Este trabalho teve como objetivo a montagem de um sistema de otimização de pulsos ultracurtos (oscilador laser modelocked de 15 fs), através de uma técnica de formatação de pulsos via absorção de dois fótons em compostos orgânicos. Está técnica utiliza uma estratégia evolucionária baseada em um algoritmo genético, onde se controla o formato do pulso pela deformação imposta a um espelho deformável, conjuntamente com o monitoramento de um sinal de realimentação. Desta forma, este sistema permite tanto a otimização do processo de absorção de dois fótons, quanto a otimização do próprio pulso do sistema laser. Após a montagem inicial do sistema de formatação de pulsos, foram implementados três métodos de otimização via monitoramento do processo de absorção de dois fótons, sendo que dois deles foram desenvolvidos nesta dissertação. Os métodos diferem entre si pelo emprego de distintos sinais de realimentação para o processo de otimização: (i) intensidade da fluorescência excitada por dois fótons; (ii) variação da transmitância não linear dos compostos orgânicos devido à absorção de dois fótons; e (iii) intensidade do efeito de lente térmica apresentada pelos compostos orgânicos após a absorção de dois fótons. Os três métodos de otimização apresentaram resultados similares e satisfatórios, aproximando a largura temporal do pulso ao final do processo de otimização da largura temporal dada pelo limite da transformada de Fourier, medidas através de técnicas de autocorrelação. Estes resultados apontam para a validade do uso dos métodos por nós desenvolvidos como alternativas para processos de otimização de pulsos ultracurtos. / In this work it is described the implementation of an ultrashort pulse optimization system (15 fs modelocked oscillator) that employs pulse shaping methods via two-photon absorption in organic materials. This technique uses an evolutionary strategy based on a Genetic Algorithm, where the pulse shape is controlled by a deformable mirror, while a feedback signal is monitored. In this way, this system allows both, the two-photon absorption process and pulse optimization. After the accomplishment of the pulse shaping system, we have implemented three distinct optimization methods via two-photon absorption monitoring, being two of them proposed in the present dissertation. These three methods differs from each other by the use of different feedback signals for the optimization process: (i) intensity of the two-photon excited fluorescence; (ii) nonlinear transmittance change in organic compounds due to the two-photon absorption; and (iii) intensity of the thermal lens effect. All optimization methods presented similar and satisfactory results, leading the ultrashort pulse, in the end of the optimization process, close to the Fourier transformed limit. In such cases, the pulse duration were determined through the autocorrelation technique. These results indicates that the new methods proposed here can be used as an alternative for both, pulse optimization and control of two-photon absorption process, specially for nonfluorescent samples.

Este trabalho demonstra o uso da fotopolimerização via absorção de dois fótons na produção de microestruturas dopadas com compostos orgânicos e nanopartículas de Au. A capacidade de produção de microestruturas com propriedades variadas é extremamente relevante, pois viabiliza o desenvolvimento de uma nova geração de dispositivos ópticos. Além disso, realizamos a conexão entre as microestruturas fabricadas e fontes de excitação, por meio de nanofibras de vidro. A integração entre essas estruturas, e destas com meios externos de excitação e detecção, é um passo essencial para o desenvolvimento de microcircuitos fotônicos, que podem representar uma nova revolução tecnológica, a exemplo do que foram os microcircuitos eletrônicos. Exploramos as possibilidades de dopagem da resina usando: (i) um composto fluorescente, (ii) um composto com birrefringência fotoinduzida e (iii) nanopartículas de ouro. Microestruturas contendo Rodamina B apresentaram boa integridade estrutural e fluorescência, tendo sido usadas para demonstrar a conexão dos microelementos com meios externos de excitação. Através de nanofibras e de micromanipuladores, comprovamos a capacidade de excitação seletiva de microestruturas através do guiamento da luz de um laser de Ar+. Estruturas birrefringentes foram obtidas pela dopagem com o azopolímero HEMA-DR13. Montamos um aparato que permite a observação da dinâmica de indução de birrefringência nas microestruturas, o qual representa um grande passo na caracterização deste tipo