Rozprawy doktorskie na temat „PDTB”

El progresivo aumento del deporte en el último tiempo ha convertido a esta actividad en una real preocupación de los Estados y en objeto de políticas de gobierno destinadas a consolidar su práctica y la actividad física en la población como parte de su cultura y de su desarrollo social. Dentro de esta realidad, es necesario crear espacios adecuados para el desarrollo de este tipo de actividades, recogiendo las inquietudes de la época y cuya utilidad sea significativa y que a su vez responda a las expectativas de la población. Por ello, la propuesta enunciada en el programa de este proyecto pretende superar los problemas que acusa la población de este sector en cuanto a actividad física o deportiva, con el consiguiente deterioro en salud y calidad de vida.

PDT, psykodynamisk psykoterapi, och KBT, kognitiv beteendeterapi, är idag de dominerande psykoterapiinriktningarna i Sverige. Metoderna har i olika skeden rekommenderats av myndigheter och meningsmotsättningar om vilken metod som är mest lämplig har förekommit. Följande studie avsåg att utforska hur sex intervjuade KBT-psykoterapeuter samarbetar med sina PDT-kollegor och utifrån patienters behov eventuellt överväger PDT. En induktiv tematisk analys tillämpades där psykoterapeuterna beskrev upplevelsen av samarbete med PDT-terapeuter i termer av ett positivt dynamiskt utbyte samtsom en negativ upplevelse av fördomsfullt bemötande från PDT-terapeuter och mindre metodologiskt utbyte. Bilden av PDT som komplementär metod hos de intervjuade KBT-psykoterapeuterna speglade ambivalens kring PDT:s effektivitet och användbarhet. Alliansens kvalitet beskrevs som avgörande för om byte sker till PDT-kollega under pågående KBT-behandling. Studiens resultat jämfördes med en tidigare enkätstudie i vilken gruppsykologiska mekanismer antagits påverka samarbetet mellan terapeuter av olika metodinriktning.En tentativ beskrivning ges av processen bakom dessa gruppmekanismer.

La photothérapie dynamique (PDT) est un nouveau traitement n’induisant potentiellement pas de mutation et utilisable pour lutter contre le rétinoblastome. Les dérivés de porphyrine utilisés comme Ps dans la Thérapie PhotoDynamique (PDT) sont largement étudiés depuis la naissance du premier Ps de synthèse (l’HpD). Une limitation importante de la PDT provient de la faible profondeur (confinée près de la surface) de pénétration de la lumière (λ <700 nm) employée pour l'excitation du Ps. Afin de fournir une énergie d’activation suffisante pour produire l’oxygène singulet dans la fenêtre thérapeutique entre 700-1300, le processus d'absorption à deux photons a été proposé. Les Ps excitables par l’absorption simultanée de deux photons conduisent au concept de PDT à deux photons (PDT-ADP). Ce processus ayant une faible probabilité, son application demande le développement de nouveaux Ps avec une section efficace importante. Une autre limitation de la PDT est la faible sélectivité et spécificité des Ps actuels pour les cellules tumorales. Un ciblage actif de récepteurs membranaires spécifiques des cellules tumorales représente une solution possible. Il a été rapporté que des récepteurs de type lectine reconnaissant certains sucres sont surexprimés par des cellules malignes. Nous présenterons la synthèse et les résultats photocytotoxiques de dimères dissymétriques de porphyrine P-Y-P', inspirée d'études précédentes du laboratoire, privilégiant l’introduction de trois chaînes para-phénoxy-diéthylène glycol mannose sur trois des positions méso de porphyrines optimisés pour l’absorption à deux photons et vectorisés vers des lectines membranaires.Pour contourner le problème de solubilité des porphyrines dimères en milieu aqueux et améliorer l’internalisation de Ps dans les cellules tumorales, nous avons analysé le comportement interfacial des porphyrines dimères à l'interface air-tampon, étudié leur incorporation dans les liposomes à l’aide de la technique de la fluorescence, et évalué l’interaction entre porphyrin dimères et la Concanavaline A (une lectine extraite de Canavalia ensiformis qui reconnaît de manière spécifique l'alpha-D-mannose)
Photodynamic therapy (PDT) is a new potential treatment against retinoblastoma, which doesn’t induce mutations. The porphyrin derivatives used as Ps in PDT are widely studied since the birth of the first synthesis Ps (HpD). An important limitation of PDT comes from low penetration of light (λ<700 nm) used for excitation of the Ps. In order to provide enough energy to enable production of singlet oxygen in the phototherapeutic window between 700-1300, the absorption of two relatively low-energy photons simultaneously has been proposed. Ps excited by simultaneous absorption of two photons leads to the concept of two-photon PDT (TPE-PDT). This process has very low probability; its application in the PDT needs develop new Ps with intensive cross section. Another limitation is the low selectivity and specificity of current Ps for tumor cells. Active targeting to appropriate receptors expressed at the tumor cells give a possible solution. It has been reported that the lectin-like receptors recognizing certain sugars are overexpressed in malignant cells. We will present the synthesis and in vitro photocytotoxical results of asymmetric porphyrin dimers P-Y-P', inspired by previous studies of our laboratory, introducing three para-phenoxy-diethylene glycol mannose chains at the meso positions of porphyrin core which optimized for two-photon absorption and targeted to membrane lectins. To circumvent the solubility problem of porphyrin dimers in aqueous medium and improve internalization of Ps into tumor cells, we analyzed the interfacial behavior of the porphyrin dimers in the air-buffer interface, studied the incorporation of porphyrin dimers in liposomes using the technique of fluorescence, and evaluated the interaction between porphyrin dimers and Concanavalin A (Canavalia ensiformis lectin derived from that specifically recognizes the alpha-D-mannose)

The Protein Data Bank (PDB) is the definitive electronic repository for experimentally-derived protein structures, composed mainly of those determined by X-ray crystallography. Approximately 200 new structures are added weekly to the PDB, and at the time of writing, it contains approximately 97,000 structures. This represents an expanding wealth of high-quality information but there seem to be few bioinformatics tools that consider and analyse these data as an ensemble. This thesis explores the development of three efficient, fast algorithms and software implementations to study protein structure using the entire PDB. The first project is a crystal-form matching tool that takes a unit cell and quickly (< 1 second) retrieves the most related matches from the PDB. The unit cell matches are combined with sequence alignments using a novel Family Clustering Algorithm to display the results in a user-friendly way. The software tool, Nearest-cell, has been incorporated into the X-ray data collection pipeline at the Diamond Light Source, and is also available as a public web service. The bulk of the thesis is devoted to the study and prediction of protein disorder. Initially, trying to update and extend an existing predictor, RONN, the limitations of the method were exposed and a novel predictor (called MoreRONN) was developed that incorporates a novel sequence-based clustering approach to disorder data inferred from the PDB and DisProt. MoreRONN is now clearly the best-in-class disorder predictor and will soon be offered as a public web service. The third project explores the development of a clustering algorithm for protein structural fragments that can work on the scale of the whole PDB. While protein structures have long been clustered into loose families, there has to date been no comprehensive analytical clustering of short (~6 residue) fragments. A novel fragment clustering tool was built that is now leading to a public database of fragment families and representative structural fragments that should prove extremely helpful for both basic understanding and experimentation. Together, these three projects exemplify how cutting-edge computational approaches applied to extensive protein structure libraries can provide user-friendly tools that address critical everyday issues for structural biologists.

A terapia fotodinâmica, do inglês photodynamic therapy (PDT) é uma fototerapia baseada na utilização de um fotossensibilizador (FS) na presença da luz em baixa intensidade em comprimento de onda ressonante à absorção do FS e que podem sensibilizar sistemas biológicos, gerando espécies reativas de oxigênio. Estudos mostram que a PDT apresenta um efeito letal em Candida albicans. O biofilme formado por C. albicans é a causa mais freqüente de infecções associadas a cateteres, possuindo uma comprovada resistência a antifúngicos, sendo que a remoção do cateter colonizado é quase sempre necessária. No entanto, poucos trabalhos na literatura relatam o comportamento e a resposta de C. albicans organizado em biofilme frente à PDT. Os objetivos deste trabalho foram desenvolver uma metodologia para formação in vitro de biofilme de C. albicans, verificar a sensibilidade do biofilme de C. albicans frente à terapia fotodinâmica antimicrobiana utilizando como FS o azul de metileno (AM) e a hipocrelina B:La+3 e analisar o biofilme através da Tomografia de Coerência Óptica (OCT) antes e depois da PDT. Para a formação do biofilme, foram confeccionados discos de silicone elastomérico oriundos de cateteres. Os fotossensibilizadores foram diluídos em solução de PBS, sendo que o AM teve duas concentrações diferentes testadas no biofilme: 100M e 1mM e a HBLa+3 somente uma de 10M. A irradiação de ambos os corantes com os microrganismos foi feita através de dois LEDs diferentes, um vermelho com = 630nm ± 20nm e outro azul com = 460nm ± 30nm. Foi realizada uma curva de fração de sobrevivência em função do tempo de irradiação de cada amostra com biofilme e de suspensão do microrganismo em formato de leveduras para verificar a susceptibilidade destes frente à PDT. As leveduras apresentaram 100% de redução em ambos os fotossensibilizadores, porém em tempos de irradiação diferentes (30s para a HBLa+3 e 6 min para o AM na concentração de 100M). Quando a terapia foi aplicada em biofilme, o AM em 100M não apresentou nenhuma redução significativa, enquanto que em concentração de 1mM houve redução de 100% após 6 min de irradiação. Já a HBLa+3 mostrou pouca redução em biofilme na concentração de 10M (menos de 1 log de redução. Concluímos que o microrganismo C. albicans se organizou em biofilme padronizado nos discos elastoméricos oriundos de cateteres e através do OCT, o biofilme medido apresentou 110m de espessura, informando uma mudança óptica ao ser submetido pela PDT com o AM 1mM.
Photodynamic therapy (PDT) is a phototherapy based on the use of a photosensitizer (PS) in the presence of low intensity light with resonant wavelength of absorption of the PS and biological systems that can raise awareness, generating reactive oxygen species. Studies show that PDT has a lethal effect on Candida albicans. The biofilm formed by C. albicans is the cause of infections associated with medical devices such as catheters, with a proven resistance to antifungal agents, and the removal of the catheter colonized almost always is necessary. However, few studies in literature report the behavior and response of biofilm organized by C. albicans against PDT. The aims of this study were to develop a methodology for in vitro biofilm formation of C. albicans, evaluate the sensitivity of the biofilm of C. albicans to antimicrobial photodynamic therapy using PS as the methylene blue (MB) and hypocrellin B: La+3 (HBLa+3) and analyze the biofilm by Optical Coherence Tomography (OCT). For biofilm formation, discs were made from elastomeric silicone catheters. The PS were dissolved in solution of PBS, and the MB had two different concentrations tested in the biofilm: 100M and 1mM; HBLa+3 only one of 10M. The irradiation of both dyes with the microorganism was done by two different LEDs, one with red emission at = 630nm ± 20nm and the other one blue emission at = 460nm ± 30nm. We performed a curve of survival fraction versus time of irradiation of each sample with biofilm and suspension of the microorganism in the yeast form to verify the susceptibility of the front PDT. The yeast showed 100% reduction using both PS, but at different times of irradiation (30s to HBLa+3 and 6 min for the MB at 100M). When the therapy was applied in biofilm, the MB 100M did not show any significant reduction, while at concentration of 1mM was reduced by 100% after 6 min of irradiation. The HBLa+3 biofilm group showed a lower reduction in the concentration of 10M in biofilm (less than 1 log reduction). OCT was performed for visualization and measurement of the thickness of the biofilm formed. The composition of the extracellular matrix of the biofilm polymer hindered the diffusion of PS inside, requiring higher concentrations of MB to disseminate it and to obtain satisfactory reduction for PDT. HBLa+3 group, in higher concentration, formed aggregates difficulting its use for PDT. We conclude that the organism C. albicans was organized in biofilms in a standardized way using elastomeric discs from catheters and the OCT showed that the biofilm measured 110m at thickness, showing an optical change when subjected to the PDT with MB 1mM.

