A terapia fotodinâmica (PDT) é uma alternativa promissora de tratamento para lesões pré-cancerosas e para câncer de pele não-melanoma, como o carcinoma espinocelular, agressivo e potencialmente metastático. Visando melhorar a eficiência da terapia fotodinâmica de carcinoma espinocelular de pele, este estudo otimizou o tempo para início da terapia, avaliou a eficácia dos fotossensibilizadores ácido aminolevulínico (ALA- 20%) e o metil-ester aminolevulínico (MEALA-10%) e verificou o coeficiente de atenuação relativo à pele normal dos grupos experimentais, por meio de Tomografia por Coerência Óptica. Para a indução do tumor foi realizada a carcinogênese química (DMBA/TPA) por um período de 28 semanas. A espectroscopia de fluorescência foi utilizada para monitoração da emissão da molécula de protoporfirina IX, induzida pelo ALA e MEALA. A aquisição de dados a cada 30 minutos totalizando um período de 360 minutos, permitiu verificar a máxima incorporação de ALA e MEALA em 300 e 330 minutos após a aplicação, respectivamente. Após a otimização foi realizada a PDT, avaliação clínica, histopatológica e análise por OCT dos grupos experimentais, por meio das quais verificou-se maior eficiência do grupo tratado com MEALA. No período de 20 dias, o percentual de lesões com redução de área maior que 50%, foi de 33% na PDT com ALA e 83% para o MEALA. Os coeficientes de atenuação ópticos para os grupos com neoplasia foram maiores do que os do grupo controle. Os grupos tratados com PDT apresentaram valores de coeficiente de atenuação que se aproximam dos valores obtidos para a pele sadia, evidenciando a resposta ao tratamento. Entretanto para ambos os fotossensibilizadores utilizados, a PDT mostrou-se eficaz quando iniciada nos tempos determinados neste estudo, e a técnica de OCT mostrou-se uma potencial ferramenta na avaliação deste tratamento. / Photodynamic therapy (PDT) is a promising alternative for pre-cancerous lesions and non-melanoma skin cancer treatment as squamous cells carcinoma, which is aggressive and potentially metastatic. Aiming the improvement of photodynamic therapy efficiency for skin squamous cells carcinoma, this study has optimized the time to start the therapy, it also evaluated the effectiveness of the aminolevulinic acid (ALA-20%) and methyl-ester aminolevulinic (MEALA-10%) as photosensitizers as well as verified the optical attenuation coefficient relative to normal skin of the experimental groups by optical coherence tomography (OCT). A chemical carcinogenesis was performed (DMBA/TPA), by a period of 28 days. Fluorescence Spectroscopy was used to monitor the emission of protoporphyrin IX induced by ALA and MEALA. Signal acquisition was performed every 30 minutes for a total period of 360 minutes, showing a maximum incorporation of ALA and MEALA, 300 and 330 minutes after the application. PDT was conducted after the optimization of the irradiation time. Clinical and histopathologic evaluation and OCT analysis of the experimental groups showed higher efficiency of the MEALA-treated group. After 20 days of treatment, the percentage of lesions with area reduction greater than 50%, was 33% for the ALA group and 83% for the MEALA group. The optical attenuation coefficients for groups with cancer were higher than those in the control group. The groups treated with PDT presented attenuation coefficient values that are close to the values obtained for healthy skin, showing the positive response to the treatment. This study showed that PDT was effective for both photosensitizers when initiated at the times determined in this study, and OCT technique proved to be a potential tool in the assessment of this treatment.

