Symmetric cell division in gram-negative bacteria is essential for generating two equal-sized daughter cells, each containing cellular material crucial for growth and future replication. The Min system, comprised of proteins MinC, MinD and MinE, is particularly important for this process since its deletion leads to minicells incapable of further replication. This thesis focuses on the interactions involving MinE that are important for allowing cell division at the mid-cell and for directing the dynamic localization of MinD that is observed in vivo. Previous experiments have shown that the MinE protein contains an N-terminal region that is required to stimulate MinD-catalyzed ATP hydrolysis in the Min protein interaction cycle. However, MinD-binding residues in MinE identified by in vitro MinD ATPase assays were subsequently found to be buried in the hydrophobic dimeric interface in the MinE structure, raising the possibility that these residues are not directly involved in the interaction. To address this issue, the ability of N-terminal MinE peptides to stimulate MinD activity was studied to determine the role of these residues in MinD activation. Our results implied that MinE likely undergoes a change in conformation or oligomerization state before binding MinD. In addition we performed circular dichroism spectroscopy of MinE. The data suggest that direct interactions between MinE and the lipid membrane can lead to conformational changes in MinE. Using NMR spectroscopy in an attempt to observe this structure change, different membrane-mimetic environments were tested. However the results strongly suggest that structural studies on the membrane-bound state of MinE will pose significant challenges. Taken together, the results in this thesis open the door for further exploration of the interactions involving MinE in order to gain a better understanding of the dynamic localization patterns formed by these proteins in vivo.

Mixed association of calix[4]resorcinarene with ethyl sulfonate groups on the lower rim and dimethylaminomethyl groups on the upper rim (CR) and cationic surfactant 4-aza-1-hexadecyl-azoniabicyclo[2.2.2]octane bromide (DABCO-16) is studied by methods of tensiometry, conductometry, potentiometry and NMR spectroscopy at fixed CR concentration and varied surfactant concentration. Beyond ca. 0.4 mM of DABCO-16, mixed aggregates enriched by CR are proved to be formed due to electrostatic forces, while beyond ca. 5 mM, aggregates enriched by surfactant occur due to the hydrophobic effect. Spectrophotometry monitoring of the solubilization of a hydrophobic dye, Orange OT, demonstrated that only the second type of mixed aggregate enriched by DABCO-16 is capable of binding the organic probe, while the mixed system where the surfactant is a minor component shows no binding capacity towards Orange OT. This finding can be used for the design of nanocontainers with controllable binding/release properties. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

This thesis deals with (i) complex nanoaggregates of cationic perfluorinated surfactant N-(1,1,2,2- tetrahydroperfluorodecyl)pyridinium chloride and of double hydrophilic block polyanions poly(ethylene oxide)-b-poly(methacrylate) and poly(ethylene oxide)-b-poly((2-sulfamate-3- carboxylate)isoprene), and with (ii) mixed micelles of amphiphilic copolymer poly(ethylene oxide)- b-poly(e-caprolactone) and nonionic perfluorinated fluorosurfactant Zonyl FSN-100. The study was aimed at the characterization of the association behavior of the block copolymer-fluorosurfactant systems in aqueous solutions depending on the amount of the added surfactant, pH of the solvent and the structure of the copolymers.

