Chemical reactions can have a staggering amount of molecular complexity. Reaction mechanisms have been proposed with over one hundred elementary reaction steps that occur in the same system simultaneously. While several methods exist to simplify and make sense of the pathways and kinetics via which these reactions proceed, e.g., reaction graphs, sensitivity or flux analysis, microkinetic analysis, and comparison of energy landscapes, etc., these methods all have limitations and are often not able to capture a comprehensive picture of the kinetics of system. It has been found useful to view these mechanisms as a network, i.e., a reaction graph. These graphs enable the visualization of the pathways of the reaction and can provide an analytical tool for pathway and kinetic analysis. However, many of the specific graph-theoretic approaches in the literature are not the most suitable for kinetic analysis of complex mechanisms; as they are simply not based on rules that are rigorous enough to fully enumerate all the pathways or provide quantitative analysis of the reaction rates. Our Reaction Route (RR) Graph approach is different in that it depicts the mechanism by a graph that is consistent with all physical and chemical laws associated with reaction networks, particularly being consistent with mass and energy conservation, i.e., Kirchoff’s Flux Law (KFL) and Kirchoff’s Potential Law (KPL). Because of their adherence to these laws, RR Graphs are able to provide an accurate graph-theoretical tool not only for depicting all reactions routes as walks (hence the name RR Graph) but also for pruning mechanisms and allowing a simplified but accurate quantitative description of reaction rates. This adherence to KFL and KPL does mean that the construction and implementation of these graphs can be prohibitively difficult for large mechanisms. For large reaction systems,especially nonlinear mechanisms, it is not realistic to generate these graphs by hand. And although there exists an analytical solution to find a determinant matrix for the RR Graph of a mechanism, the process involves an exhaustive search for a solution which experiences a combinatorial explosion as the number of steps gets very large. This leads to the idea of developing an algorithm for a computer program that can determine how to generate these graphs automatically. Unfortunately, the same combinatorial explosion is present such that for a moderately sized twenty step mechanism, it could take an average computational processor over a decade to find a solution. We have determined, however, that this brute force combinatorial approach can be avoided if heuristics could be developed to bridge gaps in our knowledge of how these graphs are constructed. Thus, developing a better analytical approach and/or a tighter set of heuristics for a computer algorithm are the overarching goals of this work. To make progress toward developing such heuristics, a set of microkinetic mechanisms were analyzed with the notion that the realization of the RR Graphs would highlight a better approach to their construction and usage. In particular, a very large linear reaction system, a smaller linear system and two non-linear reaction systems were analyzed to develop insights into how each graph is manually constructed and analyzed. Furthermore, kinetic analysis was done for these mechanisms and compared to experimental data and other analytical tools to prove not only the validity of the RR Graphs, but also how they are a significant improvement over more commonly used approaches for mechanistic and kinetic analysis. Based on the lessons learned through a consideration of these examples, a set of heuristics are established and enumerated with the ultimate goal of developing an intuitive algorithm that can help automate drawing and kinetic analysis via RR Graphs of complex mechanisms.

