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Kulkarni, Chandrashekhar V. "In-Cubo Crystallization of Membrane Proteins." Thesis, Imperial College London, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.508495.
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Svang-Ariyaskul, Apichit. "Chiral separation using hybrid of preferential crystallization moderated by a membrane barrier." Diss., Georgia Institute of Technology, 2010. http://hdl.handle.net/1853/33909.
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The major innovation of this work is an establishment of a novel chiral separation process using preferential crystallization coupled with a membrane barrier. This hybrid process was proved to be promising from a significant increase in product yield and purity compared to existing chiral separation processes. This work sets up a process design platform to extend the use of this hybrid process to a separation of other mixtures. This novel process especially is a promising alternative for chiral separation of pharmaceutical compounds which include more than fifty percent of approved drugs world-wide. A better performance chiral separation technique contributes to cut the operating cost and to reduce the price of chiral drugs.
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Liu, Wei. "Membrane protein crystallization in the lipid cubic phase testing hypotheses relating to reconstitution /." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1196274127.
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Kalakech, Carla. "Membrane crystallization by pervaporation for paracetamol production and polymorphism control." Electronic Thesis or Diss., Lyon 1, 2024. http://www.theses.fr/2024LYO10300.
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La cristallisation est une opération unitaire cruciale en ingénierie des procédés, largement utilisée dans des secteurs tels que la chimie, la pharmacie et l'électronique. Malgré son importance, les méthodes actuelles de cristallisation rencontrent diverses limitations, affectant la qualité du produit final, la constance de la production et le contrôle de la forme polymorphique. Récemment, les procédés membranaires sont apparus comme une approche prometteuse pour améliorer le contrôle de la cristallisation, en particulier la pervaporation, qui utilise une membrane dense ou composite sélective. Appliquée à la cristallisation, cette méthode permet de retirer le solvant d'un mélange solvant/antisolvant, créant ainsi la sursaturation nécessaire pour initier la cristallisation. L'objectif principal de cette thèse est de contrôler le polymorphisme du paracétamol par la cristallisation sélective et la stabilisation de la forme métastable II en utilisant la cristallisation membranaire par pervaporation. La forme II du paracétamol est privilégiée pour sa haute solubilité et compressibilité par rapport à la forme I, plus stable, mais son instabilité pendant la cristallisation, notamment sa transformation rapide vers la forme I, pose des défis importants. Pour cela, la première étude a consisté à produire la forme II en petites quantités grâce à des cycles de chauffage et de refroidissement utilisant la calorimétrie différentielle à balayage (DSC), suivie de sa caractérisation à l’aide de diverses techniques analytiques. Un modèle prédictif de polymorphisme par spectroscopie infrarouge proche (FT-NIR) hors ligne, combiné à une technique chimiométrique telle que l'analyse discriminante par moindres carrés partiels (PLS-DA), a été développé et validé lors d'une cristallisation par refroidissement en batch avec ensemencement. La cristallisation sélective et la stabilisation de la forme II en cristallisation batch ensemencée ont été optimisées en contrôlant le niveau de sursaturation et la température de fonctionnement. Les résultats ont montré que maintenir une température basse (5-10°C) et des niveaux de sursaturation faibles (β = 1,25) prolongeait la stabilité de la forme II jusqu'à 30 min. Cependant, l'augmentation de la masse de semences n'a pas amélioré la stabilité, car le stress mécanique pendant la récupération des semences générait des impuretés de la forme I. L'application de la cristallisation membranaire par pervaporation pour le contrôle du polymorphisme du paracétamol a révélé que la forme I cristallisait lors des opérations non ensemencées à différents débits de perméation et rapports surface membranaire/volume d’alimentation (S/Vc), tandis que la stabilité de la forme II était d'environ 15 min en solution sursaturée, et que sa transition était ralentie à au moins 49 min pendant les opérations de cristallisation membranaire ensemencée avec un niveau de sursaturation βs=1,1, une température de fonctionnement de 5°C et une température de semis de 7,4°C. Cependant, comparée à la cristallisation batch ensemencée conventionnelle, la stabilité de la forme II n'a pas été améliorée, ce qui suggère une préférence pour la nucléation hétérogène de la forme I qui a accéléré la transition de la forme II. La stabilisation de la forme II s'est avérée dépendre principalement des températures de fonctionnement et de semence plutôt que du débit de perméation. D'autre part, la cristallisation membranaire par pervaporation a montré des rendements de cristallisation plus élevés que la cristallisation batch par refroidissement conventionnelle. L'augmentation du rapport S/Vc et du débit de perméation a conduit à une légère amélioration de la concentration en antisolvant d'environ 5 %, ce qui n'a pas affecté le polymorphisme du paracétamol mais a augmenté le rendement de cristallisation à 43 % sans détection de vieillissement notable de la membrane ni de colmatage irréversible après 13 utilisations de la membraneCrystallization is a crucial unit operation in process engineering, widely utilized across industries such as chemical, pharmaceutical, and electronics. Despite its importance, current crystallization methods encounter various limitations, impacting the final product quality, production consistency, and the control over the polymorphic form. Recently, membrane processes have emerged as a promising approach to enhance crystallization control, particularly pervaporation, which employs a dense selective membrane. Applied to crystallization, this method allows for the removal of the solvent from a solvent/antisolvent mixture, creating the supersaturation needed for crystallization initiation. The primary goal of this PhD work is to control paracetamol polymorphism through the selective crystallization and stabilization of the metastable form II using membrane crystallization by pervaporation. Paracetamol form II is favored for its high solubility and compressibility compared to the most stable form I, but its instability during crystallization, particularly its rapid solvent-mediated phase transformation (SMPT) to form I, poses significant challenges. To do so, the initial investigation involved producing form II in small quantities through heating and cooling cycles using differential scanning calorimetry (DSC), followed by its characterization using numerous analytical techniques. An offline Fourier transform near infrared spectroscopy (FT-NIR) polymorphism prediction model supported by a chemometric technique like Partial Least Squares Discriminant Analysis (PLS-DA) was developed and validated during seeded batch cooling crystallization. The selective crystallization and stabilization of form II in seeded batch cooling crystallization was optimized by controlling the supersaturation level and the operational temperature. Results demonstrated that maintaining a low temperature (5-10°C) and low supersaturation levels (β = 1.25) extended form II stability for up to 30 min. However, increasing the seed mass did not improve stability, as mechanical stress during seed recuperation generated form I impurities. The application of membrane crystallization by pervaporation for paracetamol polymorphism control revealed that form I crystallized during unseeded operations at different permeation rates and membrane surface to feed volume S/Vc ratios whereas form II stability was around 15 min in supersaturated solution and the SMPT was slowed to at least 49 min during seeded membrane crystallization operations at a supersaturation level of βs=1.1, an operational temperature of 5°C and a seeding temperature of 7.4°C. However, when compared to conventional seeded batch cooling crystallization, form II stability was not improved suggesting a preference form I heterogeneous nucleation which accelerated form II SMPT. The stabilization of form II has been proven to be mainly dependent on the operational and seeding temperatures rather than the permeation rate. On the other hand, membrane crystallization by pervaporation exhibited higher crystallization yields than conventional batch cooling crystallization. The increase of the membrane surface to feed volume (S/Vc) ratio and the permeation rate led to a slight improvement in the antisolvent concentration of almost 5%, which did not affect paracetamol polymorphism but increased the crystallization yield to 43% with no noticeable membrane ageing and irreversible fouling detection for 13 membrane usages
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Clogston, Jeffrey. "Applications of the lipidic cubic phase from controlled release and uptake to in meso crystallization of membrane proteins /." Connect to resource, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1117564268.
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Thesis (Ph.D.)--Ohio State University, 2005.Title from first page of PDF file. Document formatted into pages; contains xxii, 352 p.; also includes graphics. Includes bibliographical references (p. 346-352). Available online via OhioLINK's ETD Center
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McGregor, Clare-Louise. "Development of lipopeptide detergents for the solubilization and crystallization of membrane proteins." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0019/MQ54093.pdf.
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Johnson, Jennifer Leigh. "The quest for a general co-crystallization strategy for macromolecules: lessons on the use of chaperones for membrane protein crystallization." Diss., Georgia Institute of Technology, 2015. http://hdl.handle.net/1853/53886.
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Crystallization is often a major bottleneck to macromolecular structure determination. This is particularly true for membrane proteins, which have hydrophobic surfaces that cannot readily form crystal contacts. Of the roughly 109,000 protein structures in the PDB, only about 539 represent unique membrane proteins, despite immense interest in membrane proteins from both a biological and therapeutic standpoint. Membrane protein crystallization has been facilitated by the development of new detergents, lipidic cubic phase methods, soluble protein chimeras, and non-covalent protein complexes. The design process of protein fusion constructs and non-covalent antibody fragments specific for each target membrane protein, however, is costly and time-consuming. An improved, more general method of membrane protein co-crystallization is needed. This dissertation details the development of two approaches for cost-effective non-covalent crystallization chaperones: (1) Engineered hypercrystallizable Fab antibody fragment with high affinity for EYMPME (EE epitope), which form complexes with EE-tagged soluble and membrane proteins. (2) Engineered monomeric streptavidin (mSA2) for complexation with biotinylated membrane proteins. Both methods are generalizable through insertion of a short epitope into a surface-exposed loop of a membrane protein by site directed mutagenesis. Crystallization trials of representative chaperone-membrane protein complexes and possible difficulties with the approach are discussed.
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Liu, Wei. "Membrane protein crystallization in the lipid cubic phase: testing hypotheses relating to reconsitution." The Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=osu1196274127.
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Misquitta, Yohann Reynold. "The rational design of monoacylglycerols for use as matrices for the crystallization of membrane proteins." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1141940412.
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Rodríguez, Banqueri Arturo. "A random approach to stabilize a membrane transport protein for crystallization studies / Un enfoque aleatorio para estabilizar un transportador de membrana para estudios de cristalización." Doctoral thesis, Universitat de Barcelona, 2013. http://hdl.handle.net/10803/109040.
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X-ray crystallography is, now at days, one of the most powerful techniques to study proteins at the atomic level. Unfortunately, obtaining high quality crystals of membrane proteins for x-ray diffraction is a difficult task due to the hydrophobic nature of these proteins. The low stability in solution of these proteins and their tendency to form aggregates are the biggest problems during crystallization studies. One of the most common strategies to overcome these problems consists on working with functional mutants of these proteins. It has been reported that single point mutations of key residues (normally within transmembrane segments) leads to a remarkable increase in the stability of some membrane proteins after detergent solubilization and extraction from the membrane. In addition, a single mutation can stabilize a specific conformer of the protein, decreasing its heterogeneity in solution. Despite this, predicting what mutations are going to improve the stabilization of a protein is virtually impossible.
The main purpose of this thesis is to build up a medium-high throughput experimental protocol with the objective to generate and characterize random mutants of a membrane protein with more stability in detergent-solubilized solution and, therefore with a better probability to crystallize. The combination of random mutagenesis with rapid and sensitive screening protocols of protein expression and stability seems to be the best approach for this goal. The use of the green fluorescent protein (GFP) as reporter has enormously facilitated the studies of expression, purification and stability of a membrane protein. Also, with the aim of minimizing undesired effects of full-length GFP, we optimized an assay based on a split GFP to build and characterized the random mutants library.
Specifically we focus on SteT, a Bacillus subtillis transporter that exchanges L-threonine by L-serine. SteT is an excellent prokaryotic model (30% of amino acid identity) of the mammalian L-amino acid transporter (LAT) family. Genetic mutations of some LATs are the direct cause of two types of aminoaciduries. Moreover, a member of this family, LAT1, is overexpressed in tumor cells, although the physiological role of this is still unknown. Unfortunately, SteT wild type solubility and stability in detergent solutions is very low and completely incompatible with crystallization tests.
Our results suggest that random mutagenesis combined with the GFP split assay, appears to be an excellent strategy to build robustness in membrane proteins for structural studies. So far, using this strategy we found a mutant of SteT that currently is undergoing for crystallization screenings to study the structure and mechanism of mammalian LATs.La cristalografía de rayos X es, hoy en día, una de las técnicas más potentes para el estudio de las proteínas a nivel atómico. Desafortunadamente, la obtención de cristales de alta calidad de proteínas de membrana para la difracción de rayos X es un desafío debido a la naturaleza hidrofóbica de estas proteínas. La baja estabilidad en solución de estas proteínas y su tendencia a formar agregados son los mayores problemas durante los estudios de cristalización. Una de las estrategias más comunes para superar estos obstáculos consiste en trabajar con mutantes funcionales de estas proteínas. Se han publicado estudios sobre mutaciones en residuos clave en proteínas de membrana (normalmente dentro de los segmentos transmembrana) que conducen a un notable incremento de la estabilidad en solución, previa extracción de la membrana y solubilización en detergente. Además, una sola mutación puede estabilizar un confórmero específico de una proteína, disminuyendo su heterogeneidad en solución. A pesar de esto, predecir qué mutaciones van a mejorar la estabilidad de una proteína es prácticamente imposible. El principal objetivo de esta tesis es la construcción de un protocolo de alto rendimiento experimental con el objetivo de generar y caracterizar mutantes aleatorios de una proteína de membrana que presenten una estabilidad adecuada después de solubilizar la proteína en detergente y, por lo tanto, con mejores garantías de cristalizar. Para conseguir estos objetivos hemos combinado técnicas de mutaciones aleatorias con métodos de cribaje rápidos y sensibles. En este sentido, el uso de la proteína fluorescente verde (GFP) ha facilitado enormemente los estudios de expresión y purificación de proteínas de membrana. Con el objetivo de minimizar los efectos no deseados de la GFP, se creó y optimizó un ensayo basado en la complementación de la GFP (GFP split system) con un fin doble: seleccionar y caracterizar los componentes de la librería de mutantes aleatorios. Este protocolo se ha puesto a punto con SteT, un intercambiador de L-serina por L-treonina de Bacillus subtilis. SteT es un excelente modelo procariota (30% de identidad de aminoácidos) de la familia de transportadores de mamíferos de amino ácidos L (LAT). Mutaciones congénitas de algunos LATs son la causa directa de dos tipos de aminoacidurias. Además, un miembro de esta familia, LAT1, se sobreexpresa en células tumorales, aunque el papel fisiológico es aún desconocido. Desafortunadamente, SteT tiene una muy baja solubilidad junto a un gran inestabilidad en detergente, propiedades totalmente incompatibles con estudios de cristalización. Nuestros resultados indican que la mutagénesis aleatoria combinada con el ensayo basado en el “GFP split system”, es una estrategia excelente para aumentar la estabilidad de proteínas de membrana en estudios estructurales. Utilizando esta metodología hemos encontrado un mutante de SteT que actualmente está siendo cristalizado. Estos estudios serán clave para conocer mejor la estructura y el mecanismo de la familia de transportadores de mamífero LAT.
