Étude structure-fonction des fructose-1,6-bisphosphate aldolases métallo-dépendantes : mécanisme catalytique et développement d’antimicrobiens

Les fructose-1,6-bisphosphate aldolases (FBPA) sont des enzymes glycolytiques (EC 4.1.2.13) qui catalysent la transformation réversible du fructose-1,6-bisphosphate (FBP) en deux trioses-phosphates, le glycéraldéhyde-3-phosphate (G3P) et le dihydroxyacétone phosphate (DHAP). Il existe deux classes de FBPA qui diffèrent au niveau de leur mécanisme catalytique. Les classes I passent par la formation d’un intermédiaire covalent de type iminium alors que les classes II, métallodépendantes, utilisent généralement un zinc catalytique. Contrairement au mécanisme des classes I qui a été très étudié, de nombreuses interrogations subsistent au sujet de celui des classes II. Nous avons donc entrepris une analyse détaillée de leur mécanisme réactionnel en nous basant principalement sur la résolution de structures cristallographiques. De nombreux complexes à haute résolution furent obtenus et ont permis de détailler le rôle de plusieurs résidus du site actif de l’enzyme. Nous avons ainsi corrigé l’identification du résidu responsable de l’abstraction du proton de l’O4 du FBP, une étape cruciale du mécanisme. Ce rôle, faussement attribué à l’Asp82 (chez Helicobacter pylori), est en fait rempli par l’His180, un des résidus coordonant le zinc. L’Asp82 n’en demeure pas moins essentiel car il oriente, active et stabilise les substrats. Enfin, notre étude met en évidence le caractère dynamique de notre enzyme dont la catalyse nécessite la relocalisation du zinc et de nombreux résidus. La dynamique de la protéine ne permet pas d’étudier tous les aspects du mécanisme uniquement par l’approche cristallographique. En particulier, le résidu effectuant le transfert stéréospécifique du proton pro(S) sur le carbone 3 (C3) du DHAP est situé sur une boucle qui n’est visible dans aucune de nos structures. Nous avons donc développé un protocole de dynamique moléculaire afin d’étudier sa dynamique. Validé par l’étude d’inhibiteurs de la classe I, l’application de notre protocole aux FBPA de classe II a confirmé l’identification du résidu responsable de cette abstraction chez Escherichia coli (Glu182) mais pointe vers un résidu diffèrent chez H. pylori (Glu149 au lieu de Glu142). Nos validations expérimentales confirment ces observations et seront consolidées dans le futur. Les FBPA de classe II sont absentes du protéome humain mais sont retrouvées chez de nombreux pathogènes, pouvant même s'y révéler essentielles. Elles apparaissent donc comme étant une cible idéale pour le développement de nouveaux agents anti-microbiens. L’obtention de nouveaux analogues des substrats pour ces enzymes a donc un double intérêt, obtenir de nouveaux outils d’étude du mécanisme mais aussi développer des molécules à visée pharmacologique. En collaboration avec un groupe de chimistes, nous avons optimisé le seul inhibiteur connu des FBPA de classe II. Les composés obtenus, à la fois plus spécifiques et plus puissants, permettent d’envisager une utilisation pharmacologique. En somme, c’est par l’utilisation de techniques complémentaires que de nouveaux détails moléculaires de la catalyse des FBPA de classe II ont pu être étudiés. Ces techniques permettront d’approfondir la compréhension fine du mécanisme catalytique de l’enzyme et offrent aussi de nouvelles perspectives thérapeutiques.

Modelo teórico e experimental da reciclagem do Carbono-13 em tecidos de mamíferos e aves

A diferença entre fontes alimentares da ordem de 14 , originárias de plantas com ciclos fotossintéticos Carbono-3 (C3) e Carbono-4 (C4) e seus subprodutos, abre novas perspectivas para o estudo do metabolismo do carbono em aves e animais de pequeno porte. Os autores propõem um modelo teórico e experimental capaz de exprimir os resultados de enriquecimento relativo, delta per mil (delta ) da razão 13C/12C versus tempo em diferentes tecidos. Utilizou-se a equação y(t) = (y0 -- q/k) e-kt + q/k onde, y(t) é a concentração isotópica no tempo desejado, y0 a concentração isotópica inicial existente no tecido, k é uma constante de troca isotópica com unidade 1/tempo, t é unidade de tempo e q é a taxa de entrada de metabólitos que contém carbono, com valores de delta /tempo. Para fígado de galinhas que tiveram a ração de ciclo fotossintético C4 substituída por dieta C3 obteve-se a equação delta13C = -24,74 + 12,37 e-0.237(nT), com meia-vida (T) de 2,9 dias. O patamar de equilíbrio de substituição do carbono foi alcançado em --24,48 , de modo que praticamente 98,4% do conteúdo isotópico do C4 no fígado foi substituído por C3 após 5,6 meias-vidas. O modelo foi adequado para determinar o tempo de reciclagem total ou parcial da concentração de carbono nos tecidos em estudo.