de microelementos. Com base nesse estudo, foi possível alcançar uma fração de birrefringência residual nas microestruturas de 35%. Por fim, propomos um método para a dopagem de microestruturas poliméricas com nanopartículas de ouro. Por se tratar de um método de dopagem indireta, ele evita interferências das nanopartículas no processo de microfabricação. Dessa forma, este trabalho abre possibilidades para a fabricação de microdispositivos funcionais com diversas propriedades especiais, bem como a integração desses microdispositivos em circuitos fotônicos. / This work demonstrates the use of two-photon photopolymerization in the fabrication of microstructures doped with organic compounds and gold nanoparticles. The ability to produce microstructures with different properties is extremely relevant, because it opens the possibility for the development of a new generation of optical devices. Besides, we have accomplished the connection between fabricated microstructures and excitation sources by means of silica nanowires. The connection among structures and with external means of detection and excitation is an essential step towards the development of new technological breakthrough in photonic microcircuits. We have explored the resin doping possibilities by using: (i) a fluorescent compound, (ii) a photoinduced birefringent compound and (iii) gold nanoparticles. Rhodamine B doped microstructures present good structural integrity and fluorescence, and were able to demonstrate the connection of microelements with external means of excitation. Through the use of nanofiber tapers and micromanipulators, we have shown the selective excitation capability of this method by guiding Ar+ laser light onto one single microstructure. Birefringent samples were obtained by doping the resin with the azopolymer HEMA-DR13. We have assembled an apparatus that allows observing the photoinduced birefringence dynamics, which represents a great step towards a better characterization of these kinds of microelements. Based on this study we were able to achieve a residual birefringence fraction of 35% in microscopic samples. Finally, we have proposed a new method for the doping of polymeric microstructures with gold nanoparticles. Because it is an indirect doping technique, it prevents gold nanoparticles from interfering with the microfabrication process. Thus, the work presented here paves the way for the fabrication of functional microdevices with a wide range of special properties, as well as for the connection of these microstructures for photonic microcircuit.

Os micro-ressonadores que suportam whispering gallery modes têm atraído a atenção da comunidade científica devido a sua grande capacidade de confinar a luz, propriedade que faz dessas estruturas plataformas ideais para o desenvolvimento de pesquisa fundamental como interação da radiação com a matéria e óptica não linear. Além disso, suas características como operação em frequências do visível e de telecomunicações, facilidade de integração e alta sensitividade os tornam extremamente flexíveis para aplicações que vão desde filtros ópticos até sensores. Neste trabalho, demonstramos a fabricação de tais micro-ressonadores via fotopolimerização por absorção de dois fótons (FA2F). Esta técnica apresenta uma série de vantagens para a confecção de micro-dispositivos, sendo elas a capacidade de resolução inferior ao limite de difração, a flexibilidade de formas e ainda, a possibilidade de incorporar compostos de interesse à matriz polimérica a fim de introduzir novas funcionalidades ao material que compõe a estrutura final. Ademais, diferentes polímeros podem ser utilizados para a fabricação das microestruturas, tornando a técnica viável para uma vasta gama de aplicações. As microestruturas poliméricas que fabricamos são micro-cilindros ocos de boa integridade estrutural com 45 μm de diâmetro externo e 100 nm de rugosidade de superfície, o que as torna potencialmente aplicáveis como micro-ressonadores para frequências de operação típicas de telecomunicações. A fim de acoplar luz nessas estruturas, em colaboração com a Universidade de Valência, na Espanha, montamos um aparato de acoplamento. Neste aparato, a luz proveniente de uma fonte de luz centrada em 1540 nm é acoplada nos micro-ressonadores via campo evanescente por meio do uso de uma fibra óptica estirada de 1.5 μm de diâmetro. A potência transmitida é guiada para um analisador de espectro óptico, onde é possível identificar os modos ressonantes, representados como picos de atenuação com free spectral range em torno de 9.8 nm. Ao término desse