Le fondaparinux (Arixtra®), anticoagulant de la classe des pentasaccharides de synthèse, est le premier inhibiteur d'origine synthétique, spécifique et indirect du facteur Xa de la coagulation. Il résulte de la synthèse chimique de l'unité pentasaccharidique des héparines, capable de se lier à l'antithrombine, une protéine endogène, inhibitrice de la coagulation. Cependant, son utilisation reste limitée par son administration uniquement possible par voie parentérale.L'objectif de ce travail de thèse est de développer une forme orale du fondaparinux en l'associant à un dérivé squalénique. Le squalène, terpénoïde naturel précurseur de la synthèse du cholestérol, possède une très bonne absorption orale (supérieure à 60 %). Dans ce contexte, deux stratégies d'association ont été développées: la première consistant à associer par liaison covalente le fondaparinux à un dérivé squalénique selon le concept de la « squalénisation » et la deuxième à associer par interactions non covalentes le fondaparinux à un dérivé squalénique cationique.Les travaux expérimentaux ont montré que la première stratégie était délicate à mettre en œuvre en raison d'une part de la difficulté à synthétiser un bioconjugué fondaparinux-squalène et d'autre part de la perte de l'activité anticoagulante du fondaparinux. En raison de ces obstacles, le concept de la « squalénisation » n'est pas adapté à ce type de molécule active. En revanche, la deuxième stratégie s'est montrée très prometteuse. Elle a consisté à formuler des nanoparticules par association non covalente du fondaparinux, chargé négativement, à un dérivé squalénique cationique. Cette approche a permis de mettre en évidence l’excellente capacité d'auto-assemblage en milieu aqueux de ces deux composés, liée à l’établissement de deux types d’interactions, électrostatiques et hydrophobes (entre les molécules de squalène). L'absorption orale du fondaparinux a été considérablement augmentée grâce à ce nouveau système nanoparticulaire. Cette nouvelle approche à base de squalène a ainsi montré son efficacité dans l'amélioration de l'administration orale du fondaparinux et pourrait représenter un système thérapeutique potentiel dans le traitement des maladies thromboemboliques. / Since its introduction in the market in 2002, fondaparinux (Arixtra®) is a drug of choice in the anticoagulant therapy. Its structure corresponds to the heparin pentasaccharide sequence that mediates its interaction with the natural plasma inhibitor of coagulation, antithrombin. However, like heparin, its application is limited due its unique administration by parenteral route. The aim of this project is to develop an efficient oral delivery system for fondaparinux by association with a squalene derivative. Squalene, a natural precursor of cholesterol in sterol biosynthesis, is well-known for its excellent oral absorption (i.e. more than 60 %). In this context, two strategies were investigated. The first consisted in achieving a covalent coupling between fondaparinux and a squalene derivative according to the concept of “squalenoylation”. The second was to associate fondaparinux to a cationic squalenoyl derivative by non-covalent association.Experimental work showed that the first strategy was delicate to implement due to the difficulty to synthesize a fondaparinux-squalene bioconjugate and, the loss of the anticoagulant properties of fondaparinux. Because of these obstacles, the concept of "squalenoylation" was not suitable for this type of active molecule. In contrast, the second strategy has been very promising. It consisted in the formulation of a nanoparticulate delivery system by ion-pairing of fondaparinux and a cationic squalenoyl derivative. This approach permitted to highlight the self-assembly of these two compounds in water as monodisperse nanoparticles thanks to electrostatic and hydrophobic interactions. Furthermore, the oral absorption of fondaparinux was significantly increased with this new nanoparticulate system. This new squalene-based approach has shown its effectiveness in improving the oral administration of fondaparinux and could be a potential delivery system in the treatment of thromboembolic diseases.

