<p>Hydrogenated amorphous silicon (a-Si:H) is second only to crystalline silicon in volume manufacturing of solar cells due to its attractive characteristics for solar panel manufacturing. These are lower manufacturing costs, and the fact that it can be deposited on any surface, and in any shape even on flexible substrates. The metal induced crystallization of hydrogenated amorphous silicon has been the subject of intense scrutiny in recent years. By combining the technology of hydrogenated amorphous silicon thin films with the superior characteristics of c-Si material, it is hoped that more efficient solar cells can be produced. In this thesis we report on the metal-mediated-thermally induced changes of the structural and optical properties of hydrogenated amorphous silicon deposited by hot-wire CVD, where aluminium and nickel were used to induce crystallization. The metal-coated amorphous silicon was subjected to a thermal annealing regime of between 150 and 520&deg / C. The structural measurements, obtained by Raman spectroscopy, show partial crystallization occurring at 350 &deg / C. At the higher annealing temperatures of 450&deg / C and 520&deg / C complete crystallization occurs. Reflection and transmission measurements in the UV-visible range were then used to extract the optical properties. By adopting the effective medium approximation a single optical model could be constructed that couldsuccessfully model material that was in different structural phases, irrespective of metal contamination. Changes in the absorption of the material in various stages of transition were confirmed with a directly measured absorption technique, and the modelled absorption closely followed the same trends This study forms part of the larger overall solar cell research project, of which the primary aim is to eventually develop a silicon solar panel that optimises the characteristics for best performance.</p>

Transdermal drug delivery systems (TDS) are pharmaceutical devices that are designed to deliver specific drugs to the human body by diffusion through skin. The TDS effectiveness suffers from crystallization in the patch when they are kept in storage for more than two years. It has been reported that there are two types of crystals in the patch: needle and aggregate, and growth of drug crystals in TDS generally occurs only in the middle third of the polymer layer. In our study, fluorescence microscopy, EDS (SEM) and Raman microspectroscopy were used to further characterize the crystals. The results show that the needle crystals most probably contain estradiol and acrylic resin conjugate. The FTIR spectrum of the model sample proved the occurrence of a reaction between estradiol and acrylic resin. Crystal growth in an unstressed matrix of a dissolved crystallizable drug component was simulated using a kinetic Monte Carlo model. Simulation using Potts model with proper boundary condition gives the crystals in the middle of matrix in the higher temperature. Bond fluctuation model is also being implemented to study representative dense TDS polymer matrix. This model can account for the size effect of polymer chain on the crystal growth. The drug release profile from TDS was also studied by simulating the diffusion of drug molecules using Monte Carlo techniques for different initial TDS microstructure. The release rate and profile of TDS depend on the dissolution process of the crystal. At low storage temperature, the grains are evenly distributed throughout the thickness of the TDS patch, thus the release rate and profile is similar to the randomly initiated system. Further work on stress induced crystallization is currently under development. Although the study was specifically done for drug in a polymer medium, the techniques developed in this investigation is in general applicable to any constrained crystallization in a polymer medium.

