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La capacité de mesurer les rapports isotopiques par spectroscopie RMN du 13C (irm-13C NMR) donne un accès direct à la distribution d’isotopes possédant une position spécifique au sein de molécules. Dans cette thèse, cette approche a été développée dans le but d’élucider le fractionnement isotopique au cours de la biosynthèse dans des plantes de différents alcaloïdes (nicotine, tropine, tramadol) qui ont certaines caractéristiques communes lors de leurs biosynthèses. L’une des caractéristiques clés de ces composés est la présence de groupes O-méthyle et/ou N-méthyle. En général, le rapport 13C/12C dans les groupes O-méthyle et N-méthyle de produits naturels est exceptionnellement faible comparé aux autres carbones dans la molécule, ces travaux ont été axés sur l’explication de ce phénomène. La grande majorité de ces groupes méthyles dans les produits naturels proviennent du transfert d’un groupe Sméthyle de L-méthionine (L-Met) via la Sadénosylméthionine (AdoMet). Il a été prouvé par irm-13C NMR que, dans la molécule donneuse, L-Met, le groupe Sméthyle est appauvrit. La cause de ce phénomène a été explorée par le biais de l’étude de petit modèle théorique pour la cobalamin-independent méthionine synthase, l’enzyme responsable du transfert du groupement méthyle durant la biosynthèse de L-met. Ces calculs ont montré une barrière d’énergie élevée pour le transfert du groupe méthyle et un fort effet isotopique cinétique du 13C y associé. De plus, une méthodologie générique de l’étude du ratio 13C/12C dans les aminoacides a été développée, ce qui permet une meilleure compréhension du fractionnement isotopique intervenant durant la biosynthèse d’acides aminés. Une caractéristique supplémentaire de ces travaux est que les données permettent : (i) une comparaison entre les produits naturels et commerciaux, permettant de distinguer ces deux sources, et (ii) une interprétation du modèle isotopique en terme d’origine biosynthétique de composés naturels. Pour le tramadol, il a été possible de proposer un chemin hypothétique pour ce produit naturel récemment découvert. / The ability to carry out isotope ratio monitoring by 13C NMR spectrometry (irm-13C NMR) gives direct access to position-specific isotope distributions in whole molecules. In this thesis this approach has been developed with the aim of elucidating isotopic fractionation during the biosynthetic pathways in plants of a number of alkaloids (nicotine, tropine and tramadol) that have certain features of their biosynthesis in common. One key common feature of these compounds is the presence of O-methyl and/or N–methyl groups. As it is generally found that the 13C/12C ratio in the O-methyl and N-methyl groups of natural products is exceptionally low relative to the other carbon positions in the molecule, the work focused on explaining this phenomenon. The vast majority of these methyl groups in natural products are derived by the transfer of the S-methyl group from L-methionine (L-Met) via S-adenosyl methionine (AdoMet). It is shown by irm-13C NMR that in the donor molecule, L-met, the S-methyl group is impoverished. The cause of this was investigated by the study of a small theoretical model for the cobalaminindependent methionine synthase, the enzyme responsible for methyl group transfer in L-met biosynthesis. These calculations showed a high energy barrier for methyl group transfer and an associated large 13C kinetic isotope effect. In addition, a generic methodology to study the 13C/12C ratios in amino acids has been developed, which allows insight into the isotopic fractionation occurring during amino acid biosynthesis. A further feature of the work is that the data allow: (i) a comparison of natural and commercial products, which enables distinguishing between sources, and (ii) an interpretation of the isotopic pattern in terms of the biosynthetic origin of natural compounds. For tramadol, this made it possible to propose a hypothetical pathway for this newly-discovered natural product.

