O objetivo deste trabalho foi investigar a variação do comprimento de onda na fotoativação de resina composta com Laser de argônio, por meio, dos testes de microdureza Vickres, variação de temperatura durante o processo de polimerização, grau de conversão e ablação. Os experimentos de microdureza foram conduzidos utilizando o aparelho MMT-3 Hardness Tester (Buehler Lake Bluff, IlIinois USA) equipado com diamante Vickers. Para medir a temperatura foi utilizado um termistor de alta precisão conectado a multímetro onde foram medidos valores de resistência em (Ohm). Para medir o grau de conversão utilizou-se o Espectrofotômetro FTIR BOMEM, modelo MB-102, na faixa espectral de 4000 a 400 cm-1. As amostras foram ablacionadas com o laser de Er:YAG pulsado, no regime de microssegundos, após a ablação os diâmetros e profundidades foram medidos com uma lupa com aumento de 40X e um relógio comparador. As amostras foram ablacionadas com Laser Libra-S (Coherent, Pala Alto, CA, EUA), no regime de femtossegundos, e medidos os diâmetros das microcavidades por meio de uma imagem gerada pelo microscópio eletrônico de varredura MEV (DSM 960, Zeiss, Jena, Germany). As imagens foram processadas em um programa matemático, onde foram calculados os diâmetros e com isso, calculada a intensidade threshold de ablação. As amostras foram confeccionadas em resina composta do tipo microhíbrida Z-250 (3M-Espe) fotoativada com o laser de argônio (Coherent, Innova 200-20, USA). Foram utilizados os comprimentos de ondas de 476.5,488.0, 501.7 e 514 nm, com tempos de exposições de 15, 40, 60, 300, 900 segundos. Considerando os testes realizados o melhor comprimento de onda para a fotoativação foi 488.0 nm, conforme pôde ser visto nos experimentos abordados nessa dissertação. / The aims this work to investigate the variation of the wavelength in the photoactivation of composite resin with argon, by means, them tests of microhardness Vikres, variation of temperature during the polymerization process, degree of conversion and ablation. The microhardness experiments were lead using device MMT-3 Microhardness Tester (Buehler Lake Bluff, Illinois the USA) equipped with Vickers diamond. To measure the temperature rise the multimeter was used a thermister of high precision hardwired where values of resistance in (Ohm) were measured. To the degree of conversion was used Spectrophotometer FTIR BOMEM, model MB-102, in the spectral band of 4000 to 400cm-1. The samples were ablateds with the Er:YAG Laser, in the regimen of microseconds, the ablation the diameter and depth has been after measured with a magnifying glass with increase of 40X and comparing a clock. The sample were ablated with Libra-S Laser (Coherent, High Palo, CA, U.S.A.), in the regiment femtosecond, and measured the diameter of the microcavities, by means, of a image generated for the electron microscope of sweepings SEM (DSM 960, Zeiss, Jena, Germany). The images were processed in a mathematical program, where the diameters were calculated and with this, calculated the threshold intensity of ablation. The samples had been confectioned in composed resin of the photo-activad Z-250 (3M-ESPE) microhibrid type with the argon laser (Coherent, Innova 200-20, the USA). The wavelength of 476.5, 488.0, 501.7 and 514.5 nm were used, with exposure times of 15, 40, 60, 300, 900 seconds. Considering the five carried through tests optimum wavelength for the photo-activation was 488.0 nm, o it could be in the boarded experiment in this dissertation.