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Moreira, Ramon Heleno. "Desenvolvimento de um processo de reúso do efluente de refinaria baseado em sistema de osmose reversa combinado com precipitação." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/3/3137/tde-14032017-141115/.
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O processo de osmose reversa produz um permeado com elevada pureza, pois a membrana é praticamente uma barreira absoluta para partículas sólidas, coloides, íons e orgânicos. No entanto, alguns parâmetros devem ser controlados para proteger a membrana de oxidações por cloro, incrustações por partículas metálicas, compostos orgânicos e por sais concentrados acima ou próximos da supersaturação. Este processo é consolidado em aplicações de dessalinização e desmineralização para geração de água de nobre, porém requer maior avanço conceitual e prático em tratamento de efluentes em função das características particulares em termos de contaminantes para cada tipo de efluente. A presença de sais de metais alcalinos terrosos pode formar inscrustação (scaling) por precipitados indesejados, tais como: sulfato de bário (BaSO4), sulfato de estrôncio (SrSO4), carbonato de estrôncio (SrCO3), carbonato de cálcio (CaCO3) e hidróxido de magnésio (Mg(OH)2), além de sílica (SiO2). Para redução do potencial de scaling, podem ser aplicados na água compostos anti-incrustantes, bem como soluções para ajuste de pH para dissolução de cristais de certos compostos incrustantes, porém estas aplicações são dependentes do estudo de impacto nos processos e equipamentos (JANG et al., 2002). Este estudo foi realizado com o efluente de uma refinaria no Brasil após ser tratado pela ETAC (Estação de tratamento de águas contaminadas). Este efluente foi submetido a um novo processo de remoção de sais dissolvidos baseado em sistema de osmose reversa integrado com precipitação. Foram também analisados os índices de saturação dos sais dissolvidos nas correntes de alimentação e de rejeito do sistema de osmose reversa, de modo a verificar a incrustação de sais nas membranas e dimensionar adequadamente a unidade sem a adição de anti-incrustantes. Também foi verificado o atendimento da qualidade da água de permeado do sistema de osmose reversa com os requisitos de qualidade de água recomendados pela ASME (American Society of Mechanical Engineers) para caldeiras a vapor. Neste estudo foi identificado que, mediante uma dosagem que eleve o pH da solução para o valor de 9,5 no precipitador, é possível precipitar 90% dois íons presentes e que em valores de pH maiores (maiores dosagens) essa eficiência de precipitação diminui. Nesta condição, o consumo de reagentes Na2CO3 e Na2SO4 foi de 3,23 mmol e para o MgCl2 foi de 0,16 mmol por litro de efluente bruto a ser tratado. O pré-tratamento de precipitação antes do sistema de osmose reversa pode reduzir a precipitação de sais superfície das membranas. Nas condições aplicadas neste estudo, foram atingidas as reduções de sais de barita em 96%,de sílica em 91,6% e complexos de apatita em 99,97%. Esta melhoria reflete diretamente na vida útil das membranas visto que a precipitação de barita e de sílica são os maiores complicadores de remoção nas membranas mesmo que seja realizada a limpeza química. Para avaliação do custo de investimento e de consumo de químicos, também foi desenvolvido um estudo de implantação de uma unidade de capacidade de tratamento de 150 m3/h. Esta vazão foi definida como referência com base no estudo de produção de efluentes pelas refinarias no Brasil. O custo de implantação e de operação foi aplicado na avaliação de três cenários distintos que poderiam haver a necessidade de implantação do sistema de reúso na refinaria, tais como: implantação em uma refinaria nova em local com baixa disponibilidade de água para captação ou com grande variação sazonal do aquífero, expansão da refinaria sem alterar as plantas com sistema de tratamento água e tratamento de água desmineralizada existentes e de expansão da refinaria com a possibilidade de implantação de novos sistemas de tratamento de água e tratamento de água desmineralizada. Os resultados apresentam que os cenários estudados foram favoráveis a iniciativa de implantar o sistema de reúso nas refinarias.The reverse osmosis process produces a permeate with high purity because the membrane is almost an absolute barrier to solid particles, colloids, and organic ions. However, some parameters must be controlled to protect the membrane from oxidation by chlorine, inlays for metal particles, organic compounds and salts above concentrated or near supersaturation. This process is consolidated in desalination and demineralization applications for boiler water generation, but requires more conceptual and practical advances in wastewater treatment according to the particular characteristics in terms of contaminants for each type of wastewater. The presence of alkaline earth metal salts may form unwanted precipitates such as barium sulfate (BaSO4), strontium sulfate (SrSO4), strontium carbonate (SrCO3), calcium carbonate (CaCO3) and magnesium hydroxide (Mg(OH)2) and silica (SiO2). This phenomenon of fouling salts is also known as \"scaling\". To reduce the scaling potential is usual to apply anti-fouling compounds, as well as pH adjustment to dissolve crystals of certain fouling compounds, but these applications are dependent upon impact study on processes and equipment (Jang et al., 2002). This study was conducted with the wastewater of a refinery in Brazil after being treated by ETAC (Wastewater Treatment Plant). The wastewater was subjected to a new process for removal of dissolved salts based on reverse osmosis system integrated with precipitation. The saturation index of the dissolved salt was also analyzed in the feed and concentrate streams of the reverse osmosis system, in order to verify salts fouling the membranes and properly sizing the unit without the addition of antifoulants. It was also checked the water quality in the permeate of the reverse osmosis with the quality requirements of water recommended by the ASME (American Society of Mechanical Engineers) for steam boilers. In this study it was identified through a dose that raise the pH to a value of 9.5, that it is possible to precipitate 90% of the ions dissolved in the wastewater and at higher pH (higher doses) this precipitation efficiency decreases. In this condition, the consumption of reagents Na2CO3 and Na2SO4 was 3.23 mmol and MgCl2 was 0.16 mmol per liter of raw wastewater to be treated. The pre-treatment with precipitation before the reverse osmosis system can reduce the precipitation on the surface of the membranes of barite salts in 96%, 91.6% to silica, and for complex apatite mineral of 99.97%. This improvement directly reflects the life-time of the membranes since the precipitation of barite and silica are the most complicating particles to the membranes even if chemical cleaning is applied. To evaluate the cost of investment and consumption of chemicals, it has also developed a study of deployment of a unit treatment capacity of 150 m3/h. This flow was defined as a reference based on the study of production of wastewater by refineries in Brazil. The cost of deployment and operation was applied in the evaluation of three different scenarios that could be a need to reuse the water in the refinery, such as: implementation of a new refinery in location with low water availability for pickup or large variation seasonal aquifer, refinery expansion without changing the plants water treatment system, and demineralized water treatment and existing refinery expansion with the possibility of developing new water treatment systems and treatment of demineralized water. The results show that the scenarios studied were favorable to initiative of introducing the system of reuse in refineries.
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Smith, Everhardus Johannes. "A numerical analysis of the hydrodynamic mixing characteristics of a rectangular versus a cylindrical mixing crystallizer tank for a membrane distillation apparatus." Thesis, Cape Peninsula University of Technology, 2018. http://hdl.handle.net/20.500.11838/2827.
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Thesis (MTech (Mechanical Engineering))--Cape Peninsula University of Technology, 2018.A membrane distillation crystallization (MDC) experimental setup was designed, constructed and commissioned with rectangular mixing crystallizer tanks. The advantages and disadvantages of a rectangular mixing tank are compared to the traditional cylindrical mixing tank with baffling by means of a computational fluid dynamic (CFD) analysis in Ansys Fluent. The effect of tank configuration and geometry on the hydrodynamic and mixing characteristics for efficient momentum, solid suspension, heat and mass transfer were investigated. The hydrodynamic conditions in a crystallizer-mixing tank determine the quality of fluid mixing essential for optimal crystallization. Forty-five degree pitched blade turbines (PBT) were used to provide the agitation in the stainless steel rectangular jacketed tanks. Clear polycarbonate replicas of the rectangular tanks were manufactured to visually observe the mixing process in the tanks. Silica particles were used to represent the calcium carbonate crystals in the experiment. The data gathered from these experiments showed that the tanks should be operated between 600 to 750 rpm in the CFD simulations to simulate partial to complete suspension. In the numerical simulations a rectangular tank was compared to a cylindrical tank with baffling of the same volume. The partial differential equations solved in the numerical simulation were the conservation of mass (continuity), conservation of momentum and additional turbulence equations. In order to solve the turbulent fluid flow characteristics, the industry standard two-equation model, namely the K-epsilon model was used. This model was refined by the addition of the Wen-Yu drag model, the Simonin turbulent dissipation and the Simonin et al. turbulence interaction models. The RANS based RNG (k-ε), derived from the instantaneous Navier-Stokes equation was selected as the preferred model to analyse the hydrodynamic flow fields in the tanks. The 3D sliding mesh method was used to compute a time accurate solution. The Eulerian-granular multiphase model was used to predict the degree of solids suspension in the tanks. The efficiency of mixing within the tank was measured by the tank’s ability to keep the crystals in suspension and preventing any particle from settling at the bottom for more than 1-2 second(s). The mixing tanks were initially loaded with 5% v/v, which equates to a loaded height of approximately 10 mm. The simulations were done with the use of the volume fraction function to visually observe the cloud height and gauge the homogeneity and distribution of the particulates within the fluid flow fields. The results from the experimental setup were compared to the CFD simulations to qualify the use of CFD simulations for the comparison of the geometrically different tanks. Lastly, the findings from the CFD simulations were used to compare the tanks and determine if the rectangular tank built for the MDC experiment perform satisfactorily to replace a standard cylindrical tank with baffling for this application.
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Clogston, Jeffrey. "Applications of the lepidic cubic phase: from controlled release and uptake to in meso crystallization of membrane proteins." The Ohio State University, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=osu1117564268.
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Kim, Laura Yaunhee. "Optimization of over-expression and purification of human leukotriene C4 synthase mutant R104A for structure-function studies by two-dimensional crystallization and electron crystallography." Thesis, Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/45820.
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Membrane proteins are involved in a number of disease pathologies and thus comprise a large number of drug targets. Determination of the high-resolution three-dimensional structure is essential for rational drug design, but several hurdles need to be overcome, primarily the over-expression and purification of said membrane proteins. Human leukotriene C4 synthase (hLTC4S), an 18 kDa integral membrane protein localized in the outer nuclear membrane of eosinophils and basophils, catalyzes the conjugation of LTA4 and reduced glutathione to produce LTC4. LTC4 and its metabolites LTD4 and LTE4 are the cysteinyl leukotrienes implicated in bronchoconstriction and inflammation pathways. The focus of my project involves optimizing the over-expression and purification of hLTC4S, which was heterologously expressed in Schizosaccharomyces pombe, purified by immobilized affinity chromatography, and finally "polished" with a buffer exchange step to remove excess co-purified lipids. The optimized protocol yielded ~1 mg of ~90% homogenous, pure protein per liter of cell culture. The finalized purified protein can then be used for further investigation of two-dimensional crystals by electron crystallography with the overall goal of structure determination.
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Li, Chia-Ling. "Preparation of poly(vinylidene fluoride) (PVDF) membrane by nonsolvent-induced phase separation and investigation into its formation mechanism." Thesis, Montpellier 2, 2010. http://www.theses.fr/2010MON20155.
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Cette thèse décrit comment la morphologie et le polymorphisme de membranes en fluorure de poly(vinylidène) (PVDF) préparées par séparation de phase induite par la vapeur d'eau (VIPS) et par un non-solvant liquide peuvent être ajustés par la température à laquelle le PVDF est dissous (Tdis) pour former la solution de coulée. Les résultats montrent que Tdis présente une transition, notée comme la température de dissolution critique (Tcri), à partir de laquelle la morphologie et le polymorphisme des membranes changent radicalement. Ce phénomène observé pour les trois solvants, N-methyl-2-pyrrolidone (NMP), dimethylacetamide (DMAc), and N,M-dimethylformamide (DMF), et les non-solvants (eau et une série d'alcools) utilisés dans cette étude peut être considéré comme général. La cristallisation a lieu avant la démixtion L-L quelle que soit Tdis. Pour une Tdis supérieure à Tcri, les membranes se présentent sous forme de nodules (cristallite forme beta) dont la taille décroît lorsque Tdis diminue. Ce domaine a été dénommé "à grossissement libre" car les chaînes de polymère peuvent cristalliser librement pendant la séparation de phase. Pour une Tdis inférieure à Tcri, des membranes avec une structure bi-continue (cristallite forme alpha) sont obtenues. Ce domaine a été appelé "à grossissement empêché" dans la mesure où la séparation de phase s'accompagne d'une gélification. Nous avons démontré que la morphologie et le polymorphisme cristallin des membranes de PVDF peuvent ainsi être contrôlés par la Tdis et la vitesse d'échange avec le non-solvant. Ces résultats sont interprétés en termes d'auto germination et de compétition entre gélification, cristallisation et démixtion L-LThis dissertation shows how the morphology and polymorphism of poly(vinylidene fluoride) (PVDF) membranes prepared by using vapor-induced phase separation (VIPS) and liquid-induced phase separation (LIPS) were tuned by varying the dissolution temperature at which PVDF was dissolved (Tdis) to form the casting solution. We observed a transition temperature denoted by critical dissolution temperature, Tcri, across which the morphology and polymorphism of membranes (obtained by VIPS) drastically changed. The phenomenon was considered as general, as a Tcri was observed for all the three solvents N-methyl-2-pyrrolidone (NMP), dimethylacetamide (DMAc), and N,N-dimethylformamide (DMF) and the non-solvents, water and a series of alcohols, used in the present study. No matter which Tdis we used, polymer crystallization occurred prior to the L-L demixing. With Tdis above Tcri, the prepared membranes were composed of nodules (mainly in beta crystalline form) and the size of polymer domains decreased as the Tdis decreased. Because the polymer chains could freely coarsen to a large domain during the phase separation, we called the system free coarsening. With Tdis below Tcri, membranes with lacy (bi-continuous) structure (mainly in alpha crystalline form) were obtained. Because the polymer solution gelled during the phase separation, we called the system hindered coarsening. It was proven that PVDF membrane morphology and crystalline polymorphs can be monitored by Tdis and the solvent-nonsolvent exchange rate. These results were discussed in terms of self-seeding effect and competition between the gelation, crystallisation and L-L demixing
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Carswell, Casey. "The Structural Characterization of Two Prokaryotic Membrane Proteins: CfrA and ELIC." Thesis, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31214.
