Presently, over 300 proteins or peptide based therapeutic medicines have been approved by the FDA owing to advances in protein engineering and technology. However, majority of these protein-based medications are unstable or have limited shelf life when in aqueous form. During pre-formulation and manufacturing, various technological processes including mixing, dissolving, filling (through pipes) can produce strong mechanical stresses on proteins. These stresses may cause the protein molecule to unfold, denature or aggregate. To improve stability upon formulation, they may be manufactured as freeze dried cakes that requires reconstitution with a buffer or water prior to administration. Although it has been successful in improving the stability of protein-based formulations, the freeze drying process itself also contributes to protein aggregation. This process introduces other stresses such as freezing, thawing and drying. In addition to these stresses, the agitation processes used during reconstitution may also destabilize the protein’s native structure. Two key processes used in preparation of protein based formulations were studied in this work; mechanical agitation and freeze drying. The aim of this project was to explore the aggregation of proteins that occur due to the various technological processes typical in the production of protein based formulations. The project has two parts that relates to liquid and solid formulations. In the first part, the effect of different methods of mechanical agitations on BSA protein was investigated. In the second part, the focus was on the effect of formulation (i.e. the application of amino acids) on aggregation of protein (BSA) in freeze dried formulations. Arginine and lysine were added individually into protein-based freeze-dried formulation to study their potential of improving the stability of the proteins during manufacturing, storage and reconstitution. In the formulation development, additional excipients were added to prevent moisture uptake due to the hygroscopic properties of the amino acids and to provide lyo- and cryo- protection for the protein molecule during freeze drying. Without further purification, BSA solutions prepared by using sonication, low shear rotor mixer or high shear tube/pipe mixing were studied using dynamic light scattering (DLS). Thioflavin T assay and turbidimetry analysis were used as complementary studies. In protein-based freeze dried formulations, at accelerated storage conditions, the presence of aggregates were studied in samples containing arginine or lysine using ThT assay and turbidimetry analysis. Characterisation of the freeze dried cakes was performed relative to their moisture sorption, cake shrinkage, mechanical properties and morphology using various analytical techniques. iv In the BSA solution studies, particle size analysis indicated two distributions for non-agitated BSA solution that corresponds to the average particle sizes of BSA molecules and their aggregates. Under mechanical stresses (all types), the intensity of distribution centered ≈ 7.8 nm reduces and broadens as the agitation time increases, indicating a reduction in the amount of “free” BSA macromolecules. The second distribution, as a result of increasing agitation time or shear intensity, reveals a significant shift towards larger sizes, or even splits into two particle size populations. These particle size growths reflect the formation of aggregates due to intensive collisions and, as a result, partial unfolding followed by hydrophobic interactions of exposed non-polar amino acids. UV spectra showed that aggregation in both low shear and mechanical vibration agitations were lower compared to the high shear stress. When compared to non-agitated BSA solution, ThT assay recorded ≈15 times higher fluorescence emission from the high shear samples, ≈2 times fluorescence emission from low shear and ≈6 times fluorescence emission from mechanical vibrations. Thus all the three agitation methods showed a good correlation between the results. The second part of this project was performed in three stages. In the initial 2 stages, 2- and 3-excipients component system were investigated to develop an optimal preliminary formulations which will be used in the final protein based 4-components formulations. From the 1st stage (ArgHCl/LysHCl + sugar/polyol), among 4 tested excipients (polyol and sugar), mannitol was observed to have resisted moisture uptake by the highly hygroscopic ArgHCl/LysHCl amino acids. However, mannitol is considered a good cryoprotector but has poor lyoprotection properties. Therefore, in the following stage, a 3rd excipient (in a 3-excipients component system) sucrose or trehalose, was introduced into the formulation. The formulation was made up of 20% ArgHCl (LysHCl), and various ratios of mannitol and sugar were explored. The criteria for selecting the best systems were based on ideal physicochemical properties i.e. moisture uptake, shrinkage, mechanical properties, matrix structure and appearance, and thermal properties. The final stage was the formulation of a 4-components system comprising the three excipients and combinations selected from the stage 2 studies, and the addition of BSA as the model protein. To study aggregation in this system, a freeze dried 4-components excipient/protein system was reconstituted and incubated at accelerated storage conditions over time. Fluorescence spectroscopy and turbidimetry were used to study aggregation of proteins, moisture uptake kinetics with gravimetric balance, and thermal analytical techniques were used to characterise the freeze dried cakes with and without BSA protein. This study represented a systematic analysis of aggregation of proteins in both liquid and solid formulations. Some of the novel aspects of this study include: v 1. The new experimental results obtained for aggregation of proteins in solution subjected to mechanical agitations. The high shear stress created by syringe agitation, simulated the real situation in post manufacturing process during filling through narrow pipes, and has been shown here to strongly affect the aggregation of protein macromolecules. 2. The development of a methodical approach for optimization of multi component (up to 4 excipients) protein based formulations. 3. The unexpected non-linear behavior of the physicochemical properties of the 3-excipients component system as a function of composition. To the best of my knowledge, this novel aspect has not been previously reported in literature. 4. Application of amino acid in protein based formulations has shown the inhibition of aggregation of BSA, with the highest effect observed with ArgHCl. The results of this study coincide with the conclusions published previously for aggregation of proteins in solution.

