La thérapie photodynamique (PDT) est un traitement alternatif à la chirurgie en oncologie utilisant un photosensibilisateur (PS), la lumière visible et l’oxygène moléculaire. La méta-tétra(hydroxyphényl)chlorine (mTHPC) est l’un des PS de deuxième génération les plus utilisés en clinique en raison de son absorption dans le rouge lointain et d’un rendement quantique en 1O2 élevé. De par sa nature hydrophobe, la mTHPC est partiellement agrégée dans la circulation sanguine diminuant sa biodistribution. Dans le but d’améliorer la sélectivité tumorale de la mTHPC, différentes stratégies de vectorisation ont été développées. La formulation liposomale de mTHPC non PEGylée (Foslip®) améliore la biodistribution ainsi que les propriétés pharmacocinétiques de la mTHPC. Cependant, une rapide destruction des liposomes en circulation ainsi qu’une rapide libération de la mTHPC sont des inconvénients majeurs. Une alternative possible est l’utilisation de vésicules extracellulaires (VE). Dérivées des cellules, les VE possèdent une stabilité naturelle dans la circulation sanguine et une capacité à transporter et délivrer leur contenu de manière spécifique aux cellules cancéreuses. Cette vectorisation est intéressante en PDT en raison d’une importante capacité d’encapsulation des porphyrines. Le but de cette étude était d’évaluer l’intérêt des VE en tant que nanovecteur de la mTHPC dans divers modèles précliniques comparé au Foslip®. Contrairement au Foslip®, l’intégrité membranaire des VE est conservée en présence de 20% de plasma. In vitro, les mTHPC-VE ont montré une internalisation cellulaire par un mécanisme actif d’endocytose. Dans un modèle cellulaire en 3D de sphéroïdes multicellulaires, les mTHPC-VE ont permis d’accroitre l’accumulation cellulaire, la diffusion au sein de ce modèle ainsi que l’efficacité PDT. In vivo, les mTHPC-VE apparaissent plus efficace au niveau PDT avec un retard de croissance tumorale significativement augmenté. En conclusion, l’intégration de la mTHPC au sein des VE améliore l’efficacité PDT dans les différents modèles d’étude. Le suivi des mTHPC-VE à l’aide d’un traceur radioactif chez la souris ainsi que l’étude du ciblage de la vascularisation tumorale seront étudiés dans la suite du travail / Photodynamic therapy (PDT) is an alternative treatment to surgery in oncology using photosensitizer (PS), light and oxygen. Meta-tetra(hydroxylphenyl)chlorin (mTHPC) is one of the most used PS in clinics due to its high absorption in the deep red and high 1O2 quantum yield. In order to improve the mTHPC tumor selectivity different attempts of nanovectorisation were conducted. Non-PEGylated liposomal mTHPC (Foslip®) increase biodistribution and pharmacokinetic properties. However, the rapid liposome destruction during circulation and rapid mTHPC release are obvious shortcomings. Alternatively, mTHPC vectorization could be realized by extracellular vesicles (EVs). Derived from the cell, EVs possess a natural stability in bloodstream and ability to transport and deliver cargo molecules into cancer cells. This formulation is interesting for PDT due to the ability to encapsulate porphyrins. The aim of the present study was to determine the interest of EVs as mTHPC nanocarriers in various preclinical models compared to Foslip®. In contrast to Foslip®, membrane integrity of mTHPC-EVs was conserved in 20% of plasma. In vitro, mTHPC-EVs showed cellular internalization by an active endocytosis mechanism. In a 3D model of spheroids, mTHPC-EVs have improved cellular uptake, better diffusion inside spheroid and increased PDT efficacy. In vivo, mTHPC-EVs appeared to be more potent in terms of PDT efficacy, with a tumor growth delay significantly higher. In conclusion, integration of mTHPC in EVs improves PDT efficacy in various preclinical models. The tracking of mTHPC-EVs using a radioactive tracer in xenografted rodents as well as the study of vascularization targeting will be studied in the next step of this work

L’insuffisance cardiaque (IC) est un problème majeur de santé publique. La pénurie des greffons cardiaques et la résistance de nombreux patients aux traitements conventionnels ont poussé les chercheurs à développer de nouvelles thérapeutiques dont la thérapie cellulaire. Bien que l’idée initiale de la thérapie cellulaire ait été de repeupler la partie nécrosée du cœur par l’administration de cellules viables et fonctionnelles, leur disparition rapide alors que les bénéfices perdurent dans le temps a conduit à l’hypothèse que les cellules agiraient via un mécanisme paracrine. Les vésicules extracellulaires (VE), incluant les exosomes et les microparticules, seraient principalement impliquées dans ce processus. Elles agiraient ainsi comme de véritables navettes transportant des biomolécules actives permettant d’activer des voies de réparation endogènes dans le tissu traité. Ce projet de thèse a pu mettre en évidence : 1) La non-infériorité des VE issues de progéniteurs cardiovasculaires (Pg) dérivés de cellules souches embryonnaires humaines par rapport à leurs cellules d’origine dans un modèle murin d’IC chronique (ICC). Ces VE activeraient des voies de signalisation endogènes impliquées dans la stimulation de la prolifération cellulaire, la survie cellulaire, la réparation de l’ADN et la diminution de la fibrose. Leur contenu moléculaire spécifique, et notamment les microARN, pourrait être impliqué dans ces phénomènes. 