Le phosphate-diphosphate de thorium (beta-PDTU) est actuellement considéré comme une matrice céramique potentielle en vue de l'immobilisation des actinides en formation géologique profonde. Les études réalisées au cours de ce travail reposent sur la synthèse du phosphate-hydrogénophosphate de thorium hydraté (PHPTH) en tant que précurseur du beta-PDT. La structure cristalline du PHPTH a été élucidée puis le mécanisme conduisant à sa transformation en beta-PDT a été établi.Ce dernier met en jeu plusieurs composés intermédiaires dont le alpha-PDT, forme monoclinique jusqu'alors inconnue. Par ailleurs l'existence d'une solution solide entre le PHPTH et le PHPUH a été démontrée. Les conditions expérimentales de frittage conduisant à une densification optimale des compacts ont été déterminées. La densité relative est toujours comprise entre 95 et 100% de la valeur calculée et une nette amélioration de l'homogénéité a été constatée. Dans le butd'incorporer simultanément des actinides tri et tétravalents, le procédé basé sur la précipitation de précurseurs cristallisés puis sur leur frittage a également été appliqué à l'élaboration de matériaux composites beta-PDTU/monazite.

A cápsula endoscópica é um instrumento revolucionário, que permite a realização de um exame livre de desconfortos para o paciente e possibilitou a técnica de endoscopia chegar a partes do sistema gastrointestinal que antes não era possível, como grande parte do intestino delgado. Este trabalho apresenta um módulo fotônico para realizar Terapia Fotodinâmica (PDT, Photodynamic Therapy) em cápsulas endoscópicas. A PDT é uma forma de tratamento para lesões que envolve a administração prévia de um medicamento fotossensível (PS, Photosensitizer) e posterior ativação com irradiação de luz em um comprimento de onda (λ) específico. Geralmente para a ativação do PS utiliza-se λ na região vermelha do espectro visível, pois a penetração nos tecidos é elevada. Células de adenocarcinoma gástrico humano (AGS, Adenocarcinoma Gastric of Stomach) foram preparadas com ácido 5-aminolevulínico (5-ALA) que é um Pró-fármaco precursor do agente fotossensível Protoporfirina IX (PpIX) com máximo de absorção nos 635 nm. Para realizar a irradiação com luz vermelha nas células, 16 LEDs do modelo LRQ396-P1Q2-1 da Osram Opto Semiconductors com dimensões muito reduzidas foram selecionados, caracterizados, montados em uma PCB (Printed Circuit Board) e alimentados por um Arduino Mega 2560. Este sistema de iluminação foi eficaz em seu propósito e causou morte celular nas células submetidas ao tratamento com o ALA em uma concentração de 2 milimol por litro e dose de luz em 5 Joules por centímetro quadrado que resulta em uma exposição de 49 minutos. Com o objetivo de se construir um módulo para controle de iluminação em cápsulas endoscópicas, foi desenvolvido em tecnologia CMOS 0.7 μm da ON Semiconductor um dispositivo capaz de controlar a intensidade dos LEDs utilizando PWM (Pulse Width Modulation). Unindo este componente com os LEDs selecionados é perfeitamente possível construir o módulo e inseri-lo CEs para realizar PDT em locais do trato gastrointestinal (GI) não acessíveis pela endoscopia convencional.
The endoscopic capsule is a revolutionary instrument that gives a free examination of discomforts for patient and allows the endoscopic technique to reach parts of the gastrointestinal system that were previously not possible, such as a large part of the small intestine. This work presents a photonic module to perform Photodynamic Therapy (PDT) in endoscopic capsules. PDT is a form of treatment for injuries involving prior administration of a photosensitive drug (PS, Photosensitizer) and subsequent activation with light irradiation at a specific wavelength (λ). Usually for PS activation, λ is used in the red region of the visible spectrum, because the tissue penetration is high. Human gastric adenocarcinoma cells (AGS) were prepared with 5-aminolevulinic Acid (5-ALA) which is a Pro-drug precursor of the photosensitive agent Protoporphyrin IX (PpIX) with maximum absorption at 635 nm. To perform red light irradiation on the cells, 16 LEDs, model LRQ396-P1Q2-1 of the Osram Opto Semiconductors with very small dimensions were selected, characterized, mounted on a PCB (Printed Circuit Board) and powered by an Arduino Mega 2560. This system was effective in its purpose and caused cell death in cells treated with ALA at a concentration of 2 mMol/L and dose of light at 5 J/cm² resulting in a 49 minutes exposure. With the objective of build a module for control of lighting in endoscopic capsules, a device able to controlling the intensity of the LEDs using PWM (Pulse Width Modulation) was developed in CMOS technology 0.7 μm of the ON Semiconductor. Combining this component with the selected LEDs, it is perfectly possible to construct the module and insert in CEs to perform PDT at sites of the gastrointestinal tract (GI) not accessible by conventional endoscopy.

Doutoramento em Quimica
O trabalho apresentado nesta dissertação descreve estudos desenvolvidos segundo três metodologias para a funcionalização de meso-tetra-arilporfirinas; uma delas baseada na funcionalização com diazo compostos, outra na reacção de Suzuki-Miyaura e a terceira na reacção de metátese. O presente documento é composto por quatro partes distintas. Na primeira parte são feitas considerações gerais sobre propriedades e aplicações de macrociclos porfirínicos. Na segunda parte é apresentado o trabalho realizado na funcionalização de porfirinas com diazo compostos através de reacções de inserção com um catalisador de ródio(II). Este estudo envolveu a preparação de duas porfirinas com grupos O-H e N-H nas posições para dum substituinte meso-fenilo. Os estudos iniciaram-se com a reacção de 5-(4-hidroxifenil)-10,15,20-trifenilporfirinatozinco(II) com diazoacetato de etilo na presença de Rh2(OAc)4. Obteve-se o produto esperado de inserção com bons rendimentos. A reacção de 5-(4-aminofenil)- 10,15,20-trifenilporfirinatozinco(II) com esse diazo composto na presença de Rh2(OAc)4 mostrou um perfil diferente do da reacção com o outro porfirinato. Foram obtidos três produtos, a saber: produto de bis-inserção e dois outros contendo o grupo funcional amida. Este capítulo descreve ainda a síntese de glicoporfirinas e de glicoclorinas através de reacções de inserção e de ciclopropanação envolvendo, respectivamente, os complexos 5-(4-hidroxifenil)- 10,15,20-trifenilporfirinatozinco(II) e 5,10,15,20- tetraquis(pentafluorofenil)porfirinatozinco(II) com α-diazoacetatos com substituintes sacarídicos. Foram usados, como catalisadores, Rh2(OAc)4 e CuCl, respectivamente, na reacção de inserção e na de ciclopropanação. Tendo em vista a sua aplicação em PDT, os novos produtos foram descomplexados e a unidade glicosídica foi desprotegida. Estudos de geração de oxigénio singuleto mostraram que os compostos obtidos são bons geradores dessa espécie citotóxica. Foram também realizados estudos de avaliação da actividade fotodinâmica em células humanas tumorais HeLa e em células humanas saudáveis HaCaT. Estudos de viabilidade celular, após tratamento fotodinâmico, mostraram que as glicoclorinas são mais eficazes na fotoinactivação das células tumorais. Apenas o derivado da clorina com a unidade de galactose mostrou selectividade para a linha celular tumoral, inibindo o crescimento desta e não causando efeito significativo na linha celular saudável. Este fotossensibilizador não foi tóxico na ausência de luz e depois do tratamento fotodinâmico e em condições sub-letais, provocou vacuolização citoplasmática e retracção pronunciada nas células tumorais, enquanto que nas células saudáveis os danos sofridos foram escassos. Nos estudos de localização celular este fotossensibilizador localizou-se na membrana citoplasmática e nos lisossomas tanto nas células HeLa como nas HaCaT. Na terceira parte deste trabalho são descritos os resultados obtidos nos estudos de funcionalização de porfirinas através da reacção de Suzuki-Miyaura. O complexo 2-(4,4,5,5,-tetrametil-1,3,2-dioxaborolan-2-il)-5,10,15,20- tetrafenilporfirinatozinco(II) foi o composto escolhido como reagente de partida. Usando a reacção de Suzuki foi possível sintetizar complexos β-bromoarilporfirínicos em bons rendimentos. Estes complexos foram sujeitos à reacção de Suzuki com pinacolborano, tendo sido possível obter os produtos de acoplamento esperados e ainda derivados diméricos de complexos porfirínicos ligados por unidades fenileno. Esta metodologia reacional, catalisada por paládio, permitiu ainda sintetizar novos derivados quinolona-porfirina através da reacção do complexo porfirínico β-borilado anteriormente referido com bromo-quinolonas Nsubstiuídas com grupos alquílicos e glicosídicos. Deste modo foram obtidos derivados quinolona-porfirina em bons rendimentos. Com vista a realizar estudos de potencial aplicação em PACT, foram hidrolisados os grupos éster da unidade de quinolona e clivados os grupos protectores das unidades glicosídicas desses derivados. No estudo por MS Tandem (MS/MS) foi possível verificar que os isómeros dos derivados quinolona-porfirina sofrem as mesmas vias de fragmentação, confirmando as suas estruturas análogas. Foi possível ainda distinguir os derivados em que a unidade de porfirina está ligada à quinolona através da posição C-6 da quinolona daqueles em que a unidade de porfirina está ligada à quinolona através da sua posição C-7. Os estudos de geração de oxigénio singuleto mostraram que as porfirinas β- substituídas com unidades de quinolona são melhores geradoras desta espécie citotóxica do que a tetrafenilporfirina e que a eficiência que têm em gerar essa espécie é afectada pela posição da ligação entre a porfirina e a quinolona, assim como com o tipo do N-substituinte da quinolona. As bases livres dos conjugados quinolona-porfirinatos foram testadas como fotossensibilizadores em PACT em células do parasita Leishmania Braziliensis, tendo-se observado que estes compostos têm capacidade para fotoinactivar este parasita. Na quarta parte deste trabalho são descritos os resultados dos estudos de funcionalização de porfirinas através da reacção de metátese com catalisador de Grubbs, visando a obtenção de novos macrociclos com grupos triazolilo. Verificouse que a eficiência da reacção de metátese cruzada entre 2-vinil-5,10,15,20- tetrafenilporfirinatozinco(II) e 4-vinil-1,2,3-triazóis N-substituídos dependia das condições reaccionais e do triazol usado em cada caso. Foi possível ainda concluir que o impedimento estéreo das espécies envolvidas é um dos responsáveis pelo ciclo catalítico prosseguir por uma via secundária, originando uma espécie de ruténio pouco eficiente como catalisador. Este estudo foi estendido à reacção dos 4-vinil-1,2,3-triazois seleccionados com 2-butadienil-5,10,15,20- tetrafenilporfirinatoniquel(II); em cada caso foi possível obter uma mistura de isómeros dos derivados triazol-porfirina, geralmente em maiores rendimentos globais do que na reacção com 2-vinil-5,10,15,20-tetrafenilporfirinatozinco(II). Este facto será indicativo de que a reacção de metátase com esse derivado porfirínico segue, predominantemente, a via mais eficiente.
The work presented in this dissertation reports studies carried out according three different methodologies for the functionalization of meso-tetra-arylporphyrins: one is related with the functionalization with diazo compounds, another one considers the Suzuki-Miyaura approach and the third one the metathesis reaction. This document has four parts. The first one relates the main properties and applications of porphyrin derivatives. The second part reports results on studies carried out for the functionalization of porphyrins with diazo compounds through insertion reactions catalyzed by a rhodium-based catalyst. This study involved the preparation of two porphyrins with OH and NH groups in the p-position of one meso-phenyl substituent of those macrocycles. Reaction of 5-(4-hydroxyphenyl)-10,15,20- triphenylporphyrinatozinc(II) with ethyl diazoacetate in the presence of Rh2(OAc)4 gave rise to the expected insertion product in good yields. In the reaction of 5-(4- aminophenyl)-10,15,20-triphenylporphyrinatozinc(II) with such diazo compound in the presence of Rh2(OAc)4 a different profile was observed. Three products were obtained: one corresponding to the bis-insertion product and the other two having each one an amide function. This part of the thesis also describes the synthesis of glycoporphyrins and glycochlorins through insertion and cyclopropanation methodologies, respectively, in reactions with 5-(4-hydroxyphenyl)-10,15,20- triphenylporphyrinatozinc(II) and 5,10,15,20- tetrakis(pentafluorophenyl)porphyrinatozinco(II) with carbohydrate substituted α- diazoacetates. As catalysts were used Rh2(OAc)4 for the insertion reactions and CuCl for the cyclopropanation ones. Having in mind their potential applications in PDT, the new derivatives obtained were demetallated and the sugar units were deprotected. Studies on the singlet oxygen generation by the new compounds have shown that these derivatives are good generators of that cytotoxic species. The new compounds were tested as photosensitisers in PDT, on a cancer cellular line, HeLa and on a normal cell line, HaCaT. Studies on cell viability after PDT treatment have shown that glycochlorins are more effective in the photoinactivation of the tumor cell line. Only the galactose-chlorin derivative showed selectivity for HeLa cells, inhibiting the growth of this tumoral cell line and causing no significant effect on the normal cell line. This photosensitiser was not toxic in the absence of light and it caused cytoplasmatic vacuolization and pronounced retraction in tumoral cell line, while in HaCaT the damages were scarce. In the study of cellular localization, this photosensitiser was located in the cytoplasmic membrane and lysosomes in both HeLa and HeCat cells. The third part of this dissertation describes the results obtained on the functionalization of porphyrins under the Suzuki-Miyaura reaction conditions. The 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,10,15,20- tetraphenylporphyrinatozinc(II) was chosen for being used in this reaction. Using the Suzuki methodology it was possible to synthesize β-bromo-arylporphyrinic complexes in good yields. These brominated porphyrinates were used in the Suzuki reaction with pinacol borane; it was possible to obtain novel coupling product derivatives and also porphyrin dimers linked by phenylene units. This reaction catalyzed by palladium also allowed us to synthesize new quinoloneporphyrin derivatives by the reaction of the β-borylated porphyrinic complex with Nsubstituted bromo-quinolones with alkyl and glycosyl groups affording quinoloneporphyrin derivatives in good yields. It was also performed the hydrolysis of the ester groups of the quinolone moieties and the deprotection of the glycosidic units of the quinolone-porphyrin derivatives, having in mind their potential application in PACT. In the electrospray tandem mass spectrometry (MS/MS) of the quinoloneporphyrin derivatives it was possible to verify that isomeric porphyrin-quinolone derivatives give rise to similar fragmentation pathways, confirming their analogous structures. Due to the relative abundance of some peaks it was possible to distinguish between derivatives with the porphyrin moiety linked at C-6 of the quinolone unit from those having the porphyrin moiety linked to the 7-position of the quinolone moiety. Singlet oxygen studies have shown that all the β-substituted porphyrin-quinolones are better singlet oxygen generators than TPP and that the efficiency of these compounds to generate singlet oxygen is affected by the linkage position between the porphyrin and the quinolone moieties, and also by the type of the N-substituent of the quinolone group. The free bases of quinolone-porphyrin conjugates were tested as photossensitizers in PACT against the Leishmania Braziliensis parasite cells; it was observed that these compounds are able to photoinactivate such parasite. photoinactivate such parasite. In the fourth part of this document it is described the results obtained in the study of the functionalization involving triazolyl groups of porphyrin macrocycles by the metathesis reaction using the Grubbs catalyst. It was found that the reaction between 2-vinyl-5,10,15,20-tetraphenylporphyrinatozinc(II) and N-substituted 4- vinyl-1,2,3-triazoles are dependent on the reaction conditions and on the triazole. The best conditions found were used for the synthesis of new triazole-porphyrin derivatives. The study of the reaction mechanism has shown that the steric hindrance between the two species involved is a main reason for the catalytic cycle to continue on a path characterized by the poor efficiency. This study of the metathesis reaction was extended to the reaction of selected N-substituted 4-vinyl- 1,2,3-triazoles with 2-butadienyl-5,10,15,20-tetraphenylporphyrinatonickel(II) (a porphyrinic complex with less steric hindrance) affording a mixture of isomeric triazole-porphyrin derivatives, in higher global yields than the reactions with 2-vinyl- 5,10,15,20-tetraphenylporphyrinatozinc(II). This might indicate that the metathesis reactions with the former porphyrin follows, predominantly, the more efficient route.

