Dissertations / Theses on the topic 'Hydrophobic interaction'

We develop a coarse grained methodology to study solvent mediated interactions between two or more hydrophobic spheres. The free energy of a configuration of two hydrophobic hard spheres is calculated as a function of their separation to understand the thermodynamic force between them mediated by water. The range of the hydrophobic interaction is found to be of the order of the equilibrium correlation length of water; beyond this range the hydrophobicity induced force is negligible. We also examine the free energy landscape corresponding to the two interacting hydrophobic spheres, and find a new intermediate state between the two states of separate and non-interacting spheres and a weakly bound cluster. The nature of this intermediate state changes depending on the size of the spherical particles, and even disappears beyond a minimum critical radius. Our results are relevant to the understanding of hydrophobic mediated interactions in coarse grained models of protein folding and protein protein interactions which, to date, have only accounted for hydrophobicity in an empirical way.

This study aims the preparation of magnetic poly(2-hydroxyethylmethacrylate-N-methacryloyl-Lphenylalanine), [poly(HEMA-MAPA)] nanoparticles for plasmid DNA separation on the basis of hydrophobic interactions. Magnetic nanoparticles will be produced emulsion polymerization of 2- hydroxyethylmethacrylate (HEMA) and N-methacryloyl-L-phenylalanine (MAPA) monomers. Nanosized particles including hydrophobic groups stemmed from polymerizable derivative of phenylalanine aminoacid were evaluated to offer surface area that is enough for the higher capacity DNA purification than commercial micronsized sorbents for DNA purification. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/34946

Rigid poly[hydroxyethyl acrylate-co-poly(ethylene glycol) diacrylate] (Poly(HEA-co-PEGDA) monoliths were synthesized inside 75-µm i.d. capillaries by one-step UV-initiated copolymerization using methanol and ethyl ether as porogens. The optimized monolithic column was evaluated for hydrophobic interaction chromatography (HIC) of standard proteins. Six proteins were separated within 20 min with high resolution using a 20 min elution gradient, resulting in a peak capacity of 54. The performance of this monolithic column for HIC was comparable or superior to the performance of columns packed with small particles. Monoliths synthesized solely from PEGDA were also found to show excellent performance in HIC of proteins. Continuing efforts showed that rigid monoliths could be synthesized from PEGDA or poly(ethylene glycol) dimethacrylates (PEGDMA) containing different ethylene glycol chain lengths for HIC of proteins. Effects of PEG chain length, bi-porogen ratio and reaction temperature on monolith morphology and back pressure were investigated. Monoliths prepared from PEGDA 258 were found to provide the best chromatographic performance with respect to peak capacity and resolution. An optimized PEGDA 258 monolithic column was able to separate proteins using a 20-min elution gradient with a peak capacity of 62. The preparation of these in situ polymerized single-monomer monolithic columns was highly reproducible. The single-monomer synthesis approach clearly improves column-to-column reproducibility.The highly crosslinked monolith networks resulting from single crosslinking monomers were found to enhance the surface area of the monolith and concentrations of mesopores. Thus, monolithic columns were developed from four additional crosslinking monomers, i.e., bisphenol A dimethacrylate (BADMA), bisphenol A ethoxylate diacrylate (BAEDA, EO/phenol = 2 or 4) and pentaerythritol diacrylate monostearate (PDAM) for RPLC of small molecules. Gradient elution of alkyl benzenes and alkyl parabens was achieved with high resolution using all monolithic columns. Porogen selection for the BADMA and PDAM was investigated in detail with the intention of obtaining data that could possiblly lead to a rational method for porogen selection.

Affibody® molecules are small affinity proteins with great opportunities in the application of biotechnological areas. Their popularity as biomolecules in pharmaceutical applications and research entails that the purity of Affibody® molecules is essential for their use as safe medical drugs. Hydrophobic Interaction Chromatography (HIC) is a purification technique that separates molecules based on their inherent difference in hydrophobicity, and the application as a purification strategy for Affibody® molecules may be possible. This thesis investigates the implementation of HIC on Affibody® molecules by screening for differences in binding, recovery, and purity influenced by different parameters, such as resin, pH, salt type, and salt concentration, have on binding, recovery, and purity. HIC presents as a viable purification method where an approximate 97% reduction in Host Cell Proteins was obtained. The yield of the purified product also presented as promising with a recovery of approximately 82%. These results indicate that further investigation and optimization of this technique may benefit the downstream process of the investigated Affibody® molecule.

The use of hydrophobic surfaces is important for many processes both in nature and industry. Interactions between hydrophobic species play a key role in industrial applications such as water-cleaning procedures and pitch control during papermaking but they also give information on how to design surfaces like hydrophobic mineral pigments. In this thesis, the influence of surface properties on wetting and interaction forces has been studied. Surfaces with close-packed particles, pore arrays, randomly deposited nanoparticles as well as reference surfaces were prepared. The atomic force microscope (AFM) was utilized for force and friction measurements while contact angles and confocal Raman microscopy experiments were mainly used for wetting studies. The deposition of silica particles in the size range of nano- to micrometers using the Langmuir-Blodgett (LB) technique resulted in particle coated surfaces exhibiting hexagonal close-packing and close to Wenzel state wetting after hydrophobization. Force measurements displayed long-range interaction forces assigned to be a consequence of air cavitation. Smaller roughness features provided larger forces and interaction distances interpreted as being due to fewer restrictions of capillary growth. Friction measurements proved both the surface structure and chemistry to be important for the observed forces. On hydrophobic pore array surfaces, the three-phase contact line of water droplets avoided the pores which created a jagged interface. The influence of the pores was evident in the force curves, both in terms of the shape, in which the three-phase contact line movements around the pores could be detected, as well as the depth of the pores providing different access and amount of air. When water/ethanol mixtures were used, the interactions were concluded to be due to ethanol condensation. Confocal Raman microscopy experiments with water and water/ethanol mixtures on superhydrophobic surfaces gave evidence for water depletion and ethanol/air accumulation close to the surface. Force measurements using superhydrophobic surfaces showed extremely long-range interaction distances. This work has provided evidence for air cavitation between hydrophobic surfaces in aqueous solution. It was also shown that the range and magnitude of interaction forces could, to some extent, be predicted by looking at certain surface features like structure,roughness and the overall length scales.<br><p>QC 20121011</p>

The interaction of a bubble with solid surfaces, hydrophobic and hydrophilic, was investigated. When a bubble approaches towards a solid surface, a thin liquid film forms between them. The liquid in the film drains until an instability forms and film ruptures resulting in a three phase contact (TPC). Following rupture, the TPC line spreads on the solid surface. In the present study, glycerol-water solutions with varying percentages of water were used to investigate the effect of viscosity. Experiments were carried out with varying bubble size. The rupture and TPC line movement were recorded by high-speed digital video camera. The dependence of the TPC line movement on different parameters was investigated. The experimental results were compared with the existing theories for the TPC line movement. An empirical equation was developed to predict the TPC line movement. Formation or rupturing of the intervening film in case of a hydrophilic surfaces, which were glass surface cleaned by six cleaning techniques, was investigated. It was shown that a stable film forms for acid or alkali cleaning.

We study the collapse transition of a lattice based protein model including an explicit coarse-grained model of a solvent. This model accounts for explicit hydrophobic interactions, and it is studied by Monte Carlo simulation. The protein is modelled as self-avoiding random walk with nearest neighbor interactions on a two dimensional lattice. Without the solvent, universal quantities of the chain around the collapse transition temperature are well known. Hydrophobicity is then modelled through a lattice of solvent molecules in which each molecule can have Q states depending of an orientation variable. Only one state is energetically favored, when two neighboring solvent molecules are both in the same state of orientation. The monomers are placed in interstitial position of the solvent lattice, and are only allowed to occupy sites surrounded by solvent cells of the same orientation. The potential of mean force between two interstitial solute molecules is calculated, showing a solvent mediated attraction typical of hydrophobic interactions. We then show that this potential increases with the energy of hydrogen bond formation as it appears in the model, while its characteristic range decreases. More importantly, we show that the chain embedded in the solvent undergoes a collapse transition, with the temperature of the transition being shifted relative to that of the chain in isolation. We calculate several critical exponents near the collapse transition, and we observe that their values are not conserved in presence of the explicit solvent.

Ce travail concerne l’altération du comportement de phase de membranes lipidiques lors de leur interaction avec des molécules hydrophiles ou hydrophobes. L’utilisation de sondes moléculaires de fluorescence sensibles à leur micro-environnement constitue un aspect majeur de ce travail. Les techniques de spectroscopie de fluorescence et de microscopie confocale ont été mises à profit pour l’étude du comportement de ces sondes, donnant accès au degré de compacité et d’ordre dans les membranes.Nos résultats montrent que le polystyrène, un plastique rencontré de façon commune dans les régions polluées des océans, présente la capacité de modifier le comportement de phase des membranes lipidiques, entrant notamment en compétition avec le cholestérol.Nous avons montré que la présence élevée de sucres, tel que l’on peut le rencontrer dans certaines situations relevant de la bio-préservation, a pour effet de rompre la qualité de compaction des lipides, et nous avons proposé un nouveau modèle thermodynamique pour interpréter nos résultats.Enfin, les effets sur la membrane de l’incorporation d’un polymère amphiphile comportant un cholestérol greffé ont été étudiés, dans le cadre de l’élaboration de nouvelles stratégies thérapeutiques à base de lipides<br>This work focuses on the alterations of lipid membrane phase behavior upon interaction with hydrophobic and hydophilic molecules. One major aspect of this thesis is the employement of environment sensitive probes to obtain information on the lipid bilayer packing by means of confocal spectral imaging and fluorescence spectroscopy. Our results show that polystyrene, a commonly found plastic in ocean wastes, has the ability to disrupt the lipid bilayer phase behavior and has a competitive interaction with cholesterol. The presence of high concentration of sugars, relevant in the field of biopreservation, has been found to alter the lipid bilayer packing and a new thermodynamics model has been proposed to complement the experimental results. Finally, the effects of an amphiphilic cholesterol-grafted polymer on model membrane was investigated, providing insight into potential new lipid therapeutic strategies