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This thesis focuses on the structural and functional characterization of two integral membrane proteins; CfrA, an outer membrane TonB-dependent transporter (TBDT) from Campylobacter jejuni, and ELIC, a pentameric ligand-gated ion channel (pLGIC) from Erwinia Chrysanthemi. The spectroscopic characterization of CfrA revealed a fold consistent with the structural and biophysical properties observed for other TBDT. Both a homology model of CfrA and sequence alignments of CfrA with other ferric-enterobactin transporters suggested a unique mode of ligand binding, thus raising the possibility that C. jejuni can be specifically inhibited. To investigate the molecular determinates of binding to CfrA, I set out to crystallize CfrA. Hundreds of crystal trials led to crystals diffracting to 3.6 Å resolution, with a complete data set acquired at 5 Å resolution that led to a structural model of the CfrA β-barrel.
In the second part of this thesis, I reconstituted ELIC into model membranes in order to test the role of intramembrane aromatic interactions in ELIC gating and lipid sensing. ELIC was reconstituted into both asolectin (aso-ELIC) and 1-palmitoyl-2-oleoyl phosphatidylcholine (PC-ELIC), membranes that stabilize the homologous nicotinic acetylcholine receptor (nAChR) in functional coupled versus non-functional uncoupled conformations, respectively. In both membrane environments, ELIC exhibits a mixed α-helical and β-sheet secondary structure, with a thermal denaturation intermediate between those of the nAChR and the close prokaryotic homolog, GLIC, in similar membranes. The data suggest that although ELIC has a decreased propensity to adopt an uncoupled conformation relative to the nAChR, its ability to undergo cysteamine-induced channel gating is sensitive to its lipid environment. The decreased propensity to uncouple may reflect an increased level of aromatics at the interface between the transmembrane α-helices, M1, M3, and M4. To test this hypothesis further, the level or aromatic residues at the M1, M3, and M4 interface in both GLIC and ELIC were varied, and in both cases the levels of intramembrane aromatic interactions correlated with the efficiency of coupling binding to gating. The data provide further evidence for a role of intramembrane aromatics in channel gating and in dictating the propensity of pentameric ligand-gated ion channels to adopt an uncoupled conformation.
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Michaud, Maïté. "Contacteur membranaire innovant pour la cristallisation : application aux systèmes de type diffusion / réaction." Thesis, Lyon, 2019. http://www.theses.fr/2019LYSE1322.
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Les procédés membranaires sont considérés comme l’une des technologies de rupture les plus prometteuses pour les opérations de cristallisation/précipitation. Les matériaux les plus exploités à ce jour sont poreux mais leurs limitations en terme de bouchage de pores et de mouillage entravent le bon déploiement du procédé. L’utilisation de matériaux denses permettrait de s’affranchir de ce phénomène de colmatage des pores tout en conservant les bénéfices apportés par les procédés membranaires. Dans une première partie expérimentale, le composé modèle choisi, le BaCO3, est précipité dans un contacteur membranaire gaz-liquide et liquide-liquide, opéré dans les deux cas en conditions statiques. Cette configuration permet de s’affranchir de l’influence de l’hydrodynamique. Les interactions membranes-cristaux sont étudiées sur divers matériaux polymères denses. La perméabilité des espèces réactives et la tension de surface sont les deux paramètres ayant un impact majeur sur la localisation de la précipitation et la capacité à décrocher les cristaux déposés de la surface du matériau. Le PDMS et le Teflon AF 2400 ont été retenus comme étant les deux matériaux les plus prometteurs pour l’application visée car ils ne présentent pas de colmatage interne et de surface. Une deuxième partie expérimentale a été menée en conditions dynamiques sur le même composé modèle, en système gaz-liquide. Des modules membranaires de fibres creuses autosupportées (PDMS) et de fibres creuses composites (PP-Teflon AF 2400) ont été utilisés. Les études réalisées sur l’influence des paramètres opératoires ont présenté des résultats semblables à ceux des contacteurs membranaires utilisés pour le captage du CO2 : la résistance au transfert de matière est majoritairement localisée dans la phase liquide. Les performances stables obtenues sur le module PP-Teflon AF 2400 d’une compacité de 10 % ont permis de valider le concept. La géométrie du module, en particulier sa compacité, est un critère primordial pour limiter le colmatage du module. Enfin, une modélisation de mécanique des fluides en 2D, par la méthode des éléments finis, a été menée. Le modèle repose sur l’ajustement d’un seul paramètre cinétique. Les profils de concentration simulés ne sont pas satisfaisants. En revanche, le modèle permet de prédire correctement la productivité des cristauxMembrane processes are considered as one of the most promising breakthrough technology for crystallization/precipitation operations. Porous materials have been extensively investigated but they have shown some serious limitations due to pore blocking and wetting phenomenon. The use of a dense membrane is expected to circumvent the pore blocking issue while keeping the advantages of membrane processes. In a first part, the model compound, BaCO3, is precipitated within a gas-liquid or liquid-liquid membrane contactor working under static conditions for both systems. In this configuration, hydrodynamic influences are avoided. The membrane-crystals interactions are studied using several dense membrane polymers. Permeability of both reactant species and surface tension are the key parameters to be considered. Indeed, these parameters greatly affect the deposit location of the crystals and their adherence on the membrane surface. Fouling within the membrane and on the surface are prevented with PDMS and Teflon AF 2400 which are thereby the two most promising materials for the given application. In a second part, the same model compound is precipitated in gas-liquid system under dynamic conditions. Self-supporting (PDMS) and composite hollow fibers (PP-Teflon AF 2400) are studied. Investigations on the operating condition influences show similar results to those obtained with membrane contactor used for CO2 capture: resistance to mass transfer is mainly located in the liquid phase. Proof of concept is supported by the stable performances obtained with the PP-Teflon AF 2400 module of 10 % packing ratio. The module geometry, and more specifically its packing ratio, is an important criterion to take into account to avoid module blocking. Finally, 2D computational fluid dynamics simulations, using the finite element method are performed. One single kinetic parameter is used to fit the experimental data. The simulated concentration profiles are not satisfactory. Nonetheless, predictability of the model seems to be promising: crystal productivities are rather well estimated
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Triger, Aurelien. "Procédé hybride cristallisation et séparation membranaire pour le traitement d'un fluide complexe (urine)." Thesis, Toulouse, INSA, 2012. http://www.theses.fr/2012ISAT0045/document.
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Le développement de systèmes d’assainissement spécifiques et décentralisés peut apporter des réponses à une double problématique : l’amélioration des conditions sanitaires dans les zones les plus pauvres du monde et le développement de sources renouvelables de nutriments essentiels pour l’agriculture. L’objectif de cette thèse est de développer des connaissances scientifiques sur une filière de traitement et valorisation de l’urine couplant une cristallisation à une séparation membranaire. Il a été démontré que la précipitation de phosphore sous forme de struvite permet de récupérer la quasi-totalité du phosphore et une partie de l’azote en 20 secondes environ suite à l’ajout de magnésium avec un ratio Mg :P=1,3 :1. L’influence des conditions d’agitation, de l’apport en magnésium, des conditions de stockage de l’urine et de la présence de matières organiques et de cristaux initiaux a été étudié en réacteur batch et continu.L’ultrafiltration de différents types d’urine (fraichement excrétée, stockée, stockée puis cristallisée) a été réalisée avec des membranes en PES, PAN et PVDF. Les mécanismes responsables de cette chute de flux ainsi que l’influence spécifique des fractions particulaires colloïdales et particulaires ont été étudiés. En se basant sur ces résultats différents procédés ont été proposés et discutésThe development of decentralized and specific sanitation system is an issue that concerns both the improvement of sanitary conditions in the poorest area of the world and the development of renewable sources of nutrients for agriculture. This study aims to provide some elements about a treatment line including crystallization and membrane separation for the treatment and valorization of urine. Crystallization allows to recover phosphorus and part of nitrogen contained in urine. Membrane separation is used in order to remove bacteria and viruses from urine. To check the potentialities of these processes some tests were performed at labscale with synthetic and real human urines.It was shown that the struvite crystallization by magnesium addition with a ratio Mg:P=1,3:1 allows recovering most of the phosphorus from urine with a very rapid kinetics (about 20s). Influence of mixing conditions, urine storage, organic matter and initial crystals in urine was studied in batch and continuous reactor. Ultrafiltrations of different pretreated urines (no pretreatment, stored urine, stored and crystallized urine) were performed with PES, PAN and PVDF membranes. Mechanisms responsible for an important flux decline during urine flitration were studied. Specific influence of particular, colloidal and soluble fraction on the flux decline was also evidenced. On these basis different possible treatment lines of urines are proposed and discussed
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Pantoja, Carlos Eduardo. "Cristalização assistida por destilação por membranas aplicada ao reuso de água: comparação com outros métodos de reuso, análise do processo e projeto hierárquico de processo." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/3/3137/tde-16062016-113017/.
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No presente trabalho foram avaliados processos alternativos de dessalinização visando a recuperação e reuso da água contida em salmouras concentradas, sendo o processo de cristalização assistida por destilação por membranas (MDC) investigado com profundidade. Foi desenvolvido um modelo diferencial para o processo de destilação por membranas por contato direto (DCMD), contemplando métodos termodinâmicos rigorosos para sistemas aquosos de eletrólitos fortes, bem como mecanismos de transferência de calor e massa e efeitos de polarização de temperatura e concentração característicos deste processo de separação. Com base em simulações realizadas a partir do modelo matemático assim desenvolvido, foram investigados os principais parâmetros que influenciam o projeto de um módulo de membranas para DCMD. O modelo foi posteriormente estendido com equações de balanço de massa e energia adicionais para incluir a operação de cristalização e desta forma representar o processo de MDC. De posse dos resultados das simulações e do modelo estendido, foi desenvolvido um método hierárquico para o projeto de processos de MDC, com o objetivo de conferir características de rastreabilidade e repetibilidade a esta atividade. Ainda a partir do modelo MDC foram discutidos aspectos importantes em MDC como a possibilidade de nucleação e crescimento de cristais sobre a superfície das membranas, bem como o comportamento do processo com sais com diferentes características de solubilidade e largura da zona metaestável. Verificou-se que para sais cuja solubilidade varia muito pouco com a temperatura e que possuem zona metaestável com pequena largura, caso do NaCl, a operação com resfriamento no cristalizador não é viável pois aumenta excessivamente o consumo energético do processo, sendo nesses casos preferível a operação \"isotérmica\" - sem resfriamento no cristalizador - e o convívio com a possibilidade de nucleação no interior do módulo. No extremo oposto, observou-se que para sais com grande variabilidade da solubilidade com a temperatura, um pequeno resfriamento no cristalizador é suficiente para garantir condições de subsaturação no interior do módulo, sem grande ônus energético para o processo. No caso de sais com pequena variabilidade da solubilidade com a temperatura, mas com largura da zona metaestável elevada, existe certo ônus energético para a operação com resfriamento do cristalizador, porém não tão acentuado como no caso de sais com zona metaestável estreita. Foi proposto um fluxograma alternativo para o processo de MDC, onde foi introduzido um circuito de pré-concentração da alimentação antes do circuito de cristalização, para o caso de alimentação com soluções muito diluídas. Este esquema proporcionou um aumento do fluxo permeado global do processo e consequentemente uma redução na área total de membrana requerida. Verificou-se que através do processo com préconcentração da alimentação de 5% até 10% em massa - no caso de dessalinização de uma solução de NaCl - foi possível reduzir-se a área total da membrana em 27,1% e o consumo energético específico do processo em 10,6%, quando comparado ao processo sem pré-concentração. Foram desenvolvidas ferramentas úteis para o projeto de processos de dessalinização por MDC em escala industrial.Alternative desalination processes aiming at the recovery and reuse of the water contained in concentrated brines were evaluated, being the membrane distillation crystallization (MDC) process investigated in depth. A differential model for the direct contact membrane distillation (DCMD) process was developed for that matter, comprising rigorous thermodynamic methods for strong electrolytes, heat and mass transfer mechanisms and temperature and concentration polarization effects. Based on simulations from the mathematical model thus developed, the main parameters that influence the design of DCMD membrane modules were investigated. The model was further extended with mass and energy balance equations in order to consider the crystallization unit operation and thus suitably represent the MDC process. Based on the simulations results and the extended model, a hierarchical method was developed for the MDC process design, adding traceability and repeatability characteristics to the design activity. Important aspects of the MDC process such as the possibility of nucleation and crystal growth on the membrane surface, as well as the behavior of the process with salts presenting different solubility characteristics and metastable zone widths were further discussed. It was observed that salts presenting negligible temperature dependence regarding their solubility and small metastable zone widths (i.e. NaCl) do not favor the operation with cooling in the crystallizer due to excessive increase in energy consumption, being the isothermal operation more indicated in such cases even at the risk of nucleation inside the membrane module. On the other hand, it was noticed that for salts whose solubility is highly temperature dependent a slight cooling in the crystallizer is enough to assure subsaturated conditions inside the membrane module with minimal energy consumption increase. In the case of salts with low temperature dependence regarding solubility but with large metastable zone widths, the operating strategy of applying cooling in the crystallizer may increase energy consumption but not as significantly as in the case of salts with small metastable zone widths. An alternative flowsheet for the MDC process was proposed, where a pre-concentration loop was introduced before the crystallization loop, showing good results for dilute feeds since it takes advantage of the higher water activity and consequently higher transmembrane fluxes due to the lower concentration. It was perceived a 27.1% reduction in the required membrane surface and a 10.6% energy consumption reduction for the modified flowsheet with the pre-concentration loop, for a feed comprised of 5% of NaCl. Useful tools aimed for the design of industrial scale processes based on MDC were developed.
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Karathanou, Argyro. "Image processing for on-line analysis of electron microscope images : automatic Recognition of Reconstituted Membranes." Phd thesis, Université de Haute Alsace - Mulhouse, 2009. http://tel.archives-ouvertes.fr/tel-00559800.
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The image analysis techniques presented in the présent thesis have been developed as part of a European projeet dedicated to the development of an automatic membrane protein crystallization pipeline. A large number of samples is simultaneously produced and assessed by transmission electron microscope (TEM) screening. Automating this fast step implicates an on-fine analysis of acquired images to assure the microscope control by selecting the regions to be observed at high magnification and identify the components for specimen characterization.The observation of the sample at medium magnification provides the information that is essential to characterize the success of the 2D crystallization. The resulting objects, and especially the artificial membranes, are identifiable at this scale. These latter present only a few characteristic signatures, appearing in an extremely noisy context with gray-level fluctuations. Moreover they are practically transparent to electrons yielding low contrast. This thesis presents an ensemble of image processing techniques to analyze medium magnification images (5-15 nm/pixel). The original contribution of this work lies in: i) a statistical evaluation of contours by measuring the correlation between gray-levels of neighbouring pixels to the contour and a gradient signal for over-segmentation reduction, ii) the recognition of foreground entities of the image and iii) an initial study for their classification. This chain has been already tested on-line on a prototype and is currently evaluated.
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Meguellati, Amel. "Synthèse de biomolécules agissant comme inhibiteurs de l'ARN polymérase ARN dépendante du virus de l'hépatite C et développement de nouveaux surfactants comme stabilisants des protéines membranaires par réseaux de ponts salins." Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GRENV001.
