Neste trabalho avaliamos a formação de oxigênio singlete in vitro em fagócitos, (células mononucleares e neutrófilos) isolados de sangue periférico humano, e eosinófilos, de lavado bronco alveolar de camundongos balb/c, ativados por estímulo partículado: zimosan opsonizado contendo o 9,10difenilantraceno (DPA) adsorvido como sonda captadora de 1O2. Por este método, a formação do 1O2 pode ser verificada pela formação do 9,10-difenilantraceno endoperóxido (DPAO2), que é detectado por HPLC. Observamos, que os fagócitos formam 1O2 e que esta formação parece ocorrer de forma diferenciada para os dois tipos celulares (neutrófilos e células mononucleares). Visando ampliar os estudos anteriores sobre o papel da melatonina (MLT) no processo inflamatório, foi testado seu efeito em fagócitos e a relação na produção de 1O2 destas células. Observamos que MLT inibe a formação de 1O2 totalmente no caso de neutrófilos e parcialmente no caso de células mononucleares e eosinófilos. Paralelamente, foi desenvolvida a síntese de um novo captador químico de 1O2, o éster 9,10-antracenil-3-bispropionato de etila (ABPE), cuja finalidade principal é o acúmulo no interior da célula, depois de sofrer hidrólise enzimática. Esta sonda, terá facil acesso ao interior das células em sua forma ester. Este novo captador de 1O2 foi testado em células mononucleares e neutrófilos estimulados de formas diferentes: via receptor independente e dependente. Os resultados demonstraram produção equivalente de 1O2 nestes fagócitos. / In this study, we evaluated the singlet oxygen (1O2) formation in vitro from phagocytes (neutrophils and mononuclear cells) isolated from human blood cells and eosinophils isolated from bronchoalveolar lavage fluid of mice balb/c activated, by opsonized zymosan. To determine whether singlet oxygen is produced by phagocytes, zymosan particles were coated with a specific chemical trap for 1O2, 9,10-diphenylanthracene (DPA). The production of 1O2 was followed using HPLC, to measure its product, 9,10-diphenylanthracene endoperoxide (DPAO2). We also noticed that the 1O2 production occurs at different levels of for two cell types, neutrophils and mononuclear cells. In order to broaden previous studies on the role of melatonin (MLT) in inflammatory processes, its effect was tested in phagocytes was tested in relation to 1O2 formation by these cells. We observed that MLT inhibits the 1O2 formation totallymt neutrophils and partiallym mononuclear cells and eosinophils. At the some time, it was also developed the synthesis of a new probe for 1O2, the 9,10-anthracene-bis-3-ethyl-propionate (ABEP), with the purpose to accumulate inside the cells, after its enzymatic hydrolysis. This probe presents easy acess to the inferior of the cells in its ester form. This new probe for trapping 1O2 was tested in mononuclear cells and neutrophils stimulated in two ways: via independent and dependent receptor. The results showed equivalent production of 1O2 for both cell types.

Estudos envolvendo o oxigênio molecular singlete (1O2) tem uma relevância biológica, uma vez que esta espécie, devido ao caráter eletrofílico, reage com moléculas ricas em elétrons como proteínas, lipídeos e DNA provocando danos que resultam em perdas de função e integridade celular. Em sistemas biológicos, a presença de outras espécies reativas de oxigênio e nitrogênio, dificultam a identificação de lesões específicas causadas por 1O2 .Neste contexto, este trabalho foi desenvolvido objetivando a síntese de endoperóxidos isotopicamente marcados com 17O para serem utilizados como fonte geradora limpa de 17[1O2] em estudos mecanísticos. A capacidade de geração de 17[1O2] pelo endoperóxido N,N\'-di(2,3-dihidroxipropil)-3,3\'-(1,4 naftilideno) dipropanamida 17O (DHPN17O2) foi confirmada utilizando o captador químico sulfato