projeto, um aparato similar foi montado no Grupo de Fotônica do IFSC/USP, a partir do qual pudemos medir os modos ressonantes tanto de fibras ópticas estiradas quanto dos micro-cilindros poliméricos. A finesse dos micro-ressonadores poliméricos caracterizados varia de 2.51 a 4.35, sendo da mesma ordem de grandeza do valor reportado na literatura para ressonadores de alta performance fabricados por FA2F a partir da mesma formulação de resina polimérica que utilizamos. / Whispering gallery modes microresonators have been attracting increasing interest due to their ability to strongly confine light within small dielectric volumes. This property is quite useful for basic research involving light-matter interaction and nonlinear optics, but their applications go beyond that. The ease of fabrication, on-chip integration and operation at telecommunication frequencies make them suitable for a variety of practical applications, including photonic filters and sensing. In the current work, we demonstrate the fabrication of such resonators via two-photon polymerization. Using this technique, complex 3D structures with submicrometer feature size can be produced. Besides, the flexibility of geometry and the possibility of incorporating a variety of additional materials, such as organic compounds make it a powerful tool for the fabrication of microresonators. The microstructures we have fabricated are 45 μm outer diameter hollow microcylinders, with good structural integrity and sidewall roughness estimated in 100 nm, which make their application as microresonators feasible in the near infrared wavelength regime. In order to couple light within these microresonators, an experimental setup was built at University of Valencia to implement the coupling. In this setup, light from a 1540 nm-centered broadband source was coupled into the fabricated microresonators via evanescent field using a 1.5 μm waist tapered fiber. The transmitted light was then guided to an optical spectral analyzer, where it was possible to measure resonances, represented as attenuation peaks, with free spectral range of about 9.8 nm. Afterwards, a similar experimental setup was assembled in the Photonics group at IFSC/USP, where we could observe resonances of both tapered optical fibers and the polymeric microresonators fabricated by means of two-photon polymerization. The finesse of the polymeric microresonators was estimated in 4.35, being in the same order of the finesse reported in the literature for high performance microring resonators fabricated using the same polymeric resin.

Microestruturas poliméricas dopadas despertam grande interesse nas áreas de óptica, fotônica e biologia, pois viabilizam a produção de dispositivos com propriedades específicas. Contudo, a dopagem de microestruturas com mais de um dopante é pouco explorada na literatura. Nesse contexto, o presente trabalho propõe o desenvolvimento de uma metodologia para fabricar microestruturas poliméricas com múltiplas dopagens, através de fotopolimerização por absorção de dois fótons. Esta técnica de microfabricação faz uso de um feixe laser pulsado (Ti:safira, 780 nm, 100 fs) que é focalizado, através de lentes de microscópio, no volume de uma resina polimérica contendo fotoiniciador composto orgânico responsável por iniciar o processo de polimerização. A intensidade dos pulsos de femtossegundos é alta o bastante para que processos não lineares absorcivos, nesse caso absorção de dois fótons, ocorram apenas no volume focal, induzindo a polimerização apenas ao seu redor. Como dopantes utilizamos os corantes fluorescentes Rodamina e Fluoresceína. Para averiguar a eficácia do sistema de fabricação desenvolvido, produzimos estruturas com apenas um dopante, e as caracterizamos utilizando microscopia óptica e eletrônica. Microestruturas contendo mais de um dopante, em regiões distintas, foram produzidas através da fabricação sequencial de estruturas dopadas. Essa metodologia permite a produção de estruturas com dupla dopagem, as quais apresentam boa integridade estrutural e preservam as propriedades ópticas dos dopantes. Por fim, visando aplicações em biologia, empregamos a metodologia desenvolvida para fabricar microambientes dopados, em sítios específicos, com o antibiótico cloridrato de ciprofloxacino. Estudos iniciais do desenvolvimento da bactéria Escherichia coli nestes microambientes foram feitos, com o objetivo de demonstrar a viabilidade para este tipo de aplicação. / Doped microstructures have attracted interest in optics, photonics and biology, because they allow the production of devices with