Les copolymères à blocs di-hydrophiles contenant un bloc thermosensible reçoivent une attention croissante grâce à leur capacité d’auto-organisation en micelles induite par une variation de température. Néanmoins, peu de travaux ont été consacrés à l’étude de leur conformation par fluorescence et de leur dynamique à l’interface air-eau et en solution aqueuse. Dans ce travail, des copolymères à blocs composés d’un bloc thermosensible deN,N-diéthylacrylamide (DEA) et d’un bloc hydrophile de N,N-diméthylacrylamide (DMA) ou d’un bloc réactif [copolymère statistique de DMA et de N-acryloxysuccinimide (NAS)] ont été synthétisés par polymérisation RAFT. Ces copolymères à blocs ont été fonctionnalisés à leur extrémité hydrophile par un chromophore, Rhodamine B ou Vert de Malachite, via une stratégie de pré- ou de postpolymérisation. Dans le premier cas, des dérivés aminés de Rhodamine B et Vert de Malachite ont été synthétisés pour l’élaboration d’agents de transfert de chaîne (ATC) marqués, ce qui permet directement l’obtention de copolymères à blocs alpha-fonctionnalisés par un chromophore. En parallèle, des copolymères à blocs ont été préparés via l’utilisation d’un ATC précurseur puis fonctionnalisés ultérieurement par les dérivés aminés des chromophores. Le comportement thermosensible de ces polymères et d’un copolymère à blocs amphiphile de DEA et de N-décylacrylamide a été étudié à l’interface air-eau et en films de Langmuir-Blodgett par AFM et microscopie confocale de fluorescence. Des études d’émission et d’anisotropie de fluorescence, de diffusion de lumière et de RMN 1H ont été réalisées pour étudier leur comportement en solution aqueuse / Double hydrophilic diblock copolymers comprising a thermoresponsive block have gained increasing attention due to their capability of self-assembling in micelles by a temperature change. However, very few fluorescence studies were devoted to investigate their conformation and dynamics both at the air-water interface and in aqueous solutions. In this work, block copolymers composed of a thermoresponsive block of N,N- iethylacrylamide (DEA) and a hydrophilic block of N,N-dimethylacrylamide (DMA) or a reactive block [statistical copolymer of DMA and N-acryloxysuccinimide (NAS)] were prepared by RAFT polymerization. These block copolymers were functionalized at the hydrophilic chain-end by a Rhodamine B or Malachite Green dye using either a pre- or a post-polymerization strategy. In the first case, Rhodamine B and Malachite Green amino derivatives were synthesized for the preparation of dyelabelled chain transfer agent (CTA), which led directly the alpha-dye-labelled block copolymers. Alternatively, the block copolymers were prepared using a precursor CTA and further functionalized with the dye amino derivative. The thermoresponsive behaviour of these polymers and of amphiphilic block copolymers of DEA and N-decylacrylamide was studied at the air-water interface and in Langmuir-Blodgett films using AFM and confocal fluorescence microscopy. Fluorescence emission and anisotropy, light scattering and 1H NMR studies were performed to investigate their behaviour in aqueous solutions.

De nombreux composés bioactifs hydrophobes sont actuellement mis en avant en raison de leurs propriétés nutritionnelles et fonctionnelles. Une attention particulière est, en conséquence, portée à leur incorporation en tant qu'ingrédients dans des aliments fonctionnels. Cependant, la majorité de ces composés bioactifs sont caractérisés par une faible solubilité en milieu aqueux, une dégradation au cours des procédés de transformation ainsi qu'une absorption limitée au niveau du tractus gastro-intestinal. La micelle de caséines, grâce à ses propriétés fonctionnelles uniques, peut être considérée comme un support d’encapsulation naturel pour ces molécules bioactives hydrophobes. En effet, une des originalités de cette suprastructure est sa dynamique dans le lait se caractérisant par des échanges réversibles de protéines et de minéraux entre le sérum et la structure micellaire interne en fonction des conditions physicochimiques, et notamment avec la température. En particulier, un stockage du lait à 4°C permet la dissociation sélective de la caséine β de la phase micellaire vers la phase soluble et un retour à température ambiante permet sa réintégration. L’objectif de cette thèse est de développer une