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Le projet de thèse se focalise sur la synthèse de biomolécules et se subdivise en deux parties. La première partie concerne la conception et la synthèse de dérivés de produits naturels d'intérêt thérapeutique nommés aurones en vue de mettre au point de nouvelles molécules à activité antivirale. Récemment, les aurones ont été identifiées comme étant des inhibiteurs de l'ARN-polymérase ARN-dépendante (NS5B) du virus de l'hépatite C (VHC). Cette enzyme, présente chez le virus mais absente chez l'homme, joue un rôle central dans la réplication virale. Suite à ces résultats antérieurs, les efforts ont été poursuivis et, dans le cadre de cette thèse, nous avons entrepris,d'une part, la synthèse d'analogues originaux dont le cycle B des aurones a été remplacé par des hétérocycles et, d'autre part, la synthèse depseudodimères d'aurones dans le but d'affiner les exigences structurales pour améliorer l'effet inhibiteur.L'activité a été évaluée selon des tests enzymatiques et cellulaires et a permis d'identifier quelques candidats doués d'une bonne activité inhibitrice et d'une faible toxicité. La deuxième partie du projet de thèse, sans lien avec la première partie,concerne des aspects plus fondamentaux et porte sur la synthèse de nouveaux surfactants agissant comme agents stabilisants lors des procédures d'extraction et de cristallisation des protéines membranaires. Les surfactants sont des composants clés dans le domaine de la biologie structurale et de la biochimie des protéines membranaire. Ils sont nécessaires pour maintenir les protéines membranaires dans leur état fonctionnel après extraction. La grande majorité des protéines membranaires est riche en résidus basiques à l'interface. Sur la base de cette caractéristique, une nouvelle famille de surfactants est développée et testée sur des protéines membranaires appartenant aux pompes d'efflux ABC multi-résistantesThe PhD project focuses on biomolecules and is divided into two parts. The first part concerns the design and synthesis of natural product derivatives with therapeutic interest in order to develop new molecules with antiviral activity. Recently, aurones were identified as new inhibitors of hepatitis C virus (HCV) NS5B polymerase. Following these results, efforts were continuedand we undertook, on the one hand,the synthesis of original analogues in which the aurone B-ring was replaced by a heterocyclic rings and, on the other hand, the synthesis of aurone pseudodimers in order to refine the structural requirements to improve the inhibitory effect. The potent NS5B inhibitory activity combined with their low toxicity make aurones attractive drug candidates against HCV infection. The second part of the PhD thesis is unrelated to the first part and concerns more fundamental aspects. It focused on the synthesis of new surfactants acting as stabilizing agents during extraction of membrane proteins (PM). Surfactants are required for maintaining PM in their functional state after extraction from membrane lipid matrix. The vast majority of PM shares a net enrichment in basic residues at the interface between membrane and cytoplasm, a property known as the positive inside rule. Based on this feature, a new family of surfactants is developed and tested on membrane proteins belonging to the multidrug ABC efflux pumps family
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Remeeva, Alina. "Développement des nouvelles approches pour nano volume cristallisation des protéines membranaires." Thesis, Grenoble, 2013. http://www.theses.fr/2013GRENY001.
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Une nouvelle méthode pour la cristallisation des protéines membranaires (MP) de bicouches amphiphiles interconnectés (IAB) a été récemment mise au point. Dans cette approche protéines cristallisées directement des membranes et il est supposé que la cristallisation est conduite principalement par les propriétés de la mésophase lipidique dans son ensemble, mais pas par des caractéristiques individuelles des détergents. Le détergent joue un rôle de modulateur de ces propriétés. L'application de ce procédé de cristallisation de la vaste gamme des protéines membranaires est très prometteuse car il facilite considérablement la cristallisation. Cependant, il faut effectuer un certain nombre d'expériences pour chaque nouvelle protéine membranaire afin de déterminer les paramètres optimaux de la mésophase lipidique pour la protéine. Pour ce faire avec des quantités limitées de protéines membranaires, en particulier des protéines humaines, qui sont généralement très difficiles à produire en quantité suffisante, l'application des systèmes robotisés de cristallisation à haut débit pour la nano volume cristallisation pourrait être la seule solution. L'objectif principal de ce travail est le transfert de l'approche actuelle pour la cristallisation des protéines membranaires de l'IAB sur les volumes nano. Pour ce faire une grande variété d'expériences cristallisations a été effectuée pour différentes protéines membranaires. Les faits, qui influent des résultats de cristallisation, ainsi que des informations structurelles obtenues ont été analysées avec soin. Il a été démontré que les gros cristaux de protéines membranaires différents adaptés pour cristallographie aux rayons X peuvent être reproductible obtenu par nano volume cristallisation. Le premier chapitre «Introduction» comprend une description générale des protéines membranaires, des approches différentes pour la cristallisation des protéines membranaires, leurs avantages et leurs limites. La vue d'ensemble des protéines membranaires, qui sont étudiés dans ce travail, les informations disponibles et les problèmes non résolus sont présentés. Le deuxième chapitre présente les matériaux et les méthodes utilisées dans l'étude. Le troisième chapitre «Résultats et discussions» est décrit les résultats obtenus et leur interprétation. Pour commencer, la nano volume cristallisation de l'IAB de la bactériorhodopsine et la comparaison avec l'approche présent de cristallisation est présenté. Nano volume cristallisations en présence d'amphiphiles fluorés et les poloxamères ont été effectuées pour la première fois et décrit en détails. L'application de l'approche du nano volume cristallisation du complexe de la rhodopsine sensorielle II de Natronomonas pharaonis avec son transducteur apparenté et les protéines du complexe avec une mutation importante dans le transducteur, ce qui élimine l'action des protéines, est présenté. Résultats de nano volume cristallisation pour un autre complexe de deux protéines membranaires - triple mutant de la bactériorhodopsine, qui fonctionne comme un transducteur de signal, avec transducteur de Natronomonas pharaonis sont décrits. Halorhopsin de Natronomonas pharaonis a également été cristallisé par la nouvelle approche et de cristallisation résultats sont présentés. La première structure de protéine membranaire - enzyme cytidine-5'-triphosphate: inositol-1-phosphate cytidylyltransférase / di-myo-inositol-1 ,3-phosphate-1-phosphate synthéase de hyperthermophiles Archaeoglobus fulgidus a été résolu en utilisant des cristaux obtenus par nano volume cristallisation. Détails de la structure, l'importance et le mécanisme proposé de l'action enzymatique sont discutées. Les conclusions finales ainsi que les perspectives de la méthodique développées sont donnés dans le dernier chapitre de la thèseA new method for crystallization of membrane proteins (MPs) from interconnected amphiphilic bilayers (IAB) was recently developed. In this approach proteins crystallized directly from the membranes and it is postulated that crystallization is driving mostly by the properties of the lipidic mesophase as a whole but not by individual features of the detergents. The detergent plays a role of the modulator of these properties. Application of this crystallization method to the wide range of MPs is very promising since it dramatically facilitates crystallization. However, one needs to perform a number of screening experiments for each new MP to determine the optimal parameters of the lipidic mesophase for certain protein. To do so with limited amounts of MPs, especially human proteins, which are usually very difficult to produce in sufficient amount, the application of the high throughput robotic systems for nano volume crystallization could be the only solution. The major goal of this work is the transfer of the present approach for large scale crystallization of MPs from IAB to the nano volumes. To do so a large variety of crystallizations experiments were carried out for different MPs. The facts, which influence the crystallization results, as well as structural information obtained were carefully analyzed. It was demonstrated that large crystals of different MPs suitable for X-ray crystallography can be reproducibly obtained using this nano volume crystallization method. The first chapter “Introduction” includes a general description of MPs, different approaches for crystallization of MPs, their advantages and limitations. The overview of membrane proteins, which are studied in this work, the information available and unsolved problems are presented. The second chapter presents the materials and methods used in the study. The third chapter “Results and Discussions” describes the results obtained and their interpretation. To begin with, nano volume crystallization from IAB of bacteriorhodopsin and the comparison with large scale crystallization approach is presented. Detailed nano volume crystallizations in the presence of fluorinated surfactants and poloxamers were performed for the first time and described in details. The application of nano volume approach for the crystallization of the complex of sensory rhodopsin II from Natronomonas pharaonis with its cognate transducer and the proteins complex with important mutation in the transducer, which eliminates the action of the proteins, is presented. Nano volume crystallization results for another complex of two MPs – triple mutant of BR, which operates like a signal transductor, together with transducer from Natronomonas pharaonis are described. A light-driven chloride pump halorhopsin from Natronomonas pharaonis was also crystallized by new approach and crystallization results are presented. The structure of new MP – bifunctional enzyme cytidine-5′-triphosphate:inositol-1-phosphate cytidylyltransferase/ di-myo-inositol-1,3-phosphate-1-phosphate synthase from hyperthermophiles Archaeoglobus fulgidus was solved using the crystals obtained by nano volume crystallization from IAB. Structure details, significance and proposed mechanism of the enzymatic action are discussed. The final conclusions as well as the perspectives of the developed methods are given in the last chapter of the thesis
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Álvarez, Marimón Mª Elena. "Structural studies of Heteromeric Amino acid Transporters (HATs): Validation of the first 3D structural model of a HAT (human 4F2hc/LAT2) and identification of new HAT targets for 3D‐crystallization." Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/284082.
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Heteromeric amino acid transporters (HATs) mediate the transport of amino acids through the plasma membrane. They are composed of two subunits (a heavy and a light one) linked by a conserved disulfide bridge. Genetic defects in the genes coding these HATs may affect its functionality or expression, leading to inherited aminoacidurias. Thus, solving the structure of the Eukaryotic HATs has become of great importance. However structural information about interactions between the heavy and light subunits of HATs is scarce.
In this work, human 4F2hc/L-type amino acid transporter 2 (LAT2) first low resolution 3D model obtained by single particle negative-staining transmission electron microscopy (TEM) was validated. In order to assess the interaction between both subunits of the heterodimer, crosslinking experiments between cysteine residues in both moieties was tried. Namely, two chemical spacers of different length (10.5 and 14.3 Å) were tested and crosslinking was observed for those mutants with pairing positions between 8 and 17.5 Å. Indeed, specific residues that crosslinked 4F2hc and LAT2 nearly completely (>80%). As a result of the positive results (as compared to the appropriate controls) the idea that 4F2hc-ED almost completely covers the extracellular surface of the transporter subunit LAT2 is reasonable. Moreover, further varied evidences (TEM, SPA and docking experiments) were in line with the obtained results, revealing that the extracellular domain of 4F2hc interacts with LAT2, almost completely covering the extracellular face of the transporter. The interaction of 4F2hc with LAT2 gives insights into the structural bases for light subunit recognition and the stabilizing role of the ancillary protein in HATs. In addition, it has been demonstrated that the ectodomain of 4F2hc suffices the stabilization of the light subunit.
The second goal of the thesis was to find a suitable HAT candidate to perform crystallization trials and posterior structure elucidation, since human 4F2h/LAT2 was not stable enough for this aim. Until now, only the human 4F2hc ectodomain atomic structure has been solved (Fort et al., 2007), and some low sequence amino acid identity prokaryotic homologues of LATs.
In order to identify putative good eukaryotic light subunits for 3D crystallization the adopted GFP‐based Saccharomyces cerevisiae protocol for our transporters resulted successful since it allowed to find three putative good candidate eukaryotic light subunits for 3D crystallization studies. GFP technology allowed quick expression screening, membrane protein-detergent solubilization screening and finally another screening step including assessment of the stability by ultracentrifugation dispersity sedimentation.
Once the candidates selected in the best conditions, further purification was required (size exclusion chromatography) before attempting crystallization. In this sense, further efforts were delivered in order to try to enhance the stability (and minimize aggregation). Thus, addition of lipids in the solubilization step and during protein purification was used to mimic the protein membrane environment and reduce the aggregation. Really interesting is the stabilizing effect that cholesterol has on almost all light subunits tested. This is in concordance with the fact that 4F2hc has been found located in lipid rafts where membrane are rich in cholesterol. Removal of reactive cysteine and generation of truncated versions of the protein in the C and N terminal were introduced to increase the crystallization probability. This work ended with the finding of 3 good candidates for crystallization screenings, and the best candidate was optimized in terms of stability and protein flexibility for crystallization studies. Just preliminary crystallization trials were done.Los transportadores heteroméricos de aminoácidos (HAT) median el transporte de aminoácidos a través de la membrana plasmática. Representan el único ejemplo de transportadores de solutos formado por dos subunidades distintas unidas por un puente disulfuro. Debido a su gran relevancia en fisiología (asociados a aminoacidurias, infección por virus, cáncer,…) el estudio de su estructura-función resulta clave. Debido a su naturaleza, son proteínas difíciles de cristalizar, de las que sólo se conoce la estructura atómica del ectodominio de 4F2hc humano. En este escenario la tesis se centró en la validación del primer modelo 3D a baja resolución de un HAT humano (4F2hc/LAT2), mediante experimentos de crosslinking entre subunidades, e identificación de nuevos candidatos para cristalización 3D. Para ello se seleccionaron 24 subunidades ligeras de distintas especies eucariotas y se testaron en un proceso de selección para determinar el/los mejores candidatos. El primer objetivo de la tesis concluyó con la determinación de residuos concretos en 4F2hc y LAT2 cercanos a una distancia de 3-14 Å mediante la utilización de crosslinkings de cisteínas. Finalmente, tres líneas distintas: el modelo 3D obtenido por microscopía electrónica de transmisión y tinción negativa de partículas individuales (en colaboración con Dr. Fotiadis), los experimentos de crosslinking, y el docking generado en colaboración con Dr. Fdz-Recio, demostraron que 4F2hc-ED cubre, casi completamente, la superficie extracelular de LAT2. Además, se demostró que el ectodominio de 4F2hc es suficiente para estabilizar LAT2. Como resultados del segundo objetivo, tres subunidades ligeras fueron seleccionadas, tras adaptar el protocolo desarrollado por Drew et al.,2008, como mejores candidatas para estudios de cristalización 3D. Posteriormente, distintas estrategias se siguieron para mejorar la estabilidad de la mejor candidata: eliminación de la cisteína reactiva, adición de lípidos a la muestra, cambio de sistema de expresión para aumentar su expresión a células de insecto Sf9. Además, se generaron mutantes delecionados en N y C terminal para reducir su flexibilidad y aumentar la probabilidad de cristalización. Se concluyó en encontrar un buen candidato para estudios de cristalización.
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Nariyoshi, Yuri Nascimento. "Estudo dos fundamentos de cristalização assistida por destilação com membranas em aplicação de dessalinização de água." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/3/3137/tde-22092016-091528/.
