Dans ce travail nous avons étudié des réactions chimiques sur la surface de deux métaux : le platine et l'or, en utilisant la microscopie ionique à effet de champ électrique (FIM) et la spectrométrie de masse de désorption par champ pulsé (PFDMS). En complément de ces données expérimentales, nous apportons des résultats obtenus par la théorie de la fonctionnelle de la densité (DFT). La taille et la morphologie de nos échantillons font qu’ils sont de bons modèles de grains de phase active dans un catalyseur réel. Sur le platine nous avons observé l'interaction du monoxyde d'azote. En présence d'un champ électrique, la dissociation du NO est promue, laissant sur la pointe une couche d'oxygène que l'on peut titrer par la suite avec du NO pour former du dioxyde d'azote. L'interaction de l'hydrogène avec une pointe de platine est différente en fonction de la température. A basse température (<200 K), l’hydrogène met en image la pointe avec un assombrissement de petites régions de la pointe. A partir de 200 K, l'hydrogène induit une transformation de la pointe : les faces denses s'élargissent au détriment des faces rugueuses, avec une rangée d'atomes le long des lignes de zone [100] et le pôle central passe d’une forme circulaire à une forme carrée. L'hydrogène est supposé être à l'état atomique sur la surface; sous sa forme moléculaire, il ne contribue qu'à la mise en image. C’est sous sa forme atomique qu’il contribue aux transformations observées. La réaction NO+H2 sur le platine conduit à l'observation d'instabilités cinétiques pour des pressions et températures données. Les régions d'amorçage ont été identifiées comme étant tout ensemble de sites de cran situé le long des lignes de zone [100]. L'observation de phénomènes oscillants n'est pas liée exclusivement à une morphologie de type polyédrique : il est possible d’observer ces instabilités cinétiques sur des pointes de rayon de courbure différent, ces pointes ne subissent pas nécessairement un changement de morphologie. L'analyse chimique au cours de la réaction a révélé la présence d'oxydes de surface ce qui nous a conduit à penser que la surface du catalyseur de platine doit être oxydée plutôt que de rester à l'état métallique. Une étape intermédiaire dans le mécanisme de site vacant a été proposée pour permettre de rendre compte de l'apparition de phénomènes oscillants. L’étape déterminante est la décomposition du monoxyde d’azote adsorbé à la surface. L’apport d’hydrogène atomique nécessaire à la formation d’eau n’est pas une étape lente, au contraire l’hydrogène semble être largement présent sous forme atomique. L’influence du champ électrique reste important, ce qui pourrait expliquer les différences de gammes de température auxquelles d’autres groupes et le nôtre observons des oscillations. L'étude de l'interaction du CO seul sur l'or a été motivée par l'observation de la réaction CO+O2 sur pointe d'or. Sous nos conditions expérimentales, il est possible de former des mono- et dicarbonyles d’or sous forme cationique. En présence d'un champ électrique, la formation de carbonyles d'or est promue au niveau des sites de cran de la surface. Les résultats obtenus par DFT sont concordants avec les résultats expérimentaux. A l'instar du Pt, nous avons étudié l'interaction du monoxyde d'azote sur pointe d'or. Nous avons observé la formation de protoxyde d'azote et d'un dimère de monoxyde d'azote. Le mécanisme de formation de ces espèces reste encore inconnu. Une couche d'oxygène adsorbée à la surface peut être titrée par du NO en formant du dioxyde d'azote. En présence d'oxygène seul sur une pointe d'or sans champ électrique, les spectres de masse ne révèlent que la présence d'oxygène moléculaire. En présence d'un champ électrique, il est possible de détecter de l'oxygène atomique sans toutefois avoir la formation d'un oxyde d'or. Il est possible de former de l'ozone à partir d'une pointe recouverte d'oxygène atomique en présence d'oxygène moléculaire gazeux. Les calculs par DFT nous laissent à penser que la formation du cation O3+ ne se fait pas à la surface mais à proximité. Ils montrent entre autres que le champ électrique déstabilise l'oxygène atomique, ce qui pourrait le pousser vers des régions où l'effet du champ est le moins intense (la tige de la pointe).

