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 presente trabalho tem como objetivo estudar propriedades ópticas e estruturais de vidros e filmes de fosfato de antimônio dopados. Os vidros foram produzidos pela fusão a 900 °C, em cadinhos de carbono vítreo, dos precursores [Sb(PO3)3]n + Sb2O3 utilizando como dopantes o PbO e Cr2O3, sendo em seguida vertidos e resfriados rapidamente em moldes de aço inox. As composições vítreas em questão são: 20% [Sb(PO3)3]n 77% Sb2O3 3%PbO, 20 %[Sb(PO3)3]n 77% Sb2O3 3%Cr2O3 e 20 %[Sb(PO3)3]n 74% Sb2O3 6%Cr2O3 (mol%). A caracterização das amostras teve início com medidas de EDX para micro - análise composicional. Em seguida para os vidros na forma de bulk foram medidos os espectros de absorção e luminescência. As medidas de luminescência foram realizadas utilizando-se o laser de Kr+ nas linhas MLUV (350 nm) e MLVI (41 5nm). Medidas de EPR foram realizadas para os vidros dopados com Cr por estes possuírem elétrons desemparelhados. Os filmes finos foram depositados em substratos de soda lime pela técnica de evaporação de feixe de elétrons. A partir do espectro de absorção, o valor do bandgap (por volta de 3 eV) foi calculado, verificando que a melhor fonte para irradiação das amostras era o laser de Kr+ em MLUV. Apenas filmes de [Sb(PO3)3]n: 3% Pb2+ apresentaram efeito fotoestrutural. Com isso um estudo variando parâmetros, como tempo de irradiação, densidade de potência e espessura dos filmes, foi realizado a fim de observar a otimização do efeito. Acompanhado das variações fotoestruturais, ocorreram variações fotocrômicas, no caso fotoclareamento, as quais se caracterizaram por um deslocamento da borda de absorção para maiores energias, observado nos espectros de absorção das áreas irradiadas. Com os resultados observados do estudo e caracterização das amostras, algumas aplicações tecnológicas foram discutidas, como no caso dos efeitos observados nos filmes que podem ser utilizados para gravação de redes de difração. / The present work is about the study of the optical and structure properties of doped antimony phosphate glasses and thin films. The glasses were produced by the melting of precursors [Sb(PO3)3]n + Sb2O3 using PbO and Cr2O3 as dopant at 900 °C in vitreous carbon crucibles, then poured and quickly cooled into steel molds. The vitreous composition are: 20% [Sb(PO3)3]n 77% Sb2O3 3%PbO, 20 %[Sb(PO3)3]n 77% Sb2O3 3%Cr2O3 e 20 %[Sb(PO3)3]n 74% Sb2O3 6%Cr2O3 (mol%). The samples characterization started with EDX measurements for compositional micro-analysis. Then for the glasses in bulks form the absorption and luminescence spectra were measured. The luminescence measurements were done using MLUV (350 nm) and MLVI (415 nm) of a Kr+ laser. EPR measurements were taken for the Cr doped glasses because they present unpaired electrons. The thin films were deposited on soda lime substrates by the electron beam evaporation technique. From absorption spectra, the bandgap value around 3 eV was evaluated, noting that the best irradiation source of the samples was using a MLUV of a Kr+ laser. Only [Sb(PO3)3]n: 3% Pb2+ films presented photostructural effects. Therefore a systematic study varying several parameters, like irradiation time, power density and film thickness was conducted to observe the effect optimization. Following the photostructural variation, occurred photocchromic effects, in this case, photobleaching effects, which were characterized by a displacement of absorption edge to lower energies, observed in the absorption spectra of the irradiated areas. With the observed results from the study and characterization of the samples, some technologies applications were discussed, like in the case of the observed films effects that can be used for recording diffraction gratings.

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.

No description available.