Yes / The 13C NMR chemical shifts of the three Edaravone tautomers (keto, enol, and amine) were calculated using a combined molecular mechanics (Pcmod 9.1/MMFF94) and ab initio (GIAO (B3LYP/DFT, 6–31 + G(d)) model. This method gave such good agreement with experiment that the assignment of the complex spectrum of Edaravone in solution, which is a mixture of the three tautomers could be made. This has been attempted previously by various methods with diverse results. In CDCl3 solution, the observed spectra show only one form, the keto tautomer, and this is also the case with acetonitrile solvent. Acetone solvent reacts with Edavarone in the NMR tube. In the other solvents studied, methanol, pyridine, DMSO, trifluoroethanol (TFE), there is a mixture of the tautomers with populations which vary with the solvent. The application of the shift predictions allows the assignment of the 13C spectra to the three tautomers and from this the proportions of the tautomers in the solution. The results at times differ significantly from previous studies, and this is discussed.

第23回名古屋大学年代測定総合研究センターシンポジウム平成22(2010)年度報告

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Metabolic imaging with hyperpolarized 13C-labeled cell substrates is a promising technique for imaging tissue metabolism in vivo. However, the transient nature of the hyperpolarization - and its depletion following excitation - limits the imaging time and the number of excitation pulses that can be used. A single-shot 3D imaging sequence has been developed and it is shown in this thesis to generate 13C MR images in tumour-bearing mice injected with hyperpolarized [1-13C]pyruvate. The pulse sequence acquires a stack-of-spirals at two spin echoes after a single excitation pulse and encodes the kz-dimension in an interleaved manner to enhance robustness to B0 inhomogeneity. Spectral-spatial pulses are used to acquire dynamic 3D images from selected hyperpolarized 13C-labeled metabolites. A nominal spatial/temporal resolution of 1.25 x 1.25 x 2.5 $mm^3$ x 2 s was achieved in tumour images of hyperpolarized [1-13C]pyruvate and [1-13C]lactate acquired in vivo. An advanced sequence is also described in this thesis in a later study to acquire higher resolution images with isotropic voxels (1.25 x 1.25 x 1.25 $mm^3$) at no cost of temporal resolution. EPI is a sequence widely used in hyperpolarized 13C MRI because images can be acquired rapidly with limited RF exposure. However, EPI suffers from Nyquist ghosting, which is normally corrected for by acquiring a reference scan. In this thesis a workflow for hyperpolarized 13C EPI is proposed that requires no reference scan and, therefore, that does not sacrifice a time point in the dynamic monitoring of tissue metabolism. To date, most of the hyperpolarized MRI on metabolism are based on 13C imaging, while 1H is a better imaging target for its 4 times higher gyromagnetic ratio and hence 16 times signal. In this thesis the world’s first dynamic 1H imaging in vivo of hyperpolarized [1-13C]lactate is presented, via a novel double-dual-spin-echo INEPT sequence that transfers the hyperpolarization from 13C to 1H, achieving a spatial resolution of 1.25 x 1.25 $mm^2$.

Les terrils du bassin minier de Provence sont implantés dans le paysage et demeurent à proximité des habitations de la métropole Aix-Marseille. Les terrils les plus anciens ont été naturellement colonisés par la végétation et des sols s’y sont développés avec une vitesse remarquable. Cette étude vise à comprendre les processus pédogénétiques en cours depuis 55 ans sur les terrils miniers. Nous avons étudié l’impact du lignite sur les caractéristiques physico-chimiques et microbiologiques d’un Rendosol naturel. Deux sols similaires, dont l’un est traversé par l’affleurement naturel d’une veine de lignite et l’autre pas, ont donc été comparés afin de caractériser les variables impactées par le lignite. L’étude du terril Armand a permis de comprendre les facteurs responsables de la formation et de la variabilité des caractéristiques des sols observés sur le terril. L’abondance de composés carbonés récalcitrants dans les sols enrichis en lignite affecte les niveaux d’activité des microorganismes responsables des processus biologiques dans les sols sans induire d’effets néfastes. Le lignite semble intervenir comme un facteur de dilution du carbone organique, diminuant ainsi la quantité de carbone disponible et donc la vitalité d’expression des fonctions microbiennes. Les activités biologiques sont diminuées en présence de lignite, mais les changements induits sur les