Completando 100 anos da descoberta da molestia, a Doenca de Chagas, causada pelo agente etiologico Trypanosoma cruzi, ainda e considerada nas Americas Central e do Sul um problema de saude publica, atingindo mais de 8 milhoes de individuos. Sua transmissao ocorre de distintas maneiras, como por vetores, transfusao sanguinea, transplante de orgaos, acidentes em laboratorios, via oral. O parasita e conhecido por sua heterogeneidade no genotipo e fenotipo, baseado em mutacoes cumulativas em diferentes sub-populacoes do parasita. A cepa CL Brener e uma cepa padrao para pesquisa com diversas peculiaridades interessantes como baixa infectividade em animais, resposta aos tratamentos existentes e seus marcadores geneticos serem estaveis. O clone CL B5 de T. cruzi e originado a partir da cepa CL Brener modificada geneticamente. A cepa possui um gene reporter, o LacZ de Escherichia coli que sintetiza uma enzima, a À-galactose que pode catalisar uma reacao colorimetrica com o substrato vermelho de clorofenol À-galactopiranosideo (CPRG). A atividade enzimatica e diretamente proporcional ao numero de parasita. No presente trabalho propomos a introducao de uma tecnica colorimetrica para caracterizacao de parametros para quantificacao do parasitismo tecidual e comparacao com a tecnica histologica e Real Time-PCR. Camundongos Balb/C machos foram divididos em grupos controle (nao infectado) e experimental variando a via de inoculacao, a saber, intraperitoneal (IP), subcutanea (SC) e oral (OR). As curvas parasitemicas foram realizadas por meio da coleta de sangue da cauda do animal. Apos morte dos mesmos, foram estudados os tecidos cardiaco, hepatico, esplenico, enterico e muscular (musculo esqueletico). Os orgaos foram retirados, divididos em tres porcoes equitativas para analise histologica, enzimatica e molecular. Na atividade enzimatica, comparando os tecidos em suas vias de inoculacao, o figado apresentou resultado significativo no grupo inoculado via IP. Tecido cardiaco apresentou diferenca estatistica (P<0,05) entre as vias de inoculacao OR e SC, apresentando maior atividade enzimatica na primeira, o que esta relacionado a uma maior quantidade de parasita tecidual. Nos cortes histologicos o grupo SC mostrou-se mais infectado com grande numero de ninhos de forma amastigota em praticamente todos os tecidos, enquanto nos demais grupos foram observados ninhos de amastigota em sua maioria no coracao e viii musculo esqueletico. A presenca do parasita nos demais tecidos foi confirmada pela eletroforese em gel de agarose 1,5% apos tecnica de PCR convencional. A Real Time-PCR nao se mostrou satisfatoria no trabalho devido a dificuldade na sua padronizacao. Concluindo, a metodologia enzimatica se mostrou favoravel e adequada na quantificacao parasitaria tecidual, podendo ser aperfeicoada para corroborar sua eficacia. / One hundred years after the discovery of Chagas Disease, an illness caused by blood born protozoan Trypanosoma cruzi, is still consider a public health problem. It is a major cause of morbidity and mortality in Central and South Americas where more than 8 million individuals are already infected. This illness can be contracted by different forms as vector transmission, blood transfusion, tissue transplant, laboratory accidents and oral route. The parasite has a heterogeneous genotype and phenotype due to accumulating mutation in their sub-populations. CL Brener is considered as a pattern strain due to some intrinsic characteristics as low infectivity, good response to therapy drugs and a stable genetic heritage. The CL B5 clone of T. cruzi was originated by a genetic modification of the CL Brener strain which has a reporter gene LacZ of Escherichia coli, which induces the syntesis of â-galactosidase which is able to catalyze a colorimetric reaction using the substrat Chlorophenol red-â-D-galactopyranoside (CPRG). The enzymatic activity is directly related with the number of the parasites. In this study we proposed a new colorimetric assay to quantify T. cruzi load in different animal tissues by comparing with other methodologies such histological and molecular assay (Real Time-PCR). Male Balb/C mice were separated into control and experimental groups according to the route of infection (IP-intraperitoneal, SC-subcutaneous, OR-oral). Parasitemic curves were made by collecting blood samples from the tail of experimental animals. Fifteen days after, animals were euthanized and their tissues removed (heart, liver, spleen, intestine and skeletal muscles) and divided in three portions for the colorimetric analyses, histopathology and molecular assay. The enzymatic activity was performed comparing the number of parasites in different tissues according to the route of infection. Comparing them, it was observed that heart and liver displayed the highest number of parasites as compared with the other studied tissues. In the liver, IP route triggered the highest number of parasites while in the heart OR and SC routes displayed enhanced parasites. The histopathology analysis revealed that SC group presented the highest number of amastigote nests. For IP and OR groups, nests were mostly observed in heart and skeletal muscles. T. cruzi DNA were detected using electrophoreses 1,5 % agarose gel after conventional PCR technique, displaying characteristic bands of DNA. The Real Time-PCR was not a satisfactory x assay for this study due to difficulties on its patronization. To conclude, the enzymatic methodology was advantageous and appropriated on quantifying tissue parasites. Some further experiments will be needed to improve methodology and its efficacy.