Smart nanomaterials can selectively respond to a stimulus and consequently be activated in specific conditions, as a result of their interaction with electromagnetic radiation, biomolecules, pH change, etc. These nanomaterials can be produced through distinct routes and be used in artificial skin, drug delivery, and other biomedical applications. In this thesis, two smart nanosystems were developed, viz., i) nanocapsules formed by aniline (A) and chitosan (CS) (A-CS) containing silver nanoparticles (AgNPs), with an average size of 78 ± 19 nm, and ii) polycaprolactone (PCL) nanofibers, fabricated by the electrospinning technique containing AgNP into their bulk, with a diameter of 417 ± 14 nm. A novel system, based on the incorporation of the as-prepared nanocapsules onto the surface of PCL nanofibers containing AgNps (antibacterial mats), was also developed. The methodology employed avoids the direct contact of silver nanoparticles with the host and optimizes its release to the surrounding environment. The AgNPs release was triggered by exposing the nanocapsules to light at 405 nm. Consequently, the electronic energy vibration resulting from the interaction of the irradiation with the surface plasmon band (SPR) of AgNps, breaking the hydrogen bonds of the nanocapsules and releasing of AgNPs at a time of 150 s. To understand the perturbation of AgNps-Nanocapsules against bacteria, membrane models using Langmuir technique with the phospholipids 1,2-dipalmitoyl-sn-glycero-3-phospho-(1\'-rac-glycerol) (DPPG) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) were employed, which are the main components of cell membrane of Escherichia coli (E. coli). The results suggest that DPPG has more influence on the incorporation of the nanoparticles on the cell membrane. The antibacterial properties of the nanofibers/nanomaterials mats towards E. coli and Staphylococcus aureus (S. aureus) were investigated using the Agar diffusion test for 8 samples. The experiments revealed that the samples based on nanofibers/nanocapsules and irradiation presented a radius of inhibition of 2.58 ± 0.28 mm for S. aureus and 1.78 ± 0.49 mm for E. coli. This nanosystem showed to be highly interesting for biomedical applications. / Nanomateriais inteligentes podem responder seletivamente a um estímulo e consequentemente ser ativados em condições específicas, como resultado da sua interação com a radiação eletromagnética, mudança do pH, campo magnético, etc. Esses materiais podem ser produzidos através de distintas rotas e utilizados em aplicações como pele artificial, liberação de fármacos, e outras aplicações biomédicas. Nessa tese, dois nanossistemas inteligentes foram desenvolvidos, a saber: i) nanocápsulas formadas por anilina (A) e quitosana (CS) (A-CS) contendo nanopartículas de prata (AgNps), com um tamanho médio de 78 ± 19 nm, e ii) nanofibras de policaprolactona (PCL), fabricadas pela técnica de eletrofiação contendo AgNps em seu interior, com diâmetro de 417 ± 14 nm. Um terceiro sistema foi desenvolvido, baseado na incorporação das nanocápsulas na superfície das nanofibras de PCL contendo AgNps (manta antibacteriana). A metodologia utilizada evita o contato direto das nanopartículas de prata com o hospedeiro e otimiza sua liberação no meio ambiente. As AgNps liberadas foram acionadas pela exposição das nanocápsulas à um fonte de luz em 405 nm. Consequentemente, a vibração da energia eletrônica resultante da interação da irradiação com a banda plasmônica de superfície (SPR) das AgNps, quebra as ligações de hidrogênio da nanocápsula e libera as AgNps no meio em um tempo de 150 s. Para entender a perturbação das AgNps-nanocapsulas contra as bactérias, modelos de membrana foram usados através da técnica de Langmuir com os fosfolipídios 1,2-dipalmitoil-sn-glicero-3- fosfo-(1\'-rac-glicerol) (DPPG) and 1,2-dimiristoil-sn-glicero-3-fosfoetanolamina (DMPE), que são os principais componentes da membrana celular de Escherichia coli (E. coli). Os resultados sugerem que DPPG tem mais influência na incorporação das nanopartículas na membrana celular. As propriedades antibacterianas das mantas de nanofibras/nanomateriais contra E. coli e Staphylococus aureus (S. aureus) foram investigadas usando o teste de difusão Agar em 8 grupos, o qual revelou que o grupo contendo a nanofibra/nanocapsula e irradiação apresentou um raio de inibição de 2.58 ± 0.28 mm para S. aureus e 1.78 ± 0.49 mm para E. coli. Este nanossistema mostrou ser altamente interessante para aplicações biomédicas.

