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<p>PEGylation, referring to the covalent attachment of poly(ethylene glycol) or PEG to protein, has become the most established technology for improving pharmacokinetic behavior of native proteins, especially the prolongation of circulation half-life <em>in vivo</em>. This thesis focuses on the synthesis and purification of PEGylated proteins.</p> <p>The conventional way to synthesize PEGylated proteins is in liquid phase batch reaction, which usually causes the formation of significant amount and high diversity of by-products (i.e. di-, tri-, and/or higher-PEGylated forms of a protein). Many chemical and physical ways have been explored to increase the specificity of mono-PEGylated protein. Chemical ways involve manipulation of operating conditions towards site-specific PEGylation. Understanding reaction kinetics is helpful in optimizing conversion and specificity of mono-PEGylation. In this thesis, the PEGylation reaction kinetics between a model protein and PEG NHS ester under various operating conditions was investigated.</p> <p>In the physical perspective, the key point is to gain degree of control on reactant addition instead of one-time addition as in liquid phase batch reaction. Herein, two novel reactor systems were developed. One is solid phase PEGylation bioreactor, bringing free protein to react with immobilized PEG on a membrane surface; the other is Hollow-fiber Membrane Reactor (HMR), distributing PEG into the fiber lumen (where protein is flowing) through the pores on the fiber wall. Greatly improved conversion and specificity of mono-PEGylated protein were observed in both systems, compared to liquid phase batch reactor.</p> <p>An effective and efficient purification technique is very essential because purification step accounts for a significant portion of total cost. In this thesis, the use of hydrophobic interaction chromatography with environment-responsive microporous membranes was examined for the fractionation of different PEGylated proteins. The capability of this technique was demonstrated by obtaining mono-PEGylated protein in a pure form and observing well-resolved chromatographic peaks for different PEGylated proteins.</p> / Doctor of Philosophy (PhD)

In general, the stability of suspension can be studied using two methods. <i>One</i> is to directly measure the forces between two interacting surfaces in media. <i>The other</i> is to study the interfacial surface free energies of the particles in suspension. Direct surface force measurements were conducted between silica surfaces in octadecyltrimetylammonium chloride (C₁₈TACl) solutions using an Atomic Force Microscope (AFM). The results showed that the hydrophobic force existed in both air-saturated and degassed C₁₈TACl solutions. The attraction decreased with NaCl addition, and was the strongest at the point of charge neutralization (p.c.n.) of silica substrate. The force measurement results obtained in C_nTACl solutions showed that the attractions decayed exponentially and became the maximum at the p.c.n.'s. The decay lengths (<i>D</i>) increased with surfactant chain length. The measured forces were fitted to a charged-patch model of Miklavic <i>et al</i>. (1994) with rather large patch sizes. It was also found that the decay length decreased linearly with the effective concentration of the CH2/CH3 groups raised to the power of -1/2. This finding is in line with the model of Eriksson <i>et al</i>. (1989). It suggested that the long-range attractions are hydrophobic forces originating from the changes in water structure across a hydrophobic surface-solution interface. For the TiO₂/water/TiO₂ system, the Hamaker constant was found to be 4±1×10^-20 J. The force curves obtained in the TiO₂/C_nTACl system showed a repulsion-attraction-repulsion transition with increasing surfactant concentration. The long-range attraction observed between TiO₂ surfaces in C_nTACl solutions reached maximum at the p.c.n., and the decay length increased with chain length. In present work, the thin-layer wicking technique was used to determine the surface free energy (γ_s) and its components of talc samples. The results showed that the basal surfaces of talc are weakly basic while the edge surfaces are acidic. The effect of chemicals on the surface free energies of talc was systemically studied. The results showed that CMC (carboxymethyl cellulose sodium salt) and EO/PO (ethylene oxide/propylene oxide) co-polymers made talc surface hydrophilic by increasing the surface free energies, especially γ^LW and γ^-. SOPA (sodium polyacrylate) increased greatly the zeta-potentials instead of the surface free energies. / Ph. D.