2) L’importance du choix du type cellulaire dans la production de VE efficaces sur le plan thérapeutique puisque ni les VE dérivées de cardiomyocytes matures ni celles de cellules souches mésenchymateuses n’ont eu d’effets bénéfiques sur la fonction cardiaque de souris en ICC. 3) L’implication des VE dans l’effet paracrine des cellules, confirmée par l’amélioration de la fonction cardiaque chez des souris présentant une ICC traitées avec des VE issues de Pg dérivés d’iPS. Des tests fonctionnels in vitro ont montré que les VE auraient un rôle pro-angiogénique, pro-prolifératif et amélioreraient la survie des cellules. Une thérapie a-cellulaire aurait une réelle pertinence clinique en réglant une partie des problèmes techniques, immunologiques et sécuritaires associés aux greffes de cellules. Si cette hypothèse est confirmée, il pourrait en résulter une simplification des problèmes réglementaires, une diminution des coûts de production et de ce fait une plus grande diffusion clinique de la méthode. / Heart failure (HF) is a major public health concern. The lack of donor hearts and the resistance of numerous patients to conventional treatments has led scientists to develop new therapies such as cell therapy. The initial goal of cell therapy was to repopulate the infarcted heart by directly injecting viable and functional cells. However, the rapid disappearance of the transplanted cells contrasts with their long term, ongoing functional benefits, suggesting that cells may act through a paracrine mechanism. Extracellular vesicles (EV), including exosomes and microparticles, may be key to this process, acting as shuttles to transport bioactive macromolecules that stimulate endogenous repair pathways in the host tissue. This PhD project demonstrates: 1) The non-inferiority of EV secreted by cardiovascular progenitors (Pg) derived from human embryonic stem cells as compared to their parent cells in a mouse model of chronic HF (CHF). These EV could act by the activation of endogenous signaling pathways implicated in cell proliferation, survival, DNA repair and decreased fibrosis. Their specific content, such as miRNA, could be involved in these benefits. 2) The importance of the cell type in the production of therapeutically effective EV, since EV derived from mature cardiomyocytes and mesenchymal stem cells did not improve cardiac function in mice with CHF. 3) The importance of EV in paracrine effects of cells, confirmed by the improvement of cardiac function in mice with CHF treated with EV secreted by Pg derived from iPS cells. In vitro data shows that EV might have pro-angiogenic, pro-proliferative and pro-survival effects. An acellular therapy should be clinically relevant by reducing technical, immunological and safety problems associated with cell transplantation. If this hypothesis is confirmed, regulatory concerns would be simplified and production costs reduced, facilitating large-scale production.

Les cellules iNKT (de l’anglais invariant Natural Killer T) constituent un sous-type particulier de lymphocytes T caractérisé par un profil de type inné. Ces cellules répondent rapidement à des antigènes lipidiques et glycolipidique présentés par le CD1d, une glycoprotéine exprimée par les différentes cellules présentatrices d'antigène. Suite à l’activation, les cellules iNKT sont capables de produire de grandes quantités de cytokines anti-inflammatoires et pro-inflammatoires et elles sont impliquées dans différentes maladies, telles que l'allergie, l'auto-immunité, le cancer et les infections, parmi lesquelles la leishmaniose. Les parasites protozoaires de les espèces Leishmania sont les agents causaux de la leishmaniose, une maladie tropicale négligée dont la manifestation la plus grave affecte les organes viscéraux et qui peut être mortelle si elle n'est pas traitée. Le succès de l'infection dépend de la capacité du parasite à maitriser la réponse immunitaire de l'hôte. Récemment, quelques groupes, y compris le nôtre, ont observé que les parasites Leishmania libèrent des vésicules extracellulaires (VE). Les VE sont formées par une bicouche membranaire lipidique, contenant des lipides, des protéines et du matériel génétique et elles peuvent transmettre des molécules dérivées des pathogènes aux cellules hôte sans contact direct entre les cellules. Les VE produites par les parasites Leishmania et aussi par d’autres protozoaires ont été associés à des effets pro-parasite car elles favorisent un environnement plus permissif à l'établissement de l'infection. Dans cette thèse, nous avons étudié l'effet du matériel extracellulaire (ME), correspondant aux VE et aux molécules non-associées aux VE, libéré par les promastigotes de L. infantum sur les cellules iNKT. Dans le début de ce travail, il a été observé que le ME de L. infantum empêche l'expansion ex-vivo des