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Ziel der vorliegenden Arbeit ist die photophysikalische Untersuchung Photosensibilisator-beladener Nanopartikel als Transportsysteme für aktives und passives Tumor-Targeting. Zu diesem Zweck wurden sowohl stationäre, als auch zeitaufgelöste spektroskopische Methoden angewandt. Der erste Teil beschäftigt sich mit der photophysikalischen Charakterisierung von Pheo-HSA-Nanopartikeln. Mittels stationärer und zeitaufgelöster Messungen konnte gezeigt werden, dass die Wechselwirkungen zwischen Phäophorbid a und den HSA-Nanopartikeln sehr stark ist. Diese Wechselwirkungen bewirken eine geringe Singulettsauerstoffquantenausbeute (0,07) in D2O verglichen mit dem von Phäophorbid a in Ethanol (0,52). Im Gegensatz dazu konnte nach der Inkubation in Jurkat- und HT-29-Zellen eine intrazelluläre Singulettsauerstoffgenerierung der Pheo-HSA-NPs nachgewiesen werden. Im zweiten Teil wurden mit den Photosensibilisatoren mTHPP and mTHPC beladene HSA- und PLGA-Nanopartikel untersucht. Es konnte gezeigt werden, dass die Photosensibilisator-Beladungsrate die photophysikalischen Eigenschaften der HSA- und PLGA-Nanopartikel stark beeinflusst. Für die HSA-Nanopartikel dominieren bei geringen Beladungsraten die Wechselwirkungen zwischen HSA und den Photosensibilisatormolekülen. Mit steigender Beladung spielen Wechselwirkungen zwischen den Photosensibilisatormolekülen eine zunehmende Rolle. Diese Wechselwirkungen verringern bei hoher Beladung der HSA-Nanopartikel die Generierung von Singulettsauerstoff. Auch für die PLGA-Nanopartikel konnte mit zunehmender Beladung ein verstärktes Singulettsauerstoffquenching nachgewiesen werden. Im dritten Teil dieser Arbeit wurden, für aktives Targeting von Tumorzellen, Oberflächenmodifizierte PLGA- und HSA-Nanopartikel untersucht. Die intrazellulären Singulettsauerstoffmessungen weisen auf eine erleichterte Aufnahme in Tumorzellen von Antikörper- und PEG-modifizierten HSA-Nanopartikeln in vitro hin. / The main goal of this PhD thesis is the photophysical investigation of biodegradable photosensitizer-nanoparticle carrier systems achieving passive and active tumour targeting strategies. For this purpose both steady state and time-resolved spectroscopic methods accompanied by data analysis were utilized. This work contains three main parts: First the photophysical properties of Pheo-HSA nanoparticles were compared to free pheophorbide a. Steady-state and time-resolved fluorescence experiments have already proved that the interaction between pheophorbide a and HSA nanoparticles is strong. This interaction leads to low singlet oxygen quantum yield (0.07) in D2O compared to free Pheo (0.52) in ethanol. But when incubated in Jurkat and HT-29 cell lines, Pheo-HSA nanoparticles have been proved to generate singlet oxygen inside cells. In the second part the well-known photosensitizers mTHPP and mTHPC were loaded to HSA- and PLGA- nanoparticles. It was found that the loading ratio determines the photophysical properties of both photosensitizer-loaded HSA and PLGA nanoparticles. For HSA nanoparticles, photosensitizer-nanoparticle interaction is the preferential mechanism in low loading ratio sample. But in high loading ratio sample, photosensitizer-photosensitizer interaction becomes the determining interaction. This interaction prevents singlet oxygen generation from high loading sample. For PLGA nanoparticles, high drug loading ratio also leads to a strong singlet oxygen quenching. At high drug loading ratio PLGA nanoparticles, some photosensitizer molecules may be localized deeply inside PLGA matrices and far away from surface. In the third part of this work, active tumour targeting behaviour achieved by surface modification of HSA and PLGA nanoparticles has been tested. Intracellular singlet oxygen measurement reveals that HSA nanoparticles, both with antibody and PEG surface modification have an enhanced targeting of tumour cells in vitro.

