Corneal cross-linking (CXL) with riboflavin and ultraviolet A light is a therapeutic procedure to restore the mechanical stability of corneal tissue. The treatment method is applied to pathological tissue, such as keratoconus and induces the formation of new cross-links. At present, the molecular mechanisms of induced cross-linking are still not known exactly. In this study, we investigated molecular alterations within porcine cornea tissue after treatment with riboflavin and ultraviolet A light by surface enhanced Raman spectroscopy (SERS). For that purpose, after CXL treatment a thin silver layer was vapor-deposited onto cornea flaps. To explore molecular alterations induced by the photochemical process hierarchical cluster analysis (HCA) was used. The detailed analysis of SERS spectra reveals that there is no general change in collagen secondary structure while modifications on amino acid side chains are the most dominant outcome. The formation of secondary and aromatic amine groups as well as methylene and carbonyl groups were observed. Even though successful cross-linking could not be registered in all treated samples, Raman signals of newly formed chemical groups are already present in riboflavin only treated corneas.

Made available in DSpace on 2017-07-24T19:22:05Z (GMT). No. of bitstreams: 1 FABIANA SIQUEIRA.pdf: 4384235 bytes, checksum: 655ceab1a73a43f410cdb4b5e3773475 (MD5) Previous issue date: 2017-04-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of the present study was evaluate the adhesive properties of eroded dentin with different pH cycling using universal adhesive systems and to evaluate the application of collagen cross-linking agent or MMP inhibitors on the resin-eroded dentin interface, through of three in vitro studies. For the experiment 1, the teeth were randomly assigned into the experimental groups according to the combination of variables: dentin surface (sound dentin and eroded dentin) vs adhesive systems (10 universal adhesives) vs Adhesive strategy [etch-and-rinse (ER) and self-etch (SE)]. The adhesive systems were applied according to the manufacturer's instructions and the teeth were restored. They were sectioned to analysis of microtensile bond strength (μTBS) and nanoleakage (NL). The surfaces of 2 tooth were examined under scanning electron microscopy (SEM) to evaluate the surface characterization after erosive-cycling. For experiment 2, the teeth were randomly assigned into 18 experimental conditions according to the variables: Erosive cycling (eroded dentin with soft drink pH ≈ 2,7 and eroded dentin with citric acid pH 2.3) vs. adhesive systems [Prime & Bond Elect (PBE); Scotchbond Universal Adhesive (SBU); Tetric N-Bond Universal (TEU)] vs Treatment agents [6.5% primer containing proanthocyanidin (PA) and 0.1% riboflavin (RI)]. The surface was treated with primers and the adhesive systems were applied and restored. The samples were sectioned and tested for μTBS, NL and degree of conversion (DC). The surfaces of 2 tooth were examined under SEM to evaluate the surface characterization after erosive-cycling. For experiment 3, the teeth were randomly assigned into 18 experimental conditions according to the variables: Erosive cycling (eroded dentin with soft drink pH ≈ 2,7 and eroded eroded with citric acid pH 2.3) vs. Adhesive Systems (PBE; SBU; TEU) vs Acid pretreatment [control with 37% phosphoric acid;Phosphoric acid containing 2% chlorhexidine (CHXacid) and phosphoric acid containing 3% PA (PAacid)]. The teeth were acid-etched according to each experimental conditions, the adhesive systems were applied and the teeth were restored. The teeth were sectioned and tested for μTBS, NL and DC. Data were submitted to appropriate statistical analysis (α = 0.05). The μTBS values in eroded dentin were statistically lower than sound dentin, independent of the adhesive strategy (p < 0.01) The