Acetoacetato (AA) e 2-metilacetoacetato (MAA) são compostos &#946;-cetoácidos acumulados em diversas desordens metabólicas como no diabetes e na isoleucinemia, respectivamente. Examinamos o mecanismo de oxidação aeróbica de AA e MAA iniciada por intermediários reativos de mioglobina de coração de cavalo (Mb) gerados pela adição de H2O2. Uma rota quimioluminescente que envolve um intermediário dioxetânico cuja termólise gera espécies &#945;-dicarbonílicas (metilglioxal e biacetilo) foi proposta e estudada. Emissão de luz ultra fraca acompanha a reação, e sua intensidade aumenta linearmente pelo aumento da concentração tanto de Mb (10-500 &#181;M) quando AA (10-100 mM). Estudos de consumo de oxigênio mostraram que MAA é, como esperado, quase uma ordem de grandeza mais reativo que AA. Estudos de EPR com captação de spin, utilizando MNP, possibilitaram detectar adutos de MAA atribuíveis a um radical centrado no C&#945; (aN = 1.55 mT) e ao radical acetila (aN = 0.83 mT). O sinal do radical acetila é totalmente suprimido por sorbato, um conhecido e eficiente supressor de espécies tripletes, o que é consistente com uma rota reacional envolvendo um intermediário dioxetânico. Clivagem-&#945; da ligação carbonila-carbonila do produto biacetilo triplete produziria, de fato, radicais acetila. Além disso, utilizando AA como substrato para Mb/H2O2, um sinal de EPR atribuível ao aduto MNP-AA&#8226; (aN = 1.46 mT e aH = 0.34 mT) foi observado e confirmado por efeito isotópico. O consumo de oxigênio e o rendimento de compostos &#945;-dicarbonílicos foram dose-dependentes à concentração de AA ou MAA (1-50 mM) bem como à concentração de H2O2 adicionado às misturas de reação contendo Mb (até 1:10 quando medido o consumo de oxigênio, e até 1:25 quando medido o rendimento de compostos &#945;-dicarbonílicos) e tert-butilhidroperóxido (até 1:200). Os perfis de pH (5,8-7,8) para consumo de oxigênio e rendimento de compostos &#945;-dicarbonílicos mostraram maiores rendimentos para baixos valores de pH, indicativo de ferrilMb formada no ciclo peroxidático da proteína. Avaliando os níveis de lesão de Mb, os &#946;-cetoácidos diminuíram o nível de desorganização protéica na estrutura secundária e terciária elicitada por H2O2. Ainda, houve maior preservação da estrutura primária da proteína, sendo que MAA protegeu mais em comparação a AA, embora quando utilizado este último composto, foi mostrado que há acetilação dose-dependente de Mb. Acetoacetato aumentou a velocidade de descoramento da hemeproteína, provavelmente por ataque de espécies tripletes geradas no sistema. Músculos de rato, plantar e sóleo, expostos ex vivo a concentrações citotóxicas de glicose oxidase (GOX, gera H2O2 em fluxo), foram protegidas pelos ésteres etílicos AAE e MAAE. Foi detectado biacetilo no meio intracelular em músculos expostos a MAAE e GOX. A concentração deste composto &#945;-dicarbonílico é claramente relacionada à abundância de Mb em cada um dos tipos de músculos estudados. Em resumo, Mb tratada com metabólitos &#946;-cetoácidos (AA e MAA) gera radicais centrados em carbono e produtos &#945;-dicarbonílicos altamente reativos no estado triplete. Experimentos realizados com tecido muscular ex vivo sugerem que esta reação possivelmente ocorra in vivo. Levantamos a hipótese de que a geração de espécies carbonílicas reativas e seus adutos em condições de desbalanço metabólico possam contribuir para a compreensão das bases moleculares de desordens cetogênicas. / Acetoacetate (AA) and 2-methylacetoacetate (MAA) are &#946;-ketoacids accumulated in several metabolic disorders such as diabetes and isoleucinemia, respectively. Here we examine the mechanism of AA and MAA aerobic oxidation initiated by the reactive enzyme intermediates formed by the reaction of muscle horse myoglobin (Mb) with H2O2. A chemiluminescent route involving a dioxetane intermediate whose thermolysis yields triplet &#945;-dicarbonyl species (methylglyoxal and diacetyl) is envisaged. Accordingly, the ultraweak light emission that accompanies the reaction increases linearly by raising the concentration of both Mb (10-500 &#181;M) and AA (10- 100 mM). Oxygen uptake studies revealed that MAA is, expectedly, almost one order of magnitude more reactive than AA. EPR spin-trapping studies with MNP detected spin adducts from MAA attributable to an &#945;-carbon-centered radical (aN = 1.55 mT) and to an acetyl radical (aN = 0.83 mT). As the acetyl radical signal is totally suppressed by sorbate, a well-known efficient triplet species quencher, the dioxetane hypothesis seems to be reliable. The &#945;-cleavage of the carbonyl-carbonyl bond of a putative excited triplet diacetyl product would, in fact, leads to an acetyl radical. Furthermore, using AA as substrate for Mb/H2O2, an EPR signal assignable to a MNP-AA&#8226; adduct (aN = 1.46 mT and aH = 0.34 mT) was observed and confirmed by isotope effect. Oxygen consumption and &#945;-dicarbonyl yield were also dependent on AA or MAA concentrations (1-50 mM) as well as on the concentration of peroxide added to the Mb-containing reaction mixtures: H2O2 (up to 1:10 when measuring oxygen uptake and up to 1:25 when measuring the &#945;-dicarbonyl yield) and t-butOOH (up to 1:200). The pH profiles (5.8-7.8) of oxygen consumption and &#945;-dicarbonyl yield show higher reaction rates at lower pHs, indicative of a ferrylMb intermediate. Evaluating Mb lesion, both &#946;-ketoacids reduced disorganization of the secondary and tertiary protein structure elicited by H2O2. Therefore, Mb primary structure