Les rhabdovirus, dont les virus de la stomatite vésiculaire (VSV) et de la rage (RAV) constituent des prototypes, sont des virus enveloppés dont le génome est constitué d'une seule molécule d'ARN simple brin de polarité négative qui font partie de l'ordre des Mononegavirales (MNV). La machinerie de transcription/réplication de ces virus est constituée de l'ARN génomique et de trois protéines qui sont communes à tous les virus de l'ordre des MNV, la (N) qui encapside le génome viral, la grande sous-unité de l'ARN polymérase ARN dépendante (L) et la phosphoprotéine (P) qui est un cofacteur non-catalytique de la L et sert de chaperonne à la N. Le premier objectif de mon travail de thèse consistait à déterminer la structure cristallographique du domaine de dimérisation de la phosphoprotéine du virus de la rage. La P des rhabdovirus est une protéine modulaire qui contient deux régions intrinsèquement désordonnée, un domaine central responsable la dimérisation et un domaine C-terminal responsable de la fixation sur la matrice N-ARN. Le modèle atomique obtenu à une résolution de 1.5A montre que la structure est très différente de celle du domaine correspondant chez VSV. Le second objectif de mon travail était la caractérisation structurale de la grande sous-unité L de la polymérase du virus de la stomatite vésiculaire. Cette enzyme de 2109,aa, possède six régions conservées. Le domaine conservé III comprend les régions impliquées dans l'activité de polymérisation et les domaines V et VI sont responsables de la formation de la coiffe des ARNm. Plusieurs stratégies ont été envisagées successivement. (1) Sur la base de prédictions de structures secondaires et de prédictions de désordre, nous avons essayé d'exprimer différents fragments en système d'expression bactérien. Les constructions testées se sont avérées insolubles et certaines d'entre elles fixaient GroEL, indiquant un problème de repliement. (2) Nous avons alors essayé d'exprimer la L seule ou en complexe avec la P en système d'expression eucaryote. La purification s'est avérée impossible, la protéine L restant toujours associées à des protéines cellulaires visibles par coloration au bleu de Coommassie. (3) Finalement nous avons réussi à purifier la polymérase à partir de virus entier. La préparation de la polymérase était très homogène et a permis d'entreprendre une caractérisation par microscopie électronique. Une classification d'images a permis de construire un premier modèle à basse résolution. Le modèle révèle la présence d'un domaine annulaire avec plusieurs domaines structurés attachés au coeur de la polymérase. La cryo-microscopie électronique et la tomographie permettront d'obtenir plus de détails sur cette protéine. / Rhabdoviruses, including vesicular stomatitis virus (VSV) and rabies virus (RAV), are enveloped viruses which genome is made of a single molecule of negative-sense RNA and are classified in the order Mononegavirales (MNV). The transcription/replication machinery of these viruses consists of the genomic RNA and of three proteins, which are common to all other viruses of the order MNV, a nucleoprotein (N) that encapsidates the viral genome, a large subunit of the RNA-dependent RNA polymerase (L) and a phosphoprotein (P) that acts as a non-catalytic cofactor of L and a chaperone of N. The first goal of my research project was to determine the crystallographic structure of the dimerization domain of the rabies virus phosphoprotein. The P protein of the rhabdoviruses is a modular protein, which contains two intrinsically disordered regions, a central dimerization domain and a C-terminal domain involved in binding to the N-RNA template. The atomic model obtained at a resolution of 1.5 A showed that the structure is different from that of the corresponding domain of VSV. The second goal was the structural characterization of the large subunit L of VSV polymerase. The enzyme of 2109 aa has six conserved regions. Conserved region III includes the residues involved in the RNA synthesis activity, whereas domains V and VI are involved in mRNA capping formation. Three strategies were successively developed: (1) On the basis of secondary structure and disorder predictions, we tried to express different fragments in bacterial expression systems. These constructions appeared to be insoluble and some of them bound GroEL suggesting a folding problem; (2) We tried to express L alone or co-express it with P in eukaryotic expression system. The purification appeared to be impossible, the L protein always remaining associated with host-cell proteins in amounts detectable by Coommassie staining; (3) We succeeded in purifying the L protein from the virus. The L samples were homogenous and allowed a characterization by electron microscopy. Image classes allowed the reconstruction of a first low-resolution model. This model revealed the presence of a large ring-like domain and several globular domains. Cryo-electron microscopy and tomography should lead to a more detailed description of this protein.

Made available in DSpace on 2014-12-17T15:42:14Z (GMT). No. of bitstreams: 1 GuymmannCS_DISSERT-.pdf: 2904070 bytes, checksum: 4cd1c00978977c422c79766db70f2678 (MD5) Previous issue date: 2011-08-12 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Alkyl polyethoxylates are surfactants widely used in vastly different fields, from oil exploitation to pharmaceutical applications. One of the most interesting characteristics of these surfactants is their ability to form micellar systems with specific geometry, the so-called wormlike micelle. In this work, microemulsions with three distinct compositions (C/T = 40 %, 30 % and 25 %) was used with contain UNITOL / butanol / water / xylene, cosurfactant / surfactante (C/S) ratio equal to 0,5. The microemulsion was characterized by dynamic light scattering (DLS), capillary viscometry, torque rheometry and surface tensiometry experiments carried out with systems based on xylene, water, butanol (cosurfactant) and nonaethyleneglycolmonododecyl ether (surfactant), with fixed surfactant:cosurfactant:oil composition (with and without oil phase) and varying the overall concentration of the microemulsion. The results showed that a transition from wormlike micelles to nanodrops was characterized by maximum relative viscosity (depending on how relative viscosity was defined), which was connected to maximum effective diameter, determined by DLS. Surface tension suggested that adsorption at the air water interface had a Langmuir character and that the limiting value of the surfactant surface excess was independent of the presence of cosurfactant and xylene. The results of the solubilization of oil sludge and oil recovery with the microemulsion: C/S = 40%, 30% and 25% proved to be quite effective in solubilization of oil sludge, with the percentage of solubilization (%solubilization) as high as 92.37% and enhanced oil recovery rates up to 90.22% for the point with the highest concentration of active material (surfactant), that is, 40%. / Os tensoativos alquil-polietoxilados s?o amplamente utilizados em diferentes campos, desde a explota??o de petr?leo at? aplica??es na ind?stria farmac?utica. Uma das caracter?sticas mais interessantes destes tensoativos ? a sua capacidade de formar sistemas micelares que apresentam uma geometria micelar espec?fica, a chamada micela wormlike, tipo verme . Neste trabalho, foram utilizados tr?s pontos de microemuls?o: C/T = 40 %, C/T = 30 % e C/T = 25 % num sistema contendo UNITOL/xileno/butanol/?gua. Esses pontos de microemuls?o foram caracterizados por espalhamento de luz din?mico (DLS), viscosimetria capilar, reometria de torque e tens?o superficial com composi??es de tensoativo, cotensoativo e ?leo fixo - 5 % (com e sem fase ?leo) e variando a concentra??o total dos pontos de microemuls?o. Os resultados mostraram que a transi??o da geometria de micela wormlike para nanogotas poderia ser caracterizada por um m?ximo na viscosidade relativa (dependendo de como a viscosidade foi definida), que foi vinculado a um m?ximo de di?metro efetivo, determinada por DLS. A tens?o superficial sugeriu que a adsor??o na interface ?gua/ar tinha um car?ter de Langmuir, o limite de excesso da superf?cie do tensoativo ? independente da presen?a de cotensoativo e do xileno, e que o valor limite do excesso superficial foi independente da presen?a de cotensoativo e xileno. Os resultados da solubiliza??o da borra de petr?leo e da recupera??o avan?ada de petr?leo com os pontos de microemuls?o C/T = 40 %, C/T = 30 % e C/T = 25 %, se mostraram bastante eficaz com solubiliza??o da borra de petr?leo, com porcentagem de solubiliza??o (% solubiliza??o) at? 92,37 %, e recupera??o avan?ada de petr?leo de at? 90,22 % para o ponto que apresenta maior concentra??o de mat?ria ativa (tensoativo), ou seja, C/T = 40 %.

