The aim of this study was to investigate the molecular interactions of the anti-cancer drug Doxorubicin (DOX) with poly(methacrylic acid) grafted starch nanoparticles (PMAA-g-St). In order to fully understand the DOX/PMAA-g-St system, we conducted in-depth studies on DOX dimer dissociation and DOX/PMAA-g-St binding interactions using various techniques such as isothermal titration calorimetry (ITC), dynamic light scattering (DLS), and fluorescence and absorption spectroscopy. Based on our experimental results, we developed a quantitative thermodynamic model with relevant parameters such as dissociation constant, Kd, as well as enthalpy of binding, ΔH, in order to explain DOX/PMAA-g-St interactions. In addition, we also studied the effect of environmental factors such as pH and NaCl on DOX self-association and DOX/PMAA-g-St complex formation. In conclusion, the combination of results obtained from various techniques as well as the multispecies equilibrium model, enables us to interpret quantitatively the data of drug loading onto and release from polymeric nanoparticles.

Polymere Nanopartikel sind ein vielversprechender Ansatz für die Diagnose und Therapie von Krankheiten. Sie ermöglichen den Einsatz von schwerlöslichen oder instabilen Wirkstoffen. Ein weiterer Vorteil ist die Möglichkeit das Targetings, durch gezielte Modifikationen des Nanopartikels wird der Wirkstoff zum Zielort transportiert und kann dort in der gewünschten Form freigesetzt werden; dadurch könnten bei erhöhter Wirksamkeit die Nebenwirkungen von Medikamenten reduziert werden. Ziel dieser Arbeit war die Untersuchung von physikalischen und biochemischen Eigenschaften von Nanopartikeln bestehend aus einem abbaustabilen Polystyren- Kern und einer biologisch abbaubaren Schale aus Polybutylcyanoacrylat. Es werden Methoden beschrieben, um die Größe, Struktur und den Abbau dieser Wirkstoffträger zu untersuchen. Die untersuchten Nanopartikel zeigen RAYLEIGH-Streuung, sowohl Größe als auch Abbau können durch Messung des Absorptionsspektrums bestimmt werden. Weiterhin konnten diese Eigenschaften mit Hilfe von dynamischer und statischer Lichtstreuung sowie Neutronenkleinwinkelstreuung untersucht werden. Bei letzterer Methode konnte gezeigt werden, dass die Schale größtenteils abgebaut werden kann, während der Kern intakt bleibt. In einem weiteren Teil der Arbeit wurde die Überwindung der Blut-Hirn-Schranke durch polymere Nanopartikel untersucht. Dabei wurde der fluoreszierende Thioflavine als Modellwirkstoffe eingesetzt. Das Durchdringen der Blut-Hirn-Schranke konnte nur mit Nanopartikeln erreicht werden, an deren Oberfläche ein Apolipoprotein E-Peptid gekoppelt war. Es konnte gezeigt werden, das die Nanopartikelschale im Gehirn abgebaut wird, der Wirkstoff freigesetzt wird und an Amyloid β, einem Marker der Alzheimer-Krankheit, bindet.