was more preserved, and MAA was more protective than AA. Moreover using the later compound, it was shown that Mb acetylation is dose-dependent. Acetoacetate increased the rate of the hemeprotein bleaching, probably due to the attack of triplet products generated in the system. Plantaris and soleous rat muscles exposed to damaging concentrations of glucose oxidase (GOX, generates H2O2 in flux), was cytoprotected by AAE and MAAE. Intracellular diacetyl was detected in muscle samples exposed to MAAE and GOX. The &#945;-dicarbonyl concentration is clearly related to the Mb abundance in the muscle types. In summary, Mb treated with peroxides reacts with &#946;-ketoacid metabolites (AA and MAA), yielding carbon-centered radicals and highly reactive &#945;-dicarbonyl products in the triplet state. Experiments carried out ex vivo with muscle tissue showed that this reaction possibly occurs in vivo. A new route for generation and accumulation of carbonyl reactive species and adducts is here proposed to occur in unbalanced metabolic situations, such as is the case of ketogenic disorders.

<p>Maternal type 1 diabetes is associated with an increased risk for foetal malformations. The mechanism by which diabetes is teratogenic is not fully known. Previous studies have demonstrated that radical oxygen species can contribute to the teratogenicity of glucose and diabetes. The aim of the present work was to study different aspects of free radical damage and antioxidant defence in experimental diabetic pregnancy.</p><p> The activity of the antioxidant enzyme catalase and the mRNA levels of antioxidant enzymes in embryos of normal and diabetic rats of two strains were measured. The catalase activity was higher in embryos of a malformation-resistant strain than in a malformation-prone strain, the difference increased further when the mother was diabetic. Maternal diabetes increased embryonic mRNA levels of catalase and manganese superoxide dismutase in the malformation-resistant strain, but not in the malformation-prone strain. Embryos of the malformation-prone rat thus had lower antioxidative defence than embryos of the malformation-resistant strain.</p><p> Administration of either vitamin E or vitamin C has previously been shown to protect embryos from maldevelopment in experimental diabetic pregnancy. The vitamins were used together in this thesis to yield protection in both the lipid and aqueous phase. The protective effect was not higher than what had been achieved using the vitamins individually. No synergistic effect was thus found using the two antioxidants together. </p><p> The urinary excretion of the lipid peroxidation marker 8-iso-PGF_2á was increased in pregnant dia-betic rats compared with non-diabetic controls, as was the plasma content of carbonylated proteins. Carbonylated proteins and TBARS concentrations were increased in foetal livers in diabetic pregnancy. However, no increased concentration of 8-iso-PGF_2á was found in the amniotic fluid of pregnant diabetic rats. Both lipids and proteins were thus oxidatively modified in experimental diabetic pregnancy. It is concluded that experimental diabetic pregnancy is associated with increased oxidative stress and that the embryonic antioxidant defence is likely to be of importance for normal development in a diabetic environment.</p>

Maternal type 1 diabetes is associated with an increased risk for foetal malformations. The mechanism by which diabetes is teratogenic is not fully known. Previous studies have demonstrated that radical oxygen species can contribute to the teratogenicity of glucose and diabetes. The aim of the present work was to study different aspects of free radical damage and antioxidant defence in experimental diabetic pregnancy. The activity of the antioxidant enzyme catalase and the mRNA levels of antioxidant enzymes in embryos of normal and diabetic rats of two strains were measured. The catalase activity was higher in embryos of a malformation-resistant strain than in a malformation-prone strain, the difference increased further when the mother was diabetic. Maternal diabetes increased embryonic mRNA levels of catalase and manganese superoxide dismutase in the malformation-resistant strain, but not in the malformation-prone strain. Embryos of the malformation-prone rat thus had lower antioxidative defence than embryos of the malformation-resistant strain. Administration of either vitamin E or vitamin C has previously been shown to protect embryos from maldevelopment in experimental diabetic pregnancy. The vitamins were used together in this thesis to yield protection in both the lipid and aqueous phase. The protective effect was not higher than what had been achieved using the vitamins individually. No synergistic effect was thus found using the two antioxidants together. The urinary excretion of the lipid peroxidation marker 8-iso-PGF2á was increased in pregnant dia-betic rats compared with non-diabetic controls, as was the plasma content of carbonylated proteins. Carbonylated proteins and TBARS concentrations were increased in foetal livers in diabetic pregnancy. However, no increased concentration of 8-iso-PGF2á was found in the amniotic fluid of pregnant diabetic rats. Both lipids and proteins were thus oxidatively modified in experimental diabetic pregnancy. It is concluded that experimental diabetic pregnancy is associated with increased oxidative stress and that the embryonic antioxidant defence is likely to be of importance for normal development in a diabetic environment.