Le photosystème II (PSII) est un complexe multi-protéique qui utilise l'énergie solaire pour oxyder l'eau et réduire des quinones. Le site catalytique d'oxydation de l'eau est localisé coté lumen du complexe, alors, que le site de réduction comprenant deux quinones (QA et QB) et un fer non-hémique est localisé sur le coté stromal du complexe membranaire. Dans cette thèse j'ai étudié les deux cotés accepteur et donneur d'électrons du PSII.QA*- et QB*- sont couplés magnétiquement au fer non-hémique donnant de faibles signaux RPE. Le fer non-hémique possède quatre ligands histidines et un ligand (bi)carbonate échangeable. Le formate peut échanger le ligand (bi)carbonate induisant un ralentissement dans le transfert d'électrons. Ici, je décris une modification du signal RPE de QB*- Fe2+ lorsque le formate est substitué au (bi)carbonate. J'ai aussi découvert un second signal RPE dû à la présence du formate à la place du (bi)carbonate lorsque QB est doublement réduit. De plus, j'ai trouvé que les signaux RPE natifs de QA*- Fe2+ et QB*- Fe2+ possèdent une signature intense encore jamais détectée. Tous les signaux RPE rapportés dans cette thèse devraient faciliter le titrage redox de QB par RPE. J'ai aussi observé que QB*- peut oxyder le fer non-hémique à l'obscurité en anaérobie. Cette observation implique qu'au moins dans une fraction des centres, le couple QB*-/QBH2 possède un potentiel redox plus haut que supposé. La quantification du nombre de centres où cette oxydation du fer se produit par le couple QB*-/QBH2 reste à faire. La réduction du PSII par le dithionite génère un signal modifié de QA*-Fe2+, un changement structural du PSII observé par électrophorèse. Cela peut indiquer la réduction d'un pont disulfure à l'intérieur du PSII. Concernant le site d'oxydation de l'eau, j'ai étudié la première étape de l'assemblage du site catalytique (Mn4Ca), en suivant l'oxydation du Mn2+ par RPE en bande X et haut champ. J'ai mis au point des conditions expérimentales permettant le piégeage du premier intermédiaire et j'ai aussi trouvé une incohérence avec des travaux publiés dans la littérature. J'ai aussi trouvé que le dithionite pouvait réduire le site catalytique Mn4Ca, en formant des états sur-réduits qui peuvent correspondre aux intermédiaires de l'assemblage du cluster Mn4Ca.

Smart nanomaterials can selectively respond to a stimulus and consequently be activated in specific conditions, as a result of their interaction with electromagnetic radiation, biomolecules, pH change, etc. These nanomaterials can be produced through distinct routes and be used in artificial skin, drug delivery, and other biomedical applications. In this thesis, two smart nanosystems were developed, viz., i) nanocapsules formed by aniline (A) and chitosan (CS) (A-CS) containing silver nanoparticles (AgNPs), with an average size of 78 ± 19 nm, and ii) polycaprolactone (PCL) nanofibers, fabricated by the electrospinning technique containing AgNP into their bulk, with a diameter of 417 ± 14 nm. A novel system, based on the incorporation of the as-prepared nanocapsules onto the surface of PCL nanofibers containing AgNps (antibacterial mats), was also developed. The methodology employed avoids the direct contact of silver nanoparticles with the host and optimizes its release to the surrounding environment. The AgNPs release was triggered by exposing the nanocapsules to light at 405 nm. Consequently, the electronic energy vibration resulting from the interaction of the irradiation with the surface plasmon band (SPR) of AgNps, breaking the hydrogen bonds of the nanocapsules and releasing of AgNPs at a time of 150 s. To understand the perturbation of AgNps-Nanocapsules against bacteria, membrane models using Langmuir technique with the phospholipids 1,2-dipalmitoyl-sn-glycero-3-phospho-(1\'-rac-glycerol) (DPPG) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) were employed, which are the main components of cell membrane of Escherichia coli (E. coli). The results suggest that DPPG has more influence on the incorporation of the nanoparticles on the cell membrane. The