Esta dissertação baseia-se na aplicação de um procedimento empírico envolvendo técnicas de preferência declarada para a estimativa do valor do tempo de viagem de motoristas em deslocamentos regionais, em um contexto de escolha entre rotas pedagiadas e não pedagiadas. Inicialmente é feita uma revisão das abordagens teórica e empírica sobre a valoração do tempo. São também revistos os conceitos básicos de outros dois elementos fundamentais na metodologia aqui utilizada: a teoria da escolha e técnicas de preferência declarada. Uma aplicação a um estudo de caso específico é feita usando informações coletadas em ampla pesquisa de preferência declarada, realizada em 2005 com motoristas de automóvel em diversos pontos da malha rodoviária do estado de São Paulo. O desenho experimental da preferência declarada envolvia três atributos: tempo de viagem por uma rota pedagiada, custo tarifário e tempo de viagem por uma rota não pedagiada. O conjunto das informações foi analisado e utilizado na obtenção de modelos de escolha discreta do tipo logit multinomial; os valores do tempo de viagem foram obtidos a partir dos coeficientes estimados em funções de utilidade aditivas e lineares nos parâmetros. Analisou-se também a variação do valor do tempo em função de características do motorista e da viagem, questão abordada através da estimação de diferentes modelos por segmentos da amostra e da especificação de funções de utilidade que incorporam variáveis dummies para representação das características analisadas. Os resultados indicaram, para a escolha específica estudada, valores de tempo médios de cerca de R$ 16/h, variando de R$12/h a R$23/h para diferentes segmentos de viajantes analisados. A duração da viagem foi uma importante característica associada a variações no valor do tempo, que decresceu na medida em que as durações aumentaram. Variações expressivas no valor do tempo de viagem foram também observadas para viagens a lazer, motoristas de renda familiar baixa e aqueles com alta posse de veículos. / This dissertation is based on the application of an empirical procedure using stated preference techniques for the estimation of the value of travel time for drivers in regional trips, in the context of a choice between tolled and non-tolled routes. It first reviews the theoretical and the empirical approaches for the valuation of travel time and then presents the basic concepts of two other topics relevant for the methodology adopted: choice theory and stated preference methods. An application to a specific case study is then presented, using information from a stated preference survey conducted in 2005 with a large sample of car drivers intercepted at several points in the highway network of the state of São Paulo. The stated preference experimental design considered three attributes: trip time on a tolled route, value of toll and trip time on a non-tolled route. Survey data were analyzed and used for the estimation of discrete choice (multinomial logit) models; values of travel time were derived from estimates of coefficients of an additive linear in the parameters utility function. The specification of the models and the segmentation of the sample allowed the estimation of the variation of travel time according to some selected driver and trip characteristics. Results indicated, for the specific choice context analyzed, an average value of travel time of approximately R$16/h, varying from about R$12/h to R$23/h for different segments of travelers. Trip length was an important characteristic influencing the variation of the value of travel time, which declined as trip length increased. Other important effects were found for leisure trips, for travelers with low income and for those with high family car ownership.