19 Dec 2004. / Published through the Information Bridge: DOE Scientific and Technical Information. "IS-T 2117" Guodong Du. 12/19/2004. Report is also available in paper and microfiche from NTIS.

Solvent effects play diverse roles in myriads of chemical, physical and biological processes. The solvent interacts with the solute by: i) non-specific (Coulombic, van der Waals interactions) and ii) specific interactions (hydrogen bonding, etc.). These interactions are responsible for solvation of the solute and are collectively termed as “solvent polarity”. Differential solvation of ground and excited electronic states is manifested in the absorption spectrum as a change in the band position, intensity or shape, which is termed as “solvatochromism”. Intermolecular hydrogen bonding (IHB) is a kind of specific solute-solvent interaction, which plays a key role in molecular or supramolecular photochemistry, as well as in photobiology. Solvation and its influence on various physico-chemical and biological processes can be understood by i) top-down; and ii) bottom-up approaches. In the top-down approach, the macroscopic properties like dielectric constant, refractive index are used to understand the microscopic solvation. This approach fails when specific interactions like hydrogen bonding interactions come into play, and furthermore it can reproduce only the macroscopic polarization of the solvent but fails miserably at the cybotactic region of solvation. With the recent advancements in the computational field, the molecular level description of solvation has been within reach for chemical physicists and experimentalists to corroborate their experimental results and in turn to visualize processes of fundamental or technologically relevant problems. The energy levels of the nπ* and ππ* singlet and triplet excited states of aromatic ketones are close-lying and therefore their energy levels can be altered by the substituents. The solvent polarity can be used as a surrogate to tune their energy levels. In certain cases, the lowest triplet or singlet excited states can switch their electronic character with increasing solvent polarity known as “electronic state switching” and thus modulate their photochemical or photophysical properties. Therefore, aromatic ketones were used as solvatochromic probes in this work. Comprehensive analyses of the solvent effects on xanthone (XT), 9,10-phenanthrenequinone (PQ) and benzophenone (Bzp) were carried out using steady-state and nanosecond time-resolved absorption, and resonance Raman spectroscopy in conjunction with ad hoc and classical-molecular dynamics and simulations generated supermolecule-continuum solvent model quantum mechanical calculations to corroborate the experimental outcomes and in turn to visualize the solvation process at the molecular level. The present thesis is divided into eight chapters and the summary of each chapter is described below: Chapter 1 provides a brief literature review of solvation effects and their influence on various physico-chemical and biological processes. Furthermore, the importance of understanding solvation at the molecular level and key concepts are discussed, which forms the heart of this thesis. Chapter 2 discusses the experimental and computational approaches used to study the solvation processes at the molecular level. A detailed explanation of spectroscopic techniques like resonance Raman (RR) and nanosecond-time resolved resonance Raman (ns-TR3) spectroscopy and their experimental and theoretical aspects are discussed, followed by a discussion on the fundamental concepts of computational methods like ab initio calculations density functional theory (DFT), and classical molecular dynamics and simulations (c-MDS) utilized in this study. Chapter 3 focuses on microscopic understanding of solvatochromic shifts observed for 9,10-phenanthrenequinone in protic solvents using UV-Vis and RR spectroscopy in conjunction with an ad hoc explicit solvation model and time-dependent density functional theory (TDDFT) calculations. The hypsochromic shift and bathochromic shift of the singlet nπ* and ππ* electronic transitions in protic solvents are due to hydrogen bond weakening and strengthening in the excited state for the corresponding electronic transitions, respectively as indicated by TD-DFT calculations and Kamlet-Taft linear solvation energy relationships. The hydrogen bond strengthening in the singlet ππ* excited