Les triterpènes sont des composés en C30 issus de la cyclisation du 2,3-epoxysqualène. L’utilisation industrielle et l’intérêt thérapeutique des triterpènes représentent un enjeu capital dans le domaine de la recherche des substances naturelles. Quant au cholestérol, il a été largement connu dans la littérature par son rôle modulateur des membranes naturelles aussi bien que synthétiques. Dans notre travail de thèse, nous nous sommes intéressés à étudier l’effet des triterpènes et du cholestérol sur la fluidité et la perméabilité des liposomes de dipalmitoylphosphatidylcholine. Deux approches sont utilisées pour réaliser ces études par le biais des liposomes charges ou des liposomes préformés. Les études de perméabilité sont réalisées par la spectroscopie de fluorescence à travers le suivi d’un fluorophore hydrophile, la sulforhodamine B en fonction du temps. Les études de la fluidité sont réalisées par la résonance paramagnétique électronique. Ces sondes permettent d’évaluer la fluidité à l’interface de la membrane ainsi qu’au cœur hydrophobe de la membrane. Les données obtenues de la cinétique de libération de la sulforhodamine sont quantifiées par des modèles mathématiques. Cers derniers serviront comme des outils pour prédire les mécanismes de libération. Nos résultats montrent que la perméabilité ainsi que la fluidité de la membrane sont réduites suite à l’ajout du cholestérol. L’effet des triterpènes sur les propriétés physiques des membranes dépend étroitement de la composition de la membrane, le taux du cholestérol, la structure du principe actif et le temps d’incubation / Triterpenes are C30 compounds derived from the cyclization of 2,3-epoxysqualene. The industrial use and the therapeutic interest of the triterpenes represent a capital importance in the field of the research of the natural substances. As for cholesterol, it has been widely known in the literature by its modulating role of natural as well as synthetic membranes. In our thesis work, we were interested in studying the effect of triterpenes and cholesterol on the fluidity and permeability of dipalmitoylphosphatidylcholine liposomes. Two approaches are used to carry out these studies via liposomes fillers or preformed liposomes. Permeability studies are carried out by fluorescence spectroscopy through the monitoring of a hydrophilic fluorophore, sulforhodamine B as a function of time. Studies of fluidity are carried out by electronic paramagnetic resonance. These probes make it possible to evaluate the fluidity at the interface of the membrane as well as at the hydrophobic core of the membrane. The data obtained from the release kinetics of sulforhodamine are quantified by mathematical models. These latter will serve as tools to predict the release mechanisms. Our results show that the permeability as well as the fluidity of the membranes are reduced following the addition of cholesterol. The effect of triterpenes on the physical properties of membranes is highly dependent on membrane composition, cholesterol level, the drug structure and incubation time

A exposição crônica à radiação ultravioleta (UV) resulta no fotoenvelhecimento da pele humana. A fim de tratá-lo, a terapia fotodinâmica (TFD) - técnica que utiliza luz, um fotossensibilizador (FS) e oxigênio molecular - tem sido utilizada em ambientes clínicos, no entanto são escassas as investigações que revelam os achados histopatológicos desse tratamento na pele fotoenvelhecida. Com isso, o objetivo deste trabalho foi analisar experimentalmente os efeitos da TFD na pele fotoenvelhecida de camundongos por meio de técnicas ópticas e histológicas. Portanto, foram utilizados camundongos hairless (sem pelo) distribuídos aleatoriamente em diferentes grupos, tais como: Controle, animais de pele sadia envelhecida intrinsecamente, não irradiados com luz UV e não tratados; UV, animais irradiados com luz UV e não tratados; UV/Luz, animais irradiados com luz UV e tratados com fototerapia; UV/TFD, animais fotoenvelhecidos (irradiados com luz UV) e tratados com a TFD; e Controle/TFD, animais de pele sadia envelhecida intrinsecamente e tratados com TFD. A indução do fotoenvelhecimento foi realizada por diferentes fontes de luz contendo, principalmente, a banda espectral UV; para a TFD, foram utilizadas fontes de luz com comprimento de onda de 415, 630 e 635 nm (azul e vermelho) juntamente com o ácido 5-aminolevulínico (ALA), precursor do FS endógeno protoporfirina IX (PpIX). Para fazer o seguimento da TFD por técnicas ópticas, foram realizadas espectroscopia de fluorescência, imagem de fluorescência de campo amplo e tomografia por coerência óptica (OCT, do inglês optical coherence tomography). A avaliação histopatológica pós-TFD foi realizada com os corantes HE, Tricrômio de Masson (TM) e Verhoeff a fim de analisar a espessura da epiderme, o infiltrado inflamatório, o conteúdo de colágeno na derme e sua espessura e a qualidade das fibras elásticas. Como resultado, observouse aumento significativo da espessura da epiderme pela regeneração dos queratinócitos e deposição de novas fibras colágenas dérmicas apenas nos animais tratados com ALA-TFD, tanto irradiados quanto não-irradiados com luz UV (grupos UV/TFD e Controle/TFD, respectivamente). Assim, torna-se evidente que a TFD trata a pele fotoenvelhecida do camundongo hairless pelos achados histopatológicos e através das imagens de OCT. Com isso, este trabalho agrega informação às investigações clínicas a respeito