A comprovação de que siRNA pode ser usado para supressão de genes em diferentes células de mamíferos atraiu grande atenção como nova possibilidade de tratamento para diversas doenças. No entanto, para aplicação terapêutica de siRNA é necessário o desenvolvimento de um sistema de liberação efetivo e não tóxico, que permita a captação celular do siRNA e também que evite a sua degradação por enzimas. A capacidade de supressão de genes promovido pelo siRNA depende tanto do número de moléculas de siRNA transfectadas quanto da taxa de duplicação da célula. Uma das formas farmacêuticas que vem sendo amplamente utilizadas na literatura com o objetivo de prolongar e proteger a liberação de fármacos são as formulações com capacidade de formação de gel in situ. Desta forma, a presente pesquisa teve por objetivo o desenvolvimento farmacotécnico de formulações líquido cristalinas com formação de gel in situ após administração por via subcutânea para veiculação sustentada de siRNA. Misturas adequadas de monoleína, propilenoglicol, tampão Tris e polietilenoimina ou oleilamina (polímero e lipídeo catiônico, respectivamente) formaram sistemas precursores capazes de se geleificar in situ com excesso de água e, como demonstrado pelo estudo de absorção de água, a formação do gel é um processo rápido. As formulações desenvolvidas também foram eficientes para complexar o siRNA comprovando a importância da incorporação dos aditivos catiônicos aos sistemas. A liberação in vitro dos sistemas líquido cristalinos mostraram que a liberação é dependente da taxa de absorção de água e os estudos in vivo em modelo animal para avaliação da formação do gel in situ e toxicidade demonstraram que o gel se forma in vivo com a absorção de água dos fluidos corporais, sendo biodegradável e biocompatível. Os sistemas desenvolvidos mostraram-se promissores para o tratamento de doenças onde a administração localizada e sustentada de siRNA é necessária. / The evidence that siRNA can be used for suppression of genes in different mammalian cells attracted wide attention as a new possibility of treatment for various diseases. However, for siRNA therapeutic application is necessary to develop an effective non-toxic delivery system, which facilitate the siRNA cell uptake and avoid its degradation by enzymes. The genes suppression promoted by siRNA depends on the number of siRNA molecules transfected as the cell replication rate. One of the dosage forms that have been widely used in literature in order to prolong and protect the drug release is the in situ gelling formulations. Thus, the present study aimed the development and characterization of the in situ gelling liquid crystal-based systems for subcutaneous application of siRNA in gene therapy. Appropriate mixtures of monoolein, propylene glycol, Tris buffer and polyethyleneimine or oleylamine (cationic polymer and lipid, respectively) was able to form precursor formulation that gelling in water excess and, as demonstrated by the swelling studies, the gel formation is a fast process. The developed formulations were also effective for complexing the siRNA, indicating the importance of the incorporation of cationic additives in the systems. The in vitro release study showed that the release is dependent on the water absorption rate. In vivo studies in animal models have shown the gel is formed in vivo after water uptake of body fluids, and it is biodegradable and biocompatible. The systems developed are promising for the treatment of diseases where the local and sustained administration of siRNA is necessary.

As doenças cardiovasculares continuam sendo a primeira causa de morte no Brasil (32%) representando a terceira maior causa de internação hospitalar. Apesar dos avanços terapêuticos das últimas décadas, estudos epidemiológicos consideram o infarto agudo do miocárdio (AMI) uma das maiores causas de morbidade e mortalidade, sendo a maioria, ligados a realização de terapias não adequadas, dos quais 50% das mortes ocorrem nas primeiras 2 horas do ocorrido e 14% morrem antes de receber atendimento médico. O objetivo deste estudo foi de avaliar a técnica de infusão transpericárdica de células mononucleares de medula óssea (CMMO) em suínos. Três suínos fêmeas, pesando 25Kg foram induzidas ao AMI, com auxilio de cateter balão colocado no 1° ramo diagonal da artéria coronária interventricular por 45 minutos, seguido por infusão de 1x108 CMMO marcadas com Hoechst® pela via transpericárdica. O grupo controle foi composto por 3 animais, os quais receberam infusão de 1x108 CMMO marcadas com Hoechst® através da mesma técnica. Os resultados revelaram distribuição homogênea das CMMO no miocárdio, concentrando-se especialmente na área infartada, enquanto que o grupo controle apresentou distribuição homogênea ao longo do miocárdio. Nós concluímos que a técnica transpericárdica é viável para infusão de CMMO em processos de isquemia cardíaca. / Cardiovascular illnesses continue to be the first cause of death in Brazil (32%), representing the third major reason of hospital internment. Although the therapeutics advances in the last decades, epidemiologic studies considered the acute myocardium infarct (AMI) to be one of the most causes of morbidity and mortality (30%), most of, related to the institution of non-adequate therapy, thereby 50% of deaths in the early two hours of the event and 14% dying before any medical assistance. Currently therapies include stent angioplasty, Thrombolytic medication and aortic-coronary venous grafting; while in experimental area the cellular therapy has been largely investigated being the cells infusion technique investigation the most enthusiastic issue. This study aimed to evaluate the transepicardic infusion technique of bone marrow mononuclear cells (BMMC) in swine. Three female swine, averaging 25 kg, were induced to AMI, with the aid of a balloon catheter displaced on the first interventricular diagonal branch of the coronary artery for 45 minutes, following to the infusion of 1x108 BMMC stained with Hoescht® by the transepicardic technique. Sham operation was carried out in three animals (Control group), which received infusion of 1x108 BMMC stained with Hoescht® by the same technique. The results revealed an inhomogeneous distribution of the BMMC in the myocardium, being more concentrated in the infarcted area, while the control group presented a homogeneous distribution along the myocardium. We concluded the transpericardic technique would be acceptable to the infusion of BMMC in cardiac ischemic processes.