state is further confirmed by Raman excitation profile (REP) analysis of PQ in different solvents. Furthermore, with increasing solvent polarity the two lowest singlet excited states undergo different hydrogen bonding mechanisms, leading to a decreasing energy gap between them. Therefore, hyperchromism of the nπ* transition has been hypothesized to be due to an increasing vibronic coupling between the lowest singlet nπ* and ππ* excited states. In Chapter 4, a real time observation of the thermal equilibrium between the lowest triplet excited states of PQ in acetonitrile solvent was carried out using ns-TR3 spectroscopy and this can explain its high reactivity towards H-atom abstraction, despite the fact that the lowest triplet excited state has ππ* character. Furthermore, extending the concept of hydrogen bonding mechanisms of the lowest singlet to the triplet excited states, the different hydrogen bonding mechanisms exhibited by them can lead to alteration of the intersystem crossing mechanisms in PQ. Chapter 5 highlights the very different role of intermolecular hydrogen bonding in the reduced reactivity of the xanthone (XT) triplet towards H-atom abstraction in protic solvents. The different hydrogen bonding mechanisms exhibited by the two lowest triplet excited states in protic solvents are derived from an ad hoc explicit solvation model, TD-DFT calculations and ns-time resolved absorption (ns-TRA): they separate them further in energy and thereby the nearest T2(nπ*) triplet state to the T1(ππ*) excited state plays an insignificant role in the reactivity towards H-atom abstraction, in contrast to the PQ triplet discussed in Chapter 4. Chapter 6 discusses the structure of XT triplet states using TR3 spectroscopy in combination with TD-DFT studies. The TR3 spectrum of the XT in acetonitrile identified a vibronic coupling mode responsible for the reactivity of XT towards H-atom abstraction, despite the fact that the lowest triplet excited state (T1) has ππ* character. This vibronic active mode is absent in the TR3 spectra of XT in protic solvents (methanol and ethanol). Furthermore, the REP analysis suggests that the nanosecond triplet-triplet absorption spectrum of XT in acetonitrile involves two different species, while in methanol it involves only one species. This observation is in agreement with the previous chapter (Chapter 5) which proposes a different hydrogen bonding mechanisms for the two lowest triplet excited states and their influence on the reduced reactivity towards H-atom abstraction. Chapters 3-6 emphasize the need for a proper solvation model at the molecular level to describe the various photophysical and photochemical processes of aromatic ketones. Therefore, Chapter 7 includes discussions on the bottom-up solvation methodology applied to benzophenone (Bzp) to understand its vibrational and electronic solvatochromic behaviour at the molecular level. Raman and UV-Vis spectroscopic techniques were used in conjunction with a c-MDS-generated supermolecule continuum solvation model DFT calculation to corroborate and to visualize the experimental outcome. The carbonyl stretching frequency of Bzp in protic solvents has two bands, corresponding to free carbonyl and hydrogen bonded carbonyl. Despite the fact that the macroscopic polarity of acetonitrile and methanol solvents are similar, the free carbonyl stretching of Bzp in methanol solvent was blue-shifted by 4 cm-1 with respect to the carbonyl stretching in acetonitrile solvent. The Gutmann’s acceptor number plot for carbonyl stretching frequencies indicates that the free carbonyl group is neighboured by a hydrophobic environment. The c-MDS-generated supermolecule-continuum solvation model DFT calculations suggest that the extended hydrogen bonding network of methanol solvent is responsible for the hydrophobic solvation around the free carbonyl. Furthermore, a linear correlation was obtained for the vibrational and electronic solvatochromism of the carbonyl frequency and energy of the singlet nπ* transition, respectively, which indicates that a variation in excitation wavelength for the singlet nπ* transition can arise from different solvation states. This can have implications for ultrafast processes associated with electron transfer, charge-transfer and also the photophysical aspects of excited states.Finally, Chapter 8 contains overall conclusions of the thesis and future directions for the present research area.

