The phenomenon of photobleaching of a photosensitizer during photodynamic therapy (PDT) is well known. For second generation photosensitizers it may be possible to exploit this effect to enhance the volume of damaged tissue and improve the efficacy of PDT. In addition, as a consequence of photobleaching, the fluorescence emitted by the photosensitizer will decrease during PDT. Mathematical models were developed which describe fluorescence emission, photobleaching and tissue necrosis resulting from the irradiation of tissue containing photosensitizer using an appropriate light source. Diffusion theory was used to model bleaching in a semi-infinite medium caused by broad-beam irradiation, and both pencil and broad-beam fluorescence excitation of the photosensitizer. In addition, models were developed for an isotropic point source imbedded in an infinite medium. Based on the relationship between the decreasing fluorescence signal and the increasing volume of tissue damage, these models allow the extent of necrosis achieved during treatment to be monitored. By analysing spatially resolved fluorescence measurements predictions about necrosis depth that are insensitive to treatment parameters such as photosensitizer concentration, tissue optical properties and bleach rate are possible. Tissue simulating optical phantoms that allow for relatively simple and accurate alteration to optical properties were developed. Photosensitizers which still undergo fluorescence and photobleaching in the solid medium were also added. Using these phantoms, treatment was simulated and spatially resolved fluorescence was measured as a function of time for a wide range of initial treatment parameters. Photobleaching of the photosensitizers was observed to occur through a decrease in fluorescence emission. Also, spatially resolved measurements provided information about the average photosensitizer depth, which was seen to increase with time, with little knowledge of initial treatment parameters. These experimental results were then compared with predictions from the mathematical theory, illustrating the validity of the models. The value and feasibility of this technique for photodynamic therapy dosimetry are discussed, along with planned improvements. / Thesis / Master of Science (MS)

Introdução: A alteração de cor dos dentes apresenta-se como um dos fatores que mais concorrem para o desequilíbrio do sorriso, sendo o clareamento dental amplamente difundido e solicitado pelos pacientes. Objetivo: Este estudo in situ teve como objetivo avaliar as mudanças morfológicas e químicas do esmalte quando submetido a três agentes clareadores à base de peróxido de hidrogênio ativados com fonte de luz híbrida e um agente placebo (gel sem peróxido de hidrogênio), por meio do uso da espectrometria de energia dispersiva de raios-X (EDS). Metodologia: Fragmentos de terceiros molares humanos foram divididos em quatro grupos (n=12), para a realização de uma sessão de clareamento com cinco aplicações de oito minutos dos géis clareadores: Placebo (Plac); Lase Peroxide Flex 35% e 15% (DMC) (LPF35LH e LPF15LH); Gel experimental a 10% (DMC) (EXP10LHV), e foram fotocatalizados com luz híbrida: LED azul/laser de diodo (LH) (Whitening Lase II, DMC) ou LED violeta/laser de diodo (LHV) (luz experimental, DMC). Após o clareamento, os espécimes foram fixados a dispositivos intraorais usados pelos participantes durante 15 dias. A composição inorgânica e topografia da superfície de esmalte foram analisadas antes e após o clareamento, e depois de 3, 7 e 15 dias de exposição à saliva. Os valores elementares da composição foram analisados por ANOVA a um critério de medidas repetidas e teste de Tukey. Para a topografia os escores foram determinados por três examinadores previamente calibrados pelo teste Kappa e foi aplicado o teste estatístico de Friedman e Kruskal-Wallis, e as comparações individuais foram realizadas pelo teste de Dunn ( = 0,05). Resultados: De maneira geral, não houve alterações significativas na porcentagem elementar do esmalte nos diferentes períodos estudados. Ao analisar os dois principais elementos, o grupo LPF35HL obteve o menor valor de cálcio (Ca), possuindo diferença estatisticamente significante quando comparado com o grupo EXP10LHV, enquanto os valores de fosfato (P) permaneceram constantes. Morfologicamente somente o grupo EXP10LHV demostrou maior planificação da superfície quando comparado o período de 7 dias com 15 dias. Conclusão: Os diferentes protocolos clareadores empregados, demonstraram alterações pontuais na variação dos elementos químicos e na morfologia do esmalte dental ao longo do período de avaliação. / Introduction: The tooth color change is one of the factors that contributes most to the smile imbalance, and dental bleaching is widely diffused and requested by the patients. Objective: The aimed of this in situ study is to evaluate the morphological and chemical changes of the enamel when submitted to three activated hydrogen peroxide bleaching agents with hybrid light source and a placebo agent (gel without hydrogen peroxide), using energy-dispersive X-ray spectroscopy (EDS). Methodology: Fragments of human third molars were divided into four groups (n = 12) to perform a bleaching session with five eight minute applications of bleaching gels: Placebo (Plac); Lase Peroxide Flex 35% and 15% (DMC) (LPF35LH and LPF15LH); 10% experimental gel (DMC) (EXP10LHV), and photocatalyzed with hybrid light: blue LED / diode laser (LH) (Whitening Lase II, DMC) or violet LED / diode laser (LHV). After bleaching, the specimens were fixed to intraoral devices used by participants for 15 days. The inorganic composition and topography