O oxigênio molecular singlete (1O2) é formado em sistemas biológicos e reage com diferentes biomoléculas. Proteínas representam um dos principais alvos de oxidação, devido as suas altas concentrações em organismos. Em pH fisiológico 1O2 reage com His, Tyr, Met, Cys e Trp. Neste trabalho investigamos a oxidação causada pelo 1O2 e a formação de dimerização em uma proteína modelo, a lisozima. A identificação dos principais produtos de oxidação e dimerização foi realizada por sequenciamento de peptídeos através de nano cromatografia acoplada a espectrometria de massas (nLC-MS/MS). A geração de 1O2 foi realizada por fotossensibilização utilizando luz e rosa bengala como fotossensibilizador, e pela decomposição térmica de endoperóxidos derivado do naftaleno DHPN16O2 e DHPN18O2, uma fonte limpa de 1O2 no meio reacional. Os resultados demonstraram que a reação do oxigênio singlete com lisozima acarreta oxidação dos resíduos de Met, His e Trp. A caracterização da estrutura primária por nLC-MS/MS dos aminoácidos confirmou a adição de átomos de oxigênio marcado (18O). A lisozima é constituída apenas de um resíduo de histidina (His15) e as oxidações identificadas foram adições de massas de +14 Da (descrita como 2-oxo-histidina), +16 e +32 Da. Os resíduos de metionina (Met12 e Met105) foram identificados como sulfóxidos (MetSO - adição de massas de +16 Da). Para os resíduos de triptofano foram identificados a formação de quinurenina (adição de massas de +4 Da), +16 e +32 Da. As oxidações levaram a formação de dimerização na proteína caracterizada por eletroforese em gel e nLC-MS/MS. O objetivo principal do trabalho foi analisar a ligação cruzada entre o resíduo de histidina 2-oxohistidina na lisozima. Entretanto, foram identificadas ligações cruzadas entre 2- oxo-histidina e resíduos de lisina, além de ligações cruzadas com resíduos de triptofano oxidado. Em consequência dos resultados obtidos com a proteína modelo, avaliamos as oxidações e formação de dímeros em proteínas extraídas do cristalino do olho bovino. Diferentes tipos de modificações foram observados, além da formação de dímeros entre resíduos de histidina (2-oxoHis-His) caracterizados por nLC-MS/MS e bioinformática. Os dados obtidos neste trabalho, fornecem evidências da ocorrência simultânea de formação de ligações cruzadas entre diferentes proteicas após exposição a 1O2. O trabalho resultou na identificação e sequenciamento através de nLC-MS/MS de peptídeos oxidados por 1O2 a partir de uma proteína modelo. Esses resultados reiteram o importante papel do 1O2 em reações com proteínas além do seu envolvimento no desenvolvimento de condições patológicas. A dimerização formada na ligação cruzada em 2-oxo-His-His representa um possível novo biomarcador para o 1O2 em sistemas biológicos / Singlet molecular oxygen (1O2) can be generated in biological systems, reacting with different biomolecules. Proteins are major target for oxidants due to higher concentration in organisms. At physiological pH, 1O2 may react with the following aminoacids: His, Tyr, Met, Cys and Trp. Here, we investigated oxidation and dimerization reactions of proteins exposed to 1O2 using lysozyme as a model. Modifications of lysozyme by 1O2 were investigated using mass spectrometry approaches. Identification of the main oxidation and dimerization products were performed by peptide sequencing by nano-chromatography coupled to mass spectrometry (nLC-MS/MS). Singlet oxygen was generated using visible light and rose Bengal as photosensitizer, and from the decomposition of thermolabile endoperoxides DHPN16O2 e DHPN18O2, clean sources of 1O2. Experimental findings showed oxidation of Met, His, and Trp residuesin lysozyme. Structural characterization by nLC-MS/MS of the oxidative modifications in lysozyme tryptic peptides showed the addition of [18O]-labeled atoms in different amino acid residues. Lysozyme has in its structure a single histidine residue (His15). We identified shifts of +14 Da (described as oxohistidine), +16 and +32 Da in this residue. Methionine residues (Met12 and Met105) were oxidized to sulfoxides (MetSO mass shift of +16 Da). Modifications in tryptophan residues were identified as kynurenine (shift mass of +4 Da), +16 and + 32 Da. Oxidized lysozyme subjected to SDS-Page showed dimmers formation. The main aim was to analyze cross-linking formation between 2-oxo-histidine residues in lysozyme. However, cross-links between 2- oxo-histidine and lysine residues, and cross-links between oxidized tryptophan residues have been identified. Following results obtained with the protein model, we evaluated oxidation and the dimers formation in proteins extracted from the lens of the bovine eye. Analysis performed in nLC-MS/MS and bioinformatics identified different types of modifications, including formation of dimers with histidine residues (2-oxo-His-His). The data provided evidence for simultaneous occurrence of protein cross-linking on exposure 1O2. These results demonstrated the important role of 1O2 in protein reactions beyond its involvement in developing of pathological conditions. In conclusion, dimerization of proteins through 2-oxo-His residues may be a possible new biomarker for 1O2 in biological systems.