Cardiovascular disease is a leading cause of morbidity worldwide. Many cardiomyopathies and developmental defects arise from misregulation of the cardiac extracellular matrix (ECM), a dynamic network of proteins, growth factors, and signaling molecules that form a protective sheath around organs and tissues. Changes in ECM composition are mediated in part by matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). ECM dysregulation leads to outcomes such as fibrotic scarring, hypertrophy, and myocardial infarction. Although fundamental to heart formation and function, the regulation of ECM integration and remodeling during growth is poorly understood. To investigate this, I developed a novel adaptation of fluorescence recovery after photobleaching (FRAP), which, for the first time, allows us to assess ECM protein incorporation during growth in live, intact Drosophila larvae. As such, recovery of fluorescently tagged proteins is a proxy for addition or relocation of ECM protein. We focus on Collagen IV (Viking), a conserved protein and major constituent of the basement membrane (BM). Integration and stabilization of Collagen IV in the BM is poorly understood, however is known to be mediated in part by Collagen modifying proteins secreted protein acidic and rich in cysteine (SPARC) and lysyl oxidase (Lox) are known. We established a time course for Vkg-GFP fluorescence accretion in the heart and body wall muscle throughout larval development, under normal conditions and those in which mmp2 or timp is overexpressed. We also observed the effects reducing the activity of SPARC and Lox Vkg dynamics in the early third instar cardiac ECM. In wildtype, we report a strong phasic pattern of Vkg accumulation at second to third instar ecdysis, potentially to support growth of the succeeding instar. Heart-specific overexpression of mmp2 and timp, the inhibitor of mmp2, perturbs net fluorescence recovery as well as estimated turnover of Vkg-GFP. Our results suggests that MMPs are positive regulators of Vkg/Col IV turnover in the ECM, which is in alignment with other recent studies (Davis et al., 2022; Töpfer et al., 2022). Loss of SPARC and Lox appears to affect estimated Vkg turnover in the cardiac ECM, consistent with a role for these proteins in integrating and stabilizing Collagen IV in the BM. These findings have implications in cardiac conditions and in other ECM-related disorders and diseases such as connective tissue disorders, muscular dystrophy, fibrosis, and cancer. / Thesis / Doctor of Science (PhD)

Fluorescence microscopy has played a pivotal role in the realm of biological and biomedical research, allowing researchers to delve into the intricacies of living organisms at the cellular and molecular levels. By using fluorescent probes, one can visualize specific molecules and structures within cells, fundamentally transforming our comprehension of biology and medicine. However, fluorescence microscopy faces its own set of challenges, namely, photobleaching and photodamage. Photobleaching involves the irreversible loss of fluorescence signal during imaging, while photodamage results in harmful effects on cells. Both severely limit fluorescence signal and observation time. Although remedies exist to mitigate these problems, most of them rely on chemical approaches. In this dissertation, to address these issues, I investigated two optical approaches that exploit control of light either in space or time. Firstly, I developed multiline scanning confocal microscopy (mLS) with a digital micro-mirror device. This method provides programmable patterns of the illumination beam as well as the detection slit. Through experimental results and optical simulations, I assessed the depth discrimination of mLS under different optical parameters and compared it with a multipoint system such as spinning disk confocal microscopy (SDCM). Surprisingly, under the same illumination duty cycle, I found that mLS offers better optical sectioning than SDCM. Importantly, the parallelized line illumination showed a much lower photobleaching rate compared to single-line scanning microscopy, while their optical sectioning capabilities remained similar. I applied this technique to visualize heterogeneous mouse epiblast stem cells, a challenging task in imaging. Secondly, I delved into low photobleaching rate two-photon microscopy (2PM). 2PM inherently provides excellent optical sectioning due to its nonlinear effects, making it suitable for high-resolution imaging within biological tissues. However, the high peak power of ultrafast pulses has always been associated with severe photobleaching, posing a longstanding challenge. I found that controlling the repetition rates of ultrafast lasers is a potential strategy to enhance photostability. Specifically, I used repetition rates lower or higher than 80 MHz in 2PM and conducted systematic experiments to investigate how optical parameters such as wavelengths, excitation powers, and pulse schemes can influence the photobleaching kinetics of fluorescent proteins and organic fluorophores. This thesis embarks on a journey to explore innovative strategies and methodologies aimed at reducing photobleaching while maintaining high-quality imaging in the realm of fluorescence microscopy.