of the enamel surface were analyzed before and after bleaching, and after 3, 7 and 15 days of exposure to saliva. The elementary values of the composition were analyzed by one-way ANOVA at a repeated measures and Tukey\'s test. For the topography the scores were determined by three examiners previously calibrated by the Kappa test and the Friedman and Kruskal-Wallis statistical test were applied, and the individual comparisons were performed by the Dunn test ( = 0.05). Results: In general, there were no significant changes in the elemental percentage of enamel in the different periods studied. When analyzing the two main elements, the LPF35HL group had the lowest calcium (Ca) value, which had a statistically significant difference when compared to the EXP10LHV group, while the phosphate (P) values remained constant. Morphologically, only the EXP10LHV group showed greater surface planning when compared to the period of 7 days with 15 days. Conclusion: The different bleaching protocols employed showed specific alterations in the variation of the chemical elements and the morphology of the dental enamel during the evaluation period.

Introdução: A alteração de cor dos dentes apresenta-se como um dos fatores que mais concorrem para o desequilíbrio do sorriso, sendo o clareamento dental amplamente difundido e solicitado pelos pacientes. Objetivo: Este estudo in situ teve como objetivo avaliar as mudanças morfológicas e químicas do esmalte quando submetido a três agentes clareadores à base de peróxido de hidrogênio ativados com fonte de luz híbrida e um agente placebo (gel sem peróxido de hidrogênio), por meio do uso da espectrometria de energia dispersiva de raios-X (EDS). Metodologia: Fragmentos de terceiros molares humanos foram divididos em quatro grupos (n=12), para a realização de uma sessão de clareamento com cinco aplicações de oito minutos dos géis clareadores: Placebo (Plac); Lase Peroxide Flex 35% e 15% (DMC) (LPF35LH e LPF15LH); Gel experimental a 10% (DMC) (EXP10LHV), e foram fotocatalizados com luz híbrida: LED azul/laser de diodo (LH) (Whitening Lase II, DMC) ou LED violeta/laser de diodo (LHV) (luz experimental, DMC). Após o clareamento, os espécimes foram fixados a dispositivos intraorais usados pelos participantes durante 15 dias. A composição inorgânica e topografia da superfície de esmalte foram analisadas antes e após o clareamento, e depois de 3, 7 e 15 dias de exposição à saliva. Os valores elementares da composição foram analisados por ANOVA a um critério de medidas repetidas e teste de Tukey. Para a topografia os escores foram determinados por três examinadores previamente calibrados pelo teste Kappa e foi aplicado o teste estatístico de Friedman e Kruskal-Wallis, e as comparações individuais foram realizadas pelo teste de Dunn ( = 0,05). Resultados: De maneira geral, não houve alterações significativas na porcentagem elementar do esmalte nos diferentes períodos estudados. Ao analisar os dois principais elementos, o grupo LPF35HL obteve o menor valor de cálcio (Ca), possuindo diferença estatisticamente significante quando comparado com o grupo EXP10LHV, enquanto os valores de fosfato (P) permaneceram constantes. Morfologicamente somente o grupo EXP10LHV demostrou maior planificação da superfície quando comparado o período de 7 dias com 15 dias. Conclusão: Os diferentes protocolos clareadores empregados, demonstraram alterações pontuais na variação dos elementos químicos e na morfologia do esmalte dental ao longo do período de avaliação. / Introduction: The tooth color change is one of the factors that contributes most to the smile imbalance, and dental bleaching is widely diffused and requested by the patients. Objective: The aimed of this in situ study is to evaluate the morphological and chemical changes of the enamel when submitted to three activated hydrogen peroxide bleaching agents with hybrid light source and a placebo agent (gel without hydrogen peroxide), using energy-dispersive X-ray spectroscopy (EDS). Methodology: Fragments of human third molars were divided into four groups (n = 12) to perform a bleaching session with five eight minute applications of bleaching gels: Placebo (Plac); Lase Peroxide Flex 35% and 15% (DMC) (LPF35LH and LPF15LH); 10% experimental gel (DMC) (EXP10LHV), and photocatalyzed with hybrid light: blue LED / diode laser (LH) (Whitening Lase II, DMC) or violet LED / diode laser (LHV). After bleaching, the specimens were fixed to intraoral devices used by participants for 15 days. The inorganic composition and topography of the enamel surface were analyzed before and after bleaching, and after 3, 7 and 15 days of exposure to saliva. The elementary values of the composition were analyzed by one-way ANOVA at a repeated measures and Tukey\'s test. For the topography the scores were determined by three examiners previously calibrated by the Kappa test and the Friedman and Kruskal-Wallis statistical test were applied, and the individual comparisons were performed by the Dunn test ( = 0.05). Results: In general, there were no significant changes in the elemental percentage of enamel in the different periods studied. When analyzing the two main elements, the LPF35HL group had the lowest calcium (Ca) value, which had a statistically significant difference when compared to the EXP10LHV group, while the phosphate (P) values remained constant. Morphologically, only the EXP10LHV group showed greater surface planning when compared to the period of 7 days with 15 days. Conclusion: The different bleaching protocols employed showed specific alterations in the variation of the chemical elements and the morphology of the dental enamel during the evaluation period.