Doutoramento em Química
This dissertation describes the synthesis and characterization of different phthalocyanine (Pc) derivatives, as well as some porphyrins (Pors), for supramolecular interaction with different carbon nanostructures, to evaluate their potential application in electronic nanodevices. Likewise, it is also reported the preparation and biological evaluation of interesting phthalocyanine conjugates for cancer photodynamic therapy (PDT) and microorganisms photodynamic inactivation (PDI). The phthalonitrile precursors were prepared from commercial phthalonitriles by nucleophilic substitution of -NO2, -Cl, or -F groups, present in the phthalonitrile core, by thiol or pyridyl units. After the synthesis of these phthalonitriles, the corresponding Pcs were prepared by ciclotetramerization using a metallic salt as template at high temperatures. A second strategy involved the postfunctionalization of hexadecafluorophthalocyaninato zinc(II) through the adequate substituents of mercaptopyridine or cyclodextrin units on the macrocycle periphery. The different compounds were structurally characterized by diverse spectroscopic techniques, namely 1H, 13C and 19F nuclear magnetic resonance spectroscopies (attending the elemental composition of each structure); absorption and emission spectroscopy, and mass spectrometry. For the specific photophysical studies were also used electrochemical characterization, femtosecond and raman spectroscopy, transmission electron and atomic force microscopy. It was highlighted the noncovalent derivatisation of carbon nanostructures, mainly single wall carbon nanotubes (SWNT) and graphene nanosheets with the prepared Pc conjugates to study the photophysical properties of these supramolecular nanoassemblies. Also, from pyridyl-Pors and ruthenium phthalocyanines (RuPcs) were performed Por-RuPcs arrays via coordination chemistry. The results obtained of the novel supramolecular assemblies showed interesting electron donor-acceptor interactions and might be considered attractive candidates for nanotechnological devices. On the other hand, the amphiphilic phthalocyanine-cyclodextrin (Pc-CD) conjugates were tested in biological trials to assess their ability to inhibit UMUC- 3 human bladder cancer cells. The results obtained demonstrated that these photoactive conjugates are highly phototoxic against human bladder cancer cells and could be applied as promising PDT drugs.
Esta dissertação descreve a síntese e caracterização de diferentes derivados de ftalocianina (Pc), assim como de algumas porfirinas (Pors), para interação supramolecular com diferentes nanoestruturas de carbono para potencial aplicação em nanodispositivos eletrónicos. Igualmente, é também reportado a preparação e avaliação biológica de interessantes conjugados de Pc para a terapia fotodinâmica (PDT) de cancro e para a fotoinativação de microrganismos (PDI). Neste trabalho científico são discutidas as propriedades gerais das Pcs e metodologias sintéticas usadas na sua preparação, bem como algumas das suas importantes aplicações. Os precursores ftalonitrilo foram preparados a partir de ftalonitrilos comerciais por substituições nucleofílicas de grupos -NO2, -Cl ou -F, presentes no núcleo ftalonitrilo, por unidades tiol ou piridilo. As correspondentes Pcs foram preparadas por ciclotetramerização dos ftalonitrilos, previamente sintetizados, na presença de um sal metálico a temperaturas elevadas. Uma segunda estratégia envolveu a pós-funcionalização na periferia do macrociclo da ftalocianina hexadecafluor de zinco(II) com unidades de mercaptopiridina ou ciclodextrina. Os diferentes compostos foram caracterizados estruturalmente por diversas técnicas espectroscópicas, nomeadamente espectroscopia de ressonância magnética nuclear de 1H, 13C e 19F (atendendo à composição elementar de cada estrutura), espectroscopia de absorção e de emissão, e espectrometria de massa. Para estudos fotofísicos específicos foram também usadas a caracterização electroquímica, espectroscopia de femtossegundo e raman, microscopia de transmissão eletrónica e de força atómica. Foi realizado a derivatização não covalente de nanoestruturas de carbono, principalmente nanotubos de carbono de parede simples (SWNT) e nanofolhas de grafeno, com os conjugados de ftalocianina preparados, para dessa forma estudar as propriedades fotofísicas dessas nanoassembleias supramoleculares. Também, a partir de Pors-piridilo e ftalocianinas de ruténio (RuPcs) foram realizadas matrizes de Por-RuPcs via química de coordenação. Os resultados obtidos mostraram interessantes interações eletrónicas doador-aceitador e podem ser considerados candidatos atrativos para diversos dispositivos nanotecnológicos. Por outro lado, os conjugados anfifílicos de ftalocianina-ciclodextrina (Pc-CD) foram testados em ensaios biológicos para avaliar a sua capacidade de inibir células cancerígenas UM-UC-3 da bexiga humana. Os resultados obtidos demonstraram que estes conjugados fotoativos são altamente fototóxicos contra este tipo de células, mostrando-se bastante promissores como agentes em PDT.

Le Phosphate-Diphosphate de Thorium (b-PDT) est actuellement considéré comme une matrice céramique potentielle en vue de l'immobilisation des actinides en formation géologique profonde. Les études réalisées au cours de ce travail reposent sur la synthèse du Phosphate-Hydrogénophoshate de Thorium Hydraté (PHPTH) en tant que précurseur du b-PDT. La structure cristalline du PHPTH a été élucidée puis le mécanisme conduisant à sa transformation en b-PDT a été établi. Ce dernier met en jeu plusieurs composés intermédiaires dont le a-PDT, forme monoclinique jusqu'alors inconnue. Par ailleurs, l'existence d'une solution solide entre le PHPTH et le PHPUH a été démontrée. Les conditions expérimentales de frittage conduisant à une densification optimale des compacts ont été déterminées. La densité relative est toujours comprise entre 95 et 100 % de la valeur calculée et une nette amélioration de l'homogénéité a été constatée. Dans le but d'incorporer simultanément des actinides tri- et tétravalents, le procédé basé sur la précipitation de précurseurs cristallisés puis sur leur frittage a également été appliqué à l'élaboration de matériaux composites b-PDTU/Monazite. Enfin, la durabilité chimique des frittés de b-PDTU a été évaluée. Les taux de lixiviation normalisés déterminés démontrent une bonne résistance des solides à l'altération. La vitesse de dissolution présente une faible dépendance vis-à-vis de la température, du pH et de plusieurs ions présents en solution. Dans tous les cas, le thorium précipite rapidement sous forme de phase néoformée à saturation du lixiviat. Celle-ci a été identifiée au PHPTH et une proposition relative au mécanisme de précipitation a été formulée
Thorium Phosphate-Diphosphate (b-TPD) is actually considered as potential host matrix for the immobilization of radionuclides, and especially actinides, in the field of an underground repository. The studies reported in this work are based on the precipitation of the Thorium Phosphate-HydrogenPhosphate Hydrate (TPHPH) as a precursor of b-TPD. The crystal structure of TPHPH was solved then the reactions involved during its transformation into b-TPD were established. It allows to put in evidence a new monoclinic variety of TPD, called a-TPD, acting as intermediate of reaction. Moreover, the existence of a complete solid solution between TPHPH and UPHPH was demonstrated. The experimental conditions of sintering leading to an optimal densification of the pellets were determined. The relative density of the samples was always between 95 and 100 % of the calculated value while a significant improvement of the homogeneity of the samples was noted. By this way, the process based on the precipitation of low-temperature crystallized precursors followed by their heat treatment at high temperature was applied to the preparation of b-TUPD/Monazite based composites in the aim to incorporate simultaneously tri- and tetravalent actinides. The chemical durability of b-TUPD sintered samples was evaluated. The normalized leaching rates determined in several experimental conditions revealed the good resistance of the solids to aqueous alteration. Moreover, the normalized dissolution rates exhibited a low dependance to temperature, pH as well as to several ions present in the leachate. For all the samples, thorium was quickly precipitated as a neoformed phosphate phase identified to TPHPH