La diffusion Raman exaltée de surface (SERS) est utilisée pour la mise au point d’un biocapteur capable de détecter des pesticides dans l’eau, en se basant sur le suivi de l’activité enzymatique de l’Acétylcholinestérase (ACHE). Les nanoparticules d’or sont utilisées comme substrats SERS actifs. Le signal Raman exalté de l’analyte est optimisé en testant plusieurs types de nanoparticules.Le Raman SERS a permis la détection directe du Paraoxon (PO) et du carbaryl (CA) et la possibilité de suivi de l’activité de l’ACHE. En absence d'inhibiteurs, la molécule d’acétylcholine (ATC) est transformée en acide acétique et en choline par l’enzyme ACHE. La mesure de l’activité de l’ACHE repose sur le suivi des concentrations en ATC car sa transformation est inhibée en présence de pesticides. Il a été ainsi possible d’établir une relation linéaire entre la concentration de pesticides et l’exaltation du signal Raman de l’ATC non transformé. La méthode a permis la détection du PO et du CA, avec une limite de détection beaucoup plus faible que la détection directe. Ce biocapteur basé sur l’activité de l’ACHE a ensuite été utilisé pour l'évaluation d’autres polluants (inhibiteurs d’ACHE) comme les additifs contenus dans les plastiques notamment. Enfin, nous avons développé une seconde approche qui consistait à mesurer l’activité de l’ACHE en utilisant la diffusion dynamique de la lumière. En effet, nous avons montré que les paramètres physicochimiques (agrégation) des AuNPs en contact avec certaines molécules, sont fortement influencés par l’activité enzymatique de l’ACHE. C’est ce phénomène d’instabilité qui nous a permis de distinguer entre les deux cas : absence et présence de PO. / Surface-enhanced Raman scattering (SERS) was used to develop a biosensor for the detection of pesticides through the monitoring of the enzymatic activity of acetylcholinesterase (ACHE). Gold nanoparticles (AuNPs) were used as an active SERS substrate. The enhanced Raman signal of the analyte is optimized by testing several types of nanoparticles. Raman SERS allowed the direct detection of Paraoxon (PO) and carbaryl (CA) pesticides and the possibility of follow-up of the activity of the ACHE. In the absence of inhibitors, the acetylcholine (ATC) is transformed into acetic acid and choline by the enzyme ACHE. The measurement of ACHE activity is performed through the monitoring of ATC concentrations because its transformation is inhibited in the presence of pesticides. Results showed a linear correlation between the concentration of pesticides and the SERS signal of the untransformed ATC. The method was optimized for the quantification of paraoxon and carbaryl with a limit of quantification much lower than the one obtained with a direct detection. Their identification was also possible using chemometrics. This biosensors, based on the ACHE activities, was applied to the evaluation of emergent pollutants: additives of commercial polymers. Our results suggested that most of the tested polymers contained molecules that act as inhibitors of the ACHE. Finally, we propose another very simple approach to measure the ACHE activity using dynamic light scattering measurements. We found that the physicochemical parameters (aggregation) of AuNPs were strongly influenced by the enzymatic activity of ACHE when in contact with specified molecules, allowing to detect the presence of PO.

Parmi les techniques d'imagerie optiques utilisées en milieu clinique, la principale limitation est la faible résolution lorsque la profondeur d'examen dépasse quelques mm. Cette limite de résolution ne permet pas à l'heure actuelle de concurrencer les techniques d'imagerie médicales permettant de réaliser un examen du corps dans son intégralité (Rayons X, IRM, Scanner). Dans ce cadre, l'imagerie acousto-optique présente plusieurs avantages: elle permet de mesurer des propriétés optiques utiles pour la détection de tumeur, à la résolution spatiale des ultrasons. Cependant, les dispositifs de détection utilisés présentent un manque de sensibilité et de rapidité qui freinent le transfert de cette technique en milieu clinique.Ce constat nous a conduit à étudier les caractéristiques intrinsèques du signal acousto-optique afin de proposer deux architectures de pixels basées sur des technologies CMOS. La première architecture, totalement analogique, présente des caractéristiques de vitesse d'acquisition compatibles avec le temps de corrélation des milieux biologiques (<1 ms)et un pré-traitement du signal utile. La seconde architecture intègre une fonction de conversion analogique-numérique de manière à simplifier le montage optique, et traiter le signal plus efficacement.Par ailleurs, le contrôle de la phase en plusieurs points du front est essentiel pour refocaliser les signaux lumineux. Pour contourner les limitations de vitesse des dispositifs de contrôle adaptatif de phase de l'état de l'art, nous avons développé un dispositif monolithique constitué de l'empilement physique d'un modulateur de lumière en phase, à cristaux liquides, sur un circuit CMOS constitué d'une matrice de photo-détecteurs et de circuits de traitement afin de permettre le contrôle de front d'onde dès son acquisition. Le dispositif opto-électronique a été proposé et couplé à la première architecture électronique. Il permet de réaliser une opération sur la phase de l'onde lumineuse en chaque pixel (conjugaison de phase par ex.) en parallèle sur les pixels d'une matrice, dans un intervalle de temps inférieur au temps de corrélation des milieux biologiques. / Among the optical medical imaging techniques used in medicine, the main limitation is the low resolution at a penetration depth greater than a few mm. This limitation does not allows competing with the standard imaging techniques such as X rays or RMI based imaging. In that scope, the acousto-optical imaging features several advantages: it allows measuring an optical contrast useful to detect tumors, in conjunction with the spatial resolution of ultrasound. However, the state of the art detecting devices feature a lack of sensitivity, which prevent its transfer to medical practitioners.This leads us to study the intrinsic features of the acousto-optical signal in order to propose two CMOS pixel architectures. The first one, fully analog, is compliant with the correlation time of biological tissue (1 ms typ.) and features an analog processing of the relevant signal. The second one is based on a digital pixel which contains an analog to digital converter, allowing simplifying the optical setup and increasing the robustness of the processing.In addition, related to the recent progress in wavefront control, an opto-electronic device, coupled with the first pixel architecture, has been proposed. It allows performing an optical phase operation (e.g. phase conjugation) in parallel on a pixels array, within the correlation time of biological media. Thus, this monolithic device circumvents the speed limitations of state of the art setup by a physical stacking of a liquid crystals spatial light modulator over a CMOS image sensor.