Στην παρούσα εργασία, γίνεται αρχικά η μελέτη τριών χρωστικών ως προς την απόδοσή τους στη διφωτονική απορρόφηση. Με βάση τα αποτελέσματα της μέτρησης αυτής, επιλέχθηκαν οι δύο χρωστικές που παρουσίασαν ισχυρότερα το φαινόμενο, ώστε να μελετηθούν σχετικά με τον αποχρωματισμό τους. Για το λόγο αυτόν, τα δείγματα που παρασκευάστηκαν από τις ουσίες, δέχτηκαν ακτινοβολία μεγάλης έντασης, μέσω πολύ στενών παλμών laser. / Photobleaching of organic dyes after two photon excitation

Upregulation of nicotinic acetylcholine receptors (nAChRs) is a well-documented response to chronic nicotine exposure. Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels consisting of alpha (α2-10) and beta (β2-4) subunits. Nicotine, an agonist of nAChRs, alters trafficking and assembly of some subtypes of nAChRs, leading to an increase in expression of high sensitivity receptors on the plasma membrane. These physiological changes in nAChRs are believed to contribute to nicotine addiction, although the mechanism of these processes has not been resolved. Recently, many studies have converged on the idea that nicotine induces upregulation by an intracellular mechanism. In this dissertation, expression levels of nAChRs were quantified upon exposure to nicotine and its primary metabolite, cotinine. A pH sensitive variant of GFP, super ecliptic pHluorin (SEP), was integrated with a nAChR subunit to study expression and trafficking of nAChRs by differentiating intracellular and plasma membrane inserted receptors. In this work, cotinine is shown to increase the number of α4β2 nAChRs within a cell. Cotinine also affects trafficking of α4β2, evident by a redistribution of intracellular receptors and an increase in single vesicle insertion events on the plasma membrane. This work shows both nicotine and cotinine alter the overall assembly of α4β2 to favor the high sensitivity (α4)2(β2)3 version. Since cotinine and nicotine induce similar physiological changes in nAChRs, the metabolite potentially plays a role in the mechanism of nicotine addiction. Although an intracellular mechanism for upregulation has been supported, a shift in assembly to the high sensitivity (α4)2(β2)3 version exclusively in the endoplasmic reticulum has not previously been detected. In order to study organelle specific changes in stoichiometry, a novel method was developed to isolate single nAChRs in nanovesicles derived from native cell membranes. Separation of nanovesicles originating from the endoplasmic reticulum and plasma membrane, encompassing isolated nAChRs, allows precise changes in stoichiometry to be monitored in subcellular regions. In this work, single molecule bleaching steps of green fluorescent protein (GFP) encoded in each alpha subunit of the pentamer are detected. The number of bleaching steps, or transitions to a nonfluorescent state upon continuous excitation, corresponds to the number of GFP-labeled alpha subunits present. Therefore, the stoichiometry can be deduced by detection of two bleaching steps, as in (α4)2(β2)3, or three bleaching steps, seen in (α4)3(β2)2. Using this method on isolated nAChRs, a shift to assembly of high sensitivity (α4)2(β2)3 receptors is detected definitively within the endoplasmic reticulum. In addition, an increase in (α4)2(β2)3 receptors located on the plasma membrane is shown when nicotine is present. This work provides convincing evidence that nicotine acts intracellularly, within the endoplasmic reticulum, to alter stoichiometry of nAChRs.