Tracing metabolic routes of feed ingredients in tissues of broiler chickens using stable isotopes

The present study aimed to quantify the proportion of 13C from energy and protein feed ingredients that follow the metabolic routing of the liver and muscle in broiler chickens. A stable isotope of carbon technique was used that is based on the isotopic discrimination that occurs in the plants during the photosynthesis process. One-day-old male chicks were subjected to treatments based on free choice of energy and protein sources. Rice bran (R) and soybean meal (S), C3 plants, have higher isotopic ratios than corn (C), a C4 plant, and corn gluten meal (G). Choices were R+S, C+G, R+G, C+S, or R+C+G+S. A complete feed (CF) was a sixth treatment. Feed intake and BW were measured at 30 d of age, when liver and breast muscle were collected for isotopic analysis. Treatments affected the amount of feed intake and the choices of energy or protein sources. Complete feed had the largest intake, differing from the other treatments that had free-choice feeding. Final BW was a direct reflection of consumption by these birds in all treatments. The isotopic results indicated that the 13C/12C ratio was generally higher in breast muscle than in liver, probably because of higher protein content. Moreover, in the liver, the proportion of 13C retained from the energy ingredient was greater than the proportion from the protein ingredient. That is in contrast to muscle, where the proportion of 13C retained from the protein ingredient was greater than from the energy ingredient that was self-selected.

Waste recovery into activated carbon for water purification from heavy metals

Dissertação apresentada para obtenção do Grau de Doutor em Engenharia Química Pela Universidade Nova de Lisboa,Faculdade de Ciências e Tecn

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A Knudsen flow reactor has been used to quantify functional groups on the surface of seven different types of combustion particle samples: 3 amorphous carbons (FS 101, Printex 60, FW 2), 2 flame soots (hexane soot generated from a rich and a lean diffusion flame), and 2 Diesel particles (SRM 2975, Diesel soot recovered from a Diesel particulate filter). The technique is based on a heterogeneous titration reaction between a probe gas and a specific functional group on the particle surface. Six probe gases have been selected for the quantification of important functional groups: N(CH3)3 for the titration of acidic sites, NH2OH for carbonyl functions of aldehydes and ketones, CF3COOH and HCl for basic sites of different strength, O3 and NO2 for oxidizable groups. The limit of detection was generally well below 1% of a formal monolayer of adsorbed probe gas. Results obtained with N(CH3)3 were higher for the FW 2 amorphous carbon (post-oxidized sample, according to the manufacturer) and the Diesel particles (between 5.2·10 13 and 5.8·10 13 molecule/cm2), indicating a higher state of oxidation than for the other samples (between 1.3·10 12 and 3.7·10 12 molecule/cm2). The ratio of uptakes of CF3COOH and HCl inferred the presence of basic oxides on the particle surface, owing to the larger stability of the acetate compared to the chloride counter ion in the resulting pyrylium salt. The reactivity of the FS 101 amorphous carbon (3.7·10 15 molecule/cm2) and the hexane flame soot (between 1.9·10 15 and 2.7·10 15 molecule/cm2) towards O3 was very high, indicating the presence of a huge amount of oxidizable or reduced groups on the surface of these samples. Besides the quantification of surface functional groups, the kinetics of reactions between particles and probe gases has also been studied. The uptake coefficient γ0 was roughly correlated with the amount of probe gas taken up by the samples. Indeed, the presence of a high density of functional groups led to fast uptake of the probe gas. These different findings indicate that the particle surface appeared multi-functional, with the simultaneous presence of antagonistic functional groups which do not undergo internal chemical reactions, such as acid-base neutralization. Results also point to important differences in the surface reactivity of the samples, depending on the combustion conditions. The relative distribution of the surface functional groups may be a useful indicator for the state of oxidation and the reactivity of the particle surface.