da TFD no tratamento da pele fotoenvelhecida, atestando seu uso para o fotorrejuvenescimento cutâneo. / The chronic exposure to ultraviolet radiation (UVR) leads to photoaging of the human skin. As a treatment, the photodynamic therapy (PDT) which brings together light, a photosensitizer, and molecular oxygen has been extensively used on clinical trials; however, there are few studies that highlight the histopathologic findings regarding this technique on photoaged skin. Thus, the aim of this study was to analyze experimentally the effects of PDT on photoaged skin of hairless mice by means of optical and histopathologic assessments. For this, hairless mice were randomly allocated in different groups, such as: Control, animals with healthy aged skin, neither exposed to an UV lamp nor treated; UV, animals exposed to an UV lamp with no treatment; UV/Light, animals exposed to an UV lamp and treated just with light; UV/PDT, animals exposed to an UV lamp and treated with PDT; and Control/PDT, animals with healthy aged skin treated with PDT. The photoaging process was induced by different light sources, which had mainly the UV spectrum; for the PDT, light sources of different wavelengths (415, 630, 635 nm) were used (blue and red light sources) and the 5-aminolevulinic acid (ALA), PS precursor of the endogenous protoporphyrin IX (PpIX). In order to follow the PDT protocol up by means of optical techniques, we used fluorescence spectroscopy, fluorescence images and optical coherence tomography. The histopathologic assessment after the PDT procedure was performed with H&E, Massons Trichrome and Verhoeff stains for epidermal thickness, inflammatory infiltrate, dermal collagen content, dermal thickness, and quality of elastic fibers. As a result, it has been observed a significant epidermal thickening due to keratinocytes regeneration and newly formed dermal collagen fibers only on the groups treated with PDT-ALA (UV/PDT and Control/PDT groups). Therefore, it is evident that PDT treats the photoaged skin of hairless mice since histopatologic findings and OCT images have shown those morphology changes. In conclusion, this work add information to the clinical trials regarding the PDT as a reliable technique to treat photoaged skin, proving its use for the skin photorejuvenation.

A terapia fotodinâmica (TFD) utilizando ácido 5-aminolevulinico (ALA) e derivados em aplicação tópica e, como precursor da protoporfirina IX (PPIX) apresenta alguns limitantes relativos a baixa permeação das substâncias na pele. Comportamento este que afeta a produção e homogeneidade da distribuição da PPIX na superfície e camadas mais profundas da pele. Para resolver essa limitação muitos autores propõem alternativas modificando a molécula do ALA e derivados, bem como modificando as propriedades químicas da fase externa da emulsão (mais hidrofílica ou hidrofóbica) ou então o sistema de entrega para a emulsão. O objetivo desse estudo é avaliar qual a proporção de ALA e metil-5-aminolevulinato (MAL) que quando misturados levam ao aumento da quantidade e uniformidade da formação da PPIX na superfície e em profundidade na pele. Para esse estudo foi realizada análises de fluorescência e histologia. O estudo foi conduzido in vivo e ex vivo usando biópsias de pele de porco cultivadas in vitro. A produção de PPIX foi monitorada utilizando espectroscopia de fluorescência, imagem de fluorescência de campo amplo e microscopia confocal de fluorescência. E para a aplicação da TFD os parâmetros usados foram de 125 mW/cm2 de intensidade e 150 J/cm2 de dose. A análise do dano causado pela irradiação foi realizada por meio de histologia da pele após 24 e 48 horas da aplicação da TFD. O ALA e MAL na concentração de 20% foram misturados nas seguintes proporções: ALA ou M, M2 (80% ALA - 20% MAL), M3 (60% ALA 40% MAL), M4 (50% ALA MAL), M5 (40% ALA 60% MAL), M6 (20% ALA 80% MAL) e MAL como M7. As diferentes proporções foram incorporadas em emulsões óleo em água (O/A) e água em óleo (A/O). De acordo com os resultados, as misturas M3, M4 e M5 mostraram maior produção de PPIX na superfície da pele segundo as medidas de fluorescência em 3h de incubação e, no estudo da cinética mostraram produzir PPIX em menor tempo. No estudo de permeação do creme in vitro em pele ex vivo, por microscopia confocal de fluorescência, observou-se que as misturas M3, M4 e M5 produziram mais PPIX nas camadas da pele do que ALA e MAL. As análises histológicas das misturas apresentaram maior dano fotodinâmico na superfície e profundidade das camadas da pele após a TFD, independente da emulsão. A análise em até 48h observou-se predominantemente a fase do processo de reparo referente à fase inflamatória, mas existem indícios ao longo das análises tanto macroscópicas e histológicas que o processo de reparo referente as fases subsequentes de proliferação e remodelamento estão iniciando-se em paralelo. A mistura M4 em ambas as emulsões apresentou elevada quantidade de formação de PPIX em menor tempo de incubação. M4 em emulsão O/A apresentou menor dano fotodinâmico já que a evolução do processo reparo foi mais rápida sugerindo-se potencial de aplicação em TFD voltado para área cosmética-estética. Já M4 em emulsão A/O levou a um maior dano fotodinâmico já que a evolução do processo de reparo foi mais lenta sugerindo-se potencial de aplicação em TFD voltado para área oncológica e de doenças de pele. De modo geral o estudo proposto apresentou impacto positivo para a