Made available in DSpace on 2014-12-17T15:42:30Z (GMT). No. of bitstreams: 1 KeilaSA_TESE.pdf: 6694216 bytes, checksum: df1754b48618e11f2ae95e003ae20c2c (MD5) Previous issue date: 2013-12-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Chitosan is a biopolymer derived from the shells of crustaceans, biodegradable, inexpensive and renewable with important physical and chemical properties. Moreover, the different modifications possible in its chemical structure generate new properties, making it an attractive polysaccharide owing to its range of potential applications. Polymers have been used in oil production operations. However, growing concern over environmental constraints has prompted oil industry to search for environmentally sustainable materials. As such, this study sought to obtain chitosan derivatives grafted with hydrophilic (poly(ethylene glycol), mPEG) and/or hydrophobic groups (n-dodecyl) via a simple (one-pot) method and evaluate their physicochemical properties as a function of varying pH using rheology, small-angle Xray scattering (SAXS), dynamic light scattering (DLS) and zeta potential. The chitosan derivatives were prepared using reductive alkylation under mild reaction conditions and the chemical structure of the polymers was characterized by nuclear magnetic resonance (1H NMR) and CHN elemental analysis. Considering a constant mPEG/Chitosan molar ratio on modification of chitosan, the solubility of the polymer across a wide pH range (acidic, neutral and basic) could only be improved when some of the amino groups were submitted to reacetylation using the one-pot method. Under these conditions, solubility is maintained even with the simultaneous insertion of n-dodecyl. On the other hand, the solubility of derivatives obtained only through mPEG incorporation using the traditional methodology, or with the ndodecyl group, was similar to that of its precursor. The hydrophilic group promoted decreased viscosity of the polymer solutions at 10 g/L in acid medium. However, at basic pH, both viscosity and thermal stability increased, as well as exhibited a pronounced pseudoplastic behavior, suggesting strong intermolecular associations in the alkaline medium. The SAXS results showed a polyelectrolyte behavior with the decrease in pH for the polymer systems. DLS analyses revealed that although the dilute polymer solutions at 1 g/L and pH 3 exhibited a high density of protonated amino groups along the polymer chain, the high degree of charge contributed significantly to aggregation, promoting increased particle size with the decrease in pH. Furthermore, the hydrophobic group also contributed to increasing the size of aggregates in solution at pH 3, whereas the hydrophilic group helped reduce their size across the entire pH range. Nevertheless, the nature of aggregation was dependent on the pH of the medium. Zeta potential results indicated that its values do not depend solely on the surface charge of the particle, but are also dependent on the net charge of the medium. In this study, water soluble associative polymers exhibit properties that can be of great interest in the petroleum industry / A quitosana ? um biopol?mero derivado de carapa?as de crust?ceos, de baixo custo, biodegrad?vel, renov?vel, que apresenta propriedades f?sico-qu?micas importantes e, ainda, proporciona diferentes possibilidades de modifica??es em sua estrutura qu?mica, gerando novas propriedades, o que torna esse polissacar?deo muito atraente do ponto de vista de aplica??o. Os pol?meros s?o utilizados em v?rias opera??es na produ??o do petr?leo. Entretanto, a crescente preocupa??o com as restri??es ambientais t?m promovido a busca por materiais ambientalmente sustent?veis pela ind?stria do petr?leo. Dessa forma, esse estudo prop?s a obten??o de quitosana graftizada com grupos hidrof?lico (poli(etileno glicol), mPEG) e/ou hidrof?bico (n-dodecila) por uma metodologia mais simples (one-pot) e a avalia??o de suas propriedades f?sico-qu?micas em fun??o da varia??o de pH, atrav?s das an?lises de reologia, espalhamento de raios-X a baixos ?ngulos (SAXS), espalhamento de luz din?mico (DLS) e potencial zeta. Os derivados de quitosana foram preparados utilizando a rea??o de alquila??o redutiva em condi??es reacionais brandas e a estrutura qu?mica dos pol?meros foi caracterizada por resson?ncia magn?tica nuclear de hidrog?nio (RMN 1H) e an?lise elementar CHN. Considerando constante a raz?o molar mPEG/Quitosana na modifica??o qu?mica da quitosana em diferentes metodologias, foi poss?vel melhorar a solubilidade da quitosana em uma ampla faixa de pH (?cido, neutro e b?sico) usando a metodologia one-pot, em que uma parte dos grupos amino foi reacetilada. Nesta condi??o, at? mesmo com a inser??o simult?nea do n-dodecila, a solubilidade se manteve. Por outro lado, a solubilidade dos derivados obtidos apenas com a incorpora??o de mPEG atrav?s de metodologia tradicional, ou com o grupo ndodecila, foi similar ao seu precursor. O grupo hidrof?lico promoveu a diminui??o da viscosidade das solu??es polim?ricas a 10 g/L em meio ?cido. Entretanto, em pH b?sico, esse grupo contribuiu para o aumento da viscosidade e da estabilidade t?rmica das solu??es, assim como, favoreceu um comportamento pseudopl?stico mais acentuado, sugerindo fortes associa??es intermoleculares no meio alcalino. Os resultados de SAXS apresentaram um comportamento de polieletr?lito com a diminui??o do pH para os sistemas polim?ricos. As