Evidências apontam para o envolvimento da peroxidação lipídica em diversas patologias. Os hidroperóxidos de lipídios (LOOH) são os produtos primários da peroxidação lipídica e sua decomposição resulta em produtos de maior reatividade e toxicidade, como os radicais peroxila. Esses radicais desempenham papel importante na propagação da peroxidação lipídica e também podem gerar oxigênio molecular singlete (1O2) por meio da combinação de dois radicais peroxila. Neste trabalho investigamos a possibilidade dos LOOH, em particular dos hidroperóxidos de ácido linoléico (LAOOH), de servirem como fonte 1O2 na presença de oxidantes de relevância biológica como metais, peroxinitrito ou ácido hipocloroso. A formação de 1O2 foi claramente demonstrada na reação de LAOOH com esses oxidantes pelas detecções (i) da emissão bimolecular na região espectral do vermelho (λ>570 nm), (ii) da emissão monomolecular no infravermelho-próximo (λ=1270 nm), (iii) do espectro de emissão no infravermelho, e (iv) da intensificação e supressão da luminescência na presença de D2O e azida, respectivamente. Além disso, os mecanismos de reação foram estudados utilizando LAOOH marcados com oxigênio-18 (LA18O18OH) e captadores químicos específicos para 1O2 aliada à tecnica de detecção por HPLC acoplada à espectrometria de massa. Os resultados mostraram a formação de 1O2 marcado [18(1O2) ] na reação de LA18O18OH com os três oxidantes, revelando que os átomos de oxigênio do 1O2 são derivados do hidroperóxido. Em conjunto, as evidências obtidas levam à conclusão de que os LOOH podem servir como fontes potenciais de 1O2 em sistemas biológicos em situações onde haja a coexistência de LOOH e metais, peroxinitrito ou ácido hipocloroso. / Evidences point to the involvement of lipid peroxidation in several diseases. Lipid hydroperoxides (LOOH) are the primary products of lipid peroxidation and their decomposition generates more reactive and toxic compounds, such as peroxyl radicals. These radicals play an important role in the propagation of lipid peroxidation and may also generate singlet molecular oxygen (1O2) by the combination of two peroxyl radicals. In this study we have investigated the possibility of LOOH, in particular linoleic acid hydroperoxide (LAOOH), to be a source of 1O2 in the presence of biologically relevant oxidants such as, metal ions, peroxynitrite or hypochlorous acid. The formation of 1O2 was clearly demonstrated in the reaction of LAOOH with all the three tested oxidants by detecting: (i) the dimol light emission in the red spectral region (λ>570 nm), (ii) the monomol light emission in the near-infrared region (λ=1270 nm), (iii) the infrared light emission spectrum, and (iv) the enhancing effect of deuterium oxide and the quenching effect of azide on light emission. Furthermore, the mechanism was studied using LAOOH labeled with 18-oxygen isotope (LA18O18OH) and specific 1O2 chemical traps in combination with HPLC coupled to mass spectrometry detection. The results have showed the formation of 18-oxygen labeled 1O2 [18(1O2) ] in the reaction of LA18O18OH with the three oxidants, indicating that oxygen atoms in 1O2 are derived from the hydroperoxide. Altogether, the obtained evidences lead to the conclusion that LOOH may serve as a potential source of 1O2 in biological systems, in situations where LOOH can interact with metals, peroxynitrite or hypochlorous acid.