Úlceras são alterações que ocorrem na integridade da pele como consequência de lesões diversas, constituindo a dificuldade de sua cura um grave problema de saúde pública. A fototerapia com laser de baixa intensidade (LLLT), através de seus efeitos biomoduladores, vem sendo apontada como uma técnica prática, rápida e eficaz para esses casos. Entretanto, aplicações de LLLT podem, em alguns casos, estimular a proliferação de microrganismos patogênicos presentes na lesão, resultando numa piora da infecção e maior retardo no processo de reparo. Nesses casos, o uso de quimioterapia fotodinâmica antimicrobiana (PACT) pode ser uma alternativa. O presente estudo testou a eficácia da PACT com Ftalocianina Cloro-Alumínio (FC-ClAl) e da LLLT na inativação de microrganismos, com uma dose energética adequada para não prejudicar o processo de cicatrização. Em biofilmes de P. aeruginosa e S. aureus previamente cultivados por um período de 24 h em microplacas de 24 e 96 poços, realizou-se etapa única de aplicação da fototerapia LLLT e PACT com 4 diferentes formulações para inativação de microrganismos: formulação vazia (ausência de fármaco fotossensível FV), formulação catiônica vazia (com alteração em sua polaridade FCV), formulação aniônica com fármaco fotossensível (apenas Ftalocianina Cloro-alumínio FC-ClAl FAPc), formulação catiônica com fármaco fotossensível (FC-ClAl + formulação com polaridade alterada FCPc). Foram feitas análises quantitativas através da contagem de células viáveis e medidas de absorbância em leitora de ELISA com sal tetrazólio, 3-(4,5-dimethylthiazol-2-yl)-2,5-difenil brometo de tetrazólio (MTT), associado. Os dados obtidos foram comparados através de análise de variância (ANOVA) com nível de confiança de 95%. Os resultados obtidos permitiram concluir que a PACT com FC-ClAl sozinha (FAPc) não apresenta ação bactericida satisfatória sobre a P. aeruginosa e a S. aureus, mostrando resultados estimulatórios a esses microrganismos quando comparada às outras 3 formulações utilizadas. A ação antimicrobiana só foi observada ao se ter a associação da PACT Ftalocianina cloro-alumínio com a formulação catiônica (FCPc), a qual mostrou intensa interação com os biofilmes microbianos, principalmente com a S. aureus, mesmo na ausência de Ftalocianina (FCV).
Ulcers are changes on the skin integrity as a consequence of several lesions, and its cure represents a serious public health problem. Phototherapy based on low level laser therapy (LLLT), through its biomodulatory effects, has been pointed as a fast, practical and effective therapy for these cases. However, LLLT applications may, in some cases, stimulate pathogenic microorganisms proliferation present in the lesion, resulting in a worst infection and longer delay on the healing process. For such cases the use of Photodynamic Antimicrobial Chemotherapy (PACT) can be an alternative. The present study tested the PACT effectiveness with a phthalocyanine-derived photosensitizer and LLLT on microorganism inactivation, with an adequate energy dose in order to not damage all the healing process. On P. aeruginosa and S. aureus mature biofilms, previously cultivated for a period of 24 h in 24 and 96 wells-plate, a single application of phototherapy based on LLLT and PACT to inactivate bacteria was performed, with four different formulations: blank formulation (FV - no photosensitive drug), cationic empty formulation (FCV - formulation with an inverted polarity), formulation with anionic photosensitive drug (FAPc - only Chloro-aluminum Phthalocyanine) and cationic formulation with photosensitive drug (FCPc - Chloro-aluminum Phthalocyanine associated with an inverted polarity formulation). Quantitative analyses were performed through the counting of colony forming units (CFU) and the absorbance analysis with MTT associated. The data were compared through variance analysis test (ANOVA), with a confidence level of 95%. The results showed that PACT with Chloro-aluminum Phthalocyanine itself does not present satisfactory bactericidal effect against P. aeruginosa and S. aureus. This photosensitizer (FAPc) presented a microorganism stimulatory action when compared with the other three formulations used. The bactericidal effect was seen only with the PACT with cationic formulation association, which demonstrated an intense interaction with microbial biofilms, especially with S. aureus, even in the absence of Phthalocyanine (FCV).

GAD är en vanligt förekommande psykiatrisk åkomma och behovet att utveckla behandlingsformer som kan nå fler ur denna patientgrupp är stort. Syftet med denna randomiserade kontrollerade studie, Origo2, var att replikera den åtta veckor långa kognitiva beteendeterapeutiska behandling som genomförts i studien Origo 2006, och att pröva om en motsvarande internetadministrerad psykodynamisk behandling skulle få lika god effekt. Utfallsmåtten var åtta självskattningsskalor samt kliniska bedömningar av diagnos och psykisk hälsa. Sammanlagt deltog 81 personer i 3 grupper med 27 personer i varje, varav en väntelistkontroll. Resultaten visade signifikanta förbättringar för båda de aktiva behandlingarna vid eftermätning samt vid 3-månadersuppföljning. Även kontrollgruppen uppvisade signifikanta förbättringar. De aktiva behandlingsgruppernas större effektstyrkor, färre diagnoser vid eftermätning samt mindre orossymptom vid 3-månadersuppföljningen, indikerar att det är möjligt att bedriva effektiv behandling av GAD över internet, inte bara med kognitiv beteendeterapi utan även på psykodynamisk grund.

Origo 2

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Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico
Experiments were carried out to adapt the technical photodynamic therapy (PDT) to the subclinical mastitis treatment in bovine. Two LEDs equipments were tested with manufactured acrylic fibers to penetration in the teats. Different energy densities were tested to development the methodology. The photodynamic therapy using low energy density was efficient to treat subclinical mastitis, it can be seen the reduction of mammary infection. The constructions the acrylic fibers to LEDs equipments were efficient, where it was possible to reach all interior of the mammary treated. The toluidine blue at 2,5% was efficient to light dispersal and in microbial activity. The energy densities (ED) used was efficient to the principal microbial agents of subclinical mastitis found in the present work. Staphylococcus coagulase positive was removed with ED 25J/cm2, Staphylococus coagulase negative were removed with ED 75J/cm2, Bacillus sp was removed with ED 100J/cm2, Streptococcus dysgalactiae bacterial total count was reduced when ED 200J/cm2 was used.
Foram realizados v?rios experimentos para adequar ? t?cnica terapia fotodin?mica (PDT) para o tratamento de mastite subcl?nica em bovinos. Foram avaliados dois aparelhos de LEDs adaptados com fibra de acr?lico para a penetra??o nos tetos. Varias densidades de energia foram testadas para o efeito desejado com o desenvolvimento de uma metodologia pr?pria. A terapia fotodin?mica utilizando baixa densidade de energia para tratar mastite subcl?nica foi eficiente posto que, houve redu??o na infec??o nos quartos mam?rios tratados. As adapta??es para adequa??o dos aparelhos LEDs para tratamento dos tetos afetados com mastite subcl?nica em bovinos foi eficiente permitindo que a irradia??o atingisse todo interior do quarto mam?rio tratado. O agente fotossensibilizante azul de toluidina a 2,5% foi capaz de desencadear o efeito desejado permitindo a dispers?o da luz e estimulando resposta antimicrobiana. As densidades de energia (DE) testadas reduziram os principais agentes microbianos isolados. Staphylococcus coagulase positivos foi eliminado com DE 25J/cm2, Estafilococos coagulase negativos com DE 75J/cm2, Bacillus sp. com DE 100J/cm2 e Streptococcus dysgalactiae diminuiu na contagem bacteriana total quando se utilizou DE 200J/cm2.

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CNPq
Experiments were carried out to adapt the technical photodynamic therapy (PDT) to the subclinical mastitis treatment in bovine. Two LEDs equipments were tested with manufactured acrylic fibers to penetration in the teats. Different energy densities were tested to development the methodology. The photodynamic therapy using low energy density was efficient to treat subclinical mastitis, it can be seen the reduction of mammary infection. The constructions the acrylic fibers to LEDs equipments were efficient, where it was possible to reach all interior of the mammary treated. The toluidine blue at 2,5% was efficient to light dispersal and in microbial activity. The energy densities (ED) used was efficient to the principal microbial agents of subclinical mastitis found in the present work. Staphylococcus coagulase positive was removed with ED 25J/cm2 removed with ED 75J/cm2, Staphylococus coagulase negative were , Bacillus sp was removed with ED 100J/cm2 , Streptococcus dysgalactiae bacterial total count was reduced when ED 200J/cm2 was used.
Foram realizados v?rios experimentos para adequar ? t?cnica terapia fotodin?mica (PDT) para o tratamento de mastite subcl?nica em bovinos. Foram avaliados dois aparelhos de LEDs adaptados com fibra de acr?lico para a penetra??o nos tetos. Varias densidades de energia foram testadas para o efeito desejado com o desenvolvimento de uma metodologia pr?pria. A terapia fotodin?mica utilizando baixa densidade de energia para tratar mastite subcl?nica foi eficiente posto que, houve redu??o na infec??o nos quartos mam?rios tratados. As adapta??es para adequa??o dos aparelhos LEDs para tratamento dos tetos afetados com mastite subcl?nica em bovinos foi eficiente permitindo que a irradia??o atingisse todo interior do quarto mam?rio tratado. O agente fotossensibilizante azul de toluidina a 2,5% foi capaz de desencadear o efeito desejado permitindo a dispers?o da luz e estimulando resposta antimicrobiana. As densidades de energia (DE) testadas reduziram os principais agentes microbianos isolados. Staphylococcus coagulase positivos foi eliminado com DE 25J/cm2, Estafilococos coagulase negativos com DE 75J/cm2, Bacillus sp. com DE 100J/cm2 e Streptococcus dysgalactiae diminuiu na contagem bacteriana total quando se utilizou DE 200J/cm

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Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia)
IPEN/D-MPLO
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP; Faculdade de Odontologia, Universidade de São Paulo, São Paulo

Internetadministrerad behandling har i många studier uppvisat samma effektstorlekar som traditionell terapi, men det finns ännu begränsade kunskaper om vilka patienter som lämpar sig bäst för behandling via Internet. Syftet med denna studie var att pröva vilka faktorer som predicerar behandlingsutfall vid Internetadministrerad kognitiv beteendeterapi och Internetadministrerad psykodynamiskt orienterad terapi för generaliserat ångestsyndrom (GAD). Underlag för studien är deltagare från en randomiserad kontrollerad studie (N = 81), vilken jämför effekten av de två behandlingarna med en kontrollgrupp (väntelista). Föreliggande studie omfattar 45 deltagare från de två behandlingsgrupperna. Som huvudutfallsmått beräknades Penn State Worry Questionnaire - residual gain scores, vilket ger ett mått på deltagarnas grad av individuell förändring. Pearson’s produktmomentkorrelationskoefficient och Spearman’s rangkorrelationskoefficient användes för att analysera vilka faktorer som predicerar utfall i de två behandlingsgrupperna. Högre utbildningsnivå (rho = .51, n = 20, p < .05) och längre duration av GAD-symtom (r = -.54, n = 20, p < .05) predicerade bättre respektive sämre utfall vid kognitiv beteendeterapi. Svårighetsgrad, kliniskt skattad (r = -.43, n = 25, p < .05), predicerade sämre utfall vid psykodynamiskt orienterad terapi. Behandlingsutfall vid Internetadministrerad behandling verkar därmed prediceras av olika faktorer vid kognitiv beteendeterapi respektive psykodynamiskt orienterad terapi för generaliserat ångestsyndrom.

La thérapie photodynamique (PDT) est une technique thérapeutique qui permet un traitement localisé par irradiation lumineuse d’un photosensibilisateur (PS) grâce à la génération d’une espèce cytotoxique, généralement de l’oxygène singulet. Cependant, de nombreux PS sont également luminescents et les deux processus sont compétitifs. L’emploi de métaux de transition est connu pour améliorer le processus de PDT mais l’impact des ions lanthanides(III) en PDT est encore peu connu. Par ailleurs, l’utilisation de l’absorption biphotonique a de nombreux avantages parmi lesquels la possibilité d’exciter le PS dans la fenêtre de transparence biologique pour des applications en milieux biologiques.Les travaux de cette thèse visent à étudier quel est l’influence de la complexation d’un atome de lanthanide(III) à un PS sur la photophysique de désexcitation de ce dernier. Les complexes synthétisés et ceux étudiés ont montré que l’effet dépend du lanthanide(III). Il est ainsi possible, avec un choix judicieux du métal, de favoriser une voie de désexcitation par rapport à une autre. En particulier, l’ion Gd(III) se révèle avoir un effet bénéfique important pour la génération d’oxygène singulet et cet effet s’ajoute à celui que des atomes lourds comme le brome peuvent avoir. L’ion Yb(III) en revanche, favorise de manière générale le transfert d’énergie par effet d’antenne et la luminescence du lanthanide est alors le processus majoritaire. Enfin, l’emploi de Gd(III) complexé à un PS excitable à deux photons ouvre la voie à des agents théranostiques moléculaires combinant l’IRM en tant que fonction d’imagerie et la PDT pour la thérapie
Photodynamic Therapy (PDT) is a therapeutic technique which consists in generating a highly reactive species, generally singlet oxygen, by shining light on a photosensitizer (PS). However, many PS are also luminescent and both processes are competitive. The use of transition metals is well known to enhance the PDT effect, but little is known about the effect of lanthanide(III) metals.On the other hand biphotonic absorption has numerous advantages, among them the possibility to excite the PS in the so-called biological transparency window for biological applications.The aim of this PhD is to get a better comprehension of the effect of complexation of a lanthanide(III) atom with a PS on the photophysics and deactivation pathways of the latter. The synthesis and conducted studies of lanthanide complexes showed that the effect is dependent on which lanthanide(III) metal is used. Thus by choosing carefully the lanthanide metal, one can favor one deactivation pathway over another. In particular, the Gd(III) ion turns out to be very efficient in promoting singlet oxygen generation and its effect is additive to the already known positive effect of heavy atoms such as bromine. On the opposite, the Yb(III) ion mainly favors the energy transfer through the antenna effect and the complex preferentially emits light.Finally, using Gd(III) linked to a two-photon excited PS opens the path to molecular theranostic probes combining MRI as a imagery technique and PDT as a therapeutic one