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Tuberculosis (TB) is the world’s most deadly infectious disease and causes enormous public health problems. The comorbidity with HIV and the rise of multidrug-resistant TB strains impede successful therapy through drug-drug interactions and the lack of efficient second-line treatments. The aim of this thesis was to support the improvement of anti-TB therapy through development of pharmacometric models, specifically focusing on the novel drug bedaquiline, pharmacokinetic interactions and methods for pooled population analyses. A population pharmacokinetic model of bedaquiline and its metabolite M2, linked to semi-mechanistic models of body weight and albumin concentrations, was developed and used for exposure-response analysis. Treatment response was quantified by measurements of mycobacterial load and early bedaquiline exposure was found to significantly impact the half-life of bacterial clearance. The analysis represents the first successful characterization of a concentration-effect relationship for bedaquiline. Single-dose Phase I studies investigating potential interactions between bedaquiline and efavirenz, nevirapine, ritonavir-boosted lopinavir, rifampicin and rifapentine were analyzed with a model-based approach. Substantial effects were detected in several cases and dose-adjustments mitigating the impact were suggested after simulations. The interaction effects of nevirapine and ritonavir-boosted lopinavir were also confirmed in patients with multidrug-resistant TB on long-term treatment combining the antiretrovirals and bedaquiline. Furthermore, the outcomes from model-based analysis were compared to results from conventional non-compartmental analysis in a simulation study. Non-compartmental analysis was found to consistently underpredict the interaction effect when most of the concentration-time profile was not observed, as commonly is the case for compounds with very long terminal half-life such as bedaquiline. To facilitate pooled analyses of individual patient data from multiple sources a structured development procedure was outlined and a fast diagnostic tool for extensions of the stochastic model components was developed. Pooled analyses of nevirapine and rifabutin pharmacokinetics were performed; the latter generating comprehensive dosing recommendations for combined administration of rifabutin and antiretroviral protease inhibitors. The work presented in this thesis demonstrates the usefulness of pharmacometric techniques to improve treatment of TB and especially contributes evidence to inform optimized dosing regimens of new and old anti-TB drugs in various clinical contexts.

L’hypoalbuminémie est une trouvaille fréquente chez le patient brulé mais sa relation avec la morbidité et mortalité n’a pas été bien établie. Objectif : Déterminer si l’hypoalbuminémie dans les premières 24 heures suivant l’admission est associée avec la dysfonction d’organes (mesurée avec le score SOFA) chez les patients présentant des brûlures graves. Méthodologie : Nous avons révisé les dossiers médicaux des patients adultes avec de brûlures de 20% ou plus de surface corporelle admis pendant les premières 24 heures à l’unité de grands brulés du CHUM entre les années 2008 et 2009. Nous avons utilisé un modèle de régression linéaire multivariée pour déterminer si l’hypoalbuminémie était un prédicteur indépendant de la dysfonction d’organes. Résultats : 56 sujets ont été analysés. L’analyse de régression linéaire multiple a montré qu’en contrôlant pour l’âge, le sexe, la surface corporelle brûlée et les brûlures par inhalation, l’hypoalbuminémie pendant les premières 24 heures suivant l’admission est un prédicteur indépendant de la dysfonction d’organes. Une concentration d’albumine ≤30g/L est aussi associée à une augmentation de la dysfonction d’organes [score SOFA au jour 0 (p = 0.005), jour 1 (p = 0.005), moyenne de la première semaine (p = 0.004)], mais n’est pas associée avec la mortalité (p = 0.061). Conclusions : L’hypoalbuminémie est associée avec la dysfonction d’organes chez les patients brulés. À la différence de facteurs non modifiables comme l’âge, le sexe, la surface corporelle brûlée et la présence de brûlures par inhalation, la correction de l’hypoalbuminémie peut être un objectif intéressant pour un futur essai clinique. / Hypoalbuminemia is a common finding in burn patients, but its association with increased morbidity and mortality has not been well established. Objective: We have assessed if hypoalbuminemia in the first 24 hours of admission is associated with organ dysfunction in patients with severe burns. Methods: For a two year period (2008-2009), we reviewed the chart of burn adult patients with a total body surface area ≥ 20% admitted in our unit in the first 24 hours following the burn injury. A multiple linear regression analysis was conducted to assess hypoalbuminemia as an independent predictor of organ dysfunction. Results: 56 patients were analyzed. Multiple linear regression analysis showed that hypoalbuminemia in the first 24 hours of admission was an independent predictor of organ dysfunction. Serum albumin concentration ≤ 30 g/L was associated with a two-fold increased in organ dysfunction [SOFA scores at day 0 (p = 0.005), day 1 (p = 0.005) and first week mean values (p = 0.004), but not with mortality (p = 0.061). Conclusion: Hypoalbuminemia is associated with organ dysfunction in burn patients. Unlike unmodifiable predictors such as age, burn surface and inhalation burn, correction of hypoalbuminemia might represent a goal for a future trial in burn patients.