Crystallization of proteins in the human body can lead to the development of diseases such as sickle cell anemia and cataract. Understanding protein crystallization can give insight into such diseases. Furthermore, protein crystallization is necessary for protein structure resolution. This is important since resolution of protein structure is the first step towards establishing structure/function relations, and possibly towards performing specific structural modifications that may change the function in desirable directions. Another important application of protein crystallization is in downstream processing in the pharmaceutical industry where it is used for separation and as a final purification step. The present study increases knowledge of interactions between protein molecules during crystallization and hence the crystallization process. Crystallization of proteins in the human body can lead to the development of diseases such as sickle cell anemia and cataract. Understanding the processes involved in protein crystallization can help us gain a better understanding of such diseases. Crystallization of human gamma D-crystallin (HGD) and apoferritin, two proteins found in the lens, was studied in relation to cataract formation. Crystallization of both proteins was studied in the presence of divalent cations which are found at elevated concentrations in cataractous lenses. Results indicate that the divalent cations studied enhance crystallization of these proteins. A thermodynamic property, the osmotic second virial coefficient, was measured in protein solutions and its value was correlated with the occurrence of crystallization. It was found that the second virial coefficient successfully predicted crystallization of both proteins. A new method was developed for indirect measurement of the second virial coefficient using dynamic light scattering. This new method is more robust and efficient than the traditional static light scattering method. Finally the ability of solubilizers to prevent crystallization in HGD solutions was studied. A commercial solubilizer, NDSB-201, was found to increase the energy barrier to nucleation. Although this did not prevent crystallization, it resulted in fewer and smaller crystals being obtained. The naturally occurring alpha A-crystallin was a superior solubilizer to NDSB-201, as it suppressed aggregation and prevented crystallization of HGD under conditions for which NDSB-201 did not. The findings in the present study provide insight into the processes by which protein crystallization occurs and hence into diseases associated with protein crystallization. The findings in the present study provide insight into the processes by which protein crystallization occurs. Using the second virial coefficient to assess whether a protein will crystallize out of solution, approaches for retardation and prevention of protein crystallization, and implications for future research, are discussed.

<p>Semi-batch crystallization experiments have been performedboth in a loop reactor and in stirred tank reactors.Hydrochloric acid was fed to a stirred solution of sodiumbenzoate, and benzoic acid immediately formed. Benzoic acid isformed in excess of the solubility making the solutionsupersaturated.</p><p>The loop reactor is U-shaped. In one leg a propeller stirrerwas placed to circulate the solution and in the other a turbinestirrer was placed in front of the feed point to vary the localmixing intensity. The objective was to analyse the relativeimportance of different levels of mixing on the product sizedistribution. The importance of mixing as well as scaling-upeffects on the product size distribution were studied in threestirred tank reactors of volumes 2.5 L, 10 L, and 200 L. Thestirred tank reactors had different geometry and were equippedwith either a marine propeller or a pitched blade turbine.</p><p>The weight mean size generally increases with increasingtotal feeding time and increasing mixing intensity. The weightmean size increases by locating an extra turbine impeller atthe feed point in the 10 L stirred tank reactor. The turbineimpeller provides the desired feed point mixing intensitywithout raising the mixing intensity of the whole tank.</p><p>The weight mean size increases with decreasing feed pipediameter in the loop reactor and for low feed rates in the 10 Lstirred tank reactor. The weight mean size increasessignificantly by changing the feed pipe opening from circularto rectangular with a constant cross-sectional area at equalfeed rates. Backmixing is visually observed in the largest feedpipe diameter in the loop reactor, thus, reducing the weightmean size. However, backmixing is not considered to be adominant phenomenon in the present work.</p><p>Mesomixing time constants have been calculated according tothe turbulent dispersion mechanism and the inertial-convectivemechanism. The time constants for mesomixing are generallylonger than the time constant for micromixing. Thus, the ratioof the mesomixing and the micromixing time constants shows aninfluence of mesomixing as is shown by the experimentalresults. The experimental results are best described by theinertial-convective disintegration mechanism showing that thefeed plume mixing increases with decreasing feed pipe diameterand increased feed point mixing.</p><p>The weight mean size is not strongly affected by the reactorvolume. However, the mixing conditions in the reactors have astrong influence on the weight mean size. No suggestedscaling-up rule can satisfactorily predict the weight mean sizein the different volumes. No single physical parameter, such asthe local energy dissipation rate, the mean energy dissipationrate or the circulation time, can satisfactorily explain theexperimental results. A new dimensionless mixing parameter, TR,has been defined as the ratio of the total feeding time and themesomixing time constant. The mesomixing time constant isdefined as the shortest dimension of the feed pipe divided bythe resultant bulk velocity passing the feed pipe entrance. Theexperimental results from both the loop reactor and the stirredtank reactors of different volumes can be correlated with TR.The weight mean size increases with increasing TR.</p><p><b>Keywords</b>: reaction crystallization, precipitation,benzoic acid, macromixing, mesomixing, micromixing,semi-batch, loop reactor, backmixing, colour experiments,scaling-up.</p><p><b>Keywords</b>: reaction crystallization, precipitation,benzoic acid, macromixing, mesomixing, micromixing,semi-batch, loop reactor, backmixing, colour experiments,scaling-up.</p>