Les ions de taille nanométrique (nano-ions), tels que les clusters ioniques de bore, les polyoxométalates (POM) et les grands ions organiques, ont suscité un intérêt remarquable ces dernières années en raison de leur capacité à s’adsorber ou se lier à des systèmes chimiques électriquement neutres, tels que les molécules hôtes macrocycliques, les nanoparticules, les tensioactifs et les polymères, etc. Il a été démontré que ces processus d'adsorption ou de liaison sont induits par un phénomène médié par solvant, l'effet chaotropique, qui pousse le nano-ion de la masse d'eau vers une interface. Ainsi, l'eau d'hydratation de l'ion et de l'interface est libérée dans la masse d'eau, ce qui entraîne une restitution de la structure intrinsèque de l'eau. Cet effet est particulièrement fort pour les nano-ions. Ils sont par conséquent appelés ions superchaotropiques ou hydrophobes dans le prolongement des ions classiques (faiblement) chaotropiques tels que le SCN-. Tous les superchaotropes couramment étudiés, bien que chimiquement divers, partagent des caractéristiques physiques telles qu'une faible densité de charge et une grande polarisabilité. Les effets des nano-ions sur les auto-assemblages de tensioactifs non ioniques éthoxylés, les phases micellaires et bicouches, sont ici élucidés pour tirer des conclusions sur leur nature chaotropique et/ou hydrophobe. En combinant la diffusion aux petits angles des neutrons et des rayons X (SANS et SAXS), et les diagrammes de phase, les systèmes tensioactifs non ioniques/nano-ion sont examinés et comparés, du nanomètre à l'échelle macroscopique. Ainsi, il est montré que tous les nano-ions étudiés induisent un chargement électrique des assemblages de tensioactifs ainsi qu'une déshydratation des têtes de tensioactif non-ionique. En outre, les ions chaotropiques ou hydrophobes diffèrent dans leurs effets sur la forme micellaire. Les ions chaotropiques entraînent les micelles allongées de tensioactif non-ionique vers les micelles sphériques (augmentation de la courbure), tandis que les ions hydrophobes provoquent une transition vers les phases bicouches (diminution de la courbure). Il est conclu que les nano-ions superchaotropiques agissent comme des tensioactifs ioniques car leur ajout à des systèmes de tensioactifs non ioniques provoque un effet de charge. Cependant, les nano-ions et les tensioactifs ioniques sont fondamentalement différents par leur association avec l'ensemble des tensioactifs non ioniques. Le nano-ion s'adsorbe sur les têtes des tensioactifs non ioniques par effet chaotropique, tandis que le tensioactif ionique s'ancre dans les micelles entre les queues des tensioactifs non ioniques par effet hydrophobe. La comparaison des effets de l'ajout de nano-ions ou de tensioactifs ioniques à des tensioactifs non ioniques a été approfondie sur les mousses. Les mousses ont été étudiées en ce qui concerne l'épaisseur du film de mousse, le drainage dans le temps et la stabilité, respectivement en utilisant la SANS, l'analyse d'image et la conductométrie. Le POM superchaotropique testé (SiW12O404-, SiW) ne mousse pas dans l'eau contrairement au SDS classique de tensioactif ionique. Néanmoins, l'ajout de petites quantités de SiW ou de SDS à une solution moussante de tensioactif non ionique a permis d'obtenir des mousses plus humides avec une durée de vie plus longue. Entre-temps, l'épaisseur du film de mousse (déterminée en SANS) est augmentée en raison de la charge électrique des monocouches de tensioactifs non ioniques dans le film de mousse. Il est conclu que le comportement remarquable des nano-ions - ici sur les systèmes tensioactifs non ioniques - peut être étendu aux systèmes colloïdaux, tels que les mousses, les polymères, les protéines ou les nanoparticules. Cette thèse démontre que le comportement superchaotropique des nano-ions est un outil polyvalent qui peut être utilisé dans de nouvelles formulations de matériaux et d'applications de la matière molle<br>Nanometer-sized ions (nano-ions), such as ionic boron clusters, polyoxometalates (POMs) and large organic ions, have spawned remarkable interest in recent years due to their ability to adsorb or bind to electrically neutral chemical systems, such as macrocyclic host molecules, colloidal nano-particles, surfactants and polymers etc. The underlying adsorption or binding processes were shown to be driven by a solvent-mediated phenomenon, the chaotropic effect, which drives the nano-ion from the water bulk towards an interface. Thus, hydration water of the ion and the interface is released into the bulk resulting in a bulk water structure recovery. This effect is particularly strong for nano-ions. Therefore, they were termed superchaotropic or hydrophobic ions as an extension to classical (weakly) chaotropic ions such as SCN-. All commonly studied superchaotropes, though chemically diverse, share physical characteristics such as low charge density and high polarizability. Herein, the effects of nano-ions on ethoxylated non-ionic surfactant self-assemblies, micellar and bilayer phases, are elucidated to draw conclusions on their chaotropic and/or hydrophobic nature. By combining small angle scattering of neutrons and x-rays (SANS and SAXS), and phase diagrams, non-ionic surfactant/nano-ion systems are examined and compared, from the nanometer to the macroscopic scale. Thus, all studied nano-ions are found to induce a charging of the surfactant assemblies along with a dehydration of the non-ionic surfactant head groups. Furthermore, chaotropic and hydrophobic ions differ in their effects on the micellar shape. Superchaotropic ions drive the elongated non-ionic surfactant micelles towards spherical micelles (increase in curvature), whereas hydrophobic ions cause a transition towards bilayer phases (decrease in curvature). It is concluded that superchaotropic nano-ions act like ionic surfactants because their addition to non-ionic surfactant systems causes a charging effect. However, nano-ions and ionic surfactants are fundamentally different by their association with the non-ionic surfactant assembly. The nano-ion adsorbs to the non-ionic surfactant heads by the chaotropic effect, while the ionic surfactant anchors into the micelles between the non-ionic surfactant tails by the hydrophobic effect. The comparison of the effects of adding nano-ions or ionic surfactant to non-ionic surfactant was further investigated on foams. The foams were investigated regarding foam film thickness, drainage over time and stability, respectively using SANS, image analysis and conductometry. The tested superchaotropic POM (SiW12O404-, SiW) does not foam in water in contrast to the classical ionic surfactant SDS. Nevertheless, addition of small amounts of SiW or SDS to a non-ionic surfactant foaming solution resulted in wetter foams with longer lifetimes. Meanwhile, the foam film thickness (determined in SANS) is increased due to the electric charging of the non-ionic surfactant monolayers in the foam film. It is concluded that the remarkable behavior of nano-ions – herein on non-ionic surfactant systems – can be extended to colloidal systems, such as foams, polymers, proteins or nanoparticles. This thesis demonstrates that the superchaotropic behavior of nano-ions is a versatile tool to be used in novel formulations of soft matter materials and applications

L’utilisation de nanoparticules (NPs) dans un milieu biologique est de plus en plus importante, alors que leur assimilation et leur toxicité reste peu maitrisée. Dans ce contexte l’objectif de ce travail est d’étudier l’interaction entre nanoparticules de dioxyde de titane (TiO2) avec des protéines ainsi que leur possible impact sur leurs propriétés structurales. Trois protéines d’intérêt ont été choisies: une protéine de la matrice extra cellulaire, le collagène, et deux protéines du plasma sanguin, l’albumine et le fibrinogène. Le choix a été basé sur l’importance biologique de ces protéines lors des interactions avec des NPs et sur la différence de leurs structures tridimensionnelles. L’étude de l’interaction protéine-NPs a été réalisée en solution et en phase adsorbée dans différentes conditions de température et de temps d’incubation. Dans un premier temps des nanoparticules de dioxyde de titane ont été synthétisées par voie solvothermale, afin d’obtenir des nanoparticules de tailles et de formes contrôlées. Deux types de nanoparticules ont été sélectionnés en vues d’étudier l’effet de forme et de taille sur l’interaction protéines-nanoparticules: des nano-sphères de diamètre de 8 à 10 nm et des nano-bâtonnets d’environ 8 nm de largeur et 23 nm longueur. Leur comportement en solution physiologique ainsi que leur réactivité ont été caractérisé par DSL et absorption UV montrant une inhibition des propriétés catalytiques ainsi qu’une forte agrégation en solution tampon phosphate saline (PBS). Dans un deuxième temps les propriétés d’adsorption du collagène en présence de nanoparticules furent étudiées sur deux types de surfaces l’une hydrophobe et l’autre hydrophile par XPS et imagerie AFM ainsi que par des mesures de forces AFM. Le comportement du collagène en solution en présence de nanoparticules a été caractérisé par ATR liquide. Les observations suggèrent que la formation des fibrilles à l’interface surface-solution de collagène est affectée par le caractère hydrophobe ou hydrophile de la surface ainsi que par la présence de nanoparticules. Enfin les propriétés d’adsorption de la HSA et du fibrinogène en présence de nanoparticules furent étudiées sur une surface hydrophile par imagerie AFM et analyses XPS. Leur comportement en solution en présence de nanoparticules fut étudié par ATR liquide. Les résultats suggèrent deux comportements différents des protéines en présence de nanoparticules pouvant être attribués propriétés physicochimiques différentes des protéines. La HSA subit d’importants changements structuraux en présence de nanoparticules contrairement au fibrinogène.L’étude en phase liquide de l’interaction protéine-nanoparticules couplé à l’étude en phase adsorbées des protéines permet de déterminer les phénomènes impliqués lors de cette interaction ainsi que leurs conséquences sur les protéines, de plus l’utilisation de nanoparticules de taille et forme différentes permet d’étudier la sensibilité des protéines à ces facteurs<br>The extensive use of NPs in a biological environment raises the problem of their assimilation or their toxicity, the main objective of this work is to study the interaction of TiO2 NPs with proteins as well as their possible impact on the structural properties of proteins. Three proteins were chosen, a protein of the extracellular matrix, the collagen, and two proteins of the plasma blood, the albumin and the fibrinogen for their biological importance as well as for their various tridimensional structures. The study of the protein-nanoparticle interaction was realized in solution and in the adsorbed phase under various condition of temperature and incubation time. First, titanium dioxide NPs were synthesized by a solvothermal method, NPs with controlled size and form were obtained. Two types of NPs were selected in order to study the effect of shape and size on the protein-NPs interaction: nano-spheres with a diameter of 8 to 10 nm and nano-rods with a width about 8 nm and a length of 23 nm. The behavior in physiological solution as well as the reactivity were characterized by DSL and UV absorption showing an inhibition of catalytic properties as well as a strong aggregation in phosphate buffer saline solution (PBS). Second, the adsorption properties of the collagen in the presence of NPs were studied on two kinds of surfaces, one hydrophobic and the other hydrophilic by XPS and AFM imaging as well as by AFM force measurements. The behavior of the collagen in solution in the presence of NPs was characterized by liquid ATR. The observations suggest that the formation of fibrils at the surface- collagen solution interface is affected by the hydrophobic or the hydrophilic character of the surface as well as by the presence of NPs. In the last part the adsorption properties of HSA and fibrinogen in the presence of NPs were studied on a hydrophilic surface by AFM imaging and XPS analyses. Their behaviors in solution in the presence of NPs were studied by liquid ATR. The results suggest two different behaviors of proteins in the presence of NPs, which can be attributed to the different physico-chemical properties of the proteins. HSA undergoes important structural changes in the presence of NPs but not fibrinogen. The study in the liquid phase of the protein-nanoparticle interaction, coupled to study of proteins in the adsorbed phase allows determining the involved phenomenon during the protein-nanoparticle interaction as well as the consequences on protein adsorption. Moreover, the use of NPs with different sizes and shapes revealed the sensitivity of proteins to these factors

Tripeptidyl peptidase II (TPPII) is an exopeptidase which cleaves tripeptides from theN-terminus of peptides. The exact functional role of TPPII is still a matter of investigation. Itis believed that the enzyme is primarily involved in intracellular protein degradation, where itcooperates with the proteasome and other peptidases to degrade proteins into free aminoacids. These amino acids can subsequently be used in the production of new proteins. The aimof this work was to express murine wild type TPPII using E. coli and thereafter purify theenzyme from the bacterial lysate. Methods used for the purification included protein andnucleic acid precipitation, anion exchange chromatography, hydrophobic interactionchromatography and gel filtration. The presence of TPPII was determined using activityassay, western blot and SDS-PAGE. Despite the fact that some modification is still needed,the purification yielded a total of 34μg TPPII with a purity of approximately 60%. Thispurified enzyme can be used for future functional characterization.

L'hydroxyéthylcellulose hydrophobiquement modifiée (HMHEC) est un polymère associatif industriel obtenu par greffage d'une très faible quantité de chaînes hexadécyle sur le polymère précurseur (HEC). En solution aqueuse, l'association des groupes hydrophobes confère au polymère modifié des propriétés qui diffèrent profondément de celles des précurseurs. En régime dilué, le caractère associatif conduit à la formation d'agrégats intermoléculaires et/ou d'interactions intramoléculaires. Au-delà d'une concentration critique, qui délimite le début du régime semi-dilué, les associations deviennent essentiellement intermoléculaires et les propriétés épaississantes augmentent considérablement. Les solutions plus concentrées présentent alors une structure enchevêtrée tridimensionnelle enrichie de zones de jonctions hydrophobes fluctuantes, responsables de l'apparition d'un caractère viscoélastique. Les associations hydrophobes, de faible énergie, sont facilement détruites sous l'influence d'une contrainte de cisaillement et on observe un caractère rhéofluidifiant. Le temps nécessaire à la reformation réversible des associations se traduit par un phénomène de thixotropie. Les associations hydrophobes sont induites par la structure de l'eau. L'amélioration de l'organisation des molécules d'eau favorise les mécanismes d'associations. Ainsi, les sels lyotropes (NaCl) renforcent les associations hydrophobes, tandis que les sels chaotropes (KSCN) les affaiblissent. La présence d'éthanol entraîne la rupture des associations par une meilleure solvatation des groupements apolaires. D'autre part, la température semble favoriser l'établissement des associations en accord avec leur caractère endothermique.