nouvelle stratégie d’encapsulation de molécules bioactives hydrophobes dans les micelles de caséines via cette dynamique de la caséine β. Dans un premier temps, l’optimisation de la dissociation de la caséine β de la micelle de caséines a été réalisée en modifiant la température et le pH, tout en portant une attention particulière au maintien de l’intégrité des micelles déplétées en caséines β. Un procédé de séparation physique de la caséine β solubilisée a été optimisé par microfiltration à l’échelle pilote. Une étude de la concentration micellaire critique de la caséine β a permis de vérifier son état monomérique à l’issue de cette séparation. Une étude de la cinétique d’interaction entre la caséine β monomérique et deux composés bioactifs hydrophobes, la curcumine et la vitamine D3, a ensuite été réalisée par résonance plasmonique de surface et par spectroscopie de fluorescence. La curcumine a été choisie pour la suite de l’étude au vu de sa bonne affinité pour la caséine β. Le complexe caséine β monomérique-curcumine a ensuite été encapsulé dans des micelles de caséines préalablement déplétées en caséines β. Les résultats de ces travaux montrent l’efficacité de cette stratégie d’encapsulation qui peut présenter un intérêt particulier pour la vectorisation de molécules bioactives hydrophobes afin d’assurer leur protection dans des produits laitiers pauvres en matière grasse.De plus, au cours de ce projet, une méthode de caractérisation des propriétés morphologiques et nano-mécaniques des micelles de caséines par microscopie à force atomique en milieu liquide a été développée. Cette méthode représente un outil intéressant de compréhension de la structure micellaire dans son environnement natif et offre la possibilité d’évaluer l’impact de certaines modifications sur les propriétés de la micelle de caséines, comme sa déplétion en caséine β ou sa réticulation. / In the last years, the number of studies highlighting the nutritional and functional properties of several hydrophobic bioactives has markedly increased. Special attention is consequently paid to their addition as ingredients to food. However, most of these hydrophobic compounds display a low aqueous solubility, poor stability during processing and low absorption in the gastrointestinal tract. Casein micelles exhibiting unique set of properties can be considered as a natural nanocarrier for these molecules. Actually, changes in environmental factors namely pH and temperature induce the dissociation of caseins and minerals from the colloidal phase to the soluble phase. Particularly, a selective dissociation of β-casein occurs at low temperatures. This effect is reversed with an increase in temperature, with a transfer of β-casein from the serum to the micelles when equilibrated at room temperature. The aim of this study is to develop a novel encapsulation strategy to incorporate hydrophobic bioactive compounds into casein micelles using the β-casein reversible dissociation. First, the β-casein dissociation from casein micelles was optimized by temperature and pH modifications while preserving the integrity of the β-casein depleted casein micelles. The separation of dissociated β-caseins from casein micelles was carried out by microfiltration at a pilot scale. The β-casein critical micelle concentration was concurrently evaluated to ensure the monomeric state of -casein after separation. Secondly, the binding kinetic between monomeric β-casein and two hydrophobic compounds, curcumin and vitamin D3, was investigated by surface plasmon resonance and fluorescence spectroscopy. Curcumin was then selected thanks to its high affinity to -casein β. The complex monomeric β-casein – curcumin was encapsulated in β-casein depleted casein micelles. The results of this study show the efficiency of this encapsulation strategy of hydrophobic bioactive compounds, which could be used to protect such molecules in low fat dairy products.Besides, during this project, a novel strategy was developed in order to evaluate the casein micelle topography and nanomechanical properties by atomic force microscopy in liquid environment. This method opens a new line of investigation to better understand the casein micelle structure in its native environment but also investigate the impact induced by the modification of physico-chemical conditions on its topography and elastic properties.