antibacterial properties of the nanofibers/nanomaterials mats towards E. coli and Staphylococcus aureus (S. aureus) were investigated using the Agar diffusion test for 8 samples. The experiments revealed that the samples based on nanofibers/nanocapsules and irradiation presented a radius of inhibition of 2.58 ± 0.28 mm for S. aureus and 1.78 ± 0.49 mm for E. coli. This nanosystem showed to be highly interesting for biomedical applications. / Nanomateriais inteligentes podem responder seletivamente a um estímulo e consequentemente ser ativados em condições específicas, como resultado da sua interação com a radiação eletromagnética, mudança do pH, campo magnético, etc. Esses materiais podem ser produzidos através de distintas rotas e utilizados em aplicações como pele artificial, liberação de fármacos, e outras aplicações biomédicas. Nessa tese, dois nanossistemas inteligentes foram desenvolvidos, a saber: i) nanocápsulas formadas por anilina (A) e quitosana (CS) (A-CS) contendo nanopartículas de prata (AgNps), com um tamanho médio de 78 ± 19 nm, e ii) nanofibras de policaprolactona (PCL), fabricadas pela técnica de eletrofiação contendo AgNps em seu interior, com diâmetro de 417 ± 14 nm. Um terceiro sistema foi desenvolvido, baseado na incorporação das nanocápsulas na superfície das nanofibras de PCL contendo AgNps (manta antibacteriana). A metodologia utilizada evita o contato direto das nanopartículas de prata com o hospedeiro e otimiza sua liberação no meio ambiente. As AgNps liberadas foram acionadas pela exposição das nanocápsulas à um fonte de luz em 405 nm. Consequentemente, a vibração da energia eletrônica resultante da interação da irradiação com a banda plasmônica de superfície (SPR) das AgNps, quebra as ligações de hidrogênio da nanocápsula e libera as AgNps no meio em um tempo de 150 s. Para entender a perturbação das AgNps-nanocapsulas contra as bactérias, modelos de membrana foram usados através da técnica de Langmuir com os fosfolipídios 1,2-dipalmitoil-sn-glicero-3- fosfo-(1\'-rac-glicerol) (DPPG) and 1,2-dimiristoil-sn-glicero-3-fosfoetanolamina (DMPE), que são os principais componentes da membrana celular de Escherichia coli (E. coli). Os resultados sugerem que DPPG tem mais influência na incorporação das nanopartículas na membrana celular. As propriedades antibacterianas das mantas de nanofibras/nanomateriais contra E. coli e Staphylococus aureus (S. aureus) foram investigadas usando o teste de difusão Agar em 8 grupos, o qual revelou que o grupo contendo a nanofibra/nanocapsula e irradiação apresentou um raio de inibição de 2.58 ± 0.28 mm para S. aureus e 1.78 ± 0.49 mm para E. coli. Este nanossistema mostrou ser altamente interessante para aplicações biomédicas.

O objetivo deste trabalho foi investigar a variação do comprimento de onda na fotoativação de resina composta com Laser de argônio, por meio, dos testes de microdureza Vickres, variação de temperatura durante o processo de polimerização, grau de conversão e ablação. Os experimentos de microdureza foram conduzidos utilizando o aparelho MMT-3 Hardness Tester (Buehler Lake Bluff, IlIinois USA) equipado com diamante Vickers. Para medir a temperatura foi utilizado um termistor de alta precisão conectado a multímetro onde foram medidos valores de resistência em (Ohm). Para medir o grau de conversão utilizou-se o Espectrofotômetro FTIR BOMEM, modelo MB-102, na faixa espectral de 4000 a 400 cm-1. As amostras foram ablacionadas com o laser de Er:YAG pulsado, no regime de microssegundos, após a ablação os diâmetros e profundidades foram medidos com uma lupa com aumento de 40X e um relógio comparador. As amostras foram ablacionadas com Laser Libra-S (Coherent, Pala Alto, CA, EUA), no regime de femtossegundos, e medidos os diâmetros das microcavidades por meio de uma imagem gerada pelo microscópio eletrônico de varredura MEV (DSM 960, Zeiss, Jena, Germany). As imagens foram processadas em um programa matemático, onde foram calculados os diâmetros e com isso, calculada a intensidade threshold de ablação. As amostras foram confeccionadas em resina composta do tipo microhíbrida