The thermal and dynamical properties of free-standing films are strongly dependent on the boundary conditions and the presence of an external field. Using distinct theoretical models and the dynamic light scattering experimental setup, we study the surface and external field effects on the relaxation dynamics, fluctuation-induced interaction energy, and phase transitions of smectic films. Using an extended McMillan model, we study as a homeotropic anchoring stabilizes the smetic order above the bulk transition temperature of the system. In particular, we determine as the transition temperature depends on the surface ordering and film thickness. We identify a characteristic anchoring for which the transition temperature does not depend on the film thickness. For strong surface ordering, we observe that the transition temperature presents a power law scaling behavior with the film thickness, in agreement with a series of existing experimental results in the literature. Within a Gaussian functional approximation, we also investigate how the fluctuationinduced interaction force depends on the nematic and smectic order parameter profiles. Close to nematic-smectic phase transition, our results indicate that the thermal Casimir force has a significant enhancement which reinforces the predominance of the fluctuationinduced interaction as compared to standard van der Waals interaction in thin smectic films. In temperatures where the smectic phase is well established, we investigate how the interplay of the surface order and the external field induced order affect the Casimirlike force. For asymmetrically anchored films, the fluctuation-induced interaction energy obeys a law of corresponding states and can change its nature from attractive to repulsive through variations of an external field. We discuss the possible relevance of this field effect in smectic wetting transitions. With concern to the dynamical properties, we investigate how surface operators modify the relaxation dynamics of hexatic-B liquid crystal films. In particular, we study how a surface ordering field modifies the damping rate of the orientational modes in the system. In a purely diffusive regime, we demonstrate that a surface tilted order drives the slowest relaxation mode from hydrodynamical to non-hydrodynamical character. In the viscous regime, the hydrodynamical character of the slowest relaxation mode persists even for a surface tilted order. However, the normal modes develop an oscillatory-exponential relaxation. Finally, we use the photon correlation spectroscopy technique to investigate the dynamic properties of free-standing films close to smectic-A - crystal-B transition. We observe that the experimental data indicate the existence of a non-hydrodynamic mode and measure the temperature dependence of the damping rate. We considered a simplified model that incorporates the gradient velocity field effects which reproduces qualitatively the experimental measurements. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / As propriedades térmicas e dinâmicas de filmes de cristais líquidos na fase esmética são extremamente dependentes das condições de contorno e da presença de campos externos. Utilizando diferentes modelagens teóricas e a técnica de espectroscopia de fotocorrelação, estudamos como as condiçõoes de contorno e a presença de um campo externo afetam a dinâmica de relaxação, a energia de interação induzida por flutuações e as transições de fase envolvendo a fase esmética. Utilizando uma extensão para o modelo de McMillan, estudamos como o ancoramento homeotrópico estabiliza a ordem esmética acima da temperatura de transição do sistema. Em particular, determinamos como a temperatura de transição depende da intensidade do ancoramento superficial e da espessura do filme. Identificamos um ancoramento característico para o qual a temperatura de transição independe da espessura. Para ancoramentos fortes, observamos que a depend encia da temperatura de transição com a espessura do filme segue aproximadamente uma lei de potência, em concordância com uma série de resultados experimentais existentes na literatura. Baseado em uma teoria funcional Gaussiana para o funcional energia livre, investigamos tamb´em como a força induzida por flutuações esméticas depende do perfil dos parâmetros de ordem nemático e esmético. Nas proximidades da transição nemática esmética, nossos resultados indicam que a força de Casimir térmica cresce significativamente, podendo ser mais relevante que a força de van der Waals usual. Em temperaturas onde a fase esmética é bem estabelecida, investigamos como a competição entre a ordem superficial e a ordem induzida por um campo externo se reflete nas características da força de