Made available in DSpace on 2014-06-11T19:33:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-13Bitstream added on 2014-06-13T21:06:45Z : No. of bitstreams: 1 hojo_etd_dr_jabo.pdf: 1539570 bytes, checksum: 9a8c4375124457a8e811faf5f3693cc6 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Visando prolongar a vida útil da lichia, principalmente quanto à manutenção da cor e da qualidade, executaram-se experimentos para avaliar a eficiência dos tratamentos hidrotérmico e com solução de ácido clorídrico (HCl); do armazenamento sob refrigeração, em atmosfera controlada e em diferentes embalagens plásticas e de coberturas com quitosana. No Experimento I, testou-se a imersão em HCl a 0,087M por 6 minutos; o tratamento hidrotérmico por imersão a 52ºC por 1 minuto, seguido de resfriamento em água a 10ºC por 6 minutos; e o tratamento hidrotérmico com resfriamento em HCl a 0,087M a 10ºC por 6 minutos. O tratamento hidrotérmico seguido de resfriamento em HCl conservou a coloração dos frutos até o 3º dia, e a polpa com qualidade adequada até o 12º dia. No Experimento II, utilizou-se o melhor tratamento do experimento anterior (hidrotérmico com resfriamento em HCl) e testaramse diferentes temperaturas de armazenamento: 2ºC (91% UR); 5ºC (98% UR); 10ºC (80% UR); e 20ºC (70% UR). Os frutos foram analisados após 1, 4, 7, 10, 13, 16, 19, 22 e 25 dias. O armazenamento de lichia a 5 ºC manteve a boa aparência por até 13 dias e a qualidade da polpa até o final do período, 25 dias. O armazenamento a 2 ºC levou a maiores prejuízos na aparência. As temperaturas, de 10 ºC e 20 ºC, não foram efetivas para a manutenção da cor vermelha da casca. No Experimento III, foi testado o efeito da atmosfera controlada, associado aos melhores tratamentos dos experimentos anteriores. Os frutos foram armazenados a 5ºC e 94% UR, em atmosfera controlada contendo 5%, 10%, 20% e 80% de O2, com avaliações após 0 (inicial), 3, 7, 14, 21, 28 dias. As lichias de todos os tratamentos mantiveram a boa qualidade da polpa por até 21 dias, com os frutos sob atmosfera com 5% de O2, apresentando menor escurecimento da casca. As lichias apresentaram escurecimento da casca... / Aiming to extend litchi life, especially regarding to color and quality maintenance, experiments were performed to evaluate the treatment efficiency under heat and using hydrochloric acid solution (HCl), refrigerated storage, controlled atmosphere, different plastic containers, and chitosan coatings. In Experiment I, it was tested immersion in 0,087M HCl for 6 minutes; hydrothermal treatment by immersion at 52ºC for 1 minute, followed by water cooling at 10ºC for 6 minutes; and hydrothermal treatment with 0,087M HCl cooling at 10 ºC for 6 minutes. Hydrothermal treatment followed by HCl cooling preserved fruit color until the 3rd day and adequate pulp quality until the 12th day. In Experiment II, it was used the best treatment in the previous experiment (hydrothermal with HCl cooling) and different storage temperatures were tested: 2ºC (91% RH), 5ºC (98% RH), 10ºC (80% RH), and 20ºC (70% RH). Fruits were analyzed after 1, 4, 7, 10, 13, 16, 19, 22, and 25 days. Storage at 5ºC kept the good fruit appearance for up to 13 days, and pulp quality until the 25th day. The 2ºC led to to ligher losses in appearance. The temperatures of 10ºC and 20ºC, were not effective for maintaining the red color of the skin. In Experiment III, the effects of controlled atmosphere combined with improved treatments of previous experiments were tested. Fruits were stored at 5ºC and 94% RH in a controlled atmosphere containing 5%, 10%, 20% and 80% O2, with evaluations after 0 (initial), 3, 7, 14, 21, 28 days. Litchis in all treatments maintained good pulp quality for up to 21 days, with the fruits under a 5% O2 atmosphere showing a lower skin browning. Litchis showed over 50% skin browning after 7 days. In Experiment IV, different concentrations of CO2 (0%, 5%, 10%, 15%, and 20%) combined with the best concentration in the previous experiment, 5% O2, were tested... (Complete abstract click electronic access below)