Les cellules sont capables de détecter les distributions spatiales de protéines et ainsi de migrer ou s’étendre dans la direction appropriée. Une compréhension de la réponse cellulaire aux modifications de ces distributions spatiales de protéines est essentielle pour l’avancement des connaissances dans plusieurs domaines de recherches tels que le développement, l’immunologie ou l’oncologie. Un exemple particulièrement complexe est le guidage d’axones se déroulant pendant le développement du système nerveux. Ce dernier nécessite la présence de plusieurs distributions de molécules de guidages étant attractives ou répulsives pour connecter correctement ce réseau complexe qu’est le système nerveux. Puisque plusieurs indices de guidage collaborent, il est particulièrement difficile d’identifier la contribution individuelle ou la voie de signalisation qui est déclenchée in vivo, il est donc nécessaire d’utiliser des méthodes pour reproduire ces distributions de protéines in vitro. Plusieurs méthodes existent pour produire des gradients de protéines solubles ou liées aux substrats. Quelques méthodes pour produire des gradients solubles sont déjà couramment utilisées dans plusieurs laboratoires, mais elles limitent l’étude aux distributions de protéines qui sont normalement sécrétées in vivo. Les méthodes permettant de produire des distributions liées au substrat sont particulièrement complexes, ce qui restreint leur utilisation à quelques laboratoires. Premièrement, nous présentons une méthode simple qui exploite le photoblanchiment de molécules fluorescentes pour créer des motifs de protéines liées au substrat : Laser-assisted protein adsorption by photobleaching (LAPAP). Cette méthode permet de produire des motifs de protéines complexes d’une résolution micrométrique et d’une grande portée dynamique. Une caractérisation de la technique a été faite et en tant que preuve de fonctionnalité, des axones de neurones du ganglion spinal ont été guidés sur des gradients d’un peptide provenant de la laminine. Deuxièmement, LAPAP a été amélioré de manière à pouvoir fabriquer des motifs avec plusieurs composantes grâce à l’utilisation de lasers à différentes longueurs d’onde et d’anticorps conjugués à des fluorophores correspondants à ces longueurs d’onde. De plus, pour accélérer et simplifier le processus de fabrication, nous avons développé LAPAP à illumination à champ large qui utilise un modulateur spatial de lumière, une diode électroluminescente et un microscope standard pour imprimer directement un motif de protéines. Cette méthode est particulièrement simple comparativement à la version originale de LAPAP puisqu’elle n’implique pas le contrôle de la puissance laser et de platines motorisées, mais seulement d’envoyer l’image du motif désiré au modulateur spatial. Finalement, nous avons utilisé LAPAP pour démontrer que notre technique peut être utilisée dans des analyses de haut contenu pour quantifier les changements morphologiques résultant de la croissance neuronale sur des gradients de protéines de guidage. Nous avons produit des milliers de gradients de laminin-1 ayant différentes pentes et analysé les variations au niveau du guidage de neurites provenant d’une lignée cellulaire neuronale (RGC-5). Un algorithme pour analyser les images des cellules sur les gradients a été développé pour détecter chaque cellule et quantifier la position du centroïde du soma ainsi que les angles d’initiation, final et de braquage de chaque neurite. Ces données ont démontré que les gradients de laminine influencent l’angle d’initiation des neurites des RGC-5, mais n’influencent pas leur braquage. Nous croyons que les résultats présentés dans cette thèse faciliteront l’utilisation de motifs de protéines liées au substrat dans les laboratoires des sciences de la vie, puisque LAPAP peut être effectué à l’aide d’un microscope confocal ou d’un microscope standard légèrement modifié. Cela pourrait contribuer à l’augmentation du nombre de laboratoires travaillant sur le guidage avec des gradients liés au substrat afin d’atteindre la masse critique nécessaire à des percées majeures en neuroscience. / Cells are able to sense spatial distribution of proteins and accordingly migrate or extend in the appropriate direction. Understanding cellular responses to modifications in molecular spatial distributions is essential for advances in several fields such as development, immunology and oncology. A particularly complex example is axonal guidance that occurs during the development of the nervous system, which relies on distributions of attractive and repulsive guidance molecules to correctly wire this intricate network. Since several guidance cues collaborate to development of the nervous system, it is particularly difficult to assess the individual contribution of each cue and the signaling cascade each trigger in vivo; therefore methods to reproduce those distributions individually in vitro are necessary to study in detail the effect of each guidance cue. Several methods exist to produce graded distributions of protein that are either soluble or substrate-bound. A few methods making solution gradients are already widely used in several laboratories to perform experiments with the guidance cues that are normally diffusing