inclusion of primers containing collagen cross-linking agents in the eroded dentin adhesive protocol improved μTBS values (p <0.001),significantly reduced NL (p <0.004) for all the adhesives tested, as well as improved DC (p <0.03). The use of phosphoric acids containing MMPs inhibitors or collagen cross-linking agent improved μTBS values (p> 0.33), produced a significant reduction of NL (p <0.004) and improved DC (p <0.03). The application of MMP inhibitors (primer or acid etching - containing) were effective to improve the properties of the resin-eroded dentin interface. / O objetivo desse estudo foi avaliar as propriedades adesivas à dentina erosionada utilizando sistemas adesivos universais e verificar o efeito da aplicação de agentes reticuladores de colágeno ou inibidores de Metaloproteinases (MMPs) na interface resina-dentina erosionada, através de três estudos laboratoriais. Para o experimento 1, os dentes foram distribuídos aleatoriamente entre os grupos experimentais de acordo com a combinação das variáveis: superfície dentinária (controle sem erosão e erosionada) vs. sistemas adesivos (10 sistemas adesivos universais) vs.estratégia adesiva [condicionamento convencional (ER) e autocondicionante (SE)].Os sistemas adesivos foram aplicados de acordo com as especificações do fabricante e os dentes foram restaurados. Depois foram seccionados para análise da resistência de união à dentina (μTBS) e nanoinfiltração (NI). A superfície de 2 dentes foi examinada sob microscopia eletrônica de varredura (MEV) para avaliar as características da superfície após ciclagem erosiva. Para o experimento 2, os dentes foram aleatoriamente alocados em 18 condições experimentais de acordo com as variáveis: Ciclagem erosiva (dentina erosionada com coca-cola pH ≈ 2,7 e dentina erosionada com ácido cítrico pH 2,3) vs. Sistemas adesivos [Prime & Bond Elect (PBE), Scotchbond Universal Adhesive (SBU) e Tetric N-Bond Universal (TEU)] vs. Agentes de tratamento [primer contendo 6,5% de roantocianidina (PA) e primer contendo 0,1% de riboflavina (RI)]. Os sistemas adesivos foram aplicados de acordo com as especificações do fabricante após aplicação do primer e restaurados. A seguir, os dentes foram seccionados e testados para análise da μTBS, NI e grau de conversão (GC). A superfície de 2 dentes foi examinada sob MEV para avaliar as características da superfície após erosão. Para o experimento 3, os dentes foram alocados aleatoriamente dentro de 18 condições experimentais de acordo com as variáveis: Ciclagem erosiva (dentina erosionada com coca-cola pH ≈ 2,7 e dentina erosionada com ácido cítrico pH 2,3) vs. Sistemas adesivos (PBE, SBU e TEU) vs. pré-tratamento ácido [controle com ácido fosfórico 37%; Ácido fosfórico contendo 2% de clorexidina (CHXacido) e ácido fosfórico contendo 3% de PA (PAacido)]. Os dentes foram condicionados de acordo com cada grupo experimental, os sistemas adesivos foram aplicados e restaurados com resina composta. Posteriormente, os dentes foram seccionados e testados para análise da μTBS, NI e GC. Todos os dados foram submetidos a ANOVA 3 fatores e teste de Tukey (α = 0,05). Os valores de μTBS em dentina erosionada foi estatisticamente inferior à dentina sadia,independente da estratégia adesiva (p > 0,61). A inclusão de primers contendo agentes reticuladores de colágeno no protocolo adesivo a dentina erosionada,melhorou os valores de μTBS (p < 0,001), diminuiu significativamente a NI (p <0,004) para os adesivos testados, além de melhorar o GC (p < 0,03). A utilização de ácidos fosfórico contendo inibidores de MMPs ou agentes reticuladores de colágeno melhorou os valores de μTBS (p > 0,33), produziu uma redução significativa da NI (p < 0,004) e melhorou o GC (p < 0,03). Os agentes reticuladores independente do veículo (primer ou condicionamento ácido) foram eficazes para melhorar as propriedades da interface resina-dentina erosionada.