La dégradation par fretting-corrosion des prothèses de hanche cimentées est l’une des principales causes de réintervention chirurgicale. L’étude du fretting-corrosion est effectuée entre un acier inoxydable 316L, matériau utilisé pour les tiges fémorales, et un polymère PMMA, matériau modèle du ciment chirurgical, dans différentes solutions, plus ou moins proches du liquide physiologique.L’étude a d'abord été réalisée à potentiel libre (OCP) pour être proche des conditions in vivo. L’influence des chlorures et le rôle de l’albumine, principale protéine du liquide physiologique, sur la dégradation du 316L et de sa couche passive sont ainsi évalués.Pour pouvoir obtenir des informations sur le courant de corrosion, des essais sont effectués à potentiel imposé. Le potentiel choisi est proche de la valeur de potentiel pendant fretting : E = -400 mV(ECS). Ce potentiel permet d’observer la transition entre courant cathodique et courant anodique en fonction de la force ionique. Lors d’un essai de fretting-corrosion, l’albumine joue le rôle d’inhibiteur de corrosion.La dégradation du 316L par fretting-corrosion est une combinaison entre l’usure corrosive, due au milieu physiologique contenant des chlorures, et l’usure mécanique. Il existe un terme de synergie entre usures corrosive et mécanique. L’influence de la force ionique et de l’albumine sur ce terme de synergie est aussi quantifiée.La forme de la trace d’usure en “W”, caractéristique du fretting-corrosion, est due à un gradient de pH et à un mécanisme de corrosion proche de la corrosion caverneuse. Une étude à pH global imposé a permis d’estimer les valeurs probables de pH dans et à une courte distance de la zone d’usure. / In case of total hip joint cemented prosthesis, one of the most important causes of reintervention is the degradation induced by fretting-corrosion. The study of fretting-corrosion mechanism is conducted between a 316L stainless steel, the same material as the femoral stem, and a polymer PMMA, a model material for bone cement, in several solutions, more or less close to physiological liquid.First, the study was investigated at Open Circuit Potential (OCP), to be close to the in vivo conditions. The influence of chlorides and the role of albumin, the principal protein in the physiological liquid, on the 316L and its passive layer degradation are evaluated.To obtain some information on corrosion current, experiments are investigated at applied potential. The chosen potential is close to the value of the potential during fretting: E = -400 mV(SCE). Besides, this potential is a threshold potential for anodic and cathodic transition of current as a function of ionic strength. One of the key points is the role of albumin as a corrosion inhibitor in the degradation by fretting-corrosion.The 316L degradation by fretting-corrosion is a combination between corrosive wear, due to the physiological liquid which contains chlorides, and mechanical wear. There is a synergy term between corrosive and mechanical wears. The influence of ionic strength and albumin concentration on this synergy term is also quantified.The shape of the worn area in “W”, typical of fretting-corrosion, is due to a pH gradient and a corrosion mechanism close to crevice corrosion. A study where the global pH of the solution is fixed allows estimating values of pH in and at a short distance from the worn area.