Isotope analysis (δ13C) of pulpy whole apple juice

The objectives of this study were to develop the method of isotope analysis to quantify the carbon of C3 photosynthetic cycle in pulpy whole apple juice and to measure the legal limits based on Brazilian legislation in order to identify the beverages that do not conform to the Ministry of Agriculture, Livestock and Food Supply (MAPA). This beverage was produced in a laboratory according to the Brazilian law. Pulpy juices adulterated by the addition of sugarcane were also produced. The isotope analyses measured the relative isotope enrichment of the juices, their pulpy fractions (internal standard) and purified sugar. From those results, the quantity of C3 source was estimated by means of the isotope dilution equation. To determine the existence of adulteration in commercial juices, it was necessary to create a legal limit according to the Brazilian law. Three brands of commercial juices were analyzed. One was classified as adulterated. The legal limit enabled to clearly identify the juice that was not in conformity with the Brazilian law. The methodology developed proved efficient for quantifying the carbon of C3 origin in commercial pulpy apple juices.

Carbon isotope analysis in apple nectar beverages

The aims of this study were to use the isotope analysis method to quantify the carbon of C3 photosynthetic cycle in commercial apple nectars and to determine the legal limit to identify the beverages that do not conform to the safety standards established by the Brazilian Ministry of Agriculture, Livestock and Food Supply. These beverages (apple nectars) were produced in the laboratory according to the Brazilian legislation. Adulterated nectars were also produced with an amount of pulp juice below the permitted threshold limit value. The δ13C values of the apple nectars and their fractions (pulp and purified sugar) were measured to quantify the C3 source percentage. In order to demonstrate the existence of adulteration, the values found were compared to the limit values established by the Brazilian Law. All commercial apple nectars analyzed were within the legal limits, which enabled to identify the nectars that were in conformity with the Brazilian Law. The isotopic methodology developed proved efficient to quantify the carbon of C3 origin in commercial apple nectars.

Warming caused by cumulative carbon emissions towards the trillionth tonne

Global efforts to mitigate climate change are guided by projections of future temperatures1. But the eventual equilibrium global mean temperature associated with a given stabilization level of atmospheric greenhouse gas concentrations remains uncertain1, 2, 3, complicating the setting of stabilization targets to avoid potentially dangerous levels of global warming4, 5, 6, 7, 8. Similar problems apply to the carbon cycle: observations currently provide only a weak constraint on the response to future emissions9, 10, 11. Here we use ensemble simulations of simple climate-carbon-cycle models constrained by observations and projections from more comprehensive models to simulate the temperature response to a broad range of carbon dioxide emission pathways. We find that the peak warming caused by a given cumulative carbon dioxide emission is better constrained than the warming response to a stabilization scenario. Furthermore, the relationship between cumulative emissions and peak warming is remarkably insensitive to the emission pathway (timing of emissions or peak emission rate). Hence policy targets based on limiting cumulative emissions of carbon dioxide are likely to be more robust to scientific uncertainty than emission-rate or concentration targets. Total anthropogenic emissions of one trillion tonnes of carbon (3.67 trillion tonnes of CO2), about half of which has already been emitted since industrialization began, results in a most likely peak carbon-dioxide-induced warming of 2 °C above pre-industrial temperatures, with a 5–95% confidence interval of 1.3–3.9 °C.

Impaired dendritic cell differentiation and maturation in the absence of C3

Human monocytes can be differentiated into immature dendritic cells (DCs) in the presence of serum and cytokines. One of the main functions of immature DCs is to capture and process antigens. Following maturation, they differentiate into antigen presenting cells. The role of complement in the differentiation process from monocytes towards immature DCs remains elusive. Here we demonstrate that complement 3 (C3) has a regulatory impact on the expression of specific DC surface molecules and DC-derived cytokine production during DC differentiation. We isolated human adherent peripheral blood mononuclear cells, which were cultured in the presence of GM-CSF plus IL-4 in medium supplemented with normal human serum or C3 deficient serum. The lack of C3 during DC differentiation negatively impacted the expression of C-type lectin receptor DC-SIGN, the antigen presenting molecules HLA-DR and CD1a, and the costimulatory molecules CD80 and CD86. Further, the spontaneous production of IL-6 and IL-12 was reduced in the absence of C3. Moreover, the maturation of immature DCs in response to LPS challenge was impaired in the absence of C3 as evidenced by reduced MHC-II, co-stimulatory molecule expression as well as modulated IL-12 and TNF-alpha production. Collectively, our results provide evidence for a novel role of C3 as a critical cofactor in human DC differentiation and maturation. (C) 2007 Elsevier Ltd. All rights reserved.