Orientador: Paulo Mazzafera / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-05T10:18:49Z (GMT). No. of bitstreams: 1 Melo_GeraldoAclecio_D.pdf: 1320706 bytes, checksum: 5f501b7dd60a44ae72341950498d75f7 (MD5) Previous issue date: 2005 / Resumo: Polifenoloxidase - PFO (EC 1.14.18.1 ou EC 1.10.3.2) é uma enzima de ampla distribuição entre as plantas e catalisa a hidroxilação de monofenóis a o-difenóis e a oxidação destes para o-diquinonas. Sua função em plantas tem sido relacionada a mecanismos de defesa contra patógenos e pragas. Em cafeeiro, o ácido 5-cafeoilquínico, também conhecido como ácido clorogênico (CGA) é o principal substrato da PFO e ambos, enzima e substrato, estão presentes em quantidades expressivas nos frutos e nas folhas desta planta. O CGA também está relacionado com mecanismos de defesas das plantas e como tal é considerando importante substrato em reações de oxidação, principalmente aquelas mediadas pela PFO. No presente estudo, com objetivo de conhecer características da PFO de folhas do cafeeiro, de averiguar sua ação em mecanismos de defesa nessa planta e de entender fatores ligados à síntese e ao acúmulo de seu principal substrato foram feitas a purificação e caracterização dessa enzima, estudos da expressão de sua atividade, bem como estudos de expressão de enzimas da via de síntese do CGA em cafeeiro. Com o uso de técnicas de precipitação com sulfato de amônio, cromatografias de troca iônica, interação hidrofóbica e exclusão molecular foi possível obter a PFO com alto grau de pureza. A enzima apresentou massa molecular de 40,5 Kda e preferência pelo ácido 5-cafeoilquínico como substrato. Seqüências de peptídeos obtidas após digestão da proteína e análise por espectrometria de massas mostraram-se homólogas a seqüências de PFO de várias outras plantas. O nível constitutivo de atividade da PFO observado para quinze genótipos de café variou de 3,8 a 88,0 unidades de atividade/mg de proteína, entretanto não teve relação direta com resistência a pragas e doenças nessa planta. A resistência ao bicho mineiro foi significativamente relacionada ao nível de compostos fenólicos, entretanto, ácido 5-cafeoilquínico, o principal substrato da PFO em café, não teve relação com essa resistência, sugerindo a importância de outros compostos fenólicos como substratos da PFO. Dano mecânico, tratamento com ácido metiljasmônico, inoculação com esporos do fungo Hemileia vastatrix e a infestação com ovos do inseto Perileucoptera coffeella levaram a respostas variadas nos níveis de atividade de PFO nos genótipos avaliados. Baseando-se nesses resultados, conclui-se que a ação da PFO na resistência do cafeeiro a pragas e doenças pode estar relacionada ao potencial oxidativo do tecido e não simplesmente uma maior atividade; que o tipo e quantidade de substrato encontrado no tecido podem ser importantes na resistência do cafeeiro e que entre os genótipos pode existir a especialização de mecanismos de resistência envolvendo a ação da PFO. Estudos de expressão por RT-PCR de fenilalanina amônia-liase (PAL), cinamato 4-hiroxilase (C4H), coumarato 3-hidroxilase (C3H), hidroxicinamoil-CoA ligase (4CL) e hidroxicinamoil-CoA:D-quinato hidroxicinamoil transferase (CQT), enzimas da via de síntese do CGA, tiveram sua expressão reduzida à medida que o tecido envelhece. No endosperma foi observado um decréscimo acentuado de expressão no final da maturação dos frutos. Plântulas estioladas obtidas pela germinação de sementes no escuro e transferidas para luz mostraram aumentos significativos no conteúdo de CGA após 24 horas. Esses aumentos foram transientes e coincidiram com a expressão da PAL, C4H, C3H, 4CL e CQT. Os resultados indicam existência de controle da síntese de CGA e a existência de mecanismos de controle da expressão em comum para as cinco enzimas estudadas / Abstract: Polyphenoloxidase - PPO (EC 1.14.18.1 ou EC 1.10.3.2) is an enzyme with broad distribution among plants and catalyzes the hydroxylation of monophenols to o-diphenols and the oxidation of these to o-diquinones. Its function on plants has been related to defense mechanisms against pathogens and plagues. 