Z-250 (3M-Espe) fotoativada com o laser de argônio (Coherent, Innova 200-20, USA). Foram utilizados os comprimentos de ondas de 476.5,488.0, 501.7 e 514 nm, com tempos de exposições de 15, 40, 60, 300, 900 segundos. Considerando os testes realizados o melhor comprimento de onda para a fotoativação foi 488.0 nm, conforme pôde ser visto nos experimentos abordados nessa dissertação. / The aims this work to investigate the variation of the wavelength in the photoactivation of composite resin with argon, by means, them tests of microhardness Vikres, variation of temperature during the polymerization process, degree of conversion and ablation. The microhardness experiments were lead using device MMT-3 Microhardness Tester (Buehler Lake Bluff, Illinois the USA) equipped with Vickers diamond. To measure the temperature rise the multimeter was used a thermister of high precision hardwired where values of resistance in (Ohm) were measured. To the degree of conversion was used Spectrophotometer FTIR BOMEM, model MB-102, in the spectral band of 4000 to 400cm-1. The samples were ablateds with the Er:YAG Laser, in the regimen of microseconds, the ablation the diameter and depth has been after measured with a magnifying glass with increase of 40X and comparing a clock. The sample were ablated with Libra-S Laser (Coherent, High Palo, CA, U.S.A.), in the regiment femtosecond, and measured the diameter of the microcavities, by means, of a image generated for the electron microscope of sweepings SEM (DSM 960, Zeiss, Jena, Germany). The images were processed in a mathematical program, where the diameters were calculated and with this, calculated the threshold intensity of ablation. The samples had been confectioned in composed resin of the photo-activad Z-250 (3M-ESPE) microhibrid type with the argon laser (Coherent, Innova 200-20, the USA). The wavelength of 476.5, 488.0, 501.7 and 514.5 nm were used, with exposure times of 15, 40, 60, 300, 900 seconds. Considering the five carried through tests optimum wavelength for the photo-activation was 488.0 nm, o it could be in the boarded experiment in this dissertation.

Tau proteins are members of the microtubule associated proteins (MAPs), and are predominantly expressed in neurons and enriched in the axonal compartment. These proteins are involved in many diseases therefore termed tauopathies . In the disease, tau is present in a hyperphosphorylated state that forms aggregates in the somato-dentritic compartment. In order to analyse the distribution of normal and tau proteins present in tauopathies in living cells, a pulse-escape fluorescence photoactivation approach was developed. A wild type wt , a R406W mutant mut , a hyperphosphorylation model PHP (pseudohyperphosphorylated), and a smaller fragment delta tau protein, and a control PA-GFPx3 were fused to the photoactivatable GFP protein. These proteins were expressed in differentiated PC12 cells and mice primary cortical cultures, and analysed in photoactivation experiments. The data showed that the wt protein was less mobile, both at the shaft or the tip of cell processes, than the delta or control proteins (higher immobile fraction, IF ). This could be attributed to the microtubule binding domain present only in the wt. Treatment of cells with drugs to disrupt microtubules, or detach tau from the filaments confirmed this interpretation. The mut mobility was comparable to that seen for the wt, suggesting that the interaction with microtubules was unaffected by the mutation. The PHP showed a lower IF compared to the wt, in agreement with a lower binding to microtubules. Furthermore, this behaviour could be reproduced by increasing the level of phosphorylation of the wt by drug treatment. A flux analysis was performed to determine the fraction of protein moving towards the distal or proximal portion of the process. Only delta and wt detached from the microtubules showed an increased flux towards the tip.The results suggest that the plasma membrane interaction is involved in the flux of wt tau proteins towards the distal portion.

No description available.