otimização da terapia fotodinâmica em aplicação tópica. / Photodynamic therapy (PDT) using 5-aminolevulinic acid and derivatives on topical application and as a precursor of protoporphyrin (PPIX) has some limitations for low permeation of substances into the skin. This behavior affects PPIX production and homogeneous distribution on the surface and deeper layers of the skin. To resolve this limitation, many authors propose alternatives such as modifying the molecule of ALA and its derivatives, as well as changing the chemical properties of the external phase of the emulsion (more hydrophilic or hydrophobic) or the delivery system to the emulsion. The aim of this study is to assess the proportion of ALA and methyl-5-aminolevulinate (MAL) that when mixed leads to an increase in the amount and uniformity of the PPIX formation on surface and deep skin. For this study we performed fluorescence analysis and histology. The studies were conducted in vivo and also using pig skin biopsies (ex vivo) cultured in vitro. The PPIX production was monitored using fluorescence spectroscopy, widefield fluorescence imaging, and fluorescence confocal microscopy. For the application of PDT an intensity of 125 mW/cm2 and a dose 150 J/cm2 were used. Analysis of the damage caused by irradiation was performed through skin histology after 24 and 48 hours after PDT application. ALA and MAL in concentration of 20% were mixed in the following proportions: ALA or M, M2 (80% ALA - 20% MAL), M3 (60% ALA - 40% MAL), M4 (50% ALA - MAL) M5 (40% ALA - 60% MAL), M6 (20% ALA - 80% MAL) MAL and as M7. Different proportions were incorporated in oil-in-water emulsions (O/W) and water-in-oil (W/O). The fluorescence measurements for 3h of incubation showed better PPIX production in the skin surface for mixtures M3, M4 and M5. Moreover, the kinetics study showed PPIX production in less time for these mixtures. In the study of cream permeation of ex vivo skin in vitro by confocal fluorescence microscopy, we observed that the mixtures M3, M4 and M5 produced more PPIX in the skin layers than ALA and MAL. The histological analyses of the mixtures showed higher photodynamic damage on the surface and deeper layers of the skin after PDT, independent of the emulsion. The analysis in 48 hours predominantly observed the phase of the healing process regarding the inflammatory phase but there are signs along both macroscopic and histological analysis that the healing process concerning the subsequent stages of proliferation and remodeling are initiating in parallel. The mixture M4 in both emulsions had high amounts of PPIX formation in shorter incubation time. M4 emulsion O/A showed a lower photodynamic damage since the evolution of the healing process was faster suggesting to potential application in PDT facing cosmetic-aesthetic area. M4 already in W/O emulsion led to a greater photodynamic damage since the evolution of the healing process was slower suggesting to potential application in PDT facing oncology and skin diseases. Overall the proposed study had a positive impact on the optimization of photodynamic therapy for topical application.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-03-15T11:39:35Z No. of bitstreams: 2 Dissertação - Diéricon Sousa Cordeiro - 2018.pdf: 6920771 bytes, checksum: 83ec23012a03b4c1207c2f24818766a5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-03-15T11:39:57Z (GMT) No. of bitstreams: 2 Dissertação - Diéricon Sousa Cordeiro - 2018.pdf: 6920771 bytes, checksum: 83ec23012a03b4c1207c2f24818766a5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-03-15T11:39:57Z (GMT). No. of bitstreams: 2 Dissertação - Diéricon Sousa Cordeiro - 2018.pdf: 6920771 bytes, checksum: 83ec23012a03b4c1207c2f24818766a5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-01-31 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Photoluminescent, electroluminescent and photoconductive materials find a wide range of scientific and technological applications, therefore, the accurate photophysics properties description is indispensable. To apply carbazole derivatives as active layers in photovoltaic devices, the steady-state and time-resolved fluorescence spectroscopy of 9-vinylcarbazole (9VCz) and its respective polymer, Poly (N-vinylcarbazole) (PVCz) in solution and thin films prepared by distinct techniques were carried out in this work. Layer-by-Layer by self-assembling and by spin-coating thin films were prepared in order to compare the effects of the distinct techniques on the final photophysics. They also had their morphology characterized by distinct techniques of microscopy. The spectroscopic fluorescence showed that the ideal solutions for producing the thin films were those at concentrations of 10-4 mol L-1 and 10-5 g L-1 for molecule and polymer, respectively. Thin films of the molecule prepared by the self-assembling technique showed structured bands, however, for the polymer, we observed that the spin-coating technique resulted in a film with less aggregation and structured spectra, thus, well defined vibrational bands could be observed. In addition, it was observed that the fluorescence intensity decreases as the number of layer deposition increases in the 9VCz thin films, however, in the self-assembling tri-layer PVCz film, spectra with higher intensity were recorded. Microscopy indicates the formation of thin films; however, it is possible to observe the formation of aggregates on the surface of