Les composés deutérés sont d’un intérêt grandissant dans des domaines variés. Par exemple, en pharmacologie, l'échange H/D peut améliorer les propriétés pharmacocinétiques de certains médicaments ou réduire leur toxicité. Les composés deuterés peuvent également être utilisés comme étalons internes en spectroscopie de masse. Il est donc important de trouver un moyen simple et sélectif d'échanger l'hydrogène avec le deutérium sur des molécules d’intérêt biologique. Les nanoparticules, de ruthénium en particulier, se sont révélées être des systèmes efficaces pour catalyser cet échange. Cependant, étant très actives, elles conduisent souvent à la réduction de substrats aromatiques. L'objectif principal du doctorat est l’élaboration de nouvelles nanoparticules permettant de contrôler la réactivité en échange isotopique H/D. Nous avons d’abord synthétisé des alliages Ru-Pt afin d’introduire du platine, moins actif en échange H/D, et donc empoisonner la surface du Ru. Nous avons montré qu’en changeant le précurseur de platine, on pouvait changer la distribution atomique de surface, et ainsi moduler la réactivité des nanoparticules. Nous avons finalement synthétisé des nanoparticules de nickel et d'iridium. Ces nanoparticules se sont avérées être des catalyseurs efficaces pour l'échange H/D, sans réduction de fonctions aromatiques. De plus, des sélectivités différentes ont été obtenues en fonction du métal utilisé. / Deuterated compounds are molecules of great interest in various fields. In pharmacology, the H/D exchange can improve the pharmacokinetic properties of some drugs or reduce their toxicity. In addition, deuterium-labelled compounds can be used as internal standards for mass spectroscopy, or as tracers for the understanding of different reaction mechanisms. Therefore, it is important to find a way to exchange hydrogen with deuterium in a simple, selective and efficient way. The main goal of the PhD project is the synthesis of novel nanoparticles for isotopic exchange (H/D). Metal nanoparticles and more particularly ruthenium nanoparticles, has shown their efficiency to catalyze this exchange. However, ruthenium nanoparticles are very active in arene hydrogenation, and often lead to the reduction of aromatic substrates. In a second part of the work, we synthesized Ru-Pt alloys in an attempt to passivate the Ru surface with platinum, which is less-active in H/D exchange. We have shown that by changing the platinum precursor, we can change the atomic distribution of the surface, and thus we were able to modulate the reactivity of nanoparticles. We finally synthesized Ni and Ir nanoparticles. These nanoparticles have proven to be efficient catalysts for H/D exchange, without reducing aromatic functions. In addition, different selectivity was obtained depending on the metal used.