Acetoacetato (AA) e 2-metilacetoacetato (MAA) são compostos &#946;-cetoácidos acumulados em diversas desordens metabólicas como no diabetes e na isoleucinemia, respectivamente. Examinamos o mecanismo de oxidação aeróbica de AA e MAA iniciada por intermediários reativos de mioglobina de coração de cavalo (Mb) gerados pela adição de H2O2. Uma rota quimioluminescente que envolve um intermediário dioxetânico cuja termólise gera espécies &#945;-dicarbonílicas (metilglioxal e biacetilo) foi proposta e estudada. Emissão de luz ultra fraca acompanha a reação, e sua intensidade aumenta linearmente pelo aumento da concentração tanto de Mb (10-500 &#181;M) quando AA (10-100 mM). Estudos de consumo de oxigênio mostraram que MAA é, como esperado, quase uma ordem de grandeza mais reativo que AA. Estudos de EPR com captação de spin, utilizando MNP, possibilitaram detectar adutos de MAA atribuíveis a um radical centrado no C&#945; (aN = 1.55 mT) e ao radical acetila (aN = 0.83 mT). O sinal do radical acetila é totalmente suprimido por sorbato, um conhecido e eficiente supressor de espécies tripletes, o que é consistente com uma rota reacional envolvendo um intermediário dioxetânico. Clivagem-&#945; da ligação carbonila-carbonila do produto biacetilo triplete produziria, de fato, radicais acetila. Além disso, utilizando AA como substrato para Mb/H2O2, um sinal de EPR atribuível ao aduto MNP-AA&#8226; (aN = 1.46 mT e aH = 0.34 mT) foi observado e confirmado por efeito isotópico. O consumo de oxigênio e o rendimento de compostos &#945;-dicarbonílicos foram dose-dependentes à concentração de AA ou MAA (1-50 mM) bem como à concentração de H2O2 adicionado às misturas de reação contendo Mb (até 1:10 quando medido o consumo de oxigênio, e até 1:25 quando medido o rendimento de compostos &#945;-dicarbonílicos) e tert-butilhidroperóxido (até 1:200). Os perfis de pH (5,8-7,8) para consumo de oxigênio e rendimento de compostos &#945;-dicarbonílicos mostraram maiores rendimentos para baixos valores de pH, indicativo de ferrilMb formada no ciclo peroxidático da proteína. Avaliando os níveis de lesão de Mb, os &#946;-cetoácidos diminuíram o nível de desorganização protéica na estrutura secundária e terciária elicitada por H2O2. Ainda, houve maior preservação da estrutura primária da proteína, sendo que MAA protegeu mais em comparação a AA, embora quando utilizado este último composto, foi mostrado que há acetilação dose-dependente de Mb. Acetoacetato aumentou a velocidade de descoramento da hemeproteína, provavelmente por ataque de espécies tripletes geradas no sistema. Músculos de rato, plantar e sóleo, expostos ex vivo a concentrações citotóxicas de glicose oxidase (GOX, gera H2O2 em fluxo), foram protegidas pelos ésteres etílicos AAE e MAAE. Foi detectado biacetilo no meio intracelular em músculos expostos a MAAE e GOX. A concentração deste composto &#945;-dicarbonílico é claramente relacionada à abundância de Mb em cada um dos tipos de músculos estudados. Em resumo, Mb tratada com metabólitos &#946;-cetoácidos (AA e MAA) gera radicais centrados em carbono e produtos &#945;-dicarbonílicos altamente reativos no estado triplete. Experimentos realizados com tecido muscular ex vivo sugerem que esta reação possivelmente ocorra in vivo. Levantamos a hipótese de que a geração de espécies carbonílicas reativas e seus adutos em condições de desbalanço metabólico possam contribuir para a compreensão das bases moleculares de desordens cetogênicas. / Acetoacetate (AA) and 2-methylacetoacetate (MAA) are &#946;-ketoacids accumulated in several metabolic disorders such as diabetes and isoleucinemia, respectively. Here we examine the mechanism of AA and MAA aerobic oxidation initiated by the reactive enzyme intermediates formed by the reaction of muscle horse myoglobin (Mb) with H2O2. A chemiluminescent route involving a dioxetane intermediate whose thermolysis yields triplet &#945;-dicarbonyl species (methylglyoxal and diacetyl) is envisaged. Accordingly, the ultraweak light emission that accompanies the reaction increases linearly by raising the concentration of both Mb (10-500 &#181;M) and AA (10- 100 mM). Oxygen uptake studies revealed that MAA is, expectedly, almost one order of magnitude more reactive than AA. EPR spin-trapping studies with MNP detected spin adducts from MAA attributable to an &#945;-carbon-centered radical (aN = 1.55 mT) and to an acetyl radical (aN = 0.83 mT). As the acetyl radical signal is totally suppressed by sorbate, a well-known efficient triplet species quencher, the dioxetane hypothesis seems to be reliable. The &#945;-cleavage of the carbonyl-carbonyl bond of a putative excited triplet diacetyl product would, in fact, leads to an acetyl radical. Furthermore, using AA as substrate for Mb/H2O2, an EPR signal assignable to a MNP-AA&#8226; adduct (aN = 1.46 mT and aH = 0.34 mT) was observed and confirmed by isotope effect. Oxygen consumption and &#945;-dicarbonyl yield were also dependent on AA or MAA concentrations (1-50 mM) as well as on the concentration of peroxide added to the Mb-containing reaction mixtures: H2O2 (up to 1:10 when measuring oxygen uptake and up to 1:25 when measuring the &#945;-dicarbonyl yield) and t-butOOH (up to 1:200). The pH profiles (5.8-7.8) of oxygen consumption and &#945;-dicarbonyl yield show higher reaction rates at lower pHs, indicative of a ferrylMb intermediate. Evaluating Mb lesion, both &#946;-ketoacids reduced disorganization of the secondary and tertiary protein structure elicited by H2O2. Therefore, Mb primary structure was more preserved, and MAA was more protective than AA. Moreover using the later compound, it was shown that Mb acetylation is dose-dependent. Acetoacetate increased the rate of the hemeprotein bleaching, probably due to the attack of triplet products generated in the system. Plantaris and soleous rat muscles exposed to damaging concentrations of glucose oxidase (GOX, generates H2O2 in flux), was cytoprotected by AAE and MAAE. Intracellular diacetyl was detected in muscle samples exposed to MAAE and GOX. The &#945;-dicarbonyl concentration is clearly related to the Mb abundance in the muscle types. In summary, Mb treated with peroxides reacts with &#946;-ketoacid metabolites (AA and MAA), yielding carbon-centered radicals and highly reactive &#945;-dicarbonyl products in the triplet state. Experiments carried out ex vivo with muscle tissue showed that this reaction possibly occurs in vivo. A new route for generation and accumulation of carbonyl reactive species and adducts is here proposed to occur in unbalanced metabolic situations, such as is the case of ketogenic disorders.