Cette these repose sur l'application de la chromatographie d'affinite (zonale) conventionnelle (cac) et haute performance (cahp) a l'etude des interactions de la lipase (ou ses isolipases, ou sa sequence c terminale) d'une part, et la colipase, d'autre part. Les constantes de dissociations k::(d) du complexe calculees en cac et cahp sont voisines des valeurs mesurees par d'autres techniques non chromatographiques dans des conditions operatoires similaires. La cahp est une technique qui permet de determiner rapidement la stabilite du complexe lipase/colipase. L'influence de parametres physiques et physico-chimiques montre que la nature des interactions lipase/colipase est mixte, de type hydrophobe et ionique. En cahp les isolipases a et c presentent une affinite plus faible que l'isolipase b pour le cofacteur immobilise. Le peptide b, region c terminale de l'enzyme, engage des interactions specifiques avec la colipase, ce qui laisse supposer que le site d'association de la lipase avec son cofacteur se situe dans la region 336-449

Previous studies in Dr. Yang’s laboratory have established a grafting, design, and subdomain approach in order to investigate the properties behind Ca2+-binding sites located in Ca2+-binding proteins by employing engineered proteins. These approaches have not only enabled us to isolate Ca2+-binding sites and obtain their Ca2+-binding affinities, but also to investigate conformational changes and cooperativity effects upon Ca2+ binding. The focus of my thesis pertains to optimizing the expression and purification of engineered proteins with tailored functions. Proteins were expressed in E. coli using different cell strains, vectors, temperatures, and inducer concentrations. After rigorous expression optimization procedures, proteins were further purified using chromatographic and/or refolding techniques. Expression and purification optimization of proteins is essential for further analyses, since the techniques used for these studies require high protein concentrations and purity. Evaluated proteins had yields between 5-70 mg/L and purities of 80-90% as confirmed by SDS-PAGE electrophoresis.

Ce travail de recherche a été consacré à la synthèse de copolymères amphiphiles cationiques à blocs, et à l'étude de leur comportement physico-chimique en milieu aqueux. Ces copolymères ont été obtenus par polymérisation radicalaire du bromure de 3-hexadecyl-1-vinylimidazol-3-ium (VIC16Br), à l'aide de macro- et poly-iniferters à base de poly(oxyethylène) (POE). Le macroiniferter est composé de deux segments POE de part et d'autre d'une fonction disulfure de thiurame ; quant au polyiniferter, sa structure consiste en une alternance de segments POE et de fonctions disulfure de thiurame. Dans notre cas, la répartition des blocs POE et poly-bromure de 3-hexadecyl-1-vinylimidazol-3-ium) (P(VIC16Br)) des copolymères synthétisés n'a pu être définie. Cependant, selon la littérature, nous pouvons suggérer l'obtention de copolymères triblocs POE-P(VIC16Br)-POE, et de copolymères alternés (-POE-P(VIC16Br)-) n lors de l'utilisation respective du macroiniferter et du polyiniferter. Ainsi, divers copolymères de viscosité intrinsèque très variable et contenant jusqu'à 40% de motifs oxyethylène ont été préparés en jouant sur les réactifs utilisés, sur leur concentration et sur le temps de polymérisation. En milieu aqueux, nous avons surtout mis en évidence l'aptitude des copolymères à s'organiser en pelotes, en créant des microdomaines hydrophobes. Ce comportement a été détecté : à l'aide de sondes fluorescentes très sensibles à leur microenvironnement, par viscosimétrie dans un mélange binaire eau/n-propanol de concentrations croissantes en eau, et par cryomicroscopie électronique en transmission. Par tensiométrie superficielle, nous avons aussi vérifié le caractère peu tensioactif de ces macromolécules. Pour toutes ces techniques, le comportement de chaque copolymère a été relié aux caractéristiques de chacun d'eux, puis comparé à ceux de l'homopolymère de VIC16Br et d'une molécule modèle amphiphile cationique tensioactive : le bromure de 3-hexadecyl-1-methylimidazol-3-ium.

Ce travail de recherche a été consacré à l'élaboration de nouveaux polymères amphiphiles cationiques dérivés de la 3-vinylpyridine. Ils sont obtenus par polymérisation radicalaire, en solution, des sels de pyridinium correspondants. Cette voie de synthèse nous a permis d'obtenir des homopolymères de structure bien définie et des copolymères finaux entièrement quaternisés. Pour mieux caractériser le comportement de ces polymères en solution aqueuse diluée, nous avons fait appel a différentes techniques physico-chimiques telles que la viscosimétrie, les effets de solvatochromie, la spectroscopie de fluorescence des rotors moléculaires, la tensiométrie et la cryo-microscopie électronique en transmission. Une corrélation entre la structure chimique de ces polymères et leurs propriétés physico-chimiques en solution aqueuse a pu être établie. Nous avons, également, étudié l'influence du milieu solvant et de la concentration en polymère sur leur comportement en milieu aqueux. Les mesures viscosimétriques révèlent la formation de microdomaines hydrophobes à partir de faibles teneurs en eau. Elles montrent un changement conformationnel qui correspond à une meilleure stabilité des microdomaines pour un taux croissant en motifs 3VPC16Br. Les mêmes effets ont été observés par fluorescence des rotors moléculaires et les effets solvatochromie qui se traduisent par un changement de la polarité du milieu environnant la sonde utilisée. Des résultats obtenus par cryo-met révèlent une organisation particulière de l'homopolymère P3VPC16Br en forme d'empreinte digitale. Enfin l'aptitude de ces microdomaines a incorporer des substances hydrophobes dans l'eau mais solubles en milieu organique nous ont conduit à les utiliser en tant que microréacteurs dans lesquels peuvent se dérouler des réactions biomoléculaires d'hydrolyse d'esters actives

Orientador: Cesar Costapinto Santana<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica<br>Made available in DSpace on 2018-07-27T02:38:10Z (GMT). No. of bitstreams: 1 Veredas_Vinicius_M.pdf: 4752847 bytes, checksum: f01c509a2798835914e0489d2f75baac (MD5) Previous issue date: 2000<br>Resumo: O crescente interesse e aplicações dos produtos biotecnológicos vem aumentando o desenvolvimento de novos processos de recuperação e purificação de proteínas. O soro de leite bovino, obtido da manufatura da caseína para a produção de queijo, é em sua maioria descartado em mananciais de água, causando sérios problemas ambientais devido a sua alta demanda biológica de oxigênio (DBO). As proteínas presentes no lactosoro apresentam um excelente valor nutritivo e farmacológico, porém, o seu uso no enriquecimento de produtos alimentícios é limitado devido a baixa concentração destas proteínas. As principais proteínas do lactosoro são: ~-Iactoglobulinas, a-Iactalbumina, albuminas de soro bovino, imunoglobulinas, lactoperoxidase, lactoferrina, lisozima e outras proteínas de menor proporção, que apresentam um alto valor agregado. A a-Iactalbumina atua no organismo estimulando os agentes do sistema imunológico por proporcionar a elevação de glutationa em vários órgãos e no sangue, resultando em benefícios para pacientes portadores de doenças degenerativas como os males de Parkinson e Alzheimer, câncer e AIDS. Neste trabalho foi estudado o processo de separação da a-Iactalbumina, através de técnicas cromatográficas empregando a metodologia de leito fixo e expandido. O leito expandido possibilita a redução nos custos do processo de purificação, eliminando etapas de separação necessárias quando o extrato apresenta material em suspensão, que é o caso dos lactosoros. Nos ensaios realizados foram estudados as melhores condições de adsorção da a-Iactalbumina visando a sua purificação empregando adsorventes de troca iônica e de interação hidrofóbica. Também foram realizados ensaios em sistemas de tanque agitados para a determinação das isotermas e cinéticas de adsorção. Neste trabalho obteve-se a a-Iactalbumina com uma pureza acima de 80% e apresentando um fator de purificação de 5 vezes utilizando as resinas de interação hidrofóbica com única etapa de purificação<br>Abstract: The interest and applications of biotechnology products has been increasing the development of new recovery and purification processes for proteins. The bovine milk serum, obtained from casein manufacture for cheese production, is mostly rejected into watercourse, causing problems to the environment due to its high biological oxygen demand (BOD). The proteins of milk serum have excellent nutritious and pharmaceutical value, h oweve r, its application for protein enrichment of food products is limited due to its low content in the milk serum. The main proteins of milk serum are: ~-Iactoglobulins, a-Iactalbumine, bovine serum albumine, immunoglobulins, lactoperoxidase, lactoferrin, lisozime and other lower content proteins which have a high aggregate value. The a-Iactalbumine acts in the human organism by stimulating the agents of the immunologycal system due to increasing on glutathione levei in several organs and blood, resulting in benefits for patients of some diseases like Parkinson and Alzheimer's iII, cancer and AIDS. It was studied in this work the separation processes of a-Iactalbumine, by chromatographic techniques making use of fixed and expanded bed methods. The expanded bed enables cost reduction on purification process by reducing separation steps used for removing suspended solids, as in case of milk serum extracts. In our experiments were studied the adsorption conditions of alactalbumine aiming at its purification by using ionic exchange and hydrophobic interaction adsorbents. Other experiments were accomplished at stirred tank systems for the determination of isotherms and adsorption kinetics. It was obtained, in this work, an a-Iactalbumine purity higher than 80%, with a five fold purification factor by using the hydrophobic interaction resins in a single purification ste<br>Mestrado<br>Desenvolvimento de Processos Biotecnologicos<br>Mestre em Engenharia Química

Sample preparation is the most time-consuming and error-prone step in chemical analysis. Miniaturization and automation of the sample preparation equipment eliminating or reducing the use of hazardous organic solvents, online hyphenation of sample preparation with analytical instruments in a cost-effective way are important factors that need to be considered to design and implement innovative sample preparation techniques and strategies. Solid-phase microextraction (SPME) is a simple, environmentally benign technique well suited for hyphenation with analytical instruments. However, poor coating stability is a significant drawback of SPME employing conventionally prepared coatings. This shortcoming arises from the lack of chemical bonding between the sorbent coating and the substrate. Introduction of sol-gel coatings in SPME greatly improved thermal stability and solvent stability in SPME, by providing direct chemical bonding between substrate and the sol-gel coating. In traditional fiber format of SPME (where the sorbent coating is placed on the outer surface of an end-segment of the fiber) the coating remains vulnerable to mechanical damage. Capillary microextraction (CME), the capillary format of SPME (also known as in-tube SPME), allows to overcome this shortcoming by securing the sorbent coating on inner walls of the capillary. This dissertation focuses on the development and systematic investigation of novel silica- and germania-based perfluorinated and non-fluorinated sol-gel sorbents in the form of CME surface coatings: their preparation, material characterization, CME performance evaluation, preconcentration and recovery of various analytes including environmental pollutants. This research established that germania-based sol-gel sorbents are characterized by superior microextraction performance than analogous silica-based sorbents. This enhanced performance provided by germania-based sol-gel sorbents may be explained based on thermogravimetric analysis suggests that higher carbon loading on germania-based sol-gel sorbents. Germania-based phenyl- (Ph), phenethyl- (PhE), octyl- (C8), octadecyl- (C18) and cyclohexenylethyl- (ChE) ligand-containing sol-gel sorbents were prepared and various pollutants with aromatic rings (such as aromatic ketones, aldehydes and polycyclicaromatic hydrocarbons) were extracted and analyzed by CME-GC and CME-HPLC. It was observed that sol-gel sorbents containing aromatic ligands (PhE and Ph) provided superior microextraction performance for the analytes with aromatic ring(s) in their structure, than the sorbents with aliphatic ligands (C8 and C18). Investigation of sol-gel sorbents containing hydrophobic perfluorooctyl (PF-C8) and perfluorododecyl (PF-C12) ligands revealed that PF-C8 and PF-C12 sol-gel sorbents provided ~ 3 times higher microextraction efficiency (measure in terms of specific extraction, SE) than corresponding non-fluorinated counterparts, C8- and C12-, respectively. The synthesis and design of silica- and germania-based dual ligands sol-gel sorbents simultaneously providing superhydrophobicity and π-π interactions with analytes represent a significant accomplishment of this research. Such sorbents contained a PF-C12 and PhE ligands incorporated in sorbent chemical structure. In this case, perfluoro- group provided enhanced hydrophobic interaction and PhE group provided π-π interaction with the analytes. Combination of such interactions proved to be quite effective in the microextraction of alkylbenzenes and related compounds. Dual-ligand sol-gel sorbents with both equimolar and non-equimolar ligand concentrations were prepared. Experimentally it was established that sorbents with higher perfluorinated alkyl ligand concentrations had higher affinity for aliphatic hydrocarbons; however; when PhE concentration was higher, the dual-ligand sorbent showed enhanced affinity for aromatic compounds. The prepared sol-gel sorbents were characterized by less than 5% run-to-run RSD values, and also less than 5% capillary-to-capillary RSD values, which indicate that the sol-gel technique used in sorbent preparation was highly reproducible. The prepared sol-gel sorbents also showed that their performance does not deteriorate under aqueous saline matrix; therefore, it could be useful in the microextraction of pollutants from ocean water.