in vivo. However, current methods allowing the fabrication of substrate-bound gradients are quite complex, which restrict their use to a few laboratories. First, we present a straightforward method exploiting photobleaching of a fluorescently tagged molecule using a visible laser to generating substrate-bound protein patterns: Laser-assisted protein adsorption by photobleaching (LAPAP). This method allows producing complex patterns of protein with micron spatial resolution and high dynamic range. An extensive characterization of the technique was performed and as proof of functionality, axons from dorsal root ganglions cells were guided on laminin peptide gradients. Secondly, LAPAP was improved in order to produce multicomponent patterns by using lasers at different wavelengths and antibodies conjugated to fluorophores corresponding to these wavelengths. Moreover, to speed-up the fabrication process and simplify the device, we developed widefield illumination LAPAP which uses a spatial light modulator, a light emitting diode and a standard microscope to directly print patterns. This patterning method is relatively simple compared to the original LAPAP setup, since it does not involve controlling the laser power or a motorized stage, but only sends an image of the desired pattern to a spatial light modulator. Finally, we used LAPAP to show how it could be used in automated high-content screening assays to quantify the morphological changes resulting from axon growth on gradients of guidance proteins. We produced thousands of laminin-1 gradients of different slopes and analyzed the variations in neurite guidance of neuron-like cells (RGC-5). An image analysis algorithm was developed to process bright field microscopy images, detecting each cell and quantifying the soma centroid and the initiation, terminal and turning angles of the maximal neurite. This data showed that laminin gradients influence the initiation angle of neurite extension of RGC-5, but does not contribute to its turning. We believe that the results presented in this thesis will facilitate the use of substrate- bound protein patterning in typical life science laboratories, since a confocal microscope or a slightly modified standard microscope is the only specialized equipment needed to fabricate patterns by LAPAP. This could increase the number of laboratories working with substrate-bound protein patterns in order to reach the critical mass necessary for major advances in neuroscience.

No description available.

O tratamento do câncer e de lesões pré-malignas é uma importante aplicação da terapia fotodinâmica. A base da técnica é o uso da luz associada a um fotossensibilizador, na presença de oxigênio, de forma a promover a formação de espécies reativas de oxigênio que culminam na morte celular. Esta técnica é altamente seletiva, e apresenta poucos efeitos colaterais. A efetividade da terapia pode ser avaliada com base na emissão de fluorescência do fotossensibilizador, uma vez que o grau de fotodegradação deste indica uma terapia mais ou menos efetiva. No caso de tumores, a reincidência de lesões, causada por células malignas remanescentes, é um problema importante e que pode piorar as condições clínicas de um paciente. Este estudo propõe, portanto, um modelo matemático baseado na aferição da fluorescência do fotossensibilizador in situ para previsão da extensão do dano promovido pela terapia fotodinâmica em tempo real. Esta aferição é feita em comparação com o parâmetro experimental de profundidade de necrose, de modo a avaliar se uma lesão pode ser completamente eliminada numa dada aplicação. O estudo utilizou fígados de ratos Wistar machos, saudáveis, como modelo inicial dada sua relativa homogeneidade óptica com relação a lesões tumorais, visando minimizar variáveis. Os fígados foram irradiados com diferentes intensidades (150, 250 e 350 mW/cm2) e com diferentes fluências (50-450 J/cm2) de luz com comprimento de onda de 630 nm, CW, para avaliação das necroses e da fluorescência (avaliada usando 532 nm para excitação). A primeira versão do modelo utilizou o conjunto de dados de 250 mW/cm2 para obtenção de um modelo inicial baseado na fotodegradação avaliada por fluorescência. Este modelo foi alterado com base em modificações biológicas observadas na literatura durante a terapia. Tais alterações estão relacionadas à modificação de aporte de oxigênio causada por vasoconstricção, pela farmacocinética do fotossensibilizador e pelas modificações nas propriedades ópticas intrínsecas ao tecido. Foi possível encontrar equivalência entre modelo e experimentação ao final das modificações propostas ao modelo inicial, embora tenha sido evidente o efeito da variação no fracionamento da fluência entregue sobre os resultados da previsão. Como forma de mostrar a capacidade do modelo não apenas para previsão da profundidade de necrose (e, portanto, da extensão do dano), mas também para avaliações de dosimetria, foi proposta uma situação hipotética onde se entrega a mesma fluência de luz com intensidades diferentes (variando linearmente). Este teste mostrou o potencial do modelo não apenas para avaliar a extensão do dano causado pela terapia em tempo real, mas também para estimar os resultados de protocolos clínicos não-testados