mono-{2-[10-(2-sulfoxi-etil)-antracen-9-il]-etil}éster de sódio e o nucleosídeo 2\'- desoxiguanosina. Os produtos isotopicamente marcados com 17O formados foram analisados por espectrometria de ressonância magnética nuclear e cromatografia líquida de alta eficiência acoplado ao espectrômetro de massa. Os lipídeos, em especial o colesterol ao reagir com o 1O2 geram hidroperóxidos de colesterol como produtos de oxidação primária e na presença de metais resulta em compostos de maior reatividade e toxicidade, como os radicais peroxila, que contribuem para a propagação da peroxidação lipídica. Neste trabalho, demonstramos que os hidroperóxidos de colesterol são capazes de gerar 1O2 na presença de metal através de medidas de luminescência, utilização de supressores e captador químico de 1O2. Os mecanismos de reação envolvidos foram estudados e determinados por espectrometria de massa acoplada a cromatografia líquida de alta eficiência. Por fim, a caracterização detalhada dos produtos formados por espectrometria de ressonância magnética nuclear e massa na reação do colesterol com 1O2 mostrou que além dos hidroperóxidos a reação também produz um aldeído, o 3&#946; -hidroxi-5&#946;-hidroxi-B-norcolestano-6&#946;-carboxialdeído. Até o momento, este composto havia sido identificado como um produto específico da ozonização do colesterol. Neste estudo, baseado nos estudos por reações de quimiluminescência, é proposto o mecanismo de formação deste aldeído em reações de oxidação de colesterol por 1O2 envolvendo intermediário dioxetano. / Studies involving singlet molecular oxygen (1O2) has biological relevance, once this species, due to its eletrophylic character, reacts with rich electron molecules such as proteins, lipids and DNA causing damages that result in loss of function and cellular integrity. In biological system, the presence of other reactive species of oxygen and nitrogen impair the identification of lesions caused by 1O2. In this context, this work was developed with the aim of synthesizing <SUP17O-labeleded endoperoxides to be used as a clean source of (1O2) in mechanistic studies. The ability of 17[1O2]generation by N,N\'-di(2,4-dihydroxypropyl)-1,4-naphthalene-dipropanamide labeled with 17O(DHNP17O2) was observed using the disodium salt of anthracene-9,10-diyldiethyl disulfate as a chemical trap and the nucleoside 2\'-deoxyguanosine. The products isotopically labeled with 17O were analyzed by nuclear magnetic resonance spectroscopy and high performance liquid chromatography coupled to a mass spectrometer. Lipds, in special the cholesterol, when reacting with singlet molecular oxygen generate cholesterol hydroperoxides as primary products and in the presence of metals result in compounds of higher reactivity and toxicity, such as peroxyl radicals which contribute to the propagation of lipid peroxidation. In this work, we demonstrated that cholesterol hydroperoxides are able to generate singlet molecular oxygen in the presence of metal by chemiluminescence measurements by testing the effect of singlet molecular oxygen quencher and by chemical trap. The involved reaction mechanisms were studied and determined by mass spectrometry coupled to the high performance liquid chromatography. Finally, we detailed characterization of the products formed in the reaction of cholesterol with 1O2 by nuclear magnetic resonance and mass spectroscopy showed that besides cholesterol hydroperoxides, the reaction also produces an aldehyde, 3&#946;hydroxy-5&#946;-hydroxy-B-norcholestan-6&#946;-carboxyaldehyde which had been identified as a specific product of cholesterol ozonization. In this study, based on the studies of chemiluminescence reactions, the mechanism of formation of this aldehyde in reaction of oxidation of cholesterol by 1O2 involving a dioxetane intermediate has been proposed.

