.S. geological survey over 96% of the earth’s water is saline. Thus, salt water desalination has emerged as the key to tackle the problem of scarcity of potable water. Reverse osmosis is a membrane-based process for water desalination wherein the membrane allows water to pass through while rejecting salts. The membranes are composed of long chain molecules called polymers. The current state of the art polymeric membrane made of polyamides show high rejection of salts with fast permeation of water. However, these membranes can be degraded by the chlorinated disinfectants added to the feed water. An alternative polymeric material, sulfonated polysulfone, can potentially be applied for reverse osmosis as these polymers are resistant to the chlorinated species. These membranes are composed of a polysulfone with sulfonate ions present randomly on the chain. This study investigates the effect of the position of the ions on the polymer chain. It is found that the membranes ability to reject salt from water can be improved by strategically placing the charged species on the polymer chain.

Bacteriophages, a promising antimicrobial alternative to modern antibiotics, can rapidly overcome bacterial resistance. However, the propagation of therapeutic phages in gram-negative bacteria leads to proinflammatory contaminants, notably endotoxins (lipopolysaccharides). To meet strict regulations on endotoxin levels, the widely accepted method for phage purification is based on polyethylene glycol precipitation followed by cesium chloride density gradient ultracentrifugation. This method, while effective, is labor-intensive and produces only small quantities of purified phage products. Moreover, the current studies on using membrane adsorbers in this application have primarily focused on removing endotoxins or recovering phages, without addressing the variability in physical properties of bacteriophages that necessitates strain-specific process development. In contrast, this thesis introduces a novel high-throughput flat sheet membrane screening approach. This approach not only aims to evaluate the effect of varying solution conditions on bacteriophage purification but also provides a comprehensive solution to the limitations of the current method, guiding larger-scale purification work. Using two clinically derived bacteriophage, this work demonstrates that the multi well device used in this work is appropriate for this application, with little background binding of bacteriophage to the device itself. Bacteriophage binding to the membrane was found to be both sensitive to the dilution of the preparation and contact time with a given membrane. The device was used to evaluate the overall binding productivity of the membranes, finding that the Natrix and Sartobind Q membranes possess superior productivity to that of Mustang Q. Furthermore, this work demonstrated that the method could be used to rapidly screen the effects of varying sodium chloride molarity on both bacteriophage and endotoxin binding, with the effect of greater NaCl molarity on both bacteriophage and endotoxin binding being generally comparable to expectations.Finally, this work demonstrates that improvements in the translatability of screened conditions to larger-scale techniques (e.g., Syringe Filtration) require a better understanding of the underlying interactions occurring between the biomolecules and membranes. / Thesis / Master of Applied Science (MASc)

The last decade has seen substantial advances in single-molecule tracking methods with nano-metre level precision. A powerful tool in single-molecule tracking is fluorescence imaging. One particular application, total internal reflection microscopy, can capture biological processes at high contrast video rate imaging at the single-particle level. This thesis presents methodologically novel methods in analysing single particle tracking data. Presented here is an application of a Bayesian statistical approach that can discriminate between the different diffusive modes that appear with the presence of membrane architecture. This algorithm is denoted BARD; a Bayesian Analysis to Ranking Diffusion. These algorithms are applied to a total internal fluorescence microscopy based experimental data of a novel membrane probe in Escherichia coli. This probe is a plasmid expressed, non-native membrane integrating trans-membrane helix and thus acts as an ideal protein based probe under no specific native control. Two experiments were performed using a combination of varying helix probe size and growth temperature experiments effectively altering the transition temperature of the membrane. These data are suggestive of a passive partitioning of the helix protein into mobile and immobile domains that emerge from the underlying phase behaviour of the membrane.

Quel que soit le domaine d'application, les membranes doivent fréquemment subir des procédures de nettoyage et/ou de désinfection afin d'éliminer le colmatage qui découle de la filtration. Ces nettoyages sont responsables d'une accélération du vieillissement des matériaux qui impacte directement les performances techniques et économiques des procédés membranaires. La pérennité de ces derniers passe par l'identification des principaux facteurs responsables du vieillissement et par la compréhension des mécanismes qui le régissent. Le vieillissement statique de membranes d'ultrafiltration spiralée en polyethersulfone et fibres creuses en polysulfone respectivement utilisées pour la filtration du lait et pour la production d'eau potable a été étudié. Ce vieillissement a été mis en place selon la méthodologie des plans d'expériences qui a permis l'étude simultanée de la concentration, de la température et du temps de contact membrane/détergents et ce, pour différents détergents formulés couramment utilisés sur les sites de production (alcalin, acide, enzymatiques, oxydant chloré et oxydant non chloré). Les résultats ont permis de modéliser statistiquement les modifications membranaires induites par chaque détergent. Ces modifications ont été suivies aussi bien à l'échelle microscopique que macroscopique afin d'appréhender le phénomène de vieillissement dans sa globalité : propriétés de transport, propriétés mécaniques, propriétés de surface, ainsi que les modifications de la composition chimique du polymère constitutif de la membrane. Des préconisations d'utilisation des différents produits sont proposées : véritable carnet de bord les industriels. / In Food Industries, using membrane processes, contact with cleaning chemicals is believed to play an important role in membrane ageing. In this thesis, polysulfone hollow fibers and polyethersulfone spiral wound membranes were used to simulate the industrial cleaning in static conditions. Ageing of the membrane was mimicked by immersing samples in solutions containing commercial detergents with various concentrations, temperatures and soaking times defined by experimental designs. In an innovative way in the chemical membranes ageing research, an approach based on methodological tools has been realized for different formulated detergents (alkaline, acidic, enzymatic, oxidant and biocide). The main interest is to achieve a relevant ageing pattern without using an accelerated ageing protocol which has been proved to be not relevant. Results allow modeling macroscopic ageing involved by each detergent. Those modifications have been studied as well at the macroscopic scale as at the microscopic scale in order to put membrane ageing in a global perspective: flux, mechanical properties, surface properties, polymer modifications. Specifications of using can be advised according to membranes and products which represent a real oversight handbook about membrane cleaning for industrial users.