Neste trabalho, utilizamos a técnica de espalhamento de raios-X a baixos Ângulos (SAXS) para estudar a influência de dois derivados fenotiazínicos na estrutura de sistemas micelares, assim como suas propriedades de auto-associação, além de investigar a influência da variação de pH e de concentração nas interações entre proteínas em solução. Para tanto, utilizamos dois fármacos fenotiazínicos, (Trifluoperazina, TFP e a Clorpromazina, CPZ), em presença de L--fosfatidilcolina (LPC), um surfactante zwiteriônico (30 mM), a pH 4.0 e 7.0. Os resultados de SAXS indicam que a micela de LPC, em ausência de fenotiazina, pode ser representada por uma micela com forma elipsoidal (com razão axial 1.6 0.1). No entanto, em presença de TFP e de CPZ a forma da micela se altera, passando para um cilindro (com razão axial 2.5 0.1). Este efeito é acompanhado por uma diminuição do raio parafínico da micela (22.5 0.3 Å), em ausência de fármaco, para 20.0 0.5 em presença de 10 mM de fármaco. Em paralelo, realizamos medidas de EPR (Ressonância Paramagnética eletrônica) destes sistemas. Combinando os resultados de SAXS e de EPR, propusemos um sítio para a localização destes compostos nas micelas de LPC, que seria na interface polar/apolar da mesma. Em um segundo momento, utilizamos as técnicas de SAXS e de EPR para investigar as características estruturais dos agregados formados por TFP e CPZ (a 20 e 60 mM, a pH 4.0 e 7.0). As curvas de SAXS são compatíveis com o espalhamento de agregados pequenos com diferentes geometrias: elipsoidal, cilíndrico e tipo-paralelepípedo. Devido à resolução da técnica, dentro do intervalo de vetores de espalhamento utilizada (até cerca de 0.3 Å-1), não é possível determinar, de forma absoluta, a correta geometria dos agregados, ou seja, todas as geometrias citadas acima ajustam de forma satisfatória as curvas de SAXS. As análises dessas curvas também não excluem a possibilidade de que estes fármacos mantenham-se como nano-cristais em solução (compostos por cerca de 10 celas unitárias, empilhadas na direção-z), seguindo sua estrutura cristalográfica. Medidas de EPR indicam que os auto-agregados a pH 4.0 possuem características semelhantes às micelas, mas a pH 6.5 este efeito não foi evidenciado, uma vez que ocorre uma forte interação entre a sonda e os agregados. Este fato indica que os agregados, a pH 6.5, têm um maior empacotamento, em comparação aos sistemas a pH 4.0. Por fim, utilizamos a Albumina de Soro Bovina (BSA, a 10 50 mg/ml), em diferentes pHs (2.0 9.0), para investigar os efeitos de concentração e de pH nos potenciais de interação das macromoléculas em solução. O fator de forma da proteína foi obtido através da estrutura cristalográfica da HSA (Human Serum Albumine, proteína humana homóloga a BSA), enquanto que as interações proteína-proteína foram calculadas através da relação de fechamento RPA (Random Phase Approximation). Nossos dados indicam que a BSA mantém sua estrutura terciária inalterada de pH 4.0 a 9.0, independente de sua concentração. No entanto, a pH 2.0 a proteína sofre um processo de desenovelamento, indicado pelo aumento da dimensão máxima da mesma. Nossos dados dão suporte para concluir que as interações entre as proteínas, a 10 mg/ml, são praticamente desprezíveis, exceto para os sistemas compostos a pH 2.0 (onde a proteína está desenovelada) e a pH 4.0 (onde evidenciamos a presença de interferência atrativa entre as proteínas). Entretanto, a medida em que aumentamos a concentração proteica, uma função de interferência do tipo repulsiva aparece na curvas de SAXS (para os sistemas de pH 4.0 a 9.0). Além disso, no sistema composto por BSA, pH 5.4 e 50 mg/ml, evidenciamos a existência de monômeros e dímeros em solução, provavelmente devido a proximidade do ponto isoelétrico da proteína (entre 4.8 5.6). Este efeito não foi evidenciado para os outros pHs, nesta mesma concentração. A pH 2.0 (25 e 50 mg/ml) evidenciamos uma compactação da proteína, sendo que sua forma