cellules iNKT humaines à partir de cellules mononucléaires du sang périphérique. Cela a mis en évidence la communication entre les cellules iNKT et le ME de L. infantum, ce qui a été exploré par la suite. Le ME de L. infantum module la capacité très importante des cellules iNKT à produire des cytokines. En effet, le ME de L. infantum empêche la production des différentes cytokines par les cellules iNKT, comme par exemple IL-4 et IFNγ. De plus, nous avons aussi démontré que le ME de L. infantum compète avec l’α-GalCer, un agoniste très puissant des cellules iNKT, pour la liaison à la molécule CD1d, ce qui justifie l’effet inhibiteur dans l'activation des cellules iNKT. Nous avons aussi montré que les lipides qui sont présents dans chaque fraction du ME de L. infantum ont un rôle très important dans l’inhibition de l'activation et l'expansion des cellules iNKT. Ainsi, le ME de L. infantum, par ces lipides, peut participer à l’altération de l’activation des cellules iNKT dépendante du CD1d. Cela ajoute une nouvelle évidence de la contribution du ME de L. infantum dans la subversion de la réponse immunitaire de l’hôte. La communication entre le ME libéré par un pathogène et les cellules iNKT a été étudié pour la première fois, ce qui a suggéré un mécanisme de modulation de ces cellules qui n’avait jamais été décrit. Ce travail ouvre des perspectives pour l'étude de l'interaction de ME libéré par d'autres pathogènes avec des cellules iNKT. De plus, l'analyse des lipides contenus dans le ME de L. infantum pourra aboutir à la découverte de nouvelles molécules spécifiques pour inhiber les cellules iNKT. Cela apporterait des avantages significatifs dans les approches cliniques ciblant la modulation de l'activation des cellules iNKT. / The invariant natural killer T (iNKT) cells constitute a particular subset of T lymphocytes characterized by an innate-like profile. These cells rapidly respond to lipid and glycolipid antigens bound by the glycoprotein CD1d expressed by different antigen presenting cells. Once activated, they release large amounts of anti- and proinflammatory cytokines. Thus, iNKT cells are endowed with a remarkable immunomodulatory potential and they have been implicated in different disorders, such as allergy, autoimmunity, cancer and infection, among which is leishmaniasis. Leishmania spp. are a group of protozoan parasites that includes the causative agents of leishmaniasis. This is a neglected tropical disease in which the most severe form of manifestation affects visceral organs and could be fatal if left untreated. Importantly, the success of Leishmania infection relies on the capacity of the parasite to subvert host immune responses. Recently, a few groups, including ours, observed that Leishmania parasites release extracellular vesicles (EVs). EVs are vesicles formed by a lipid bilayer membrane, containing other lipids, proteins and genetic material on their surface as well as in their lumen. Due to their potential to transmit messages between pathogens and host cells without a direct cell contact, they have been a focus of great interest regarding infection. EVs derived from Leishmania and other protozoan parasites have been associated with pro-parasite effects, by creating a more permissive environment to the establishment of the infection. Herein, we studied the effect of the extracellular material (ExM), which encloses both EVs and vesicle-depleted material, released by L. infantum promastigotes in iNKT cells. In the first steps of this work, it was observed that L. infantum ExM is capable of impairing the expansion of human iNKT cells ex vivo from peripheral blood mononuclear cells. This evidenced the cross-talk between iNKT cells and L. infantum ExM that we explored further. L. infantum ExM also modulates the important capacity of iNKT cells to release cytokines, impairing the production of different cytokines, such as IL-4 and IFNγ by these cells. Furthermore, we also show that L. infantum ExM competes with α-GalCer, a potent iNKT cell agonist, for CD1d binding, which justifies its effect in the impairment of iNKT cell activation. Additionally, we also proved the lipids present in each fraction of L. infantum ExM take an important role in the inhibition of iNKT cell activation and expansion. Thus, L. infantum ExM, through their lipids, is suggested to participate in the impairment of CD1d-mediated activation of iNKT cells, adding a new evidence regarding the contribution of the parasite ExM to subvert host immune responses. To the best of our knowledge, this is the first time that the cross-talk between the ExM released by a microbe and iNKT cells was assessed, shedding light on a mechanism of iNKT cell modulation that remained unexplored so far. This opens new perspectives regarding the study of the interaction of the ExM released by other pathogens with iNKT cells. Moreover, a further analysis of the lipid content of L. infantum ExM might allow the finding of new inhibitory molecules specific to iNKT cells, which can bring significant advantages in clinical approaches targeting the modulation of iNKT cell activation.

