Dans un contexte de réduction des intrants chimiques, l’utilisation des phytosanitaires naturels stimulant l’immunité végétale ouvre la porte vers une nouvelle approche de protection des plantes. Ces composés éliciteurs regroupés sous le terme de «Stimulateurs des Défenses naturelles des Plantes» (SDP) activent le système défensif de la plante la rendant plus résistante aux bio-agresseurs. Les SDP, de nature diverse, se présentent sous forme de composés uniques ou en mélange dans les extraits végétaux. Au cours de ma thèse, nous avons démontré l’activité SDP des extraits de marc de raisin. Les extraits issus de sous-produits de la vigne, marc de raisin rouge, marc de raisin blanc et pépins de raisin induisent diverses réactions de défense au sein de plantes modèles. Nous avons focalisé notre étude sur l’extrait de marc de raisin rouge (EMR) stimulant l’immunité chez le tabac. Infiltré sur feuilles, l’EMR induit la réponse de type HR caractérisée par l’apparition de lésions chlorotiques et accumulation de composés autofluorescents dans les tissus infiltrés. Ces réactions de défense locales ont été observées également chez l’arabette et la tomate. L’EMR déclenche les réponses LAR et SAR avec l’accumulation des transcrits des gènes de défense dans les feuilles de tabac et ce quelque soit son mode d’application (infiltration ou pulvérisation). Le mode d’action de l’EMR a été abordé sur cultures cellulaires de tabac BY-2. L’EMR induit une forte alcalinisation du milieu extracellulaire avec une mobilisation du calcium (Ca2+), l’expression des gènes de défense et la mort cellulaire. Une étude pharmacologique de la mort cellulaire suggère la mise en place de mort cellulaire programmée (PCD) dans les cellules de tabac. La caractérisation de la voie de signalisation activée par l’EMR a été étudiée avec le mutant NahG de tabac incapable d’accumuler l’acide salicylique (SA). Les réponses de défense (HR, LAR et SAR) sont faiblement induites par l’EMR chez le mutant nahG. L’EMR provoque une réponse de type HR fortement réduite avec une faible accumulation des composés autofluorescents et une diminution drastique de l’accumulation des transcrits des gènes PR suggérant l’intervention du SA dans l’induction des réactions de défense. Le degré de protection induit par l’EMR a été déterminé sur le pathosystème tabac/Phytophthora parasitica. Pulvérisé sur feuilles, l’EMR réduit de 45% les zones infectées par l’oomycète. Ce degré de protection semble être le résultat de l’activité antimicrobienne de l’EMR combinée à l’activité SDP. Aucune protection n’a été observée chez le mutant nahG confirmant l’implication de SA dans la résistance induite par l’EMR. Le fractionnement de l’EMR a permis de simplifier la formule active des extraits de raisin et d’identifier un mélange de molécules potentiellement capables d’induire l’activité SDP. Les composés actifs sont de nature polyphénolique et contiennent de la procyanidine B2 capable à elle seule d’induire la réponse de type HR et l’expression de l’antimicrobien PR1. Cependant, il semble que cette molécule agisse en association avec d’autres composés polyphénoliques pour stimuler le système défensif de la plante. / In order to reduce chemical inputs, the use of natural phytosanitary products stimulating plant immunity are emerging approaches in phytoprotection. These elicitor compounds known as "Plant Defense Inducers" (PDI) activate the plant defense system and improve their resistance to pests attack. PDI are single molecule or mixture of compounds extracted from plant. In my thesis, we demonstrated the PDI activity of different grape marc extracts. The winery byproducts, red grape marc extract, white grape marc extract and grape seed extract all induced various defense reactions in several plant models. We focused our study on the red grape marc extract (GME) which stimulates the immunity system in tobacco plants. When infiltrated into tobacco leaves, GME induced HR-like response characterized by the appearance of chlorotic lesions and accumulation of autofluorescent compounds in infiltrated tissues. Similar local defense reactions have been observed in Arabidopsis thaliana and tomato. GME also triggered LAR and SAR responses and induced defense gene transcript accumulation in tobacco leaves after infiltration or spraying. The GME mode of action was studied using the suspension-cultured cells of tobacco BY-2. GME induced rapid alkalinization of extracellular medium with calcium mobilization, expression of defense genes and cell death. A pharmacological approach of this defensive phenomenon suggests the establishment of programmed cell death (PCD) in tobacco cells. The characterization of the signaling pathway activated by GME was studied using tobacco nahG mutant unable to accumulate salicylic acid (SA). Defense responses (HR, LAR and SAR) induced by GME were impaired in the nahG mutant. GME drastically reduced HR-like response symptoms and PR transcript accumulation. These data suggest the implication of SA in the GME-induced plant defense reactions. The GME-induced protection was evaluated in the model pathosystem of compatible interaction between Nicotiana tabacum and Phytophthora parasitica var. nicotianae (Ppn). GME could reduce by 45% the infected areas induced by the oomycete on tobacco leaves. This level of protection was the result of the combined antimicrobial and PDI actions of GME. GME had no protecting effect against Ppn on NahG leaves evidencing the involvement of SA in the GME-induced resistance. GME fractionation led to identification of a bioactive molecule mixture capable of inducing the PDI activity. The active compounds are polyphenolics and involve procyanidin B2 which is by itself able to induce the HR-like response and PR1 transcript accumulation. This compound should act in combination with other polyphenolic molecules to stimulate the full plant defense reactions.