specific proprieties. However, doping microstructures with more than one dopant is not much exploited in the literature. In this work we demonstrate the development of a method to fabricate multi-doped microstructures by two-photon absorption polymerization. For the microfabrication we used a femtosecond laser (Ti:sapphire laser, 780 nm, 100 fs) that is focused, by a microscope objective, in the volume of a polymeric resin containing a photoinitiator organic compound responsible to initiate the polymerization. The intensity of the femtosecond pulses is high enough to induced two-photon absorption, and consequently polymerization, only around the focal volume. As dopants we employed the fluorescent dyes Rhodamine and Fluorescein. In order to verify the microfabrication system, we have initially fabricated microstructures with only one dopant, which were characterized using optical and electron microscopies. Microstructures containing more than one dopant, in distinct regions, were produced by sequential fabrication of single doped structures. Such method allowed the fabrication of double doped structures, which presents good structural integrity and maintain the characteristic optical properties of the dyes. Finally, aiming at biological applications, we employed the developed method to fabricate micro-environments doped, in specific sites, with the antibiotics ciprofloxacin hydrochloride. Initial studies on the growth of the bacterium Escherichia coli, on such microstructures, were carried out to demonstrate the feasibility for such type of application.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-08-18T12:43:12Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE MESTRADO JESUS PAVON.pdf: 10424016 bytes, checksum: e34855e82c43c610bfad8eca83df6ebe (MD5) / Made available in DSpace on 2016-08-18T12:43:12Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE MESTRADO JESUS PAVON.pdf: 10424016 bytes, checksum: e34855e82c43c610bfad8eca83df6ebe (MD5) Previous issue date: 2015-09-03 / CNPq / Neste trabalho apresentamos um estudo da geração de luz no azul, em vapor de rubídio, devido à ação combinada de um trem de pulsos ultracurtos (fs) e um laser contínuo (diodo). Os dois feixes com polarizações paralelas e lineares, operando em 780 nm (diodo) e 776 nm (fs), são focalizados no centro da célula de Rb, e são responsáveis por cada etapa da transição de dois fótons 5S1/2 - 5P3/2 - 5D. Na configuração de feixes co-propagantes, uma coerência entre as transições 5S1/2 - 5P3/2 - 5D - 6P é induzida e, na condição de casamento de fase, um feixe azul, coerente, é gerado em 420 nm, através de um processo de mistura de quarto ondas. Medidas do espectro de excitação em função da frequência do laseres de diodo, para diferentes temperaturas da amostra e intensidades dos laseres incidentes, indicam uma forte competição entre os efeitos de saturação, ganho e absorção. Estudos de polarização e uma comparação com os recentes resultados de fluorescência permitem caracterizar para que parâmetros o mecanismo de mistura de ondas é responsável pelo sinal observado. Utilizando um laser de femtosegundos com uma taxa de repetição da ordem de 76 MHz, é possível observar, dentro do perfil Doppler, a contribuição dos diferentes modos do pente de frequência na geração do feixe coerente. / We present a study of blue light generation, in rubidium vapor, due to the combined action of a train of ultrashort pulses (fs) and a continuous laser (diode). The two beams with parallel and linear polarizations, operating at 780 nm (diode) and 776 nm (fs) are focused on the center of the Rb cell and are responsible for each step of the two photon transition from 55S1/2 - 5P3/2 - 5D. In the configuration of the co-propagating beams, a coherence between the 5S1/2 - 5P3/2 - 5D - 6P transitions is induced and, in phase matching condition, a blue coherent beam is generated at 420 nm through a process of four wave mixing. The measurements of excitation spectrum as a function of the frequency of the diode laser, for different temperatures of the sample and intensities of the incident laseres, indicate a strong competition among saturation, gain and absorption effects. The polarization studies and a comparison with recent results of fluorescence are used to characterize the parameters for which the wave mixing mechanism is responsible for the observed signal. Using a femtosecond laser with a repetition rate of about 76 MHz, it is possible observe, within the Doppler profile, the contribution of the different modes of the frequency comb in the generation of the coherent beam.