the thin films, which are also evidenced by steady-state fluorescence. / Materiais com propriedades fotoluminescentes, eletroluminescentes e fotocondutoras encontram aplicações científicas e tecnológicas bastante diversas, para isso, é indispensável executar o estudo detalhado da fotofísica dos materiais de interesse. Com o objetivo de aplicar derivados de carbazol como camada ativa em dispositivos fotovoltaicos, o estudo de espectroscopia de fluorescência fotoestacionária e com resolução temporal do 9-vinilcarbazol (9VCz) e seu respectivo polímero o Poli(N-vinilcarbazol) (PVCz) foi realizado nesse trabalho, quando em solução e quando na forma de filmes finos produzidos por métodos distintos: Layer-by-Layer por automontagem e por spin-coating, afim de comparar o efeito na fotofísica das técnicas de confecção de filmes finos, que também tiveram sua morfologia caracterizada por distintas técnicas de microscopia. Os resultados espectroscópicos das soluções evidenciaram as soluções 10-4 mol L-1 e 10-5 g L-1 para a molécula e para o polímero, respectivamente, como as soluções ideais para produção dos filmes finos. Os filmes finos preparados pela técnica de automontagem apresentaram bandas estruturadas para a molécula, no entanto para o polímero, observou-se que a técnica por spin-coating apresenta menor agregação e espectros mais estruturados, com isso, bandas vibracionais melhor definidas foram visualizadas. Além disso, notou-se que a intensidade de fluorescência apresentou uma diminuição à medida que se aumenta o número de camadas depositadas umas sobre as outras na produção dos filmes finos do 9VCz, no entanto, no filme de PVCz em tricamada, obtido por automontagem a fluorescência tem maior intensidade. A morfologia registrada indica a formação de filmes finos, porém, é possível observar a formação de agregados na superfície dos filmes finos, agregação essa que também é evidenciada pelos dados de espectroscopia de fluorescência fotoestacionária e resolvida no tempo.

A laranja é uma das frutas mais produzidas e consumidas no mundo, sendo o Brasil o maior produtor e exportador do seu suco concentrado. Entretanto, pragas e doenças comprometem consideravelmente sua produção. Atualmente, a doença mais preocupante é o Greening, também conhecida mundialmente como Huanglongbing (HLB). A doença não possui cura, apresenta longa fase assintomática e não possui um método eficiente de controle. Além disso, não existem métodos de diagnóstico aplicáveis em larga escala. Neste trabalho são propostas as técnicas fotônicas de fluorescência induzida por laser e de infravermelho por transformada de Fourier para o diagnóstico do HLB. Para a realização das medidas, foram coletadas folhas de árvores saudáveis, doentes com HLB e doentes com a clorose variegada dos citros, sendo esta incluída nos estudos para verificar a capacidade de diferenciação entre as doenças. Foram coletadas quatro classes de folhas nessas plantas: sadia, HLB-sintomática, HLB-assintomática e CVC-sintomática. As folhas foram medidas em laboratório e seus espectros foram pré-processados para indução de um classificador via regressão por mínimos quadrados parciais. Além das folhas, foram medidas amostras dos seguintes metabólitos primários e secundários para entendimento espectral: amido, glicose, sacarose, hesperidina, naringina e umbeliferona. Taxas de acerto de superiores a 89% foram obtidas na classificação das folhas nas técnicas de fluorescência e infravermelho, sendo superior às taxas dos métodos de manejo empregados atualmente no campo. A fluorescência induzida por laser apresenta um grande potencial para uso em campo devido a possibilidade de miniaturização de seus componentes. Os espectros dos metabólitos secundários apresentam fortes indícios de que a alteração de suas concentrações podem contribuir na detecção de doenças pelas técnicas fotônicas. / Sweet orange is one of the most produced and consumed fruit in the world, and Brazil is the largest producer and exporter of this fruit. However, pests and diseases significantly reduce the worldwide production. Currently, the most destructive disease in the field is called greening, also known as huanglongbing (HLB). There is no control for HLB. In addition, the disease presents a long asymptomatic phase. Furthermore, no diagnostic methods are available to use in large scale. In this study are proposed fluorescence and infrared spectroscopy for the HLB diagnosis. For the measurements were collected leaves from healthy, HLB- and citrus variegated chlorosis-infected plants, being the last one to comparison between the diseases. It were collected four classes of leaves: healthy, HLB-asymptomatic, HLB-symptomatic and CVC-symptomatic. The leaves were measured and their spectra were pre-processed for the induction of classifier via partial least squares regression. In addition, samples of plant metabolites were measured for leaves spectral interpretation: starch, glucose, sucrose, hesperidin, naringin and umbelliferone. Success rates above 89% were obtained through both photonic techniques, higher compared to the sucess rates of the actual management methods. The metabolites spectra have shown strong evidence that their concentrations changes could contribute to the diagnosis of the diseases by photonic techniques. Particularly, the fluorescence spectroscopy seems interesting because it has a great potential for field application due to the existence of portable photonic devices.