Les protéines à radical S-adénosyl-L-méthionine (SAM) utilisent un centre [Fe4S4] réduit pour initier le clivage réductive homolytique de la SAM et la formation d'une espèce hautement réactive - le radical 5'-déoxyadénosyl ou 5'-dA•. Dans la quasi-totalité de cas ce radical alkyl va arracher un atome d'hydrogène sur le substrat et déclencher ainsi sa conversion en produit. On trouve ces enzymes au niveau d'étapes clé de la synthèse de certaines vitamines, antibiotiques, précurseurs de l'ADN ou encore cofacteurs protéiques où elles sont souvent impliquées dans le clivage ou la formation des liaisons C-C, C-N, C-S ou encore C-P. Les travaux réalisés au cours de cette thèse ont été focalisés sur l'étude structurale et fonctionnelle de la protéine HydE ; une enzyme à radical SAM, qui intervient dans la biosynthèse du site actif organométallique de l'hydrogénase à [FeFe]. L'objectif principal était d'identifier le substrat de HydE et d'étudier les détails du fonctionnement d'une protéine à radical SAM. Nous avons réussi à identifier un groupe de molécules, dérivées de la cystéine, contentant un cycle thiazolidine avec un ou deux groupements carboxylates, qui ont une très bonne affinité pour le site actif de HydE. Certains de ces ligands se sont montrés d’être des substrats non physiologiques de l’enzyme. Grâce à ces substrats nous avons pu mettre en évidence un nouveau mécanisme d’attaque radicalaire dans les protéines à radical SAM. En effet, dans HydE nous avons observé une attaque directe du radical 5'-dA• sur l’atome soufre du thioéther appartenant au cycle thiazolidine. Cette réaction constitue un exemple pas comme les autres d’une insertion d’un atome de soufre (ou de sélénium) catalysée par une enzyme à radical SAM. Il s'agit également d'une première observation d'une réaction radicalaire dans les cristaux protéiques d'une enzyme à radical SAM et également un premier suivi en temps réel par la RMN du 13C et 1H de l'accumulation d'un des produits de la réaction catalysée par ces enzymes. Les résultats de calculs théoriques basés sur nos structures cristallographiques de haute résolution suggèrent que dans le cas de cette superfamille de protéines le radical 5'-dA• serait plutôt un état de transition et donc pas une espèce intermédiaire isolable. / Radical S-adenosyl-L-methionine (SAM) proteins use a reduced [Fe4S4] cluster to initiate homolytic reductive cleavage of SAM, which leads to the formation of highly reactive 5'-deoxyadenosyl radical species or 5'-dA•. In almost all cases this alkyl radical will abstract a hydrogen atom from the substrate and thus trigger its conversion into product. These enzymes are found in key steps of the synthesis of certain vitamins, antibiotics, DNA precursors or protein cofactors. They are often involved in the cleavage or formation of C-C, C-N, C-S or C-P bonds. The present thesis work has been focused on the structural and functional study of HydE protein; a radical SAM enzyme, involved in the biosynthesis of the organometallic active site of [FeFe]-hydrogenase. The main goal was to identify the substrate of HydE and to study details of how radical SAM proteins control the highly oxidizing 5'-dA• species. We managed to identify a group of molecules, derived from cysteine, containing a thiazolidine ring with one or two carboxylate groups, which have a very good affinity for the active site of HydE. We have demonstrated some of these ligands are non-physiological substrates of the enzyme. With these substrates we could highlight a new radical attack mechanism in radical SAM proteins. Indeed, in HydE we observed a direct attack on the 5'-dA • radical on the sulfur atom of the thioether belonging to the thiazolidine ring. This is an unprecedented reaction that contrasts with sulfur (or selenium) atom insertion reactions catalysed by some radical SAM enzymes. This is also the first observation of a radical reaction in the protein crystal of a radical SAM enzyme and also the first real-time monitoring by 1H- & 13C-NMR spectroscopy of the accumulation of products of the reaction catalysed by these enzymes. Theoretical calculations based on our high-resolution crystal structures suggest that in the case of this protein superfamily the 5'-dA• radical, which triggers the reaction in radical SAM enzymes, is a transition state and therefore not an isolable intermediate species.