5-Caffeoylquinic acid, also known as chlorogenic acid (CGA), is the main PPO substrate in coffee tissues and both, enzyme and substrate are present on substantial quantities in fruits and leaves. CGA is also referred to having connection with plants defense mechanisms and it is also an important substrate on oxidation reactions, mainly those mediated by PPO. Therefore, in order to increase our knowledge on the coffee PPO characteristics, to verify its role in defense mechanisms and also to understand the factors connected to the synthesis of CGA, coffee leaf PPO was purified and characterized regarding kinetic parameters and its activity in leaves of several coffee species exposed or not to pest (leaf miner) and disease (leaf rust). Also studies on the expression of the enzymes of CGA synthesis were carried out. By using ammonium sulfate precipitation followed by chromatographic steps on ionic exchange, hydrophobic interaction and molecular exclusion resins it was possible to purify PPO to homogeneity. The enzyme presented a molecular mass of 40,5 Kda and used 5-cafeoylquinic acid as the preferred substrate. Peptide sequences obtained after digestion of the purified PPO and analysis through mass spectrometry were homologous to PPO sequences of several other plants. The constitutive level of PPO activity observed for 15 coffee genotypes varied from 3,8 to 88,0 units of activity/mg of protein, but did not have a direct relationship with resistance to plagues in this plant. Resistance to leaf miner was significantly related to the level of phenolic compounds. However, 5-caffeoylquínic acid, the main substrate of PPO on coffee, was not related with resistance, suggesting the importance of other phenolic compounds as PPO substrates. Mechanical damage, treatment with methyljasmonic acid, inoculation with spores from Hemileia vastatrix and the infestation with the insect Perileucoptera coffeella led to varied results of the PPO activity in the evaluated genotypes. Based on these results, we conclude that the PPO role in the coffee resistance to plagues and diseases might be related to the oxidative potential of the tissue and not only on the PPO activity; that the kind and quantity of PPO substrate found in the tissue might be important for the resistance of the coffee tree and that there may be specific mechanisms of resistance involving PPO action among the genotypes. RT-PCR studies of the expression of phenylalanine ammonia-lyase (PAL), cinnamate 4-hyroxylase (C4H), coumarate 3-hydroxylase (C3H), hydroxycinnamoyl-CoA ligase (4CL) and hydroxycinnamoyl-CoA:D-quinate hydroxycinnamoyl transferase (CQT), which code for enzymes of the CGA biosynthetic pathway, showed that the expression of these enzymes decrease with tissue aging. In the endosperm, an evident decrease on the expression was observed in the end of the fruit ripening. Etiolated seedlings obtained by germination of coffee seeds in the dark and transferred into light showed significant increasing on the CGA content after 24 hours. The increase was transient and followed the expression pattern of PAL, C4H, C3H, 4CL and CQT. The results indicate that CGA biosynthesis is coordinately regulated by the expression of the five enzymes / Doutorado / Biologia Vegetal / Doutor em Biologia Vegetal

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Phenolic compounds are secondary metabolites, widely distributed in the plant kingdom, which present antiinflamatory, antibacterial, antiviral, antialergical and antitumoral activities, in addition to antioxidant activities. Even in low concentrations compared to the oxidant substrate, the antioxidant compounds can delay or inhibit free radical oxidation rates. Thus, this study aimed to quantitatively determine and classify the compounds as natural antioxidants isolated and its contribution to the antiradical activity from the chromatographic analysis (HPLC-DAD) and a systematic study of the antiradical activity of antioxidant compounds isolated (patterns reference) and natural (herbal extracts) using in vitro and ex vivo.bem. Furthermore, the antioxidant species bioactivity was assessed from cells of the cortex and permeation cell biomembranes, artificial lipid. Among the antioxidant compounds studied, quercetrin presented protection action against free radicals on cell-based studies (cortex cells), high in vitro and cell-based