an?lises de DLS revelaram que as solu??es dilu?das dos pol?meros a 1 g/L em pH 3, embora apresentem uma alta densidade de grupos amino protonados ao longo da cadeia polim?rica, o alto grau de cargas contribuiu significativamente para a agrega??o, promovendo o aumento do tamanho das part?culas com a diminui??o do pH. Al?m disso, o grupo hidrof?bico tamb?m contribuiu para aumentar o tamanho dos agregados em solu??o no pH 3 e o grupo hidrof?lico favoreceu para reduzi-los em toda faixa de pH. Entretanto, a natureza de agrega??o foi dependente do pH do meio. Os resultados do potencial zeta indicaram que seus valores n?o dependem apenas da carga da superf?cie da part?cula, mas ? resultante da carga l?quida do meio. Os sistemas polim?ricos associativos em solu??o aquosa obtidos neste estudo apresentam propriedades que podem ser atraentes em v?rias aplica??es na ind?stria do petr?leo

This thesis has the dual purpose of raising awareness of the importance of the mixing protocol on the end products of polyelectrolyte-oppositely charged surfactant systems, and to contribute to a better understanding of the properties of bottle-brush polyelectrolytes when adsorbed onto interfaces. In the first part of this thesis work, the effects of the mixing protocol and the mixing procedure on formed polyelectrolyte-oppositely charged surfactant aggregates were investigated. It was shown that the initial properties of the aggregates were highly dependent on the mixing parameters, and that the difference between the resulting aggregates persisted for long periods of time. The second part of the studies was devoted to the surface properties of a series of bottle-brush polyelectrolytes made of charged segments and segments bearing poly(ethylene oxide) side chains; particular attention was paid to the effect of side chain to charge density ratio of the polyelectrolytes. It was shown that the adsorbed mass of the polyelectrolytes, and the corresponding number of poly(ethylene oxide) bearing segments at the interface, went through a maximum as the charge density of the polyelectrolyte was increased. Also, it was found that bottle-brush polyelectrolyte layers were desorbed quite easily when subjected to salt solutions. This observation was rationalized by the unfavourable excluded volume interactions between the side chains and the entropic penalty of confining them at an interface, which weaken the strength of the binding of the polyelectrolytes to the interface. However, it was shown that the same side chains effectively protect the adsorbed layer against desorption when the layer is exposed to solutions containing an oppositely charged surfactant. Investigation of the lubrication properties of the bottle-brush polyelectrolytes in an asymmetric (mica-silica) system also related the observed favourable frictional properties to the protective nature of the side chains. The decisive factor for achieving very low coefficients of friction was found to be the concentration of the side chains in the gap between the surfaces. Interestingly, it was shown that a brush-like conformation of the bottle-brush polyelectrolyte at the interface has little effect on achieving favourable lubrication properties. However, a brush-like conformation is vital for the resilience of the adsorbed layer against the competitive adsorption of species with a higher surface affinity. / QC 20100830

This thesis describes bioanalytical methods for drug determination in biological matrixes, with drugs in focus used against diseases largely affecting low-income countries. Solid-phase extraction is used for sample cleanup, and processed samples are analyzed by liquid chromatography. Developed bioanalytical methods are validated according to international guidelines. Eflornithine (DFMO) is a chiral drug, used for treating human African trypanosomiasis. A bioanalytical method for determination of DFMO enantiomers in plasma is presented. The enantiomers are detected by evaporative light-scattering detection. The method has been applied to determination of D-DFMO and L-DFMO in rats, after intravenous and oral administration of racemic DFMO. It is concluded that DFMO exhibits enantioselective absorption, with the more potent enantiomer L-DFMO being less favored. Sulfadoxine (SD) and sulfamethoxazole (SM) are sulfa-drugs used for malaria and pneumonia respectively. Two methods are described for simultaneous determination of SD and SM in capillary blood sampled on filter paper. The former method allows direct injection of extracts from dried blood spots (DBS), while for the latter method solid-phase extraction is added. Pre-analytical factors contributing to measurement uncertainty is also discussed, and it is concluded that it is of high importance that homogeneity in type of sampling paper and sampling volume is assured. Piperaquine (PQ) is an antimalarial, increasingly used in artemisinin combination therapy. A method for determination of piperaquine in DBS is presented. By using a monolithic LC column, a very short LC analysis of two minutes per sample is achieved. A method for simultaneous determination of three antiretroviral drugs i.e. lamivudine (3TC), zidovudine (AZT) and nevirapine (NVP), in DBS samples is described. The method is applied to drug determination in two subjects after receiving standard antiretroviral treatment. Conclusion is that the method is suitable for determination of 3TC and NVP, and to some extent for AZT.