Le projet de thèse se focalise sur la synthèse de biomolécules et se subdivise en deux parties. La première partie concerne la conception et la synthèse de dérivés de produits naturels d'intérêt thérapeutique nommés aurones en vue de mettre au point de nouvelles molécules à activité antivirale. Récemment, les aurones ont été identifiées comme étant des inhibiteurs de l'ARN-polymérase ARN-dépendante (NS5B) du virus de l'hépatite C (VHC). Cette enzyme, présente chez le virus mais absente chez l'homme, joue un rôle central dans la réplication virale. Suite à ces résultats antérieurs, les efforts ont été poursuivis et, dans le cadre de cette thèse, nous avons entrepris,d'une part, la synthèse d'analogues originaux dont le cycle B des aurones a été remplacé par des hétérocycles et, d'autre part, la synthèse depseudodimères d'aurones dans le but d'affiner les exigences structurales pour améliorer l'effet inhibiteur.L'activité a été évaluée selon des tests enzymatiques et cellulaires et a permis d'identifier quelques candidats doués d'une bonne activité inhibitrice et d'une faible toxicité. La deuxième partie du projet de thèse, sans lien avec la première partie,concerne des aspects plus fondamentaux et porte sur la synthèse de nouveaux surfactants agissant comme agents stabilisants lors des procédures d'extraction et de cristallisation des protéines membranaires. Les surfactants sont des composants clés dans le domaine de la biologie structurale et de la biochimie des protéines membranaire. Ils sont nécessaires pour maintenir les protéines membranaires dans leur état fonctionnel après extraction. La grande majorité des protéines membranaires est riche en résidus basiques à l'interface. Sur la base de cette caractéristique, une nouvelle famille de surfactants est développée et testée sur des protéines membranaires appartenant aux pompes d'efflux ABC multi-résistantes. / The PhD project focuses on biomolecules and is divided into two parts. The first part concerns the design and synthesis of natural product derivatives with therapeutic interest in order to develop new molecules with antiviral activity. Recently, aurones were identified as new inhibitors of hepatitis C virus (HCV) NS5B polymerase. Following these results, efforts were continuedand we undertook, on the one hand,the synthesis of original analogues in which the aurone B-ring was replaced by a heterocyclic rings and, on the other hand, the synthesis of aurone pseudodimers in order to refine the structural requirements to improve the inhibitory effect. The potent NS5B inhibitory activity combined with their low toxicity make aurones attractive drug candidates against HCV infection. The second part of the PhD thesis is unrelated to the first part and concerns more fundamental aspects. It focused on the synthesis of new surfactants acting as stabilizing agents during extraction of membrane proteins (PM). Surfactants are required for maintaining PM in their functional state after extraction from membrane lipid matrix. The vast majority of PM shares a net enrichment in basic residues at the interface between membrane and cytoplasm, a property known as the positive inside rule. Based on this feature, a new family of surfactants is developed and tested on membrane proteins belonging to the multidrug ABC efflux pumps family.