There is a great diversity of vines in the world, generating high variability characteristics of the wines produced. With the increased interest motivated by health benefits, making room for the growth of consumption and the grape and wine market potential, the search for varieties able to adapt to different climatic conditions and the growing consumer demand for wine quality, introduces the 'Marselan' piecemeal viticulture scenario. 'Marselan' is a red grape vinifera, obtained by crossing the 'Grenache' and 'Cabernet Sauvignon'. The Rio Grande do Sul has the largest percentage of cultivated area with grape in Brazil, and is the largest producer of wine. The quality of products derived from the grape is dependent on its physicochemical properties and phenolic composition characteristics of cultivars. This study aims to characterize physicochemical the cultivate and the varietal wine, and also describe the sensory profile of the varietal wine Marselan obtained from grapes produced in the regions of the Campanha Gaúcha, Serra Gaúcha and Serra do Sudeste of Rio Grande do Sul. Six samples of grapes produced different regions were collected. Part of berries was frozen in liquid nitrogen and stored in a freezer for further analysis and preparation of extracts, the remainder was used for the microvinification. Analysis of total polyphenols, total flavonoids, procyanidins, tannins, anthocyanins, polymeric pigments, intensity and color tone, color characteristics by the CIE L* a* b* space, acidity, pH, total soluble solids, total sugars (reducing and non-reducing) and antioxidant capacity were performed. The sensory characteristics of the wines were determined by quantitative descriptive analysis with a team of 10 trained panelists. The grape and wine samples Marselan differ in physicochemical parameters evaluated and sensory analysis. Industrial maturation, phenolic ripeness, phenolic compounds and antioxidant capacity data of 'Marselan' demonstrated their adaptability to all regions of Rio Grande do Sul, but are highly dependent on the weather, which provide different features. All red wines are in accordance with the standards established by Brazilian law. The total polyphenol content (2047.07 to 4777.98 mg L-1) and anthocyanins (645.20 874,67mg the L-1) found in a young wine, Marselan presents a good potential for development of guard wines. The antioxidant capacity follows the trend of phenolic compounds. Regarding the color parameters of the wine samples are somewhat different, but it is not possible to characterize differences in the area and its color is characteristic of young wines. The method of quantitative descriptive analysis revealed that the studied samples have distinct sensory profiles, except in the aroma, which indicates the typical characteristics of the cultivar studied for the aromas of red fruits, dried fruits, woody and sweet. The attributes violet color, translucent, fruity flavor, astringency and persistence are the most important to discriminate samples of red wine, however the other attributes as herbaceous aromas, alcohol and floral, bitter flavors, acid and woody, pungency and body help to describe sensorily the cv. Marselan. It is possible to discriminate sensory samples of red wine based on the production region. / Existe uma diversidade muito grande de videiras no mundo, gerando elevada variabilidade de características dos vinhos produzidos. Com o aumento de interesse motivado pelos benefícios à saúde, abrindo espaço para o crescimento do consumo e do potencial do mercado da uva e do vinho, a busca de variedades capazes de se adaptar as condições climáticas diferenciadas e a procura crescente do consumidor por vinhos de qualidade, introduz a Marselan aos poucos ao cenário da viticultura. Marselan é uma uva tinta vinífera, obtida pelo cruzamento da Grenache e Cabernet Sauvignon . O Rio Grande do Sul detém a maior percentagem de área cultivada com uva no Brasil, além de ser o maior produtor de vinho. A qualidade dos produtos derivados da uva é dependente das suas propriedades físico-químicas e da composição fenólica características das cultivares. O presente trabalho tem como objetivo caracterizar físico-quimicamente a cultivar e o vinho varietal, e também descrever o perfil sensorial do vinho varietal Marselan obtido de uvas produzidas nas regiões da Campanha Gaúcha, Serra Gaúcha e Serra do Sudeste, do Rio Grande do Sul. Seis amostras de uvas produzidas diferentes regiões foram coletadas. Parte das bagas foi congelada em nitrogênio líquido e armazenada em freezer para posterior elaboração de extratos e análise, o restante foi destinado à microvinificação. Foram realizadas análises de polifenóis totais, flavonóides totais, procianidinas, taninos, antocianinas, pigmentos poliméricos, intensidade e tonalidade de cor, características cromáticas pelo espaço CIE L*a*b*, acidez, pH, sólidos solúveis totais, açúcares totais (redutores e não redutores) e capacidade antioxidante. As características sensoriais dos vinhos foram determinadas por Análise Descritiva Quantitativa com uma equipe de 10 provadores treinados. As amostras de uva e vinho Marselan apresentam diferenças nos parâmetros físico-químicos avaliados e na análise sensorial. Os dados de maturação industrial, maturação fenólica, compostos fenólicos e capacidade antioxidante da Marselan demonstraram a sua adaptação a todas as regiões do Rio Grande do Sul, porém é altamente dependente das condições climáticas, as quais conferem características bem diferenciadas. Todas as amostras de vinho tinto encontram-se de acordo com os padrões estabelecidos na legislação brasileira. Pelo teor de polifenóis totais (2047,07 a 4777,98 mg L-1) e antocianinas (645,20 a 874,67mg L-1) encontrados no vinho ainda jovem, a Marselan apresenta um ótimo potencial para elaboração de vinhos de guarda. A capacidade antioxidante acompanha a tendência dos compostos fenólicos. Em relação aos parâmetros de cor dos vinhos as amostras apresentam diferenças entre si, mas não é possível caracterizar diferenças quanto à região e sua cor é característica de vinhos jovens. O método da Análise Descritiva Quantitativa revelou que as amostras estudadas apresentam perfis sensoriais distintos, exceto no aroma, o que indica a tipicidade da cultivar estudada para os aromas de frutas vermelhas, frutas secas, madeirizado e doce. Os atributos cor violeta, translúcido, sabor frutado, adstringência e persistência são os mais importantes para discriminar as amostras de vinho tinto, no entanto os outros atributos como aromas herbáceo, alcoólico e floral, sabores amargo, ácido e madeirizado, pungência e corpo auxiliam a descrever sensorialmente a cv. Marselan. É possível discriminar sensorialmente as amostras de vinho tinto com base na região de produção.