A candidíase vaginal (CV) é uma doença causada por fungos do gênero Candida spp. que acomete milhares de mulheres no mundo. Estima-se que 75% das mulheres sofrerão CV pelo menos uma vez durante a vida fértil, 40 a 50% terão o segundo episódio e 5 a 8% sofrerão a forma recorrente, caracterizada pela ocorrência de quatro ou mais episódios durante um ano. O tratamento ainda permanece um desafio para as candidíases complicadas. A terapia fotodinâmica (TFD) é uma modalidade terapêutica que utiliza substâncias fotossensibilizadoras (FS) e uma fonte de luz, que juntas, produzem espécies reativas de oxigênio, tóxicas para micro-organismos e inofensivas para a célula animal hospedeira. O objetivo deste trabalho foi avaliar os efeitos antifúngicos e anti-inflamatórios da TFD mediada por azul de metileno (AM) e laser de emissao vermelha (LEV) no tratamento da CV em modelo animal. Fêmeas de camundongos BALB/c, com 6 a 10 semanas foram estrogenizadas e, 72h após, receberam via intravaginal 20μL de suspensão contendo 107 UFC/mL de C. albicans ATCC 90028. Cinco dias após, a TFD foi aplicada na vagina das fêmeas, utilizando AM 1000μM e laser (100mW, 660nm, energia de 36J por 6 min ou duas aplicações de 18J por 3min, com intervalo de 24h entre sessões). Após 0, 24 e 96h foram feitas lavagens vaginais para recuperação fúngica, cultivo microbiológico, eutanásia para remoção das vaginas e estudo histológico. Lâminas coradas por hematoxilina e eosina foram utilizadas para contagem da área de células inflamatórias (ACI), utilizando o programa ImageJ. Os resultados mostraram que TFD in vivo reduziu a carga fúngica em aproximadamente 1,6 log UFC/mL e, quando aplicada em duas sessões de 18J por 3min, diminuiu a ACI. A TFD mediada por LEV e AM 1000μM mostra-se como alternativa promissora para o desenvolvimento de novas modalidades terapêuticas para vaginites fúngicas.
Vaginal candidiasis (VC) is a disease caused by fungi of the genus Candida spp. that affects thousands of women worldwide. It is estimated that 75% of women in childbearing age will have VC at least once, 40 to 50% will have a second episode and 5 to 8 % will suffer recurrent form, characterized by the occurrence of four or more episodes during one year. Treatment remains a challenge for complicated candidiasis. Photodynamic therapy (PDT) is a therapeutic modality that uses photosensitizing (FS) substances and a light source, which together produce reactive oxygen species, toxic to microorganisms and harmless to host animal cell. The aim of this study was to evaluate antifungal and anti - inflammatory effects of PDT mediated by methylene blue (MB) and red laser (RL) in the treatment of CV in an animal model. Female BALB/c mice 6 to 10 weeks were estrous and 72h after received intravaginally 20μL of suspension containing 107 CFU/mL of C. albicans ATCC 90028. Five days after, PDT was applied in the vagina of females using MB 1000μM and laser (100mW, 660nm, 36J for 6 min or two applications of 18J for 3 min, with an interval of 24h between sessions). Vaginal washes for fungal recovery and microbiological culture, and euthanasia for removal of vaginas and histological study were made after 0, 24 and 96h. Glass slides stained by hematoxylin and eosin were used for counting the area of inflammatory cells (AIC) using ImageJ software. PDT in vivo reduced the fungal burden in approximately 1.6 log CFU/mL, and, when applied in two sessions with 18J for 3min, decreased the AIC. PDT mediated by RL and MB 1000μM shown as a promising alternative for the development of new therapeutic modalities for yeast vaginitis.

Cette thèse de doctorat a pour dessain d’évaluer d’un point de vue chimique, mais surtout biologique les complexes polypyridyle Ru (II). Ces complexes métalliques peuvent être utilisés comme photosensibilisateurs (PS) pour la thérapie photodynamique (PDT), ou encore comme agents chimiothérapeutiques dans le traitement du cancer. La PDT est un traitement alternatif ou complémentaire à la chirurgie, la chimiothérapie ou la radiothérapie.Ses nombreux avantages lui confèrent un intêret dans le traitement actuel du cancer. Son contrôle spatial et temporel est particulièrement intéressant, ce qui conduit à cibler les tumeurs tout en préservant les tissus sains. De plus, les résistances à répétition et les effets secondaires graves provoqués par la chimiothérapie incitent le monde scientifique à rechercher de nouveaux médicaments candidats anticancéreux. Les complexes de ruthénium sont l'un des groupes les plus prometteurs de médicaments candidats à base de métaux (comme chimiothérapeutiques ou PS) en raison de leurs multiples états d'oxydation stables. Cette thèse décrit un aperçu des modes d'action connus des complexes polypyridyle Ru (II) comme PS pour la PDT et introduit de nouveaux complexes, qui peuvent être utilisés pour des traitements PDT réguliers et ciblés. En outre, cette thèse se concentre également sur la caractérisation d'une nouvelle classe de complexes Ru générés comme agents anticancéreux potentiels pour la chimiothérapie, par coordination de différents dioxoligands au noyau métallique
Nowadays chemotherapy is the most common therapy used to treat cancer. Currently employed drugs are not ideal, and there is a need for new chemotherapeutics. Photodynamic therapy (PDT) is a succesful alternative or complimentary treatment to chemotherapy, radiotherapy and surgery. This medical technique involves non-toxic photosensitiser (PS) which after irradiation with specific and defined wavelenght can react with molecular oxygen or other biological substrates. As a result very reactive oxygen species or radicals are created, leading to impairment of metabolic pathways and eventually to cell death. Ru(II) polypyridyl complexes are promising chemotherapeutics as well as photosensitisers for PDT. Aim of this thesis is to understand how Ru(II)complexes excert their action in living cells. Understanding mechanisms in which photosensitizers or chemotherpeutics are acting is an important step in designing new, better compounds suitable for chemotherapy and/or for PDT

Ce travail concerne le développement de nanoparticules de TiO2 et de SiO2 sensibilisées aux photosensibilisateurs pour application dans la photocatalyse et la thérapie photodynamique (PDT). Les NP ont été soit recouverts d'une coquille de polysiloxane, soit modifiés par l'aminopropyltriéthoxysilane (APTES) seul. Les PSs de tétraphényl monocarboxylphosphine (P1-COOH) ou de chlorine e6 (Ce6) ont été couplés aux NP par liaison amide. En photocatalyse, les NP hybrides modifiées par l'APTES, en particulier TiO2-APTES-Ce6, présentent une activité photocatalytique supérieure vis-à-vis de la dégradation du bleu de méthylène et de l’orange de méthyle sur les systèmes cœur-coquille sous lumière solaire et visible. Pour la PDT, des tests in vitro ont été désignés sur la lignée cellulaire de glioblastome U87 à différentes concentrations de NP éclairées à 652 nm. TiO2-APTES-Ce6 a révélé une bonne phototoxicité car la viabilité cellulaire a diminué de 89% après illumination. L'incorporation cellulaire et la localisation de ces NP et de leurs analogues de la silice ont été explorées. Les ROS impliqués dans la photocatalyse et la PDT ont été étudiés
This work addresses the development of dye-sensitized TiO2 and SiO2 nanoparticles (NPs) for application in photocatalysis and photodynamic therapy (PDT). The NPs were either coated with a polysiloxane shell or modified by aminopropyl triethoxysilane (APTES) alone. Monocarboxylic tetraphenyl porphyrin (P1-COOH) or chlorin e6 (Ce6) PSs were coupled to the NPs by amide bond. In photocatalysis, The APTES-modified sensitized NPs, particularly TiO2-APTES-Ce6, exhibit a superior activity towards the degradation of methylene blue and methyl orange over the core-shell systems under solar and visible light. For PDT, in vitro tests were conducted on the glioblastoma cell line U87 at different NPs’ concentrations illuminated at 652 nm. TiO2-APTES-Ce6 revealed a good phototoxicity as the cell viability decreased by 89% after illumination. The cellular uptake and localization of those NPs and their silica analogues were explored. The ROS involved in photocatalysis and PDT were investigated

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina
Os carcinomas pouco diferenciados (PDTC) e anaplásicos (ATC) da tiróide são tumores muito agressivos, para os quais não existe actualmente uma forma de tratamento eficaz. Neste sentido, é importante identificar os mecanismos moleculares responsáveis pelo desenvolvimento destes tumores. Em estudos dos perfis de expressão génica globais, realizados pelo nosso grupo, foram identificadas assinaturas moleculares comuns em PDTC e ATC, relacionadas com a proliferação celular, ciclo celular, adesão celular e metastização. Com o objectivo de identificar alvos terapêuticos para PDTC e ATC, pesquisaram-se mutações em genes relacionados com as assinaturas moleculares identificadas (TP53, CTNNB1, AXIN1, PIK3CA, N- H- e K-RAS, BRAF, CDKN2A, CDKN2B, CDKN2C, CDKN1A, CDKN1B) numa série de 49 tumores primários da tiróide (26 ATC e 23 PDTC) e em 6 linhas celulares (2 PDTC e 4 ATC). Analisaram-se também os níveis de expressão de um gene envolvido na invasão/metastização (SNAI2). Nos tumores primários, identificaram-se mutações nos genes BRAF (PDTC=4%; ATC=4%), RAS (PDTC=14%; ATC=31%),TP53 (PDTC=22%; ATC=42%), PIK3CA (PDTC=13%; ATC=4%), CTNNB1 (PDTC=5%; ATC=0%), CDKN2A (PDTC=15%; ATC=5%), CDKN2B (PDTC=10%; ATC=0%) e CDKN2C (PDTC=0%; ATC=6%). Este estudo mostrou que os genes TP53 e RAS apresentavam as mutações mais frequentes em PDTC e ATC. As mutações no gene TP53 e as mutações nos genes RAS ou BRAF, apresentaram evidências de mútua exclusividade (P=0,0193). A presença de mutações nestes genes estava associada a um menor tempo de sobrevivência global dos doentes com PDTC e ATC (P=0,0222). Observou-se ainda que a sobre-expressão do gene SNAI2 estava associada ao desenvolvimento de um subgrupo de ATC. Os resultados do estudo das alterações moleculares na série de PDTC e ATC, confirmaram o envolvimento dos genes BRAF, RAS, TP53 e PIK3CA e revelaram a contribuição de genes envolvidos na regulação do ciclo celular (CDKN2A, CDKN2B, CDKN2C) e invasão/metastização (SNAI2). Estes genes poderão representar alvos para intervenção terapêutica com impacto no seguimento clínico e sobrevivência destes doentes.

In the past few years, the solution-processed organic based solar cells gained more importance by meeting the demands for cost effective photovoltaic devices. To date, the focus of the organic photovoltaic devices has been on the optimization of the processing the materials to improve photo conversion efficiency and also by modifying the active components of the organic materials. Recently, it has been recognized that the deposition techniques also plays a major role in enhancing the power conversion efficiencies. Currently, though the most common deposition technique for organic solar cells is spin coating, which does not allow scaling up of the large device area. As an alternative method, a simple airbrush spray deposition technique has been developed to fabricate the test devices. The film thickness of the layers was characterized under scanning electron microscope. Devices with different thickness (1000 nm, 500 nm, 240 nm) of poly(3,4-ethylenedioxythipohene) polystyrene sulfonate (PEDOT.PSS) and active layers are prepared and their photovoltaic performances have been evaluated and compared by plotting the IV curves with respect to each thickness. Maintaining the distance between the substrate and the airbrush nozzle the thickness of the layers was controlled. From the results, we found that the test devices with 500 nm thickness of PEDOT.PSS and active layers shows the best device performance with highest current density of 3.97 mA/cm2, open circuit voltage of 1.3 V and power conversion efficiency of 2.34%. As a control experiment, devices were also developed using the standard poly(3- hexylthiophene-2,5-diyl):phenyl-C61-butyric acid methyl ester (P3HT:PCBM) system, but the power conversion efficiencies of these devices were not promising with respect to the literature results. Future studies of this project will focus on improving the power conversion efficiency of poly(3-hexylthiophene-2,5-diyl)/perylenediimide bridged system (P3HT/PDIB) by developing a new device architecture called “tandem solar cells” which consists of multiple layers of different donor and acceptor blends with inorganic transition metal oxides such as zinc oxide and molybdenum oxides.