Casimir. Obtivemos que, para filmes com ancoramentos superficiais assimétricos, a força de Casimir térmica obedece a uma lei de estados correspondentes e pode mudar sua natureza de atrativa para repulsiva através de variações de um campo externo. Discutimos como este comportamento pode influenciar as transições de umedecimento da fase esmética. No que diz respeito às propriedades dinâmicas, investigamos como operadores de superfície modificam a dinâmica de relaxação de filmes na fase hexática-B. Em particular estudamos como um campo de ordenamento superficial modifica a taxa de amortecimento dos modos orientacionais do sistema. Num regime puramente difusivo, nós demonstramos que uma ordem superficial com o vetor diretor não paralelo à direção normal das camadas faz com que os modos de relaxação tornem-se não-hidrodinâmicos. No regime viscoso, sempre existirão modos hidrodinâmicos e a dinâmica de relaxação é sempre lenta. Entretanto, os modos normais podem apresentar um decaimento oscilatório. Por fim, usamos a técnica de espectroscopia de fotocorrelação para investigar as propriedades dinâmicas de filmes livremente suspensos nas proximidades da transição esmética-A - cristal-B. Observamos que os dados experimentais indicam a existência de um modo não-hidrodinâmico e medimos a dependência da taxa de relaxação com a temperatura. Propusemos um modelo que incorpora a influência do gradiente do campo de velocidades que reproduz qualitativamente as observações experimentais.

Cette thèse porte sur l’étude expérimentale de l’extraction liquide-liquide au sein d’une colonne de laboratoire reproduisant de manière très simplifiée les appareils utilisés pour retraiter le combustible nucléaire (procédé hydro-métallurgique PUREX). Une technique optique non intrusive, dite de réfractométrie ou diffractométrie arc-en-ciel, a été spécifiquement développée pour caractériser la granulométrie (et donc l’aire interfaciale) et la composition de la phase dispersée. Cette dernière est composée de gouttes millimétriques de mélanges d’alcanes en ascension dans une colonne remplie d’eau au repos. Les spécificités de l’arc-en-ciel produit par ces gouttes à faible indice de réfraction relatif ont été étudiées à l’aide de différents modèles de diffusion asymptotiques de la lumière et la théorie électromagnétique de Lorenz-Mie. Grâce au développement de différentes approches directes et inverses (paramétriques), il a été démontré numériquement et expérimentalement que cette technique optique permettait d’estimer individuellement (ou collectivement) diamètre (moyen) et fraction de mélange de quelques dizaines de milliers de gouttes à quelques pour cents près. Des travaux préliminaires ont été réalisés sur l’extraction, résolue dans le temps de l’acétone, d’un milieu continu aqueux vers une goutte sessile composée initialement de toluène pur. Ils ont montré que l’on pouvait réellement estimer les constantes de transfert (de diffusion et de distribution moléculaire) à partir de l’analyse de l’évolution temporelle des arcs-en-ciel et en utilisant une méthode inverse qui intègre un modèle de diffusion moléculaire à symétrie radiale et un modèle électromagnétique ad hoc. / This thesis deals with the experimental study of liquid-liquid extraction within a laboratory column reproducing in a simple way the apparatuses used in a nuclear fuel reprocessing process (PUREX). A non-intrusive optical technique, called rainbow refractometry or diffractometry, has been specifically developed to characterize the size (and hence the interfacial area) and the composition of the dispersed phase. The latter is first time composed of millimeter droplets of alkanes mixtures free rising in a column filled with water at rest. Specificities of the rainbow produced by these droplets with a low relative refractive index were studied using different asymptotic light scattering models and the Lorenz-Mie electromagnetic theory. Thanks to the development of different direct and reverse (parametric) approaches, it was demonstrated both numerically and experimentally that this optical technique allows estimating individually (or collectively) the diameter (in average) and the mixing fraction of a few tens of thousands of droplets with a few percent of accuracy. Preliminary works have been carried out on the time-resolved extraction of acetone from an aqueous continuous medium to a sessile droplet initially composed of pure toluene. They have revealed that the transfer (diffusion and molecular splitting) constants can be estimated from the analysis of the temporal evolution of the rainbow signals using an inverse method that integrates a molecular diffusion model with a radial symmetry and an electromagnetic light scattering model.