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Rosmarinus officinalis L., popularly known as rosemary, is a medicinal and aromatic species used in the cosmetics and food processing industries, with interest for the herbal industry. This study aimed to determine the biomass production and volatile oil during the growth of rosemary plants cultivated under different salinity (nutritious solution concentrations) and different nitrogen (N) concentrations, and to evaluate the effect of the extracts and volatile oil of the species on the Allium cepa L. cell cycle and genetic material, as well as knowing the phenolic compounds present in the extracts and the chemical composition of volatile oil. The work was divided into two stages: the first aimed evaluating the production of biomass and volatile oil during the rosemary plants growth cultivated under different nutritious solution concentrations, as well as the effect of oil and extracts on the A. cepa cell cycle and genetic material, besides determining the chemical composition of the oil and the phenolic compounds present in the extracts; the second stage aimed evaluating biomass and volatile oil production during the rosemary plants growth cultivated under different N concentrations, as well as the effect of oil and the extracts on the A. cepa cell cycle and genetic material, and determining the chemical composition of the oil and the phenolic compounds present in the extracts as well. The rosemary leaves were collected and analyzed for dry matter, volatile oil extraction and preparation of aqueous extracts by infusion at 5, 20, and 50 g.L-1 concentrations for plants cultivated under different salinities, and 5 and 50 g.L-1 concentrations for plants cultivated at different N concentrations. The volatile oil was analyzed by Gas Chromatography to knowledge of its constitution, and the aqueous extracts were analyzed by High Performance Liquid Chromatography for phenolic compounds determination. The oil and extracts effect was evaluated on the A. cepa cell cycle and genetic material. The completely randomized design was used, and the biomass production and volatile oil yield data were subjected to analysis of variance with polynomial regression and the remaining data were compared by the Scott-Knott test at the 5% level of probability. The nutritious solution concentration, the N concentration and the plant age interfere into the biomass production, and consequently into the rosemary volatile oil yield. However, do not alter the oil and extracts effect on the A. cepa cell cycle and genetic material. The volatile oil and extracts inhibited cell division in all concentrations, and this effect is dose-dependent. In addition, extracts did not show genotoxicity, in spite of oil concentration of 10% being genotoxic. The majority compounds camphor, 1.8 cineole, α-pinene, β-myrcene and verbenone, beyond of β-pinene compounds only controlling the oil crop plants in the concentrations of 8:05, 13:05 and 15:55 mmol.L-1. The phenolic compounds present in the extracts were rosmarinic acid, chlorogenic acid, caffeic acid, carnosic acid, kaempferol, quercetin and rutin. / Rosmarinus officinalis L., popularmente conhecida como alecrim, é uma espécie medicinal e aromática utilizada na indústria de cosméticos e agroalimentícia, com interesse para a indústria de fitoterápicos. Objetivou-se determinar a produção de fitomassa, óleo volátil, composição química do óleo e compostos fenólicos presentes nos extratos, durante o crescimento de plantas de alecrim cultivadas com diferentes salinidades (concentrações de solução nutritiva) e diferentes doses de nitrogênio (N), além de avaliar o efeito dos extratos e óleo volátil da espécie sobre o ciclo celular e material genético de Allium cepa L.. Foram coletadas as folhas de alecrim para determinação da fitomassa, extração de óleo volátil e preparo de extratos aquosos por infusão. Avaliou-se a produção de fitomassa e óleo volátil durante o crescimento de plantas de alecrim cultivadas com diferentes salinidades (1.0, 2.0, 3.0, 4.0 e 5.0 dS.m-1) e em diferentes doses de N (5.55, 8.05, 10.55, 13.05 e 15.55 mmol.L-1), além de avaliar o efeito do óleo (3 e 10%) e dos extratos (nas concentrações de 5, 20, e 50 g L-1 para as plantas cultivadas em diferentes salinidades e nas concentrações de 5 e 50 g L-1 a partir das plantas cultivadas em diferentes doses de N), sobre o ciclo celular e material genético de A. cepa. O óleo volátil foi submetido à análise por Cromatografia Gasosa para conhecimento de sua constituição, e os extratos aquosos à análise por Cromatografia Líquida de Alta Eficiência para a determinação dos compostos fenólicos. As plantas cultivadas em diferentes salinidades foram coletadas aos 100 e aos 160 dias após o plantio (DAP) e as cultivadas em diferentes doses de N aos 90, 180, 270 e 365 DAP. Foi utilizado o delineamento experimental inteiramente casualizado, os dados de produção de fitomassa e rendimento de óleo volátil foram submetidos à análise de variância com regressão polinomial e os demais dados foram comparados pelo teste Scott-Knott (p<0,05). A salinidade, a dose de N e a idade fisiológica da planta interferem na produção de fitomassa e consequentemente no rendimento de óleo volátil de alecrim e não alteram o efeito do óleo e dos extratos sobre o ciclo celular e material genético de A. cepa. O óleo volátil e os extratos inibiram a divisão celular em todas as concentrações estudadas e esse efeito é dose dependente, mas sem apresentar genotoxicidade, exceto na concentração de 10% quando o óleo foi genotóxico. Os compostos majoritários do óleo foram cânfora, 1.8 cienol, α-pineno, β-myrceno e verbenona, além dos compostos β-pineno majoritários apenas no óleo de plantas cultivadas nas concentrações de 8.05, 13.05 e 15.55 mmol.L-1. Os compostos fenólicos presentes nos extratos foram ácido rosmarínico, ácido clorogênico, ácido cafeico, ácido carnósico, canferol, quercetina e rutina.

Vanilla planifolia, orchidée épiphite florifère, est la principale source naturelle de l'arôme de vanille. Largement utilisé dans les produits laitiers, les boissons, les pâtisseries et les parfums, cet arôme est le résultat d'un processus complexe : de huit à neuf mois après la fécondation des fleurs, les gousses matures sont récoltées et traitées pendant environ un an afin de libérer leur bouquet aromatique. Aujourd'hui, plus de la moitié de la production mondiale de vanille provient de Madagascar. Pour faire face à cette concurrence, les producteurs de la Réunion se tournent vers la production de vanille "haut de gamme". L'exploitation des vanilliers les plus intéressants du point de vue aromatique est donc favorisée. Toutefois, les programmes d'amélioration se heurtent au manque de connaissances sur la physiologie de la plante. Il devient alors essentiel de mieux comprendre les mécanismes physiologiques et biochimiques impliqués dans la production aromatique des gousses de V. planifolia. Dans ce travail de thèse, une analyse des métabolites présents dans les gousses vertes et les feuilles de vanille a été effectuée par Résonance Magnétique Nucléaire. Cette technique permet l'évaluation qualitative et quantitative des métabolites primaires (sucres, acides animés et organiques...) et secondaires (composés phénoliques...) présents dans la plante dans diverses conditions physiologiques : au cours du développement de la gousse, lors d'une infection virale, selon les saisons ou encore sur différentes accessions. / Vanilla planifolia, a flowering epiphitic orchid, is the major natural source of vanilla flavour. Largely used in dairy products, beverages, bakeries and perfume, vanilla flavour is obtained after a long process: from eight to nine months after flower pollinisation, mature pods are harvested and then prepered during about one year in order to release the characteristic vanilla aroma. Nowadays, more than half vanilla pods world production comes from Madagascar. To face the concurrence, a solution could be develop higher quality pods. Selection of the most aromatic vanilla plant is then preferred. Nevertheless, amelioration program are facing up to a lack of knowledge in vanilla plant physiology. It is now essential to understand more the physiological and biochemical mechanisms implied in the aromatic production of V. planifolia pods. In this thesis, a metabolomic analysis of vanilla green pods and leaves has been performed by nuclear magnetic resonance. This technique has allowed the qualitative and quantitative analysis of primary (sugar, amino and organic acids...) and secondary metabolites (phenolic compounds...) present in vanilla plant according to various physiological conditions: developing pods, viral infection, inter-accession or seasonal variation.