Whole grains are an increasingly popular health food in America. However, shelf life of whole grains is compromised due to the presence of lipoxygenases in the bran and germ, which lead to rancidity and generation of oxidative byproducts. These byproducts reduce sensory quality and may have a degradative effect on vitamins in whole grain products. The purpose of this study was to determine the degree of lipid and vitamin degradation during long-term storage of three whole grains: whole wheat flour, brown rice, and rolled oats. We also examined vitamin loss after cooking to determine if oxidative byproducts had an effect on vitamins during typical household cooking. Whole wheat flour, brown rice, and rolled oats were stored for 12 months and periodically analyzed for conjugated dienes, free fatty acids, tocopherols, thiamin, and riboflavin. Whole wheat bread, steamed brown rice, and oat porridge were made from samples stored for 0 months and 12 months and were analyzed for thiamin and riboflavin. Conjugated dienes increased significantly only in rolled oats, while tocopherols decreased significantly in whole wheat flour and rolled oats and insignificantly in brown rice. Free fatty acids increased significantly in whole wheat flour and brown rice. Thiamin and riboflavin were stable in raw stored grains and cooked products made from stored grains with the exception of brown rice, in which we observed a significant decrease in thiamin after 12-month storage and cooking. These results suggest whole wheat flour, brown rice, and rolled oats experience significant lipid and tocopherol degradation, but it does not appear to affect thiamin and riboflavin in raw stored products. Cooking appears to cause degradation of thiamin after storage of brown rice, but thiamin and riboflavin were otherwise stable in these whole grains.

A new Laser Induced Fluorescence Capillary Zone Electrophoresis (LIF-CZE) bioassay to detect and study the catalytic activity of the sulfur assimilating enzyme commonly found in E. coli species; alkanesulfonate monooxygenase (EC 1.14.14.5) is described for the first time. This technique enables the possibility for direct injection onto a capillary for detection without the need for pre-concentration of sample and with minimal sample preparative steps prior to analysis. In this bioassay, a group of Fischer based cyanine dyes and two Oxazine (Nile red) derivatives were designed for further optimization as key Vis-NIR fluorescent substrate. In developing this technique, the test dyes were first assessed for their photophysical properties, based on four criteria; (1) photostable (2) solvatochromism (3) binding affinity towards both the monooxygenase active site and serum albumin and (4) chemical stability in strong electric field strength. Applying key dye characterization procedures including; molar absorptivity determination, quantum yield determination, photostability, solvatochromism and protein interaction studies it was determined that the Fischer indolium cyanine dyes were most suitable for the method development. The data revealed that under the test conditions, reduced flavin, the oxidative monooxygenase catalytically specifically converts the alkylsulfonate substituted cyanine dyes to the corresponding aldehyde. This new bioassay has proven to be quick, portable, sensitive, reliable and the exhibit the possibility of ‘on-the-spot’ detection; advantages not readily realized with other commonly applied techniques such as PCR, SPR, ELISA and GC used to study bacterial sulfur assimilation processes. In addition, recent literature results proposed by other research groups developing similar techniques showed strong reliance on GC analyses. Those assays involve the use of low molecular weight straight chain non-emissive alkanesulfonate substrates. Once enzyme catalysis occurs the aldehyde is formed becomes rather volatile and requires complex and tedious headspace sampling for GC analyses. This feature limits the in vitro applicability and eliminated the possibility in vivo development. Our goal is to further develop, optimize and present this CZE based bioassay as a suitable alternative to the current trends in the field while creating a more robust and sensitive in vitro monooxygenase detection method with the possibilities of in vivo application.