antiradical actions, and also biomembrane permeations. These responses, when added, can confer to quercitrin higher relevance as a antioxidant at physiological levels when compared to others poliphenols. This work presents a systematic study on antiradical activities of isolated antioxidant compounds (from reference standards) and natural antioxidant compounds (medicinal plant extracts), by using in vitro and ex vivo methods. / Os compostos fenólicos constituem-se por um grupo de metabólitos secundários, amplamente distribuídos no reino vegetal, que apresentam propriedades antinflamatórias, antibacterianas, antivirais, antialérgicas e antitumorais, além de possuírem propriedades antioxidantes. Mesmo presentes em baixas concentrações em relação ao substrato oxidante, os antioxidantes podem atrasar ou inibir as taxas de oxidação dos radicais livres. Desta forma, este estudo teve como objetivo classificar e determinar quantitativamente os compostos antioxidantes isolados e naturais quanto a sua contribuição na atividade antirradicalar a partir da análise cromatográfica (HPLC-DAD) e de um estudo sistemático da atividade antirradicalar de compostos antioxidantes isolados (padrões de referência) e naturais (extratos de plantas medicinais) usando métodos in vitro e ex vivo.bem. Além disso, a bioatividade das espécies antioxidantes foi avaliada a partir de células de córtex e da permeação por biomembranas celulares artificiais lipídicas. Dentre os antioxidantes estudados, a quercitrina apresentou ação protetora contra radicais livres em nível celular (células de córtex), alta ação antirradicalar in vitro e em nível celular (ex vivo), além de permear biomembranas. Essas respostas, quando somadas, podem credenciar a quercitrina com um dos antioxidantes da classe dos polifenóis de maior relevância em nível fisiológico.

Les architectures moléculaires qui se forment exclusivement sur une surface sont encore rares. L’électrodéposition est un procédé exploitant des « signaux » électriques afin de déclencher et contrôler l’assemblage de films. Récemment, une nouvelle méthode : l’autoconstruction de films en « une étape » par l’utilisation d’un morphogène (un gradient de catalyseur généré depuis une électrode), a attiré l’attention de la communauté scientifique. En effet, elle permet l’auto-assemblage rapide de films polymériques robustes. Cependant, cette technique était limitée à des systèmes basés sur la chimie click du Cu (I). Le but de ce travail était d’étendre cette stratégie à d’autres systèmes en utilisant une approche bio-inspirée. Le concept du morphogène a été appliqué pour développer deux nouveaux systèmes d’autoconstruction déclenchées par électrochimie. Le premier système est basé sur l’autoconstruction covalente de films polymériques induite par l’oxydation d’une molécule organique, inspirée de la moule. Le deuxième est basé sur l’auto-assemblage de films de polyphénols par électro-assemblage par liaisons de coordinations. Enfin, nous avons appliqué ces deux concepts pour immobiliser électrochimiquement une enzyme sur une électrode afin de créer un biosenseur. / Molecular architectures that spontaneously grow exclusively near a surface are rare. Electrodeposition is a process in which imposed electrical « signals » are employed to direct the assembly of thin films. Recently, a new method based on the one-pot self-construction of films by means of a morphogen (a catalyst gradient generated from a surface) has attracted attention since it allows the quick self-assembly of robust films. Nevertheless, this technique was quite limited to systems based on click chemistry.The purpose of this work was to extend this strategy to other systems using a bio-inspired approach. The one-pot morphogen concept was applied to design two new electro-triggered self-construction concepts. The first one is based on the self-construction of covalent polymer films triggered by mussel-inspired molecule oxidation. The second one is based on the electro-self-assembly of polyphenols films based on ionic bonds coordination. Finally, we tried to apply these concepts in order to electrochemically immobilize an enzyme on an electrode to create a biosensor.