L’imagerie par résonance magnétique du 13C (13C-IRM) hyperpolarisée est une technique émergente pour le suivi métabolique in vivo en vue de la détection précoce de cancers. L’optimisation de la sensibilité de la mesure IRM nécessite l’injection de molécules endogènes marquées au carbone-13 et deutérium. Dans ce contexte, le sodium L-[1-13C,U-D] lactate est un agent prometteur pour le diagnostic et le suivi thérapeutique des patients atteints du cancer de la prostate. Pendant cette thèse, la synthèse de cette molécule a été réalisée à partir de la L-[1-13C] alanine. Pour réaliser cette synthèse, deux méthodes pour la deutération des positions C2 et C3 de l’alanine ont été développées. La deutération en position C2, basée sur la technique de l’échange hydrogène-deutérium metallo-catalysé, a permis de préparer différents acides-aminés marqués en cette position à l’échelle du gramme. La deutération en position C3 est réalisée via fonctionnalisation C-H pallado-catalysée. Cette technique permet d’obtenir un marquage sélectif de certains des 20 acides-aminés naturels en position C3. Un procédé pour le marquage sélectif de la position C1 de l’alanine avec du carbone-13 a été établi à partir de l’acétaldéhyde et du K13CN selon une modification de la procédure de Strecker. L’utilisation de l’acétaldéhyde perdeutéré rend ainsi possible une synthèse à large échelle du sodium L-[1-13C,U-D] lactate. / Hyperpolarized 13C magnetic resonance imaging (13C-MRI) is a promising emerging tool to follow the metabolic routes in vivo in view of early stage detection of cancer. Optimization of the sensitivity of the MRI measurement requires injection of carbon-13 or deuterium labelled endogenous molecules. In this context, sodium L-[1-13C,U-D] lactate was individuated as a promising probe for the detection and prediction of treatment-response in patients with prostate cancer. We realized the first chemical synthesis of such probe starting from L-[1-13C] alanine by developing two distinct strategies for the selective deuteration of the position C2 and C3. The method of C2 deuteration relies on the technique of the metal-catalyzed hydrogen-deuterium exchange and can be applied to the synthesis of several deuterated amino-acids to an industrial-scale. The method of C3 deuteration was realised by Pd(OAc)2 catalyzed C-H activation. It provides a unique route to obtain amino-acids selectively deuterated in position C3. We also developed a robust route for the 13C labelling of the C1 position of L-alanine from acetaldehyde and K13CN by a modification of the Strecker’s synthesis. By using deuterated acetaldehyde, a scalable synthesis of L-[1-13C,U-D] lactate could now be possible.

La majorité des composants internes de la cuve des réacteurs à eau pressurisée (REP) est fabriquée en acier inoxydable austénitique (304L ou 316L). Ces matériaux sont exposés à un milieu oxydant sous irradiation et subissent des contraintes mécaniques. Dans ces conditions, ils sont susceptibles de subir un endommagement par corrosion sous contrainte assistée par irradiation (IASCC ). La première étape des phénomènes de fissuration par IASCC est l'amorçage qui implique une rupture du film passif. La nature et la structure de l'oxyde formé sur ces aciers sont donc des paramètres clés vis-à-vis de l'amorçage de la fissuration par IASCC. Dans ce contexte, l'objectif de ce travail est d'une part de mieux appréhender les mécanismes d'oxydation des aciers inoxydables en milieu primaire et d'autre part d'étudier les effets des défauts créés par irradiation sur le film d'oxyde formé sur ces aciers. Des ions xénon ou des protons ont été implantés dans des échantillons d'acier inoxydable austénitique de type 316L, respectivement à une énergie de 240 et 230 keV, afin de simuler les défauts d'irradiation. Ces échantillons ainsi que des échantillons non implantés ont été exposés dans une boucle de corrosion, à 325°C en milieu aqueux, contenant 1000 ppm de Bore, 2 ppm de Lithium, et 1,19.10-3 mol.L-1 d'hydrogène dissous. Les échantillons ont été analysés par MET avant et après exposition en milieu primaire afin de caractériser, d'une part les défauts engendrés par l'implantation et d'autre part la nature, la structure et la morphologie de l'oxyde formé. La comparaison des échantillons implantés et non implantés a permis de montrer que la nature et la densité de défauts en sub-surface de l'alliage jouent un rôle sur la composition (principalement sur la teneur en Cr et en Mo) et l'épaisseur de la couche d'oxyde interne. L'étude de la cinétique d'oxydation par couplage de deux techniques d'analyse par faisceau d'ions (NRA et RBS) a permis de révéler des comportement différents entre les deux catégories d'échantillons : non implantés et implantés. Des essais de traçage isotopique (D et 18O) ont été menés afin d'étudier le mécanisme de formation de la couche interne ainsi que les mécanismes de transports associés. L'étude du transport de l'oxygène et de l'hydrogène à travers la couche