Algal infestation in water bodies causes the release of soluble organic compounds that impact negatively on the taste and odour of the water. With increasing pollution in water bodies and increasing nutrient loading from agricultural activities, most water reservoirs in South Africa and around the world have become affected by this problem. In this study, an advanced oxidation process (AOP), namely, photocatalysis was evaluated for its potential to degrade aromatic compounds; and taste and odour causing bi-cyclic compounds originating from algae. Semiconductor photocatalysis is an environmentally friendly technology requiring no chemical inputs which is capable of completely mineralising organic pollutants to CO2 and H2O thereby eliminating production of unwanted by-products. Although processes involved in the photo-degradation have been reported for a wide range of pollutants, the degradative pathway in this process has not been fully established. In this study, compounds including phenol, 2-chlorophenol, 4-chlorophenol and nitrophenol were successfully eliminated from simulated wastewater. Degradation of geosmin at an environmentally significant initial concentration of 220 ng/L to levels below the lowest detectable concentration was achieved with an optimum catalyst concentration of 60 mg/L at a rate of 14.78 ng/L/min. Higher catalysts loading above 60 mg/L resulted in a decrease in degradation rates. An increase in initial geosmin concentration resulted in a decrease in rates. Ionic species commonly found in surface waters (HCO3 -, and SO4 2-) significantly reduced the efficiency of geosmin degradation. Degradation of geosmin produced acyclic intermediates from ring fission tentatively identified as 3,5-dimethylhex-1-ene, 2,4-dimethylpentan-3-one, 2-methylethylpropanoate and 2-heptanal. The results obtained indicate that the degradation of organic pollutants in aqueous solution is as a result of synergic action from hydroxyl radicals, positive holes and direct photolysis by UV radiation, though the predominant pathway of degradation is via hydroxyl radicals in solution. Major aromatic intermediates of phenol degradation include catechol, resorcinol and hydroquinone produced in the order catechol > resorcinol > hydroquinone. All three are produced within 2 minutes of photocatalytic reaction of phenol and remain in solution until all phenol is degraded in aerated systems. Production of resorcinol in non-aerated systems is transient, further supporting the hydroxyl radical dominant reaction pathway. / Thesis (PhD)--University of Pretoria, 2014. / gm2014 / Chemical Engineering / unrestricted

Photo-initiated reactions involve the use of ultraviolet (UV) or visible light radiation to effect chemical transformations. Some of the advantages of photo-initiated reactions over thermal or high pressure reactions include mild reaction conditions like ambient temperature and pressure, good control over the reaction by the simple switching on/off the light source, and faster reaction kinetics. Usually, semiconductor photocatalysts or oxidizing agents are used to enhance the rate of photo reactions. “Photocatalysis” involves the generation of valence band holes and conduction band electrons by the band gap excitation of a semiconductor photocatalyst. These charge carriers produce reactive hydroxyl and superoxide radicals, which mediate oxidation and reduction reactions. However, the oxidizing agents are decomposed by the incident radiation to generate reactive radicals, which accelerate the photo reaction. Today, photocatalysis and photo-oxidative reactions are widely being practiced for environmental pollution abatement, synthesis of fine chemicals, synthesis of polymers, generation of hydrogen as a clean energy carrier, and in anti-fogging and self-cleaning surface treatments. The present investigation focuses on elucidating the mechanism and kinetics of environmentally and synthetically relevant photo-initiated reactions for a better understanding of the fundamental aspects of the photo processes. The different photo-initiated reactions studied in this dissertation can be grouped under the broad categories of (i) photocatalytic degradation of organic compounds like dyes and phenols, and reduction of metal ions, (ii) photocatalytic degradation of polymers, (iii) selective photocatalytic oxidation of cyclohexane, (iv) sonophotocatalytic degradation of dyes, (v) photopolymerization, and (vi) sonophotooxidative degradation of polymers. Nano-sized TiO2, synthesized by solution combustion technique (henceforth denoted as CS TiO2), was used as the photocatalyst for most of the above reactions, except for the last two polymer reactions, where organic initiators were used. Invariably, the photocatalytic activity of CS TiO2 was compared with the commercially available Degussa P-25 TiO2 (DP25). Based on the experimental results, detailed mechanisms were proposed for the different reactions, kinetic models were derived, and the rate coefficients signifying the importance of the underlying reaction steps were evaluated. Pd2+ substituted and Pd0 impregnated TiO2 were synthesized by solution combustion and reduction techniques, respectively, and characterized by powder XRD, XPS, TEM, BET surface area, UV/visible, TGA, FT-IR and photoluminescence measurements. While the above catalysts are known to be more active compared to CS TiO2 for the gas phase NO reduction and NO decomposition reactions, it was found in this study, that these catalysts exhibit lower activity for the degradation of organic compounds like dyes, phenol and 4-chlorophenol, in the aqueous phase. The decrease in activity was correlated with a reduction in surface area and photoluminescence intensity of these catalysts, compared to CS TiO2. Ag+ substituted (Ag sub) and Ag0 impregnated (Ag imp) nano-TiO2 were synthesized by solution combustion and reduction techniques, respectively, and characterized by the above standard measurements. These catalysts were used for the photodegradation of dyes, and the selective photooxidation of cyclohexane to cyclohexanone. For the photocatalytic degradation of dyes, unsubstituted CS TiO2 exhibited the highest activity, followed by 1% Ag imp and 1% Ag sub. However, for the photooxidation of cyclohexane, the total conversion of cyclohexane and the selectivity of cyclohexanone followed the order: 1% Ag sub > DP-25 > CS TiO2 > 1% Ag imp. The kinetics of photodegradation of the dyes and the photooxidation of cyclohexane was modeled using Langmuir-Hinshelwood rate equation, and a free radical mechanism, respectively. This study proves that the photoactivity of a catalyst is not solely determined