<p>Naturally occurring carbohydrates polymers and their functional derivatives play important roles in the research and technology development in the food, nutrition, and pharmaceutical areas. A major property of these polymeric materials is to associate, enable, enhance, and/or deliver small-molecule active compound such as phytochemicals, nutraceuticals, and active pharmaceutical ingredients (APIs). The goal of this project was to synthesize and characterize phytoglycogen-based materials and study their structure-function relationships in association with selected small-molecule active compounds, including resveratrol, a food-related poorly water-soluble phenolic compound, griseofulvin, an insoluble API, and CCVJ (9-(2-carboxy-2-cyanovinyl) julolidine) a molecular rotor used as a structural probe of polymeric materials. </p><p>In this study, phytoglycogen (PG) was derivatives to phytoglycogen octenyl succinate (PG-OS), hydroxypropyl phytoglycogen (HPP), and octenylsuccinate hydroxypropyl phytoglycogen (OHPP). PG, HPP, and OHPP were evaluated for their efficacy in improving the solubility and Caco-2 permeation of resveratrol and griseofulvin, and using CCVJ, PG-OS was evaluated on its performance at oil-water interface in comparison with OSA-starch, acacia gum, and sodium caseinate. The results showed that: 1) PG, HPP, and OHPP substantially improved the soluble amount and Caco-2 monolayer permeation of resveratrol and griseofulvin, and anti-fungal efficacy of griseofulvin in the aqueous system were significantly enhanced; suggesting that the active ingredients were effective solubilized and released to become bioavailable, 2) among all PG-based biopolymers, OHPP showed superior performance in solubilizing resveratrol and griseofulvin, and 3) in the oil-water two-layer model system, PG-OS, OSA-starch, acacia gum, and sodium caseinate all affected the transferring of CCVJ from oil to aqueous phase, and the effect was monitored and interpreted by the emission spectra of molecular rotor; in the emulsion system, the emission peak wavelength of CCVJ was correlated with the amount of biopolymer adsorbed at the interface of emulsion droplets, and the molecular rotor-based method can be used to characterize the interfacial adsorption of biopolymer at the interface in oil-in-water emulsion.</p><p>This study provides information on the interactions between phytoglycogen-based biopolymers and poorly water-soluble active ingredients, and may potentially supports the study of new functional ingredients interaction with phytoglycogen-based biopolymers in aqueous system. Furthermore, this work allowed us to advance the use of molecular rotor as new analytical tool to study the physicochemical properties of biopolymer.</p>

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Les polyphénols et les phénols sont des molécules organiques largement présentes dans le règne végétal et souvent évaluées pour leur potentiel pharmacologique. Ces travaux de thèse concernent la synthèse totale de gallotannins, une classe importante de polyphénols, et l’hémisynthèse de la norbergénine, un C-arylglucoside naturel. Les gallotannins font partie des tannins hydrolysables dont la biosynthèse conduit à des structures chimiques caractérisées par la présence, sur un cœur glucopyranose, de plusieurs unités galloyle liées les unes aux autres par des liaisons méta-depside. Aucune synthèse chimique de ces composés n’ayant été décrite à ce jour, nous avons réalisé la synthèse totale de gallotannins naturels et de leurs anomères non naturels porteurs de motifs di- ou tri-galloyle depside. Ces travaux ont également permis d’étudier l’équilibre méta-para de ces motifs en solution. Quant à l’hémisynthèse de la norbergénine, elle a été réalisée avec succès en une seule étape à partir de la bergénine, un C-arylglucoside commercial, par réaction de O-déméthylation oxydante au SIBX (version stabilisée commerciale du iodane_λ5 IBX). Cette réaction chimiosélective a également été appliquée à des 2-méthoxyphénols et a notamment permis l’obtention de l’hydroxytyrosol à partir de l’alcool homovanillique. / Polyphenols and phenols are organic molecules widely found in the plant kingdom and very often evaluated for their pharmacological potential. This thesis work describes the total synthesis of gallotannins, an important class of polyphenols, and the hemisynthesis of norbergenin, a natural C-arylglucoside. The gallotannins belong to the hydrolysable tannins whose biosynthesis leads to chemical structures characterized by the presence, on a glucopyranose core, of several galloyl units linked ones to the others by meta-depside bonds. No chemical synthesis of these compounds have been reported to date, so we completed the total synthesis of several naturally occurring meta-depsidic gallotannins and their non natural anomers that contain di- or tri-galloyl motifs. This work also allowed the study of the meta-para equilibrium of these motifs in solution. For the norbergenin hemisynthesis, it was successfully achieved in a one step reaction from bergenin, a commercial C-arylglucoside, by a SIBX-mediated oxidative O-demethylation (SIBX = commercially available stabilized version of the λ5 _iodane IBX). This chemoselective reaction was also applied to 2-methoxyphenols and notably allowed the hemisynthesis of hydroxytyrosol from homovanillyl alcohol.