Les groupements protecteurs photolabiles sont utilisés pour de nombreuses applications, notamment en neuroscience pour la libération de neurotransmetteurs avec un contrôle spatio-temporel très fin. La démocratisation des lasers pulsés infrarouges a permis la mise au point de nouveaux composés sensibles à l’excitation à deux photons, de plus en plus efficace. Cependant, l’ingénierie moléculaire des cages n’est pas la seule méthode qui peut être utilisée pour améliorer l’efficacité des de ces composés. Mon travail de thèse a ainsi abordé cette problématique sous l’angle de la nanostructuration de groupements protecteurs photolabiles sensibles à l’excitation à deux photons. Nous avons débuté cette étude avec des composés de petite taille (dimère et tétramère) basés sur des groupements photosensible type ortho-nitrophénéthyle. Nous avons ensuite fonctionnalisé des dendrimères de type polyamidoamine, cependant les propriétés photophysiques et photochimiques de ces nouveaux composés suggèrent que des interactions intramoléculaires perturbent l’efficacité de la réaction de photolibération. Des dendrimères partiellement fonctionnalisés ont ensuite été envisagés. / Photosensitive protecting groups have been used for many applications, including neuroscience for the release of neurotransmitters with an amazing spatio-temporal control. New compounds sensitive to two-photon excitation were developed with the spread out of pulsed infrared lasers. However, cages molecular engineering is not the only way to improve such compounds. This manuscript focuses on nanostructuration to improve the overall efficiency of two-photon sensitive protecting groups. We started our study with small compound as dimer and tetramer based on the ortho-nitrophenethyl architecture. Next, polyamidoamine dendrimers were tested, but photophysics and photochemistry properties of these new compounds suggest that intramolecular interactions disturb photochemical reaction. Synthesis of partially functionalized dendrimer was considering in a next step.

Au cours du développement cérébral des vertébrés, le processus permettant à des neurones nouveaux-nés de s'incorporer dans des réseaux déjà établis est mal compris. En effet, la majorité des études ayant été réalisées à l'échelle de la cellule, une description détaillée de la dynamique des circuits au cours de ce phénomène est manquante. Pour l'étudier, j'ai développé une méthode innovante utilisant la larve de poisson zèbre comme modèle expérimental et une approche pluridisciplinaire combinant la génétique, la microscopie bi-photonique et l'optogénétique pour suivre le développement de l'activité de neurones nouveaux-nés et des réseaux matures voisins dans un vertébré intacte et non-anesthésié. En utilisant cette technique j'ai décrit pour la première fois, pendant plusieurs jours consécutifs, le développement des propriétés fonctionnelles de neurones nouveaux nés avant et pendant leur incorporation dans les circuits du toit optique, la structure cérébrale la plus complexe du poisson zèbre permettant l'intégration l'information visuelle. Les résultats obtenus suggèrent une séquence de développement durant laquelle les neurones morphologiquement immatures spontanément actifs se connectent en premier à la rétine. Dans un second temps, ces neurones s'incorporant graduellement au circuit mature en montrant des corrélations avec des neurones matures éparses. Troisièmement, l'organisation spatiale des corrélations entre les neurones nouveaux-nés est raffinée et devient plus dense. Ces résultats suggèrent que les neurones nouveaux-nés se connectent dans un premier temps a une population éparse de neurones matures avant que les connections a longue distance disparaissent permettant aux neurones en développement d'obtenir une signature fonctionnelle robuste (ex. réponses restreintes spatialement). Récemment, des traitements basés sur la transplantation des tissues neuronaux ont été développées pour certaines maladies neuro-dégénératives (ex. maladie de Parkinson). Cependant ces thérapies sont actuellement limitées par le faible taux de survie et l'incorporation des neurones injectés. Ces travaux apportent une meilleure compréhension des mécanismes à l’œuvre lors de la formation de circuits neuronaux et pourront peut-être permettre d'améliorer l'efficacité des traitements utilisant des cellules souches pour réparer le cerveau humain. / In the vertebrate brain, mechanisms leading to the incorporation of newborn neurons into already functional networks still remain poorly understood. Indeed, since most of the studies have been performed at the single-cell level, a detailed description of the circuit dynamics is lacking. To investigate this phenomenon, I have developed a pioneer methodology using the zebrafish larva as an experimental model and a multidisciplinary approach combining genetics, two-photon microscopy and optogenetics to monitor the developing activity of genetically targeted newborn neurons and the surrounding matured networks, in an intact and non-anesthetized vertebrate. Using this technique I have described for the first time, and in the time course of several days, the developmental dynamics of the functional properties of newborn neurons before and during their incorporation into the mature tectal circuit, the zebrafish most complex layered structure and highest visual center. Overall, these results suggest a developmental sequence of events during which newborn neurons capable of generating intrinsic activity dynamics first connect to their pre-synaptic sensory organ (the retina). At a second stage, the newborn neurons gradually incorporate into the tectal mature circuit showing sparse correlations with mature neurons. At a third stage, the spatial organization of the correlation between the newborn and the mature neurons is refined, becoming denser. I thus suggest that the newborn neurons first connect to a large population of sparsely located mature neurons and subsequently distant connections are pruned, permitting the newborn-labeled neuron to acquire a stable and robust functional signature (e.g. sharp receptive fields). In the recent years, treatments based on the transplantation of neural tissue have been developed to target neurodegenerative diseases such as Parkinson's disease. Because these therapies face the problem of poor survival and long-term functional incorporation, this study may provide better understanding of neuronal circuits formation and might pave the way to improve the efficiency of stem-cells-based treatments for human-brain reparation.