Superhydrophobic (SHS) and lubricant-infused surfaces (LIS) exhibit exceptional non-wetting characteristics that make them attractive for energy production applications including steam condensation and fouling mitigation. The dissertation work focuses on application of non-wetting surfaces to energy production using a systematic approach examining each component of surface fabrication in three functional areas. First, SHS and LIS are fabricated using robust, scalable methods and tested for durability in heated, wet conditions and under high-energy water jet impingement. Clear performance differences are shown based on surface texturing, functionalizing agent, and infused lubricant. Second, SHS and LIS are applied to tube exteriors and evaluated for their ability to produce sustained dropwise condensation in a typical power plant condenser environment. The surfaces are shown to produce heat transfer coefficients up to 7-10 times that of film-wise condensation, with condenser effectiveness of 0.92 or better compared to effectiveness of about 0.6 in conventional condensers. Third, LIS on the interior of tubes are assessed in accelerated mineral fouling conditions. LIS are shown to mitigate calcium sulfate and calcium carbonate fouling under laminar conditions. The results of the study bear profound benefits to reducing the levelized cost of condensers and water uptake in thermoelectric power plants, that currently consume about 50% of the total water use in the U.S. / Doctor of Philosophy / Creating durable, hybrid surfaces for improved steam condensation and fouling mitigation would provide substantial impact to power generation worldwide. Bioinspired, non-wetting surfaces, such as superhydrophobic (SHS) and lubricant-infused surfaces (LIS) exhibit exceptional non-wetting characteristics that make them attractive for energy applications. Each of these non-wetting technologies, however, faces durability and scalability challenges that make them unfeasible for widespread, practical adoption. As a result, decades of materials science research have stagnated in the research laboratories with limited demonstrations of dropwise condensation and fouling mitigation in static situations. The dissertation work focuses on application of SHS and LIS to energy production using a systematic approach examining each component of surface fabrication in three functional areas. First, SHS and LIS are fabricated using robust, scalable methods and tested for durability using ASTM standard static and dynamic evaluation methods. Clear performance differences are shown based on surface texturing, functionalizing agent, and infused lubricant. Second, dropwise steam condensation on the surfaces are shown to exhibit heat transfer performance an order of magnitude greater than film-wise condensation in a typical power plant condenser environment. The surfaces are shown to produce heat transfer coefficients up to 7-10 times that of film-wise condensation, with condenser effectiveness of 0.92 or better compared to effectiveness of about 0.6 in conventional condensers. This work presents for the first time, a non-dimensional correlation for a priori prediction of LIS heat transfer performance given known qualities of the LIS. Third, challenges of fouling mitigation in power plants have been studied for over a decade. This work demonstrates for the first time that LIS applied to the interior of tubes mitigate calcium sulfate and calcium carbonate fouling in both static and laminar flow conditions.

Thin liquid films (TLFs) of water are ubiquitous in daily lives as well as in many industrial processes. They can be formed between two identical phases, as in colloid films between two macroscopic surfaces and foam films between two air bubbles; and between two dissimilar phases, as in wetting films. Stability of the colloids, foams, and wetting films is determined by the surface forces in the TLFs. Depending on the nature of the surfaces involved, the stabilities can be predicted using combinations of three different forces, i.e., the van der Waals, electrical double layer (EDL), and hydrophobic forces. The objective of the present work is to study the roles of these forces in determining the stabilities of the TLFs of water confined between i) an air bubble and a hydrophobic surface and ii) an oil drop and a hydrophobic surface, with particular interest in studying the role of the hydrophobic force. The first part of the study involves the measurement of the surface forces in the TLFs confined between bitumen drops and mineral surfaces. Deformation of bitumen drops has been monitored by interferometry while it approaches a flat surface. By analyzing the spatiotemporal film profiles, both the capillary and hydrodynamic forces have been calculated using the Young-Laplace equation and the Reynolds lubrication approximation, respectively, with the surface forces being determined by subtracting the latter from the former. The results are useful for better understanding the effects of electrolyte and pH on bitumen liberation and recovery by flotation and for developing a filtration model from first principles. The second part of the study involves the surface force measurement in wetting (flotation) films. Surface forces in the TLFs of water on silica surfaces have been measured using the force apparatus for deformable surfaces (FADS) using an air bubble as a force sensor. The