Orientador: Lucilene Delazari dos Santos / Resumo: O uso de animais de grande porte como sorodoadores tem se mostrado de grande importância para a produção do selante de fibrina do Centro de Estudos de Venenos e Animais Peçonhentos (CEVAP). O objetivo deste trabalho foi certificar um plantel de bubalinos, realizar uma investigação sobre os potenciais candidatos à biomarcadores de brucelose em soro de búfalos e evidenciar o isolamento e a quantificação pioneira de exossomos (EVs) do soro de bubalinos com theileriose e babesiose. Realizaram-se os seguintes testes sorológicos: brucelose (BRU), leptospirose (LEP), febre aftosa (FMD), rinotraqueíte infecciosa bovina (IBR), diarréia viral bovina (BVD), varíola bovina (Pox) e tuberculose, língua azul (BTV), estomatite vesicular (EV) - sorotipos cocal (COCV) e alagoano (VSAV), leucose bovina (BLV), neospora (NC), toxoplasmose (TX) e testes bioquímicos laboratoriais. Todas as amostras de soro foram submetidas ao diagnóstico sorológico para detecção de brucelose, diagnóstico de reação em cadeia da polimerase para detecção de theileriose e babesiose, cromatografia líquida de afinidade, isolamento de exossomos, digestão de proteínas em solução, análise de espectrometria de massa e ferramentas de bioinformátca. Observamos um enriquecimento de 90,5% de proteínas nos soros de búfalos após este protocolo de depleção. A análise MS/MS evidenciou que as principais diferenças no proteoma dos animais com brucelose. No que se refere ao exossosmos isolados e quantififcados neste trabalho, nossa met... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The use of large animals as sorbozers has been shown to be of great importance for the production of fibrin sealant from the Center for the Study of Venoms and Poisonous Animals (CEVAP). The objective of this work was to certify a flock of buffaloes, to conduct an investigation of the potential candidates for brucellosis biomarkers in buffalo serum and to evidence the isolation and the pioneer quantification of buffalo serum exosomes (EVs) with theileriose and babesiosis. The following serological tests were carried out: brucellosis (BRU), leptospirosis (LEP), foot-and-mouth disease (FMD), infectious bovine rhinotracheitis (IBR), bovine viral diarrhea (BVD), bovine pox (Pox) and tuberculosis, blue tongue (BTV), vesicular stomatitis (EV) - cocal (COCV) and alagoan (VSAV) serotypes, bovine leukosis (BLV), neospora (NC), toxoplasmosis (TX) and laboratory biochemical tests. All serum samples were submitted: 1) serological test; 2) Polymerase chain reaction; 3) Depletion; 4) Isolation of exosomes; 5) Mass spectrometry and 6) Analysis and interpretation of the data. The MS / MS analysis showed that the major differences in the proteome of animals with brucellosis. Regarding the isolated and quantified exosmos in this work, our methodology is strongly encouraged for the isolation and characterization of EVs in Apicomplexa infections in farm animals. We conclude that this study on different fronts such as management, infectious diseases and parasitic diseases can be used in small pro... (Complete abstract click electronic access below) / Doutor

O sucesso no desenvolvimento de tumores não dependente somente de mutações, mas também é influenciado pelo microambiente do tumor; nele ocorre a interação entre as células tumorais e o estroma. Essa interação pode ser mediada por vesículas liberadas por essas células para o meio extracelular. Essas vesículas atuam na comunicação celular que pode influenciar a progressão tumoral. O objetivo deste estudo foi analisar as interações mediadas por vesículas entre células tumorais e fibroblastos normais. As células tumorais foram plaqueadas sobre a monocamada de fibroblastos e carregadas com diferentes corantes vitais. Nossos resultados evidenciaram a presença e a troca de vesículas entre as células em co-cultura. Vesículas isoladas mostraram tamanhos heterogêneos. Células tumorais possuem mais vesículas que as células normais. As vesículas são compostas pelas proteínas AHNAK e Anexinas. AHNAK foi detectada em vesículas trocadas e estava aumentada em tumores. AHNAK é molécula estrutural das vesículas extracelulares que pode influenciar a biologia dos tumores de mama. / The successful development of tumors is not only dependent on cell mutations, but also driven by the tissue microenvironment; relies on interaction of cells and their surrounding stroma. Some cell types release vesicular structures into the extracellular space that would be involved in cellular communication and tumor progression. The aim of this study was to analyze vesicle-mediated interactions between tumor cells and normal fibroblasts. Tumor cells were plated above fibroblasts monolayer and both loaded with different vital dyes. Our results evidenciated presence and exchange of vesicles between breast tumor cells and fibroblasts in co-culture. Vesicles isolated showed heterogeneous sizes. Tumor cell showed more vesicles than normal cells. These vesicles were composed of AHNAK and Annexins proteins. The protein AHNAK was detected in exchanged vesicles and was increased in tumors when compared to normal breast tissues. AHNAK could represent a vesicle structural molecule that would influence breast tumor biology.