by a single physical property, but rather by a number of variables including the surface area, band gap, surface hydroxyl content, oxide ion vacancy and surface charge of the catalyst. The photocatalytic degradation of five anionic, eight cationic and three solvent dyes, containing different functional groups, was evaluated. The degradation of the dyes was quantified using the initial rate of decolorization and overall percent mineralization. The decolorization of the anionic dyes with CS TiO2 followed the order: Indigo Carmine > Eosin Y > Amido Black 10B > Alizarin Cyanine Green > Orange G. The decolorization of the cationic dyes with DP-25 followed the order: Malachite Green > Pyronin Y > Rhodamine 6G > Azure B > Nile Blue Sulfate > Auramine O ≈ Acriflavine ≈ Safranin O. CS TiO2 exhibited higher rates of decolorization and mineralization for all the anionic dyes, while DP-25 was better in terms of decolorization for most of the cationic dyes. The solvent dyes exhibited adsorption dependent decolorization. The observed results were rationalized based on the molecular structure and degradation pathway of the dyes. The simultaneous photocatalytic degradation of phenolic compounds like phenol and 4-nitrophenol, and the reduction of metal ions like copper (Cu2+) and chromium (Cr6+) were studied. It was found that the presence of phenol accelerated the reduction of Cu2+ to Cu+, and the presence of phenol and 4-nitrophenol accelerated the adsorption of Cr6+ onto CS TiO2. A detailed dual-cycle, multi-step reaction mechanism was proposed for the simultaneous degradation and reduction, and a model was developed using the network reduction technique. The kinetic rate constants in the model were evaluated for the systems studied. The simultaneous UV and ultrasound (US) degradation of anionic dyes was carried out in presence of CS TiO2. The rates of degradation and mineralization of the dyes were higher for the sonophotocatalytic process compared to the individual photo-and sonocatalytic processes. The effect of dissolved gases and US intensity on the sonophotocatalytic degradation of the dyes was evaluated. A dual-pathway network mechanism of sonophotocatalytic degradation was proposed for the first time, and the rate equations were modeled using the network reduction technique. The kinetic rate coefficients of the individual steps were evaluated for all the systems by fitting the model with the experimental data. Eosin Y and Fluorescein dye sensitized visible light degradation of phenol, 4chlorophenol, 2,4-dichlorophenol and 2,4,6-trichlorophenol was studied. A detailed mechanism of sensitized degradation was proposed, and a mechanistic model for the rate of degradation of the phenolic compound was derived by using the pyramidal network reduction technique to evaluate the rate coefficients. An important conclusion of this study indicates that at low initial dye concentrations, the rate of degradation of the phenolic compound is first order in the concentration of the dye, while at high initial dye concentrations, the rate is first order in the concentration of the phenolic compound. The different phenolic and dye intermediates that were formed during degradation were identified by mass spectrometry, and a most probable pathway of degradation was proposed. The solution photopolymerization of methyl-, ethyl-, butyl-and hexylmethacrylates in presence of benzoyl peroxide as the initiator was studied. The effect of initiator and monomer concentrations on the time evolution of polymer concentration, number average molecular weight (Mn) and polydispersity (PDI) was examined. The reversible chain addition and β-scission, and primary radical termination steps were included in the mechanism along with the classical initiation, propagation and termination steps. The rate equations were derived using continuous distribution kinetics and solved numerically to fit the experimental data. The model predicted the instantaneous increase of Mn and PDI of the polymers to steady state values. The rate coefficients exhibited a linear increase with the size of the alkyl chain of the alkyl methacrylates. Poly(acrylamide-co-acrylic acid) copolymers of different compositions were synthesized and characterized. The copolymers were statistical with a relatively high percentage of acrylamide units, as determined by 13C-NMR. The aqueous phase photolytic and photocatalytic degradation of the copolymers and the homopolymers was conducted. The degradation was modeled using continuous distribution kinetics. The degradation followed a two step mechanism, wherein the rapid first step comprised of the scission of weak acrylic acid units along the chain, which was followed by the breakage of the relatively strong acrylamide units. The rate constants for the weak and strong links followed a linear trend with the percentage of acrylic acid and acrylamide in the copolymer, respectively. The photocatalytic degradation of the copolymers of methyl methacrylate with butyl methacrylate (MMA-BMA), ethyl acrylate (MMA-EA) and methacrylic acid (MMA-MAA) was carried out in toluene. The copolymers and the corresponding homopolymers degraded randomly along the chain. The degradation rate coefficient was determined using continuous distribution kinetics. The time evolution of the hydroxyl and hydroperoxide stretching vibration in the FT-IR spectra of the copolymers indicated that the degradation rate follows the order: MMA-MAA > MMA-EA > MMA-BMA. The photodegradation rate coefficients were compared with the activation energy of pyrolytic degradation. The observed contrast in the order of thermal stability compared to the photostability of these copolymers was attributed to the two different mechanisms governing the scission of the polymers and the evolution of the products. The mechano-chemical degradation of poly(methyl methacrylate), poly(ethyl methacrylate) and poly(n-butyl methacrylate) using US and UV radiation, in presence of benzoin as the photoinitiator, was carried out. A degradation mechanism that included the decomposition of the initiator, generation of polymer radicals by hydrogen abstraction of the initiator radicals, and reversible chain transfer between the stable polymer and the polymer radicals, was proposed. The mechanism assumed mid-point chain scission due to US and random chain scission due to UV radiation. The steady state evolution of PDI was successfully predicted by the continuous distribution kinetics model. The rate coefficients of polymer scission due to US and UV radiation exhibited a linear increase and decrease with the size of the alkyl group of the poly(alkyl methacrylate)s, respectively.