e, portanto, para a proposta de melhorias a protocolos de terapia fotodinâmica. / Malignant and pre-malignant cancer lesions treatment is an important application of photodynamic therapy. It is based on the use of light associated to a photosensitizer, in the presence of oxygen, so that reactive oxygen species may be formed, resulting in cell death. This is a highly selective technique, and few side effects are reported. The therapy efficacy may be evaluated through the photosensitizer fluorescence emission, since its level of bleaching indicates a more or less effective therapy application. Concerning cancer, recurrent lesions, caused by remaining malignant cells, is an important problem which may cause the worsening of patient´s clinical condition. Therefore, this study proposes a mathematical model based on the assessment of photosensitizer fluorescence in situ for prediction of the damage extent caused by photodynamic therapy. This evaluation is performed by comparison with the experimental parameter of depth of necrosis, as to evaluate if a lesion can be completely eliminated during an application. This study used male Wistar rats healthy livers as an initial model due to its relative optical homogeneity when compared to tumor lesions, hence minimizing variables. Livers were irradiated using different light fluence rates (150, 250 and 350 mW/cm2) and fluences (50-450 J/cm2) with 630 nm wavelength, CW, to perform evaluation of fluorescence (wit 532 nm excitation) and depth of necrosis. A first model was obtained from the 250 mW/cm2 dataset, based on fluorescence-assessed photobleaching. Such model was modified according to literature observations on biological changes during therapy. Those changes are related to the oxygen arrival, promoted by vessels shutdown, by the photosensitizer pharmacokinetics and by the changes in the intrinsic optical properties of liver tissue. Correspondence between model and experimentation was achieved after the changes proposed to the initial model, although the effect of different fractioning of light fluences on prediction has become evident. To show the model ability not only to predict depth of necrosis (and, hence, damage extent), but also for dosimetry studies, an hypothetical situation in which the same fluence has been delivered in different (linearly varying) fluence rates. This test result showed the potential of the model not only to evaluate the damage extension by the therapy in real-time, but also to investigate untested clinical protocols results and, therefore, to allow proposals of improvement to photodynamic therapy protocols.

O objetivo deste estudo in vivo, internacional, randomizado e duplo cedo foi avaliar comparativamente a efetividade e o pH de diferentes géis clareadores na técnica de clareamento em consultório, com e sem o emprego de fonte de luz híbrida em função do grau de alteração de cor, sensibilidade e manutenção do tratamento ao longo de 12 meses de acompanhamento. Foram selecionados 48 voluntários de acordo com os critérios de inclusão e exclusão. Os pacientes foram divididos, de forma randomizada, em 4 grupos de 12 participantes cada, onde: Grupo EXP10 5 aplicações do gel de peróxido de hidrogênio a 10% (Gel Experimental DMC Equipamentos) e ativação de luz híbrida de LED (violeta)/Laser (Experimental DMC Equipamentos) com 7′ e 30″ por aplicação, com tempo total de 37′30; Grupo LP15 5 aplicações do gel de peróxido de hidrogênio 15% (Lase Peroxide Lite DMC Equipamentos) seguindo mesmo protocolo do grupo EXP10; Grupo TB35LH 3 aplicações do gel de peróxido de hidrogênio a 35% (Total Blanc Office - DFL) e ativação de luz híbrida de LED (azul)/Laser (Whitening Lase II DMC Equipamentos) de 7′ e 30″ por aplicação, com tempo total de 22′30″; Grupo TB35 3 aplicações do gel de peróxido de hidrogênio a 35% (Total Blanc Office - DFL) sem ativação com fonte de luz, totalizando 45″. A determinação dos valores de pH foi realizada com o peagômetro digital (Sentron Model 1001, Sentron) nos tempos inicial e após o término do protocolo clareador. A aferição da cor foi feita com espectofotômetro VITA Easyshade antes do clareamento, após 24 horas, 1 semana, 1, 6 e 12 meses. A sensibilidade dentária e grau de satisfação dos pacientes foram avaliados por meio do questionário VAS e IPS antes, imediatamente após o clareamento, 24 horas e uma semana após. Os resultados da alteração do pH receberam tratamento estatístico pela ANOVA e teste de Bonferroni a 0,05%. Os resultados indicaram que o pH aumentou do momento inicial para o final para todos os protocolos. Não houve diferenças significativas entre os protocolos TB35 e TB35LH em nenhum dos momentos, e o pH médio do grupo EXP10 foi maior em comparação aos outros três grupos nos dois momentos avaliados. Os resultados do ΔE receberam tratamento estatístico pela ANOVA e teste de Bonferroni a 0,05%. Os resultados indicaram que não houve diferença significativa entre os grupos LP15, TB35 e TB35LH. O ΔE médio observado após 24 horas foi estatisticamente maior que para os outros tempos (inicial, 1 semana, 1 mês, 6 e 12 meses). Para análise da sensibilidade foi construído um modelo linear misto e atribuídos postos (ranks) aos valores de Δ e teste de Bonferroni a 0,05% para comparações pareadas. Não houve