A Anfotericina B (AB) é um antibiótico antifúngico natural, do grupo dos polienos, produzido por cultura de bactérias Streptomyces nodosus. A AB foi isolada pela primeira vez em 1953, e desde então tem sido utilizada extensivamente no tratamento clínico de infecções sistêmicas. Como a AB é altamente insolúvel em meio aquoso, a formulação de escolha tem sido dispersões aquosas de micelas mistas de AB e desoxicolato de sódio (Fungizon). Entretanto, a alta toxicidade deste fármaco tem estimulado a proposição de várias outras formulações. O presente trabalho estuda uma dessas formulações alternativas propostas: agregados de AB e Brometo de dioctadecildimetilamônio (DODAB). O objetivo principal deste trabalho foi tentar avaliar, numa visão estrutural, as propriedades e peculiaridades envolvidas na interação da AB com as dispersões de DODAB. Neste trabalho, centrou-se no estudo de dispersões de DODAB, sonicadas (DODABS) e não sonicadas (DODABNS), onde o antibiótico estivesse, principalmente, na forma monomérica, tendo em vista a proposta baixa toxicidade da AB enquanto monômero. Para avaliar estes sistemas utilizaram-se as técnicas de absorção óptica, calorimetria diferencial de varredura (DSC) e a Ressonância Paramagnética Eletrônica (RPE) de marcadores de spin incorporados nos agregados lipídicos. Mostrou-se que a AB monomérica incorpora-se tanto em vesículas de DODABNS, como nos pequenos agregados de DODABS, sendo maior sua incorporação nestes últimos. Foi possível mostrar que até a fração molar (AB/DODAB) de 1,2 mol% a AB está totalmente monomérica, incorporada em agregados de DODABS, mas somente parcialmente em vesículas de DODABNS. Nas bicamadas de DODABNS, a preferência da AB monomérica é pela fase gel, 40 % incorporada, comparada com 23 % na fase fluida. As várias técnicas indicam que a AB encontra-se na superfície da bicamada, com seu cromóforo hidrofóbico enxergando um meio de polarizabilidade maior do que a da água, tanto em DODABS como em DODABNS. Marcadores de spin mostram a coexistência de domínios lipídicos em fases diferentes, gel e fluida, a baixas temperaturas, em DODABS. Curiosamente, indicam que a AB monomérica interage, preferencialmente, com os domínios mais fluidos, em desacordo com a observada maior afinidade da AB pela fase gel de bicamadas lipídicas de DODABNS. O estudo termo-estrutural de dispersões de DODABNS e DODABS aqui apresentado, na presença e ausência de AB, pode ser relevante na proposta de novas formulações farmacológicas. / Amphotericin B (AB) is a natural antibiotic, produced by Streptomyces nodosus, with a very potent antifungal activity. It is a polyene macrolide molecule, isolated in 1953. Since then, it has been extensively used in the treatment of systemic mycotic infections. Considering AB very low solubility in aqueous medium, it has been largely used as an aqueous dispersion of sodium deoxycholate-AB mixed micelles (Fungizon). However, the high toxicity of this preparation has led to the proposition of different other formulations. The present work focuses on one of the proposed preparations, namely aggregates formed by AB and the cationic lipid dioctadecyldimethylammonium (DODAB). Considering the proposed low toxicity of monomeric AB, the present work studies DODAB dispersions, sonicated (DODABS) and non-sonicated (DODABNS), where AB is mostly monomeric. To the structural analysis, optical absorption, differential scanning calorimetry (DSC) and electron spin resonance of spin labels incorporated in the aggregates were used. It was found that AB as a monomer incorporates in aggregates present in both dispersions, DODABS and DODABNS, but the incorporation of monomeric AB in the small fragments present in DODABS is much larger. It was shown that AB incorporates in DODABS aggregates, as a monomer, up to AB/DODAB concentration of 1.2 mol%. At this concentration, only 40 % and 23 % of AB molecules are in the monomeric state in the gel and fluid phases of DODABNS bilayers, respectively. Hence, the gel phase of DODABNS bilayers favors the monomerization of AB, as compared to the bilayer fluid phase. All the applied techniques point to a superficial interaction of monomeric AB with DODAB aggregates, with the hydrophobic polyene chromophor positioned relatively inside the bilayer, in a region of polarizability higher than that of water, in both dispersions, DODABS and DODABNS. Spin labels indicate the coexistence of gel and fluid domains in DODABS aggregates, at low temperatures, supporting the proposed hypothesis of the presence of bilayer fragments in the dispersion. Contrarily to the higher detected affinity of monomeric AB for the gel phase, as compared to the fluid phase of DADABNS bilayers, spin labels indicate that monomeric AB is mostly adsorbed at the fluid domain of the fragments, possibly corresponding to the periphery of the fragments. The thermal-structural study presented here of DODABNS and DODABS, in the presence and absence of AB, can be relevant in the design of new pharmaceutical formulations.