This thesis reports the results of light-scattering measurements and visual investigations of critical phenomena in the complex mixture 1-propanol (1P) + water (W) + potassium chloride (KCl) which has a special critical point (or a special thermodynamic state) known as the double critical point (DCP). The main theme of the thesis is the critical behavior on approaching a special critical point (i.e., the DCP) in a complex or associating mixture in contrast with that in simple, nonassociating mixtures. The asymptotic critical behavior in complex or associating fluids, such as polymer solutions and blends, ionic and nonionic micellar solutions, microemulsions, aqueous and nonaqueous electrolyte solutions, protein solutions, etc., is now commonly accepted to belong to the 3D-Ising universality class. However, the temperature range of the asymptotic regime in these fluids, with universal behavior, has a nonuniversal width and is, in general, smaller than that in simple or nonassociating fluids. In complex mixtures, which are made up of relatively large molecules or particle clusters of mesoscopic range, the coupling between the conventional correlation length of the critical fluctuations ( ξ) and an additional length scale associated with the mesoscale structures (ξD) is known to modify the approach towards the universal nonclassical critical behavior near their critical points. Nevertheless, the generality of this approach needs to be confirmed. There are also instances of a pure classical or close to classical behavior being observed in the critical domain of complex mixtures, although recent experimental results contradict the earlier observations. Therefore, further experimental evidences than that presently available are necessary before one can say how far the analogy between simple and complex fluids can be pushed. Variations in the effective dielectric constant of a mixture have been known to affect the critical behavior. Furthermore, we anticipate the presence of special critical points in complex mixtures to cause nontrivial modifications in the approach towards the universal asymptotic critical behavior. Special thermodynamic states are characterized by critical fluctuations with exceptionally large correlation length, and are displayed by multicomponent liquid mixtures, in which there are a multitude of thermodynamic paths by which a critical point can be approached, and offers rich information about the critical phenomena. These issues are being addressed in this research work. This thesis is organized into 7 Chapters. Chapter 1 begins with an account of the historical development of the field of critical point phenomena with a brief introduction to critical phenomena in simple fluids. Critical phenomena observed in various complex systems such as aqueous and nonaqueous ionic fluids, polymer solutions and blends, micellar and microemulsion systems, etc., are discussed, with particular attention to investigations into crossover from Ising to mean-field critical behavior observed in these systems, which are relevant to the present work. Theoretical attempts at modeling ionic criticality are cited and summarized. This is followed by a discussion of re-entrant phase transitions in multicomponent liquid systems. An account of the various types of special critical points, such as double critical point, critical double point, critical inflection point, quadruple critical point, etc., highlighting the critical behavior on approaching these special critical points, and some of the models of reentrant miscibility are briefly given. The Chapter ends with a statement on the goals of the present research work. Chapter 2 describes the instrumentation developed and the data acquisition procedures adopted for the study. Details of the thermostats and precision temperature controllers used for visual and light-scattering measurements are provided. The important design considerations relating to the achievement of a high degree of temperature stability (~ ±1 mK in the range 293-383 K) are elucidated clearly. The temperature sensors used in the present experiments and their calibration procedures are discussed. The light-scattering instrumentation is discussed in depth. The problems associated with the light-scattering techniques when it is used to study critical point phenomena, and the strategies adopted to overcome them are discussed. The sample cells used for visual investigations and light- scattering experiments, along with the procedure adopted for cleaning and filling of sample cells are also described. Chapter 3 essentially deals with the characterization of the system 1P + W + KCl. It begins with a brief introduction to the critical behavior in complex mixtures, and the motivation behind choosing the present system. The phase behavior in the present mixture, the generation of the coexistence curves and the line of critical points in the mixture, and the method used for preparation of the samples are described. The criticality of the samples is judged by the equal volume phase separation criterion through visual investigations. Addition of a small amount of salt (i.e., KCl) to the 1P + W solution induces phase separation in the mixture as a result of a salting-out process. Decreasing the salt concentration has the same effect as that of increasing pressure on the liquid-liquid demixing of this mixture. Therefore, KCl may be considered as an appropriate field variable analogous to pressure in this mixture. The mixture 1P + W + KCl exhibits reentrant phase transitions and has an array of lower (TL) and upper (TU) critical solution temperatures. It is found that the line of TL’s and TU’s, known as the line of critical points, merge (TU - TL = ΔT → 0) to form a special thermodynamic state known as the DCP. The DCP is approached as close as 509 mK (i.e., ΔT ~ 509 mK) in this work. An analysis of the critical line shows that it is roughly parabolic in shape, which is in consonance with the predictions of the lattice models and the Landau-Ginzburg theory of phase transition. In addition to the presence of a special critical point, various structure probing techniques like small angle X-ray scattering (SAXS), small angle neutron scattering (SANS), etc., indicate the presence of large-scale density inhomogeneities or clusters in 1P + W solution and its augmentation on adding small amount of KCl. Therefore, the present mixture provides a unique possibility to investigate the combined effects of molecular structuring as well as a special critical point on the critical behavior. Only a section of the coexistence surface of the mixture could be generated, owing to various experimental limitations and other problems inherent to the system. This limited further studies on the coexistence curves in the mixture. Chapter 4 reports the critical behavior of osmotic susceptibility in the present mixture. The behavior of the susceptibility exponent is deduced from static light-scattering measurements, on approaching the lower critical solution temperatures (TL’s) along different experimental paths by varying t [ =| (T - T TL)/ TL|] from the lower one-phase region. The light-scattering data analysis emphasizes the need for correction-to-scaling terms for a proper description of the data over the investigated t range. Renormalization of the critical exponents is observed as the critical line is approached along certain special paths. Experimental evidence for the doubling of the extended scaling exponent Δ1 near the DCP is shown. There is no signature of Fisher renormalization in the values of the critical exponents. The data analysis yields very large magnitudes for the correction amplitudes A1 and A2, with the first-correction amplitude A1 being negative, signifying a nonmonotonic crossover behavior of the susceptibility exponent in the mixture. The magnitudes of the correction amplitudes are observed to increase gradually as TL approaches the DCP. The increasing need for extended scaling in the neighborhood of special critical points has been noted earlier in several aqueous electrolyte solutions, in polymer-solvent systems, etc. However, the magnitudes of the correction amplitudes were not as large as that in the present case. Analysis of the effective susceptibility exponent