Estudos com tratamento hipertérmico de tumores utilizando nanopartículas metálicas têm sido realizados durante as últimas décadas e mostram resultados bons quanto à remissão de tumores, por vezes chegando à cura completa. O mesmo acontece em relação aos tratamentos baseados em ação fotodinâmica de fotossensibilizadores. Tratamentos aliando a terapia hipertérmica com nanopartículas de ouro e a terapia fotodinâmica com diversos fotossensibilizadores tem efeito sinérgico e apresenta excelente potencial terapêutico, em que pese serem necessários mais estudos para que uma nova terapia conjunta possa ser implementada. A proposta deste trabalho foi investigar esse efeito sinérgico utilizando nanobastões de ouro complexados com fotossensibilizadores. Após a síntese dos nanobastões pelo método de seeding, a eficácia do tratamento fotodinâmico e da terapia hipertérmica, separadamente, foi investigada. A metodologia do recobrimento dos nanobastões por fotossensibilizador, em um primeiro momento, não logrou êxito com a porfirina, porém com a ftalocianina tetracarboxilada se mostrou mais eficaz. A taxa de fotodegradação da ftalocianina em solução foi investigada como parâmetro para a eficiência em geração de oxigênio singlete. Após centrifugação e lavagem das nanopartículas, no entanto, evidenciou-se por espectrofotometria que o fotossensibilizador não permaneceu aderido aos nanobastões. Em um segundo momento, optamos por recobrir os nanobastões por porfirinas tetrassulfonadas, com ou sem grupamentos metil-glucamina. Após o processo de recobrimento, essas ftalocianinas formaram complexos iônicos com o CTAB que recobre os nanobastões. Os complexos nanobastões-ftalocianinas foram analisados por microscopia eletrônica de transmissão e as taxas de geração de oxigênio singlete e de radical hidroxil foram investigadas. Além disso, foram utilizadas para testes in vivo e in vitro com células de melanoma melanótico (B16F10) ou amelanótico (B16G4F). As células tumorais em cultura ou os tumores em camundongos C57BL6 foram irradiados com luz em 635 nm e os tumores foram observados por 15 dias após o tratamento. Houve evidente aumento na geração de oxigênio singlete por ambos fotossensibilizadores, e maior geração de radicais livres por parte do fotossensibilizador metilglucaminado. O oposto ocorre com o fotossensibilizador sem metilglucamina. Houve, também, moderada citotoxicidade no escuro quando células foram incubadas com nanopartículas recobertas por ftalocianinas ou não. Quando ativados pela luz, os complexos ftalocianinas-nanobastões desencadearam um aumento de 5ºC no meio de cultura das células, e a morte celular observada foi extensa (91% para a linhagem B16G4F e 95% para a linhagem B16F10). Tanto os resultados in vitro quanto os in vivo indicam que as propriedades das ftalocianinas testadas são melhoradas significativamente quando elas estão complexadas aos nanobastões. Este é um estudo pioneiro por utilizar duas porfirinas tetrassulfonadas específicas e por utilizar o mesmo comprimento de onda para a ativação dos fotossensibilizadores e nanobastões.
Studies with hyperthermic tumor ablation using metallic nanoparticles have been performed on the last decades, and show promising results on tumor remission, sometimes achieving the complete cancer elimination. The same occurs regarding on treatments based on photodynamic activity of photosensitizer compounds. Studies indicate that those therapeutic interventions - hyperthermic therapy using gold nanorods and photodynamic activity with many photosensitizers - together can present a synergistic effect, and offer a great therapeutic potential, although more investigation needs to be performed before such approach could be implemented. We proposed to investigate the effect of the attachment of photosensitizers onto the surface of gold nanorods (well-characterized devices for hyperthermia generation). After nanorods synthesis through a seed-mediated method, the PDT and hyperthermia\'s efficacy was assessed separately. The method used for covering the gold nanorods with photosensitizers did not permit, in a first approach, the attachment of porphyrins onto the nanoparticles surface, but the attachment of tetrasulfonated phthalocyanines was more successful. The phthalocyanine\'s degradation rate was assessed as an indirect parameter of singlet oxygen generation. After centrifuging and washing the nanoparticles, we saw that the photosensitizers do not keep attached to the nanorods. On a second approach, we chose to recover the nanorods with two zinc phthalocyanines - with or without methyl-glucamine groups. After the recovering process, the phthalocyanines formed ionic complexes with the CTAB that is previously recovering the nanoparticles. The nanorod-phthalocyanines complexes were analyzed by TEM, and their singlet oxygen and hydroxyl radical generation yield were assessed. Furthermore, they were tested in vitro in melanoticB16F10 and amelanotic B16G4F melanoma cells, and in vivo. The tumor cells (in vitro) and the tumor tissue (in vivo) with nanoparticles were irradiated with laser (at 635 nm), and the tumor growth in mice was observed for 15 days after the laser irradiation. It is evident the increase in the singlet oxygen generation, and higher HPF activation for the glucaminated Pc, but the inverse for the other photosensitizer. It seems like there is a type I to type II switch on the action mechanism of the latter Pc. A mild cytoxocity was observed with the nanorods conjugated with photosensitizer in the dark, but when they are activated by light (and taken into account a 5ºC rise in the temperature because of the surface plasmon resonance from the gold nanorods), the cell killing is intense (91% for B16G4F cell line, and 95% for B16F10 cell line). Both in vitro and in vivo results indicate that the photodynamic properties of the phthalocyanines tested are enhanced when they are attached onto the nanorods surface. This is a novel study because we used two tetrasulfonated phthalocyanines and because we used the same wavelength to activate both the nanorods and the photosensitizers.

A Terapia Fotodinâmica (do inglês, Photodynamic Therapy - PDT) é uma técnica indicada para o tratamento local de câncer que vem tendo grandes avanços ao longo dos anos. A PDT consiste na interação entre luz e uma substância fotossensibilizadora, resultando na transformação do oxigênio molecular em oxigênio singleto, altamente reativo e tóxico para a célula, levando à destruição do tecido. Nesse contexto, o uso do modelo de Membrana Corioalantóica (CAM) é uma opção para o estudo dos efeitos vasculares envolvidos nessa terapia, além de permitir o estudo na variação de diversos parâmetros associados com a PDT e seus efeitos. Nesse estudo foram investigados um composto derivado de porfirina e um derivado de clorina. Esses fotossensibilizadores foram administrados topicamente e por via intravenosa, sendo variados diversos parâmetros. No primeiro caso, o tempo de incubação foi variado entre 20 e 80 minutos e a concentração de área da droga foi variada entre 0,1 e 100 g/cm2. Quanto à dose de luz, o intervalo foi entre 4,8 e 60 J/cm2, empregando lasers de diodo em 635 nm para Photogem® e 660 nm para Photodithazine®. Depois de estabelecido 30 J/cm² para a aplicação tópica, foi usada a aplicação intravenosa com essa dose, a fim de comparar os resultados. Após iluminação, foram feitas imagens da membrana imediatamente após a iluminação até 300 minutos pós-terapia. Com ajuda de softwares de processamento de imagem, foi feita a transformação da imagem: os pixels referentes aos vasos sanguíneos foram transformados em preto e o restante (clara, gema e embrião) em branco. Esse processo levou à obtenção de um valor relacionado à área de vasos sanguíneos em função do tempo pós-terapia, quantificando e mostrando o comportamento do efeito vascular da PDT. A comparação entre os dois fotossensibilizadores mostrou que o uso da mesma concentração (1 g/cm²) pode levar à destruição da rede vascular tratada para a porfirina e à dilatação dos vasos periféricos com destruição dos vasos mais calibrosos, para a clorina. Entretanto, variando as concentrações de ambos, obtemos respostas contrárias. A análise dos resultados obtidos, observando a diferença de ação dos fotossensibilizadores, permitiu a obtenção de uma relação quantitativa do comportamento vascular além do estudo individualizado do efeito da terapia fotodinâmica para análise do dano tecidual induzido.
Photodynamic Therapy is a local treatment of cancer that has great advances over the years. To obtain an efficient process that leads to tissue destruction, a dynamic interaction of three factors is required: photosensitizing agent, light, and molecular oxygen. When there is the appropriate illumination of tissue with photosensitizer, this molecule is excited and produces singlet oxygen. This product is reactive and cytotoxic, and it results in cell death. In this context, the CAM model allows studies with variation in many parameters linked with PDT and its effects and it is an option to study of vascular effects. A compound of porphyrin (Photogem®) and a compound of chlorine (Photodithazine®) were investigated in this study. These fotosensitizers were topically administrated and the parameters were ranged between 20-80 minutes for incubation, and 0.1-100 g/cm2 for concentration. About light dose, the range was between 4.8-60 J/cm2, with diode laser at 635 nm (Photogem®) and 660 nm (Photodithazine®). After established that light dose of 30J/cm² as ideal for topical application, intravenous application was used with that dose in order to compare the results. After illumination, the membrane images were made from zero to 300 minutes. At image processing, the pixels corresponding to vessels were defined as black and the remaining structures as white pixels. Calculating the percentage area of the black pixels as a function of the treatment time, it is possible to quantify the vascular effect of this therapy. The comparison between two fotosensitizers (1 g/cm²) can lead to vascular network destruction to porphyrin and can lead to dilation of peripheral vessels with destruction to larger vessels, for chlorine. However, increasing the porphyrin concentration and decreasing the chlorine concentration, we have opposite responses. With varying PDT parameters, it was possible to obtain the best assay for PDT. The results obtained by observing the difference of action of photosensitizers, allows obtaining a quantitative relationship of vascular behavior in addition to individual study of the effect of photodynamic therapy for analysis of tissue damage induced.