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A cristalização assistida por destilação com membranas (membrane distillation crystallization, MDC) se destaca como uma alternativa aos processos convencionais de cristalização evaporativa com múltiplos estágios e/ou recompressão mecânica de vapor para dessalinização de soluções aquosas concentradas com descarga zero de líquido (zero liquid discharge, ZLD) no meio ambiente. Os principais atrativos da MDC são as condições operacionais mais brandas de temperatura e pressão, o que possibilita o emprego de fontes de calor de baixa entalpia e instalações menos requisitadas mecanicamente. Entretanto, por ser um processo de separação que envolve membranas, a formação de incrustação se destaca como inconveniente. Assim sendo, grande parte dos estudos em MDC têm sido voltados para essa questão, com foco na operação de destilação com membranas (membrane distillation, MD), quando o produto de interesse é a água recuperada. Nesse contexto, esta tese amplia o conhecimento na área, sendo estudados os fundamentos de cristalização e a sua relação com parâmetros selecionados do processo. Dessa forma, estudos teórico-experimentais foram conduzidos investigando os mecanismos de cristalização predominantes nesse, de maneira a ampliar a abrangência das teorias clássicas de cristalização. No primeiro capítulo, realizou-se uma revisão bibliográfica a fim de apresentar os fundamentos tecnológicos, bem como as características, limitações e desafios para consolidação em escala industrial da MDC. No segundo capítulo, foi definido o conhecimento científico produzido através da especificação de objetivos. No terceiro capítulo, a operação MD na configuração DCMD (direct contact membrane distillation) foi caracterizada utilizando equações matemáticas para o cálculo do fluxo de vapor. Os valores calculados foram validados com experimentos em escala de bancada. Foi possível identificar que a difusão ordinária molecular é o mecanismo de transporte de massa dominante nos poros da membrana, e quantificar os fenômenos de polarizações de temperatura e de concentração adjacentes à superfície da membrana. No quarto capítulo, a cristalização foi integrada com a DCMD e explorada em uma unidade de bancada. Os processos elementares e acessórios de cristalização predominantes foram esclarecidos como sendo nucleação primária heterogênea (incrustação por cristalização), nucleação secundária (abrasão de cristais) e crescimento cristalino molecular (aumento de tamanho). O quinto capítulo versou sobre uma estratégia proposta para reduzir a nucleação primária heterogênea, força motriz da incrustação por cristalização na membrana. Com base nos processos elementares e acessórios de cristalização identificados, foi avaliada uma modificação na MDC, a submersão de membranas em cristalizador, a qual foi implementada em escala de bancada. Essa modificação se mostrou mais sensível à formação de incrustação, com possibilidade de se encontrar condições favoráveis, uma vez que foi possível operar o processo durante três horas sem desenvolvimento de incrustação na membrana (tempo máximo investigado), mas precisa ser melhor investigada. Por último, no sexto capítulo abordou-se a nucleação primária heterogênea, relacionando esta com a queda de fluxo de vapor e com a distribuição de sólidos formados. O equacionamento do sistema validado no terceiro capítulo foi aplicado para quantificação da supersaturação local na membrana e respectiva associação com os mecanismos de cristalização. Observou-se que o aumento de fluxo aumenta a supersaturação local, que aumenta a nucleação primária heterogênea, sendo essa responsável pela formação de cristais que permaneceram aderidos na membrana (incrustação por cristalização) e que foram soltos em solução (suspensão). Os cristais soltos em solução são predominantes. A fluidodinâmica de escoamento (geometria dos módulos de membranas) associada à supersaturação local (fluxo de vapor) impactam no desprendimento de cristais na membrana originados por nucleação primária heterogênea. Com o aumento de supersaturação local, o desprendimento de cristais em suspensão é favorecido em módulo de membranas do tipo fibras ocas, enquanto não afeta o módulo do tipo tubular. Em seu conjunto, esta tese contribui para a melhoria do entendimento de aspectos fundamentais selecionados do processo MDC e para o emprego deste conhecimento em situações de interesse prático.Membrane distillation crystallization (MDC) stand as an alternative to conventional evaporative crystallization processes with multiple-stage evaporator and/or mechanical vapor recompression towards concentrated brine desalination aiming at zero liquid discharge (ZLD) in the environment. The major advantages of MDC are the moderate temperature and pressure conditions, which allow the use of low enthalpy heat sources and facilities less mechanically required. However, as in membrane separation processes, membrane fouling plays an important role in MDC. Therefore, most MDC studies have been focused on that, with emphasis on membrane distillation (MD) operation, when the product of interest is the recovered water. In this context, this thesis extends the knowledge in the field, wherein the crystallization fundamentals and its relation with selected process parameters were studied. Thus, theoretical and experimental approaches were carried out in order to investigate the prevailing crystallization mechanisms in the process, so the range of the current crystallization theories could be extended. In the first chapter, a literature review was carried out in order to introduce selected fundamental concepts, as well as the process characteristics, limitations and challenges for industrial consolidation. In the second chapter, the scientific knowledge produced by this work was defined in its objectives. In the third chapter, the MD operation in direct contact configuration (DCMD) was characterized using mathematical equations for the calculation of vapor flux and validated with experimental data in a bench scale unit. The dominant mechanism of mass transport in porous media was found to be the ordinary molecular diffusion and the temperature and concentration polarization effects were quantified in the vicinity of membrane surface. In the fourth chapter, the crystallization operation was integrated with DCMD and investigated in a bench scale unit. The elementary and accessory crystallization mechanisms were highlighted as heterogeneous primary nucleation (crystallization fouling), secondary nucleation (crystal abrasion) and crystalline molecular growth (increase in size). The fifth chapter describes a strategy proposed in order to reduce heterogeneous primary nucleation, the driving force of membrane crystallization fouling. Based on the featured crystallization mechanisms, a modification in the conventional MDC operation was evaluated, the submersion of membranes into the crystallizer vessel, which was implemented in a bench scale unit. The preliminary results showed that this modification is more sensitive to crystallization fouling, with possibility to find promising conditions, once it was possible to operate during three hours without development of crystallization fouling (maximum period of time investigated), but further investigation is needed. Lastly, in the sixth chapter, the primary heterogeneous nucleation mechanism was explored, associating it to flux decay and solid distribution in the unit. The system equations validated in the third chapter were applied in order to quantify the supersaturation ratio generated in the vicinity of membrane surface and respective association with crystallization mechanisms. It was observed that increasing the vapor flux, the local supersaturation ratio also increase and, as consequence, the primary heterogeneous nucleation as well. This mechanism is responsible for the formation of crystals that remained adhered on membrane surface (crystallization fouling) and released in solution (suspension). The portion of crystals loose in solution was predominantly formed. The fluid dynamic of flow (geometry of membrane module) associated with the local supersaturation ratio (vapor flux) impact in the detachment of crystals in the membrane surface, originated by heterogeneous primary nucleation. With an increase in the local supersaturation ratio, the detachment of crystals is increased in the hollow fiber membrane module, while in the tubular module the detachment of crystals does not change. As a whole, this thesis contributes to a better understanding of MDC selected fundamental aspects and to the use of this knowledge in practical situations.
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Johnson, Matthew C. "Identifying key factors in two-dimensional crystal production and sample preparation for structure-function studies of membrane proteins by cryo-EM." Diss., Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/52974.
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Electron crystallography of two-dimensional crystals is a structure-determination method well suited to the study of membrane protein structure-function. Two-dimensional crystals consist of ordered arrays of protein within reconstituted lipid bilayers, an arrangement that mimics the natural membrane environment. In this work we describe our recent progress in the use of this method with three different proteins, each providing a window into a separate paradigm in the electron crystallographic pipeline. Specific crystallization conditions for human leukotriene C₄ synthase (LTC₄S) have previously been determined, but our continued refinement of purification and crystallization has identified a number of additional parameters that greatly affect crystal size and quality, and we have developed a protocol to rapidly and reproducibly grow large, non-mosaic crystals of LTC₄S. The human gamma-glutamyl carboxylase (GGCX) has also been crystallized, but is sensitive to cryo-EM sample preparation conditions and we present here the successful reproduction of crystallization and refinement of cryo-EM sample preparation conditions. Lastly, we describe our crystallization screens with the Vibrio cholerae sodium-pumping NADH:ubiquinone reductase complex (Na⁺-NQR), and identify the factors critical to membrane reconstitution of the complex, a necessary first step towards crystallization. We also describe a semi-quantitative crystal screening protocol we have developed that provides quick and accurate method to assess two- dimensional crystallization trials, and discuss some general observations in optimization of membrane protein purification and two-dimensional crystallization for electron crystallography.
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Panwar, Pankaj. "Relations structure-fonction des transporteurs nucléotides." Phd thesis, Université de Grenoble, 2012. http://tel.archives-ouvertes.fr/tel-00684264.
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Le transporteur NTT1 est responsable pour l'import d'ATP dans les chloroplastes afin de maintenir le métabolisme en période d'activité réduite ou nulle de la photosynthèse. Cependant, le mécanisme moléculaire de ce transporteur est encore méconnu, essentiellement du à la difficulté de manipulation des protéines membranaires. Nous avons réussi à développer un protocole pour la production de ce transporteur, permettant une bon rendement de solubilisation et obtention de protéines purifiées pour des études structurales. Combinant des caractérisations biochimiques et biophysiques, nous avons pu identifier des conditions de préparation d'échantillons qui ont mené aux premiers cristaux. Afin d'étendre notre connaissance sur les transporteurs de nucléotides, nous avons également entrepris des études structurales et fonctionnelles sur AAC, le transporteur ADP/ATP des mitochondries. AAC et NTT1 appartiennent à des familles de protéines différentes mais ont des fonctions voisines. À partir de la première structure d'AAC déjà connue, nous avons recherché par des criblages in silico de nouvelles molécules se liant au transporteur de façon compétitive avec le nucléotide et pouvant ainsi inhiber le transport. Les outils de docking ont été mis en place et ont permis à partir d'une librairie de 75000 composés d'identifier 17 molécules. Ensuite, nous avons testés ces molécules expérimentalement et montré qu'une d'entre elles inhibent le transport. De plus, trois nouveaux analogues d'ADP ont également été identifiés comme inhibiteurs.
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Armstrong, Shannon Renee. "Novel Applications of Co-Extruded Multilayer Polymeric Films." Case Western Reserve University School of Graduate Studies / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=case1370531152.
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Matar, Merheb Rachel Rima. "Caractérisation d’une nouvelle génération de détergents stabilisateurs des transporteurs abc en solution : cristallisation de BmrA, transporteur ABC bactérien." Thesis, Lyon 1, 2010. http://www.theses.fr/2010LYO10303.
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En raison de leur résistance aux agents chimiothérapeutiques, les transporteurs ABC de phénotype MDR ont attiré l'attention de la communauté scientifique. Notre projet vise à trouver des conditions dans lesquelles les transporteurs ABC restent fonctionnels en solution pour aboutir à la cristallisation de ces protéines dans une conformation active. Dans ce but, nous avons conçu et développé une nouvelle classe de détergents, à base de calix[4]arène, qui stabilisent ces protéines. Afin de résoudre la structure 3D à résolution atomique du transporteur ABC bactérien "BmrA", responsable de la résistance aux antibiotiques, nous avons utilisé une approche classique utilisant des détergents commerciaux en parallèle à nos détergents innovants. En présence de la Foscholine 12, nous avons obtenu des cristaux diffractant jusqu’à 5 Å de résolution. Cependant, les données de diffraction n’étaient pas suffisantes pour déterminer la structure tridimensionnelle complète de la protéine, seuls les domaines transmembranaires ont été résolus. D'autre part, nous avons atteint l'objectif de l'extraction, la purification et la stabilisation de ce transporteur à l'aide des détergents à base de calix [4] arène. Nous avons également montré que ces détergents promeuvent et améliorent la cinétique de cristallisation de BmrA, une étape que nous sommes en train d’optimiser, pour obtenir des cristaux de meilleure résolution, pour résoudre la structure 3D de BmrA qui sera utilisé pour concevoir des inhibiteurs adaptésDue to their preponderance in the resistance to chemotherapies, the MDR ABC transporters have drawn the attention of the scientific community. Our project aimed at finding conditions in which ABC transporters are active in solution to lead the crystallization of these proteins in an active conformation. In this purpose, we conceived and developed a new class of detergents, based on calix[4]arene ring, that stabilize these proteins. In order to solve the 3D-structure to atomic resolution of bacterial ABC transporter “BmrA” responsible for antibiotic resistance, we used a classical approach with commercial detergents in addition to the innovative ones. We have crystallized the protein in presence of Foscholine 12 with a diffraction resolution up to 5 Å. The data was incomplete; solving partially the structure of the transmembrane domains. On the other hand, we have reached the objective of extraction, purification and stabilization of this transporter by using calix[4]arene-based detergents. We have also shown that these detergents promote and enhance the kinetics of crystallization of BmrA, a step that we are improving, to get crystals of better resolution, for resolving the BmrA 3D-structure which will be used to design adapted inhibitors
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Boukra, Nouara. "Structure de la rhodopsine bovine : analyse par microscopie electronique et essai de cristallisation en trois dimensions." Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13126.
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Kilburg, Arnaud. "Cristallisation du transporteur ABC BmrA de Bacillus subtilis : développement d’une nouvelle méthode de dosage des détergents par Matrix-Assisted Laser Desorption Ionization (MALDI)." Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10116/document.
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Notre projet vise à déterminer la structure 3D du transporteur BmrA de Bacillus subtilis. La protéine a été purifiée dans six détergents différents. L'utilisation de foscholine 12, a conduit à cristalliser OmpF, une porine de la membrane externe d'E. coli. Nous montrons que les conditions de cristallisation influencent directement l'empilement cristallin d'OmpF. Le protocole de purification de BmrA, optimisé en utilisant du triton X100 à l'extraction puis un mélange β-D-dodecyl maltoside-cholate pour les étapes chromatographiques nous a permis d'obtenir à 4°C des cristaux, pour lesquels nous avons vérifié qu'ils sont constitués de BmrA. Ces cristaux ont permis d'obtenir un jeu complet jusqu'à 7 Å. Ces données de diffraction constituent une avancée significative pour résoudre à court terme la structure 3D de BmrA. Nous avons développé une nouvelle méthode de dosage des détergents qui est basée sur la détermination par spectrométrie de masse de type MALDI du ratio d'isotopes deutérés/ protonés. La méthode a été validée avec la FC12, le DDM, le β-OG, le LMNG, le CHAPS, le cholate et des détergents calix[4]aréniques, en mesurant la concentration de ces détergents dans différentes conditions d'extraction/purification, de concentration, dialyse et gel filtration, de différentes protéines membranaires. Cette méthode nous a permis (i) d'estimer la taille de la ceinture de détergent associée à BmrA et d'autres protéines membranaires (ii) de moduler cette taille en fonction de mélange de détergents et (iii) d'apporter des informations sur le comportement des complexes protéine-détergentOur project aims to determine the 3D structure of BmrA from Bacillus subtilis. The protein was purified in six different detergents. Using foscholine 12, led to crystallize OmpF, an outer membrane porin of E. coli. We show that the crystallization conditions directly influence the crystal packing of OmpF. The BmrA purification protocol optimized by using Triton X100 at the extraction and a mixture β-D-dodecyl-maltoside cholate for chromatographic steps allowed us to get to 4°C crystals, for which we verified they consist of BmrA. These crystals have yielded full data to 7 Å. These diffraction data are a significant advance in the short term to resolve the 3D structure of BmrA. We have developed a new detergents dosage assay which is based on the determination by MALDI-type mass ratio of deuterated isotopes / protonated. The method was validated with the FC12, the DDM, the β-OG, the LMNG, CHAPS, cholate detergents and calix [4] aréniques by measuring the concentration of these detergents in different conditions of extraction/ purification, concentration, dialysis and gel filtration, of different membrane proteins. This method allowed us (i) to estimate the size of the detergent belt associated to BmrA and other membrane proteins (ii) to modulate this size in terms of the detergent mixture and (iii) to provide information on the behavior of complex protein-detergent
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Shih, Wen-Yi. "Formation and control of calcium sulfate dihydrate (gypsum) crystallization on RO membranes and surrogate polymeric surfaces." Diss., Restricted to subscribing institutions, 2007. http://proquest.umi.com/pqdweb?did=1383480211&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Narducci, Riccardo. "Membranes conductrices ioniques pour piles à combustible." Thesis, Aix-Marseille, 2014. http://www.theses.fr/2014AIXM4764.