Les protéines d’adhésions homophiles N-cadhérine jouent un rôle majeur dans le développement du cerveau, notamment en agissant sur la croissance et la plasticité synaptique. Au cours de ma thèse, j’ai étudié le rôle de la N-cadhérine dans ces deux processus en utilisant des neurones issus de cultures primaires déposés sur des substrats micropatternés. Ces substrats sont recouverts de N-cadhérine purifiée afin d’induire des adhésions N-cadhérines sélectives au niveau de micro-motifs régulièrement espacés. Mes deux premières études sont basées sur le modèle d’embrayage moléculaire, décrivant le processus par lequel la motilité du cytosquelette d’actine se couple aux adhésions au niveau de la membrane cellulaire afin de générer des forces de traction aux zones de contact avec le substrat, permettant ainsi l’avancée cellulaire (Giannone et al., 2009). Plusieurs études ont mis en avant l’existence d’un tel modèle (Mitchison et Kirschner, 1988 ; Suter et Forscher, 1998), cependant le mécanisme exact permettant d’expliquer ce couplage mécanique de l’actine aux protéines d’adhésions reste mal connu. Via des techniques de pinces optiques, des travaux précédemment menés dans l’équipe ont prouvé l’existence d’un couplage entre le flux d’actine et les adhésions N-cadhérine permettant la migration du cône de croissance (Bard et al., 2008). Cette technique n’a cependant pas permis la visualisation directe de l’engagement d’un tel mécanisme. Nous avons donc couplé l’utilisation des substrats micro-patternés à la microscopie haute résolution sptPALM/TIRF afin de visualiser directement la dynamique des protéines impliquées dans l’embrayage moléculaire. Dans le premier article, j’ai montré pour la première fois l’existence d’interactions transitoires entre le flux d’actine et les adhésions N-cadhérines au niveau du cône de croissance, reflétant un embrayage glissant à l’échelle de la molécule unique (Garcia et al., en préparation). Dans le second article, en travaillant sur des neurones plus matures, nous avons pu montrer l’engagement d’un embrayage moléculaire trans-synaptique entre adhésions N-cadhérines et flux d’actine permettant la stabilisation du filopode dendritique et ainsi sa transition en épine mature (Chazeau/Garcia et al., en préparation). J’ai également participé à une troisième étude dans laquelle j’ai observé l’effet des substrats micropatternés recouverts de N-cadhérine, sur la synaptogenèse. J’ai ainsi pu prouver que la N-cadhérine déposée sur les micro-motifs, stimule la croissance dendritique et axonale et joue un rôle prépondérant dans la maturation morphologique des neurones. Cependant, la N-cadhérine est incapable d’induire la formation de synapses contrairement aux protéines d’adhésion neurexine/neuroligine ou SynCam (Czöndör et al., 2013). / The homophilic adhesion molecule N-cadherin plays major roles in brain development, notably affecting axon outgrowth and synaptic plasticity. During my PhD work, I addressed the role of N-cadherin in these two processes, using primary neurons cultured on micro-patterned substrates. These substrates are coated with purified N-cadherin to trigger selective N-cadherin adhesions in a spatially controled manner. My two first studies are based on the “molecular clutch” paradigm, by which the actin motile machinery is coupled to adhesion at the cell membrane to generate forces on the substrate and allow cells to move forward (Giannone et al., 2009). Many publications have provided evidence for such a mechanism (Mitchison et Kirschner, 1988 ; Suter et Forscher, 1998), but the exact mechanisms underlying the molecular coupling between the actin retrograde flow and adhesion proteins remain elusive. The team previously inferred, using optical tweezers, that a molecular clutch between the actin flow and N-cadherin adhesions drives growth cone migration (Bard et al., 2008), but could not achieve a direct visualization of the engagement process with this technique. Here, we combined the use of micropattern substrates with high resolution microscopy sptPALM/TIRF to visualize directly the dynamics of the main proteins involved in the molecular clutch. In my first paper, I reveal for the first time transient interactions between the actin flow and N-cadherin adhesions in growth cones, reflecting a slipping clutch process at the individual molecular level (Garcia et al., in preparation). In a second study, working with more mature neurons, we revealed that engagement of a molecular clutch between trans-synaptic N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines (Chazeau/Garcia et al., in preparation). I also participated to a third study, where I observed the effect of N-cadherin coated substrates on synaptogenesis. I showed that, although N-cadherin on micro-patterned substrates stimulated axonal and dendritic elongation and played a major role in morphological maturation, it was not able to induce synapse formation like neurexin/neuroligin or SynCAM adhesions (Czöndör et al., 2013).