Este trabalho consiste na análise da modificação superficial da liga armazenadora de hidrogênio, MmNi3,4Co0,8Al0,8, através de óxidos de Cobre, Níquel e Cobalto, utilizando-se para tanto o método sol-gel. As técnicas de caracterização usadas para o recobrimento obtido, Microscopia Eletrônica de Varredura (MEV) e Redução Térmica Programada (RTP), permitiram a observação de uma \"rede\" formada pelos óxidos presentes nos recobrimentos que proporcionam a aglomeração das partículas da liga, sem contudo, impedir a interação de hidrogênio com o material. O estudo do comportamento eletroquímico do recobrimento foi realizado pelas técnicas de Voltametria Cíclica , Ciclos Galvanostáticos de Carga e Descarga e Espectroscopia de Impedância. Foi possível verificar que a camada de óxidos formada pelo recobrimento através do método sol-gel melhora o desempenho da liga: em relação a sua capacidade de descarga que é significativamente aumentada, principalmente no recobrimento por óxido de cobalto e também em relação a proteção contra a pulverização do material, que proporciona maiores números de ciclos de carga e descarga / This work consists in the analysis of the surface modification of the hydrogen storage alloy, MmNi3,4Co0,8Al0,8, threugh Copper, Nickel and Cobalt oxides, using for this the sol-gel method. The characterization techniques used for the obtained surfase modification (SEM and TPR) allowed the observation of a \" net \" formed by the presents oxides in the surface modification that provides the gathering of the alloys particles, without however, to harm the hydrogen interaction with the material. The study of the electrochemical behavior of the surface modification was carried out by the techniques of Cyclic Voltammetry, Charge/Discharge cycles and Electrochemical Impedance. It was possible to verify that the oxides of layer formed by the surface modification for the sol-gel method improves the alloy performance: in relation to its discharge capacity that is significantly increased, mainly in the surface modification by oxide of cobalt, and also in relation to the protection against the deterioration of the material, that provides higher numbers of cycles charge and discharge

Este trabalho apresenta uma análise sobre a Arquitetutra PCE (Path Computation Element), como alternativa para o roteamento inter-domínios em redes heterogêneas, ou seja, em ambientes onde ocorre a integração do domínio óptico, das redes de transporte ópticas, com o domínio eletrônico, das redes convencionais, tratando inclusive com questões de qualidade de transmissão do sinal óptico, ou QoT (Quality of Transmission). Os objetivos e o funcionamento do mecanismo do PCE em redes integradas são detalhados. São apresentados também os conceitos e definições das redes ópticas, bem como seus principais elementos, a proposta de interconexão das mesmas com as redes convencionais provendo uma rede única e integrada, o papel dos planos de controle, e os principais mecanismos de roteamento. A análise do PCE foi direcionada para o comportamento do principal protocolo utilizado na sua arquitetura, denominado PCE Protocol. De forma mais específica, foi realizada a análise de suas mensagens de requisição de rotas e das mensagens de respostas a estas requisições, e sua interferência no tráfego dos enlaces das redes onde são introduzidas. A partir daí é apresentada uma proposta de extensão de parâmetros para o protocolo PCE Protocol, com o objetivo de atender necessidades específicas de QoT, baseado em estudos das particularidades das características das transmissões ópticas. A partir destas definições, foram realizadas análises adicionais a fim de verificar o impacto com relação ao tráfego gerado em enlaces de diferentes larguras de banda, com a definição original do protocolo, e com a extensão proposta, permitindo uma comparação entre ambos. / This thesis