Made available in DSpace on 2015-07-22T20:49:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-05-30. Added 1 bitstream(s) on 2015-08-11T03:26:18Z : No. of bitstreams: 1 Publico-13221.pdf: 17673997 bytes, checksum: 5eccd3ef771fd6553a1cc32756fcef7a (MD5) / Objetivos: 1) Avaliar se o cross-linking do colágeno corneano antes da inserção de segmentos de anéis corneanos intraestromais altera o tratamento do ceratocone. 2) Avaliar se o cross-linking do colágeno corneano modifica as características citológicas da superfície ocular em portadores de ceratocone. Métodos: Trinta e nove olhos de 31 pacientes foram alocados em 2 grupos: 19 olhos foram submetidos ao tratamento do cross-linking com riboflavina e luz ultravioleta A, e 20 olhos receberam colírio de riboflavina 0,1% (w/v) em solução de 20% dextran 4 vezes ao dia por 30 dias. Após 3 meses, todos os pacientes foram submetidos à inserção de segmentos de anéis corneanos intraestromais pela técnica do laser de femtosegundo. Avaliações foram realizadas nos momentos pré-operatório, com 1 e 3 meses após o cross-linking ou colírio de riboflavina, e também com 1, 3, 6, 12 e 24 meses após a inserção de segmentos de anéis corneanos intraestromais. Os pacientes foram submetidos aos seguintes exames: acuidade visual sem correção e acuidade visual com correção; refração manifesta; biomicroscopia; sensibilidade ao contraste; topografia corneana; Orbscan IIz; Pentacam; tonometria de aplanação; tonometria dinâmica de contorno; paquimetria ultrassônica; biomecânica da córnea; tomografia de coerência óptica; microscopia especular da córnea; citologia de impressão e mapeamento de retina. Análise de covariância e o teste de Mann-Whitney foram realizados para comparação das variáveis do estudo entre os 2 grupos. Teste de Friedman foi utilizado para avaliação da citologia de impressão antes do tratamento, e com 1 e 3 meses após o cross-linking ou colírio de riboflavina, e novamente com 6, 12 e 24 meses após a inserção de segmentos de anéis corneanos intraestromais. Resultados: Todos os pacientes foram acompanhados por 24 meses. A média e variação de idade dos pacientes foram de 28,30 ± 9,3 anos (17-55 anos) no grupo cross-linking e 30,40 ± 9,1 anos (22-55 anos) no grupo colírio de riboflavina. Não houve diferença estatisticamente significante entre os grupos em relação à acuidade visual sem correção (p=0,70) ou acuidade visual corrigida (p=0,78). Com 24 meses de seguimento, não houve diferença estatística entre os grupos em relação ao equivalente esférico (p=0,94). Também não houve diferença estatística para os 3 parâmetros topográficos (mais plano-K1 [p=0,81], mais curvo-K2 [p=0,68] e curvatura média [p=0,52]). Os exames de sensibilidade ao contraste, microscopia especular, paquimetria, tonometria e propriedades biomecânicas da córnea não apresentaram diferença estatística entre os grupos com 24 meses de seguimento. Na citologia de impressão, alguns parâmetros conjuntivais (ex.: adesividade das células epiteliais, proporção núcleo:citoplasma, nível de organização da cromatina nuclear, células caliciformes e queratinização [p≥0,001]) apresentaram diferenças estatisticamente significantes. Conclusões: O cross-linking do colágeno corneano não altera o efeito de segmentos de anéis corneanos intraestromais para ceratocone antes de sua inserção em relação à refração, topografia, sensibilidade ao contraste, microscopia especular, paquimetria, tonometria e biomecânica da córnea com 24 meses de seguimento. A comparação do escore total de citologia de impressão entre os grupos não mostrou diferença estatisticamente significante, apesar de diferenças em alguns parâmetros conjuntivais. / Purpose: To report the impression cytologic results after corneal cross-linking and insertion of intrastromal corneal ring segments for keratoconus. Methods: Thirty-nine eyes were distributed into two groups: 1) cross-linking group (patients underwent corneal cross-linking procedure), and 2) riboflavin eyedrops group (patients received riboflavin 0.1% (w/v) eyedrops in 20% dextran solution for 1 month). After 3 months, all patients underwent insertion of intrastromal corneal ring segments. Impression cytologic specimens were obtained from all eyes at baseline, at 1 month and 3 months after cross-linking or riboflavin eyedrops, and again at 6 months, 1 year, and 2 years after intrastromal corneal ring segment insertion. Results: Patients in the cross-linking group demonstrated improvement in the cell-to-cell contact of epithelial cells and the nucleusto- cytoplasm ratio on the temporal conjunctiva after treatment (P = 0.008 and P = 0.047), respectively. On the superior conjunctiva, increases in goblet cell density (P = 0.037) and level of organization of nuclear chromatin (P = 0.010) after treatment were noted. Patients in the riboflavin eyedrops group demonstrated improvement in the cellto- cell contact of epithelial cells on the superior conjunctiva after treatment (P = 0.021). On the temporal conjunctiva, an improvement in the cell-to-cell contact of epithelial cells (P < 0.001) and increases in the nucleus-to-cytoplasm ratio (P < 0.001), goblet cell density (P = 0.001), and less keratinization (P = 0.011) were noted. No changes were identified on the cornea for either group. Fisher’s exact test comparison of the impression cytologic total scores after treatment revealed no difference between groups. Conclusion: Despite changes in some conjunctival parameters (e.g., cell-tocell contact of epithelial cells, nucleus-to-cytoplasm ratio, level of organization of nuclear chromatin , goblet cell density, and keratinization), comparison of the total impression cytologic scores revealed no difference between groups. / TEDE / BV UNIFESP: Teses e dissertações

No description available.