O tratamento radioterápico para pacientes com neoplasias de cabeça e pescoço pode trazer consequências secundárias graves como alterações da estrutura dental, com conseguinte prejuízo da função oral, a qual influencia negativamente a qualidade de vida. Recentemente trabalhos de pesquisa tem demonstrado que a radiação induz a expressão e ativação das metaloproteinases da matriz (MMPs), consideradas as principais enzimas responsáveis pela remodelação da matriz orgânica, incluindo os componentes e estruturas da junção amelodentinária (JAD). Questiona-se então se as alterações dentais observadas em pacientes pós-radioterapia poderiam ser causadas também pela ativação das MMPs que se encontram na JAD. O presente estudo apresentou três avaliações: a ativação e expressão das MMPs, a implementação de inibidores de proteases como método de inibição das MMPs e a ativação das MMPs devido a um desafio ácido. Para as medições foram utilizados 178 fragmentos dentais de molares, divididos aleatoriamente em 2 grupos (decíduos e permanentes) / 4 subgrupos experimentais (irradiados e não-irradiados). Os fragmentos foram expostos à radiacao com Co-60, com fracao de dose de 2 Gy, 5 dias consecutivos, ate atingirem a dose total de 60 Gy, com um total de 30 ciclos, durante 6 semanas. Com o objetivo de determinar a expressão e atividade das MMPs, foram realizados os ensaios de imunofluorescência e zimografia in situ, nos fragmentos dentais de 0,6mm, analisando os tecidos duros do esmalte, dentina e JAD. Para avaliar se produtos odontológicos inativam as MMPs, os dentes foram imersos em 0,5ml de digluconato de clorexidina a 0,12%, fluoreto de sódio a 0,05%, polifenol epigalocatequina 3-galato 400&mu;M e água destilada (grupo controle), por 1 hora. Assim também com objetivo de avaliar se em um ambiente ácido, as MMPs apresentariam maior atividade, os dentes foram colocados em contato com 20&mu;l de solucao desmineralizadora com pH de 4,8, por um minuto, e posteriormente lavados com 20&mu;l de água deionizada, por um minuto. De maneira geral pudemos observar que a irradiação ativa as MMPs na JAD e estes efeitos foram mais evidentes nos dentes permanentes que nos decíduos. Com relação à expressão das diferentes MMPs, foi observada uma maior expressão das MMPs-9 e -20 para dentes decíduos, e para dentes permanentes as MMPs-2, -9 e -20 apresentaram expressão semelhante. Tendo em vista que a irradiação foi capaz de ativar as MMPs expressas na JAD de dentes permanentes, e em busca de soluções capazes de inibi-las, observamos que o Digluconato de Clorexidina, o Fluoreto de Sódio e o Polifenol Epigalocatequina 3-galato inibiram a atividade das MMPs na JAD em dentes permanentes. Por último ao investigar o efeito de um desafio ácido, na atividade das MMPs, observamos que a desmineralização não aumentou a atividade das MMPs em dentes não irradiados, porém aumentou a atividade das MMPs em dentes irradiados. Comparando dentes irradiados submetidos ou não à desmineralização, observou-se que a desmineralização incrementou a atividade das MMPs, já induzida pela irradiação. / Radiotherapy for patients with head and neck cancer can have serious secondary consequences such as changes in tooth structure, with consequent loss of oral function which negatively influences an individual\'s quality of life. Recently research work has shown that radiation induces the expression and activation of matrix metalloproteinases (MMPs) which are considered the major enzymes responsible for the remodeling of the organic matrix, including the components and structures of the dentinoenamel junction (DEJ). It is questionable if the dental changes observed in post-radiotherapy patients could also be caused by the activation of MMPs that are in the DEJ. The present study has three assessments: the activation and expression of MMPs, the implementation of protease inhibitors such as method of inactivating MMPs and the activation