measurements have been conducted in the presence of various cationic surfactants such as dodecylamine hydrochloride (DAH), and alkyltrimethylammonium chloride (CnTACl), electrolytes, and polymers. The results show that film stability and hence the kinetics of film thinning can be greatly improved by the control of bubble ζ-potentials, whose role in flotation has long been neglected in flotation studies. Force measurements have also been conducted in the TLFs of water confined between oil drops and hydrophobic surfaces. Stability of this type of film plays an important role in a process of using oil drops rather than air bubbles to collect hydrophobic particles from aqueous phase. The force measurements conducted in the present work show that hydrophobic forces are much stronger in water films formed between oil drops and hydrophobic surfaces than in water films formed between air bubbles and hydrophobic surfaces, which can be attributed to the differences in the Hamaker constants involved. / Doctor of Philosophy / When two macroscopic surfaces in water are brought to a close proximity, a thin liquid film (TLF) is formed in between, with its stability being determined by the surface forces present in the film. TLFs are ubiquitous in daily lives and play a decisive role in many industrial processes such as mineral flotation, food processing, oil extraction, heat transfer, etc. In the present work, the surface forces present in wetting films have been measured by approaching an air bubble (or an oil drop) slowly toward a flat surface while monitoring the curvature changes during film thinning by interferometry and calculating the capillary forces using the Young-Laplace equations. By analyzing the results in view of the Frumkin-Derjaguin isotherm and the extended DLVO theory, it was possible to determine the changes in the van der Waals, electrical double-layer (EDL), and hydrophobic forces during film thinning. The results show that both the EDL and the long-range component of the hydrophobic force control the kinetics of film thinning and rupture while the contact angle formation is controlled by the van der Waals force and the short-range hydrophobic force. It has been found also that n-alkane drops form substantially larger contact angles than air bubbles on a hydrophobic surface due to the fact that the van der Waals force is attractive in the drop-surface interactions while the same is repulsive in the bubble-surface interactions. These observations have a profound implication in flotation, that is, oil drops can recover hydrophobic particles from an aqueous phase better than air bubbles.

Comprehension of reactive chemical dynamics in the gas phase and at the gas/organic-surface interface and non-reactive dynamics at the interface between hydrophobic surfaces and water requires an understanding of the fundamental atomic and molecular interactions that undergird these important phenomena. In an effort to study these regimes of chemical interaction, we have performed computational simulations that probe the dynamics of chemical systems that exemplify each of these domains. To study gas-phase chemical dynamics, we reparametrized semiempirical Hamiltonians so that they can accurately describe the potential energy surfaces for two distinct atom+alkane reactions. In addition to their demonstrated accuracy, these methods possess the attractive quality of being computationally inexpensive enough to afford extensive direct-dynamics trajectory studies. Our results on the dynamics of atom+alkane hydrogen-abstraction reactions have shown good agreement with experimental metrics that are as diverse as product velocity distributions, excitation functions, angular distributions and rovibrational state distributions for diatomic products of the abstraction. We have demonstrated that our reparametrized Hamiltonians are suitable for investigating gas-phase reactions with up to 15 (5 heavy) atoms and that they are appropriate for studying reactions beyond the gas phase, especially gas/surface reactions. By employing our semiempirical methods within a quantum-mechanics/molecular-mechanics hybrid scheme we are able to examine hydrogen-abstraction reactions of fluorine atoms with alkanethiolate self-assembled monolayers. Our simulations reproduce the general trends of experimental results for the cousin F+squalane reaction. Our simulations also probe the role that secondary collisions play in determining the final internal and translational energy of the product HF molecules. For instance, we determined that very few interactions with the SAM surface were required to cool rotational and translational modes of the HF product, while its vibrational energy remains unchanged on the time scale that HF molecules trap on the SAM surface. Moving beyond the gas/organic surface interface, we have also performed molecular-dynamics simulations of thin water films confined between hydrophobic SAM surfaces. These simulations illuminated the structural and dynamics behavior induced in the water films by confinement in hydrophobic environments. While most effects of the surface do not penetrate deep into the water layers we have noted that enhanced lateral diffusion of water molecules can persist in these films with > 1 nm length scales. We have elucidated a possible mechanistic precursor for the attractive forces seen in experimental measurement of the hydrophobic effect. / Ph. D.