Oscillations of the electrical potential and interfacial tension have been studied during mass transfer in water/dichloromethane biphasic systems: CTAB/picric acid and SDS/TAAB for which, the chain length of the tetraalkylammonium was varied (from ethyl to butyl). A detailed analysis of the signals recorded allowed us to confirm the hydrodynamic origin (Marangoni instability) of the oscillations. We have determined the physico-chemical properties of all species involved (partition and adsorption constant, molecular area, and association constant of the ion pairs). For this, measurements of the surface tension, UV/Visible spectrophotometry and mass spectrometry have been performed. For the SDS/TAAB system, we have observed increasing association of TAADS ion pairs with chain length. We have also carried out theoretical calculations (DFT, Ab-initio) to highlight a geometry supporting the hydrophobic interactions between the two ions.

Les propriétés associatives de dérivés de la carboxyméthylcellulose à hydrophobie variable ont été étudiées en solution diluée et semi diluée. Deux types de réactions de modification chimique, par greffage d'une alkylamine, ont été réalisés sur la fonction acide de la CMC. Le premier greffage de chaînes alkyle fait intervenir l'activation des fonctions acide par la dicyclohexylcarbodiimide (L-CMC). La seconde réaction consiste à former le sel d'ammonium de la CMC qui est ensuite deshydraté à 140°C (I-CMC). Des dérivés mono et bigreffés ont ainsi pu être obtenus. Une étude RMN 1H et 13C a permis de confirmer les taux de greffage déterminés par dosage conductimétrique et d'établir la répartition des substituants sur le motif anhydroglucose. Il a été montré que la phase d'activation des fonctions acides par la dicyclohexylcarbodiimide s'accompagne d'une dégradation de la chaîne principale (L-CMC). Les études de diffusion de la lumière et de viscosimétrie ont mis en évidence la présence de macromolécules agrégées en régime dilué. Il existe un taux de greffage critique (4 %) en hexadecylamide à partir duquel les associations hydrophobes de type intermoléculaire deviennent majoritaires, ce qui permet une amélioration des propriétés épaississantes (L-CMC). Le système bigreffé présente, en régime semi dilué, une augmentation spectaculaire des propriétés associatives liée à l'augmentation de l'hydrophobie (présence de 30 % de groupes butyle) et à la diminution de la densité de charge. Les systèmes bigreffés (I et L) et monogreffés (I) ont des propriétés rhéologiques particulières qui suggèrent l'existence de plusieurs niveaux d'organisation hydrophobe. L'influence de la force ionique sur le pouvoir viscosifiant des solutions en régime dilué et semi dilué varie en fonction de l'hydrophobie des systèmes. L'incorporation d'un tensioactif neutre (Triton X100) dans les solutions de polymères mono et bigreffés en régime dilué et concentré, dans l'eau et dans NaCl 0,05 M, a confirmé l'existence de zones de jonctions hydrophobes très denses.

প্লাজমা ঝিল্লি একটি প্রাথমিক কার্যকরী ইউনিট হিসাবে একটি কোষ দক্ষ কার্যকরী জন্য অপরিহার্য হিসাবে গণ্য করা হয়। এই ঝিল্লিগুলি বহিরাগত কোষের কোষের ভিতরের অংশটিকে পৃথক করে এবং পাশাপাশি এটি জুড়ে চলমান নিয়ন্ত্রনের বাধা হিসাবে কাজ করে। প্লাজমা ঝিল্লিগুলি বিভিন্ন বিভিন্ন উপাদানের সাথে গঠিত, তবে সবার মধ্যে, ঝিল্লি প্রোটিনগুলিকে বৈজ্ঞানিক সম্প্রদায়ের দ্বারা প্লাজমা ঝিল্লির প্রধান কাঠামোগত এবং কার্যকরী স্তম্ভগুলির মধ্যে সর্বসম্মতিক্রমে গ্রহণ করা হয়। বৈজ্ঞানিক গবেষণায়, ঝিল্লী প্রোটিনগুলির কার্যকারিতা গুরুতর রোগের জন্য দায়ী বলে মনে করা হয়েছে। সুতরাং, এটি কৃত্রিম ট্রান্সমেম্রেন প্রোটিন ডোমেনগুলি ডিজাইন এবং বিকাশের জন্য একটি দুর্দান্ত বৈজ্ঞানিক আগ্রহ রয়েছে যা স্বাভাবিকের ত্রুটিগুলির সমাধান করতে সক্ষম। এই প্রোটিন ডোমেনগুলির ভাঁজ গঠন এবং ট্রান্সমেমব্রেন গতিবিদ্যা পিছনে আণবিক শক্তি এবং অন্যান্য পদার্থ-রাসায়নিক প্রক্রিয়াগুলি বোঝা হালনাগাদকৃত কৃত্রিম ট্রান্সমিম্ব্রেন প্রোটিন ডোমেনগুলি বিকাশের প্রক্রিয়ার অবিচ্ছেদ্য অংশ। গত দুই দশক ধরে, বিটা-পেপটাইডগুলি আরও প্রতিশ্রুতিশীল পেপটিডোমিম্যাটিক মোটিফগুলির মধ্যে একটি হিসাবে বিবর্তন হয়েছে। প্রোটিলাইটিক হ্রাসের বিরুদ্ধে অসাধারণ স্থিতিশীলতা এবং স্থিতিশীল হেলিকাল সেকেন্ডারি স্ট্রাকচার যেমন 14 -12- এবং বিকল্প 10 / 1২-হেলিসেসগুলি 4-6 এমিনো এসিডগুলি তৈরি করার ক্ষমতা, এর পিছনে দুটি প্রধান কারণ peptidomimetics মধ্যে β-peptides এর বিমোচন এন্ট্রি। অন্যান্য গুরুত্বপূর্ণ প্যারামিটারগুলির পাশাপাশি পেপটাইডের হেলিক্যাল ম্যাক্রো-ডিপোল মুহূর্তটি ট্রান্সমেম্রেন সন্নিবেশ এবং বিস্তারের ক্ষেত্রে গুরুত্বপূর্ণ ভূমিকা বলে মনে করা হয়। পেপটাইডগুলির হেলিক্যাল ম্যাক্রো-ডিপোল মুহূর্তের সরাসরি পরীক্ষামূলক সিদ্ধান্ত অত্যন্ত চ্যালেঞ্জিং হচ্ছে, হেলিক্যাল ম্যাক্রো-ডিপোল মুহূর্তের সম্ভাব্য প্রভাবগুলি কেবলমাত্র তাত্ত্বিকভাবে প্রস্তাবিত। অতএব, এই থিসিসের প্রধান উদ্দেশ্যগুলি ট্রান্সমেম্রেন সন্নিবেশ এবং বিস্তারের পাশাপাশি পরোক্ষ পরীক্ষার মাধ্যমে সেলুলার উপসর্গের মধ্যে হেলিক্যাল ম্যাক্রো-ডিপোল মুহূর্তের সম্ভাব্য ভূমিকা পালন করা। সাধারণভাবে, β-peptides নির্দিষ্ট হেলিক্যাল ম্যাক্রো-ডিপোল মুহূর্ত থাকে তবে প্রাকৃতিকভাবে ঘটমান α-peptide analogues এর তুলনায় বিপরীত দিকে থাকে। ধারণাটি হল বিটা-পেপটাইডের একটি ধরণের সনাক্তকরণ এবং সংশ্লেষ করা যার প্রায় মোট কোনও হেলিক্যাল ম্যাক্রো-ডিপোল মুহূর্ত নেই এবং β-peptides সহ এবং হেলিক্যাল ম্যাক্রো-ডিপোল ছাড়া ট্রান্সমেমব্রেন সন্নিবেশ স্টাডিজগুলি অন্যান্য সমস্ত পরামিতিগুলিকে ধ্রুবক রাখে। ক্ষেত্রে, তারা ঝিল্লি সন্নিবেশের জন্য কোন ডিফারেনশিয়াল ক্ষমতা প্রদর্শন করে, এটি পরীক্ষামূলকভাবে নিজ নিজ পদার্থ-রাসায়নিক ঘটনায় হেলিক্যাল ম্যাক্রো-ডিপোলের ভূমিকা নির্দেশ করবে। ব্যাপক গবেষণার পরে, বিকল্প β3 / β2-amino অ্যাসিডের সংযোজিত বিকল্প 10/12-হেলিকাল β-peptides তাদের অনন্য রূপান্তরিত অভিযোজন কারণে সামগ্রিক অলঙ্কৃত হেলিক্যাল ম্যাক্রো-ডিপোল পাওয়া যায় নি। অতএব, বিভিন্ন ধরণের β-peptides সহ 14-, 12- এবং বিকল্প 10/12-হেলিক্যাল পেপটাইড তুলনীয় ট্রান্সমেম্রেন দৈর্ঘ্য এবং ক্রম সহ বিভিন্ন সিন্থেটিক কৌশল মিশ্রিত করে সংশ্লেষিত করার পরিকল্পনা করা হয়েছে, যেমন মাইক্রোওয়েভ সহায়তায় ম্যানুয়াল SPPS, অ- মাইক্রোওয়েভ সহায়তায় ম্যানুয়াল SPPS, এবং ফ্লুরোস-ট্যাগ সংযুক্ত তরল ফেজ পেপটাইড সংশ্লেষণ। পরের ধাপে, পেপাইডাইডগুলির ট্রান্সমেমব্রেন সন্নিবেশ হাইড্রোফোবিক মাইক্রো-এনভায়রনমেন্ট সংবেদনশীল ট্রপ-ফ্লোরেসেন্স স্পেকট্রোস্কপি দ্বারা পরীক্ষা করা হবে। তিনটি ভিন্ন লিপিড, ডিএলপিসি / ডিএমপিসি / পিওপিসি এর একই গোষ্ঠীটি বিভিন্ন 14-, 12-এবং বিকল্প 10/12-হেলিক্যাল পেপাইডাইডের জন্য তুলনামূলক দৈর্ঘ্যের সাথে এইভাবে নির্বাচিত হয় যে নেতিবাচক হাইড্রোফোবিক মেলেম্যাচ ধীরে ধীরে একটি প্রায় পুরোপুরি hydrophobic ম্যাচিং পরিস্থিতি। এটি ভালভাবে জানা গেছে যে নেতিবাচক হাইড্রোফোবিক মেলেম্যাচের থ্রেশহোল্ড মানের উপরে ট্রান্সমেমব্রেন সন্নিবেশ সম্ভব নয়। অন্য দিকে, ইথানল মত শর্ট চেইন অ্যালকোহল, অ্যাসিড চেইন interdigitating দ্বারা লিপিড ঝিল্লি বেধ কমানোর একটি উচ্চারণ প্রভাব ভোগ করতে পরিচিত। অতএব, ETOH এর ক্রমবর্ধমান বৃদ্ধি ঘনত্বটি বিভিন্ন পেপটাইডের জন্য ব্যবহার করা হবে এবং একই লিপিডের জন্য একই পেপাইডাইডগুলির জন্য প্রতিটি পেপাইডাইডের জন্য প্রয়োজনীয় ন্যূনতম থ্রেশহোল্ড ঘনত্বের অনুরূপ নেতিবাচক হাইড্রোফোবিক মেলেম্যাচটি সাবধানে ন্যূনতম ক্ষতিপূরণ নেতিবাচক ক্ষতিপূরণ হিসাবে পর্যবেক্ষণ করা হবে। Trp-fluorescence বর্ণালী ক্রিয়ার সাহায্যে সফল ট্রান্সমেমব্রেন সন্নিবেশের জন্য অপরিসীম প্রয়োজন। এই পরীক্ষামূলক ফলাফল থেকে, এই সিদ্ধান্তে পৌঁছানো সম্ভব হবে যে পেপাইডাইডটি ETOH- এর আরো কম ঘনত্বের প্রয়োজন, যা নেতিবাচক মেলামেশের উচ্চতর ক্ষতিপূরণ, লিপিড ঝিল্লিতে পুনর্গঠন করতে সফলভাবে, ট্রান্সমেম্রেন সন্নিবেশ এবং বিস্তারের দিকে কম প্রবণ। ক্ষেত্রে, হেলিক্যাল ম্যাক্রো-ডিপোল মুহূর্তের সাথে এবং পেপাইডাইডগুলি এই আচরণের প্রতি কোনও ডিফারেনশিয়াল প্রবণতা প্রদর্শন করে, এটি পরোক্ষভাবে নির্দেশ করে এবং পরীক্ষামূলকভাবে ট্রান্সমেম্রেন সন্নিবেশ এবং স্প্যানিংয়ের মধ্যে হেলিক্যাল ম্যাক্রো-ডিপোল মুহূর্তের উল্লেখযোগ্য ভূমিকা যাচাই করবে (যেহেতু পেপাইডাইডগুলির মধ্যে প্রধান পার্থক্য হল হেলিক্যাল ম্যাক্রো-ডিপোল মুহূর্তের উপস্থিতি এবং অনুপস্থিতি)। তাছাড়া, লিপিড পরিবেশের অভ্যন্তরে যখন চারিত্রিক হেলিক্যাল প্যাটার্নটি রক্ষণাবেক্ষণ করা হয় কিনা তা ব্যাখ্যা করার জন্য, বিভিন্ন পেপাইডাইডগুলির দ্বিতীয় হেলিক্যাল কাঠামো সমাধান এবং পাশাপাশি অভ্যন্তরীণ লিপিড ভিসিক্যালগুলিতে নির্ধারণ করা হবে। তাপমাত্রা নির্ভর সিডি-স্পেকট্রসকপি দ্বারা সমাধান হিসাবে তুলনায় লিপিড vesicles ভিতরে যখন 14- এবং 10/12-হেলিক্যাল পেপটাইড স্থিতিশীলতা পরিবর্তন করা হয় কিনা তা পরীক্ষা করা হবে। হেলিক্যাল ম্যাক্রো-ডিপোল মুহূর্তটি সমাধান বা অভ্যন্তরীণ লিপিড vesicles মধ্যে সেকেন্ডারি হেলিকাল কাঠামো স্থিতিশীল করতে কোনো প্রভাব আছে কিনা তাও ইঙ্গিত করে। অবশেষে, 6-অ্যামিনো অ্যাসিড দীর্ঘ শৃঙ্খল চেইন 14-হেলিকাল এবং 10 / 1২-হেলিকাল 5 (6) -ফ্যাম সংযুক্ত পেপটাইডগুলি মানব ব্রোঞ্চিয়াল এডেনোকার্কিনোমা সেল লাইন A549 ব্যবহার করে সেলুলার আপটেক স্টাডিজের জন্য সংশ্লেষিত হয়। প্রথমটি ক্লোজোজেনিক অ্যাস এবং এমটিটি-অ্যাস দ্বারা একই কোষ লাইনে সাইটোটক্সিসটিটি পরীক্ষা করা হয়। যদি 1 μM ঘনত্ব না হওয়া পর্যন্ত অ-সাইটোটক্সিক পাওয়া যায়, তাহলে ফ্লোরোসেন্স অ্যাক্টিভেটেড সেল সোর্সিং (FACS) দ্বারা পরিমাণগত সেলুলার উত্তোলনের দক্ষতার দিকে আরও গবেষণা 14- এবং বিকল্প 10/12-হেলিক্যাল পেপাইডাইডগুলি হয়। একটি সুপরিচিত কোষ তীক্ষ্ণ পেপটাইড, এইচআইভি -1 ট্যাট, একটি রেফারেন্স মান হিসাবে ব্যবহৃত হয়। দুটি লক্ষ্য পেপটাইডগুলির মধ্যে সেলুলার আপটেক কার্যকারিতাগুলির মধ্যে কোন পার্থক্য পরীক্ষামূলকভাবে নির্দেশ করবে যে হেলিক্যাল ম্যাক্রো-ডিপোল মুহূর্তটি ট্রান্সমেমব্রেন সন্নিবেশ এবং বিস্তারকে প্রভাবিত করে না তবে সেলুলার অ্যাকটেককেও নিয়ন্ত্রণ করে। FACS ফলাফলগুলি নিশ্চিত এবং সমর্থন করার জন্য, পেপাইডাইডগুলি কন confocal লেজার স্ক্যানিং ফ্লুরোসেন্স মাইক্রোস্কপি অধীনে দৃশ্যমান হবে। মাইক্রোস্কোপি ইমেজিং প্রদর্শন করবে যে টার্গেট পেপাইডগুলি প্রকৃতপক্ষে সেল অনুপ্রবেশের মাধ্যমে অভ্যন্তরীণ হয় কিনা বা শুধুমাত্র ঝিল্লিতে আটকা পড়ে। উপরন্তু, যদি কোন লক্ষ্য β-peptides উল্লেখযোগ্য কোষ অনুপ্রবেশ ক্ষমতা পাওয়া যায়, এটি নতুনত্ব, হাইড্রোফোবিক, uncharged সেল ভেতরে পেপটাইড (সিপিপি) প্রার্থী যারা proteases উপস্থিতি স্থিতিশীল স্থিতিশীল দিকে একটি নতুন বর্ণমালা খুলতে হবে। অবশেষে, এই সমস্ত গবেষণাগুলি পরীক্ষামূলকভাবে ট্রান্সমেম্রেন সন্নিবেশ, বিস্তার এবং সেলুলার উপসাগরীয় অঞ্চলে ঝিল্লি প্রোটিন ডোমেনের হেলিক্যাল ম্যাক্রো-ডিপোল মুহূর্তের নিয়ন্ত্রক প্রভাবের উপর আলোকপাত করবে। এই তথ্যটি এই গুরুত্বপূর্ণ পদার্থ-রাসায়নিক ঘটনাগুলিতে পেপটাইড হেলিক্যাল ম্যাক্রো-ডিপোল মুহূর্তের প্রভাবকে মোকাবেলা করবে এবং β-পেপটাইড ভিত্তিক মডেল ট্রান্সমেম্রেন ডোমেন সিস্টেমগুলি পাশাপাশি β-পেপটাইড-ভিত্তিক নতুন প্রজন্মের কোষ তীব্র পেপটাইডগুলি ডিজাইনে সহায়তা করবে।