Estudos envolvendo o oxigênio molecular singlete (1O2) tem uma relevância biológica, uma vez que esta espécie, devido ao caráter eletrofílico, reage com moléculas ricas em elétrons como proteínas, lipídeos e DNA provocando danos que resultam em perdas de função e integridade celular. Em sistemas biológicos, a presença de outras espécies reativas de oxigênio e nitrogênio, dificultam a identificação de lesões específicas causadas por 1O2 .Neste contexto, este trabalho foi desenvolvido objetivando a síntese de endoperóxidos isotopicamente marcados com 17O para serem utilizados como fonte geradora limpa de 17[1O2] em estudos mecanísticos. A capacidade de geração de 17[1O2] pelo endoperóxido N,N\'-di(2,3-dihidroxipropil)-3,3\'-(1,4 naftilideno) dipropanamida 17O (DHPN17O2) foi confirmada utilizando o captador químico sulfato mono-{2-[10-(2-sulfoxi-etil)-antracen-9-il]-etil}éster de sódio e o nucleosídeo 2\'- desoxiguanosina. Os produtos isotopicamente marcados com 17O formados foram analisados por espectrometria de ressonância magnética nuclear e cromatografia líquida de alta eficiência acoplado ao espectrômetro de massa. Os lipídeos, em especial o colesterol ao reagir com o 1O2 geram hidroperóxidos de colesterol como produtos de oxidação primária e na presença de metais resulta em compostos de maior reatividade e toxicidade, como os radicais peroxila, que contribuem para a propagação da peroxidação lipídica. Neste trabalho, demonstramos que os hidroperóxidos de colesterol são capazes de gerar 1O2 na presença de metal através de medidas de luminescência, utilização de supressores e captador químico de 1O2. Os mecanismos de reação envolvidos foram estudados e determinados por espectrometria de massa acoplada a cromatografia líquida de alta eficiência. Por fim, a caracterização detalhada dos produtos formados por espectrometria de ressonância magnética nuclear e massa na reação do colesterol com 1O2 mostrou que além dos hidroperóxidos a reação também produz um aldeído, o 3&#946; -hidroxi-5&#946;-hidroxi-B-norcolestano-6&#946;-carboxialdeído. Até o momento, este composto havia sido identificado como um produto específico da ozonização do colesterol. Neste estudo, baseado nos estudos por reações de quimiluminescência, é proposto o mecanismo de formação deste aldeído em reações de oxidação de colesterol por 1O2 envolvendo intermediário dioxetano. / Studies involving singlet molecular oxygen (1O2) has biological relevance, once this species, due to its eletrophylic character, reacts with rich electron molecules such as proteins, lipids and DNA causing damages that result in loss of function and cellular integrity. In biological system, the presence of other reactive species of oxygen and nitrogen impair the identification of lesions caused by 1O2. In this context, this work was developed with the aim of synthesizing <SUP17O-labeleded endoperoxides to be used as a clean source of (1O2) in mechanistic studies. The ability of 17[1O2]generation by N,N\'-di(2,4-dihydroxypropyl)-1,4-naphthalene-dipropanamide labeled with 17O(DHNP17O2) was observed using the disodium salt of anthracene-9,10-diyldiethyl disulfate as a chemical trap and the nucleoside 2\'-deoxyguanosine. The products isotopically labeled with 17O were analyzed by nuclear magnetic resonance spectroscopy and high performance liquid chromatography coupled to a mass spectrometer. Lipds, in special the cholesterol, when reacting with singlet molecular oxygen generate cholesterol hydroperoxides as primary products and in the presence of metals result in compounds of higher reactivity and toxicity, such as peroxyl radicals which contribute to the propagation of lipid peroxidation. In this work, we demonstrated that cholesterol hydroperoxides are able to generate singlet molecular oxygen in the presence of metal by chemiluminescence measurements by testing the effect of singlet molecular oxygen quencher and by chemical trap. The involved reaction mechanisms were studied and determined by mass spectrometry coupled to the high performance liquid chromatography. Finally, we detailed characterization of the products formed in the reaction of cholesterol with 1O2 by nuclear magnetic resonance and mass spectroscopy showed that besides cholesterol hydroperoxides, the reaction also produces an aldehyde, 3&#946;hydroxy-5&#946;-hydroxy-B-norcholestan-6&#946;-carboxyaldehyde which had been identified as a specific product of cholesterol ozonization. In this study, based on the studies of chemiluminescence reactions, the mechanism of formation of this aldehyde in reaction of oxidation of cholesterol by 1O2 involving a dioxetane intermediate has been proposed.