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Dans cette thèse, les membranes perfluorosulfoniques (PFSA) et les polymères aromatiques sulfonés (SAP) sont étudiés en vue d'une meilleure compréhension de leurs propriétés thermodynamiques, d'hydratation, mècaniques et électriques.Concernant les PFSA: 1) Préparation de membranes Nafion ayant diverses morphologies et structures (amorphe, semi-cristalline, stratifiée) et relation avec les propriétés, comme la transition vitreuse, la fusion, la conductivité protonique. 2) Divers traitements de recuit ont été appliqués et analysés par une nouvelle méthode quantitative appelé INCA (Ionomère nc analyse), utilisant aussi des agents de recuit spéciaux. Concernant les SAP: 1) Synthèse in situ de polymères réticulés et clarification du mécanisme. 2) Optimisation du degré de reticulation en vue de la meilleure conductivité protonique. 3) Obtention d'ionomères conducteurs cationiques par échange de cations du SPEEK et détermination des propriétés de ces nouveaux ionomèresIn this thesis, perfluorosulfonic acid membranes (PFSA) and sulfonated aromatic polymers (SAP) are studied to better understandtheir thermodynamic, hydration, mechanical and electrical properties. The following main points were addressed:Regarding PFSA:1) Nafion membranes with various morphology and microstructure (amorphous, semi-crystalline, layered) were prepared and the relation to the properties, such as glass and melting transitions, and proton conductivity, was established.2) Various annealing treatments were performed and analyzed by the quantitative INCA (Ionomer nc Analysis) method using also special annealing agents. Regarding SAP:1) The in situ synthesis of cross-linked polymers was studied and the mechanism was clarified. 2) The degree of cross-linking was optimized for best proton conductivity.3) Cation-conducting ionomers were obtained by cation exchange of SPEEK and the properties of these new ionomers were determined
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Cravillon, Janosch [Verfasser]. "Microporous metal imidazolates : synthesis and characterization of nano- and microcrystals as well as membranes and investigation of the mechanisms of crystallization / Janosch Cravillon." Hannover : Technische Informationsbibliothek und Universitätsbibliothek Hannover (TIB), 2012. http://d-nb.info/1019943602/34.
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Penel, Simon. "Organisation du détergent dans les cristaux de protéines membranaires : analyse des cristaux des porines de Rhodobacter capsulatus et de Escherichia coli." Université Joseph Fourier (Grenoble), 1997. http://www.theses.fr/1997GRE10132.
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Le nombre de proteines membranaires dont la structure est connue est tres faible en regard du nombre de structures resolues, ceci en raison des difficultes a obtenir des cristaux de qualite suffisante pour les etudes par diffraction des rayons x. La finalite de ce travail est de comprendre l'implication du detergent, utilise pour solubiliser puis cristalliser les proteines membranaires, dans le processus de cristallisation et dans la qualite des cristaux obtenus. En particulier, on s'est interesse a l'organisation prise par le detergent dans les cristaux, et aux differentes interactions auxquelles il participe. Les structures du detergent dans les cristaux ont ete resolues par cristallographie des neutrons a basse resolution. Les micelles de detergent, pures ou complexees avec les proteines, ont elles aussi ete etudiees, en solution, par diffusion neutronique aux petits angles. Ces deux techniques sont utilisees en combinaison avec la methode de variation de contraste. Les structures formees par le detergent dans deux cristaux de porine (de rhodobacter capsulatus et de escherichia coli) de groupes d'espace differents ont ete modelisees, ainsi que les micelles de detergent presentes dans la solution de cristallisation de l'une de ces porines. On a essaye d'en tirer une analyse generale du role du detergent, dans l'optique d'optimiser la formation de cristaux de proteines membranaires de bonne qualite. En outre les interactions entre la membrane et la proteine, mises en lumiere par la localisation du detergent au niveau de la zone transmembranaire de la proteine, ont ete abordees.
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Barret, Laurie-Anne. "Influence des tensioactifs dans la cristallisation du complexe photosynthétique RC-LH1-pufX de Rhodobacter blasticus." Phd thesis, Université d'Avignon, 2013. http://tel.archives-ouvertes.fr/tel-01017895.
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Ce projet vise à étudier, par une approche pluridisciplinaire, l'influence des la cristallisation des protéines membranaires (PM) en prenant pour protéine modèle le complexe photosynthétique RC-LH1-pufX de Rhodobacter blasticus. Des cristaux de ce complexe avaient été obtenus en présence de dodécyl-!-maltoside (DDM) et avaient diffractés à 8 Å de résolution. L'objectif final est de pouvoir améliorer, de façon rationnelle, la qualité des cristaux du complexe RC-LH1-pufX grâce à une meilleure compréhension des mécanismes mis en jeu. Dans un premier temps, trois tensioactifs dérivés du DDM ont été conçus et synthétisés. L'intérêt est d'augmenter la rigidité et le caractère lipophobe des parties hydrophobes des tensioactifs par rapport au DDM, pour les rendre moins déstabilisants envers la protéine: soit par l'incorporation d'un groupement bicyclohexyle (PCC-maltoside), soit par l'ajout d'un segment fluoré de longueur modulable (F4H5- et F2H9-maltoside). Nous avons inclus également le F8TAC, tensioactif fluoré utilisé depuis une vingtaine d'années pour le maintien en solution des PM, et les "tripodes", amphiphiles faciaux dont la géométrie particulière n'avaient jamais été testée. Nous avons ensuite réalisé la caractérisation physico-chimique, en solution, de ces tensioactifs et du DDM en terme de CMC (concentration micellaire critique), nombre d'agrégation, taille (par diffusion de la lumière dynamique, DLS), facteur de forme (par diffusion des rayons X aux petits angles, SAXS) et facteur de structure (par mesure du second coefficient du viriel, indicateur du potentiel des tensioactifs à initier la cristallisation)afin de déterminer les caractéristiques importantes au maintien en solution et à la cristallisation des PM. Le PCC-malt présentant le même comportement que le DDM,nous l'avons sélectionné pour réaliser une étude en présence de la protéine.Après avoir mis au point une méthode de dosage des tensioactifs par HPTLC (HighPerformance Thin Layer Chromatography) et identifier les lipides présents dans les de Rhodobacter blasticus, nous avons pu quantifier les quantités de lipides et de tensioactifs associés à la protéine en présence de DDM et de PCC-malt.Enfin, dans une dernière partie, nous avons réalisé des essais de cristallisation du complexe RC-LH1-pufX en présence des tensioactifs sélectionnés pour faire le lien entre les conditions de cristallisation et l'étude physico-chimique des micelles en solution.
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Ko, Chia-Chieh, and 柯家傑. "Preparation of Hydrophobic Inorganic Membranes for VacuumMembrane Distillation and Vacuum Membrane Crystallization." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/zfrfwu.
Full textAbstract:
博士國立臺灣大學
化學工程學研究所
107
Lack in pure water and raw material is an urgent problem accompanying with the rise in life quality requirement and the rapid development in industrial technology. Furthermore, the treatment of the discharge from technology plants, seawater desalination plants and wastewater treatment plants is another issue. Membrane distillation (MD) and membrane crystallization (MCr) are relatively emerging membrane separation processes with potential to recover pure water and minerals from non-volatile concentrated aqueous solution that offers exciting opportunities to achieve the target, “Zero Liquid Discharge, (ZLD).” In recent years, inorganic membranes including metal, ceramic membranes have attracted great of interest due to its high mechanical strength, good thermal stability and chemical durability that can be used in extreme membrane separation process for various application such as ultra-pure water or organic solvent production and heavy-metal wastewater treatment, however industrialization might be a challenge. Tubular and hollow fiber membrane are more attractive because of their large-scale production and high packing density, especially hollow fiber which provides packing density greater than 1000 m2/m3, as respect to flat sheet membrane. This study is dedicated to preparation of hydrophobic tubular and hollow fiber membranes, investigation of fabricated parameter effect on membrane structures, applicability confirmation of ceramic membranes for MD/MCr process. In this study, PMSQ tubular aerogel membrane and hydrophobic alumina hollow fiber membrane were applied to VMD and VMCr processes. The former was prepared by coating PMSQ aerogel on alumina tubular support via sol-gel method, and the latter was fabricated by combined phase-inversion and sintering method and hydrophobilization. To optimization, we explored the influence of preparation parameters on the structures of both membranes and VMD performance. Our findings led us to give following conclusions: (1) Growth quality of PMSQ aerogel on alumina tubular support was depended on solvent/precursor molar ratio. (2) Even though the raise in modified frequencies improved the growth quality, it caused the decay in average pore size and porosity. (3) Pore size of PMSQ tubular aerogel membrane can be tuned by changing the HCl concentration used in sol-gel method. (4) Air gap was important for making alumina hollow fiber with symmetric cross-sectional geometry. (5) Suspension flow rate affected the microstructures in alumina hollow fiber. (6) Raise in sintering temperature, 1400 to 1500℃, caused significant drop in average pore size and porosity of alumina hollow fiber. Finally, the alumina hollow fiber membrane which prepared with the parameters of 20 cm, 15 ml/min and 1400℃ in air gap, suspension flow rate and sintering temperature, exhibited 220 nm in average pore size and high pure water flux as 3000 Lm−2h−1bar−1. After hydrophobilizaiton, the hydrophobic alumina hollow fiber membrane showed 60 Lm-2h-1 in permeate flux and over 99.9% in rejection in VMD using 3.5 wt% NaCl aqueous solution at 70℃ at 3 kPa. The high porosity of 55% and thin thickness of hollow fiber made the permeate flux exceeded that of PMSQ tubular aerogel membrane that also overtook the literature data. Furthermore, well-VMD-performance ceramic membranes were applied to VMCr and eventually produced NaCl and LiCl crystals from highly concentrated aqueous solution. It is the first time applying ceramic membrane to MCr process.
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Su, Po-Chen, and 蘇柏丞. "Operation and Kinetic Modeling of Membrane-assisted Crystallization." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/789h5b.
Full textAbstract:
碩士國立臺灣大學
化學工程學研究所
106
Membrane-assisted crystallization processes have the potential to reduce energy consumption and equipment size (footprint) compared to evaporative crystallization. In this thesis, reverse osmosis membranes and porous hydrophobic membranes are considered for the removal of water from solution to facilitate crystallization. Experiments using both types of membranes are performed and mathematical models of membrane-assisted crystallization processes are also constructed. Simulation of the process is also conducted to identify conditions that minimize the nucleated crystal volumetric ratio (μ_(3,n)⁄(μ_3)). Adipic acid and potassium chloride are used in the experimental work for the reverse osmosis membrane and the porous hydrophobic membrane respectively. Preliminary experimental work has been done including solubility measurements, data collection and regression for relation of temperature and conductivity measurements for online measurement of concentration, and salt rejection tests to measure membrane selectivity. Five substances are chosen for the membrane-assisted crystallization simulation: potassium nitrate, potassium sulfate, pentaerythritol, succinic acid, and potassium alum; the kinetic parameters from literature are used. Two limiting cases of crystallizer design are considered: one in which only clear solution is circulated from the crystallizer to the buffer tank, and one in which a crystal slurry is circulated. In the simulation, the effect of seed loading and seed size on the objective function is discussed. Furthermore, in order to understand the influence of membrane water removal rate on the product nucleation volumetric ratio, different water removal trajectories as function of time were considered.
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Quist-Jensen, Cejna Anna, Enrico Drioli, Francesca Macedonio, and Raffaele Molinari. "Membrane crystallization for recovery of valuable compounds from waste streams." Thesis, 2016. http://hdl.handle.net/10955/1290.