This article focuses on a systems biology approach to studying the pathophysiology of cigarette smoking during pregnancy. Particular emphasis is given to the damaging role of oxidative stress. Cigarette smoking exerts multiple adverse affects but abundant evidence, mostly in adults, suggests that oxidative stress and free radical damage is a major pathophysiological factor. Smoking during pregnancy is known to contribute to numerous poor birth outcomes, such as low birth weight, preterm birth as well as life-long health and developmental problems. It is clinically important to know the separate contributions that cigarette derived-nicotine and smoking-induced free oxidative stress make to these poor outcomes. Surprisingly, the extent to which smoking dependent oxidative stress contributes to these poor outcomes is not well studied but the application of redox proteomics should be useful. Considerable biochemical evidence suggests that antioxidants, such as tocopherols and ascorbate, could be useful in minimizing oxidative stress induced pathology to the developing fetus in those women who, despite medical advice, continue to smoke. Nevertheless, this suggestion has yet to be tested in well-designed clinical studies.

O presente estudo teve como objetivo avaliar os compostos carbonílicos na atmosfera da cidade de São Paulo. Para tal medidas de carbonílicos foram feitas empregando metodologia analítica já existente. A influência do ozônio na geração de artefatos durante a amostragem de compostos carbonílicos empregando sorventes sólidos, como C18 e sílica, observada em amostragens diurnas e noturnas ( ausência de O3) permitiu estabelecer um protocolo de amostragem que recomenda a utilização de um coletor de O3 na entrada do cartucho de sílica impregnado com 2,4-dinitro-fenil-hidrazina (DNPH) para evitar possíveis artefatos de amostragem. Os compostos carbonílicos presentes na fase gasosa da atmosfera da cidade de São Paulo foram medidos durante o inverno de 1999, em dois sítios distintos, Cidade Universitária (CID) e Água Funda (AF). Semelhante a outras regiões urbanas do mundo, os carbonílicos majoritários foram formaldeído (0,6-46,7 ppbv) e acetaldeído (1,2-75,3 ppbv). Os carbonílicos de peso molecular maior (>C2) tiveram abundância relativa de 20-40%, sendo acetona (0,3-13,0 ppbv), acroleína (0,1-5,2 ppbv), propionaldeído (0,5-8,5 ppbv), isômeros C4 (0,1-2,4 ppbv), benzaldeído (0,1-5,4 ppbv), valeraldeído (0,1-3,5 ppbv), m-tolualdeído (0,1-2,4 ppbv) e hexaldeído (0,1-4,5 ppbv). As razões de concentração para acetaldeído/formaldeído > l em julho sugerem que a contribuição das emissões diretas é predominante. Já no mês de agosto, foi observada uma diminuição das razões médias, 1,1-1,3 (manhã), 0,7-0,8 (meio-dia) e 0,6 (tarde), indicando que há contribuição de emissões diretas na manhã e nos outros períodos prevalece a formação fotoquímica in situ. Neste trabalho, a influência de parâmetros meteorológicos nos níveis dos compostos carbonílicos foi verificada em alguns eventos. Foram propostas fontes de emissão através de correlações com CO e O3. As correlações no sítio CID em agosto mostraram que a contribuição das emissões veiculares é muito importante pela manhã, mas a influência dos processos fotoquímicos foi observada nos períodos seguintes. Finalmente, considera-se que este trabalho contribuiu para uma compreensão atual sobre o panorama dos carbonílicos na cidade e para o estudo em grande escala que permitirá diagnosticar e prever os episódios de poluição na cidade de São Paulo. / The goals of this study were to evaluate the carbonylic compounds in the atmosphere of São Paulo City. To accomplish this, measures were carried out using the pre-existent analytical methodology. The ozone influence in artifacts generation during carbonyls sampling using solid sorbents such as C18 and silica, observed through day and night samples, allowed to establish a sampling protocol which recommends the utilization of an ozone scrubber at the upstream end of the DNPH-impregnated silica cartridge to avoid eventual sarnpling artifacts. Carbonyl compounds present in the gaseous phase of São Paulo atmosphere were measured during winter, 1999, at two different sites, Cidade Universitária (CID) and Água Funda (AF). As observed in other urban regions of the world, the major carbonyls were formaldehyde (0,6-46, 7 ppbv) e acetaldehyde (1,2-75,3 ppbv). Toe higher molecular weight carbonyls (>C2) showed relative abundances of 20-40%, being