Dans ce travail de thèse, nous avons étudié l'effet des rayonnements ionisants (rayons X et γ) jusqu'à une dose maximale de 1 Grad sur différents types de fibres multimodes (dopées -P, -P-Ce, -Ge, -Ge-F, -Ge-Ce et -N). Les caractérisations ont été réalisées principalement avec trois techniques expérimentales : online Atténuation Induite par Radiation en temps réel (RIA), Résonance Paramagnétique Electronique (EPR), Micro-Luminescence (ML). Dans la première partie du travail de cette thèse, nous avons étudié la réponse aux radiations de différents types de fibres optiques. L'absorption liée aux défauts du phosphore induits par irradiation a été étudiée par des mesures RIA dans le domaine spectral UV-Visible. Les mesures EPR nous permis de détecter les défauts POHC, P1 et P2. En particulier, pour la détection de P1 et P2, nous avons utilisé le mode de détection de la seconde harmonique pour déterminer la cinétique de croissance des P1 et P2 en fonction de la dose. Nous avons également étudié les effets dus au changement des conditions de fibrage et ceux liés à la variation de la température d'irradiation (25-280 ° C). Nous avons aussi étudié l’effet du codopage du coeur de la fibre avec du Cérium. Dans ce cas, nous avons observé une production moins importante de centres POHC et P2 sous irradiation. De plus, les mesures EPR ont montré que la génération des défauts P1 n’est pas sensiblement influencée par le codopage avec le Cérium. En ce qui concerne les fibres optiques dopées Ge, on a étudié trois types de dopage : Ge seul, co-dopage Ge-F et Ge-Ce. Pour chaque type, nous avons examiné trois conditions de fibrage. La réponse à l’irradiation de ces fibres a été étudiée par les trois techniques utilisées. Plus particulièrement la ML, nous a permis d'obtenir une vision plus complète du rôle du codopant et des précurseurs dans la formation des défauts induits par l'irradiation. Nous avons également étudié la réponse au rayonnement de la fibre dopée N avec trois différentes conditions de fibrage. Les réponses à l’irradiation dans les régions spectrales UV-visible ont été obtenues par des mesures RIA. Par EPR, nous avons pu détecter deux défauts liés à l'azote pour les doses élevées de radiation. Enfin, les mesures ML sur les fibres irradiées ont montré trois bandes d'émission dans le visible qui ont été attribuées clairement à des centres émetteurs liés à l'azote. Dans la deuxième partie de la thèse, nous avons étudié les effets liés au chargement en oxygène des fibres étudiées. Par des mesures en microspectroscopie Raman, nous démontrons qu'un traitement à haute température et haute pression peut favoriser l’introduction d’une grande quantité de O2 dans les fibres optiques à cœur de silice pure (PSC) ou dopées F, Ge ou P. Les réponses à l’irradiation de certaines des fibres optiques chargées en O2 ont été étudiés (et en particulier PSC et celle dopée F. Sur la base des données de la littérature, nous avons effectué les décompositions des spectres RIA en fonction de la dose. De plus, l'étude EPR des fibres optiques dopées P et chargées en O2 a montré une forte réduction des défauts P1 et P2 comparées aux fibres non traitées. Dans cette partie de la thèse, j’ai également présenté les résultats concernant la radioluminescence infrarouge (1272 nm) des molécules O2 dans la fibre optique. La faisabilité d'un capteur de radiation pour des environnements sous fortes doses et forts débits de dose a été discutée / In this Thesis work we have investigated the effect of ionizing irradiation (X and γ rays) up to 1 Grad on different types of multimode optical fibers (P-doped, P-Ce-doped , Ge-doped, Ge-F-doped, Ge-Ce-doped, and N-doped). The experiments were carried out by three main experimental techniques: online Radiation Induced Attenuation (RIA), Electron Paramagnetic Resonance (EPR) and Confocal Micro-Luminescence (CML). In the first part of the Thesis work we report on the radiation response of several types of optical fibers. The absorption due to radiation induced P-related defects was studied by RIA in the UV-Visible domain. Moreover, by EPR measurements we were able to detect POHC, P1 and P2 defects. In particular, for the detection of P1 and P2 defects we have validated the use of EPR second-harmonic detection mode which allowed us to obtain the growth kinetics of P1 and P2 with the dose. The effects due to the variation of the drawing conditions of the fibers were investigated as well as the ones due to the change of the temperature of irradiation (from 25 to 280 °C). Finally, concerning the P-doped OFs, we report on the effects due to the Cerium codoping of the core of the optical fiber. We observed a reduced generation of POHC and P2 centers under irradiation. However, EPR investigation has shown that the generation of P1 defects is essentially unaffected by the Ce-codoping. Regarding Ge-doped optical fibers we report on three basic typologies: Ge-doped, Ge-F-doped and Ge-Ce-doped. For each fiber typology we