γeff in terms of t indicate that, for the TL far away from the DCP, γeff displays a nonmonotonic crossover from its single limit 3D Ising value (~ 1.24) towards its mean-field value with increase in t. While for that closest to the DCP, γeff displays a sharp, nonmonotonic crossover from its nearly doubled 3D-Ising value (~ 2.39) towards its nearly doubled mean-field value (~ 1.84) with increase in t. For the in-between TL’s, the limiting value of γeff in the asymptotic as well as nonasymptotic regimes gradually increases towards the DCP. The renormalized Ising regime extends over a relatively larger t range for the TL closest to the DCP, and a trend towards shrinkage in the renormalized Ising regime is observed as TL shifts away from the DCP. Nevertheless, the crossover behavior to the mean-field limit extends well beyond t > 10¯2 for the TL’s studied. The crossover behavior is discussed in terms of the emergence of a new lengthscale ξD associated with the enhanced ion-induced clustering seen in the mixture, as revealed by various structure probing techniques, while the observed unique trend in the crossover is discussed in terms of the varying influence of the DCP on the critical behavior along the TL line. The discussion is extended to explain the observed critical behavior in various re-entrant systems having other special critical points. The extended renormalized Ising regime towards the DCP is also reflected in a decrease in the correlation length amplitude (ξ0) as TL approaches the DCP. It is observed that the first-correction amplitude A1 corresponding to fit using two correction terms becomes more negative as TL approaches the DCP, implying an increase in the value of the parameter ū of the crossover model [by Anisimov et al., Phys. Rev. Lett. 75, 3146 (1995)] as the DCP is approached. This increase in reflected in a trend towards a relatively sharp crossover behavior of γeff as TL shifts towards the DCP, i.e., towards the high temperature critical points. The significance of the field variable tUL in understanding different aspects of reentrant phase transitions is manifested in the present system as well. Analysis of the data in terms of tUL led to the retrieval of universal values of the exponents for all TL’s. The effective susceptibility exponent as a function of tUL displays a nonmonotonic crossover from its asymptotic 3D-Ising value towards a value slightly lower than its nonasymptotic mean-field value of 1. The limited (TL _ T) range restricted such a behavior of the effective exponent (in terms of t as well as tUL) for the lowest TL. This feature of the effective susceptibility exponent is interpreted in terms of the possibility of a nonmonotonic crossover to the mean-field value from lower values in the nonasymptotic, high tUL region, as foreseen earlier in micellar systems. The effective susceptibility exponent in terms of tUL also indicates an increase in the sharpness of crossover towards the high temperature TL’s. An increase in the sharpness of crossover with polymer chain length has been observed in polymer solutions. Therefore, our results suggest the need for further composition and temperature-dependent study of molecular structuring in the present mixture. There is also a large decrease in the dielectric constant of the mixture towards the high temperature TL’s. In Chapter 5 the light-scattering measurements are performed on approaching the DCP along the line of the upper critical solution temperatures (i.e., TU’s), by varying t [ = (T - TU )/ TU ] from the high temperature one-phase region in the mixture. A trend towards shrinkage in the simple scaling region is observed as TU shifts away from the DCP. Such a trend was not visible in the data analysis of the TL’s using the correction terms, due to the varying (TL - T) ranges. The light-scattering data analysis substantiates the existence of a nonmonotonic crossover behavior of the susceptibility exponent in the mixture. As with the TL’s, for the TU closest to the DCP, γeff displays a nonmonotonic crossover from its 3D-Ising value towards its nearly doubled mean-field value with increase in t. While for that far away from the DCP, γeff displays a nonmonotonic crossover from its single limit Ising value towards a value slightly lower than its mean-field value of 1 with increase in t. The limited (TL – T) range restricted such a behavior of γeff for the TL far away from the DCP, This feature of γeff in the nonasymptotic, high t region is yet again interpreted in terms of the possibility of a nonmonotonic crossover to the mean-field value from below. Unlike TL’s, the crossover behavior in the present case is pronounced and more sharp for all TU’s. However, the variation in the width of the renormalized Ising regime on approaching the DCP along the TU line is quite similar to that observed along the TL line. The crossover behavior is attributed to the strong ion-induced structuring seen in the mixture, while the observed trend in the crossover as TU shifts towards/away from the DCP is attributed to the varying influence of the DCP. The influence of the DCP on the critical behavior along the TU (or TL) line decreases as TU (or TL) shifts away from the DCP. Our observations indicate an increase in the sharpness of crossover as the critical temperature shifts from TL towards TU, or in other words, as the critical point shifts towards higher temperatures. SANS measurements on the present mixture indicate no difference in the growth of mesoscale clusters in the lower and upper one-phase regions in the mixture. Hence, the observed increase in the sharpness of crossover towards the TU’s is very puzzling. The dielectric constant of the major constituent (i.e., water, ~ 62 %) of the present mixture decreases from around 80 to 63 as the critical temperature shifts from TL towards TU. Therefore, our results suggest the need to look at the crossover phenomena probably from two perspectives, namely, the solvent or dielectric effect and the clustering effect. The increase in the sharpness of the crossover behavior on approaching the high temperature critical points is probably related to the macroscopic property of the mixture, i.e., to the decrease in the dielectric constant of the mixture, while the actual nonmonotonic character of the crossover behavior is related to the microscopic property of the mixture, i.e., to the clustering effects, the extent of which determines the width of the asymptotic critical domain. However, this conclusion is somewhat subtle and calls for rigorous theoretical and experimental efforts to unravel the exact dependence of the crossover behavior on the dielectric constant. Analysis using the field variable tUL in lieu of the conventional variable t led to the retrieval of unique, universal exponents for all TU’s irrespective of the ΔT value. For all TU’s, the effective susceptibility exponent in terms of tUL displays a nonmonotonic crossover from its asymptotic 3D-Ising value towards a value slightly lower than its nonasymptotic mean-field value of 1, as that observed in the t analysis of the effective exponent for the TU far away from the DCP. Like with the TL’s, the crossover behavior extends over nearly the same tUL range for the TU’s studied. However, the crossover is again sharper when compared to the TL’s. Chapter 6 reports light-scattering measurements (by heating as well as cooling) on a non phase-separating 1P + W + KCl mixture in the vicinity of the DCP. The results indicate that despite the lack of phase-separation or critical points, critical-phenomena-like fluctuations can still occur in homogeneous mixtures if they reside in some other direction than temperature or composition (like, pressure or salt concentration) of the phase diagram. Unlike earlier studies on non phase-separating mixtures, our results indicate a crossover behavior of the effective susceptibility exponent, in addition to the power-law behavior. Chapter 7 sums up the major findings of the work reported in this thesis. It also presents a range of open problems that need to be explored further in order to fully understand the results that are reported in this thesis, especially, regarding the exact dependence of dielectric constant of the mixture on the character of the crossover behavior.