acetone (0,3-13,0 ppbv), acrolein (0,1-5,2 ppbv), propionaldehyde (0,5-8,5 ppbv), isomers C4 (0,1-2,4 ppbv), benzaldehyde (0,1-5,4 ppbv), valeraldehyde (0,1-3,5 ppbv), m-tolualdehyde (0,1-2,4 ppbv) and hexaldehyde (0,1-4,5 ppbv). The acetaldehyde/formaldehyde (A/F) ratio >1 in July suggests that direct emissions influence is predominant. In August, a decrease in average concentration was observed, 1,1-1,3 (morning), 0,7-0,8 (midday) and 0,6 (evening), indicating the direct emissions contribution in the morning, while for the other periods the in situ photochemical formation prevails. At the present work, the influence of meteorological parameters in carbonyls levels was also pointed out for some events. The emission sources are proposed by calculating the correlations between carbonyls and CO and O3. The correlations found for the CID site in August showed that the vehicular emissions are very important in the morning, but the influence of photochemical processes was observed in the subsequent periods. Finally, this work contributed to a current comprehension of the carbonyls panorama in the city and for the large scale study that will allow the diagnosis and mimic of the urban chemical environment in São Paulo City.

Nous avons mis en place un dispositif permettant l’acquisition d'échos de photons stimulés infrarouges à l’échelle femtoseconde. Le but est d'examiner la dynamique vibrationnelle aux temps courts de métaux carbonyles (W(CO)₆ and Fe(CO)₅) piégés en matrice cryogénique (4-50 K). Cet environnement solide, issu de la condensation d'un mélange gazeux contenant une impureté et un gaz inerte (N₂, CH₄, Ar, etc.), est propice à l’étude de systèmes dans leur état fondamental. L’excitation d’une vibration moléculaire s’atténue toujours temporellement, ce qui correspond dans le domaine spectral à un élargissement des raies d’absorption. L’étude de la dynamique vibrationnelle vise à examiner les causes physiques à l’origine de cet élargissement spectral. Typiquement, elles sont de trois sortes : phénomènes intramoléculaires, interactions entre molécules piégées et interactions entre la molécule piégée et l’environnement. Les échos de photons permettent de distinguer les contributions homogènes et inhomogènes de l’élargissement spectral et de caractériser les processus de déphasage, de relaxation des populations et de diffusion spectrale. Parmi les résultats obtenus, nous avons mis en évidence l'influence des phonons spécifiques aux matrices moléculaires (ex : libration de N₂ et rotation de CH₄ ) sur le déphasage vibrationnel ainsi que l’influence de la transition de phase du méthane solide à 20 K sur la dynamique vibrationnelle. Nous avons également montré que la dynamique vibrationnelle était dépendante du site cristallographique dans lequel est piégée la molécule. Enfin, en excitant plusieurs modes de vibration simultanément, nous avons pu examiner les couplages intramoléculaires. / We built an experimental set-up in order to generate infrared stimulated photon echoes at the femtosecond timescale. The purpose is to examine the short time vibrational dynamics of metal carbonyls (W(CO)₆ and Fe(CO)₅) trapped in cryogenic matrices (4-50 K). This environment, resulting from the condensation of a gas mixture containing the impurity and an inert gas (N₂, CH₄, Ar, etc.), is well suited to study systems in their ground state. An excited molecular vibration is always damped in the time domain. It corresponds in the frequency domain to a broadening of the absorption line. The study of the vibrational dynamics aims at examining the physical causes of this spectral broadening. Typically, there are three kinds of causes: intramolecular phenomena, interactions between trapped molecules and interactions between the impurity and the environment. Photon echoes allow distinguishing between the homogeneous and the inhomogeneous contributions of the spectral broadening and characterizing dephasing process, population relaxation and spectral diffusion. Among the obtained results, we highlighted the influence of phonons that are specific to molecular matrices (ex: N₂ libration and CH₄ rotation) on the vibrational dephasing. Moreover, we observed the influence of the phase transition of solid methane at 20 K on the vibrational dynamics. We also showed that the vibrational dynamics depends on the site in which the molecule is trapped. Finally, when exciting several vibrational modes, we are also able to study intramolecular couplings.