Les ressources renouvelables sont considérées comme des alternatives durables, et parfois moins chères, aux matières premières d'origine pétrochimique. Dans cette thèse, on a utilisé du tanin provenant d’écorces de Mimosa comme précurseur de matériaux carbonés. La synthèse de ces nouveaux matériaux carbonés comprend un processus en deux étapes: la première étape est un traitement hydrothermal à des températures modérées, de 180 à 200°C, ce qui permet l'enrichissement en carbone ainsi que la fixation des hétéroatomes et la production de différentes morphologies: microsphères ou gels. La deuxième étape est une carbonisation à une température plus élevée, à savoir 900°C, ce qui permet d'augmenter la surface spécifique, la porosité ainsi que la conductivité électrique. La préparation d’un carbone poreux présentant des mésopores parfaitement définis en taille et dans leur organisation a été réalisée en associant le tanin, précurseur de carbone, à un tensio-actif structurant de la porosité, le copolymère Pluronic F127. Ces nouveaux matériaux carbonés ont été testés comme des électrodes de supercondensateur / Renewable resources are considered as sustainable, and sometimes cheaper, alternatives to substitute raw materials of petrochemical origin. We used tannins extracted from Mimosa barks. Tannin is an attractive precursor to synthesize new organic porous materials due to the presence of highly reactive phenolic molecules. The synthesis of these new carbon materials from tannin comprises a two-step process: the first step is a hydrothermal carbonization at moderate temperatures, 180-200°C, which allows obtaining a hydrochar, richer in carbon, and fixing other heteroatoms. These hydrochars present different morphologies as powder or gel. The second step is a carbonization at higher temperature (900°C), which allows increasing surface area (SBET), porosity as well as improving conductivity. The preparation of a porous carbon presenting mesopores perfectly defined in size and in their organization was realized by associating tannin, as carbon precursor, with a surfactant for porosity templating, the copolymer Pluronic F127. These new carbon materials were tested as electrodes of supercapacitor

Les nématodes parasites du tube digestif demeurent une contrainte majeure pesant sur la santé, le bien être et les productions des petits ruminants élevés à l’herbe. Le mode usuel de maîtrise de ce parasitisme repose sur l’emploi répété de molécules chimiques anthelminthiques. Toutefois, le développement et la diffusion généralisée de résistances à ces molécules dans les populations de vers imposent désormais d’utiliser ces traitements avec discernement et de trouver des solutions complémentaires ou alternatives. L’incorporation dans la conduite d’élevage (ration des moutons ou des chèvres) de légumineuses fourragères riches en tannins condensés dotées de propriétés anthelminthiques s’est avérée une option prometteuse pour réduire le recours aux molécules chimiques. Toutefois, une des difficultés d’application de ces plantes tient à la variabilité des résultats observés. En prenant le sainfoin comme modèle de légumineuse contenant des tannins et en s’appuyant essentiellement sur des méthodes in vitro basées sur les larves 3 infestantes, l’objectif général de cette thèse est d’analyser le rôle respectif de facteurs liés à l’environnement, aux variétés génétiques ou aux modes de préservation technologiques sur les propriétés anthelminthiques. Quelque soit le critère envisagé, une forte variabilité a été observée. Les principales variations liées à l’environnement dépendaient de l’année et du cycle de coupe, ainsi que du site d’exploitation. Parmi les 38 variétés testées, 9 se sont avérées à forte activité AH, alors que 22 étaient à très faible activité. Enfin, les résultats ont aussi surtout souligné une plus forte activité antiparasitaire dans des formes séchées ou ensilées par comparaison à des échantillons frais. La comparaison des profils biochimiques liés à cette variabilité a permis une exploration des composés phénoliques expliquant l’activité anthelminthique. L’existence d’une relation dose-réponse a été précisée. Le rôle des tannins condensés a été confirmé, notamment ceux à faible degré de polymérisation et à faible poids moléculaire. L’importance des prodelphinidines qui seraient plus actives que les procyanidines reste à confirmer. De plus, il a été montré que d’autres flavonoides peuvent jouer un rôle, notamment les flavan-3-ols et les flavonols. La différence d’activité entre les échantillons de sainfoin ensilés ou fanés par comparaison aux échantillons frais s‟expliquerait en partie par la présence de formes non glycosidés de flavonols. Ces résultats devraient conduire à développer des méthodes de dosage pour identifier les échantillons de sainfoin, et plus généralement de légumineuses riches en tannins, dotés de propriétés anthelminthiques significatives. / Gastrointestinal nematodes remain a major constraint on the health, welfare and production of small ruminants. Over the past decades, the usual mode of control of this parasitism has mainly relied on the repeated use of chemical anthelmintics. However these treatments are nowadaysfacing some limits among which the most important is the development and widespread diffusion of resistance to these chemical molecules within worm populations. Consequently, the need to find complementary or alternative solutions is becoming urgent. The possible exploitation of forage legumes, rich in condensed tannins, with anthelmintic properties, by incorporation in the diet of sheep or goats, seems a promising option to reduce the reliance on chemical molecules. However, one of the main difficulties to use these plants as nutraceuticals relates to the variations in results. By using sainfoin (Onobrychis viciifoliae) as a model of tannin-containing legume and based on in vitro methods on the infective third stage larvae, the main objectives of this PhD were i) to examine the influence of environmental, genetic (40 different varieties) and technological (mode of preservation) factors on the anthelmintic properties of sainfoin and ii) to analyse whether differences in phenolic compounds might explain the variations. Whatever the factor considered, a high variability in results was observed. The main variations due to the environmental factors depended on the year and the cycle of cutting, as well as on the site of cultivation. Among the 38 varieties tested, only 9 have shown AH activity over 50 %. Last, a higher antiparasitic activity was found in the dried or ensiled forms compared to the fresh samples. A comparison of the biochemical profiles associated with these variations indicated a role of proanthocyanidins plus other phenolic compounds in the anthelmintic properties. The dose-response relationship between the AH activity and the ability to form complex with proteins was defined. The role of condensed tannins was confirmed, particularly those with a low degree of polymerization. The respective importance of prodelphinidins vs procyanidins remains to be further investigated. Moreover, the possible role of other flavonoids, in particular of flavan-3-ols and flavonols was also confirmed. The difference in activity between dried or ensiled forms compared to fresh sainfoin samples was partly explained by the presence of flavonol aglycosides. These results should favour the development of measurements in order to identify sainfoin samples with higher anthelmintic properties.