By flavin's unique structure, nature predestined riboflavin and its derivatives to the participation in redox processes within the bodies of all the living organisms. These biomolecules draw attention with intriguing optical properties and photosensitising abilities. Nature-inspired flavin derivatives share these qualities, and there is also a possibility of fine-tuning for the particular application from the chemical point of view. The thesis deals with two main aims. The first aim handles the synthesis of the trimer heteroaromatic precursor and 1,2-diketone. These key intermediates are essential for the future synthesis of the central aromatic core of the novel NH-free non-fused flavin derivative. The thesis introduces and verifies three approaches, including oxidation of diarylalkynes, nucleophilic addition of a corresponding organolithium compound to a Weinreb amide and benzoin condensation. The second aim covers the properties customization of NH-free fused systems by implementation of linear and bulky alkyl side-chains on the nitrogen atoms N1 and N3 of the alloxazine dilactam. N,N-alkylation introduced an increase in solubility in common organic solvents dichloromethane and chloroform. For the derivatives with 2-(adamantan-1-yl)ethyl substituents, high thermal stability was observed via TGA.

No description available.

Hemocompatibility tuning was adopted to explore and refine an innovative, GA-free preparation strategy combining decellularization, riboflavin/UV crosslinking, and low-energy electron irradiation (SULEEI) procedure. A SULEEI-protocol was established to avoid GA-dependent deterioration that results in insufficient long-term aortic valve bioprosthesis durability. Final SULEEI-pericardium, intermediate steps and GA-fixed reference pericardium were exposed in vitro to fresh human whole blood to elucidate effects of preparation parameters on coagulation and inflammation activation and tissue histology. The riboflavin/UV crosslinking step showed to be less efficient in inactivating extracellular matrix (ECM) protein activity than the GA fixation, leading to tissue-factor mediated blood clotting. Intensifying the riboflavin/UV crosslinking with elevated riboflavin concentration and dextran caused an enhanced activation of the complement system. Yet activation processes induced by the previous protocol steps were quenched with the final electron beam treatment step. An optimized SULEEI protocol was developed using an intense and extended, trypsin-containing decellularization step to inactivate tissue factor and a dextran-free, low riboflavin, high UV crosslinking step. The innovative and improved GA-free SULEEI-preparation protocol results in low coagulant and low inflammatory bovine pericardium for surgical application.