<p>Photodynamic therapy (PDT) is a rapidly developing clinical treatment modality involving a light-activatable photosensitizer, tissue oxygen and light of an appropriate wavelength to generate cytotoxic reactive molecular species - primarily singlet oxygen (¹O₂). Singlet oxygen readily reacts with surrounding biomolecules leading to different biological effects and subsequent therapeutic outcomes. Over the last decades, many standard PDT treatments have been approved worldwide to treat different medical conditions ranging from a variety of cancer conditions to age-related macular degeneration (AMD). Meanwhile, many active clinical trials and pre-clinical studies are underway for other clinical indications. The therapeutic outcomes of PDT are difficult to predict reliably even with many years of research. The fundamental cause for this is the inherent complexity of PDT mechanisms. As PDT involves three main components, the outcomes of PDT are determined by the combination of all components. Each component varies temporally and spatially during PDT, and the variations are mutually dependent on each other. Moreover, components such as the photosensitizer can have great variations in their initial distribution among patients even before PDT treatment. Given this, no well accepted standard PDT dose metric method has been recognized in clinics, although different approaches including explicit, implicit and direct dosimetry have been studied. To tackle the inherently complicated PDT mechanism in order to provide insights into PDT and PDT dosimetry, a theoretical one-dimensional model for aminolevulinic acid (ALA) induced protoporphyrin IX (PpIX)-PDT of human skin was developed and is presented in this thesis. The model incorporates major photophysical and photochemical reactions in PDT, and calculated temporal and spatial distributions of PDT components as well as the detectable emission signals including both sensitizer fluorescence and singlet oxygen luminescence (SOL) using typical clinical conditions. Since singlet oxygen is considered to cause PDT outcomes, the correlations of different PDT dose metrics to average reacted (¹O₂) "dose" and "dose" at different depths were examined and compared for a wide range of varied treatment conditions. The dose metrics included absolute fluorescence bleaching metric (AFBM), fractional fluorescence bleaching metric (FFBM) and cumulative singlet oxygen luminescence (CSOL), and the varied treatment conditions took into account different treatment irradiances and wavelengths, varied initial sensitizer concentration and distribution, and a wide range of optical properties of tissue. These investigations and comparisons provide information about the complicated dynamic process of PDT such as the induction of tissue hypoxia, photosensitizer photobleaching and possible PDT-induced vascular responses. It was also found that the CSOL is the most robust and could serve as a gold standard for the testing of other techniques. In addition to these theoretical studies, recent progress on the assessment of a novel, more efficient superconducting nanowire single photon detector (SNSPD) for singlet oxygen luminescence detection will be introduced and the current photomultiplier tubes (PMT) system will be briefly described as well. The author participated in the experimental assessments of the SNSPD and analyzed the results shown in this thesis.</p> / Doctor of Philosophy (PhD)

Ce mémoire rapporte deux approches de la synthèse des protéines à l'échelle de la molécule unique. Nous utilisons la microscopie de fluorescence en onde évanescente pour sonder l'activité traductionnelle de deux types de ribosomes. Les premiers, issus d'E. Coli (organisme procaryote), sont mutés afin de les marquer d'un nanocristal semiconducteur (QD). La fin de la traduction, qui correspond au décrochage du ribosome de l'ARNm lorsque celui-ci atteint le codon stop, est alors mise en évidence par la disparition du QD de la surface de l'échantillon. Le deuxième type de ribosome étudié est quant à lui extrait de cellules de lapins (organisme eucaryote) et est dit "sauvage", c'est à dire qu'il n'a pas subi de modification, tandis qu'un oligonucléotide marqué d'un fluorophore est hybridé à l'ARNm. L'activité hélicase du ribosome lui permettant de séparer deux brins complémentaires, l'oligonucléotide et donc le fluorophore disparaissent en même temps que le ribosome parcourt l'ARNm, permettant ainsi de sonder l'activité du ribosome. Nous donnons pour ces deux types de ribosomes une vitesse moyenne de la traduction dans des milieux contenant les facteurs de la traduction issus d'extraits cellulaires.La deuxième approche de la synthèse des protéines porte sur les propriétés de l'ARNm, support de l'information génétique codant pour la séquence des protéines. Nous avons développé un montage de pince optique permettant de manipuler et caractériser les propriétés mécaniques d'oligonucléotides, ainsi qu'une méthode originale de calibration de ce piège optique. La cohérence de nos mesures sur l'étirement d'un double brin d'ADN avec la littérature nous permettra de poursuivre notre étude sur la mesure des forces nécessaires pour ouvrir une structure secondaire de l'ARNm. / We hereby report two approaches of the protein synthesis at the single molecule level. We use total internal reflection fluorescence microscopy to study the translation kinetic of two different types of ribosomes. The first ones, extracted from E. Coli (prokaryotic organism), are mutated in order to label them with a quantum dot (QD). The end of translation, which corresponds to the dissociation of the ribosome from the mRNA when the stop codon has been reached, is highlighted by the disparition of the QD from the surface. The second type of ribosome is extracted from rabbit cells (eukaryotic organism) and has not been modified (wild type), while a labeled oligonucleotide is hybridized on the mRNA. The helicase activity of the ribosome allowing the dissociation of two complementary strands, the oligonucleotide and so the label disappear at the same time while the ribosome moves along the mRNA and thus inform us about its activity. For these two types of ribosomes we measure their average translation speed in cell extracts.The second approach focuses on the properties of the mRNA, carrying the genetic code for the protein sequence. We developped an optical tweezer setup in order to manipulate and characterize the mechanical properties of nucleotides, as well as an original method to calibrate this optical trap. The consistency of our measurements with the litterature on the properties of a double stranded DNA will allow us to study secondary structures of mRNA.