Investigou-se o efeito do \"caráter iônico\" de interfaces zwitteriônicas, formalmente neutras, representadas por micelas e vesículas obtidas de monômeros com cabeça hidrofílica dipolar, nas propriedades de acumulação e troca de espécies iônicas. Os sistemas estudados incluíram, micelas de 1-(N-hexadecil-N,N-dimetilamônio) propanossulfonato (HPS), Hexadecil fosforil colina (C16PN), 3-hexadecil glicerofostidil colina (lisoPC) e vesículas de fosfatidil colina (PC). O estudo da ligação e troca iônica nestas interfaces foi monitorado a partir de efeitos sobre reações prototrópicas de sondas como a 8-hidroxi-1,3,6-pirenotrisulfonato de sódio (piranina). Observou-se a ligação crescente da piranina na seguinte ordem lisoPC < C16PN < HPS e troca iônica com sais adicionados (função do ânion) na ordem inversa, isto é, lisoPC &gt; C16PN &gt; HPS. Estudos de reprotonação de 1 e 2 naftóis incluídos nestes agregados, via salto de pH induzido por laser, revelaram um perfil de concentração de prótons inverso àquele das cargas do dipolo, isto é, concentração nos polos negativos e exclusão nos positivos. Este resultados foram ainda reforçados por estudos de excitação de volume, via espalhamento de luz, com micelas de HPS em função de sal adicionado. A partir destes resultados propôs-se um modelo de capacitor esférico imerso em meio eletrolítico para estas interfaces, o qual se mostrou bastante versátil na análise e previsão de resultados experimentais. Em suma, estabeleceu-se no presente trabalho a visão de que interfaces dipolares são geradoras de assimetria iônica na circunvizinhança da interface, assimetria esta de polaridade invertida àquela do dipolo radialmente disposto à interface. A generalidade deste modelo pode ser extrapolado para outras interfaces e colaborar na compreensão de diversos processos que dependem da acumulação e troca iônica. / The effect of ionic domains of formally neutral zwitterionic interfaces, represented by micelles and vesicles obtained with dipolar headgroups amphiphiles, on the binding and exchange of ionic species were investigated. The systems studied included: aqueous micelles of 1-(N-hexadecyl-N,N-dimethylammonio) Propane Sulfonate (HPS), Hexadecyl Phosphoryl Choline (C16PN), and 1-hexadecylglycerophostidyl Choline (lisoPC) and vesicles of Phosphatidylcholine (PC). Binding and ion-exchange processes were assessed from the effect of these agreggates on prototropic reactions of probes such as 8-hydroxy-1,3,6-pyrenetrisulfonate pyranine). Binding of pyranine increased in the following order lisoPC < C16PN < HPS. The effectiveness of anion dependent salt displacement of the probe from the aggregates was opposite to that of affinities, i.e. lisoPC &gt; C16PN &gt; HPS. Laser pH jump studies with 1 and 2 naphtols incorporated in these aggregates revealed a proton concentration profile inverse to that of the charged dipole, that is, accumulation in the region vicinal to the negatively charged group and exclusion from the positive end of the dipole. This finding were substantiated by HPS micelle excluded volume data, obtained from light scattering studies as function of added salt. A model of a spherical capacitor immersed in an electrolyte media for these interfaces was proposed. This model accounts for satisfactorily the observed characteristics of these interfaces. The model establishes that dipolar interfaces generate ionic asymmetries in the neighborhood of the interface that opposes the radially extended monomer dipole. The generality of the model can be very helpful in the analysis and understanding of several processes occurring in/at zwitterionic interfaces.

Regulation of the actin cytoskeleton is key to cellular function and underlies processes including cell migration, mitosis and endocytosis. Motile cells send out dynamic actin protrusions that enable them to sense and interact with their environment, as well as generating physical forces. Linking of the actin cytoskeleton to the cell membrane is essential for the formation of these protrusions. The proteins that are thought to fulfil such a role have a membrane interacting domain (such as the PH domain in lamellipodin, or I-BAR protein in IRSp53) and a domain which interacts with actin regulatory proteins (such as the SH3 domain of IRSp53, which binds Ena and VASP). I investigated the contribution of the F-BAR protein Toca-1 in linking actin polymerisation to membranes, by characterising a new protein-protein interaction and the interaction of Toca-1 with giant unilamellar vesicles. FBP17, a homologue of Toca-1, can oligomerise to form 2D flat lattices and 3D tubules on membranes. Proteins of the Toca-1 family have previously been implicated in actin polymerisation in cell-free systems and during endocytosis. However, there is emerging evidence that Toca-1 family proteins could also be involved in the formation of outward facing protrusions, lamellipodia and filopodia. In an in vitro system that recapitulates the formation of filopodia-like structures (FLS) on supported lipid bilayers, Toca-1 is recruited early, suggesting a Toca-1 scaffolding mechanism could precede the recruitment of other actin regulators. One prediction of this model is that Toca-1 would bind proteins previously implicated in filopodia formation, such as formins. I found that extracts depleted of Toca-1 binding partners no longer forms filopodia-like structures and subsequently optimised pull-down assays to identify Toca-1 binding partners by mass-spectrometry. I identified four formins, Diaph1, Diaph3, FHOD1 and INF2, and as well as the actin elongation factors and filopodia proteins, Ena and VASP. I further characterised these interactions and found that Toca-1 binds Ena and VASP via its SH3 domain. The interaction is direct and is strongly reduced if the proline-rich region in Ena is deleted. VASP was still able to bind without its proline