Processos que envolvem sensibilização são extremamente importantes para diversas áreas do conhecimento, incluindo a biologia, a química e a medicina. A aplicação de sensibilização em medicina tem se destacado, especialmente, em face de uma modalidade alternativa de tratamento de câncer denominada terapia fotodinâmica (TFD). Uma das linhas de pesquisas fundamentais para a evolução da terapia fotodinâmica é o desenvolvimento de novos fotossensibilizadores (Fs) com composição definida, que absorvam na janela terapêutica (600- 800nm) e que apresentem maior eficiência na indução de apoptose. Os Fs que apresentam cargas positivas e que são relativamente lipofílicos, permeiam membranas e são atraídos pelo potencial negativo das mitocôndrias, que tem papel central no controle da vida e da morte celular. Neste trabalho foi realizado um estudo da influência dos grupos funcionais na atividade dos Fs, através da funcionalização da protoporfirina IX (Pp IX). Foram estudadas três diferentes rotas sintéticas. Na rota I (esquema 14), utilizou-se como composto de partida a Hematoporfina IX (Hp IX), a qual foi funcionalizada com grupos aminas e amidas, respectivamente, nas hidroxilas e nas carboxilas. Esta rota forneceu baixo rendimento global (20%), e compostos de difícil purificação, no entanto obteve-se 1 composto puro. Na rota II (esquema 15), utilizou-se como composto de partida a Pp IX, a qual foi funcionalizada nos grupos vinílicos com grupos aminas. Obteve-se 4 compostos, com rendimento global de reação superior a 50%, mas observou-se que ocorre uma reação de eliminação que impede a quartenarização das aminas localizadas nas posições α ao anel porfirínico. Na rota III (esquema 16), os grupos carboxílicos de Pp IX foram transformados em cloreto de ácido, que foram substituídos por compostos bifuncionais, amina primária e um segundo grupo. Esta rota possibilitou a obtenção de 7 compostos, inclusive compostos quaternários com rendimento global de reação superior a 70%. Dois derivados da clorofilina, que apresentam a banda QIV com um ε elevado em 650nm, foram também sintetizados. Todos os compostos, foram caracterizados estruturalmente de forma inequívoca através do espectro eletrônico (UV-vis e fluorescência), vibracional no infravermelho, RMN de 1H e 13C (1D e 2D) e espectrometria de massa. A série de compostos obtidos permitiu um estudo da relação entre a estrutura química do Fs com a sua fotoatividade. As propriedades fotofísicas foram caracterizadas por espectro de absorção e de emissão, fotólise de relâmpago a laser, eficiência quântica de fluorescência e de geração de oxigênio singlete. Estas determinações indicaram que as propriedades fotofísicas dos Fs não foram consideravelmente alteradas no processo sintético. Foi determinada a formação de agregados em solução aquosa e o equilíbrio monômero agregado foi deslocado no sentido da formação do monômero na presença de micelas de CTAB e SDS e de soro fetal. Observou-se, que a contribuição para desagregação é mais eficiente quando a carga da micela é oposta à do Fs. Foi determinado o coeficiente de partição octanol/água (logPo/a) em função do pH e constatou-se que os compostos que têm carga líquida apresentam valores de logPo/a entre -1 e 1 na faixa de pH entre 3 e 10. A incorporação destes compostos em lipossomos, seguiu perfil esperado considerando a carga, o logPo/a e a característica anfifílica dos compostos sintetizadas. Genericamente a eficiência de morte celular fotoinduzida seguiu o perfil de incorporação das drogas em células HeLa. Os estudos comparativos da citolocalização foram realizados por co-encubação das porfirinas sintetizadas com rodamina 123 (Rd), que se concentra em mitocôndrias. A localização em organelas citoplasmáticas foi determinada através de imagens obtidas por microscopia de fluorescência confocal. A sobreposição da emissão da Rd foi de 80% e 31% com o composto catiônico PpNpNI e aniônico PpNetPO3, respectivamente, comprovando a localização mitocondrial do composto positivo.
Processes that involve sensitization are extremely important for several areas of knowledge, including biology, chemistry and medicine. The application of sensitization in medicine is especially important, in face of an alternative modality of cancer treatment called Photodynamic Therapy (PDT). One of the lines of basic research important for the evolution of PDT is the development of new photosensitizers (PS) with defined composition, that absorb in the therapeutical window (600-800nm) and that present greater efficiency in the induction of apoptosis. Positively charged PS with the proper lipophilic/hidrophilic balance permeate membranes and are attracted by the negative potential of mitochondria, which is an organelle that has central roles in the control of cell life and death. In this work the influence of the functional groups in the activity of PS was carried out, through the functionalization of protoporphyrin IX (Pp IX). Three different synthetic routes were used. In route I (scheme 14), Hematoporphyrin IX (Hp IX), was used as departure compound and it was modified with amino and amide groups in hydroxyls and carboxyls, respectively. We found low yield in the synthetic (20%) and purification steps. However 1 pure PS was obtained. In route II (scheme 15), Pp IX was used as departure compound, which was modified in the vinilic groups with amino groups. We obtained 4 compounds, with global yield superior than 50%, but it was observed that an elimination reaction occurs that hinders the quarternization of the amino groups located in position α to the porphyrin ring. Into route III (scheme 16), the carboxilic groups of Pp IX had been transformed into chloride acid, which was substituted by bi- functional groups, primary amine in one side and another group in the other. This route made it possible to obtain 7 compounds, including quaternary ammonium compounds with global yield superior than 70%. Two derivatives of chlorophyllin, that present QIV band with large extinction coefficient in 650nm, also were synthesized. All compounds were characterized structurally through the electronic and vibrational spectra (UV-vis and fluorescence, IR), RMN of 1H and 13C (1D and 2D) and mass spectrometry. This series of compounds allowed us to study the relation between the chemical structure of the Fs with its photoactivity. The photophysical properties were characterized by emission and absorption spectra, laser flash photolysis, fluorescence emission in the visible and NIR regions (generation of singlet oxygen). These determinations indicated that the photophysical properties of the PS were not modified in the synthetic process. The formation of aggregates were characterized in aqueous solution and the balance between aggregate and monomer species was dislocated in the direction of the formation of monomers in the presence of CTAB, SDS micelles and fetal serum. It was observed, that the disaggregating efficient is larger with micelles that have the opposite charges to that of the PS. The Octanol water partition coefficient (logPo/a), was determined as a function of pH. logPo/a values between -1 and 1 were observed for charged PS in the pH range of 3-10. The incorporation of these compounds in liposomes, followed the expected profile considering the charge, logPo/a and the amphifilic nature. Generically, the photoinduced cell death efficiency followed the profile of incorporation of the PS in HeLa cells. Comparative studies of cytolocalization were carried out by co-incubation of the cells with porphyrins and Rhodamine 123 (Rd), which localizes in mitochondria. The localization in cytoplasmic organelles was determined through fluorescence confocal microscopy images. The overlapping emission of the Rd was of 80% and 31% with the cationic PpNpNI and the anionic PpNetPO3 compounds, respectively, proving the mitochondrial localization of the positive PS.

La thérapie photodynamique (PDT), une thérapie basée sur l'irradiation de molécules photosensibilisatrices pour générer un stress oxydant, est déjà utilisée comme traitement de certaines pathologies. Très souvent, les photosensibilisateurs utilisés sont des molécules fortement hydrophobes qui s'agrègent en milieux aqueux. De ce fait, utilisées seules, elles nécessitent d'être injectées à des concentrations élevées, entraînant un risque de photosensibilisation générale. Pour diminuer cet effet secondaire et rendre le traitement plus efficace, il est possible d'encapsuler ces molécules. Des travaux précédant au sein du laboratoire des IRMCP ont permis le développement de vecteurs à base de copolymères à blocs pour l'encapsulation d'un photosensibilisateur, le phéophorbide-a. Ces travaux ont montré une efficacité de certains de ces vecteurs en conditions PDT sur des cultures cellulaires et ont ouvert des questions sur les processus de réponse cellulaire. L'objectif de ce doctorat était de développer des outils permettant de mieux comprendre les mécanismes ayant lieu lors de l'utilisation de nanovecteurs à base de copolymères à blocs encapsulant du phéophorbide-a et lors de l'irradiation lumineuse du photosensibilisateur. Les nanovecteurs étudiés étaient des micelles formulées à base de trois copolymères différents, le PEO-PCL, le PEO-PLA et le PEO-PS. Pour simplifier le système étudié, nous avons choisi d'utiliser des modèles de membranes pour simuler la cible biologique, des liposomes (ou vésicules lipidiques). En utilisant les propriétés de fluorescence du phéophorbide-a, nous avons pu obtenir les constantes d'affinité du photosensibilisateur pour les micelles et pour les vésicules lipidiques, puis évaluer le passage du phéophorbide-a des micelles vers les vésicules. Dans un second temps, nous nous sommes intéressés aux phénomènes en jeu lors de l'irradiation du photosensibilisateur. Nous avons pu estimer la production relative d'oxygène singulet en fonction du type de micelles utilisé. En suivant la fuite d'une sonde fluorescente contenue dans les liposomes, permettant ainsi de remonter à leur perméabilité, il a été possible mesurer les effets de la production d'oxygène singulet sur l'intégrité de la membrane des liposomes. De manière complémentaire, nous avons suivi l'oxydation des lipides constituant les liposomes durant l'irradiation du phéophorbide-a par spectrométrie de masse. Ces résultats combinés nous ont permis de voir quels étaient les paramètres influençant l'efficacité des micelles encapsulant un photosensibilisateur après irradiation et d'établir un classement de ceux ayant le plus d'effets sur l'intégrité de membranes modèles parmi ceux étudiés
Photodynamic therapy (PDT), a therapy based on the irradiation of photosensitizing molecules to generate an oxidative stress, is already used as a treatment of some pathologies. The photosensitizers used are often highly hydrophobic molecules that aggregate in aqueous medium. Therefore, used by themselves, they require to be injected at high concentrations, leading to a risk of global photosensitization. To reduce this secondary effect and increase the effectiveness of the treatment, it is possible to encapsulate those molecules. Previous work in the IMRCP laboratory has led to the development of block copolymer-based carriers to encapsulate a photosensitizer, pheophorbide-a. This work has showed superior efficiency of some type of carriers compared to others under PDT conditions on cell culture. The aim of this project was to develop tools to better understand the mechanisms occurring when using block copolymers-based nanocarriers encapsulating pheophorbide-a and during the irradiation of the photosensitizer. The nanocarriers studied were block copolymer-based micelles made of PEO-PCL, PEO-PLA and PEO-PS. To simplify the system studied, we chose to use liposomes as membrane models to simulate the biological target. Using the fluorescence properties of pheophorbide-a, we were able to obtain the affinity constants of the photosensitizer for the micelles and the lipid vesicles, and then evaluate the transfer of pheophorbide-a from the micelles to the vesicles. Following that, we investigated the phenomena occurring during the irradiation of the photosensitizer. We were able to estimate the relative production of singlet oxygen depending on the type of micelles used. By monitoring the leakage of a fluorescent probe contained in the liposomes, allowing us to evaluate their permeability, it was possible to measure the effects of singlet oxygen production on the integrity of the liposome membrane. Complementarily, we followed the oxidation of the lipids of the liposomes during the irradiation of pheophorbide-a by mass spectrometry. These results combined together allowed us to see what were the parameters influencing the PDT efficiency of micelles encapsulating a photosensitizer. We managed to classify those with the greatest effect on the integrity of model membranes among those studied

DAISY has published a Specification for DAISY Protected Digital Talking Book. This paper discusses why such a specification is useful, not only for rightsholders but also for readers with print disabilities. An implementation of PDTB2 is proposed, called dtbprotect. It makes possible to simply produce an encrypted book from a book in DAISY format. It is currently experimented on the Helene Digital Library for the blind. It will be made available open source as to facilitate its implementation by other digital libraries.

Phthalocyanines are a group of molecules, which have been studied extensively due partly to their use and potential in a wide variety of applications, but also on account of the seemingly endless list of differently substituted moieties, which in principle may be prepared. The history of the compounds in general are discussed briefly in Chapter 1 of this thesis, while Chapter 2 focuses on Photodynamic Therapy, an area in which they have more recently been found to exhibit excellent potential. The synthesiso of novel phthalocyanines for potential application in PDT is reported in Chapter 3. The current research investigated the synthesis of binuclear Pcs from the linking of two preformed phthalocyanine rings by means of an oxalyl bridge functionality and also from the mixed cyclisation of phthalonitriles with bisphthalonitriles, which proved to be extremely problematic. The synthesis of water-soluble phthalocyanine derivatives was investigated as a sideline, which resulted in the synthesis of a novel phthalocyanine monomer substituted with eight triethylene glycol chains. Efforts to synthesise a phthalocyanine linked to a glucose unit were unsuccessful. Chapter 4 describes research into the photophysical properties of a number of mononuclear and binuclear phthalocyanines synthesised both in the current work and also compounds synthesised by co-workers. The electronic absorption and fluorescence spectra of the compounds are investigated, and the absorption spectrum of a purely cofacial dimeric structure is obtained by calculation from the spectrum of one of the binuclear derivatives. Laser Flash Photolysis was performed on a number of the samples, determining triplet lifetimes, triplet quantum yields and singlet oxygen quantum yields. The effect of aggregation on the photoproperties of phthalocyaninesis investigated in Chapter 5. The current work involved low temperature absorption and fluorescence spectroscopy on a number of mononuclear and binuclear phthalocyanine derivatives. The nature of the aggregated structures, which were observed to form at 77K, are then rationalised with reference to previous research into phthalocyanine aggregates and crystal structures.