diferença nos valores da sensibilidade imediatamente e 24 horas após o tratamento, com relação ao momento inicial. Houve diferença significativa entre Δ1 e Δ3 indicando que a sensação de dor após uma semana do tratamento foi menor do que as observadas nos instantes imediato e após 24 horas. Para os resultados de satisfação foi construído um modelo linear misto e atribuídos postos (ranks) e o Método de Bonferroni (0,05%) foi utilizado para as comparações pareadas do efeito de tempo. Os resultados indicam queda nos níveis de satisfação entre os períodos imediato e um ano e entre os períodos 24 horas e um ano. Todos os géis clareadores apresentaram mínima variação do pH nos tempos avaliados, entretanto houve um aumento do pH da primeira para a última aplicação em todos os grupos estudados e o grupo EXP10 apresentou os maiores valores de pH seguido do LP15, TB35LH e TB35 apresentaram os valores mais baixos de pH. Os grupos LP15, TB35 e TB35LH apresentaram menor variação da cor ao longo de 12 meses de acompanhamento. O efeito do protocolo clareador não influenciou a sensibilidade dos pacientes e após uma semana a sensibilidade retornaram aos níveis normais. O nível de satisfação dos pacientes foi significativo em relação ao tempo e não aos protocolos clareadores, os pacientes do grupo TB35 mostraram-se mais insatisfeitos ao longo da pesquisa. / The aim of the present in vivo study, international, randomized and double early was to comparatively evaluate the effectiveness and pH of different bleaching agents for in office bleaching techniques, with and without the use of a hybrid light source depending on the degree of color change, sensitivity and maintenance treatment over a 12-month follow-up. Selected were 48 volunteers according to the inclusion and exclusion criteria. The patients were randomly divided into 4 groups of 12 participants each, where: EXP Group10- 5 applications of 10% hydrogen peroxide gel (Experimental Gel - Equipment DMC) and activation of hybrid LED light (violet) / Laser (Experimental - Equipment DMC) with 7 ″and 30″ per application, with a total time of 37′30; LP15 Group- 5 applications of 15% hydrogen peroxide gel (Lase Peroxide Lite - Equipment DMC) following the same protocol of the EXP10 Group; TB35LH Group- 3 applications of 35% hydrogen peroxide gel (of Blanc Office - DFL) and activation of hybrid LED light (blue) / Laser (Whitening Lase II - DMC Equipment) 7 ″and 30″ per application, with a total time of 22′30; TB35 Group - 3 applications of 35% hydrogen peroxide gel (of Blanc Office - DFL) without light activation, totaling 45 ″. The determination of pH was carried out with a digital pH meter (Sentron Model 1001, Sentron) in the initial times and after the bleaching protocol. The color measurement was made with a VITA Easyshade spectrophotometer before the treatment, after 24 hours, 1 week, 1, 6 and 12 months. Tooth sensitivity and degree of patient satisfaction were assessed by the VAS IPS questionnaire before, immediately after bleaching, 24 hours and one week after. The pH change results were statistically processed by the ANOVA and Bonferroni tests at 0.05%. The results indicated that the pH increased from baseline to the end for all protocols. There were no significant differences between the TB35 and TB35LH protocols in any of the times, and the average pH of the EXP10 group was higher compared to the other three groups in the two periods evaluated. The results of the ΔE received statistical analysis by ANOVA and Bonferroni testing at 0.05%. The results indicated that there was no significant difference between the LP15, TB35 and TB35LH Groups. The average ΔE observed after 24 hours was statistically greater than for the other times (initial, 1 week, 1 month, 6 months and 12 months). For the sensitivity analysis, a mixed linear model was constructed and assigned points (ranks) for Δ values and the Bonferroni test at 0.05% was used for paired comparisons. There was no difference in sensitivity values immediately and 24 hours after treatment with the initial time. There was a significant difference between Δ1 and Δ3 indicating that the sensation of pain after one week of treatment was lower than those observed in the immediate times and after 24 hours. For the results of satisfaction, a mixed linear model was constructed and assigned points (ranks) and the Bonferroni method (0.05%) was used for paired comparisons of time effects. The results indicate a reduction in the levels of satisfaction between the immediate and one year periods and periods between 24 hours and one year. All bleaching gels showed minimal pH variation in the evaluated times, however, there was an increase in pH from the first to the last application in all groups and the EXP Group showed the highest pH followed by the LP15, TB35LH and TB35 Groups which presented lower pH values. The LP15, TB35 and TB35LH Groups showed less color variation over a 12 month follow-up. The effect of the bleaching protocol did not affect the sensitivity of patients and after a week, and the sensitivity returned to normal levels. The level of patient satisfaction was significant in relation to the time and not the bleaching protocols. The patients in the TB35 Group were more dissatisfied during the research.