é diferente da forma nativa da BSA. Nestas condições, é possível que a proteína tenha alcançado um estado molten globule, como evidenciado em outros trabalhos. Acreditamos que os efeitos de volume excluído são de grande importância para a estabilidade da proteína in vivo. / In this work we study, mainly by means of small angle X-ray scattering (SAXS), the influence of two phenothiazine derivatives on biomimetic systems as well as the self-assembly features. At the same time, the conformational stability of proteins in the presence of denaturant agents (pH and concentration) was evaluated. First of all, the phenothiazine compounds trifluoperazine (TFP) and chlorpromazine (CPZ) with micelles of the zwitterionic surfactant L--lysophosphatidylcholine (LPC), at pHs 4.0 and 7.0, are reported. The SAXS results demonstrate that, upon addition of both phenothiazines, the LPC micelle of prolate ellipsoidal shape changes into a cylindrically shaped micelle, increasing its axial ratio from 1.6 0.1 (in the absence of drug) to 2.5 0.1 (for 5 and 10 mM of phenothiazine). Such an effect is accompanied by a shrinking of the paraffinic shortest semiaxis from 22.5 0.3 to 20.0 0.5 Å. Besides, EPR (Electronic Paramagnetic Resonance) evidenced a bigger motion immobilization of the nitroxe probe, in the presence of phenothiazines. Our results provide evidence that the positively charged phenothiazine molecule must be accommodated near the hydrophobic/hydrophilic inner micellar interface. Furthermore, SAXS and EPR experiments were carried out to investigate the structure of the self-aggregates of CPZ and TFP, in aqueous solution. SAXS studies (drug solutions of 20 and 60 mM, at pH 4.0 and 7.0) evidenced that several different particle form factors with a homogeneous electron density distribution, in respect to the water environment, could reproduce the scattering curves. Due to the limitation of scattering intensity in the q range above 0.15 Å-1, precise determination of the aggregate shape was not possible and all of the tested models for ellipsoids, cylinders, or parallelepipeds fitted the experimental data equally well. The SAXS data allows inferring, however, that CPZ molecules might self-assemble in a basis set of an orthorhombic cell, remaining as nanocrystallites in solution. Such nanocrystals are composed of a small number of unit cells (up to 10, in c-direction), with CPZ aggregation numbers of 60-80. EPR spectra of 5- and 16-doxyl stearic acids bound to the aggregates were also performed, indicating a micelle-like aggregate at pH 4.0, and a significant motional restriction of the nitroxide was observed at pH 6.5. This implies that the aggregate is densely packed at this pH and that the nitroxide is tightly bound to it producing a strongly immobilized EPR spectrum. Finally, the effect of concentration and pH on the protein-protein interactions of BSA (Bovine Serum Albumin, from 10 up to 50 mg/ml) was evaluated by SAXS. Our results give support to infer that BSA keeps its native shape (similar to the Human Serum Albumin, HSA, crystallographic structure) unaltered at middle-acid (pH 4.0) up to basic pHs (9.0). At pH 2.0, however, BSA undergoes an unfolding process, indicated by a non globular shape. The protein-protein interactions were analysed into the Random Phase Approximation. The results show that at smaller amounts of BSA (10 mg/ml) the interference effects are not significative over the SAXS curve for pH 5.4 up to 9.0. At pH 4.0 and 10 mg/ml, however, an attractive potential takes place over the SAXS curves, that becomes repulsive with increasing BSA concentration. Besides, at pH 5.4 and 50 mg/ml, we evidenced a dimer-monomer co-existence in the solution. At pH 2.0 and 25 and 50 mg/ml, BSA undergoes to a compact conformation. Probably, BSA is in a molten globule state. Our results give also support to infer that probably, the exclude volume effect plays an important role on the protein stability in vivo.