Les ions lanthanides possèdent des propriétés photophysiques particulières, qui ont suscité une forte attention dans le développement de sondes biologiques luminescentes. En effet, les avantages que présentent leurs émissions en raies fines et caractéristiques, ainsi que leurs temps de vie de luminescence longs, permettent diverses applications en imagerie. Cependant, le faible coefficient d’absorption molaire de ces éléments conduit à préférer une excitation par le biais d’une antenne capable de transférer l’énergie au métal.Par ailleurs, l’utilisation de l’absorption biphotonique a montré de nombreux avantages, puisqu’elle permet une excitation de la molécule à une longueur d’onde deux fois plus grande que l’excitation à un photon : cette longueur d’onde peut alors se situer dans la fenêtre de transparence biologique (650 - 1100 nm).Les travaux de cette thèse visent à combiner les avantages des lanthanides avec ceux de l’excitation biphotonique. Les complexes synthétisés sont basés sur une plateforme triazacyclononane substituée par des antennes à deux-photons. Leurs propriétés spectroscopiques à un photon ont été étudiées, et ont alors permis de réaliser des images cellulaires en multiplexing à deux photons utilisant Eu3+ et Tb3+. Des sondes biphotoniques de brillance optimisée ont également été obtenues pour Sm3+, Tb3+ et Dy3+, et appliquées à l'imagerie. L’étude approfondie d’un des complexes de terbium(III) a de plus mis en évidence sa sensibilité à la viscosité. L’analyse des propriétés spectroscopiques de cette sonde a permis d'en rationaliser le comportement photophysique, ouvrant ainsi la voie à des applications originales en imagerie fonctionnelle. Enfin, nous avons montré une internalisation rapide de complexes de lanthanide cationiques de type cyclen ou cyclam par des cellules vivantes, et l'étude de ces nouvelles structures offre de nouvelles perspectives dans le développement de bio-sondes de lanthanides. / Lanthanide ions demonstrate peculiar photophysical properties that has attracted a substantial attention in the development of luminescent bioprobes. Indeed, the advantages stemming from their sharp and characteristic emissions, as well as their long luminescence lifetimes, enable various imaging applications. However, the weak molar extinction coefficient of these elements results in a preferential excitation through an antenna, which transfers its energy to the metal.On the other hand, biphotonic absorption showed numerous advantages, since excitation of a molecule can be achieved at a wavelength twice as high as using one-photon excitation. This wavelength may therefore reach the optical transparency window (650 - 1100 nm).The work carried out during this PhD thesis aims at combining the advantages of lanthanides with those of biphotonic excitation. The synthesized complexes are based on a triazacyclononane platform substituted with two-photon antennae. Their one-photon spectroscopic properties have been studied, and enabled to perform two-photon multiplexed cellular imaging, using Eu3+ and Tb3+. Biphotonic probes with an optimized brightness have been obtained for Sm3+, Tb3+ and Dy3+, and applied to cell imaging. The thorough study of one of the terbium(III) complexes has moreover displayed evidence of a sensitivity towards viscosity. Analyzing the spectroscopic properties of this probe has therefore allowed to rationalize its photophysical behavior, paving the way for original functional imaging applications. Lastly, a rapid internalization of cationic lanthanide complexes with a cyclen or cyclam framework by living cells has been proved. Thus, the study of these new structures presents new perspectives for the development of lanthanide bioprobes.