<p>The two techniques aqueoustwo-phase partitioning and expanded bed adsorption that bothare suitable for primary protein recovery were studied. Most ofthe work was focused on partition in aqueous two-phase systemsand in particular on the possibility to effect the partitionbehaviour by fusion of short peptide tags or protein domains tothe target protein.</p><p>The partitioning of fusionproteins between different variants of the domain tag Z and thenaturally occurring protein DNA Klenow polymerase were studiedin Breox/Reppal aqueous two-phase systems. Most studies wereperformed with cell homogenate. The Breox/Reppal system was infocus because if the fusion protein can be partitioned to theBreox-rich top phase the next step can be a thermoseparatingaqueous two-phase system. When the Breox phase is heated to50°C it switches from a one-phase system to a two-phasesystem resulting in an almost pure water rich top phase andhighly concentrated Breox-rich bottom phase. The Breox can thenbe reused and the protein recovered from the water phase. TheZ-domain was genetically modified in different ways to Z_basic1, Z_acid2and Z_trp12and fused to the Klenow protein to try toenhance partitioning to the Breox-rich phase. From theexperiments it was not possible to observe any effects on thepartition behaviour irrespectively of tested properties of thedomain tag. Despite the absence of domain tag effects highK-values, i.e. partition to the Breox-rich top phase, wereobserved in the Breox/Reppal system. However, the proteinK-values seemed to be rather sensitive to the cell homogenateload and showed a tendency to decrease with increased cellhomogenate load. Also increased phosphate concentration reducedthe K-values. The partitioning of cell debris also seemed todependent on the cell homogenate load. At higher homogenateload (<=20g DW/L) clear Breox-rich top phases were observedwith the cell debris collected in Reppal-rich bottomphases.</p><p>Two different tetrapeptides,AlaTrpTrpPro and AlaIleIlePro were inserted near the C-terminusof the protein ZZT0. The Trp-rich peptide unit stronglyincreased both the partitioning of ZZT0 into the poly(ethyleneglycol) (PEG)-rich phase in a PEG/potassium phosphate aqueoustwo-phase system and its retention on PEG and propylhydrophobic interaction chromatographic columns with potassiumphosphate as eluent in isocratic systems. Both the partitioningand the retention increased with increasing number of Trp-richpeptide units inserted into ZZT0. Insertion of Ile-richtetrapeptide units affected the partitioning and retention to amuch lesser extent. Partition and modelling data also indicateda folding of inserted Trp and Ile tetrapeptide units, probablyto minimise their water contact. It was also investigated howto predict the partitioning of proteins in isoelectricPEG/phosphate aqueous two-phase systems.</p><p>The capture ofß-galactosidase from<i>E. coli</i>cell homogentate (50g DW/L) by metal chelatexpanded bed adsorption was studied. These experiments showedthat capture, with a certain degree of selectivity, andclarification of ß-galactosidase could be achieved from acell homogenate. However, a rather low recovery of about 35 %was obtained at a capacity of 0.25mg/mL of gel. Thus, severalparameters remain to be optimised like the load buffercomposition and the cell homogenate load.</p><p><b>Keywords:</b><i>E. coli</i>, aqueous two-phase systems, fusion proteins,hydrophobic interaction chromatography, expanded bedadsorption, ß-galactosidase, Klenow polymerase, Z-domain,peptide tags</p>