rich region, suggesting there could be additional binding sites. I discovered that the binding of Ena and VASP was dependent on the clustering state of Toca-1, whilst the binding of the previously identified Toca-1 binding partner N-WASP was not. This further supports the importance of Toca-1 oligomerisation in actin polymerisation. I tested these interactions in the FLS system and found that increasing Toca-1 concentration leads to increased recruitment of N-WASP, as well as the novel binding partner Ena to the structures, whereas an increase in VASP was not observed. SH3-domain mediated interactions are required for Toca-1 recruitment to FLS, suggesting that its membrane and protein binding activities act cooperatively. I showed that unlike N-WASP, which promotes the formation of branched actin, Ena and VASP are not required for actin polymerisation on supported lipid bilayers, suggesting that they are redundant with other factors in the elongation step of FLS formation. Ena and VASP are known to be important for the formation of neuronal filopodia and so I began to further test the role of these interactions in a cellular context using a neuronal cell culture system. As well as recruiting protein binding partners, F-BAR family proteins are implicated in stabilising lipid microdomains and can induce the clustering of phosphoinositides. I investigated the role of Toca-1 in actin polymerisation on PI(4,5)P2-rich giant unilamellar vesicles (GUVs). Actin-rich tails formed on the GUVs only when excess Toca-1 was supplemented into the extracts, and I propose that this is due to lipid organisation by Toca-1. In summary, my work suggests a model in which Toca-1 clusters, stabilises the membrane lipids and recruits regulators of actin polymerisation, such as Ena. This mechanism could be used to link actin polymerisation to the membrane in cellular protrusions, such as filopodia.

Lipossomos de cloreto de dioctadecildimetilamonio (DODAC) com 0,51&#181; de diâmetro externo médio e transição de fase ocorrendo em uma faixa estreita de temperaturas (0,5-1,0&#176;C) foram obtidos por vaporização de clorofórmio e comparados com vesículas sonicadas de DODAC (cerca de 250 &#197; de diâmetro externo). Sacarose foi impermeante através de lipossomos grandes de DODAC ou de vesículas sonicadas de DODAC podendo ser utilizada para determinações de volume interno em ambas as preparações. Volume interno aparente para lipossomos grandes de DODAC foi 9,7 &#177; 1,3 1/mol e para vesículas sonicadas de DODAC, 0,33 &#177; 0,20 1/mol (adsorção de sacarose à bicamada de, respectivamente, 0,64 &#177; 0,30 e 0,20 &#177; 0,08 1/mol). Lipossomos grandes de DODAC comportaram-se como osmômetros em relação à sacarose e ao KCl (0-50mM KCl). Já as vesículas sonicadas de DODAC foram osmoticamente não-responsivas em relação à sacarose, floculando em presença de KCl. Essa não-responsividade osmótica foi interpretada como decorrente da presença única e exclusiva de água de hidrataçao no compartimento aquoso interno das vesículas sonicadas. Outras propriedades de lipossomos grandes de DODAC foram análogas às apresentadas por lipossomos de fosfolípides. Permeabilidades relativas (KCl como referência incorporado ao compartimento aquoso interno) do NaCl, HCl e sacarose foram similares às descritas para lipossomos de fosfatidilcolina; NaCl e sacarose sendo tão impermeantes quanto o KCl e HCl, sendo ligeiramente mais permeante que o KCl. Nas vizinhanças da temperatura de transição de fase, ocorreu um acentuado aumento da velocidade inicial de encolhimento e a extensão total de encolhimento chegou a um mínimo. A comparaçao de algumas propriedades físicas e funcionais de vesículas sonicadas e de lipossomos grandes de DODAC permitiu concluir que lipossomos grandes de DODAC constituem um modelo mais adequado para estudos de transporte. Adicionalmente, o método de vaporização de clorofórmio foi testado para o dihexadecilfosfato de Na+ (DCP) sendo obtidas dispersões homogêneas desse anfifílico capazes de incorporar um volume de 13 &#177; 4 1/mol e de responderem como osmômetros a gradientes osmóticos de sacarose, um soluto que praticamente nao é adsorvido à bicamada de DCP e que resultou impermeante através da mesma. / Dioctadecyldimethylammonium chloride (DODAC) liposomes with 0.51&#181; mean external diameter and sharp phase transitions were obtained by chloroform vaporization and compared with (small) sonicated DODAC vesicles. Sucrose was impermeant through large DODAC liposomes and sonicated vesicles and was used for internal volume determinations. The apparent internal volumes for large DODAC liposomes and sonicated DODAC vesicles were, respectively, 9.7 &#177; 1.3 and 0.33 ± 0.20 1/mol. (External sucrose adsorption were, respectively, 0.64 &#177; 0.30 and 0.20 &#177; 0.08 l/mol). Ideal osmometer behaviour towards KCl over the 0-50mM concentrations range and towards sucrose were observed only for large DODAC liposomes. Sonicated DODAC vesicles were osmotically non responsive towards sucrose and floculate with KCl. Other properties of large DODAC liposomes closely resembled those of phospholipid liposomes. At temperatures near the phase transition temperature, a steep increase in the initial shrinkage rate and a minimum for the total extent of shrinkage occured. Relative permeabilities (KCl as reference entrapped inside) to NaCl, HCl and sucrose were similar to those of phosphatidyl choline liposomes. NaCl and sucrose were as impermeant as KCl, and HCl slighthly more permeant than KCl. Large DODAC liposomes are proposed as an adequate synthetic membrane model, in contrast to sonicated DODAC vesicles. In addition, the chlroform vaporization method was tested for sodium dihexadecylphosphate (DCP). Large DCP liposomes were demonstrated to be impermeant towards sucrose entrapping 13 ± 4 l/mol and behaving as an osmometer towards this solute.