Proton exchange membrane fuel cells (PEMFCs) have been extensively studied as clean, sustainable and efficient power sources for electric vehicles, and portable and residential power sources. As one of the key components in PEMFC system, proton exchange membranes (PEMs) act as the electrolyte that transfers protons from the anode to the cathode. The state-of-art commercial PEM materials are typically based on perfluorinated sulfonic acid containing ionomers (PFSAs), represented by DuPont's Nafion®. Despite their good chemical stability and proton conductivity at high relative humidity (RH) and low temperature, several major drawbacks have been observed on PFSAs, such as high cost, high fuel permeability, insufficient thermo-mechanical properties above 80°C, and low proton conductivity at low RH levels. Therefore the challenge lies in developing alternative PEMs which feature associated ionic domains at low hydration levels. Nanophase separated hydrophilic-hydrophobic block copolymer ionomers are believed to be desirable for this purpose Three series of hydrophobic/hydrophillic, partially fluorinated/sulfonated multiblock copolymers were synthesized and characterized in this thesis. The hydrophilic blocks were based upon the nucleophilic step polymerization of 3, 3′-disulfonated, 4, 4′-dichlorodiphenyl sulfone (SDCDPS) with an excess 4, 4′-biphenol (BP) to afford phenoxide endgroups. The partially fluorinated hydrophobic blocks were largely based on 4, 4′-hexafluoroisopropylidenediphenol (6F-BPA) and various difluoro monomers (excess). These copolymers were obtained through moderate temperature (~130-150°C) coupling reactions, which minimize the ether-ether interchanges between hydrophobic and hydrophilic telechelic oligomers via a nucleophilic aromatic substitution mechanism. The copolymers were obtained in high molecular weights and were solvent cast into tough membranes, which had nanophase separated hydrophilic and hydrophobic regions. The performance and structure-property relationships of these materials were studied and compared to random copolymer systems. NMR results supported that the multiblock sequence had been achieved. They displayed superior proton conductivity, due to ionic, proton conducting channels formed through the self-assembly of the sulfonated blocks. The nano-phase separated morphologies of the copolymer membranes were studied and confirmed by transmission electron microscopy (TEM) and small angle X-ray scattering (SAXS). Through control of a variety of parameters, including ion exchange capacity and sequence lengths, performances as high, or even higher than those of the state-of-the-art PEM, Nafion®, were achieved. Another series of semi-crystalline hydrophobic poly(ether ether ketone)-hydrophilic sulfonated poly(arylene ether sulfone) (PEEK-BPSH100) multiblock copolymers was first synthesized and characterized. However due to their semi-crystalline structure, PEEK blocks are insoluble in most organic solvents at relatively low reaction temperatures, which prevents the coupling reaction between PEEK and BPS100. In order to facilitate the synthesis and processing, removable bulky ketimine was introduced to synthesize amorphous pre-oligomers poly(ether ether ketimine) (PEEKt). The synthetic procedure first involves the synthesis of hydrophobic poly(ether ether ketimine)-hydrophilic sulfonated poly(arylene ether sulfone) (PEEKt-BPS100) multiblock pre-copolymers via coupling reactions between phenoxide terminated hydrophilic BPS100 and fluorine terminated hydrophobic PEEKt blocks. The membranes cast from PEEKt-BPS100 were boiled in 0.5M sulfuric acid water solution to hydrolyze the amorphous PEEKt blocks to semi-crystalline PEEK blocks and acidify BPS100 blocks to BPSH100 blocks simultaneously. FT-IR spectra clearly showed the successful hydrolysis and acidification. The proton conductivity, water uptake and other membrane properties of the acidified semi-crystalline PEEK-BPSH100 membranes were then evaluated and compared with those of the state-of-the-art PEM, Nafion®. / Ph. D.