interne et dans l'alliage sous-jacent (par SIMS et SDL) a permis d'aboutir à l'écriture d'un mécanisme de corrosion des aciers inoxydables austénitiques en milieu primaire. De plus, l'impact des défauts d'implantation sur ces phénomènes de transport a été étudié, mettant en évidence le rôle des défauts sur les propriétés de l'oxyde formé engendrant des modifications de transport de l'oxygène au sein de cette couche d'oxyde. Ces études de traçage isotopiques ont également permis de mettre en évidence une accumulation d'hydrogène dans l'alliage sous-jacent à l'oxyde. L'ensemble de ces résultats a permis d'apporter de nouveaux éléments de compréhension relatifs à la formation de la couche d'oxyde et à l'absorption d'hydrogène dans les aciers inoxydables austénitiques exposés en milieu primaire, et de mettre en avant l'effet des défauts d'implantation sur les propriétés de l'oxyde formé. / Internal parts of pressurized water reactor (PWR) vessels are often made of austenitic stainless steels (304L and 316L). These structural materials are exposed to an oxidizing medium under irradiation and mechanical stresses. Under these conditions, they can suffer damages by IASCC (Irradiation-Assisted Stress Corrosion Cracking). The first step in this cracking phenomenon is the initiation, which implies the breakdown of the passive layer. The nature and the structure of the oxide film formed on these steels are key factors in initiation of IASCC cracks. In this context, the objective of this work is first to better understand the oxidation mechanisms of stainless steels in primary medium and second to study the effects of irradiation induced defects on the oxide film formed on stainless steels in primary medium. Xenon ions and protons, were implanted in 316L-type austenitic stainless steel samples, respectively at an energy of 240 and 230 keV in order to simulate the irradiation defects. Implanted and non-implanted samples were exposed in a corrosion loop at 325°C to an aqueous medium containing 1000 ppm of boron, 2 ppm of lithium and 1,19.10-3 mol.L-1 of dissolved hydrogen. The samples were analyzed by TEM before and after exposure to primary medium in order to characterize both the defects generated by the implantation and the nature, structure, and morphology of the formed oxide. Comparing implanted and non-implanted samples has shown that the nature and the density of defects in the alloy subsurface played an important role on the composition (mainly on the content of Cr and Mo) and on the thickness of the inner layer. The study of the oxidation kinetics by coupling two ion beam analysis techniques (NRA and RBS) has revealed different behavior between the two types of samples: non-implanted and implanted. Tracer experiments (using D and 18O) were conducted to study the growth mechanism of the inner oxide layer and the associated transport mechanisms. The study of the oxygen and hydrogen transport through the inner layer and the underlying alloy, by SIMS and GD-OES, has resulted in writing a corrosion mechanism for austenitic stainless steels exposed to primary medium and linking this mechanism to hydrogen absorption in the alloy. Furthermore, the impact of implantation defects on these transport phenomena has been studied, highlighting the role of defects on oxide layer properties generating modification of the oxygen transport in the oxide scale. These results have helped to shed some light on the mechanism and kinetics involved in the formation of the oxide layer and on the hydrogen absorption in austenitic stainless steels exposed to primary medium and to point out the effect of implantation defects on the oxidation processes.

Mitochondrial isocitrate dehydrogenase 2 (IDH2) catalyzes reductive carboxylation (RC, reverse Krebs cycle pathway) and 2HG synthesis (2HG) - metabolite of which many scientists are interested. 2HG may be concurrently synthetized in cytosol by IDH1. RC is involved in anabolic reactions necessary for cell proliferation - produces citrate, fatty acid precursor - especially in hypoxia. IDH2 and IDH1 are not the only enzymes that are involved in 2HG synthesis. Recently, several enzymes, which participate in 2HG production, have been discovered. 2HG is useful in cancer diagnostics due to its overproduction by transformed cells. Moreover, 2HG may cause epigenetic changes via inhibition of 2-oxoglutarate dependent dioxygenase. In this work, the importance of RC and 2HG synthesis in cancer and healthy cells was investigated by gas chromatography with mass spectrometry detection as well as IDH2 influence. We found that IDH2 significantly participates in reverse RC and 2HG synthesis in breast cancer cell lines and uses glutaminolysis as a supplementary anaplerotic pathway. RC is increased by hypoxia, inhibition of respiration, and decreased by activation of respiration or hypocapnia. We confirmed 2HG synthesis and RC in healthy cells (fibroblasts, breast epithelial cells etc.) as well as in cancer cells....