Muscle delta C-13 change in nile tilapia (Oreochromis niloticus) fingerlings fed lants grain-based diets

The effects of grain-based diets from C3 or C4-cycle plants on muscle delta C-13 change process in Nile tilapia (Oreochromis niloticus) fingerlings were investigated. Two groups of sex reversal males Nile tilapia fingerlings were fed with isoproteic (32.0% DP) and isocaloric (3200 kcal DE/kg) diets, differing from each other by their delta C-13. Muscle samples were collected and the carbon isotopic composition was measured. For C4 diet, the formula for the muscle delta C-13 change related to the intake time of a new diet was delta C-13=- 14.88 - 9.2 1 e(-0.0209t) and the half-life (T) of the muscle carbon was 33.2 days. For C3 diet, the formula was delta C-13 = - 25.43 + 8.59e(-0.0533), with T = 13 days. The C3 diet was considered more appropriate based on its palatability and consequent larger food intake than the C4 diet, resulting in an increased muscle delta C-13 change rate. However, for future studies, would be necessary to mix both the C3 and C4 feedstuffs to formulate diets nutritionally appropriated, with contrasting stable isotopes signatures. Tissue delta C-13 change rate is therefore indicated as a promising tool to better understand the biotic and abiotic factors that influence nutrients utilization from the diet and animal growth. (c) 2006 Elsevier B.V. All rights reserved.

Método de análise isotópica (δ13C) e limite de legalidade em néctar de laranja

Os objetivos deste trabalho foram desenvolver o método de análise isotópica para quantificar o carbono do ciclo fotossintético C3 em néctares de laranja comerciais e mensurar o limite de legalidade, baseado na legislação brasileira, para identificar as bebidas que não estão em conformidade com o Ministério da Agricultura, Pecuária e Abastecimento (MAPA). As bebidas foram produzidas em laboratório, conforme a legislação brasileira. Também foram produzidos néctares adulterados com quantidade de suco de laranja abaixo do limite mínimo permitido pelo MAPA. Na análise isotópica, foi mensurado o enriquecimento isotópico relativo dos néctares de laranja e também de suas frações, sólidos insolúveis (polpa) e açúcar purificado. Com esses resultados, foi estimada a quantidade de fonte C3 por meio da equação da diluição isotópica. Para determinar a existência de adulteração, foi necessária a criação do limite de legalidade de acordo com a legislação brasileira. Oito marcas comerciais de néctar de laranja foram analisadas. Todas foram classificadas como legais. O limite de legalidade foi uma importante inovação metodológica, que possibilitou identificar as bebidas que estavam em conformidade com a legislação brasileira. A metodologia desenvolvida provou ser eficiente para quantificar o carbono de origem C3 em néctares de laranja comerciais.

Carbon isotope analysis in apple nectar beverages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação do Metabolismo Nutricional em Poedeiras pela Técnica dos Isótopos Estáveis do Ccarbono (13C/12C)

Os isótopos estáveis do carbono que eram utilizados em estudos ecológicos e paleoecológicos apresentaram um incremento nos últimos trinta anos, na utilização em estudos dietéticos em animais. Entretanto, existem poucas informações sobre o padrão metabólico e sobre as taxas de turnover do 13C em aves. O presente experimento estabeleceu curvas de substituição e taxas de movimentação do 13C no ovo e no fígado de aves de postura adultas, pela substituição da ração comercial por dietas compostas de grãos dos ciclos fotossintéticos C3 e C4, durante 50 dias. A diferença no conteúdo isotópico do delta per mil do carbono-13 (delta 13C) entre as duas dietas foi de 16,13 . A taxa de substituição do 13C das dietas, nos tecidos, adequou-se num modelo exponencial, descrevendo o turnover do carbono nos tecidos analisados. As taxas de movimentação do 13C, nas aves alimentadas com dieta baseada em grãos C3, foi maior no fígado em relação ao ovo, com valores para a meia-vida de 2,9 e 3,7 dias, respectivamente. As aves que receberam ração com grãos C4 apresentaram uma taxa de turnover no ovo superior àquela obtida para o fígado, com meia-vida de 4,0 e 5,3 dias, respectivamente. Os valores do delta 13C observados para ovo e fígado diferiram em aproximadamente 2 daqueles referentes às dietas.

Qualidade de compostos orgânicos produzidos com resíduos do processamento de plantas medicinais

Pós-graduação em Agronomia (Energia na Agricultura) - FCA