of MMPs due to an acid challenge. The measurements that were used were 178 molar dental fragments randomly divided into 2 groups (deciduous and permanent) / 4 experimental subgroups (irradiated and non-irradiated). The samples were exposed to radiation using Co-60 at a cumulative dose of 2 Gy fraction, 5 consecutive days, until they reached a total dose of 60 Gy, with a total of 30 cycles for 6 weeks. In order to determine the expression and activity of MMPs immunofluorescence assays were performed and in situ zymography, the dental fragments of 0.6mm, analyzing the DEJ in three areas of the tooth (cervical, cuspal and groove of pit). To assess whether MMPs inactivate dental products, the teeth were immersed in 0.5 ml of chlorhexidine digluconate at 0.12%, sodium fluoride 0.05%, polyphenol epigallocatechin-3 gallate 400&mu;M and distilled water (control group) for 1 hour. To evaluate effects in an acidic environment, MMPs have higher activity, the teeth were put in contact with 20&mu;l of demineralizing solution with pH 4.8, for a minute, and then washed with 20&mu;l of deionized water, one minute. In general we observed that the radiation active MMPs in DEJ and these effects were more evident in the permanent teeth than in the primary teeth. Regarding the expression of different MMPs, showed the greatest expression of MMP-9 and -20 for deciduous teeth, and permanent teeth MMPs-2, -9 and -20 showed similar expression. Given that the irradiation was able to activate MMPs expressed in the DEJ permanent teeth, and looking for solutions that inactive them, we observed that the digluconate Chlorhexidine, the Sodium Fluoride and Polyphenol Epigallocatechin-3-gallate inhibited activity of MMPs in the DEJ in permanent teeth. Finally, to investigate the effect of an acid challenge, in the activity of MMPs, we observed that the demineralization did not increase the activity of MMPs in non-irradiated teeth but increased the activity of MMPs in irradiated teeth. Comparing irradiated whether subjected to demineralization teeth or not, it was found that demineralization increased activity of MMPs, induced by the radiation.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2019-02-01T09:56:12Z No. of bitstreams: 2 Dissertação - Thaís Forest Giacomello - 2019.pdf: 3222145 bytes, checksum: 30b7cb27105455314142954d1f75610f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2019-02-01T10:05:20Z (GMT) No. of bitstreams: 2 Dissertação - Thaís Forest Giacomello - 2019.pdf: 3222145 bytes, checksum: 30b7cb27105455314142954d1f75610f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2019-02-01T10:05:20Z (GMT). No. of bitstreams: 2 Dissertação - Thaís Forest Giacomello - 2019.pdf: 3222145 bytes, checksum: 30b7cb27105455314142954d1f75610f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2019-01-17 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Chalcone is a class of natural products that has a lot of interest, mainly pharmaceutical because of its biological actions. They are several classes and in addition they are non rigid and complex molecules making their structural characterizations become difficult task in experimental techniques. Spectroscopic techniques, in the last years, have developed very fast, greatly aiding the elucidation of natural products. However, several cases of review of natural product structures have been found in the literature due to erroneous elucidations in analytical techniques of experimental routines. Thus, it is extremely important to develop protocols that can assist in determining the correct structures of these molecules. In this work aimed to develop a parameterized protocol for NMR 13C chemical shift calculations with the purpose of assisting in the correct determination of polyphenol type molecules. Thus, a group of polyphenols, specifically a subclass of these, chalcones, having varied substituents and experimental structural