Les études épidémiologiques ont montré qu'une consommation régulière en tomate et ses produits dérivés de tomate permet de lutter contre diverses pathologies dégénératives associées notamment au stress oxydant (maladies cardiovasculaires, cancers etc..). Les effets bénéfiques pourraient être dus au lycopène pigment rouge de la tomate et/ou à ses métabolites qui interviendraient dans ce processus soit de part leurs capacités antioxydantes, soit au travers de la régulation de l’expression de gènes. Dans ce contexte, quatre familles de molécules dérivées du lycopène, pouvant être des métabolites potentiels, ont été ciblées pour la synthèse organique : les apo-11-lycopénoïdes, les apo- 14’-lycopénoïdes, les apo-12’-lycopénoïdes et les apo-10’-lycopénoïdes. Chacune des familles a été synthétisée, via des réactions de couplages tels que Wittig et Horner-Wadsworth-Emmons, avec quatre fonctions chimiques terminales : ester, acide carboxylique, alcool et aldéhyde. Par la suite deux types de propriétés physico-chimiques des composés synthétisés ont été étudiés : mesure du pouvoir antioxydant dans des conditions expérimentales mimant un stress oxydant dans le compartiment gastro-intestinal (inhibition de la peroxydation lipidique initiée par la metmyoglobine en milieu micellaire) et une étude d’interaction avec l'albumine de sérum humain, protéine impliquée dans le transport des acides gras dans le plasma. / Epidemiological studies have shown that regular consumption of tomatoes and its derived products participate to the prevention of degenerative pathologies associated with oxidative stress (cardiovascular disease, cancers). The beneficial effects could come from lycopene and/or its metabolites. In this context four families of lycopene derived compounds, mimicking possible metabolites, were targeted to be synthesized: the apo-11- lycopenoids, the apo-14’- lycopenoids, the apo-12’- lycopenoids and the apo-10’-lycopenoids. For each family, Wittig and Horner- Wadsworth-Emmons coupling reaction were used and four different ending functions were obtained: ester, carboxylic acid, alcohol and aldehyde. Then two physico-chemical properties were studied: antioxidant effect mimicking oxidative stress in the gastro-intestinal tract (inhibition of lipidic peroxidation initiated by metmyoglobin protein in micellar medium) and study of the interaction with human serum albumin, a protein involved in the transport of fatty acid in the plasma.

Estudos anteriores mostram que o polímero Nafion® pode causar deslocamento do equilíbrio conformacional de proteínas em valores de pH que não o fisiológico. Nesse sentido, o Nafion® não só pode ser utilizado como uma sonda interessante para estudos estruturais de proteínas, mas, também, é importante entender seu papel na conformação da proteína. Portanto, a Albunina do Soro Bovino (BSA) foi escolhida como modelo para o estudo dos efeitos do Nafion® na conformação helicoidal de proteínas. A finalidade deste trabalho é entender as alterações na conformação e vizinhanças aromáticas da BSA, na faixa de pH de 2 a 12, na presença e ausência de Nafion®, que pode também revelar o papel do polímero na exposição dos aromáticos e nos processos de transferência de energia. As alterações da estrutura secundária foram medidas por Dicroísmo Circular e os espectros de fluorescência no estado estacionário foram usados para analisar as mudanças nas vizinhanças dos aromáticos. Os resultados mostraram a diminuição discreta do conteúdo helicoidal da conformação da BSA na região extremamente básica, pH 11 em relação à conformação em pH 7. Já na região ácida, pH 2, embora haja considerável diminuição do conteúdo helicoidal, a BSA ainda mantém quase 50% de sua conformação secundária regular. Em relação aos ambientes dos aromáticos triptofano e tirosina, a eficiência quântica da emissão de fluorescência diminui em regiões ácidas e básicas, indicando que, nessas estruturas, os aromáticos encontram-se em restrição conformacional em relação ao observado na proteína nativa. Estes resultados apontam para a mudanças na conformação da BSA em ambas as regiões: ácidas e básicas, incluindo mudanças das estruturas secundárias e nas vizinhanças dos aromáticos. A adição do Nafion®, por outro lado, acentua o deslocamento para o azul e diminuição da exposição dos aminoácidos, tanto em solução quanto em estado sólido. A estrutura secundária da proteína é completamente modificada pelo polímero na região ácida, e esta conformação é mantida nas regiões neutra e básica, sugerindo que o Nafion® não estabiliza estruturas helicoidais / Previous studies have shown that Nafion® can disturb the conformational equilibrium of some proteins when at pH other than physiological ones. In this sense, Nafion® can used to study protein conformation, but is also important to understand its interaction with the proteins. In this work, Bovine Serum Albimun (BSA) was chosen as a model to understand the modifications caused by Nafion® at helicoidal proteins conformation. More specifically, the aim encloses the understanding of changes in BSA secondary conformation and aromatic vicinities, at pH range from 2 to 12, in the Nafion®s presence and absence. Secondary changes were measured by Circular Dichroism and steady-state fluorescence was used to study the aromatic vicinities. Results have shown small differences at helix content in the extremely basic pH (pH 11) when compared to BSA conformation at pH 7 (native one). At pH 2, on the other hand, although a decreasing in helical content was observed, BSA was able to keep almost 50% of secondary regular conformation. Regarding the aromatic vicinities (tryptophans and tyrosines) the fluorescence emission quantum eficience decreased in both regions (acid and basic), suggesting that the aromatics in these conformations are found in a more restrict environment. Nafion®, when added, promoted a decreasing in aromatic exposition, both in solution and solid state, while the secondary structure is completelu modified by its presence in all pH range, suggesting that helical conformations are not stabilized by Nafion®