presents an analysis of Path Computation Element Architecture (PCE) as an alternative to be implemented in heterogeneous network inter-domain routing, in other words, environments where optical domains are integrated with the electronic domains of conventional networks. Also, the ability of PCE to manage issues regarding the transmission quality of the optical signal, the QoT (Quality of Transmission), is presented. Objectives and functionality of the PCE mechanism in integrated networks is detailed. Concepts and definitions regarding optical networks and their main elements, the aim of interconnection with conventional networks to build a unified and integrated network, the role of control planes, and the main routing mechanisms are also presented. The analysis of the PCE was focused on the behavior of its main protocol, called PCE Protocol. Specifically, the analysis was performed on both its request and response messages, in addition, the interference in the traffic of the links where these messages had been introduced was also analyzed. Afterwards, an extension of the PCE Protocol parameters was proposed based on the study of optical transmission characteristics, with the aim of meeting specific QoT requirements. From these definitions, further analyses of the impact on the traffic in links with different bandwidths were performed, using the PCE protocol, with and without the proposed extension, allowing a comparison between them.

La voie d’administration d’un vaccin et le modèle préclinique dans le lequel il est évalué sont des facteurs majeurs qui contribuent à son succès chez l’homme. Dans ce contexte, la découverte que la voie transcutanée (TC) induit une réponse immunitaire puissante a fait de la vaccination antitumorale TC une stratégie prometteuse. Une évaluation complémentaire du candidat vaccin dans un modèle de souris humanisée (Hu-SPL-NSG), plus prédictif de la réponse humaine, est aussi nécessaire. L’objectif de cette thèse est i) d'optimiser des constructions liposomiques peptidiques incorporant un agoniste de TLR pour la voie TC et ii) d’évaluer leur immunogénicité dans le modèle Hu-SPL-NSG. Ainsi, nous avons fait varier la nature de l’agoniste de TLR et la déformabilité de la vésicule liposomique, et avons rajouté une molécule de ciblage des cellules dendritiques. L’immunogenicité de ces formulations par voie TC a ensuite été évaluée chez la souris. Enfin, nous avons testé la capacité d’une construction liposomique modèle à induire une réponse cellulaire et humorale dans le modèle Hu-SPL-NSG. L’ensemble de ces travaux a fourni une première preuve de concept sur la faisabilité de la vaccination antitumorale TC par des liposomes et de son applicabilité chez l’homme. / A vaccine administration route and the preclinical model in which it is evaluated are major factors that contribute to its success in humans. In this context, the discovery that the transcutaneous (TC) route induces a powerful immune response has made the TC tumor-specific vaccination a promising strategy. Further evaluation of candidate vaccines in a humanized mouse model (Hu-SPL-NSG), more predictive of the human response, is also needed. The objective of this thesis is to (i) optimize liposomal constructs incorporating peptides and a TLR agonist for the TC pathway and (ii) evaluate their immunogenicity in the Hu-SPL-NSG model. Thus, we have varied the nature of the TLR agonist and the deformability of the liposomal vesicle, and have added a dendritic cell targeting molecule. Immunogenicity of these formulations by the TC route was then evaluated in mice. Finally, we tested the ability of a model liposomal construct to induce a cellular and humoral response in the Hu-SPL-NSG model.All of this work provided a first proof of concept on the feasibility of TC tumor-specific vaccination by liposomes and its applicability in humans.