Orientador: Helena Teixeira Godoy / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-24T06:47:07Z (GMT). No. of bitstreams: 1 Ballus_CristianoAugusto_D.pdf: 2840544 bytes, checksum: cd74911d057ffa04ee6e1e5b54ebc8e9 (MD5) Previous issue date: 2014 / Resumo: O consumo de azeite de oliva extravirgem (EVOO) é altamente recomendado por seus benefícios à saúde humana. No Brasil, a ingestão de EVOO, o qual é importado de outros países, vem aumentando anualmente. Nos últimos anos, o Brasil começou a produzir EVOO, porém de maneira experimental. No capítulo 1 foi apresentada uma revisão bibliográfica destacando os estudos mais relevantes acerca do composição química e dos benefícios à saúde do azeite de oliva extravirgem. No capítulo 2, o objetivo foi determinar o teor de fenólicos totais (TFT) e a capacidade antioxidante (CA), bem como a correlação entre o TFT e cada um dos quatro métodos de CA, de 15 marcas de EVOO, cada qual em três lotes, resultando em 45 amostras. O TFT foi avaliado pelo método do reagente de Folin-Ciocalteu, enquanto a CA foi determinada pelos ensaios de FRAP, ABTS, DPPH¿ e ORAC. O TFT variou de 70 a 297 mg EAG kg-1, FRAP de 114 a 1557 µmol ET kg-1, ABTS de 0,5 a 1,9 mmol ET kg-1, DPPH¿ de 72 a 1129 µmol ET kg-1, e ORAC de 1,1 a 12,9 µmol ET g-1. Houve elevada e significativa correlação entre o TFT e cada um dos métodos de CA (FRAP, r2 = 0,8904; p < 0,001; ABTS, r2 = 0,7837; p < 0,001; DPPH¿, r2 = 0,7908; p < 0,001; ORAC, r2 = 0,7431; p < 0,001). Portanto, a maioria das marcas de EVOO apresentaram considerável TFT e elevados valores de CA. No capítulo 3, o objetivo foi otimizar a separação de 17 compostos fenólicos previamente detectados em EVOO. Foi utilizado um planejamento experimental Doehlert, avaliando-se o pH e a concentração do eletrólito. Resolução, tempo de corrida e coeficientes de variação dos tempos de migração foram as respostas. A função de desejabilidade de Derringer foi utilizada para otimizar simultaneamente as 37 respostas. Os 17 picos dos compostos foram separados em 19 minutos em capilar de sílica fundida (50 µm diâmetro interno, 72 cm comprimento efetivo) com bulbo estendido e eletrólito ácido bórico 101,3 mmol L-1 (pH 9,15, 30 kV). O método foi validado e aplicado em 15 amostras comerciais de EVOO. No capítulo 4, o objetivo foi determinar o teor de compostos fenólicos, tocoferóis e ácidos graxos de 17 EVOO monovarietais produzidos em Minas Gerais, durante duas colheitas. Foram quantificados os ácidos palmítico (6-12,6%), palmitoleico (0,2-2,5%), esteárico (1,6-2,2%), oleico (70,8-84,3%), linoleico (3,2-11,7%), a-linolênico (0,6-1,4), araquídico (0,4-0,8%), 9-eicosenoico (0,4-0,9%) e os compostos tirosol (NQ-155,21 mg kg-1), (+)-pinoresinol (2,89-22,64 mg kg-1), hidroxitirosol (ND-37,74 mg kg-1), luteolina (ND-2,23 mg kg-1), a-tocoferol (28,92-232,93 mg kg-1), ß-tocoferol (ND-9,56 mg kg-1) e ?-tocoferol (ND-18,75 mg kg-1). Em geral, os resultados foram similares aos descritos na literatura. O objetivo do capítulo 5 foi determinar o teor de compostos fenólicos em EVOO brasileiros por cromatografia líquida de rápida resolução acoplada à espectrometria de massas por tempo de voo com ionização por electrospray (RRLC-ESI-TOF-MS). Foram analisadas 25 amostras de EVOO do Rio Grande do Sul, Santa Catarina e Minas Gerais e duas colheitas. Foram identificados e quantificados 20 compostos fenólicos das classes dos alcoóis fenólicos, secoiridoides, lignanas e flavonoides. Os teores de compostos fenólicos totais nos EVOOs destacaram-se nas variedades Coratina (364 mg kg-1), Arbosana (255 mg kg-1) e Grappolo (228 mg kg-1). Desta forma, os EVOOs brasileiros são promissores no que se refere ao teor de compostos fenólicos totais, visto que os valores são comparáveis àqueles dos EVOOs de elevada qualidade produzidos em outros países / Abstract: Consumption of extra-virgin olive oil (EVOO) is highly recommended for its benefits to human health. In Brazil, consumption of EVOO, which is imported from other countries, is increasing annually. In the last years, Brazil started to produce EVOO,although in an experimental way. In chapter 1, a literature review highlighting the most relevant studies on the chemical composition and the health benefits of the extra-virgin olive oil is presented. In chapter 2, the aim was to determine the total phenolic content (TPC) and the antioxidant capacity (AC), as well as the correlation between TPC and each one of the four AC methods, of 15 EVOO brands, each one in three batches, resulting in 45 samples. TPC was evaluated by Folin-Ciocalteu reagent method, while the AC was assessed using FRAP, ABTS, DPPH¿ and ORAC assays. The TPC varied from 70 to 297 mg GAE kg-1, FRAP from 114 to 1557 µmol TE kg-1, ABTS from 0.5 to 1.9 mmol TE kg-1, DPPH¿ from 72 to 1129 µmol TE kg-1, and ORAC from 1.1 to 12.9 µmol TE g-1. High and significant correlation was found between the TPC and each one of the AC methods evaluated in this study (FRAP, r2 = 0.8904; p < 0.001; ABTS, r2 = 0.7837; p < 0.001; DPPH¿, r2 = 0.7908; p < 0.001; ORAC, r2 = 0.7431; p < 0.001). Therefore, most of the EVOO brands presented a considerable TPC and high AC values. In chapter 3, the aim was to optimize the separation of 17 phenolic compounds already detected in EVOO. A Doehlert matrix experimental design was used, evaluating the effects of pH and electrolyte concentration. Resolution, runtime and migration time relative standard deviation values were used as responses. Derringer¿s desirability function was used to simultaneously optimize all 37 responses. The 17 peaks were separated in 19 minutes using a fused-silica capillary (50 mm internal diameter, 72 cm of effective length) with an extended light path and 101.3 mmol.L-1 of boric acid electrolyte (pH 9.15, 30 kV). The method was validated and applied to 15 EVOO samples found in Brazilian supermarkets. In chapter 4, the aim was to determine the phenolic compounds, tocopherols and fatty acids contents of 17 monovarietal EVOOs produced in Minas Gerais state, during two crop years. Compounds identified comprised palmitic acid (6-12.6%), palmitoleic acid (0.2-2.5%), stearic acid (1.6-2.2%), oleic acid (70.8-84.3%), linoleic acid (3.2-11.7%), a-linolenic acid (0.6-1.4), arachidic acid (0.4-0.8%), 9-eicosenoic acid (0.4-0.9%), tyrosol (NQ-155.21 mg kg-1), (+)-pinoresinol (2.89-22.64 mg kg-1), hydroxytyrosol (ND-37.74 mg kg-1), luteolin (ND-2.23 mg kg-1), a-tocopherol (28.92-232.93 mg kg-1), ß-tocopherol (ND-9.56 mg kg-1), ?