A laranja é uma das frutas mais produzidas e consumidas no mundo, sendo o Brasil o maior produtor e exportador do seu suco concentrado. Entretanto, pragas e doenças comprometem consideravelmente sua produção. Atualmente, a doença mais preocupante é o Greening, também conhecida mundialmente como Huanglongbing (HLB). A doença não possui cura, apresenta longa fase assintomática e não possui um método eficiente de controle. Além disso, não existem métodos de diagnóstico aplicáveis em larga escala. Neste trabalho são propostas as técnicas fotônicas de fluorescência induzida por laser e de infravermelho por transformada de Fourier para o diagnóstico do HLB. Para a realização das medidas, foram coletadas folhas de árvores saudáveis, doentes com HLB e doentes com a clorose variegada dos citros, sendo esta incluída nos estudos para verificar a capacidade de diferenciação entre as doenças. Foram coletadas quatro classes de folhas nessas plantas: sadia, HLB-sintomática, HLB-assintomática e CVC-sintomática. As folhas foram medidas em laboratório e seus espectros foram pré-processados para indução de um classificador via regressão por mínimos quadrados parciais. Além das folhas, foram medidas amostras dos seguintes metabólitos primários e secundários para entendimento espectral: amido, glicose, sacarose, hesperidina, naringina e umbeliferona. Taxas de acerto de superiores a 89% foram obtidas na classificação das folhas nas técnicas de fluorescência e infravermelho, sendo superior às taxas dos métodos de manejo empregados atualmente no campo. A fluorescência induzida por laser apresenta um grande potencial para uso em campo devido a possibilidade de miniaturização de seus componentes. Os espectros dos metabólitos secundários apresentam fortes indícios de que a alteração de suas concentrações podem contribuir na detecção de doenças pelas técnicas fotônicas. / Sweet orange is one of the most produced and consumed fruit in the world, and Brazil is the largest producer and exporter of this fruit. However, pests and diseases significantly reduce the worldwide production. Currently, the most destructive disease in the field is called greening, also known as huanglongbing (HLB). There is no control for HLB. In addition, the disease presents a long asymptomatic phase. Furthermore, no diagnostic methods are available to use in large scale. In this study are proposed fluorescence and infrared spectroscopy for the HLB diagnosis. For the measurements were collected leaves from healthy, HLB- and citrus variegated chlorosis-infected plants, being the last one to comparison between the diseases. It were collected four classes of leaves: healthy, HLB-asymptomatic, HLB-symptomatic and CVC-symptomatic. The leaves were measured and their spectra were pre-processed for the induction of classifier via partial least squares regression. In addition, samples of plant metabolites were measured for leaves spectral interpretation: starch, glucose, sucrose, hesperidin, naringin and umbelliferone. Success rates above 89% were obtained through both photonic techniques, higher compared to the sucess rates of the actual management methods. The metabolites spectra have shown strong evidence that their concentrations changes could contribute to the diagnosis of the diseases by photonic techniques. Particularly, the fluorescence spectroscopy seems interesting because it has a great potential for field application due to the existence of portable photonic devices.