O câncer de mama é a segunda principal causa de morte entre as mulheres nos países em desenvolvimento, devido ao seu alto grau de malignidade. O tratamento baseia-se, principalmente, em terapias hormonais, uma vez que as células deste tipo de tumor expressam, em sua maioria, um elevado número de receptores hormonais, responsáveis pela regulação do crescimento do mesmo. O tamoxifeno é um fármaco da classe dos moduladores seletivos de receptores de estrógeno, que atua através do antagonismo à ativação de tais receptores por este hormônio, reduzindo, assim, a taxa de crescimento celular do tecido tumoral. Embora o tratamento com tamoxifeno seja altamente efetivo, este se relaciona a severos efeitos colaterais dosedependentes. O objetivo central deste trabalho foi desenvolver sistemas micelares de longo tempo de circulação contendo tamoxifeno, preparados à base do fosfolipídeo DSPE-PEG(n), associado ou não ao derivado de vitamina E TPGS, para administração intravenosa, capazes de permitir um acúmulo maior do fármaco no sítio tumoral devido a suas dimensões nanométricas, permitindo, desta forma, a redução da dose e a consequente redução dos efeitos colaterais. A determinação da eficiência de encapsulação e a quantificação do tamoxifeno no estudo de liberação in vitro a partir dos sistemas obtidos foram realizadas por CLAE, utilizando métodos previamente validados. Os melhores resultados foram alcançados com as formulações à base de DSPE-PEG(2000) e TPGS, preparadas pelo método de evaporação do solvente, as quais apresentaram diâmetro médio inferior a 20 nm, baixo índice de polidispersividade e eficiência de encapsulação entre 70 e 95%. A análise por microscopia eletrônica de transmissão evidenciou o formato esférico e comprovou a homogeneidade do tamanho das partículas. Os sistemas foram caracterizados, ainda, por espectrofotometria no infravermelho para avaliação de possíveis interações entre os componentes das formulações. O perfil de liberação in vitro demonstrou que após 168 h, no máximo cerca de 30% do fármaco foi liberado, verificando-se que o aumento na quantidade de TPGS na formulação reduziu a porcentagem de tamoxifeno liberado. A baixa taxa de liberação in vitro sugere que a maior parte do fármaco mantenha-se no interior da estrutura micelar durante o período de permanência no sangue, favorecendo a chegada da nanoestrutura íntegra ao sítio tumoral. No estudo do perfil de concentração plasmática em ratas Wistar, não foi possível detectar o fármaco e seu principal metabólito pelo método por CLAE desenvolvido, sugerindo que os sistemas micelares tenham extravasado rapidamente para os órgãos. / Breast cancer is the second main cause of death among women in development countries due to their high malignance grade. The treatment is mainly based on hormonal therapies, once the cells of the majority of mammary tumors express a high number of hormone receptors, responsible for the tumor growth. Tamoxifen is a selective estrogen receptor modulator drug, acting through the antagonism of the activation of the estrogen receptor, reducing thus the tumor growing rate. Despite the treatment with tamoxifen is highly effective, it is related to severe dose-dependent side effects. The central objective of this work was the development of long time circulation micelles containing tamoxifen, prepared with the phospholipid DSPEPEG(n) and TPGS, a vitamin E derivative, by the method of solvent evaporation, for intravenous administration, able to allow a higher accumulation of the drug at the tumoral site due to their nanometric dimensions, leading to a reduction in the dose and consequently in the side effects. The determination of the encapsulation efficiency and the quantification of tamoxifen in the in vitro release profile study from the micellar systems were carried out by HPLC, using methods previously validated. The best results were achieved with the formulations based on DSPE-PEG(2000) and TPGS, which showed mean particle diameter less than 20 nm, low polydispersity index and encapsulation efficiency ranging from 70 to 95%. The transmition electronic microscopy pointed the spherical shape and proved the homogeneity of particle size. The systems were also characterized by infrared spectrophotometry to identify eventual interactions among the components of the formulations. The in vitro release profile study showed that after 168 h, a maximum of about 30% of tamoxifen was released, evidencing that the increase of the TPGS amount in the formulation reduced the amount of tamoxifen released. The low rate of in vitro release drug suggests that the major part of the drug will remain encapsulated during the period of blood permanence, favoring the arrival of the intact nanostructure at the tumoral site. During the evaluation of the plasmatic concentration profile, conducted with Wistar rats, it was not possible to detect neither the tamoxifen nor its main metabolite, suggesting that the intact micelles may have quickly accumulated in the organs.