Full textAbstract:
Scuola di Dottorato "Pitagora" in Scienze Ingegneristiche, Dottorato di Ricerca in Ingegneria Chimica e dei Materiali, Ciclo XXVIII, a.a. 2015-2016Sustainable development and Process intensification strategy are guidelines for industrial processes in perspective. It is becoming more and more common that industry wants to fully exploit their resources due to environmental regulations, economic gain, sustainable standpoint, etc. In this perspective, waste streams have to be turned into resources in the most environmental friendly, economic and sustainable way. Membrane Engineering is already a key-figure to realize this objective. Novel membrane technologies such as membrane distillation (MD), membrane crystallization (MCr), pressure retarded osmosis (PRO), reverse electrodialysis (RED) and forward osmosis (FO), are evolving and are being suggested for a better exploitation of waste streams. This Ph.D. study focusses, particular, on Membrane crystallization (MCr), which is a novel technology for simultaneously production of water and minerals. It has several advantages with respect to conventional crystallizers in terms of purity, controlled kinetics and crystal morphology. Moreover, MCr is able to treat high concentration solutions, which are challenging for other traditional membrane operations. The current Ph.D. work emphasizes on various aspects of membrane crystallization for approaching zero-liquid discharge in industrial processes. Improved membranes, specifically developed for MCr applications, have to be manufactured. In this study, preliminary suggestions on membrane features are given for the requirements in MCr. Lab-made PVDF membranes with different characteristics have been tested and evaluated for their performance in MCr. This study, suggests that membranes with symmetric sponge layer structure and low thickness are favorable. Membrane of asymmetric structure with many macrovoids seems more pronounced to suffer from wetting. Moreover, it has been shown that, membrane crystallization is able to treat several kinds of feed solutions including RO brine, produced water and wastewater containing high amounts of sodium sulfate. The recovered crystals exhibit high purity, good size distribution and controlled growth. Na2SO4 can be recovered as different polymorphs and in this study it has been crystallized in the anhydrous form (Thenardite). Moreover, the process has shown excellent stability in terms of transmembrane flux and maintenance of hydrophobicity of the membrane. In some cases the treatment has been continued for more than 90 hours by only slight cleaning with distillate water. Membrane crystallization, in the direct-contact membrane distillation configuration, can normally treat solutions with very high concentrations. However, its limitations in the recovery of lithium from single salt solutions have been highlighted in this study. Vapor pressure, due to increase in concentration, is reduced significant, that it is not possible to reach LiCl saturation by this configuration. Likewise, combined direct-contact and osmotic distillation configuration have not been able to increase the driving force enough in order to exceed saturation. Instead vacuum membrane distillation has been introduced to eliminate the osmotic phenomena. This configuration has been able to recover LiCl in two different polymorph structures depending on the utilized operative conditions. Furthermore, integrated membrane system, including membrane crystallization, has shown excellent capability to treat orange juice. The quality of the juice has been maintained through ultrafiltration, membrane distillation and membrane crystallization treatment. In this study, the MD/MCr feed temperature is kept below 30 °C causing a relatively low flux. However, it has still been possible to reach from a concentration of 9 °brix to 65 °brix using MD/MCr. The advantages of MD/MCr with respect to isothermal osmotic membrane distillation configuration, is the elimination of the reconcentration stages of the draw solution. All the carried out case studies show that MD/MCr is able to reduce the volume of the waste stream significantly. The obtained results might be used as guidelines for practical application. Moreover, the low temperatures and atmospheric pressures utilized, makes it possible in real industrial processes to use waste or low-grade heat. Unlike other processes, MCr is able to produce two high quality products (i.e. water and salts) and will therefore not produce any additional waste. Hereby, the extended treatment by means of MCr will only positively influence the overall “sustainability” of the entire industrial process.
Università degli Studi della Calabria
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Ghanei, Hamed. "Biophysical and Structural Studies of Lipopeptide Detergents." Thesis, 2013. http://hdl.handle.net/1807/43565.
Full textAbstract:
Biochemical and structural studies of membrane protein usually require their stabilization in solution with detergents. However, purified membrane proteins often show reduced activity and stability in traditional detergents. Lipopeptide detergents (LPDs) are a new class of engineered amphiphiles that from small micelles and mimic the lipid bilayer more closely than traditional detergents. An LPD molecule consists of an α-helical peptide with fatty acyl chains covalently attached to both ends. These molecules self-assemble into cylindrical micelles with a hydrophilic exterior and a hydrophobic interior made up of the fatty acyl chains. Here we present the biophysical and biochemical properties of a model bacterial ABC transporter, MsbA, in different LPD variations.
Four types of LPD molecules have been synthesized and have been categorized as original LPD, LPD5Q, LPD2, and LPD4 based on the peptide sequence. Dynamic light scattering, thermal aggregation, and ATPase activity were used to measure the biophysical properties of MsbA in LPDs and in a traditional detergent, n-decyl-β-D-maltoside (DM). The results show that MsbA-LPD particles are monodisperse with small hydrodynamic radii. When compared to DM, MsbA is thermodynamically more stable and has higher catalytic activity in LPDs. Membrane proteins have favorable biophysical properties in LPDs, suggesting that these detergents resemble the native lipid bilayer environment more closely.
We also present crystal structures of three LPDs: LPD-12, LPD5Q-14 and LPD- 14. These structures reveal that LPD micelles are highly ordered with varying oligomeric states. The octomeric structure of the LPD-12 micelle is composed of four sets of antiparallel coiled-coil dimers, while the LPD5Q-14 micelle assembles as a nonamer of three trimers each with an “up-up-down” topology. The LPD-14 micelle, on the other hand, is a dodecamer of three tetramers with all helices assuming an antiparallel orientation. Overall, the structures of LPDs show highly ordered detergent micelles that are made up of repeated building blocks. Based on these results, we propose that LPDs can sample multiple conformational states, but the number of accessible conformations is significantly reduced relative to traditional detergents.
Our results show that LPDs are an alternative platform for in vitro studies of membrane proteins. Future studies will focus on the crystallization of membrane proteins in LPDs and the further characterization of these complexes.
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Liu, Che-Wen, and 劉哲文. "The Preparation of Cobaltous Sulfate Crystals by Air-Gap Membrane Distillation Crystallization Technology." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/g2r577.
Full textAbstract:
碩士國立臺北科技大學
化學工程研究所
105
Membrane distillation is a new type of separation and purification technology, which combined with evaporation process and membrane separation technology both two methods advantages. The driving force is use the saturated vapor pressure difference between the hot fluid side and the cool fluid side temperature difference. Let the vapor of hot water side can pass through a porous hydrophobic membrane. To achieve the effect of separation of the solvent and solute. In this study, explore the cobaltous sulfate solution was concentrated and crystallized by use of the material for the polytetrafluoroethylene (PTFE) membrane in using air gap membrane distillation, because the AGMD advantages is the equipment is simple, low cost, easy to operate. We have variables, Such as the different concentration of the hot fluid, and the different volumetric flow rate of the hot and cold fluid. The collected samples were analyzed for permeability, rejection efficiency and crystal yield. We find the optimal operating conditions in the AGMD system is hot flow 9 L/min, cold flow 5 L/min. And the AGMD crystallization, the average energy efficiency of the feed solution 4.8 M is 84.92% and the average energy efficiency of the feed solution 5.2 M is 83.09%. The rejection efficiency will vary with feed solution concentration, when the low concentration (below 2.5 M), the rejection efficiency are more than 99.9%, at high concentration (above4.8 M), the rejection efficiency decreases with increasing feed concentration.
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PRUDNIKOVA, Tatyana. "Crystallization, structural and modeling studies of photosynthetic membrane protein and water soluble haloalkane dehalogenase." Doctoral thesis, 2011. http://www.nusl.cz/ntk/nusl-71479.
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This PhD thesis is devoted to study of a) water soluble protein haloalkane dehalogenase DbeA (wild type enzyme) and its mutant variant DbeA1 from bacteria Bradyrhizobium elkani USDA94 and also b) the inner membrane protein complex of Photosystem II (PSII) from Pisum sativum L. An active site architecture of the haloalkane dehalogenase enzymes DbeA, DbeA1, stabilization by loop insertion in a mutant form, and structure-function relationships of haloalkane dehalogenases have been studied by using experimental approaches protein crystallization and crystallography. The protein crystals have been grown and X-ray diffraction data on these crystals have been collected and 3D molecular structure has been solved for both dehalogenases DbeA and DbeA1 at 2.2 A? resolution, for DbeA crystals soaked with 1-fluoropentane as a substrate at 1.65 A? resolution, and with 1,3-dichloropropane substrate at 2.15 A? resolution. Crystallization and molecular modelling methods were applied on membrane protein PSII core complex from higher plants in order to find optimal conditions for the 3D crystals formation, to establish the crystallization protocol and to study light-induced processes in PSII reaction center.
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Lai, Yan-Syun, and 賴彥熏. "The Preparation of Manganese(II) Acetate Crystals by Air-Gap Membrane Distillation Crystallization Technology." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/k3d5p2.
Full textAbstract:
碩士國立臺北科技大學
化學工程與生物科技系化學工程碩士班
106
The discharge of waste water from the factory causes ecological pollution and also poses a risk to human health. The membrane distillation technology combined with the crystallization system can distill pure water from the wastewater and recover valuable crystals. This method not only reduces the pollution level of discharged wastewater but also recovers available resources from it. There is no doubt that there are promising wastewater treatment solutions. In this study, an air gap membrane distillation combined with a crystallization system was used to concentrate the manganese acetate solution and obtain a crystalline product. We changed operating conditions such as temperature, concentration, and flow rate. The feed solution and crystalline product were analyzed to investigate the effects of concentration and crystallization. The experimental results show that when we increase the feed concentration and reduce the flow rate, both the permeation flux and the manganese ion barrier effect will decrease. In addition, when the operating conditions are feed concentration 2.5 M and volume flow rate 1 L/min, we have the lowest permeate flux 0.246 kg/m2×h and the highest conductivity 446.4 μS/cm. Another result shows that increasing the temperature difference between the cooling water and the hot feed has a positive correlation with the permeate flux, and a lower feed temperature can increase the acetic acid ion rejection efficiency. When we fix the temperature difference between the cooling water and the hot feed, raising the water temperature of the cooling water and the hot feed will result in a larger permeation flux due to the increase of the kinetic energy of the water molecules. We used the Laser Diffraction Particle Size Analyzer (LPSA), Scanning Electron Microscope (SEM), and X-ray Diffraction Analyzer (XRD) to observe the surface and analysis structural of the crystallized product. The analysis results indicate that the increase of feed concentration will cause the XRD pattern of the crystalline product to have more miscellaneous phases. In addition, the change of the feed temperature also affects the particle size distribution. These results demonstrate that operating conditions can control the formation of crystals.
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SANTONICOLA, MARIAGABRIELLA. "Molecular self-assembly and interactions in solutions of membrane proteins and surfactants." Doctoral thesis, 2007. http://hdl.handle.net/11573/506839.
Full textAbstract:
Membrane proteins are amphiphilic proteins that are associated with biological membranes. They regulate critical functions between the cells and their surroundings, yet the relationship between structure and functionality for most of these proteins is still elusive. Integral membrane proteins often span the entire bilayers, and they are characterized by a hydrophobic domain that rests in the membrane and hydrophilic regions on either side of the membrane. These proteins are removed from their native membranes for purification and characterization, and surfactants are typically used to solubilize the hydrophobic portion of the molecule. Unfortunately, membrane proteins often exhibit poor stability when solubilized in surfactant solutions and they are very difficult to crystallize. The goal of this dissertation is to contribute to the understanding of how the self-assembly of surfactants in solution affects the stability of solubilized membrane proteins.
Liquid-liquid phase boundaries in surfactant solutions have been suggested to have a prominent role in promoting the crystallization of protein-surfactant complexes. Medium-chain alkyl monoglucosides are highly soluble nonionic surfactants that are widely used for the solubilization of membrane proteins. In combinations with more hydrophobic surfactants or water-soluble polymers such as polyethylene glycol (PEG), solutions of these alkyl monoglucosides exhibit miscibility gaps that can be shifted rationally in the temperature and concentration windows. Isothermal titration calorimetry is used to quantify the effect of PEG on the micellization properties of the alkyl monoglucosides, whereas small-angle neutron scattering gives insight into the microstructure of the mixtures near liquid-liquid phase boundaries. Analysis of the scattering profiles using indirect Fourier transformation methods reveals that the role of PEG is to drastically change the strength and range of intermicellar interactions with minimal impact on the geometry of the micelles. These observations confirm the role of the surfactant phase boundary on tuning attractive micellar interactions, and can be related to current protein crystallization strategies.
Understanding how surfactants bind to membrane proteins and affect their stability is essential for the manipulation of these proteins outside native membranes. Contrast variation studies by analytical ultracentrifugation and small-angle neutron scattering enable measurement of the composition of the protein-surfactant complexes and determination of the thickness of the surfactant shell bound to the protein. When bacteriorhodopsin is solubilized in solutions of alkyl polyglucosides, the surfactant layer around the protein has a thickness equal to a single amphiphile molecule or larger. The thickness of the surfactant shell increases with increasing surfactant length, and it is generally unrelated to the aggregation number of the micelles even for small and predominantly hydrophobic membrane proteins. Studies of bacteriorhodopsin activity by absorption spectroscopy show that the surfactant arrangement as a single layer directly correlates with a limited stability of the protein over time. A similar connection between surfactant binding and protein stability is observed when bacteriorhodopsin is illuminated and active in pumping protons. These results are useful to guide the choice of surfactant solutions for optimal solubilization of membrane proteins, which is the key to increasing success rates in crystallization and functional studies of these proteins.
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Caridi, Antonella, Enrico Drioli, Profio Gianluca Di, and Raffaele Molinari. "An insight on pharmaceutical crystallization process by using membrane technology: PVDF-based mixed matrix membranes and PP grafted membranes as new tools for controlling the supersaturation rate and the heterogeneous nucleation mechanism." Thesis, 2014. http://hdl.handle.net/10955/1197.
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Dottorato di Ricerca in "Ingegneria Chimica e dei Materiali" Ciclo XXVI, a.a. 2013-2014Questo elaborato finale del progetto di dottorato tratta lo studio del processo di cristallizzazione a membrana finalizzato alla produzione di composti farmaceutici in forma cristallina. Lo studio ha come obiettivo quello di dare una visione globale del processo di cristallizzazione a membrana andando oltre lo stato dellate, bensì popoedo lipleetazione della tecnica di cristallizzazione a membrana di base. A tal proposito il progetto è stato sviluppato seguendo in due diverse direzioni: da una parte la tecnica di istallizzazioe a eaa di ase ha isto lappliazioe ad uo speifio settore dellidustia faaeutia, dallalta pate lo studio è poseguito investigando i meccanismi di cristallizzazione indotti dalla stessa membrana e successivamente ha visto una vera e propria progettazione di membrane opportunamente pensate per la cristallizzazione. Duue, il aloe aggiuto di tale studio osiste ellaee diostato la possibilità di ampliare il campo di applicazione del processo a membrana, di aver esteso la conoscenza di base dei meccanismi di nucleazione eterogenea sottesi dalla membrana e di aver progettato, prodotto e caratterizzato delle membrane con differenti materiali e strutture appositamente per essere testati nella tecnica di cristallizzazione. In dettaglio, il lavoro presenta uno studio iniziale sul processo di nucleazione eterogenea che parte da particelle libere in soluzione per poi continuare studiando il processo di nucleazione eterogenea sullle membrane stesse. Ua seoda sezioe tatta lappliazioe del processo a membrana alla cocristallizzazione farmaceutica. Successivamente inizia la parte di disegno e realizzazione di membrane eterogenee sia dal punto di vista chimico che strutturale: membrane fabbricate con tecniche e materiali differenti e membrane commerciali che sono state opportunamente funzionalizzate. Infine il lavoro si conclude con i tests di cristallizzazione condotti su tali membrane.
Università degli Studi della Calabria
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McQuade, David Tyler. "Further studies of 1,6 methano[10]annulene-derived contrafacial amphiphiles and synthesis of tripod detergents for membrane protein solubilization and crystallization." 1998. http://catalog.hathitrust.org/api/volumes/oclc/43611309.html.
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Aleid, Sara. "Design and Fabrication of Multi-functional Photovoltaic-Membrane Distillation-Evaporative Crystallizer for Water Desalination, Electricity Generation, Salt Crystallization and Solar Cell Cooling." Thesis, 2019. http://hdl.handle.net/10754/660160.