propriétés physico-chimiques semblent améliorer la fertilité du sol. Les sols du terril Armand demeurent cependant encore à un stade d’évolution trop jeune pour préjuger de leur évolution future. / Spoil heaps are scattered over the coal basin of Provence: they are inserted in the landscape and often located close to urban areas of the Aix-Marseille Metropole. The oldest spoil heaps have been naturally colonized by local vegetation and soils have simultaneously quickly developed. This study aims at understanding the processes involved in soil forming on undisturbed lignite-rich spoil heaps since 55 years. We studied the impact of lignite on the physico-chemical and microbiological characteristics of an undisturbed soil: we compared two similar Rendosols, except that one was developed in a natural lignite outcrop. Then we studied on the 55-year-old Armand spoil heap the factors responsible for soil genesis and variability of soil characteristics. Recalcitrant carbon compounds found in soils enriched with lignite modify microbial activity but do not induce negative effects. It seems that lignite acts as a diluting factor of the organic carbon that decreases the available carbon pool and consequently on the vitality of the expression of the microbial functions. Enzymatic activities and basal respiration decrease while changes observed on physico-chemical properties tend to improve soil fertility. Some characteristics of the soils developed on the spoil heap are similar to those of the soil developed from the lignite outcrop, while others are more related to the way the spoil heap was set up. Although these results have shed light on some of the processes involved in soil formation on spoil heaps in a carbonated environment, soils on Armand spoil heap are still at an early stage of development that precludes conclusion on their future evolution.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Little is known about the decomposition and stabilization of shoots and intact roots derived carbon (C) under no-tillage (no-till) field conditions. The present study was designed with following objectives: 1) evaluation of 13C pulse labeling method to label crop plants under field conditions for subsequent C dynamics studies, 2) estimation of the actual rates of mineralization of intact roots and shoot residues, decomposing simultaneously, and 3) finally to determine the fate of shoot vs root residues derived C in soil, under no-till field conditions. The experiment was conducted at Federal University of Santa Maria , Rio Grande de Sul, Brazil in 2013-2014 in an loam textured Typic Paleudalf. Wheat (Triticum aestivum L.), pea (Pisum sativum L.) and vetch (Vicia sativa L.) plants were grown inside polyvinyl chloride (PVC) cylinders and were pulse labeled weekly with 13C in the field until the flowering stage. At plant harvest, the biomass of shoots and roots and chemical composition and isotopic enrichment was determined. In order to achieve second objective, paired treatments were designed by combining 13C labeled shoots with unlabeled roots+soil and unlabeled shoots with 13C labeled roots+soil for each crop, plus a control treatment. For the third objective, soil cylinders were excavated after 60, 180 and 365 days for the determination of distribution of shoot and root derived Cnew in soil aggregates. The chemical composition of plant tissues (shoot and roots) were not modified by 13C labeling. The maximum level of 13C enrichment in plants at harvest, was +495 in wheat, +426 in pea and +378 in vetch plants. Our results though demonstrated heterogeneity of 13C among plant parts particularly between stems and leaves however, it was far less than reported in other field and controlled conditions experiments. The mineralization of roots+soil C was higher than shoot-C residues for the three species (73 vs. 45 % initial C for wheat, 76 vs. 48 % for pea and 73 vs. 51 % for vetch). Remaining 13C in root and soil organic matter (SOM) at day 180 indicated both a higher rate of root-C decomposition and a higher rate of root- derived C in SOM compared to shoots. Greater proportion of the shoot and root derived Cnew of three crops was associated with large macroaggregates (>2000 μm) in 0-5 cm soil layer which declined with time. The content of root and shoot derived C microaggregates (53 250 μm) increased gradually with time in all the three soil for all crops. After 365 days, 30% of the root derived C was present in soil compared to 5 % (average of three crops) of the shoot derived C. The mean relative contribution of root vs shoot derived Cnew