MS Analysis of Allergenic Proteins. Food allergy is a significant worldwide public health issue. Proteins from cow's milk, chicken eggs, soybean and peanuts are the most frequent allergens contained in the complex foods prepared by industrial processes. Allergenic proteins can induce allergic reaction in their native structural state or upon chemical or conformational changes induced by the industrial treatments. Nowadays, the identification of allergenic proteins in foods is conducted by using immunochemical methods such as ELISA tests, but these techniques suffer from several limitations due to cross-reactivity and false negative results. Indeed, alterations in the allergen’s structure or chemical modifications can prevent the interaction with the antibody, thus causing misleading data. Since allergens are toxic even in trace amounts, there is a need for reliable and sensitive analytical methods for allergenic proteins. The purpose of this PhD project was to develop procedures for the identification of these proteins in food samples by using mass spectrometry (MS), likely overcoming some limitations of the immunochemical assays. Indeed, the MS approach for identifying proteins makes use of data pertaining to the amino acid sequence of the protein, while immunochemical methods are linked to the integrity of the three-dimensional structure of proteins. In order to test immunochemical approaches, polyclonal antibodies raised against the main allergenic proteins of milk (α-lactalbumin and β-lactoglobulin) and eggs (ovomucoid, ovalbumin and lysozyme) were purchased. Preliminary studies were performed in order to check the quality of the antibodies, in terms of specificity of recognition and cross-reactivity. Moreover, the responses of the antibodies using as antigens the purified commercial proteins and the same proteins contained in complex food matrices after thermal treatment were checked. Since allergenic proteins usually are contained in complex mixtures of huge amounts of other proteins, the methodology nowadays named “targeted proteomics” was considered very appropriate. By this approach, a protein contained in a complex mixture can be identified by a MS analysis of a peptide fragment that is specific for the protein of interest and contained in the very complex mixture of a tryptic digest of a protein sample. The procedure involves specific labelling and isolation of the specific peptide, named “proteotypic”. To this aim, tryptophan (Trp) residues in proteins were modified by reaction with 2,4-dinitrophenyl-sulfenyl chloride (DNPS-Cl), that leads to a Trp-derivative with the DNPS label attached at 2-position of the indole nucleus. The selection of Trp(DNPS)- peptides from the complex mixture of a tryptic digest of a protein sample was achieved by exploiting the significant change in hydrophobicity and retention time of DNPS-modified peptides in a reverse-phase HPLC column. Moreover, DNPS-labelled Trp-peptides were isolated by hydrophobic interaction chromatography, as well as by immunoaffinity chromatography using a column prepared with anti-DNP antibodies. The “targeted proteomics” procedure was optimised using a mixture of model proteins and then applied to identify a protein allergen contained in a raw bakery product. Overall, it was demonstrated that the novel procedure of selective labelling and isolation of Trp-peptides allows a considerable simplification of the fingerprinting/MS approaches nowadays used for the identification of proteins in proteomics research. Other Research Activities. During the PhD course I had the opportunity to collaborate with other members of the lab in a couple of additional projects, partly as a continuation of previous research conducted for the doctoral thesis. Documentation of this activity is herewith included as an Appendix at the end of this PhD Thesis. The molecular properties of the complex formed by α-lactalbumin with oleic acid were investigated in detail. This complex appears to be very interesting, since it has been shown to display cellular toxicity specifically for cancer cells. It was shown that the protein in the complex is in an oligomeric state, at variance from previous statements that the protein was monomeric. Moreover, it was shown the oleic acid can interact also with other proteins, including apomyoglobin. The main conclusion of this work was that the protein moiety serves as a carrier of the otherwise poorly soluble fatty acid, thus leading to an enhancement of its water solubility and consequently of its intrinsic cytotoxic properties. A manuscript rescrubbing these results is in an advanced state of preparation. Enterocin AS-48 is a 70-residue circular polypeptide produced by Enterococcus faecalis displaying a wide antibacterial activity. Limited proteolysis of AS-48 was used to prepare a linear form of this enterocin, as well as 38- and 55-residue fragments. Nicked AS-48 showed a lower helicity by far-ultraviolet circular dichroism and a reduced stability to thermal denaturation, but it was active against the sensitive bacteria assayed. The fragments also partly retained the biological activity of the intact protein. These results indicate that the circularization phenomenon is not required for the antibacterial activity, but it is crucial for the stabilization of the native structural state. This research was published in FEBS Lett. (2008).<br>Analisi di proteine allergeniche mediante spettrometria di massa. Le allergie alimentari rappresentano ormai una problematica clinica di livello mondiale. Tra i prodotti alimentari considerati pericolosi per il loro elevato contenuto in proteine allergeniche troviamo il latte bovino, le uova, la soia e le arachidi. Le proteine allergeniche possono scatenare reazioni allergiche sia mantenendo la loro struttura nativa, sia in seguito a modifiche chimiche e conformazionali indotte dai processi industriali. I metodi d’elezione applicati per l’identificazione di proteine allergeniche negli alimenti sono rappresentati dai saggi immunochimici come i test ELISA. Tali metodi presentano però numerose limitazioni causate da fenomeni di cross reattività e da falsi positivi. Inoltre, alterazioni nella struttura delle proteine allergeniche o eventuali modifiche chimiche possono modificare l’interazione con gli anticorpi specifici, invalidando i risultati. Dal momento che gli allergeni sono tossici anche in tracce, è necessario sviluppare dei metodi analitici efficaci e affidabili per la loro identificazione. Lo scopo di questo progetto di tesi è stato quello di sviluppare delle procedure per l’identificazione di proteine allergeniche mediante spettrometria di massa (MS) che possano superare i limiti metodici dei saggi immunologici. Oltretutto, l’identificazione delle proteine mediante MS si basa sull’analisi della sequenza amminoacidica di quest’ultime, mentre i saggi immunochimici sono strettamente dipendenti dall’integrità della struttura tridimensionale della proteina antigenica. Al fine di testare la validità dell’approccio immnuchimico, sono stati testati alcuni anticorpi policlonali diretti contro le principali proteine allergeniche di latte α-lattalbumina e β-lattoglobulina) e uova (ovomucoide, ovalbumina e lisozima). Sono stati condotti alcuni studi preliminari per validare la qualità di questi anticorpi, in termini di specificità di riconoscimento della proteina antigenica e della presenza di eventuali fenomeni di cross reattività. Inoltre, è stata valutata la risposta anticorpale usando come antigeni sia le proteine commerciali purificate, sia le stesse proteine contenute in prodotti alimentari prima e dopo trattamento termico. Dato che le proteine allergeniche sono contenute in miscele complesse costituite da altre proteine, è stata considerata estremamente appropriata l’applicazione di una tecnica detta “targeted chromatography”. Secondo questo strategia, è possibile identificare mediante MS una proteina contenuta in una miscela complessa attraverso l’analisi di alcuni frammenti peptidici derivati dalla digestione triptica, che sono specifici della proteina stessa. Questa procedura prevede la modifica chimica e il successivo isolamento di specifici peptidi detti “prototipici”. A tale scopo, i residui di triptofano contenuti nelle proteine sono stati chimicamente modificati mediante una reazione con il composto 2,4- dinitrofenilsulfenil cloruro (DNPS-Cl), che porta alla formazione di un derivato triptofanilico, con il DNPS legato in posizione 2 dell’anello indolico. La selezione dei peptidi modificati con il DNPS-Cl contenuti in una miscela triptica è stata effettuata sfruttando l’aumento di idrofobicità e del tempo di ritenzione di questi peptidi modificati in una colonna HPLC a fase inversa. Inoltre, gli stessi peptidi modificati con DNPS-Cl sono stati isolati mediante cromatografia per immunoaffinità utilizzando una resina derivatizzata con anticorpi monoclonali diretti contro il gruppo DNP. La strategia di ”targeted proteomics” è stata ottimizzata utilizzando una miscela modello di sette proteine e successivamente è stata applicata per l’identificazione di una proteina allergenica contenuta in un prodotto dolciario. È stato inoltre dimostrato che queste nuove procedure di modifica selettiva e di selezione dei peptidi triptofanilici permette di semplificare considerevolmente l’analisi di fingerprinting/MS che è solitamente utilizzata per l’identificazione di proteine nei protocolli di proteomica. Altre attività di ricerca. Durante il periodo di dottorato, ho avuto l’opportunità di collaborare con altri membri del laboratorio in due progetti addizionali come continuazione di un progetto di ricerca precedente. La documentazione relativa a queste attività è riportata in appendice alla tesi di dottorato. Sono state investigate le proprietà molecolari del complesso formato da α-lattalbumina con l’acido oleico. Il complesso appare interessante poiché ha mostrato avere tossicità cellulare diretta selettivamente contro cellule tumorali. È stato dimostrato che la proteina nel complesso ha una struttura oligomerica, diversamente da quanto riportato nelle prime osservazioni, che ipotizzavano fosse in uno stato monometrico. Inoltre, è stato osservato che l’acido oleico interagisce anche con altre proteine, come l’apomioglobina. La principale conclusione di questo lavoro è stata che il motivo oligomerico della proteina veicola l’acido oleico, normalmente poco solubile, favorendo quindi la solubilizzazione dell’acido grasso e conseguentemente della sua proprietà citotossica. È in preparazione un articolo riguardante questi risultati. L’enterocina AS-48, è un polipeptide circolare di 70 residui prodotto da Enterococcus faecalis che mostra a vere attività antibatterica. La proteolisi limitata è stata usata per preparare una forma lineare e due frammenti di questa enterocina. Misure di dicroismo circolare nel lontano ultravioletto hanno dimostrato che la proteina ha una bassa ellitticità e una ridotta stabilità alla denaturazione termica, ma mantiene la sua attività antibatterica., mentre i frammenti presentano un’attività ridotta. Questi risultati indicano che la circolarizzazione è un fenomeno che non è richiesto per l’attività antibatterica, ma è cruciale per la stabilizzazione della struttura nativa. Questa ricerca è stata pubblicata in FEBS Lett. (2008).

Un système microfluidique à onde élastique de surface a été développé dans le but de réaliser une réaction d'amplification de brins d'ADN par PCR. Nous avons principalement étudié la température et l'uniformité de l'échauffement des gouttes irradiées par des ondes de type Rayleigh. Ces dernières sont générées à la surface d'un substrat piézoélectrique de Niobate de Lithium (LiNbO3). Nous avons pensé un système consommant le moins d'énergie électrique possible pour atteindre les températures désirées et permettant une meilleure uniformité de la température des gouttes. Pour cela, un dispositif à transducteur enterré a été réalisé sous une couche isolante de silice. Les gouttes sont ainsi directement posées sur le transducteur ce qui limite les pertes et améliore la répartition de la chaleur au sein des gouttes. Nous avons ensuite vérifié que les réactifs de la PCR ne sont pas affectés par les ondes de Rayleigh ce qui laisse présager que la PCR peut être réalisée à l'aide d'un système d'échauffement par ondes de Rayleigh. Par ailleurs, le déplacement de goutte sur ce type de substrat de LiNbO3 est important pour des applications de type laboratoire sur puce. Ce substrat ayant des propriétés hydrophiles, des revêtements ont été développés afin de minimiser la force nécessaire à l'actionnement des gouttes. Dans ces travaux, nous proposons un nouveau type de couche à base de copolymère P(VDF-TrFe) dont la fabrication est simplement réalisée par dissolution et étalement par spin-coating. Nous avons montré que ce type de couche n'affecte que très peu la propagation des ondes de Rayleigh et les propriétés hydrophobes sont équivalentes à d'autres revêtements<br>In this work, a microfluidic system based on surface acoustic wave has been developed in order to achieve the amplification of DNA strands by temperature cycling (PCR). We studied mainly the temperature and the heat uniformity of microdroplets irradiated by Rayleigh waves. These waves are generated at the surface of a lithium niobate substrate. We propose a system allowing better temperature uniformity within microdroplets with an optimal energy consumption. For this, a device with buried transducer has been developed under an insulating layer (Silice). The droplets are then placed directly on the transducer which limits losses and improves the distribution of heat within the microdroplets. We then verified that the PCR reagents are not affected by the Rayleigh waves which suggests that PCR can be performed using a heating system by Rayleigh waves. Moreover, the move of microdroplets on this kind of LiNbO3 substrate is important for lab on chip applications. This substrate having hydrophilic properties, some coatings have been developed to minimize the required force to actuate the microdroplets. In this work, we developed a new hydrophobic layer based on copolymer P(VDF-TrFe) whose production is simply made by dissolving and spreading by spin-coating. We have shown that this kind of layer is compatible with Rayleigh waves and that the hydrophobic properties are equivalent to other coatings

Trimethylamine-N-oxide (TMAO) and guanidinium chloride (GdmCl) are both highly studied molecules in the field of protein folding/unfolding. Thermodynamic studies have shown that TMAO, an organic osmolyte, is a strong stabilizer of the protein folded state, while GdmCl is known to be one of the most effective protein denaturants. Although TMAO and GdmCl are well studied the mechanism by which they stabilize and denature proteins, respectively, is not well understood. In fact there are few studies looking at their effects on hydrophobic interactions. In this work we determine the effect of TMAO and GdmCl on hydrophobic interactions, by looking at the model system of phenyl and alkyl hydrophobic contact pairs. Contact pair formation is monitored through the use of fluorescence spectroscopy, i.e., measuring the intrinsic phenol fluorescence being quenched by carboxylate ions. Hydrophobic interactions are isolated from other interactions through a developed methodology. The results show that TMAO addition to the aqueous solvent destabilizes hydrophobic contact-pairs formed between phenol and carboxylate ions. The TMAO acts as a “denaturant” for hydrophobic interactions. For GdmCl the data shows that for small alkyl groups, acetate and propionate, hydrophobic contact-pairs are slightly stabilized or are not affected, respectively. For the larger alkyl groups GdmCl disrupts contact pair formation and destabilizes them. GdmCl’s effect on hydrophobic interactions shows a size dependence on carboxylate ion size, i.e., as carboxylate ion tail length increases the contact pair formed with phenol is destabilized to a greater degree.