O Melanoma é um tipo de neoplasia que se origina de melanócitos normalmente presentes na epiderme. Uma das características do melanoma é a capacidade de adquirir resistência a terapias. As células de melanoma podem aumentar a liberação de vesículas extracelulares (VEs) em resposta ao tratamento com quimioterápicos. A cisplatina (CDDP) e a temozolomida (TMZ) são drogas utilizadas para o tratamento de tumores. Ambas as drogas formam adutos no DNA, mas as vias de sinalização que deflagram a morte celular são distintas. O objetivo desse estudo é investigar a morte celular da linhagem B16-F10 na presença de VEs oriundas de células B16-F10 tratadas com cisplatina CDDP ou TMZ. Inicialmente as VEs oriundas de células de melanoma murino, B16-F10, tratadas com CDDP ou TMZ e seus controles, foram isoladas por ultracentrifugações sucessivas. Para os experimentos in vitro, as células foram tratadas com as drogas em combinação com as respectivas VEs. As amostras foram realizados avaliações de ciclo celular e de morte e ensaio clonogênico. Para os experimentos in vivo, as células B16-F10 foram pré-tratadas com VEs, e posteriormente, as células foram inoculadas via subcutânea em camundongos C57BL/6 e os tumores foram mensurados diariamente. Em nosso estudo concluimos que a metodologia do isolamento de VEs é eficiente. Além disso, observamos que o tratamento com CDDP ou TMZ aumenta a liberação de VEs por células tumorais. Apesar do resultado contraditorio, as VEs liberadas por células tumorais tratadas com quimioterápicos aumentam a capacidade de sobrevivência das células de melanoma in vitro. VEs oriundas de células de melanoma não participam inicialmente da sensibilização à morte de células tumorais causada pelas mesmas drogas, mas a longo prazo, as VEs oriundas de células tratadas com a TMZ podem conferir uma resposta celular de sobrevivência às células tumorais in vitro. In vivo, o resultado é inconclusivo, uma vez que para confirmar se as VEs fazem parte da adaptação tumoral conferindo fenômenos de sobrevivência celular in vivo, é necessário avaliar em outros modelos celulares e animais / Melanoma is a neoplasm derived from melanocytes normally present in the skin specifically in the epidermis. One of the malignancies of melanoma is the ability to acquire chemoresistance. Cisplatin (CDDP) and temozolomide (TMZ) are drugs used for the treatment of tumors. Both drugs can form alkylating adducts in DNA, however, the pathways that trigger cell death are distinct. Tumor cells, including melanoma, may increase the release of extracellular vesicles (EVs) in response to chemotherapeutic treatment. The aim of this study is to investigate the cell death phenomenon in B16-F10 cell line in presence of EVs derived from chemotherapeutic-treated B16-F10 cells. For in vitro experiments, the cells were treated with CDDP or TMZ in combination with EVs from chemotherapictreated samples. For in vivo experiments, B16-F10 cells were exposed to EVs and inoculated subcutaneously in C57BL/6 mice. The growth was measured daily. In this work, we established and characterized VEs released by melanoma cells treated with chemotherapics and we established chemotherapics treatments to isolate EVs for next EVs isolation. Our results showed that CDDP or TMZ treatment increase the release of EVs by tumor cells. The EVs released by melanoma cells after CDDP or TMZ treatment seem to increase the survival capacity of melanoma cells. Thus, we concluded that EVs derived from melanoma cells do not participate in the cell death sensitization induced by CDDP or TMZ. However, EVs derived from TMZ treated cells may offer a survival effect to tumor cells in vitro a long term. In vivo, The result is inconclusive since to confirm how VEs are part of the tumor adaptation conferring cellular survival phenomena in vivo, it is necessary to evaluate in other cellular and animal models