Os Pactos para o Desenvolvimento e Coesão Territorial apresentam-se como uma relevante inovação no período de programação 2014-2020, dado que evoluíram qualitativamente a partir da figura dos contratos de delegação de competências com a subvenção global que existiram no período anterior. Também no atual período de programação, o enfoque nos resultados é uma forte preocupação da União Europeia, pelo que os sistemas de monitorização são uma ferramenta essencial para a persecução dos objetivos da Política de Coesão 2014-2020. Este relatório de estágio divide-se em três capítulos principais, primeiramente procura-se abordar um conjunto de temas que conduzam, numa ordem lógica, a um enquadramento teórico das políticas de base territorial e, consequentemente, dos Pactos para o Desenvolvimento e Coesão Territorial, com propósito de entender o racional das políticas de base territorial que são a base para estes instrumentos territoriais. O segundo capítulo acontece devido à experiência obtida durante o estágio curricular realizado na Comunidade Intermunicipal da Alentejo Central e resulta no enquadramento prático deste relatório, constituído por uma contextualização e análise detalhadas das atividades desenvolvidas. O último capítulo, pretende apresentar uma proposta de Sistema de Monitorização para o Pacto para o Desenvolvimento e Coesão Territorial – Alentejo Central, visto que a monitorização é um processo fundamental para uma boa gestão e deve constituir-se como um instrumento tão importante como o próprio desenvolvimento da intervenção, apenas com o correto acompanhamento se consegue garantir o avanço em direção aos resultados esperados; ABSTRACT: PACT FOR DEVELOPMENT AND TERRITORIAL COHESION OF THE ALENTEJO CENTRAL – MONITORING SYSTEM PROPOSAL The Pacts for Development and Territorial Cohesion are a significant innovation in the programming period 2014-2020, since they have evolved qualitatively from the figure of the delegation of competences contracts with the global subvention that existed in the previous period. Also, in the current programming period the focus on results is a strong concern of the European Union, and monitoring systems are therefore an essential tool for achieving the objectives of Cohesion Policy 2014-2020. This internship report is divided into three main chapters. It is firstly intended to address a set of themes that will lead, in a logical order, to a theoretical framework of integrated approaches for territorial development and, consequently, the Pacts for Development and Cohesion Territorial, with the purpose of understanding the rational territorial-based policies that are the basis for these territorial instruments. The second chapter happens due to the experience obtained during the internship in the Intermunicipal Community of Alentejo Central and results in the practical framework of this report, constituted by a contextualization and detailed analysis of the activities developed. The last chapter intends to present a proposal for a monitoring system for the Pact for Development and Territorial Cohesion - Alentejo Central, monitoring is a fundamental process for good management and should be as important an instrument as the development of the intervention, only with the correct accompaniment can we guarantee the progress towards the expected results.

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.

A Terapia Fotodinâmica (TFD) é uma técnica que provoca dano celular pela ação de um fotossensibilizador (FS), com seletividade de localização em tecido tumoral; a luz que, absorvida pelo FS, leva-o a um estado tripleto metaestável; e oxigênio molecular, o qual recebe a energia absorvida pelo FS, passando a um estado singleto de alta capacidade oxidativa. A técnica é bem sucedida no tratamento de lesões como câncer, mas enfrenta, entretanto, dificuldades para a determinação de sua dosimetria. Uma delas é a quantificação da distribuição do FS no tecido tratado. Este trabalho tem três objetivos: a obtenção de informação quantitativa por espectros de fluorescência de fluoróforos em meios turvos; a demonstração da distribuição do FS Photogem® em fígados sadios de ratos Wistar e suas implicações na dosimetria; e a melhoria de um dos modelos existentes para previsão da profundidade de necrose (Ynec), importante parâmetro no estudo da TFD. Realizaram-se os experimentos em três fases: na primeira, tentou-se reconstruir o espectro do fígado sadio a partir de uma composição de espectros isolados de fluoróforos endógenos do fígado. Na segunda, realizaram-se estudos com corantes alimentícios Coralim-Mix® nas cores azul, verde e vermelho e com corantes Exciton® (Coumarin-480 e LDS-722) in vitro e in vivo, visando identificar os espectros dos corantes em misturas com meios turvos e entre eles. Na terceira, aplicou-se Photogem® em ratos Wistar e se coletou a fluorescência do FS nos fígados, relacionando-se a variação na intensidade de fluorescência com a concentração de FS presente e com perfis de necrose obtidos por TFD. Aplicou-se, por fim, os resultados obtidos na melhoria do modelo para previsão da Ynec. A reconstrução dos espectros não foi bem sucedida, tal qual a recuperação dos espectros dos corantes. Os resultados mostraram que muitos fatores contribuem para a distorção da fluorescência coletada, e que a informação obtida é prejudicada se estes forem ignorados. Verificou-se que a distribuição do FS não é homogênea num órgão fotossensibilizado. Obteve-se uma função para distribuição do FS no tecido e, através dela, foi possível melhorar o modelo para a previsão da Ynec. Observou-se que a turbidez do meio afeta de maneira complexa a coleta de fluorescência, criando obstáculos para a quantificação direta de fluoróforos nele inseridos. Fica evidente a necessidade do aprofundamento dos estudos sobre a interação da luz com as partículas dos meios turvos para remover as distorções geradas por estas. Demonstrou-se ainda a importância do mapeamento da distribuição do FS num tecido fotossensibilizado como parte da dosimetria da TFD, e que a espectroscopia de fluorescência é forte candidata à técnica mais apropriada para este mapeamento, desde que dominados os obstáculos à coleta de fluorescência.
Photodynamic Therapy (PDT) is a technique that implies in cell damage by the action of a photosensitizer (PS) with tumor tissue localization selectivity; light at PS absorption spectrum wavelengths, which leads the PS to a metastable triplet state; and molecular oxygen, which earns the energy absorbed by the PS, reaching a high oxidative potential singlet state. The technique has found sucess on the treatment of lesions as cancer. However, it finds difficulties for its dosimetry stablishment, like the quantification of PS distribuition in a photosensitized tissue. This work has three purposes: obtainance of fluorophores quantitative information into turbid media through fluorescence spectroscopy; to show the distribution of the PS Photogem® in healthy Wistar rats’ livers and its consequences on dosimetry; and the upgrade of an existing model for depth of necrosis (Ynec) forecast. There were three experimental stages: the first one was an attemp to rebuild a healthy liver spectrum from a composition using mathematical weights for isolate liver endogenous fluorophores spectra. On the second stage, in vitro and in vivo studies were performed using Coralim-Mix® blue, green and red food dyes and the Exciton® dyes Coumarin-480 and LDS-722, aiming to recover dyes spectra from dyes in turbid solutions and dyes mixtures. On the third one, Photogem® was administered to Wistar rats and fluorescence was collected on rats’ livers, and a relationship was stablished between the changes on fluorescence intensity, PS concentration in the tissue and necrosis profiles obtained via PDT. Results were applied to the upgrade of the Ynec forecast model. Spectra rebuilding, as well as dyes spectra recovering, were not completely reached. Results showed that a great deal of factors contribute to distortions at the collected fluorescence. It was verified that PS distribution is inhomogeneous in a photosensitized organ. It was found a function for the PS tissue distribution and it made possible to upgrade the Ynec forecast model. It was showed that medium turbidity affects in a complex manner the collected fluorescence, making difficult to quantify directly fluorophores in such medium. A need to go deeper into the investigation of light interactions with turbid media so that we may remove distortions they introduce into fluorescence spectra became evident. It was also showed how important is to track PS distribuition in a photosensitized tissue as a part of PDT dosimetry, and how fluorescence spectroscopy seems to be appropriate to perform such tracking, as long as the difficulties on fluorescence collection are overcome.

Photodynamic therapy (PDT) has shown to be a promising technique to treat various forms of malignant neoplasia. The photodynamic eradication of the tumor cells is achieved by applying a photosensitizer either locally or systemically and following local activation through irradiation of the tumor mass with light of a specific wavelength after a certain time of incubation. Due to preferential accumulation of the photosensitizer in tumor cells, this procedure allows a selective inactivation of the malignant tumor while sparing the surrounding tissue to the greatest extent. These features and requirements make the PDT an attractive therapeutic option for the treatment of retinoblastoma, especially when surgical enucleation is a curative option. This extreme solution is still in use in case of tumours that are resistant to conventional chemotherapy or handled too late due to poor access to medical care in less advanced country. In this study we initially conducted in-vitro investigations of the new cationic water-soluble photo sensitizer tetrahydroporphyrin-tetratosylat (THPTS) regarding its photodynamic effect on human Rb-1 and Y79 retinoblastoma cells. We were able to show, that neither the incubation with THPTS without following illumination, nor the sole illumination showed a considerable effect on the proliferation of the retinoblastoma cells, whereas the incubation with THPTS combined with following illumination led to a maximal cytotoxic effect on the tumor cells. Moreover the phototoxicity was lower in normal primary cells from retinal pigmented epithelium demonstrating a higher phototoxic effect of THPTS in cancer cells than in this normal retinal cell type. The results at hand form an encouraging foundation for further in-vivo studies on the therapeutic potential of this promising photosensitizer for the eyeball and vision preserving as well as potentially curative therapy of retinoblastoma.

L'objectif poursuivi au cours de ce travail est l'élaboration de nanoparticules biocompatibles à visée diagnostique (IRM) et thérapeutique (PDT). Dans ce but, un protocole de nanoprécipitation a été optimisé pour obtenir de façon quantitative et reproductible, des nanoparticules de PLGA de diamètre compatible avec une injection par voie parentérale. Cette formulation a été employée avec succès pour l'encapsulation d'un chélate lipophile de Gd(III), pour l'encapsulation d'un photosensibilisateur (m-THPC) et pour la co-encapsulation de ces deux substances actives. Les formulations optimales permettent d'obtenir des efficacités d'encapsulation de 7 et 46 % en chélate de gadolinium et m-THPC respectivement. La cytotoxicité et la photocytotoxicité des GdDO3AC12-mTHPC@PLGA ont été testées sur deux lignées cellulaires (C6 et fibroblastes) et les résultats obtenus montrent que les propriétés photocytotoxiques du m-THPC sont maintenues après l'encapsulation. L'efficacité IRM de ces nanoparticules a aussi été évaluée et les mesures NMRD et IRM à 3T montrent que l'encapsulation des chélates de gadolinium améliore leur capacité à générer un contraste en mode T1 et donc la qualité des images
This work aimed at the synthesis of biocompatible nanoparticles for PDT and MRI applications. To reach this goal, a nanoprecipitation technique was optimized using only biocompatible starting materials. This technique allowed the preparation, in a reproducible and quantitative manner of PLGA nanoparticles, compatible with parenteral injections. This formulation was successfully applied to encapsulate a lipophilic Gd(III) chelate, a photosensitizer (m-THPC) and to co-encapsulate these two substances. Optimal formulations showed encapsulation yields of 7 and 46 % for the gadolinium chelate and m-THPC, respectively. Cytotoxicity and photocytotoxicity experiments performed for two different cell lines (C6 cells and fibroblasts) incubated with GdDO3AC12-mTHPC@PLGA nanoparticles showed that m-THPC photocytotoxicity was maintained after its encapsulation. MRI efficacy of GdDO3AC12-mTHPC@PLGA nanoparticles was also evaluated by NMRD measurements and 3T MRI images. The corresponding results indicated that gadolinium chelate encapsulation improved its tendency to generate an efficient T1 contrast and consequently, enhanced the image contrast

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L'objet d'étude du travail est l’analyse de la conception de la Politique éducationnelle de Partis Politiques qui opèrent dans le quadre politique Brésilien: PFL, PRN, PDT ET PT Le travail a été divisé en quatre parts. D'abord c'est détaillé le quadre théorique que ira traverser l'analyse des partis cités a partir de deux points. Le premier s'agit des rapports théoriques utilisé dans études autour des politiques sociaux et spécifiquement éducationnelles. En suite aborde la relation éducation et travail dans le mode de production capitaliste. La deuxième part du travail est relatif aux présuppositions théoriques que fond les Partis Politique: Néolibéralisme et socialisme. C'est delinée les points principaux du concept d'éducation de chaque versant. La troisième part sont fait considérations sur les Partis partisans aux libéralisme: PFL et PRN et au socialisme: PT et PDT. Sont sintentizer les points principaux des Programmes de ces Partis la. Enfin c'est procedée l'analyze du concretizacion de deux politiques éducationnelles: Du gouvernement Collor et les proposition du Parti des Ouvriers dans les municipes sur son gouvernement.
O objeto de estudo desse trabalho é a análise da concepção de política educacional de partidos políticos atuantes no cenário político brasileiro: PFL, PRN, PDT e PT. O trabalho foi subdividido em quatro partes. Na parte inicial é detalhado o quadro teórico que permeará a análise dos referidos partidos a partir de dois enfoques. O primeiro trata dos referenciais teóricos utilizados nos estudos acerca das políticas sociais e especificamente das educacionais. O segundo aborda a relação entre educação e trabalho no modo de produção capitalista. A segunda parte do trabalho é relativa aos pressupostos teóricos que embassam os partidos políticos: Neoliberalismo e Socialismo. Nela são delineados os pontos principais do conceito de educação de cada vertente. Na terceira parte são efetuadas considerações sobre os partidos adeptos do neoliberalismo (PFL e PRN) e do Socialismo (PT e PDT). São sintetizados os pontos principais dos programas desses partidos. Na última parte é procedida a análise da concretização de duas políticas educacionais: a do Governo Collor e a do Partido dos Trabalhadores nos municípios sob sua administração.