L'influence du désordre sur les phénomènes critiques est jusqu'à présent essentiellement étudiée théoriquement. La motivation principale de ces études est de comprendre dans quelle mesure la différence de comportement entre les systèmes réels et les modèles théoriques idéaux résulte du désordre. Cette thèse aborde ce problème expérimentalement en étudiant la condensation de l'hélium 4 dans un aérogel de silice. En effet, ce système offre la possibilité de contrôler continûment l'influence du désordre. Plus précisément, nos résultats indiquent que ce phénomène est une réalisation expérimentale du modèle d'Ising avec champ aléatoire (Random Field Ising Model, RFIM) athermique étudié par Sethna et coll. (Phys. Rev. Lett., 70, 3347 (1993)). Pour cela, nous observons deux propriétés spécifiques à ce modèle, en accord avec les simulations numériques effectuées par Detcheverry et coll. sur un modèle de gaz sur réseau (Phys. Rev. E, 72, 051506 (2005)) : l'existence de points critiques contrôlés par le désordre ainsi que la mémoire du point de demi-tour (Return Point Memory, RPM). Les points critiques se manifestent par un changement de forme des isothermes d'adsorption mesurées par diffusion de la lumière. Nous avons mis au point une technique utilisant la grande sensibilité du speckle à la configuration microscopique locale des domaines liquide-gaz en vue de détecter les avalanches prédites par le modèle de Detcheverry et coll. Bien qu'aucune avalanche individuelle n'ait pu être isolée de façon claire, cette méthode a permis la première mise en évidence du RPM microscopique dans un système expérimental, justifiant ainsi le caractère athermique du système. Ces mesures sont les premières à observer le comportement du RFIM athermique dans un système non-magnétique. / Up to now, the effect of disorder on critical phenomena has been mainly studied theoretically. The motivation of these studies is to understand to what extent the difference between the behavior of the real systems and the ideal theoretical models is due to disorder. This thesis addresses this problem experimentally through the condensation of helium 4 in silica aerogels. This system provides the opportunity to tune the disorder strength. More precisely, we show that this phenomenon is an experimental realization of the athermal Random Field Ising Model (RFIM) studied by Sethna et al. (Phys. Rev. Lett., 70, 3347 (1993)). To this end, we observe two properties of this model, in agreement with numerical simulations done by Detcheverry et al. on a lattice gas model (Phys. Rev. E, 72, 051506 (2005)): the existence of disorder driven critical points and the Return Point Memory (RPM). The change of shape of the adsorption isotherms measured by light scattering is interpreted as the signature of the disorder driven critical points. In order to detect the avalanches predicted by the Detcheverry et al.'s model, we have developed a technique based on the great sensitivity of the speckle patterns to the local microscopic liquid-gas domains. Although no single avalanche could be clearly resolved, this method allowed the first observation of microscopic RPM in an experimental system, showing the athermal character of the system. These are the first measurements to exhibit the athermal RFIM behavior in a non-magnetic system.