was 2.1 ranging from 1.5 (pea) to 2.5 (wheat). Our findings suggest that, crop residues location and contact with soil and, the soil moisture and temperature, are important factors that significantly promoted roots decomposition and root derived C in soil in situ, compared to shoots, erasing the consequences of their different initial chemical composition. The 13CO2 labeling technique used under the field appeared to be a practical approach with respect to resource demand and is suitable for in situ labeling. This work provides a framework for further studies focusing on the interactions between aboveground and belowground crop residues and environmental factors under no-till field conditions. / Pouco é conhecimento sobre a decomposição e a estabilização no solo do carbono (C) proveniente da parte aérea e de raízes in situ em sistema de plantio direto (SPD). O presente estudo foi desenvolvido com os seguintes objetivos: 1) avaliar o método de marcagem de plantas com 13C através da aplicação de pulsos de 13C no enriquecimento e distribuição do 13C nas plantas cultivadas em condições de campo para posterior estudo da dinâmica de C; 2) estimar as taxas reais de mineralização do C de raízes in situ e da parte aérea na superfície do solo; e 3) determinar o destino do C da parte aérea e de raízes no solo, em condições de campo em SPD. O experimento foi conduzido na Universidade Federal de Santa Maria, Rio Grande do Sul, Brasil, em 2013-2014 em um Argissolo vermelho distrófico arênico. Plantas de Trigo (Triticum aestivum L.), ervilha (Pisum sativum L.) e ervilhaca (Vicia sativa L.) foram cultivadas em condições de campo dentro de cilindros de PVC, nas quais foram aplicados semanalmente pulsos de 13C até o estágio de floração. A biomassa da parte aérea e das raízes, a composição química e o enriquecimento isotópico foram determinados no momento da colheita das plantas. A fim de alcançar o segundo objetivo, para cada cultura foi montado tratamentos pareados combinando a parte aérea marcada com 13C com as raízes sem marcação e a parte aérea não marcada com 13C com as raízes marcadas. Além desses foi utilizado um tratamento controle. Para o terceiro objetivo, os cilindros com solo foram escavados depois de 60, 180 e 365 dias após a instalação dos tratamentos pareados e realizada a determinação da distribuição nos agregados do Cnovo derivado da parte aérea e das raízes. A composição química das plantas (parte aérea e raízes) não foi modificada pela marcagem com 13C. O nível máximo de enriquecimento de 13C nas plantas, no momento da colheita, foi de 495 no trigo, 426 na ervilha e 378 na ervilhaca. Os resultados demonstraram a heterogeneidade na distribuição do 13C entre as partes da planta, particularmente entre caules e folhas, no entanto, esse nível de heterogeneidade é inferior aos reportados na literatura para experimentos de campo e em condições controladas. A mineralização de C das raízes das três espécies foi maior do que aquela observada para os resíduos da parte aérea (73 x 45 % no trigo, 76 x 48 % na ervilha e 73 x 51 % na ervilhaca). O 13C remanescente nas raízes e na matéria orgânica do solo (MOS) aos 180 dias indicou elevada decomposição das raízes e alta taxa de C derivado das raízes na MOS comparado à parte aérea. Maior proporção do Cnovo derivado da parte aérea e das raízes das três culturas foi associada aos macroagregados (>2000 μm) na camada de 0-5 cm, a qual diminuiu com o passar do tempo. O C derivado das raízes e da parte aérea nos microagregados (53 250 μm) aumentou gradualmente com o passar do tempo em todas as camadas do solo para todas as culturas. Aos 365 dias, 30% do C das raízes estava presente no solo, comparado aos 5% (média das três culturas) do C da parte aérea. A contribuição relativa média do Cnovo derivado da raiz x parte aérea foi de 2,1 variando de 1,5 (ervilha) a 2,5 (trigo). Os resultados do presente trabalho sugerem que a localização dos resíduos culturais, o contato com o solo e a umidade e a temperatura do solo, são fatores importantes que promovem maior decomposição das raízes in situ e Cnovo das raízes no solo, comparado com a parte aérea, reduzindo o efeito das diferenças na composição química inicial. A técnica de enriquecimento das plantas através da aplicação de pulsos de 13C em condições de campo parece ser viável em relação à demanda de recursos é adequada para a marcagem in situ. Este trabalho fornece informações de suporte para estudos futuros, com enfoque nas interações entre os resíduos culturais da parte aérea e raízes e os fatores ambientais em condições de campo em SPD.