La necesidad de polímeros bioestables para fabricación de implantes protésicos queda patente, entre otros indicadores, por la proliferación de dispositivos actualmente comercializados. La caracterización físico-química así como la respuesta biológica de un conjunto de materiales poliméricos bioestables es el objetivo último de esta tesis. En este trabajo se han sintetizado diferentes materiales poliméricos de la familia de los acrilatos y metacrilatos variando sutilmente sus características superficiales, como el grado de hidrofilia o la distribución de cargas eléctricas. El procedimiento consistió en la copolimerización via radical de acrilato de etilo, EA, acrilato de 2-hidroxietilo, HEA, y ácido metacrílico, MAAc. Se ha caracterizado los materiales en estado seco y en presencia de diferentes contenidos de agua mediante calorimetría diferencial de barrido, DSC, análisis dinámico-mecánico, DMA, microscopía de fuerza atómica, AFM, análisis dieléctrico, DRS, contenido de agua en equilibrio, EWC, y energía superficial, SE, persiguiendo el objetivo de dilucidar si el agua es capaz de inducir cambios conformacionales en las cadenas poliméricas que den lugar a una separación de fases. Sobre los materiales en forma de scaffold poroso con poros esféricos interconectados se ha cultivado fibroblastos y endoteliales. La compatibilidad de las células endoteliales se midió en términos de viabilidad celular y la adecuada diferenciación endotelial y su funcionamiento. Se han realizado cultivos de células endoteliales humanas primarias, HUVEC, y se ha determinado si su morfología y función se vio afectada por el material. Se examinó la adhesión y proliferación de las mismas, así como un marcador importante de activación endotelial, la E-selectina. Se evaluó si se mantuvieron los fenotipos endoteliales normales y sus funciones observadas in vivo mediante análisis de los contactos célula-célula y la regulación de la expresión génica del marcador de activación E-selectina cuando se añadió un estímulo (LPS). Además, como posible aplicación de estos materiales en una prótesis de córnea artificial, y dado que los fibroblastos del estroma de la córnea (es decir, los queratocitos) son de relevancia en la cicatrización de la córnea se determinó cómo afectaba la hidrofilicidad del substrato a la adhesión celular de la línea de fibroblastos humanos MRC-5, como modelo celular para estudiar la disposición del citoesqueleto tras la adhesión a los diferentes soportes mediante la detección de F-actina. Asimismo, se ha sembrado células epiteliales evaluando su comportamiento/funcionamiento celular ya que uno de los requisitos esenciales para que un implante de queratoprótesis tenga éxito es que se cree y mantenga una capa de células epiteliales que impidan entrar a las bacterias al interior del ojo y permita la difusión la capa lagrimal de manera estable en el tiempo. Así, se han analizado parámetros celulares como adhesión, proliferación y viabilidad de una línea de células epiteliales de conjuntiva humana, NHC, cultivada sobre substratos poliméricos con diferentes grados de hidrofilia y cargas eléctricas superficiales buscando qué grado de hidrofilicidad permite la epitelización del substrato y podría darle al material flexibilidad y la hidrofilicidad necesaria para un mejor contacto con los párpados y lágrima. Los resultados obtenidos se han correlacionado con la adsorción y conformación de una proteína de la matriz extracelular, la fibronectina. / Campillo Fernández, AJ. (2014). Polímeros bioestables para fabricación de implantes protésicos: caracterización físico-química y respuesta biológica in vitro [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/44232 / Premios Extraordinarios de tesis doctorales

This thesis presents investigations on air-cooled air gap membrane distillation for desalination and the application of radial waveguides based on total internal reflection for solar thermal desalination. Using an air-cooled design for an air gap membrane distillation (AGMD) process may result in significantly lower energy requirements for desalination. Experiments were conducted on AGMD module to study the effect of air gap, support mesh conductivity and hydrophobicity, condensing surface hydrophobicity. A novel modular design was used in which modules could be used in a series configuration to increase the flux value for the distillate. The output from the series configuration was found to have about three times the production from a single pass water-cooled system with the same temperature difference between the saline and clear water streams. The results also indicated that the mesh conductivity had a favorable effect on the flux value whereas the hydrophobicity of the mesh had no significant effect. The hydrophobicity of the condensing surface was favorable on two accounts: first, it led to an increase in the flux of the distillate at temperatures below 60 °C and second, the temperature difference of the saline feed when it enters and leaves the module is lower which can lead to energy savings and higher yields when used in a series configuration. The second part of the thesis considers use of low-cost radial waveguides to collect and concentrate solar energy for use in thermal desalination processes. The optical-waveguide-based solar energy concentrators are based on total internal reflection and minimize/eliminate moving parts, tracking structures and cost. The use of optical waveguides for thermal desalination is explored using an analytical closed-form solution for the coupled optical and thermal transport of solar irradiation through a radial planar waveguide concentrator integrated with a central receiver. The analytical model is verified against and supported by computational