Les déshydrogénases sont faiblement actives en présence de fortes concentrations (> 50 % (v/v)) de liquides ioniques (LIs) miscible à l’eau et les raisons précises de cette inactivation ne sont pas connues. La structure de la formiate déshydrogénase de Candida boidinii (FDH) en présence de ces LIs miscibles à l’eau a été étudiée par atténuation de fluorescence par de l’iode ou de l’acrylamide. Une concentration critique, la CILc ("Critical Ionic Liquid concentration"), au dessus de laquelle la fluorescence n’est pas exploitable, est déterminée. Elle se situe entre 30 et 40 % (v/v) des LIs de cette étude. Les LIs s’avèrent être de forts agents dénaturants : les constantes d’atténuation de fluorescence en présence de LI sont de 1,4 à 2 fois plus importantes qu’en présence d’urée. La FDH a été modifiée chimiquement par des cations analogues aux cations des LIs afin de la préserver de l’interaction avec les LIs. Les enzymes modifiées par des cations de plus petite taille présentent une importante activité résiduelle (30-45%) en présence de 70% (v/v) de LI tandis que l’enzyme sauvage est inactive. En présence de 30% (v/v) de LI, l’efficacité catalytique (kcat/KM) des enzymes modifiées augmente de 1,3 à 3,6 fois et la constante de Michaelis (KM) diminue de 1,7 à 4,6 fois suivant le cation utilisé. Selon la taille du cation greffé, le temps de demi-vie des enzymes modifiées augmente de 3 à 5 fois en solution tamponnée. Enfin, la structure des enzymes modifiées est préservée en présence de 40% (v/v) de LI tandis que l’enzyme native commence à se dénaturer. La technique d’évolution dirigée a également été utilisée. A ce jour, 987 mutants ont été criblés. Un mutant (M60) présente une activité résiduelle de 35% à 70% (v/v) de LI tandis que l’enzyme sauvage est inactive. Ce dernier porte deux mutations, N187S et T321S, situées à la surface de la structure protéique. En présence de 30 % (v/v) de LI, le mutant 60 fixe les substrats avec des valeurs de de KMNAD et de KDN3 (N3 est l’azide, un inhibiteur compétitif du formiate) 2 fois plus faibles que celles de l’enzyme sauvage. / The dehydrogenases are weakly active in the presence of high concentration (> 50% (v/v)) of water-miscible ionic liquids (ILs) and the mechanism of enzyme inactivation in ILs is not fully understood. The structure of the formate dehydrogenase Candida boidinii (FDH) in ILs has been studied by quenching of fluorescence experiments in the presence of iodide or acrylamide. A critical concentration, the CILc (“Critical Ionic liquid concentration"), is defined as the concentration of IL above which the fluorescence is not relevant. In this work, the CILc is comprised between 30 and 40 % (v/v) of the ILs. The ILs have revealed to be denaturing agents which increase the quenching of fluorescence efficiency by 1.4 to 2 times more than in urea. The FDH is chemically modified by analogous cations found in ILs in order to preserve the biocatalyst from the direct interaction with ILs. The enzymes modified by the smaller cations shown 30 45% residual activity at 70% (v/v) of ILs while the native enzyme is fully inactive. In the presence of 30% (v/v) of ILs, the kinetic efficiency (kcat/KM) of the grafted enzymes is improved by a 1.3-3.6 fold factor and the constant of Michaelis (KM) is reduced by a 1.7-4.6 fold factor depending on the grafted cations. In aqueous solution, the half-lives of modified enzymes are 3 to 5 fold higher than the native FDH depending on the size of the cation grafted. Finally, the structure of the grafted enzymes is somehow maintained in the presence of 40% (v/v) of ILs while the native FDH begins to unfold. We also used directed evolution to improve the FDH activity in the presence of ILs. At this stage, 987 mutants have been screened and one mutant (M60) shows 35% of residual activity at 70% (v/v) of ILs. In the presence of 30% (v/v) of ILs, the mutant binds the substrates in a greater extent than the native FDH, the values of KMNAD and the KDN3 (N3 is the azide, a competitive inhibitor of formate) are reduced by a 2 fold factor by comparison to the native enzyme.