L'objectif de ce travail est de développer et d'évaluer de nouvelles résines originales biosourcées pour l'application lithographique. Pour atteindre cet objectif, nous avons sélectionné différents types de lignine et étudié l'influence du fractionnement de la lignine sur les propriétés finales de la résine obtenue. Le fractionnement de la lignine est réalisé à l’aide de solvants organiques sélectionnés pour leur polarité. Nous avons choisi ce polymère naturel en raison de la présence de nombreux groupes fonctionnels directement responsables de sa réactivité : molécules photosensibles et potentiellement réticulables. La lignine est également une bonne candidate car abondante sur Terre et sa valorisation actuelle en tant que matière première chimique est négligeable. Nous avons étudié la photoréactivité de la résine à base de lignine à 395 nm en utilisant des sources de lumière LED. Sur la base des observations FTIR et UV-VIS, nous avons étudié et quantifié les changements structurels survenus lors de l'exposition de la photorésine à la lumière. Cependant, ces changements étaient plus intenses au cours de l'irradiation conduite dans l'air par rapport à une atmosphère inerte. De plus, nous avons établi le lien entre la structure chimique de la lignine et sa réactivité décrite par les tests de sensibilité, de contraste et de résolution. Nous avons proposé des mécanismes cohérents basés sur des données de la littérature. Ainsi, la réticulation de la lignine se produit sous les photons des rayonnements UV-VIS entraînant la formation de structures intermédiaires, de chromophores ou de composés volatils de bas poids moléculaire. Enfin, nous avons démontré qu'il était possible de fabriquer par écriture laser directe des nanostructures mono-, bi- et tridimensionnelles dans la photorésine à base de lignine "juste fractionnée" par polymérisation à deux photons. / The goal of this work was to develop and to evaluate new, original and bio-based resist for lithographic application. To reach this purpose, we have selected various types of lignin and study the influence of lignin fractionation in different organic solvents on the final resist properties. We have chosen this photosensitive and crosslinkable polymer due to its great abundance and to its currently poor valorisation as a chemical feedstock, as well as the versatility of the functional groups that were directly responsible for its reactivity. We have investigated the photoreactivity of lignin-based resist at 395 nm, using LED light sources. Based on the FTIR and UV-vis monitoring, we have investigated and quantified the structural changes occurred during photoresist exposure to the light. However, they were more visible during the irradiation conducted in the air than in the inert atmosphere. Moreover, we have established the link between chemical structure of lignin and its reactivity observed by sensitivity, contrast and resolution tests. Additionally, we have proposed reasonable mechanisms based on literature data according to which lignin crosslinking occur under UV-visible photons. They comprised the formation of intermediates structures, chromophores, or low molecular weight volatile compounds, as well and crosslinking reactions. Finally, we have demonstrated that it was possible to fabricate one-, two- and three-dimensional structures from "just-fractionated" lignin photoresist by two-photon polymerisation achieved by direct laser writing.