In der vorliegenden Arbeit wurde der lichtinduzierte Reaktionszyklus des bakteriellen Phytochroms Agp2 aus Agrobacterium tumefaciens mit FTIR‑ und UV‑Vis‑Spektroskopie untersucht. Der Photorezeptor besteht aus einem photosensorischen Modul und einer signalgebenden Histidin-Kinase-Domäne. Das photosensorische Modul bindet das Tetrapyrrol Biliverdin als Chromophor. Der Grundzustand von Agp2 (Pfr, 750 nm) ist gegenüber dem lichtaktivierten Zustand (Pr, 700 nm) rotverschoben, weshalb Agp2 den Bathyphytochromen zugeordnet wird. Die Untersuchungen erfolgten unter Verwendung von Isotopenmarkierung, H/D-Austauschexperimenten und ortsspezifischer Mutagenese. Daraus ließen sich folgende molekulare Änderungen charakterisieren, welche im Reaktionszyklus von Agp2 erfolgen: Die lichtinduzierte Isomerisierung des Chromophors führt zu einem Übergang vom Pfr- in den Pr-Zustand, wobei zwei Intermediate, Lumi‑F und Meta‑F, durchlaufen werden. Neben der Konformationsänderung des Chromophor‑D‑Rings ist auch die C‑Ring-Propionsäureseitenkette an der Photoreaktion beteiligt. Die C-Ring-Propionsäureseitenkette ist im Pfr-Zustand protoniert und wird im Übergang von Meta-F zu Pr deprotoniert. Der Pr-Zustand weist eine pH-Abhängigkeit auf, welche auf die pH-abhängige Ladung des Histidins 278 der Chromophortasche zurückzuführen ist. Je nach Ladung des Histidins 278 wird die Keto‑ bzw. Enolform der C(19)=O‑Gruppe des D‑Rings stabilisiert. Die Keto/Enol-Tautomerie ist auf eine innerhalb des Chromophors erfolgende Protontranslokation zurückzuführen und moduliert die Relaxation in den Pfr-Zustand. Änderungen der Amid-I-Absorption im Pfr-Pr-Übergang werden der Umstrukturierung der Tongue-Region des photosensorischen Moduls von einer Alpha-helikalen zu einer Beta‑Faltblatt-Struktur zugeordnet. Diese Strukturänderung wird als möglicher Weg der proteininternen Signaltransduktion zwischen photosensorischem und signalgebendem Modul vorgeschlagen. / In this thesis the light-induced reaction cycle of the bacterial phytochrome Agp2 from Agrobacterium tumefaciens was investigated using FTIR and UV‑vis spectroscopy. The photoreceptor comprises a photosensitive module and a signalling histidine kinase domain. The photosensitive module binds the biliverdin tetrapyrrol as chromophore. The Agp2 ground state (Pfr, 750 nm) is red-shifted in comparison with its light-activated state (Pr, 700 nm). Therefore, Agp2 is assigned to the group of bathy phytochromes. The investigations were conducted using isotopically labelled protein, labelled chromophore as well as hydrogen‑deuterium (H‑D) exchange and site-directed mutagenesis. Based on these the following molecular changes could be characterized that occur in the reaction cycle of Agp2: The light-induced isomerization of the chromophore leads to a transition from the Pfr to the Pr state, involving two intermediates, Lumi-F and Meta-F. Besides conformational changes of the chromophore D-ring, the C-ring propionic side chain is involved in the photoreaction as well. The C-ring propionic side chain is protonated in the Pfr state and gets deprotonated in the Meta-F to Pr transition. The Pr state exhibits pH‑dependent alterations which can be explained by pH dependent polarity changes of histidine 278 in the chromophore pocket. Depending on the charge of histidine, the D‑ring C(19)=O group is stabilized either in keto or enol form. The keto/enol tautomerism involves a proton translocation within the chromophore and modulates the relaxation to the Pfr state. The changes in the amide I region in the Pfr-Pr transition are associated with an alpha‑helix to beta‑sheet secondary structure change of the PHY domain tongue‑region. This structural change is proposed as the potential path of signal transduction between the photosensitive and the signalling module.