In der gartenbaulichen Forschung gewinnen zerstörungsfreie Produktmonitoringverfahren im Hinblick auf ein verbessertes Prozessmanagement an Bedeutung. Optische Methoden werden bereits in mobilen Systemen und Sortieranlagen zur Produktbewertung in Nachernteprozessen eingesetzt. In der vorliegenden Arbeit wurde ein Beitrag zur quantitativen Bestimmung ernährungsphysiologisch bedeutender Fruchtpolyphenole auf der Basis laser-induzierter Fluoreszenzspektroskopie geleistet. An gelagerten Äpfeln und Möhren wurde die Varianz der Produktfluoreszenz bei verschiedenen Lagerbedingungen mit Hilfe der Hauptkomponentenanalyse ausgewertet, um die Produktentwicklung zerstörungsfrei aufzuzeigen. Für eine angepasste Methode der Datenauswertung wurden hierbei verschiedene Signalvorverarbeitungsmethoden getestet. Die quantitative Bestimmung einzelner Inhaltsstoffe wird in der komplexen pflanzlichen Matrix sowohl beeinflusst durch die Fluoreszenzquantenausbeute als auch Reabsorptions- und Löschungseffekten. Aufbauend auf Untersuchungen an Phenolstandards, Fruchtextrakten und geschnittenem Fruchtgewebe zu Einflussparametern und fluoreszenzspektrokopisch messbaren Konzentrationsbereichen wurden neuere Datenvorverarbeitungsmethoden zur Korrektur angewendet. Kalibriermodelle wurden auf der Basis der fluorimetrisch und chromatographisch ermittelten Werte von Hydroxyzimtsäurederivaten bei Apfel und Erdbeere erarbeitetet und hinsichtlich der Messungenauigkeit in der Kalibrierung und Kreuzvalidierung verglichen. Aufgrund der hohen Variabilität gartenbaulicher Produkte wurden diese Modelle auf einem unabhängigen Datensatz getestet. Mit Hilfe mathematischer orthogonaler Signalkorrektur konnte die für den Polyphenolgehalt nicht relevante Varianz aus den spektralen Daten entfernt und verringerte Kalibrierungs- und Validierungsfehler erzielt werden. Der in der Fluoreszenzanalyse übliche empirische Ansatz mit reflexionskorrigierten Fluoreszenzspektren zu arbeiten führten hingegen zu keiner Fehlerverminderung. / During recent years several research groups focussed on the development of non-destructive product monitoring methods to improve the process management for horticultural products in the entire supply chain. Optical methods have been applied for fruit monitoring in production and postharvest processes using mobile measuring systems or NIR sorting lines. The aim of the present study was to quantitatively determine health promoting native fruit polyphenols by means of laser-induced fluorescence spectroscopy. The variance in the fluorescence signal was detected on apples and carrots stored under different conditions. With the help of principal component analysis the fluorescence spectra were evaluated to visualize senescence effects during storage. Different data pre-processing methods were tested for a descriptive factor analysis regarding the wavelength-dependent intensities as variables. However, in a complex fruit matrix the quantitative determination of fruit compounds is influenced by its fluorescence quantum yield as well as reabsorption and quenching effects. The influence of side-effects was studied in phenol standards, fruit extracts and sliced fruit tissue and spectral data was corrected using new data pre-processing methods.. Calibration models for the polyphenol analyses were built on the fruit fluorescence spectra (apples, strawberries) using the chromatographically analysis of hydroxycinnamic acids as a reference. The uncertainty of the models was evaluated by their root mean squares errors of calibration and cross-validation. The feasibility of the non-destructive analysis in practice is influenced by the high variability of horticultural products. Therefore, the models were validated on an independent test set. The mathematical data pre-processing method of direct orthogonal signal correction removed the non relevant information in the spectral data and resulted in the lowest errors. In comparison, the often applied empirical approach in fluorescence spectroscopy to correct with simultaneously recorded reflectance spectra did not improve the calibration models.

<p>In the pentose phosphate pathway, carbohydrates such as glucose and ribose are degraded with production of reductive power and energy. Another important function is to produce essential pentoses, such as ribose 5-phosphate, which later can be used in biosynthesis of nucleic acids and cofactors. </p><p>This thesis presents structural and functional studies on three enzymes involved in ribose metabolism in <i>Escherichia coli</i>. </p><p>Ribokinase is an enzyme that phosphorylates ribose in the presence of ATP and magnesium, as the first step of exogenous ribose metabolism. Two important aspects of ribokinase function, not previously known, have been elucidated. Ribokinase was shown to be activated by monovalent cations, specifically potassium. Structural analysis of the monovalent ion binding site indicates that the ion has a structural rather than catalytic role; a mode of activation involving a conformational change has been suggested. Product inhibition studies suggest that ATP is the first substrate to bind the enzyme. Independent K_d measurements with the ATP analogue AMP-PCP support this. The results presented here will have implications for several enzymes in the protein family to which ribokinase belongs, in particular the medically interesting enzyme adenosine kinase. </p><p>Ribose 5-phosphate isomerases convert ribose 5-phosphate into ribulose 5-phosphate or <i>vice versa</i>. Structural studies on the two genetically distinct isomerases in <i>E. coli</i> have shown them to be fundamentally different in many aspects, including active site architecture. However, a kinetic study has demonstrated both enzymes to be efficient in terms of catalysis. Sequence searches of completed genomes show ribose 5-phosphate isomerase B to be the sole isomerase in many bacteria, although ribose 5-phosphate isomerase A is a nearly universal enzyme. All genomes contain at least one of the two enzymes. These results confirm that both enzymes must be independently capable of supporting ribose metabolism, a fact that had not previously been established.</p>