The majority of fast excitatory neurotransmission in the brain occurs at glutamatergic synapses. The extensive dendritic arborisations of pyramidal neurons in the neocortex and hippocampus harbor thousands of synaptic connections, each formed on tiny protrusions called dendritic spines. Spine synapses are rapidly established during early postnatal development – a key period in neural circuit assembly – and are subject to dynamic activity-dependent plasticity mechanisms that are believed to underlie neural information storage and processing for learning and memory. Recent decades have seen remarkable progress in identifying diverse plasticity mechanisms responsible for regulating synapse structure and function, and in understanding the processes underlying computation of synaptic inputs in the dendrites of individual neurons. These advances have strengthened our understanding of the biological mechanisms underlying brain function but, not surprisingly, they have also raised many new questions. Using a combination of whole-cell electrophysiology, 2-photon imaging and glutamate uncaging in rodent brain slice preparations, I have helped to document the subtype-specific regulation of glutamate receptors during a homeostatic form of synaptic plasticity at CA1 pyramidal neurons of the hippocampus, and have discovered novel synaptic calcium dynamics during a critical period of neural circuit formation. First, we found that during a homeostatic response to prolonged inactivity, both AMPA and NMDA subtypes of glutamate receptors undergo a switch in subunit composition at synapses, but exhibit a divergence in their subcellular localization at extrasynaptic regions of the plasma membrane (this work was published in the Journal of Neuroscience in 2013). In separate series of experiments using 2-photon calcium imaging, I discovered a functional coupling between NMDA receptor activation and intracellular calcium release at dendritic spines and dendrites that is selectively expressed during a critical period of synapse formation. This synaptic calcium signaling mechanism enabled the transformation of distinct spatiotemporal patterns of synaptic input into salient biochemical signals, and is thus apt to locally regulate synapse development along individual dendritic branches. Consistent with this hypothesis, I found evidence for non-random clustering of synapse development between neighboring dendritic spines. Together, these experimental results expand the current understanding of the dynamics of synapse function during homeostatic plasticity and early postnatal development. --- Les synapses glutamatergiques soutiennent la majorité de la neurotransmission excitatrice rapide du cerveau. Des milliers de ces synapses, localisées sur de minuscules saillies appelées épines dendritiques, décorent les vastes arborisations dendritiques des neurones pyramidaux du néocortex et de l'hippocampe. Ces synapses sont formées tôt lors du développement postnatal et sont soumises à des mécanismes dynamiques de plasticité qui sous-tendent, croit-on, les capacités d'apprentissage et de mémoire du cerveau. Les dernières décennies ont vu des progrès remarquables dans l'identification de divers mécanismes de régulation de la structure et de la fonction des synapses sur différentes échelles de temps, et dans la compréhension des processus qui régissent l’intégration des inputs synaptiques au niveau des dendrites individuelles. Ces progrès ont renforcé notre compréhension des éléments fondamentaux régissant la fonction cérébrale et ont ouvert de nouvelles voies d’investigations neurophysiologiques. En utilisant une combinaison d’électrophysiologie cellulaire, d'imagerie à deux-photons et de photolibération de glutamate sur des neurones pyramidaux de la région CA1 de l'hippocampe de rats, j’ai contribué à la découverte et à la caractérisation de nouvelles régulations des récepteurs du glutamate durant la plasticité synaptique homéostatique. J’ai également découvert un nouveau type de dynamique de calcium synaptique relié à une organisation spatiale du développement des synapses pendant une période critique de l’ontogénie des circuits neuronaux. Dans la première étude, nous avons constaté que lors d'une plasticité de type homéostatique induite par une inactivité prolongée, les récepteurs de glutamate de types AMPA et NMDA sont soumis à un changement important dans la composition de leurs sous-unités. De plus, nous avons observé un ciblage différentiel de ces récepteurs vers des compartiments subcellulaires spécifiques des neurones. Dans une série d'expériences séparée utilisant l’imagerie calcique à deux-photons, j’ai découvert un couplage fonctionnel durant le développent entre l'activation des récepteurs NMDA et une libération de calcium intracellulaire qui envahit tant les épines dendritiques que les dendrites. J’ai également trouvé que ce mécanisme de signalisation de calcium synaptique transforme des motifs spatiotemporels d’activités synaptiques spécifiques en signaux biochimiques post-synaptiques de manière à potentiellement réguler l’organisation spatiale des synapses durant le développement. Conformément à cette hypothèse, j’ai observé des manifestations fonctionnelles claires de regroupement dans l’espace de synapses de forces similaires le long de branches dendritiques individuelles. Ensemble, ces résultats expérimentaux élargissent notre compréhension actuelle de de la fonction des synapses durant la plasticité homéostatique ainsi que durant le développement postnatal du cerveau. En étudiant les mécanismes neurophysiologiques de base, il sera possible d'avoir un aperçu plus profond du fonctionnement du cerveau et de ses pathologies.