Les complexes de lanthanides, grâce à un temps de décroissance de luminescence élevé, sont parmi les sondes les plus sensibles puisqu'ils permettent une mesure de luminescence en temps résolu. Ces complexes sont à la base de certaines technologies permettant, in vitro et/ou in cellulo, la détection d'interactions entre biomolécules et/ou leur quantification et localisation. Elaborer des complexes de lanthanides présentant un maximum d'absorption au-delà de 350 nm reste un des défis majeurs alors qu'à ces rayonnements UV le matériel biologique se dégrade moins. Un chélate luminescent d'Eu(III) fondé sur une structure original bis-pyridinylpyrazine possédant un maximum d'absorption à 345 nm, stable jusqu'à 355 nm, a été synthétisé. Un analogue portant une fonction bioconjugable a permis, après activation par un réactif de couplage homo-bifonctionnel, de marquer des anticorps anti c-myc et anti HA. La deuxième partie du projet porte sur le développement de sondes lanthanidiques photoréactives capables de détecter spécifiquement, puis de s'accrocher de façon covalente à des protéines d'intérêt taguées (polyHis ou polyAsp), afin de suivre leur trafic cellulaire et leurs interactions avec d'autres biomolécules. Idéalement, l'étape de reconnaissance devrait avoir lieu entre l'étiquette et le cation métallique suivi par l'accrochage covalent initié par photoactivation. Ceci devrait conduire à une modulation du signal émis par le marqueur lanthanidique. Pour ce faire, différents complexes comportant des benzophénones, azido-coumarines ou quinolinones ont été synthétisés.

Cancer is responsible for about 25% of deaths in developed countries and for 15% of all deaths worldwide. Cancer is a devastating disease and while there have been great advances in the development of anticancer drugs, off-target toxicity is a major limitation with conventional cancer chemotherapy. The consequence of this form of treatment results in the killing of healthy and rapidly-growing non-cancerous cells including cells in the bone marrow; hair follicles; and cells in the mouth, digestive tract, and reproductive system. In addition to these general effects, certain anticancer drugs can have other associated toxicities. For instance, the anticancer drug doxorubicin (Dox), which my research has focused around, has dose-limiting cardiotoxicity. For these reasons, there has been much research focused on improving the selectivity of anticancer drugs in order to lower their toxicity to normal cells and decrease associated side effects. To circumvent the problem of non-selectivity, many methods have been developed to target cancer cells regionally at the site of the tumor. One such method is photodynamic therapy (PDT) which relies on a photosensitizer that is activated using light directed towards the tumor. Once activated, the photosensitizer creates singlet oxygen, which is cytotoxic to the illuminated tumor cells. PDT has shown profound effects in the treatment of many cancers including head and neck, lung, bladder, prostate, and esophagus. As PDT continues to be streamlined, it has the potential to serve as a standalone modality in the treatment and management of cancer at different stages. However, there are many inadequacies in the use of PDT. The fundamental problem lies in the inability of PDT to treat solid, bulky tumors or deep-seated tumors. This is partly due to the fact that current PS used cannot effectively kill cancer cells because with increasing tissue thickness the number of hypoxic cells increases. We have designed a new light based drug delivery which will allow for drugs to be released on the surface of the tumor, allowing the drug to freely diffuse through the tumor without the need of O2 and also, due to the manner our drug is attached, a more potent form the pre-attached drug is released. In this methodology, drug delivery will only be specific to an area of interest and the potential for side effects emerging from chemotherapy should be limited. Here we have shown, for the first time the generation of highly potent drug in a light dependent manner via a photocaging molecule and the commonly used chemotherapy agent Dox. We designed our photocage to generate a latent reactive form of Dox after illumination with UV light. This intermediate reacts in an intramolecular fashion to generate a highly potent form of the drug compared to its previous unattached form. Because of its high potency we have named the cleaved drug Super-Dox. Once the synthesis of the photocleavable drug conjugate was complete and we confirmed the photorelease of Super-Dox, via photolytic assay, and confirmed our drug conjugate is 80% cleaved from its photocage after 30 minutes of irritation with UV light. Next, we performed cell viability assays using MCF7 breast cancer cells to determine the efficiency of our drug conjugate to induce cell death. Our drug conjugate was able to induce significant cell death in the presence of light when compared to the dark. When compared to Dox, our cleaved drug conjugate is 26 folds more potent with light illumination an when compared to toxicity in the dark our drug conjugate was 374 fold less potent when conjugated to the photocage. However, our drug conjugate was not completely benign in the dark due to cell permeability, evidenced by confocal microscopy, we have worked equip our linker with sulfated cell impermeable group that will lower its background toxicity in the dark and allow us to achieve even higher enhancements in activity with light. This type of innovation creates a new avenue into cancer treatment which can limit adverse side effects and improve overall treatment.