<p>X-ray and neutron crystallography are the predominant methods for obtaining atomic-scale information on bimolecular macromolecules. Despite the success of these techniques, generating well diffracting crystals critically limits going from protein to structure. In practice, the crystallization process proceeds through knowledge-informed empiricism. Better physico-chemical understanding remains elusive because of the large number of variables involved, hence little guidance is available to systematically identify solution conditions that promote crystallization. </p><p>The fields of structural biology and soft matter have independently sought out fundamental principles to rationalize protein crystallization. Yet the conceptual differences and limited overlap between the two disciplines may have prevented a comprehensive understanding of the phenomenon to emerge. Part of this dissertation focuses on computational studies of rubredoxin and human uniquitin that bridge the two fields.</p><p>Using atomistic simulations, the protein crystal contacts are characterized, and patchy particle models are accordingly parameterized. Comparing the phase diagrams of these schematic models with experimental results enables the critical review of the assumptions behind the two approaches, and reveals insights about protein-protein interactions that can be leveraged to crystallize proteins more generally. In addition, exploration of the model parameter space provides a rationale for several experimental observations, such as the success and occasional failure of George and Wilson's proposal for protein crystallization conditions and the competition between different crystal forms.</p><p>These simple physical models enlighten the connection between protein phase behavior and protein-protein interactions, which are, however, remarkably sensitive to the protein chemical environment. To help determine relationships between the physico-chemical protein properties and crystallization propensity, statistical models are trained on samples for 182 proteins supplied by the Northeast Structural Genomics consortium. Gaussian processes, which capture trends beyond the reach of linear statistical models, distinguish between two main physico-chemical mechanisms driving crystallization. One is characterized by low levels of side chain entropy and has been extensively reported in the literature. The other identifies specific electrostatic interactions not previously described in the crystallization context. Because evidence for two distinct mechanisms can be gleaned both from crystal contacts and from solution conditions leading to successful crystallization, the model offers future avenues for optimizing crystallization screens based on partial structural information. The availability of crystallization data coupled with structural outcomes analyzed through state-of-the-art statistical models may thus guide macromolecular crystallization toward a more rational basis.</p><p>To conclude, the behavior of water in protein crystals is specifically examined. Water is not only essential for the correct functioning and folding of proteins, but it is also a key player in protein crystal assembly. Although water occupies up to 80% of the volume fraction of a protein crystal, its structure has so far received little attention and it is often overly simplified in the structural refinement process. Merging information derived from molecular dynamics simulations and original structural information provides a way to better understand the behavior of water in crystals and to develop a method that enriches standard structural refinement.</p> / Dissertation

Hydrophobic membrane proteins perform a variety of important functions in the cell, but their structures are notoriously difficult to solve. Thus, new strategies to obtain crystals of membrane proteins for structure determination are critical. We aim to develop a toolbox of peptide specific single-chain antibody fragment chaperones engineered for hyper-crystallizability. These peptide sequences can be introduced into various regions of membrane proteins without interfering with protein function. The resulting protein-chaperone complex is expected to form a crystal lattice mediated by chaperone interactions. We have developed candidate scFv chaperone proteins binding hexa-histidine (His6) and EYMPME (EE) tags with improved biophysical features influencing crystallization propensity, including peptide affinity, stability and solubility. The scFv libraries were generated using a novel ligation-free technique, MegAnneal, allowing us to rapidly generate large libraries based on 3D5 scFv. We identified two candidate chaperones, 3D5/His_683, specific for His6 and 3D5/EE_48, specific for EE tags. Variants exhibit high solubility (up to 16.6 mg/ml) and nanomolar peptide affinities; complexes of 3D5/EE_48 with EE-tagged proteins were isolated by gel filtration. We have developed design rules for EE peptide placement at terminal, inter-domain or internal loop regions of the target protein to balance peptide accessibility for chaperone binding while retaining rigid protein-chaperone complexes suitable for crystallization. The 3D5/ His_683 crystallized in four different conditions, utilizing multiple space groups. The 3D5/EE_48 scFv was crystallized (3.1 Å), revealing a ~52 Å channel in the crystal lattice, which may accommodate a small peptide-tagged target protein. Our evolution experiments altered scFv surface residues, resulting in use of different crystallization contacts. Analysis of these crystal contacts and those used by crystallized 14B7 scFv variants, led us to postulate that lattice formation is driven by strong crystal contacts. To test this hypothesis, we introduced amino acid changes expected to reduce the affinity of the 3D5/EE_48 energetically dominant crystal contacts. This approach to crystal contact engineering may allow semi-rational control over lattice networks preferred by scFv chaperones. Co-crystallization trials with model proteins are on-going. These engineered scFvs represent a new class of chaperones that may eliminate the need for de novo identification of candidate chaperones from large antibody libraries. / text