At the most basic level viruses are biological nano-containers constituted by genetic material enclosed in a protein shell, capsid. A peculiar feature of viruses, both bacterial and some eukaryotic viruses, lies in the high packaging density of the genome in order to fit itself in the small capsid and hence the high internal osmotic pressure. Virus is a relatively stable particle equipped with fascinating mechanical properties of the capsid that are crucial for the virus lifecycle. Viruses have only one purpose: infect a host cell for reproducing themselves in order to generate new viral progeny (Roos et al. 2007). Therefore, the first and foremost consideration arising from the concept of virus reflects its pathogenesis and virulence that can ultimately result in many important infectious diseases such as common cold, influenza, hepatitis, rabies, measles, cancer and AIDS. As a consequence, pathogenic viruses represent a heavy hurdle for the global health and there is a strong need for developing robust strategies such as vaccines or antiviral drugs against virus infections (Baram- Pinto et al. 2010). On the other hand, viruses in the course of evolution have become efficient specialized gene delivery agents. Therefore they represent powerful tools in biomedicine for gene therapy and vaccine purposes (Schaffer et al. 2008). For successful gene therapy and immunization programs, the efficiency and stability of viral vectors are fundamental aspects (Jorio et al. 2006). To address this challenge, in the present research project we have investigated the interaction between viruses and nanomaterials. In the last years materials on the nanoscale for their unique properties have provided a broad range of potential biomedical uses (Verma et al. 2008) and for that reason we decided to explore their application with viruses. More specifically, we have examined three types of sulfonate- functionalized gold nanoparticles (AuNPs), namely, MUS:OT, MUS and MUS:brOT NPs, which are less than 5 nm in size, negatively charged and poorly cytotoxic (Verma et al. 2008). The NPs are coated with self-assembled monolayer (SAM) of thiolated organic molecules and one of the ligand is a sulfonated molecule, MUS (Verma et al. 2008). The MUS ligand itself was tested in our experiments as well. As virus models we focused on human recombinant adenovirus type 5 (Ad), one of the most promising viral vector as vaccine and gene therapy carrier and two picornaviruses of the genus enterovirus, namely, EV1 and CVB3, important human pathogens associated with several infectious diseases (e.g. myocarditis, aseptic meningitis, encephalitis, paralysis)(Kossila et al. 2002)(Marjomäki et al. 2014a). In spite of their medical impact, there are no therapeutic treatments available against picornavirus infections and the only vaccine products are against three types of poliovirus and hepatitis A virus (Merilahti et al. 2012). Two sets of experiments were carried out: (1) Short-term incubation of Ad with nanomaterials for 1 h at 37°C prior transducing HeLa cells or before in vivo administration in zebrafish and mice. The results demonstrated that Ad shortly pre-treated with nanomaterials showed a significant increase in the gene expression in vitro and in vivo The NPs’enhanced adenovirus transduction aims to reduce Ad vector doses in vivo thereby minimizing the adverse reactions of the immune response due to high vector dosage; (2) Long-term thermostabilization studies of Ad, EV1 and CVB3 in vitro in the presence and in the absence of our nanomaterials and other substances such as sugars (sucrose, glucose, glycerol) and Polyethylene glycol (PEG) molecules at 37°C or room temperature for extensive periods of time. Our results showed the capability of the nanomaterials and sucrose to increase substantially the heat stability of the viruses. In order to elucidate the thermal inactivation mechanism of viral particles and the stabilizing effect provided by some compounds on viruses we set out to formulate an analytical theory. This line of research fits in the context of developing more thermo-stable viral vector preparations for vaccine purposes that do not require the maintenance of the challenging cold chain system in order to preserve the effectiveness of viral vaccines during the storage, shipment and administration to the patients and hence to ensure the success of global immunization programs (Alcock et al. 2010).

The dominating factors affecting sorption of per- and polyfluoroalkyl substances (PFAS) remain subject of research and debate. Traditionally, distribution coefficients (e.g., Kd and KOC) are used to calculate the fractionation of the contaminant between soil and water, to estimate leaching and subsequently the risks it imposes reaching water reservoirs. Research has aimed to establish the sorption mechanisms for PFAS but, due to the complexity of interactions between the substance specific physiochemical properties and geochemical sorbent characteristics, it has shown to be a complicated task. For PFOS, one of the most commonly encountered PFAS, the Swedish Geotechnical Institute (SGI) recommends using the 10th percentile of a small data set for the organic carbon-water distribution coefficient KOC (500 L/kg) and multiply this with the organic content of the in-situ soil to obtain the soil-water distribution coefficient (Kd). The result of this study shows that this method is insufficient to obtain a good approximation of the mobility of PFOS at a contaminated site. With a review of recent research on PFAS sorption and a case study performed at Stockholm Arlanda Airport, this study concludes that as of today, and due to PFAS potent mobility, well measured field coefficients for each soil type present in the soil profile and an elaborate geohydrological model is necessary to estimate PFAS environmental transport, fate and associated risks. It also concludes that parameters such as anionic exchange capacity and soil protein content may be highly relevant to estimate PFAS sorption.

La synthèse de polymères hydrosolubles présentant un caractère associatif a été effectuée, par réaction de diverses alkylamines à chaine longue (Cn-NH2, 8≤n≤16) sur l'alginate de propylène glycol (PGA), un dérivé partiellement estérifié d'un polysaccharide extrait des algues. Les propriétés physico-chimiques des dérivés hydrophobisés résultants ont été étudiées, comparativement à celles des polymères parents, en rhéologie, en spectrophotométrie de fluorescence et par des mesures de tension superficielle, dans l'eau pure ou en présence de sels. De façon générale, on observe que le caractère associatif des dérivés synthétisés augmente avec le taux et la longueur des segments hydrophobes immobilisés sur le PGA. Ainsi, si les effets d'interactions hydrophobes ne sont pas mis en évidence pour le dérivé en C8, les dérivés hydrophobisés par des chaines alkyles longues (C12 et C14) présentent, quant à eux, d'importantes variations de comportement par rapport aux polymères parents. Celles-ci résultent de la mise en place d'interactions hydrophobes entre les chaines alkyles immobilisées sur le squelette polysaccharidique. Ces interactions sont essentiellement de nature intramoléculaire en solution diluée tandis que les associations intermoléculaires deviennent prépondérantes en régime semi-dilué. Des entités de volume hydrodynamique élevé apparaissent alors et conduisent à une augmentation spectaculaire de la viscosité, pouvant aller jusqu'à la formation d'un réseau tridimensionnel ayant une structure de gel physique

An understanding of the reactivity of hydrophobic organic micropollutants (HOMs) is of paramount importance to water quality managers because of their toxicity, persistence, and liability to bioaccumulate. In this study, the role played by the main estuarine variables (organic matter, suspended particulate matter [SPM], particle type and salinity) on HOM behaviour was investigated by employing samples from estuaries with different geochemical signatures (Chupa, Russia, and the Dart, Plym, Beaulieu and Carnon, U K ) . A laboratory-based technique was developed for the determination of the solubility and sorptive behaviour of HOMs using 14C-labelled, beta-emitting organic compounds (2,2\5,5'-tetrachlorobiphenyl (2,2’5,5'-TCB), bis(2-ethylhexyl)phthalate ester (DEHP), and benzo[a]pyrene (BaP)) coupled with liquid scintillation counting. The results indicate that relative solubility is mainly dependent upon the type of dissolved organic carbon (DOC) present, not its concentration, and is reduced with increasing salinity. The uptake of 2,2’5,5'-TCB and BaP by particles is time dependent with a system response time (the time required to achieve 63% of the new equilibrium) of about 0.37 hours for 2,2',5,5’-TCB and 0.02 hours for BaP. The adsorption, expressed as particle-water partition coefficients, KDS, is to a varying extent dependent on DOC, salinity and particle characteristics (iron/manganese hydroxides, particulate carbon and specific surface area). Adsorption is best defined by a linear isotherm and is enhanced in sea water compared with river water owing to a reduction in charge on particle surfaces at high ionic strengths. This effect has been quantified using an adsorption salting constant, Gp, whose values are typically in the range 0.4-2 L mol-2 The inverse relationship between KD and SPM concentration, an effect well documented in the literature, has been defined by a simple power law (KD = a SPM-b where a and b are site and compound-specific constants). Typical values for a and b are approximately 4x10^ and 0.6 for 2,2',5,5'- TCB, 50x105 and 1.0 for DEHP, and 2x105 and 0.5 for BaP, respectively. Empirical parameterisation of these effects are extremely useful for encoding into numerical transport and distribution models, and their application is demonstrated in this thesis by calculating the retention of HOMs by estuaries.

Les matériaux carbonés présentent de nombreux avantages pour les domaines des nanotechnologies et de l'environnement.La mixité de chiralité des nanotubes de carbone limite leur application dans les appareils électroniques et le nano-confinement. Dans une première partie, ce travail de thèse s'est concentré sur la séparation en chiralité de nanotubes de carbone de type CoMoCAT, afin d'élaborer de nouveaux nano-conteneurs.Après sélection en chiralité, nous avons évalué les propriétés sous hautes pressions des fagots de nanotubes sélectionnés, et leur interaction avec l'eau. Les résultats ont montré que les fagots supportent des pressions jusqu'à 17 GPa, avant de subir un effondrement radial réversible, permettant de les utiliser en tant que nano-enclumes.L'élaboration d'une éponge de carbone (super)hydrophobe pour le traitement des eaux après pollution aux hydrocarbures a été décrite dans une seconde partie. La pyrolyse de mousses polymères a permis de conserver la très grande porosité de la mousse (&gt; 99%), tout en lui conférant des propriétés proches de la superhydrophobie et de grandes capacités d'absorption de pétrole et solvants organiques (85-200 g/g). L'élasticité du matériau permet sa régénération par simple compression mécanique : récupération du polluant et réutilisation de l'absorbant. De plus, cette caractéristique reste valable même après une centaine de cycles de compression-décompression, en conservant 81% de sa capacité d'absorption dans le cas du pétrole brut<br>Carbon materials present many advantages for the nanotechnology and environment fields. The chirality mixity of carbon nanotubes limits their application in electronic devices and the nano-confinement. In the first part, this thesis has focused on the chirality separation of CoMoCAT carbon nanotubes, in order to elaborate new nano-containers.After the chirality selection, the properties of selected nanotubes bundles under high pressure were evaluated, as well as their interaction with water. The results show that the bundles support pressures until 17 GPa, before to undergo a reversible collapse, allowing their application as nano-anvils.The (super)hydrophobic carbon sponge elaboration for the clean-up of water polluted by oils was described in the second part. The polymeric foams pyrolysis allows to keep the very high foam porosity (&gt; 99%), to give properties next to the superhydrophobicity and large absorption capacities in oils and organic solvents (85-200 g/g). The material elasticity allows its regeneration by simple mechanical compression : recovery of pollutant and re-use of the absorbant. Moreover, this feature remains valid after a hundred compression-decompression cycles, with 81% of the initial crude oil absorption capacity

A better understanding of the factors that determine the affinity for ligand-protein interactions remains an important goal in biophysical and medicinal chemistry. Three-dimensional structures reveal a significant amount of information about a complex, but these static structures only represent the lowest energy conformation. Molecules are mobile. Their conformational changes and internal dynamics are an integral part of their function. Linking these motions with architecture is not always straightforward, but is essential to understanding what drives the association between two molecules.

Most of the processes in a living cell are carried out by proteins. Depending on the needs of the cell, different proteins will interact and form the molecular machines demanded for the moment. A subset of proteins called integral membrane proteins are responsible for the interchange of matter and information across the biological membrane, the lipid bilayer enveloping and defining the cell. Most of these proteins are co-translationally integrated into the membrane by the Sec translocation machinery. This thesis addresses two questions that have emerged during the last decade. The first concerns membrane proteins: a number of α-helices have been observed to span the membrane in the obtained three-dimensional structures even though these helices are predicted not to be hydrophobic enough to be recognized by the translocon for integration. We show for a number of these marginally hydrophobic protein segments that they indeed do not insert well outside of their native context, but that their local sequence context can improve the level of integration mediated by the translocon. We also find that many of these helices are overlapped by more hydrophobic segments. We propose, supported by experimental results, that the latter are initially integrated into the membrane, followed by post-translational structural rearrangements. Finally, we investigate whether the integration of the marginally hydrophobic TMHs of the lactose permease of Escherichia coli is facilitated by the formation of hairpin structures. However our combined efforts of computational simulations and experimental investigations find no evidence for this. The second question addressed in this thesis is that of the interpretation of the large datasets on which proteins that interact with each other in a cell. We have analyzed the results from several large-scale investigations concerning protein interactions in yeast and draw conclusions regarding the biases, strengths and weaknesses of these datasets and the methods used to obtain them.<br>At the time of the doctoral defense the following publications were not published and had a status as follows: Paper 2: In press; Paper 4 Manuscript.