elucidation were selected in the literature. This base of chalcones was submitted to randomized conformational searches using Monte Carlo method and MMFF force field. In addition, the configurators with energy of up to 3 kcal/mol of each chalcone were calculations optimization of geometry and frequency. The chemical shift of 13C was calculated after assuming Boltzmann statics. All of these calculations were performed using the mPW1PW91 / 6-31G (d) level. After that, the scaled chemical shifts (δesc) was defined. This was obtained using the expression δ𝑒𝑠𝑐 = 𝑎 𝑥 δ𝑐𝑎𝑙𝑐 + 𝑏, where a and b are the coefficients of linear regressions obtained between calculated (δcalc) versus experimental chemical shift. In order to validate the protocol, the scaling factor were used to obtain δesc values for chalcones different from those used in the base. The result shows that the level of theory applied reproduced excellently the experimental data. Calculations performed with a scaling factor lead to a better result than when there is no use of this factor. In addition, the applicability of the scaling factor allows the cancellation of systematic errors, which make δesc are closer to the experimental ones. Thus, the parameterized protocol was shown to be an important tool for the structural elucidation of polyphenols through theorotical calculations of ¹³C NMR chemical shifts. / Chalcona é uma classe de produtos naturais que tem muito interesse, principalmente farmacêutico, devido as suas ações biológicas. São moléculas não rígidas e complexas fazendo com que suas caracterizações estruturais se tornem tarefa difícil em técnicas experimentais. As técnicas espectroscópicas, nos últimos anos, tiveram um desenvolvimento muito rápido auxiliando bastante a elucidação de produtos naturais. No entanto, vários casos de revisão de estruturas de produtos naturais foram encontrados na literatura devido a ter elucidações errôneas em técnicas analíticas de rotinas experimentais. Com isso, é de extrema importância desenvolver protocolos que podem auxiliar na determinação de estruturas corretas dessas moléculas. Este trabalho buscou desenvolver um protocolo parametrizado para cálculo de deslocamento químico de RMN 13C com o intuíto de auxilar a determinação correta de moléculas tipo polifenóis. Assim, selecionou-se um grupo de polifenóis, especificamente uma subclasse desses, as chalconas, que possuissem substituintes variados e elucidação estrutural experimental na literatura. Essa base de chalconas foi submetida a buscas conformacionais estocásticas, onde usa-se o método Monte Carlo e campo de forças merck. Então, os confôrmeros com energia de até 3 kcal/mol de cada chalcona foram selecionados e assim feito cálculos de otimização de geometria e frequência. O deslocamento químico de 13C foi calculado após, considerando a distribuição populacional de Boltzmann. Todos esses cálculos foram realizados utilizando o nível mPW1PW91/6-31G(d). Após, com esses dados foi definido o deslocamento químico escalonado (δesc). Esse, foi obtido utilizando a expressão 𝛿𝑒𝑠𝑐=𝑎 𝑥 𝛿𝑐𝑎𝑙𝑐+𝑏, onde a e b são os coeficientes de regressões lineares obtidas entre os deslocamentos químicos calculados (δcalc) e experimentais. Para validação do método, o fator de escalonamento foi utilizado para obter os valores de δesc em outras chalconas diferentes das utilizadas na base. O resultado mostra que o nível de teoria aplicado permite uma boa reprodução dos dados experimentais. Os cálculos realizados com fator de escalonamento levam a um melhor resultado do que quando não há o uso deste fator. Além disso a aplicabilidade do fator de escalonamento permite o cancelamento de erros sistemáticos, o que faz com que os valores de δesc sejam mais próximos aos experimentais. Assim, o protocolo parametrizado mostrou-se uma importante ferramenta para a elucidação estrutural de polifenois através de cálculos de deslocamentos químicos de RMN ¹³C.