The interaction between two proteins into salt aqueous solutions is investigated throughout this thesis. Experiments, modeling and molecular simulations were carried out to get a better understanding of the phenomenon. Bovine serum albumin was used as a model protein. An analytical expression for the structure factor for globular proteins in aqueous solution is presented in this work. This expression was obtained considering an intermolecular potential given by the sum of a hard core, a van der Waals attractive and a screened Coulomb contribution. Experimental data of Small Angle X-Ray Scattering for bovine serum albumin in aqueous solutions containing sodium salts at different protein concentrations and pH values are also presented. The expression developed for the structure factor describes accurately these experimental data provided a dependence of the attractive parameter on protein concentration is established. An expression for the osmotic pressure was derived from the structure factor. With attractive parameters adjusted from X-ray scattering data, the osmotic pressure of bovine serum albumin aqueous solutions could be predicted with very good agreement with experimental data. A derivation of the thermodynamic potentials, such as the chemical potential, using the new osmotic equation of state is presented. Applying the phase equilibrium criterion, the fluid-fluid phase equilibrium for bovine serum albumin in salt aqueous solution was calculated. Although such separation was not experimentally observed at the isoelectric point, it was indeed experimentally observed for a pH value below the isoelectric point. The predictions seem to be valuable to discuss how ion specificity affects the phase diagram of proteins. To apply molecular dynamic techniques to simulate how proteins interact to each other in salt aqueous solutions, two new coarse-grained force fields are proposed. The first one, meant for sodium sulfate aqueous solution, avoids the unphysical association observed for non-polarizable atomistic force fields; and allows the prediction of thermodynamic and dynamic properties. The second one, meant for bovine serum albumin in aqueous solution, is used as a new strategy to evaluate the scattering form factor of proteins as a low resolution technique for protein structure prediction. / Nesta tese apresenta-se uma investigação sobre a interação entre duas proteínas em soluções aquosas salinas. Experimentos, modelagem e simulações moleculares foram realizadas para conseguir um melhor entendimento do fenômeno. Albumina de soro bovina foi usada como proteína modelo. Uma expressão para o fator de estrutura de proteínas globulares em solução aquosa é apresentada neste trabalho. Esta expressão foi obtida considerando-se um potencial intermolecular dado pela soma de um núcleo duro, uma contribuição atrativa tipo vander Waals e uma contribuição de potencial coulômbico blindado. Dados experimentais de espalhamento de raios-X a baixos ângulos para a albumina de soro bovino em soluções aquosas contendo sais de sódio com diferentes concentrações de proteína e valores de pH também são apresentados. A expressão desenvolvida para o fator de estrutura descreve com precisão estes dados experimentais, desde que uma dependência entre o parâmetro atrativo com a concentração de proteína seja estabelecida. Uma expressão para a pressão osmótica foi derivada do fator de estrutura. Com parâmetros atrativos ajustados aos dados de espalhamento de raios-X, a pressão osmótica da albumina de soro bovino em solução aquosa pôde ser predita com grande correlação com os dados experimentais. Uma derivação dos potenciais termodinâmicos usando a nova equação osmótica de estado é apresentada. Aplicando o critério de equilíbrio de fases, foi possível calcular o equilíbrio fluido-fluido para a albumina de soro bovino em solução aquosa. Embora tal separação não tenha sido observada experimentalmente em um pH igual ao ponto isoelétrico, ela foi de fato observada experimentalmente para um valor de pH menor do que o ponto isoelétrico. As predições parecem ser valiosas para discutir como a especificidade iônica afeta o diagrama de fases de proteínas. De modo a avaliar como proteínas interagem umas com as outras usando técnicas de dinâmica molecular, dois novos campos de força coarse-grained são propostos. O primeiro, para o sulfato de sódio em solução aquosa, evita a associação não-física que é observada para campos de força atomísticos não-polarizáveis. Este modelo é capaz de prever propriedades dinâmicas e termodinâmicas. O segundo, para a albumina de soro bovino em solução aquosa, é usado como uma nova estratégia para avaliar o fator de forma de espalhamento de proteínas como uma ferramenta de baixa resolução na predição de estruturas proteicas.