The membrane proteins signal peptide peptidase, signal peptide peptidase like and presenilin are intramembrane aspartyl proteases located in the endoplasmic reticulum, plasma membrane and organelle. These membrane proteins are able to catalyze a hydrolytic reaction in a hydrophobic space. The downstream consequences of these reactions impact a variety of cellular functions such as cytokine production, inflammatory responses, embryogenesis, and immune system regulation. Additionally, the aspartyl proteases such as signal peptide peptidase and presenilin, a part of the γ-secretase complex, hydrolyze peptides leading to pathogen maturation and Alzheimer’s disease, respectively. Electron crystallography offers the unique aspect of studying membrane proteins in a near native state. Determining the structures of Haloarcula morismortui and Methanoculleus marisnigri JR1 signal peptide peptidases by electron crystallography may provide insight into how a hydrolysis reaction occurs in a hydrophobic environment and how the protein determines which transmembrane signal peptides to cleave. Additionally, structure determination may help answer questions regarding why human presenilin, part of the γ-secretase complex, incorrectly processes amyloid precursor protein into amyloid-beta peptides leading to Alzheimer’s disease. Such structural data may not only shed light on how amyloid precursor protein is processed but how other proteins are processed by signal peptide peptidase leading to immune responses, cell signaling, and pathogen maturation. In addition, structure-function data may have an impact on pharmaceutical drug designs that targets signal peptide peptidase, signal peptide peptidase like, and/or presenilin. To determine the structure of aspartyl proteases, two archaeal signal peptide peptidases were used for two-dimensional crystallization trials to be able to study their structure by electron crystallography. Haloarcula morismortui and Methanoculleus marisnigri JR1 signal peptide peptidases, both human signal peptide peptidase homologues, were recombinantly over-expressed and purified. During dialysis trials, various lipid-to-protein ratios, sodium chloride concentrations, temperatures, detergents and a variety of other variables were tested. Methanoculleus marisnigri JR1 signal peptide peptidase showed the most promising results in terms of crystallinity. Optimizing dialysis conditions, specifically narrowing the lipid to protein ratio, resulted in two-dimensional crystals. Ordered arrays measuring up to 200 nm x 200 nm were observed. These ordered arrays have been shown to be reproducible amongst multiple batches of purified Methanoculleus marisnigri JR1 signal peptide peptidase. Preliminary projection maps of negatively stained ordered arrays show unit cell dimensions of a = 178 Å, b = 160 Å, γ = 92.0 Å and a = 175 Å, b = 167 Å, γ = 92.0 Å. The monomer measurements are approximately 70 Å by 80 Å. This is the first time a signal peptide peptidase homologue has been crystallized by two-dimensional crystallization.

No description available.

<p>Hydrogenated amorphous silicon (a-Si:H) is second only to crystalline silicon in volume manufacturing of solar cells due to its attractive characteristics for solar panel manufacturing. These are lower manufacturing costs, and the fact that it can be deposited on any surface, and in any shape even on flexible substrates. The metal induced crystallization of hydrogenated amorphous silicon has been the subject of intense scrutiny in recent years. By combining the technology of hydrogenated amorphous silicon thin films with the superior characteristics of c-Si material, it is hoped that more efficient solar cells can be produced. In this thesis we report on the metal-mediated-thermally induced changes of the structural and optical properties of hydrogenated amorphous silicon deposited by hot-wire CVD, where aluminium and nickel were used to induce crystallization. The metal-coated amorphous silicon was subjected to a thermal annealing regime of between 150 and 520&deg / C. The structural measurements, obtained by Raman spectroscopy, show partial crystallization occurring at 350 &deg / C. At the higher annealing temperatures of 450&deg / C and 520&deg / C complete crystallization occurs. Reflection and transmission measurements in the UV-visible range were then used to extract the optical properties. By adopting the effective medium approximation a single optical model could be constructed that couldsuccessfully model material that was in different structural phases, irrespective of metal contamination. Changes in the absorption of the material in various stages of transition were confirmed with a directly measured absorption technique, and the modelled absorption closely followed the same trends This study forms part of the larger overall solar cell research project, of which the primary aim is to eventually develop a silicon solar panel that optimises the characteristics for best performance.</p>