Ces travaux s’inscrivent dans le cadre des recherches sur l’optimisation du séchage par atomisation de suspensions colloïdales employées pour la production de supports de catalyseurs. Pour mieux appréhender les phénomènes fondamentaux qui régissent ce procédé, le problème a été ramené à celui de l’étude expérimentale et la modélisation du séchage d’une goutte unique en lévitation dans un champ acoustique sous flux gazeux. L’expérience permet de contrôler les différents paramètres de séchage dans la chambre d’évaporation. Le suivi du séchage a été réalisé à l’aide de techniques optiques (vélocimétrie laser, imagerie en transmission et diffractométrie à l’angle d’arc-en-ciel) et des analyses post-mortem complémentaires. L’emploi de la diffractométrie arc-en-ciel a nécessité le développement d’un modèle d’optique géométrique avancé capable de décrire la diffusion de gouttes ellipsoïdales et les effets des suspensions nanoparticulaires sur le phénomène d’arc-en-ciel. Le modèle de séchage est un modèle à symétrie radiale. Il permet de prédire les taux d’évaporation, les profils internes de concentration et la déformation finale du grain. Les comparaisons expérimentales ont montré qu’il prédit très correctement le taux de séchage des gouttes colloïdales pour des nombres de Reynolds compris entre 100 et 230, des températures comprises entre 25°C et 55°C et des taux d’hygrométrie compris entre 2.5% et 70%. Il ressort également que le seuil de croûtage, identifié ici à partir du changement de régime du taux d’évaporation, semble intervenir pour des concentrations volumiques locales en nanoparticules de l’ordre de 12.3% bien inférieures aux concentrations de blocage des pâtes. / This PhD work takes place in the framework of researches on the optimization of the spray drying of colloidal suspensions used for catalyst support production. To better understand fundamental phenomena governing this process, the problem is reduced to the experimental study and modelling of the drying of a single droplet levitated in an acoustic field with an external gas flow. The experiment allows also controlling parameters such as composition of the suspension, temperature or humidity inside evaporation chamber. The drying is monitored using in situ optical diagnostics (particle image velocimetry, shadowgraphy and rainbow diffractometry) as well as post-mortem analyzes. The use of rainbow diffractometry has required the development of advanced light scattering models accounting for the droplet non-sphericity and heterogeneity. The drying model is a model with radial symmetry. It predicts various quantities such as the droplet evaporation rate, internal concentration profile or the deformation of the final grain. Experimental comparisons show that this model can accurately predicts the drying rate of colloidal droplet for Reynolds numbers ranging from 100 to 230, temperatures between 25°C and 55°C and relative humidity between 2.5% and 70%. It is also shown that the crust compactness factor, about 12% when identified from the change in the rate of evaporation, is much lower than that reported classically for the jamming of dense suspensions.

A proteína P21 do Trypanosoma cruzi participa no processo de infecção da célula hospedeira, desse modo é de grande importância: elucidar as vias de sinalização induzidas pela proteína, bem como caracterizar a nível molecular e estrutural a P21. O que vai auxiliar no entendimento da função biológica da P21 e sua participação no processo de infecção. A P21 recombinante é expressa Escherichia coli em sua maioria na fração insolúvel. Visando aumento de proteína na fração solúvel, foi realizada a clonagem do gene da P21 em vetor pSMT3, bem como testes de expressão subsequentes em diferentes cepas de expressão de E. coli, em vetor pET-28 e pSMT3 e variadas condições de expressão e lise celular. Desse modo obtiveram-se as condições que permitissem uma maior concentração da P21 na fração solúvel. A expressão foi realizada no vetor pET-28, cepa BL21, a 37&deg;C com meio 2xYT a 0.5 mM de IPTG, utilizando a técnica de sonicação como lise. A P21 foi purificada em cromatografia de afinidade e posteriormente em coluna de exclusão molecular. Foram realizados estudos de modelagem por homologia levaram a elaborar a hipótese de que a P21 tem a função de inibidor de serinoprotease do tipo kunitz. Posteriormente essa hipótese foi confirmada com ensaios de avaliação da atividade inibitória da P21 frente à tripsina, quimiotripsina e elastase neutrofílica, o qual a P21 mostrou capaz de inibir a elastase neutrofílica. Estudos com espalhamento dinâmico da luz (DLS) revelaram que as amostras testadas de P21 contém diferentes concentrações de agregados de alto peso molecular em todos os pHs avaliados. Outras medidas foram realizadas para avaliar o estado de agregação da P21 de acordo com a temperatura e verificou-se que entre 32-52 &deg;C, a proteína apresenta menor raio