Resumo O presente trabalho teve como objetivo sintetizar e caracterizar um novo complexo trinuclear de rutênio, [Ru3O(CH3OO)6(3-pic)2(NO)]+, através de rotas sintéticas previamente descritas na literatura para complexos análogos. Para a obtenção deste complexo, foram necessárias cinco etapas de síntese, cada qual gerando o precursor da etapa seguinte. Os complexos precursores [Ru3O(CH3OO)6(3-pic)3]+, [Ru3O(CH3OO)6(3-pic)2(CO)] e [Ru3O(CH3OO)6(3-pic)2(H2O)]+, foram caracterizados por técnicas espectroscópicas e voltamétricas. Para o complexo de interesse, [Ru3O(CH3OO)6(3-pic)2(NO)]+, além da caracterização por técnicas espectroscópicas e voltamétricas, foram realizados estudos de fotólise e de interação com albumina de soro humano (HSA). Pelas técnicas espectroscópicas, pudemos ratificar as estruturas propostas tanto para o nitrosilo quanto para os precursores estudados. Para a caracterização do complexo [Ru3O(CH3OO)6(3-pic)2(NO)]+, utilizando a técnica de espectroscopia de infravermelho verificamos a coordenação do ligante NO ao centro metálico [Ru3O], devido ao estiramento característico deste ligante. Os espectros de absorção UV-vis e RMN mostraram que existe uma forte interação entre o elétron desemparelhado do centro metálico e o elétron do ligante NO. Pelos estudos de voltametria cíclica, observamos que os processos redox envolvendo o ligante NO, são compartilhados com o centro metálico. A fotólise do complexo [Ru3O(CH3OO)6(3-pic)2(NO)]+, mostrou-se eficiente, uma vez que a liberação fotoinduzida do NO ocorreu na região do visível e em pH fisiológico. Pelos estudos de supressão de fluorescência, observamos que o complexo realmente interage com a HSA na proporção de 1:1, na região em que se encontra o resíduo de triptofano. / The aim of the present study was synthesize and characterize a new trinuclear ruthenium complex, [Ru3O(CH3OO)6(3-pic)2(NO)]+, via synthetic routes previously described in the literature for analogous complexes. To obtain this complex, it took five synthetic steps, each one yielding a precursor for the next step. The precursor complexes [Ru3O(CH3OO)6(3-pic)3]+, [Ru3O(CH3OO)6(3-pic)2(CO)] and [Ru3O(CH3OO)6(3-pic)2(H2O)]+, were characterized by spectroscopic and voltammetric techniques. For the complex of interest, [Ru3O(CH3OO)6(3-pic)2(NO)]+, in addition to characterization by spectroscopic and voltammetric studies, it was carried out photolysis and interaction with human serum albumin. By spectroscopic techniques, we could confirm the proposed structures for both the nitrosyl and precursors. For characterization of the complex [Ru3O(CH3OO)6(3-pic)2(NO)]+, infrared spectroscopy allowed us to verify that the ligand coordination to the metal center [Ru3O], due to the characteristic stretching band of that ligand. The absorption spectra of UV-vis and NMR showed that there is a strong interaction between the unpaired electron of the metal center and the NO ligand. By cyclic voltammetry studies, we observed that the redox processes involving the NO ligand are shared with the metal center processes. The photolysis of the complex [Ru3O (CH3OO) 6 (3-pic) 2 (NO)] +, was efficient, since the photoinduced release of NO occurred in the visible region and at physiological pH. By fluorescence quenching studies, we observed that the complex actually interacts with the HSA in a 1:1 ratio, in the region which is the residue of tryptophan.