Thesis (Ph.D)--Chemical Engineering, Georgia Institute of Technology, 2010.<br>Committee Chair: Breedveld, Victor; Committee Chair: Hess, Dennis; Committee Member: Aidun, Cyrus; Committee Member: Deng, Yulin; Committee Member: Singh, Preet. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Des polymeres hydrosolubles a faibles teneurs en groupements hydrophobes (0. 75 a 3% mol. ) ont ete synthetises par copolymerisation radicalaire de l'acrylamide et du n-ethylphenylacrylamide. Une etude du mecanisme de polymerisation indique que la distribution des sequences hydrophobes dans le copolymere depend du mode de synthese: polymerisations micellaires (en presence de tensio-actif), heterogene ou en melange de solvant. Seule la polymerisation micellaire conduit a des copolymeres presentant de fortes interactions hydrophobes. Les proprietes rheologiques des copolymeres ont ete etudiees en regime semi-dilue en fonction de la concentration en copolymere, la vitesse de cisaillement, la teneur en groupements hydrophobes et en tensio-actif. Les copolymeres prepares par voie micellaire ont un pouvoir epaississant superieur a celui d'homopolymeres de meme masse moleculaire (2. 10#6). Sous l'action de contraintes de cisaillement, il y a dissociation reversible des liaisons interchaines. L'addition de tensio-actif permet de regler les interactions hydrophobes et donc la viscosite des solutions. L'influence de l'histoire de l'echantillon a ete examinee en detail. La formation de microdomaines hydrophobes a ete suivie par une technique de fluorescence et les resultats obtenus confirment les donnees rheologiques

Comme de nombreux colloïdes, des nanoparticules métalliques recouvertes de ligands en suspension s’organisent au-delà d’une fraction volumique seuil et forment ce que l’on appelle un « supracristal ». Ce sont ainsi des systèmes modèles, déjà largement étudiés à partir de suspensions dans des solvants volatils, qui permettent de mieux comprendre l’auto-assemblage de sphères déformables. Les interactions qui conduisent à l’auto-assemblage sont couramment décrites par une compétition entre une attraction de van der Waals entre les cœurs métalliques et une répulsion entre les ligands qui va dépendre de l’affinité entre les ligands et le solvant. Un effet du solvant a déjà été observé sur l’auto-organisation de nano-objets. En mesurant par diffusion de rayons X aux petits angles le facteur de structure de suspensions de nanoparticules d’or greffées, j’ai pu sonder de façon systématique les interactions entre des nanoparticules en suspension avec plusieurs tailles de cœur, des ligands alcane-thiols de longueur différente et dans différents solvants à la fois volatils et non volatils. J’ai ainsi pu mettre en évidence une interaction attractive inattendue dans des alcanes linéaires flexibles et dont l’intensité augmente avec la longueur de l’alcane. Pour corréler les interactions entre particules à leur diagramme de phase, j’ai suivi le processus de cristallisation dans des suspensions en solvant volatil ou partiellement volatil ainsi qu’en émulsion, techniques qui permettent d’augmenter lentement la concentration en nanoparticules. Les interactions attractives induites par le solvant contribuent ainsi à la formation de supracristaux à de très faibles fractions volumiques. A de fortes concentrations, la structure des supracristaux ne dépend pas du solvant utilisé mais, à forte densité de greffage, du rapport R entre la longueur des ligands et le diamètre du cœur d’or. Pour un rapport R voisin de 0.7, la structure finale observée est cubique centrée, la structure à concentration intermédiaire étant cubique à faces centrées. Pour un rapport R deux fois plus petit, une structure originale a été mise en évidence. Il s’agit d’une structure hexagonale de grand paramètre de maille, analysée comme une phase de Frank et Kasper de type MgZn2 ou C14. C’est la première fois qu’une telle phase à empilement local tétraédrique est observée dans un système de sphères monodisperses molles. L’existence de cette phase ainsi que le rôle du rapport R a pu être interprétée en estimant quantitativement la compétition entre l’attraction de van der Waals forte et l’entropie des ligands<br>As many colloids, metallic nanoparticles grafted with hydrophobic ligands self-assemble above a volume fraction threshold and thus build superlattices. These model systems, which are widely studied when suspended in volatile oils, enable a better understanding of soft spheres self-assembly.Interactions which lead to self-assembly are commonly described by the combination of van der Waals attraction with interaction between the ligand shells. The shell behavior is controlled by the ligand affinity with the solvent. An effect of the solvent on the self-assembly of nanoparticles has already been observed. Using a small angle X-ray scattering, I measured, through the structure factor, the interactions between gold nanoparticles grafted with alkanethiols in different oils, at various concentrations, for different lengths of ligands and core diameters. I noticed an attractive interaction when using flexible linear alkanes as solvent. It has also been shown that the attraction intensity increases with the solvent length.In order to correlate the interactions between particles to their phase diagram, I studied the crystallization process by concentrating nanoparticles using evaporation in capillaries or Ostwald ripening in emulsions. I showed that attractive interactions induced by the solvent lead to superlattices formation at very low volume fractions.At high concentrations, the superlattice structure depends on the ratio of the ligand length over the gold core diameter. For a ratio around 0.7, the final structure observed is body centered cubic, whereas at lower concentration, it is face centered cubic. When this ratio is halved, an unexpected structure is observed. It is a hexagonal structure with a large lattice parameter. It has been analyzed as a Frank and Kasper’s phase named MgZn2 or C14. It is the first time that this topologically close-packed structure is observed for monodisperse soft spheres. The existence of this phase and the role of the ratio R have been interpreted by considering quantitatively the competition between ligands entropy and the strong van der Waals attraction

La compréhension du transport et du dépôt bio-colloïdal dans un milieu poreux présente un grand intérêt dans les applications environnementales, en particulier pour le contrôle de la bio-remédiation des sols et la protection des ressources en eau souterraine. Afin de mieux évaluer et prévenir les risques de contamination de la nappe phréatique et de proposer des solutions adéquates de remédiation, il est nécessaire d’avoir une bonne compréhension des mécanismes qui contrôlent le transport et le dépôt des bactéries dans les milieux poreux saturés et non saturés. L’objectif des ces travaux de thèse est d’étudier le rôle de l’hétérogénéité physique du milieu poreux (distribution granulométrique, porosité…) et de l’hydrodynamique du milieu sur les mécanismes de transport et de dépôt de particules bio-colloïdales, tout en prenant en compte l’impact des propriétés de cellules bactériennes sur ces mécanismes. Des expériences de traçage et d’injection de suspensions bactériennes ont été menées à l’échelle de colonnes de laboratoire dans trois milieux poreux avec une porosité et une distribution de taille de pore distincte. Afin de caractériser l’écoulement dans les milieux poreux, un soluté non-réactif a été utilisé comme traceur de l’eau. Trois souches bactériennes ont été utilisées pour préparer les suspensions bactériennes : une bactérie mobile (Escherichia coli), et deux non mobiles (Klebsiella sp. et R. rhodochrous). La caractérisation des propriétés cellulaires (telles que la taille et la forme des cellules, le potentiel zêta, la motilité et l'hydrophobicité) a été effectuée pour chaque souche. Des simulations numériques ont été réalisées en utilisant le code de calcul HYDRUS-1D afin de modéliser l’écoulement et d’estimer les paramètres de transport et de dépôt des bactéries. Ces derniers ont été explorés afin d'identifier le mode de transport bactérien et les mécanismes physico-chimiques ou physiques impliqués dans le dépôt des bactéries. Des expériences supplémentaires à l'échelle des pores ont été réalisées à l'aide de dispositifs microfluidiques conçus à cet effet. Un calcul théorique des différentes interactions entre les bactéries et le milieu poreux aux interfaces air/eau/solide a été effectué pour compléter les résultats expérimentaux ainsi que ceux issus de la modélisation numérique. Ainsi, les énergies d’interactions telles que les forces de van der Waals, électrostatiques de double couche, hydrophobes, stériques, capillaires et hydrodynamiques, impliquées dans le dépôt de bactéries ont été calculées pour décrire les interactions bactéries-interfaces afin d'identifier leur impact relatif sur le dépôt physico-chimique et physique des bactéries. Les résultats expérimentaux et la modélisation numérique ont mis en évidence un écoulement non uniforme, dépendant de la taille des grains ainsi que de la distribution de la taille des pores du milieu poreux. Pour un milieu poreux donné, l’écoulement devient plus dispersif quand la teneur en eau du milieu diminue. Ceci est dû à l’augmentation de la tortuosité du milieu, du fait de la présence de l’air dans les pores. Le transport des bactéries diffère de celui du traceur de l’eau. Le dépôt bactérien a été fortement influencé par la géométrie du réseau poral du milieu, les propriétés cellulaires et le degré de saturation en eau. Le piégeage physique et physico-chimique sont des mécanismes qui doivent être pris en compte pour bien décrire le dépôt bactérien, mais leur importance sur les mécanismes de dépôt est étroitement liée aux propriétés du milieu poreux et des cellules. Ces travaux mettent en évidence l’effet simultané des propriétés cellulaires, des propriétés physiques (granulométrie et distribution de taille de pores) et de l'hydrodynamique du milieu poreux sur les mécanismes de transport et de dépôt bactérien. Le calcul des différentes énergies d’interaction a permis d’identifier leur importance sur les mécanismes de dépôt bactérien<br>The investigation of the transport and retention of bacteria in porous media has a great practical importance in environmental applications, such as protection of the surface and groundwater supplies from contamination, risk assessment from microorganisms in groundwater, and soil bioremediation. The aim of this study is to gain a fundamental understanding of the mechanisms that control bacteria transport and deposition in saturated and unsaturated porous media. Laboratory tracer and bacteria transport experiments at Darcy scale were performed in three porous media with distinct pore size distribution in order to investigate and quantify water and bacteria transport process under steady state flow conditions. A conservative solute was used as water tracer to characterize water flow pathways through porous media. A gram negative, motile Escherichia coli, a gram negative, non-motile Klebsiella sp. and a gram positive, non-motile R. rhodochrous were selected for the transport experiments. Characterization of cell properties (such as cell size and shape, zeta potential, motility and hydrophobicity) was performed for each strain. Numerical simulations with HYDRUS-1D code were performed to characterize water flow and to estimate bacteria transport and deposition parameters. The later were explored to identify bacteria flow patterns and physicochemical or physical mechanisms involved in bacteria deposition. To provide a better understanding of the mechanisms involved on bacteria transport and deposition, pore scale experiments were carried out by using microfluidic devices, designed for this purpose. The information obtained from laboratory experiments and numerical modeling was improved by theoretical calculation of different interactions between bacteria and porous media at air/water/solid interfaces. DLVO and non-DLVO interactions such as hydrophobic, steric, capillary and hydrodynamic forces involved in bacteria deposition were considered to describe bacteria-interface interactions in order to identify their relative impact on physicochemical and physical deposition of bacteria. Results obtained through both laboratory experiments and numerical simulationsoutlined non-uniform flow pathways, which were dependent on both grain/pore size as well as pore size distribution of the porous media. For a given porous medium, water flow patterns became more non-uniform and dispersive with decreasing water saturation due to the presence of air phase, which lead to an increase of the tortuosity of the flow pathways under unsaturated conditions. Bacteria transport pathways were different from the tracer transport, due to size exclusion of bacteria from smaller pore spaces and bacteria motility. Bacteria deposition was greatly influenced by pore network geometry, cell properties and water saturation degree. Both physical straining and physicochemical attachment should be taken into account to well describe bacteria deposition, but their importance on bacteria deposition is closely linked to porous media and cell properties. The results obtained in this work highlighted the simultaneous role of cell properties, pore size distribution and hydrodynamics of the porous media on bacteria transport and deposition mechanisms. The calculation of DLVO and non-DLVO interactions showed that bacteria deposition in saturated and unsaturated porous media was influenced by both kinds of interactions