Neuf maladies neurodégénératives sont le produit de l’expression de gènes mutés, dans lesquels le codon CAG est répété au-delà d’un seuil pathologique. Ceci produit des protéines mutantes dans lesquelles sont insérés des segments de polyglutamines (polyGln), qui perdent leur activité et acquièrent une nouvelle fonction, ce qui est toxique pour le neurone. Ces altérations sont attribuables aux propriétés particulières de la polyGln. En effet, ces dernières possèdent la capacité de s’assembler pour former des corps d’inclusion intracellulaires. Cette propension à l’agrégation de la polyGln rend difficile l’étude de ces pathologies. C’est ainsi que l’utilisation de peptides peut s’avérer une approche avantageuse. Toutefois, la synthèse de polyGln est associée à de nombreuses délétions et nécessite l’ajout de groupements chargés afin de permettre leur purification. Cependant, ce prérequis donne lieu à des interactions électrostatiques qui biaisent la structure et la cinétique d’agrégation de ces peptides, en plus d’interférer avec l’évaluation d’éventuels agents thérapeutiques. L’objectif du projet est de développer un système permettant l’étude de la polyGln en s’affranchissant des effets de charges. Pour ce faire, deux approches ont été explorées, la première utilise la polyGln non chargée et la seconde utilise une structure polyGln-morpholine ayant des charges labiles en fonction du pH. Ces peptides ont été produits en utilisant une approche linéaire de synthèse peptidique sur support solide avec protection maximale des chaînes latérales. La purification a été effectuée par chromatographie de haute performance en phase inverse en milieu acide. Ces stratégies ont permis de produire des peptides de polyGln de grande pureté avec des rendements acceptables. Une procédure de solubilisation des peptides alliant sonication et lyophilisation a été développée afin d’étudier chacun de ces peptides à l’aide de diverses techniques physicochimiques, telles que la diffusion de la lumière, la spectroscopie de résonance magnétique nucléaire, Raman et UV-visible, le dichroïsme circulaire et la microscopie optique polarisée. La polyGln non chargée solubilisée dans le trifluoroéthanol-eau a montré que la taille des particules et la vitesse d’agrégation sont proportionnelles à la fraction volumique en eau. De plus, la structure secondaire en solution est à prédominance alpha et semble être peu sensible à la fraction d’eau jusqu’à un certain seuil (25%) après lequel la structure aléatoire prédomine. L’analyse des agrégats à l’état solide montre des structures hélicoïdales > aléatoires et ont les caractéristiques des fibrilles amyloïdes. Le peptide de polyGln-morpholines a un pKa de 7,3 en milieu aqueux. Il demeure en solution lorsque le pH < pKa et à faible force ionique, alors qu’il s’autoassemble lorsque ces conditions ne sont pas respectées. Ceci suggère que la répulsion électrostatique est responsable de la stabilisation du peptide en solution. La dimension fractale nous indique que le peptide forme des agrégats compacts dont les constituants ont une taille de 2,5 nm, compatibles avec une conformation aléatoire compacte, en coude bêta ou hélicoïdale. Ceci est en accord avec l’étude structurale des peptides en solution qui a montré des espèces aléatoires > bêta > alpha. De plus, en RMN, l’élargissement des signaux du 1Hγ en cours d’agrégation suggère une interaction via les chaînes latérales. Les analyses en phase solide ont plutôt montré une prédominance de structures bêta et alpha. L’inhibition de l’agrégation à pH 8 varie selon rouge de Congo > tréhalose, alors que le peptide liant la polyGln 1 et la thioflavine T ne semble pas avoir d’effet. Ces approches ont donc permis pour la première fois de s’affranchir des effets de charges auparavant inhérents à l’étude de la polyGln en solution et par conséquent d’obtenir des informations inédites quant à la solubilité, la structure et la cinétique d’agrégation. Enfin, le dispositif à charges labiles permet d’évaluer l’efficacité d’éventuels agents thérapeutiques à pH quasi physiologique. / Nine neurodegenerative diseases come from mutated genes expression in which the CAG codon is repeated above a pathological threshold. This is producing mutant proteins, in which are inserted polyglutamine (polyGln) segments, which lose their activity and acquire a new function that is toxic for the neuron. These alterations are related to the peculiar properties of the polyGln. Indeed, these polypeptides have the capacity of autoassemble to form intracellular inclusion bodies. This aggregation tendency of polyGln makes difficult the study of these pathologies. Thus, the use of peptides could constitute an advantageous approach. However, the synthesis of polyGln is associated with numerous deletions and necessitates the addition of charged moieties to achieve purification. Unfortunately, this requirement creates electrostatic interactions that modify the structure and aggregation kinetics of these peptides, in addition to interfering with the evaluation of potential therapeutical agents. The aim of this project is to develop a system to study polyGln without the charge effects. To do so, two approaches were explored, the first used uncharged polyGln and the second used a polyGln-morpholine structure bearing pH-dependent labile charges. These peptides were produced by solid-support synthesis using a linear and maximal protection approach. Purification was performed by reverse phase high-performance liquid