A via de administração vaginal pode ser considerada uma alternativa para diversos tratamentos, tanto de ação farmacológica local como sistêmica. No entanto, o tempo de permanência do fármaco no local da aplicação e a eficácia esperada representam um desafio para o desenvolvimento de formulações. O objetivo deste trabalho foi desenvolver nanocápsulas de superfície catiônica (EUDRAGIT® RS 100) ou aniônica (EUDRAGIT® S 100), contendo ou não o marcador de fluorescência Vermelho do Nilo como um modelo de fármaco lipofílico, e incorporar essas partículas em hidrogéis de quitosana, a fim de aumentar o tempo de residência da formulação na mucosa vaginal, devido às propriedades mucoadesivas desse polímero. Diversas formulações foram preparadas com concentrações crescentes de quitosana e analisadas em termos de pH e comportamento reológico, a fim de selecionar a mais adequada para aplicação vaginal. Os hidrogéis foram produzidos com quitosana 2,5% p/p, com ou sem nanocápsulas. Foi avaliada a aderência (mucoadesividade e perfil lavabilidade) e a capacidade de penetração (microscopia confocal e extração seguido de quantificação do vermelho do nilo) das formulações quando aplicadas em mucosa vaginal de porcas. As suspensões de nanocápsulas apresentaram diâmetro em torno de 200 nm e potencial zeta entre +13 mV (NC-RS) e -13 mV( NC-S) e valores de pH entre 5,1 e 6,2. A formulação de quitosana apresentou viscosidade característica e pH ácido (em torno de 4,5), ideal para aplicação vaginal. Os testes de mucoadesão mostraram que as formulações propostas contendo nanocápsulas poliméricas apresentaram maior adesividade em mucosa vaginal em comparação com a formulação composta somente de quitosana. Através do experimento de lavabilidade não foram encontradas diferenças significativas entre as formulações. No entanto, as técnicas de microscopia confocal e a quantificação após a extração de fluorescência a partir da mucosa demonstraram uma maior penetração de vermelho do nilo quando nanoencapsulado, especialmente em nanocápsulas catiônicas. As formulações desenvolvidas com base no veículo do hidrogel de quitosana e nanocápsulas poliméricas, especialmente as nanocápsulas catiônicas, demonstraram aplicabilidade para a entrega de substâncias hidrofóbicas pela via vaginal. Palavras-chave: quitosana, nanocápsuas, via vaginal, EUDRAGIT® RS100, EUDRAGIT® S100. / The vaginal route of administration might be an alternative for several treatments, either for local or systemic pharmacological effect. However, the permanence of the drug at the site of application and its expected effectiveness represent a challenge in the development of formulations. Thus, the objective of this work was to develop nanocapsules with cationic or anionic surface charge, containing or not nile red as a model of lipophilic substance, and to incorporate such particles into chitosan vehicle in order to increase the residence time of the formulation due to chitosan mucoadhesive properties. Several formulations prepared with increasing chitosan concentrations were analyzed in terms of pH and rheological behaviour in order to select the most suitable one for vaginal application. Gel formulations were produced with chitosan at 2.5% w/w, with or without nanocapsules. The adhesion (tensile stress test and washability profile) and penetration enhancement properties (confocal microscopy- CLSM and extraction followed by quantification) of the formuations, when applied on porcine vaginal mucosa, were evaluated. The nanocapsule suspensions presented adequate properties and pH values around 5.1 and 6.2. The chitosan formulation presented a characteristic viscosity and an acid pH (around 4.5), which is suitable for vaginal application. Mucoadhesion tests showed that the proposed formulations containing polymeric nanocapsules had higher adhesion to the vaginal mucosa in comparison with the formulation containing only chitosan. The washability evaluation showed no significant differences between the formulations. However, the confocal microscopy and the fluorescence quantification after extraction from the mucosa showed higher penetration of nile red when nanoencapsulated, especially into cationic-charged nanocapsules. The formulations developed, based on chitosan gel vehicle and polymeric nanocapsules, especially the cationic nanocapsules, demonstrated applicability for the vaginal delivery of hydrophobic substances.