hidrodinâmico, indicando menor agregação nesse intervalo. Estudos de dicroísmo circular revelaram que a P21 apresenta por volta de 20% de &alpha; hélice, 32% de folha-&beta;, 22% de volta e 23 % estrutura randômica. De acordo com a curva de desnaturação referente ao espectro de CD obtido, a P21 se mostra desnaturada a partir de 64&deg;C. / The Trypanosoma cruzi protein P21 participates in the host cell infection process, but its specific function is poorly described. Thus it is important to elucidate the signaling pathways induced by the protein and characterize the P21 at the structural and molecular levels, as a contribution towards the understanding of the P21 biological function and its role in the infection process. The Escherichia coli recombinant P21 is expressed mostly in the insoluble fraction. Aiming to increase protein in the soluble fraction, we performed the cloning of the P21 gene in pSMT3 vector and subsequent expression tests in different expression strains of E. coli in pET-28 and pSMT3 vectors and varied expression conditions and cell lysis. Thus we obtained conditions that allow a greater concentration of the P21 in the soluble fraction. The expression vector was performed using vector pET-28, in Bl21 strain at 37&deg;C in 2xYT culture medium with 0.5 mM IPTG, using the sonication technique in cell lysis. The recombinant P21 was purified by Ni affinity chromatography and subsequently a molecular exclusion column. We performed homology modeling studies which led to assume that P21 can be a serinprotease inhibitor of Kunitz type. Furthermore, this hypothesis was confirmed in the experiments testing the P21 inhibitory activity against the trypsin, chymotrypsin and elastase. We found the P21 exclusively inhibit neutrophil elastase. Studies using dynamic light scattering (DLS) revealed that the samples containing P21 contained large molecular weigth aggregates in different concentrations at all evaluated pHs. Others measurements were performed to assess the P21 aggregation state according to the temperature and we found that between 32-52 &deg;C the protein had a smaller hydrodynamic radius, indicating less aggregation in this range. Circular dichroism (CD) studies revealed that P21 has about 20% &alpha;-helix, 32% &beta;, -sheet, 22% turn and 23% of random structure. According to the denaturation curve for the CD spectrum obtained, the P21 was denatured from 64&deg;C.

O desenvolvimento de algoritmos computacionais para a obtenção de distribuições de tamanho de partícula em dispersões e que utilizam dados espectroscópicos em tempo real e in-line a partir de sensores permitirá uma variedade de aplicações, como o monitoramento de propriedades em fluidos de corte industriais, acompanhamento de processos de polimerização, tratamento de efluentes e sensoriamento atmosférico. O presente estudo tem como objetivo a implementação e comparação de técnicas para resolução de problemas de inversão, desenvolvendo algoritmos que forneçam distribuição de tamanho de partículas em dispersões a partir de dados de espectroscopia UV-Vis-Nir (Ultravioleta, Visível e Infravermelho próximo). Foram implementadas quatro técnicas, sendo uma delas um método alternativo sem a presença de etapas de inversão. Os métodos que utilizaram alguma técnica de inversão evidenciaram a dificuldade em se obter distribuições de tamanho de gotas (DTG) de boa qualidade, enquanto o método alternativo foi aquele que se mostrou mais eficiente e confiável. Este estudo é parte de um programa cooperativo entre a Universidade de São Paulo e a Universidade de Bremen chamado programa BRAGECRIM (Brazilian German Cooperative Research Initiative in Manufacturing) e é financiado pela FAPESP, CAPES, FINEP e CNPq (Brasil) e DFG (Alemanha). / The development of computer algorithms to obtain the particle size distributions in dispersions using spectroscopic data in real time and in-line from sensors enable a variety of applications such as monitoring properties in industrial cutting fluids, monitoring of polymerization processes, wastewater, atmospheric sensing and other applications. The aim of this study is to implement techniques for inversion problem solving, by testing algorithms that provide particle size distribution in dispersions from UV-Vis-Nir (Ultraviolet, Visible and Near-Infrared) spectroscopic data. Four techniques have been implemented, one of them being an alternative method without the inversion step. The methods using inversion techniques showed difficulties to obtain droplet size distributions (DSD) with good quality, while the alternative method was the one that was more efficient and reliable. This study is part of a cooperative program between the University of São Paulo and the University of Bremen, within the BRAGECRIM program (Brazilian German Cooperative Research Initiative in Manufacturing) and is financially supported by FAPESP, CAPES, FINEP and CNPq (Brazil) and DFG (Germany).