A técnica de fluorescência óptica tem sido aplicada em diversas áreas médicas, como no acompanhamento da degradação de drogas e na detecção de câncer, por apresentar alta sensibilidade, simplicidade e rapidez na obtenção de dados. A avaliação não-invasiva e não-destrutiva é um grande atrativo que esta técnica oferece para o diagnóstico clínico. Assim, o objetivo deste projeto consistiu no desenvolvimento de um sistema de fluorescência óptica por imagem de campo amplo e avaliação do sistema no monitoramento da fotodegradação da Protoporfirina XI, utilizada na Terapia Fotodinâmica (TFD), e na visualização da presença de microrganismos presentes na microbiota bucal. O sistema desenvolvido é constituído de um sistema óptico, mecânico, eletrônico e de detecção. O sistema óptico é composto por LEDs de alta intensidade, com emissão centrada em 405nm e 450nm e 3 filtros ópticos: 1. passa-banda: utilizado na excitação; 2. dicróico; e 3. passa-alta: utilizados para excitação e emissão da fluorescência. O sistema mecânico foi desenvolvido em alumínio, possuindo as funções de dissipação de calor do sistema de iluminação e estrutural. O sistema eletrônico possui a função de controle e fornecimento de energia ao sistema de iluminação. O sistema de detecção é composto por uma câmera CCD e fotográfica, acoplada ao sistema de fluorescência desenvolvido. Um dos principais fatores para obtenção de bons sinais de fluorescência é a potência óptica obtida no sistema de iluminação, que neste caso foi de 200 mW. Outro fator é o comprimento de onda da iluminação; neste sistema foi obtida uma banda de iluminação muito eficaz entre 390 nm e 460 nm. O sistema de filtros proporcionou um sinal de fluorescência bastante satisfatório, bem como um bom contraste na visualização das imagens de fluorescência. Com este sistema foi possível acompanhar a fotodegradação da Protoporfirina IX, em função do tempo de iluminação durante a TFD. Proporcionou-se, assim, uma nova ferramenta para o avanço na avaliação da dosimetria da TFD, podendo otimizar e personalizar a TFD para cada paciente, já que o sistema desenvolvido permite a visualização da presença do agente fotossensível na terapia. Outra contribuição relevante do trabalho alcançada foi a visualização da presença de microrganismos da microbiota bucal, já que estes são os grandes responsáveis pelas doenças bucais como é o caso da cárie dental. Desta forma, conclui-se que foi possível desenvolver um sistema para auxílio da dosimetria na TFD e na visualização de microrganismos presentes na microbiota bucal. O sistema desenvolvido se mostrou compacto, agregando iluminação e visualização, tornando-o num protótipo com interface para uso clínico. O protótipo foi testado em pacientes para a visualização da microbiota bucal, tratamento de pré-câncer de pele e de vulva. / Optical fluorescence has been applied to several medical areas, as to monitor drug degradation and cancer detection, due to its high sensitivity, simplicity and fast response. Non-invasive and non-destructive assessment of tissues using this technique is very attractive for clinical diagnosis. Hence, the aim of was the development of an optical fluorescence wide-field imaging system, and the evaluation of its performance on monitoring protoporphyrin IX (PpIX) during Photodynamic Therapy (PDT), and one visualization of microrganisms present in oral microbiota. The developed device is composed by optical, mechanical, electronic, and detection systems. Optical system is composed by high-intensity light-emitting diodes (LED), with emission centered at 405 nm and 450 nm, and 3 optical filters: a bandpass filter for excitation; and set of dichroic and long wave pass filter, for fluorescence excitation and emission. The mechanical system was built in aluminum for structural and ilumination systems heat dissipation. The electronic system provides the control of the illumination system. The detection system is composed by a CCD and a conventional didgital camera, coupled to the developed device. One of the main factors for good fluorescence signals is the achieved optical power - in our case, it was of 200 mW. Another factor is excitation wavelength; for this system, a very efficient illumination band was achieved between 390-460 nm. The optical filters allowed a very satisfying fluorescence signal, with good contrast for fluorescence images visualization. The developed system enabled the monitoring of PpIX photobleaching as a function of illumination time during PDT. Therefore, a new tool to improve PDT dosimetry is offered by the use this system, allowing a more customized dosimetry each patient, since the device allows visualization of the photosensitive agent during the therapy. Visualization of oral microrganisms was also achieved, which was another relevant contribution of the developed instrumentation because they are the main cause of oral diseases such as caries. Thus the development of a system to both improve PDT dosimetry and oral microrganisms visualization was achieved as a compact device, joining illumination and visualization. These characteristics shows a good interface for clinical use. The prototype was tested in patients for oral microbiota visualization, and skin/vulva pre-cancer lesions treatment.