O ácido clavulânico (AC) é um potente inibidor de β-lactamases, sendo utilizado em associação com antibióticos β-lactâmicos. Atualmente, a purificação industrial do AC envolve, principalmente, processos de extração líquido-líquido com solventes orgânicos e etapas cromatográficas. Assim, métodos alternativos como os sistemas micelares de duas fases aquosas (SMDFA), os quais oferecem seletividade na partição de biomoléculas de acordo com sua hidrofobicidade, são de grande interesse. O presente trabalho teve como objetivo estudar a partição do AC em sistemas micelares não iônicos de duas fases aquosas, puros e com adição do sal (NH4)2SO4 ou do polímero sulfato de dextrana (Dx-S). Os estudos de estabilidade do AC mostraram que o fármaco é mais estável em pH 6,5 e temperaturas mais baixas (5 - 20 ºC). Em relação à presença dos aditivos, foi verificado que a adição do Dx-S acarretou em menor perda da estabilidade do AC quando comparado ao (NH4)2SO4, com valor residual ≥ 90% a 35 °C. Na presença dos tensoativos Triton X-114 e Triton X-100, o AC apresentou-se estável, com valor residual de aproximadamente 100%. De acordo com os ensaios de partição, o AC foi recuperado preferencialmente na fase pobre em micelas, tanto nos sistemas TX/tampão quanto TX/sal para ambos os tensoativos, com valores de coeficiente de partição (KAC) ~ 0,7 e rendimento na fase diluída (Yclavd) ~ 75%. A adição do polímero em maiores concentrações (≥ 8% p/p) proporcionou um pequeno aumento nos valores de KAC, porém com valores ainda próximos a 1 - 1,5. Portanto, os resultados demonstraram que a presença dos aditivos não influenciou suficientemente a partição do AC para a fase micelar e, desta maneira, os sistemas TX/tampão mostraram ser mais eficientes para a recuperação do ácido clavulânico na fase pobre em micelas, podendo ser empregados como etapa prévia de extração em um processo biotecnológico. / Clavulanic acid (CA) corresponds to a potent β-lactamase inhibitor that is used in association with β-lactamic antibiotics. The industrial purification of CA usually involves liquid-liquid extraction processes employing organic solvents followed by several chromatographic steps. Therefore, new purification alternatives such as aqueous two-phase micellar systems (ATPS) are of great interest. These systems can provide selectivity in biomolecule partitioning according to hydrophobicity and other molecular properties. Within this context, the main goal of this study was to investigate CA partitioning in aqueous two-phase micellar (nonionic) systems, with and without the addition of (NH4)2SO4 or dextrane sulfate (Dx-S). Stability studies performed with CA indicated that the drug is more stable at pH 6.5 and lower temperatures (5 - 20 ºC). In addition, it was demonstrated that Dx-S addition led to a lower loss of CA stability in comparisson to (NH4)2SO4, with residual values ≥ 90% at 35 °C. The drug was found to be very stable in the presence of the surfactants Triton X-114 and Triton X-100, with residual values around 100%. Regarding CA partitioning in the ATPMS, the drug partitioned preferentially to the micelle-poor phase, irrespective of the surfactante employed and of the presence of (NH4)2SO4,with partition coefficient (KAC) ~ 0.7 and yield in the poor phase (Yclavd) ~ 75%. Nonetheless, the addition of Dx-S in concentrations (≥ 8.0% p/p) resulted in a discrete increase in KAC, with values around 1 - 1.5. Therefore, the results obtained in this work demostrated that the addition of (NH4)2SO4 or Dx-S to ATPMS did not significantly influenced CA partitioning to the micelle-rich phase and, in this context, the systems investigated could be considered more eficiente for CA recovery in the micelle-poor phase, as a previous extraction step of a biotechnological process.