investigated three drawing conditions. The radiation responses of these fibers were characterized by RIA and EPR measurements. Furthermore, performing CML measurements we were able to obtain further insight on the role of the co-dopants and of the defect precursors in determining the radiation induced defects. We have also investigated the radiation response of N-doped OFs (three drawing conditions). The radiation responses in the UV-Visible domains were obtained by RIA, and by EPR measurements we were able to detect the signals of two N-related defects at high radiation doses. Finally, CML measurements on irradiated samples have shown three emission bands in the visible domain which are tentatively assigned to N-related centers. In the second part of the Thesis we report on the effects of an O2 loading treatment produces on some of the investigated samples. By micro-Raman measurements we demonstrate that a high pressure high temperature treatment can incorporate high quantity of O2 into Pure-Silica-Core (PSC), F, Ge and P doped optical fibers. The radiation responses of some of the O2-loaded optical fibers were investigated with particular regard to the fluorine doped and pure-silica-core optical fibers. On the basis of literature data we performed band decompositions of the RIA spectra as a function of the dose. Moreover, the EPR study of the O2 loaded P-doped optical fiber have shown a strong reduction of the signals associated to the P1 and P2 defects as compared to the untreated fibers. In this part of the thesis we also report on the characterization of the near infrared radioluminescence (1272 nm) of O2 molecules embedded in the optical fiber matrix and the feasibility of a radiation sensor based on this phenomenon for environments characterized by high radiation doses and high dose-rates

This diploma thesis is focused on the charge exchange processes between projectile and target studied by the Low Energy Ion Scattering (LEIS) technique. Basic premise to investigate charge exchange processes is correct cleaning processes and proper settings of experimental instrument Qtac 100 placed in the Central European Institute of Technology (CEITEC) in Brno. Ion fraction expresses neutralization rate of the projectile. The parametr is investigated for clean and oxidized polycrystalline copper. Oxygen presence performs a significant part of reionization of backscattered neutralized projectiles.

No description available.

L’objectif de ce travail de thèse était la préparation et la caractérisation des hétéropolyanions anhydres sur supports oxydes par la stratégie de chimie organométallique de surface. Les acides anhydres H3PMo12O40 et H3PW12O40 ont été préparés sur silice partiellement déshydroxylée. Cette réaction conduit à une interaction ionique par protonation des silanols de surface. La réactivité de ces hétéropolyacides anhydres en présence d’alkylsilanes a été étudiée en milieu homogène et a conduit à la formation d’espèces silylées cationiques [Et2MeSi+]3[HPA3-] et au dégagement d’hydrogène. Cette réactivité a ensuite été appliquée en milieu hétérogène en fonctionnalisant la surface de la silice par des groupements [(≡SiO)SiMe2H] et a conduit à la formation d’une espèce de surface polyoxometalate liée de manière covalente au support. L’introduction de fonction chloroalkylsilane à la surface de la silice [(≡SiO)SiMeCl2] et [(≡SiO)2SiMeCl] a également permis de former des liaisons covalentes Si Support-O-M HPA. Par ailleurs, Par ailleurs, l’activation du méthane a été observée sur tous les solides HPA/SiO2 à travers le dégagement de CO2, H2O, H2. L’activation C-H a lieu sur ces systèmes même à basse température et les données obtenues suggèrent la formation d’une espèce méthoxy de surface par réaction des protons acides avec le méthane / The aim of this work was the preparation and characterization of anhydrous heteropolyanions on oxide supports using surface organometallic chemistry approach. Anhydrous H3PMo12O40 and H3PW12O40 were prepared on partially dehydroxylated silica. This reaction led to an ionic interaction by protonation of surface silanols. The reactivity of these heteropoly compounds with alkylsilanes was studied in homogeneous conditions and led to the formation of cationic silicon species [Et2MeSi+]3[HPA3-] and release of hydrogen. This reactivity was then applied in heterogeneous conditions by introduction of silane groups [(≡SiO)SiMe2H] at the silica surface and led to the formation of a surface polyoxometalate species covalently bonded to the support. The introduction of chloroalkylsilane groups [(≡SiO)SiMeCl2] and [(≡SiO)2SiMeCl] has also enabled the formation of covalent bonds Si Support-O-M HPA. In addition, methane activation was observed on all HPA/SiO2 solids through the releases of CO2, H2O, H2. The C-H activation takes place on these systems even at low temperature and obtained data suggest the formation of a methoxy surface species by reaction of stronf acidic protons with methane