-tocopherol (ND-18.75 mg kg-1). Some of these monovarietal EVOOs presented results similar to those described in the literature. The aim of chapter 5 was to determine the phenolic compound contents of Brazilian EVOO, using rapid-resolution liquid chromatography coupled to electrospray ionization time-of-flight mass spectrometry (RRLC-ESI-TOF-MS). A total of 25 EVOO samples from Rio Grande do Sul, Santa Catarina and Minas Gerais states and two crops, were analyzed. It was possible to identify and quantify 20 phenolic compounds, belonging to the phenolic alcohol, secoiridoid, lignan and flavonoid classes. EVOOs from Coratina (364 mg kg-1), Arbosana (255 mg kg-1) and Grappolo (228 mg kg-1) varieties presented the highest total phenolic contents. The results showed that Brazilian EVOOs are promising concerning the total phenolic contents, since the values were comparable to those from high-quality EVOOs produced in other countries / Doutorado / Ciência de Alimentos / Doutor em Ciência de Alimentos

In the present study, compounds derived from β-lapachone were electrochemically studied in protic (phosphate and acetate buffer) and aprotic (DMF + TBAP) media, using glassy carbon as working electrode. The electroreducedintermediatesof the quinones reacted with oxygen, indicative of the generation of reactive oxygen species. The interaction of the quinones β-lapachone, α-lapachone and nor-β-lapachone with DNA, was investigated using the technique of quartz crystal microbalance.To increase the solubility of the quinones, in order to allow in vivo studies, the formation of inclusion complexes with β-cyclodextrin was evaluated. Positive results were obtained, leading to a viable formulation alternative for further biological studies with the compounds. β-lapachone was studied spectroelectrochemically through the techniques of Polarization Modulation Infrared Reflection Absorption Spectroscopy + Cyclic Voltammetry to confirm the formation of the hydrogen bondfrom the electroreduced β-lapachone and the secondary hydroxy group of β-cyclodextrin. The inclusion complex betweenmangiferin, an antioxidant polyphenoland β-cyclodextrin, was confirmed using the techniques of Fourier Transform Infrared Spectroscopy, Hydrogen Nuclear Magnetic Resonance, Differential Scanning Calorimetry and Polarization Modulation Infrared Reflection Absorption Spectroscopy. Also, the increase of the antioxidant activity of mangiferin was demonstrated when in presence of β-cyclodextrin, by means of radical scavenging activity toward 2,2’-diphenyl-1-picrylhydrazyl radical (RSA-DPPH ), with additional analysis of solvent effects, being demonstrated that below 50% of organic solvent, it is impossible to work with this method. The assay of the capacity of oxygen radical absorbance capacity-fluorescein (ORAC-FL) was also used, showing that the complex mangiferin: β-cyclodextrin is fifteenfold more potent than the standard molecule,Trolox. Using Electrophoresis and Differential Pulse Voltammetry, it was possible to emphasize that the method used by our research group for the ssDNA production (treatment acid-base with heating) is useful, simple, cheap and completely reproducible, being this, one of the methods used to show the interaction between DNA and free or complexed thymol with cyclodextrin. This interaction was still evaluated using dsDNA biosensor through Differential Pulse Voltammetry, Electrochemistry Quartz Crystal Microbalance and Scanning Electrochemistry Microscopy. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Neste trabalho, derivados da nor-β-lapachona e nor-α-lapachona, foram estudados eletroquimicamente nos meios prótico (tampão fosfato e acetato) e aprótico (DMF + TBAP), utilizando carbono vítreo como eletrodo de trabalho.Os intermediáriosda eletro-redução das quinonas reagiram com oxigênio, indicativo da geração de Espécies Reativas de Oxigênio. A interação das quinonas β-lapachona, α-lapachona e nor-β-lapachona com DNAfoi investigada,utilizando-sea técnica de microbalança de cristal de quartzo. Com o objetivo de aumentar a solubilidade das quinonas, para posteriores estudos in vivo, foi avaliada a formação de complexos de inclusão com β-ciclodextrina, sendo a β-lapachona estudada espectroeletroquimicamente através das técnicas de Espectroscopia de Reflexão-Absorção no Infravermelho com Polarização Modulada + Voltametria Cíclica para confirmar a formação da ligação hidrogênio entre a hidroquinona gerada a partir da β-lapachonae as hidroxilas secundárias da β-ciclodextrina. A formação do complexo de inclusão entre a mangiferina, um composto antioxidante polifenólico, e a β-ciclodextrina foi confirmado utilizando-se as técnicas de Infravermelho com transformada de Fourier, Ressonância Magnética Nuclear de Hidrogênio, Calorimetria Diferencial e Espectroscopia de Reflexão-Absorção no Infravermelho com Polarização Modulada. Foi,também, demonstrado o aumento da atividade antioxidante da xantona supracitada quando em presença de β-ciclodextrina através da medida da atividade sequestradora de radicais frente ao radical 2,2’-difenil-1-picrilidrazil (RSA-DPPH ),com análise adicional do efeito do solvente sendo assim demonstrado que abaixo de 50% de solvente orgânico, é impossível trabalhar com este método. O ensaio da capacidade de absorbância do radical oxigênio-fluoresceína (ORAC-FL) também foi utilizado, mostrando que o complexo mangiferina:β-ciclodextrina é quinze vezes mais potente que a molécula padrão trolox. Com a utilização de ensaios de Eletroforese e Voltametria de Pulso Diferencial, foi possível enfatizar que o método usado por nosso grupo de pesquisa para a produção de ssDNA (tratamento ácido-base com aquecimento) é útil, simples, barato e completamente reprodutível,sendo este um dos métodos utilizados para mostrar a interação entre DNA e timol livre e complexado com ciclodextrina. Esta interação foi ainda avaliada usando biossensor de dsDNA por Voltametria de Pulso Diferencial, Microbalança Eletroquímica de Cristal de Quartzo e Microscopia Eletroquímica de Varredura.