chromatography. These strategies allowed the production of peptides of high purity in good yields. A solubilization procedure combining sonication and lyophilization was developed to study each of these peptides by physicochemical techniques such as light scattering, magnetic resonance, Raman and UV-visible spectroscopies, circular dichroism and polarized optical microscopy. The uncharged polyGln solubilized in trifluoroethanol-water showed that particle size and aggregation kinetics are proportional to volumetric water fraction. Furthermore, the secondary structure in solution is alpha-predominant and seems rather insensitive to water fraction up to a threshold (25%) above which random coil structure predominates. The analysis of solid-state aggregates showed that helicoidal structures are more abundant than random structures and have the characteristics of amyloid fibrils. The polyGln-morpholines peptide has a pKa of 7.3 in aqueous media. It is soluble when pH < pKa and at low ionic strength, but it autoassociates when these conditions are not respected. This suggests that electrostatic repulsion is responsible for the stabilization of the peptide in solution. The fractal dimension indicates that the peptide forms compact aggregates whose constituents are 2.5 nm in size, in agreement with compact random coil, beta-hairpin or helicoidal structures. This is in agreement with the results of solution peptide structure studies showing that random coil > beta > alpha. Furthermore, the broadening of 1Hγ NMR signals while the peptide is aggregating suggests an interaction between side-chains. Solid-phase studies showed predominant beta and alpha structures. The aggregation inhibition at pH 8.0 was higher for Congo red than for trehalose, while polyglutamine binding peptide 1 and thioflavine T did not seem to be effective. These approaches permitted for the first time to overcome the charge effects that were previously inherent to polyGln solution studies and to obtain new information about solubility, structure and aggregation kinetics. Finally, the labile charge groups allow the evaluation of the efficiency of potential therapeutic agents at near physiological pH.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Στην παρούσα διπλωματική εργασία θα γίνει μελέτη και εφαρμογή μεθόδων επίλυσης του προβλήματος αναγνώρισης γεωμετρικών χαρακτηριστικών ανθρώπινων ερυθρών αιμοσφαιρίων από προσομοιωμένες εικόνες σκέδασης ΗΜ ακτινοβολίας ενός He-Ne laser 632.8 μm. Στο πρώτο κεφάλαιο γίνεται μια εισαγωγή στις ιδιότητες και τα χαρακτηριστικά του ερυθροκυττάρου καθώς, επίσης, παρουσιάζονται διάφορες ανωμαλίες των ερυθροκυττάρων και οι μέχρι στιγμής χρησιμοποιούμενοι τρόποι ανίχνευσής των. Στο δεύτερο κεφάλαιο της εργασίας γίνεται μια εισαγωγή στις ιδιότητες της ΗΜ ακτινοβολίας, περιγράφεται το φαινόμενο της σκέδασης και παρουσιάζεται το ευθύ πρόβλημα σκέδασης ΗΜ ακτινοβολίας ανθρώπινων ερυθροκυττάρων. Το τρίτο κεφάλαιο αποτελείται από δύο μέρη. Στο πρώτο μέρος γίνεται εκτενής ανάλυση της θεωρίας των τεχνητών νευρωνικών δικτύων και περιγράφονται τα νευρωνικά δίκτυα ακτινικών συναρτήσεων RBF. Στη συνέχεια, αναφέρονται οι μέθοδοι εξαγωγής παραμέτρων και, πιο συγκεκριμένα, δίνεται το θεωρητικό και μαθηματικό υπόβαθρο των μεθόδων που χρησιμοποιήθηκαν οι οποίες είναι ο αλογόριθμος Singular Value Decomposition (SVD), o Angular Radial μετασχηματισμός (ART) και φίλτρα Gabor. Στο δεύτερο μέρος περιγράφεται η επίλυση του αντίστροφου προβλήματος σκέδασης. Παρουσιάζεται η μεθοδολογία της διαδικασίας επίλυσης όπου εφαρμόστηκαν ο αλογόριθμος συμπίεσης εικόνας SVD, o περιγραφέας σχήματος ART και ο περιγραφέας υφής με φίλτρα Gabor για την εύρεση των γεωμετρικών χαρακτηριστικών και νευρωνικό δίκτυο ακτινικών συναρτήσεων RBF για την ταξινόμηση των ερυθροκυττάρων. Στο τέταρτο και τελευταίο κεφάλαιο γίνεται δοκιμή και αξιολόγηση της μεθόδου και συνοψίζονται τα αποτελέσματα και τα συμπεράσματα που εξήχθησαν κατά τη διάρκεια της εκπόνησης αυτής της διπλωματικής. / In this thesis we study and implement methods of estimating the geometrical features of the human red blood cell from a set of simulated light scattering images produced by a He-Ne laser beam at 632.8 μm. Ιn first chapter an introduction to the properties and the characteristics of red blood cells are presented. Furthermore, we describe various abnormalities of erythrocytes and the until now used ways of detection. In second chapter the properties of electromagnetic radiation and the light scattering problem of EM radiation from human erythrocytes are presented. The third chapter consists of two parts. In first part we analyse the theory of neural networks and we describe the radial basis function neural network. Then, we describe the theoritical and mathematical background of the methods that we use for feature extraction which are Singular Value Decomposition (SVD), Angular Radial Transform and Gabor filters. In second part the solution of the inverse problem of light scattering is described. We present the methodology of the solution process in which we implement a Singular Value Decomposition approach, a shape descriptor with Angular Radial Transform and a homogenous texture descriptor which uses Gabor filters for the estimation of the geometrical characteristics and a RBF neural network for the classification of the erythrocytes. In the forth and last chapter the described methods are evaluated and we summarise the experimental results and conclusions that were extracted from this thesis.