Full textAbstract:
Ensuring electricity availability and acquiring access of potable water during emergencies in remote areas are becoming a global challenge around the world. Utilizing solar energy electricity generation by photovoltaics and clean water production by solar distillation have shown its great potential to meet the world’s future energy and water demands. In this work, we fabricated a photovoltaic-membrane distillation-evaporative crystallizer device (PV-MD-EC), in which high electricity generation efficiency (~15%), clean water production rate (~2.66 kg/m2 h) and salt crystallization from seawater can be achieved in such an integrated system under one sun illumination. In addition, the solar cell operates in a much lower temperature at around 48 oC, which is much lower than previous work. The advanced performance is attributed to the utilization of a highly porous and thinner hydrophobic membrane. This design provides a new strategy to address the challenge of water-energy nexus.
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(8715135), Siddhi-Santosh Hate. "DISSOLUTION AND MEMBRANE MASS TRANSPORT OF SUPERSATURATING DRUG DELIVERY SYSTEMS." Thesis, 2020.
Find full textAbstract:
Supersaturating drug delivery systems are an attractive solubility enabling formulation strategy for poorly soluble drugs due to their potential to significantly enhance solubility and hence, bioavailability. Compendial dissolution testing is commonly used a surrogate for assessing the bioavailability of enabling formulations. However, it increasingly fails to accurately predict in vivo performance due its closed-compartment characteristics and the lack of absorptive sink conditions. In vivo, drug is continually removed due to absorption across the gastrointestinal membrane, which impacts the luminal concentration profile, which in turn affects the dissolution kinetics of any undissolved material, as well as crystallization kinetics from supersaturated solutions. Thus, it is critical to develop an improved methodology that better mimics in vivo conditions. An enhanced approach integrates dissolution and absorption measurements. However, currently-used two-compartment absorptive apparatuses, employing a flat-sheet membrane are limited, in particular by the small membrane surface area that restricts the mass transfer, resulting in unrealistic experimental timeframes. This greatly impacts the suitability of such systems as a formulation development tool. The goal of this research is two-fold. First, to develop and test a high surface area, flow-through, absorptive dissolution testing apparatus, designed to provide in vivo relevant information about formulation performance in biologically relevant time frames. Second, to use this apparatus to obtain mechanistic insight into physical phenomenon occurring during formulation dissolution. Herein, the design and construction of a coupled dissolution-absorption apparatus using a hollow fiber membrane module to simulate the absorption process is described. The hollow fiber membrane offers a large membrane surface area, improving the mass transfer rates significantly. Following the development of a robust apparatus, its application as a formulation development tool was evaluated in subsequent studies. The dissolution-absorption studies were carried out for supersaturated solutions generated via anti-solvent addition, pH-shift and by dissolution of amorphous formulations. The research demonstrates the potential of the apparatus to capture subtle differences between formulations, providing insight into the role of physical processes such as supersaturation, crystallization kinetics and liquid-liquid phase separation on the absorption kinetics. The study also explores dissolution-absorption performance of amorphous solid dispersions (ASDs) and the influence of resultant solution phase behavior on the absorption profile. Residual crystalline content in ASDs is a great concern from a physical stability and dissolution performance perspective as it can promote secondary nucleation or seed crystal growth. Therefore, the risk of drug crystallization during dissolution of ASDs containing some residual crystals was assessed using absorptive dissolution measurements and compared to outcomes observed using closed-compartment dissolution testing. Mesoporous silica-based formulations are another type of amorphous formulations that are gaining increased interest due to higher physical stability and rapid release of the amorphous drug. However, their application may be limited by incomplete drug release resulting from the adsorption tendency of the drug onto the silica surface. Thus, the performance of mesoporous silica-based formulations was also evaluated in the absorptive dissolution testing apparatus to determine the impact of physiological conditions such as gastrointestinal pH and simultaneous membrane absorption on the adsorption kinetics during formulation dissolution. Overall, the aim of this research was to demonstrate the potential of the novel in vitro methodology and highlight the significance of a dynamic absorptive dissolution environment to enable better assessment of complex enabling formulations. In vivo, there are multiple physical processes occurring in the gastrointestinal lumen and the kinetics of these processes strongly depend on the absorption kinetics and vice-a-versa. Thus, using this novel tool, the interplay between solution phase behavior and the likely impacts on bioavailability of supersaturating drug delivery systems can be better elucidated. This approach and apparatus is anticipated to be of great utility to the pharmaceutical industry to make informed decisions with respect to formulation optimization.
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Polino, Mariella. "Ion-exchange membranes for protein crystallization and protein crystals’ derivatization." Doctoral thesis, 2019. http://hdl.handle.net/10362/73017.
Full textAbstract:
This PhD thesis is focused on the application of ion-exchange membranes for protein
crystallization and protein crystals derivatization. The experimental work is divided in
three parts. The first part of the work is focused on the understanding of the effect of
topography on nucleation. Soft lithography is used to modify the surface topography
of Nafion® membranes with target designs, avoiding changes of surface chemistry that
might mask the effect of topography on nucleation. The imprinted membranes are characterized
by Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM) and
contact angle and tested for the crystallization of Trypsin from Bovine Pancreas. Nucleation
and crystals growth are followed over time by optical microscope. Experimental
results are compared with theoretical calculations of the ratio of change of Gibbs free
energy of heterogeneous to homogeneous nucleation. The second part of the work is
focused on the development of a method for performing a gentle derivatization of protein
crystals using ion-exchange membranes. Nafion® and Neosepta 01 were selected
after an initial screening of several membranes, due to their ability of promoting nucleation.
The kinetics of ion-transport for Br, PtCl2
4 and Hg2+ is evaluated and used for
modelling the transport in the derivatization set-up. Stability of crystals derivatized by
ion-exchange membranes over time is compared with the stability of crystals derivatized
by the conventional soaking method. The crystals derivatized by the help of the ionexchange
membranes are analysed by synchrotron and protein structure resolved with
the Isomorphous Replacement technique. The third part of the work involves the integration
of the ion-exchange membrane derivatization concept in a Polydimethylsiloxane
(PDMS) microdevice. A microdevice composed of two compartments, one with channels
and one with wells is designed and built by photolithography and soft-lithography.
Bonding of the membrane to the PDMS parts is done by grafting. Transport modelling
of water, NaCl and Hg2+ transport in the microdevice, crystallization experiments where supersaturation is achieved by osmosis and evaluation of the crystals’ diffraction quality
are performed.
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Sharma, Maya. "Microporous Membranes Derived using Crystallisation Induced Phase Separation in PVDF/PMMA (Polyvinylidene Fluoride/ Polymethyl Methacrylate) Blends in Presence of Multiwalled Carbon Nanotubes." Thesis, 2017. http://etd.iisc.ac.in/handle/2005/3266.
Full textAbstract:
Segmental chain dynamics in polymer blends is a very important topic, not only from a fundamental point of view but also from technological applications. Because of the difficulties in the commercialization of new polymers, industries have turned increasingly towards blending of polymers to optimise their end use (mechanical, rheological) properties. The design of tailor-made materials would be enormously facilitated by the understanding of the blending phenomena at a molecular level. The key question to address is to understand the dynamics of each component of the blend modified by blending? The thesis has systematically studied the effect of multiwalled carbon nanotubes on the chain dynamics, demixing temperature, structural properties and evolution of morphology in a classical miscible polymer blend system (PVDF/PMMA).
The thesis comprises of six chapters, Chapter 1 is an introductory chapter that outlines the fundamentals of polymer blends, crystallisation in polymer blends and the basics of dielectric spectroscopy. As one of the rationales of this work is to systematic study whether phase separated in these blends can be used as a tool to develop membrane for water purification. This chapter also gives an overview of the reported studies of ultrafiltration membrane fabrication, factors affecting membrane morphology and flux. In Chapter 2, the materials and methodology used to carry out experiments and the experimental procedures are discussed.
Chapter 3 discusses the effect of concentration of PMMA and amine functionalized multiwalled carbon nanotubes (MWNTs) on the crystallisation induced phase separation using FTIR, XRD, POM and shear rheology. Electron microscopy and selective etching confirmed the localisation of MWNTs in the PVDF phase of the blends. Blends with MWNTs facilitated in heterogeneous nucleation manifesting in an increase in crystallisation temperature. The crystallisation induced phase separation in PVDF/PMMA blends was observed to influence the interconnected network of MWNTs in the blends.
Chapter 4 discuss the effect of concentration of PMMA and MWNTs on the miscibility and the segmental relaxations was probed in situ by DSC and dielectric relaxation spectroscopy (DRS). The dynamic heterogeneity in the blends as manifested by the presence of an extra relaxation at a higher frequency at or below the crystallisation induced phase separation temperature was also discussed. We found that PVDF/PMMA blend (PVDF ≥ 80 wt%) exhibits three distinct relaxations; αc corresponding to crystalline PVDF, αβ segmental relaxation of PMMA and αm of amorphous miscibility whereas all relaxations overlap and constitute a single broad relaxation in PVDF/PMMA blend (PVDF ≤ 70 wt%). This confirms that there is a certain composition width in this blend wherein three distinct relaxations can be traced. This could due to many reasons like the width of crystal-amorphous interphase in the crystal lamellae, crystal size and morphology is strongly contingent on the concentration of PMMA. Relaxations are not very distinct in presence of MWNTs due to defective spherulites that shift the relaxations towards a higher frequency.
Chapter 5 has attempted to tune the microporous morphology of PVDF membranes using crystallisation induced phase separation in PVDF/PMMA blends. As PVDF/PMMA is a melt-miscible blend, the samples were allowed to crystallise and the amorphous PMMA phase, which isolates in the interlamellar or inter-spherulitic regions in the blends, was etched out to generate
microporous structures. The pore sizes can be tuned by varying the PMMA concentration in the blends. We observed that 60/40 PVDF/PMMA blends showed larger pores as compared to 90/10 PVDF/PMMA blends. We further modified PVDF membranes by sputtering silver on the surface. The bacterial cell viability was distinctly suppressed (99 %) in silver sputtered membranes. The ICP analysis suggests that slow Ag+ ions release from the sputtered membrane surface assisted in developing antibacterial surface. Our findings open new avenues in designing water filtration membranes and also help in understanding the crystallisation kinetics for tuning pore size in membranes.
Chapter 6 summarises the important results of this work. MWNTs act as hetero nucleating agent and specifically interact with PVDF thereby influences the dynamics of PVDF chains. MWNTs can also restrict the amorphous segmental mobility and can influence the intermolecular cooperativity and coupling. The crystallisation induced phase separation in various blends can result in various crystalline morphologies depending on the PVDF concentration. By selectively etching PMMA from the phase-separated blends, microporous morphology can be generated
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Sharma, Maya. "Microporous Membranes Derived using Crystallisation Induced Phase Separation in PVDF/PMMA (Polyvinylidene Fluoride/ Polymethyl Methacrylate) Blends in Presence of Multiwalled Carbon Nanotubes." Thesis, 2017. http://hdl.handle.net/2005/3266.
Full textAbstract:
Segmental chain dynamics in polymer blends is a very important topic, not only from a fundamental point of view but also from technological applications. Because of the difficulties in the commercialization of new polymers, industries have turned increasingly towards blending of polymers to optimise their end use (mechanical, rheological) properties. The design of tailor-made materials would be enormously facilitated by the understanding of the blending phenomena at a molecular level. The key question to address is to understand the dynamics of each component of the blend modified by blending? The thesis has systematically studied the effect of multiwalled carbon nanotubes on the chain dynamics, demixing temperature, structural properties and evolution of morphology in a classical miscible polymer blend system (PVDF/PMMA).
The thesis comprises of six chapters, Chapter 1 is an introductory chapter that outlines the fundamentals of polymer blends, crystallisation in polymer blends and the basics of dielectric spectroscopy. As one of the rationales of this work is to systematic study whether phase separated in these blends can be used as a tool to develop membrane for water purification. This chapter also gives an overview of the reported studies of ultrafiltration membrane fabrication, factors affecting membrane morphology and flux. In Chapter 2, the materials and methodology used to carry out experiments and the experimental procedures are discussed.
Chapter 3 discusses the effect of concentration of PMMA and amine functionalized multiwalled carbon nanotubes (MWNTs) on the crystallisation induced phase separation using FTIR, XRD, POM and shear rheology. Electron microscopy and selective etching confirmed the localisation of MWNTs in the PVDF phase of the blends. Blends with MWNTs facilitated in heterogeneous nucleation manifesting in an increase in crystallisation temperature. The crystallisation induced phase separation in PVDF/PMMA blends was observed to influence the interconnected network of MWNTs in the blends.
Chapter 4 discuss the effect of concentration of PMMA and MWNTs on the miscibility and the segmental relaxations was probed in situ by DSC and dielectric relaxation spectroscopy (DRS). The dynamic heterogeneity in the blends as manifested by the presence of an extra relaxation at a higher frequency at or below the crystallisation induced phase separation temperature was also discussed. We found that PVDF/PMMA blend (PVDF ≥ 80 wt%) exhibits three distinct relaxations; αc corresponding to crystalline PVDF, αβ segmental relaxation of PMMA and αm of amorphous miscibility whereas all relaxations overlap and constitute a single broad relaxation in PVDF/PMMA blend (PVDF ≤ 70 wt%). This confirms that there is a certain composition width in this blend wherein three distinct relaxations can be traced. This could due to many reasons like the width of crystal-amorphous interphase in the crystal lamellae, crystal size and morphology is strongly contingent on the concentration of PMMA. Relaxations are not very distinct in presence of MWNTs due to defective spherulites that shift the relaxations towards a higher frequency.
Chapter 5 has attempted to tune the microporous morphology of PVDF membranes using crystallisation induced phase separation in PVDF/PMMA blends. As PVDF/PMMA is a melt-miscible blend, the samples were allowed to crystallise and the amorphous PMMA phase, which isolates in the interlamellar or inter-spherulitic regions in the blends, was etched out to generate
microporous structures. The pore sizes can be tuned by varying the PMMA concentration in the blends. We observed that 60/40 PVDF/PMMA blends showed larger pores as compared to 90/10 PVDF/PMMA blends. We further modified PVDF membranes by sputtering silver on the surface. The bacterial cell viability was distinctly suppressed (99 %) in silver sputtered membranes. The ICP analysis suggests that slow Ag+ ions release from the sputtered membrane surface assisted in developing antibacterial surface. Our findings open new avenues in designing water filtration membranes and also help in understanding the crystallisation kinetics for tuning pore size in membranes.
Chapter 6 summarises the important results of this work. MWNTs act as hetero nucleating agent and specifically interact with PVDF thereby influences the dynamics of PVDF chains. MWNTs can also restrict the amorphous segmental mobility and can influence the intermolecular cooperativity and coupling. The crystallisation induced phase separation in various blends can result in various crystalline morphologies depending on the PVDF concentration. By selectively etching PMMA from the phase-separated blends, microporous morphology can be generated