Neste trabalho foram estudados os fenômenos fotoinduzidos apresentados pelos filmes oxisulfetos de composição: 90% GeS2 + 10% Ga2 O3. Os filmes foram depositados em substrato de borosilicato pela técnica de evaporação por feixe de elétrons. A partir dos espectros de transmissão, a energia do bandgap, o índice de refração e a espessura foram determinados por diferentes métodos de análise. Para determinar as condições que otimizamos o efeito da fotoexpansão, as amostras foram expostas à radiação UV com energia acima do bandgap (3,5 eV), variando a densidade de potência (7,1 - 47,2 mW/mm2), tempo de exposição (30 180 min) e a espessura do filme (0,37 4,80 m). As áreas expostas foram analisadas usando um perfilômetro e valores de fotoexpansões variando de 0,03 a 0,16 m foram obtidos, cujo valor máximo foi encontrado para um filme com 1,80 m de espessura após iluminação com 24,3 mW/mm2 durante duas horas. Medidas da borda de absorção óptica revelaram um deslocamento para menores comprimentos de onda após a iluminação. O efeito de fotoclareamento foi acompanhado por uma diminuição do índice de refração, medido pela técnica de acoplamento de prisma. Os resultados revelaram a influência do oxigênio incorporado na matriz vítrea quando comparado ao Ga10Ge25S65. Consideramos que as mudanças fotoinduzidas são causadas por mudanças estruturais, como pôde ser verificado por medidas de espalhamento Raman nas configurações HH e HV. A dependência dos espectros Raman com a polarização da luz, observada em filmes iluminados e não-iluminados, é uma evidência direta para a ocorrência de importantes mudanças estruturais causadas por irradiação óptica, principalmente nas ligações Ge-S. As composições químicas foram determinadas por EDX e indicaram um aumento de oxigênio na superfície iluminada que pode estar associado ao aumento das ligações Ga-O-Ga. Como aplicação destes fenômenos fotoinduzidos, a fotoexpansão foi usada para a produção de redes de difração. As medidas de eficiência de difração e as imagens de microscopia de força atômica demonstraram que a fotoexpansão cria uma rede de relevo na superfície do vidro. / Photoexpansion and photobleaching effects were observed in 90% GeS2 + 10% Ga2O3 films. The films were deposited onto borosilicate substrates by electron beam evaporation technique. From transmission spectra, their bandgap energy, refractive index and thickness were determined by different analysis methods. To evaluate the photoinduced effects and find the optimal conditions to get the largest photoexpansion, the samples were exposed above bandgap light (~ 351 nm), varying power density (7.1- 47.2 mW/mm2), exposure time (30 120 min) and film thickness (0.37 4.80 m). The exposed areas were analyzed using profilemeter and photoexpansions from 0.03 to 0.16 m were obtained, whose maximum value was found for a 1.80 m thick film after 24.3 mW/mm2 illumination during 120 min. Fractional expansion (_V/V) from 8% to 30% was obtained and optical absorption edge measurements revealed a blue shift after illumination. This photobleaching was accompanied by a decrease in refractive index, as measured with the prism-coupling technique. The results reveal the influence of incorporated oxygen in the glass matrix when compared with Ga10Ge25S65 [1]. The chemical compositions were measured using an energy dispersive analyzer (EDX) and no significant difference could be observed between the compositions of illuminated and nonilluminated samples. So, we supposed that the photoinduced changes are caused by photostructural changes as well observed with Raman-scattering measurements in HH and HV configurations. The dependence of Raman spectra with the polarization of the light, observed in illuminated and non-illuminated films, is a direct evidence for the occurrence of important structural changes in local bonding configuration caused by optical irradiation. As application of the induced phenomenon, photoexpansion effect has been used to produce diffraction gratings. Atomic microscopy images and diffraction efficiency data indicate that photoexpansion leads to relief gating on the glass surface.