In this study, membranes were made from three polyvinylidene fluoride (PVDF) polymers individually and the blend systems of high (H) and low (L) molecular weight PVDF by phase inversion process. After investigating membrane casting solutions’ viscous and thermodynamic properties, the membranes so fabricated were characterized by scanning electron microscopy, gas permeation tests, porosity measurement, contact angle (CA) and liquid entry pressure of water (LEPw) measurement, and further subjected to vacuum membrane distillation (VMD) in a scenario that was applicable for cooling processes, where the feed water temperature was maintained at 27℃. It was found that PVDF solutions’ viscosities and thermodynamic instabilities were determined by the types of PVDF employed in single polymer systems and the mixing ratios of two PVDF polymers in blend systems. Thus the membrane properties and performances were influenced by the aforesaid factors as well. In single polymer systems, it was found that the membrane surface roughness and porosity increased with an increase in molecular weight. Among all the membranes casted in this study, the water vapor flux of VMD was found to be the highest at the intermediate range of H:L ratio, i.e., 4:6, at which the thickness of the sponge-like layer showed a minimum, the finger-like macro-voids formed a more orderly single-layer structure, and the LEPw showed a minimum. A conclusion can be made that blend systems of high molecular weight PVDF polymers and low molecular weight PVDF polymers could be used to optimize membrane performance in vacuum membrane distillation.

Mesoporous silica materials are versatile platforms for biological catalysis, isolation of small molecules for detection and separation applications. The design of mesoporous silica supports for tailored protein and biomolecule interactions has been limited by the techniques to demonstrate biomolecule location and functionality as a function of pore size. This work examines the interaction of proteins and lipid bilayers with engineered porous silica surfaces using spherical silica particles with tunable pore diameters (3 – 12 nm) in the range relevant to biomolecule uptake in the pores, and large particle sizes (5 - 15 µm) amenable to microscopy imaging The differentiation of protein location between the external surface and within the pore, important to applications requiring protein protection or catalytic activity in pores, is demonstrated. A protease / fluorescent protein system is used to investigate protein location and protection as a function of pore size, indicating a narrow pore size range capable of protein protection, slightly larger than the protein of interest and approaching the protease dimensions. Selective functionalization, in this case exterior-only surface functionalization of mesoporous particles with amines, is extended to larger pore silica materials. A reaction time dependent functionalization approach is demonstrated as the first visually confirmed, selective amine functionalization method in protein accessible supports. Mesoporous silica nanoparticles are effective supports for lipid bilayer membranes and membrane associated proteins for separations and therapeutic delivery, although the role of support porosity on membrane fluidity is unknown. Transport properties of bilayers in lipid filled nanoparticles as a function of pore diameter and location in the particle are measured for the first time. Bilayer diffusivity increases with increasing pore size and is independent of bilayer location within the core, mid or cap of the particle, suggesting uniform long range bilayer mobility in lipid filled pores. Application of lipid bilayers on mesoporous silica was examined for membrane associated proteins A unique method to adhere functional proteins in lipid bilayers on mesoporous silica particles is established using vesicles derived from cell plasma membranes and their associated proteins. This method of membrane protein investigation retains proteins within native lipid membranes, stabilizing proteins for investigation on supports.

In this study, membranes were made from three polyvinylidene fluoride (PVDF) polymers individually and the blend systems of high (H) and low (L) molecular weight PVDF by phase inversion process. After investigating membrane casting solutions’ viscous and thermodynamic properties, the membranes so fabricated were characterized by scanning electron microscopy, gas permeation tests, porosity measurement, contact angle (CA) and liquid entry pressure of water (LEPw) measurement, and further subjected to vacuum membrane distillation (VMD) in a scenario that was applicable for cooling processes, where the feed water temperature was maintained at 27℃. It was found that PVDF solutions’ viscosities and thermodynamic instabilities were determined by the types of PVDF employed in single polymer systems and the mixing ratios of two PVDF polymers in blend systems. Thus the membrane properties and performances were influenced by the aforesaid factors as well. In single polymer systems, it was found that the membrane surface roughness and porosity increased with an increase in molecular weight. Among all the membranes casted in this study, the water vapor flux of VMD was found to be the highest at the intermediate range of H:L ratio, i.e., 4:6, at which the thickness of the sponge-like layer showed a minimum, the finger-like macro-voids formed a more orderly single-layer structure, and the LEPw showed a minimum. A conclusion can be made that blend systems of high molecular weight PVDF polymers and low molecular weight PVDF polymers could be used to optimize membrane performance in vacuum membrane distillation.

Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 161-183). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 148-159). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Conçues à partir d’une approche rationnelle, nous avons créé de nouvelles sondes membranaires permettant l’imagerie de l’organisation de la membrane plasmique cellulaire. Dans ce travail, nous avons d’abord développé un groupe d’outils, à partir du fluorophore solvatochrome Nile Red et de Black Hole Quencher-2, capable de marquer spécifiquement les domaines ordonnés et désordonnés (radeaux) en les identifiant par leur couleur d’émission. Les études cellulaires, à l’aide de ces sondes, suggèrent que la membrane plasmique est composée de deux phases distinctes. Puis dans le but de créer de nouvelles sondes basées sur Nile Red compatibles avec le sérum et fixables par formaldéhyde/glutaraldéhyde, nous avons modifié la sonde, préalablement développée, NR12S avec un groupement PEG ou amino, respectivement. Etonnamment, la sonde PEGylée est rapidement internalisée dans la cellule et le dérivé animo agrège avec l’agent fixant. D’un autre côté,basée sur Nile Red, nous avons conçu une sonde capable de détecter un récepteur donné et de visualiser son environnement lipidique. Initialement, nous avons obtenu des sondes capables d’allumer leur fluorescence en se liant sur le RCPG à l’ocytocine. Puis, nous avons conjugué NR12Spar l’intermédiaire d’un espaceur PEG(12) au ligand de l’intégrine, RGD. Les résultats préliminaires montrent que la molécule peut se lier à la membrane et détecter l’ordre lipidique, cependant les études cellulaires nécessitent un achèvement. Nous avons aussi travaillé sur des sondes membranaires fluorogéniques (turn-on) pour de l’imagerie multi-couleurs. Basées sur le fluorophore3-méthoxychromone, nous avons obtenu des sondes plus brillantes et plus photostables que la sonde développée originellement à partir de 3-hydroxychromone (F2N12S). Grâce à l’important déplacement de Stokes, elles permettent une imagerie de la membrane cellulaire avec une autofluorescence minimale dans la région spectrale bleue, compatible avec les marqueurs communs verts et rouges. Pour finir, basées sur le fluorophore squaraine, nous avons développé trois nouvelles sondes opérant dans la région rouge lointain, qui est particulièrement intéressante pour l’imagerie in vitro et in vivo. Ces sondes montrent une orientation parallèle avec la membrane lipidique, alors que les expériences cellulaires indiquent que seule la sonde avec deux ancres lipidiques est capable de marquer de façon stable la membrane plasmique. Ces sondes développées ici sont prévues pour être utilisées dans la recherche des radeaux lipidiques aussi bien que pour l’imagerie super-résolution et multi-couleurs de cellules vivantes. / Based on rational molecular design, we design new membrane probes that enable fluorescence imaging of cell plasma membrane organization. In this work, we first synthesized a toolkit, based on solvatochromic Nile Red dye and Black Hole Quencher-2, that can stain specifically ordered and disordered lipid domains (rafts) and identify them by the emission color. Cellular studies with these probes suggested that the plasma membrane is composed of two distinct phases. Then,with the idea to make Nile Red-based probes compatible with serum medium and fixable by formaldehyde/glutaraldehyde, we modified previously developed probe NR12S with PEG and aminogroups, respectively. Surprisingly, the PEGylated probe is quickly internalized inside the cell and the amino-derivative aggregates with the fixing agent. On the other hand, based on Nile Red we designed probes capable to detect a given receptor and visualize its lipid environment. Initially, we obtained probes that can turn-on fluorescence on binding to the oxytocin GPCR receptor. Then, we conjugated NR12S through a PEG(12) spacer to the ligand of intergrin, RGD. The first data show that the molecule can bind to the membrane and detect the lipid order, though cellular studies have to be completed. We also worked on fluorogenic (turn-on) membrane probes for multi-color imaging. Based on blue 3-methoxychromone dyes, we obtained probes that are brighter and more photostable than the originally developed probe based on 3-hydroxychromone (F2N12S). Due to large Stocks shift, they enabled cell membrane imaging with minimal auto-fluorescence in the blue spectral region, compatible with common green and red probes. At the end, based on squaraine fluorophore, we developed three new probes operating in the far red region, which is also very interesting for in vitro and in vivo imaging. These dyes show a parallel orientation with the lipid membrane, while the cellular experiments point out that only the probe with two anchor groups is able to stain stably the plasma membrane. The probes developed here are expected to be used for lipid rafts research as well as for super-resolution and multi-color imaging of living cells.