optical ray tracing simulations. The effects of various design and operating parameters are systematically investigated on the system performance, which is quantified in terms of net thermal power delivered, aperture area required and collection efficiency. Design constraints like thermal stress, maximum continuous operation temperature and structural constraints have been considered to identify realistic waveguide configurations which are suitable for real world applications. The study provides realistic estimates for the performance achievable with radial planar waveguide concentrator-receiver configuration. In addition to this, a cost analysis has been conducted to determine the preferred design configurations that minimize the cost per unit area of the planar waveguide concentrator coupled to the receiver. Considering applications to thermal desalination which is a low temperature application, optimal design configuration of waveguide concentrator-receiver system is identified that result in the minimum levelized cost of power (LCOP). / Master of Science / Depleting reserves of fresh water and deteriorating quality of naturally occurring water reserves has led to growing scarcity of potable water. The severity of this water crisis has made it necessary to explore other sources of potable water. The abundance of seawater makes it rewarding to explore desalination of seawater as a source of potable water. This thesis presents investigations on the use of air-cooled air gap membrane distillation (AGMD), which is a filtration technique which can be used to remove salt and other impurities from seawater, for desalination. Radial waveguide can be used for concentrating solar energy on a smaller surface, which in turn can be used to raise the temperature of a fluid passing through that surface. These waveguides can be used to heat up the seawater for the solar thermal desalination process. Using an air-cooled design for an air gap membrane distillation process may result in significantly lower energy requirements for desalination. A novel modular design was used in which modules could be used in a series configuration to increase the output of the potable water. The output from the series configuration was found to be about three times the output from a single pass water-cooled system with the same temperature difference between the saline and clear water streams. The second part of the thesis considers use of low-cost radial waveguides to collect and concentrate solar energy for use in thermal desalination processes. The optical-waveguide-based solar energy concentrators minimize/eliminate moving parts, tracking structures and cost. The use of optical waveguides for thermal desalination is explored using an analytical closed-form solution for the coupled optical and thermal transport of solar irradiation through a radial planar waveguide concentrator integrated with a central receiver. The analytical model is verified against and supported by computational optical ray tracing simulations. The effects of various design and operating parameters are systematically investigated on the system performance. Design constraints like thermal stress, maximum continuous operation temperature and structural v constraints have been considered to identify realistic waveguide configurations which are suitable for real world applications. In addition to this, a cost analysis has been conducted to determine the preferred design configurations that minimize the cost per unit area of the planar waveguide concentrator coupled to the receiver. Considering applications to thermal desalination which is a low temperature application, optimal design configuration of waveguide concentrator-receiver system is identified that result in the minimum levelized cost of power (LCOP).

Yes / Protein quality control mechanisms are essential for cell health and involve delivery of proteins to specific cellular compartments for recycling or degradation. In particular, stray hydrophobic proteins are captured in the aqueous cytosol by a co-chaperone, the small glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA), which facilitates the correct targeting of tail-anchored membrane proteins, as well as the sorting of membrane and secretory proteins that mislocalize to the cytosol and endoplasmic reticulum-associated degradation. Full-length SGTA has an unusual elongated dimeric structure that has, until now, evaded detailed structural analysis. The Cterminal region of SGTA plays a key role in binding a broad range of hydrophobic substrates, yet in contrast to the well-characterized N-terminal and TPR domains, there is a lack of structural information on the C-terminal domain. In this study, we present new insights into the conformation and organization of distinct domains of SGTA and show that the C-terminal domain possesses a conserved region essential for substrate processing in vivo. We show that the C-terminal domain region is characterized by α-helical propensity and an intrinsic ability to dimerize independently of the N-terminal domain. Based on the properties of different regions of SGTA that are revealed using cell biology, NMR, SAXS, Native MS, and EPR, we observe that its C-terminal domain can dimerize in the full-length protein and propose that this reflects a closed conformation of the substrate-binding domain. Our results provide novel insights into the structural complexity of SGTA and provide a new basis for mechanistic studies of substrate binding and release at the C-terminal region. / MRC New Investigator Research Grant: G0900936; BBSRC grants: BB/L006952/1 and BB/L006510/1; BBSRC grant: BB/N006267/1; Wellcome Trust Investigator Award in Science: 204957/Z/16/Z; BBSRC grant: BB/J014567/1