Introduction : Chez les nouveau-nés prématurés, l’hyper-alimentation intraveineuse (HAIV) contribue à leur survie, mais elle est aussi une source importante de molécules oxydantes. L’absence d’une protection adéquate contre la lumière ambiante génère in vitro, via la photo-excitation de la riboflavine, du H2O2, des peroxydes organiques et un dérivé peroxydé de la vitamine C, l’ascorbylperoxyde (AscOOH). Plusieurs données du laboratoire associent l’infusion d’HAIV à des désordres lipidiques dans notre modèle animal. L’hypothèse est donc que l’AscOOH a un pouvoir oxydant et est responsable de certains des effets biologiques observés. Mes objectifs sont les suivants : 1) développer une méthode de dosage de l’AscOOH; 2) démontrer, à l’aide du modèle animal bien établi au laboratoire, des relations entre la concentration tissulaire de cette molécule et des paramètres métaboliques et l’état redox au foie et dans la circulation; et 3) confirmer l’effet physiologique de l’AscOOH dans un modèle cellulaire. Méthode : Différents étalons internes potentiels ont été testés pour le dosage de l’AscOOH par spectrométrie de masse après séparation sur HPLC (LC-MS). Les phases mobiles et conditions chromatographiques ont été optimisées. Pour l’objectif 2, des cobayes de 3 jours de vie (n=11) ont reçu par voie intraveineuse une dose d’AscOOH (entre 0 et 3,3mM). Les animaux ont été sacrifiés au 4e jour de traitement pour le prélèvement de tissus. Les concentrations tissulaires d’AscOOH ont été déterminées au LC-MS. La triglycéridémie et la cholestérolémie ont été mesurées à l’aide d’un kit commercial par spectrophotométrie. Le glutathion oxydé et réduit ont été mesurés par électrophorèse capillaire. Les relations linéaires obtenues sont exprimées par le ratio des carrés (r2), et traitées par ANOVA. Résultats : La validation du dosage de l’AscOOH par LC-MS a été réalisée. Chez les animaux, la concentration urinaire d’AscOOH par créatinine corrèle positivement avec la dose reçue, négativement avec la lipidémie, et négativement avec le redox sanguin et érythrocytaire, indiquant un milieu moins oxydé. Conclusion : La concentration urinaire d’AscOOH peut donc être un reflet de l’oxydation de l’HAIV en clinique. Nos données chez l’animal suggèrent une interaction de l’AscOOH avec le métabolisme hépatique produisant une chute de la concentration plasmatique de cholestérol et de triglycérides. Le modèle cellulaire n’a pas permis d’élucider le mécanisme moléculaire de l’action de l’AscOOH sur le métabolisme. / Introduction: Intravenous hyperalimentation (IVHA) often contributes to the survival of preterm newborns, but it is also an important source of oxidizing molecules. The lack of adequate protection from ambient light generates, in vitro, through the photo-excitation of riboflavin, H2O2, organic peroxides and a peroxidated derivative of vitamin C: ascorbylperoxide (AscOOH). Certain data from our laboratory linked the infusion of IVHA to lipid disorders in our animal model. The hypothesis is that AscOOH is an oxidant that is responsible for some of the biological effects observed. My objectives are: 1) to develop a method for quantitation of AscOOH, 2) to demonstrate, using the guinea pig model used by our laboratory, relations between the tissue concentration of this molecule and metabolic and redox parameters in the liver and plasma, and 3) to confirm the physiological effect of AscOOH in a cell culture model. Method: Different promising internal standards were tested for AscOOH quantitation by mass spectrometry after HPLC separation (LC-MS). Mobile phases and chromatography conditions have been optimized. For objective #2, 3 days old guinea pig pups (n = 11) received an intravenous dose of AscOOH (between 0 and 3.3mM). Animals were sacrificed on the 4th day of treatment for tissue gathering. Tissues AscOOH concentrations were determined by LC-MS. The triglyceride and cholesterol levels were measured by spectrophotometry using a commercial kit. The oxidized and reduced glutathione were measured by capillary electrophoresis. The linear relations obtained are expressed by the square of the correlation coefficient (r2), and processed by ANOVA. Results: The validation of the LC-MS method for AscOOH quantification has been achieved. In animals, the concentration of urinary AscOOH by creatinine correlates positively with the dose received, negatively with blood lipids, and negatively with blood and erythrocyte redox, indicating a less oxidized environment. Conclusion: The urinary AscOOH concentration may be a good indicator of the oxidation state of clinical IVHA. Our data in animals suggest an interaction between AscOOH and liver metabolism producing a drop in plasma concentration of cholesterol and triglycerides. The cell model was not able to clarify the molecular mechanism of AscOOH action on metabolism.