Cette thèse aborde un point clé du couplage entre ces cycles: la dynamique des molécules azotées (AAs) des matières organiques du sol (MOS). Par des expériences d'incubation, nous avons estimé que les flux de biosynthèse des AAs par les micro-organismes du sol lors du processus de décomposition sont de l'ordre de 25% de la biomasse nouvellement formée. Le profil des AAs individuels biosynthétisés de novo est plus dépendant du type de sol que de la nature du substrat. Dans chaque sol, il est très similaire à celui des AAs des MOS. La biodégradation de matériaux végétaux marqués en 13C a révélé la transformation rapide des protéines végétales en matériaux microbiens. Ces résultats montrent que les AAs des MOS sont d'origine microbienne. Nous avons mesuré le renouvellement du C des AAs à long terme dans les horizons de surface de neuf sites présentant des végétations, climats et types de sol variés, en utilisant la technique de traçage par les abondances naturelles en 13C. L'âge moyen du carbone des AAs varie de 50 à 200 ans. Un modèle simple permet de discuter les hypothèses du recyclage des AAs des MOS par les micro-organismes. Les rapports isotopiques stables des AAs individuels ont été mesurés par chromatographie en phase gazeuse couplée à la spectrométrie de masse isotopique. À cette fin, nous avons développé une méthode d'étalonnage générique pour la détermination du rapport isotopique des composés spécifiques, par analyse de cultures microbiennes uniformément marquées. Au-delà des résultats présentés, l'étude apporte un large ensemble de données des AAs et examine les variations de l'abondance naturelle en 13C entre les AAs individuels. / We analyzed the coupled dynamics of C and N in Soil Organic Matter (SOM) through the dynamics of N-containing soil organic compounds (amino acids (AAs)) by tracing their carbon atoms. Stable isotope ratios of individual amino acids were measured by gas chromatography coupled with isotope ratio mass spectrometry. For this purpose, we developed a generic calibration method for compound-specific stable isotope ratio analysis, based on the analysis of uniformly labelled microbial cultures. We quantified the biosynthesis of AAs associated with the biodegradation process in four contrasted topsoils through short-term incubation experiments of 13C-labelled substrates. Amino acids-C accounts for ca. 25% of the newly-formed microbial biomass-C. The composition of the de novo biosynthesized individual amino acids was dependent on the soil type, and in each soil was similar to that of SOM amino acids. Biodegradation of 13C-labelled plant materials revealed the rapid conversion of plant proteins into microbial materials. These results together demonstrate that SOM amino acids are of microbial origin. We measured the dynamics of amino acids-C on the long term (decades to centuries) in nine sites using the natural 13C-labelling technique. On average, the age of AAs was equal or slightly inferior to that of bulk soil organic carbon, with mean ages ranging from 50 to 200 years. We built a conceptual model of AAs dynamics to discuss various hypotheses of AAs stabilization. Beyond these perspectives on C and N coupling in soil processes, the overall study brings a broad dataset of amino acids, as well as discuses variations of 13C natural abundance (δ13C) in-between individual amino acids.

In this study of energy pathways in karst groundwater the first chapter examines spatial and temporal patterns of bacterial density and activity in the Dorvan-Cleyzieu karst aquifer, France. During baseflow, bacterial density and activity in the water column was similar in upper and lower zones of the aquifer. Floods apparently scoured inactive bacteria from the aquifer matrix but had little effect on respiring cells. Dissolved organic carbon was more abundant at the base of the aquifer, probably because of patchy distribution of particulate organic matter in upper aquifer zones that leached dissolved organic carbon. The temporal sequence of flooding and drying in the aquifer appears to play an important role in the maintenance of biofilms which should be important energy sources to higher trophic levels in the aquifer. The ecosystem expansion and contraction model, originally developed to describe surface streams, may be a good descriptor of spatial and temporal patterns of microbial films in karst aquifers. The process of leaf and wood breakdown in cave streams in Organ Cave, West Virginia is examined in Chapter 2. Leaf and wood breakdown rates and microbial biomass and respiration on leaves and wood were compared between cave streams with and without coarse particulate organic matter (CPOM) input from the surface to examine the role of CPOM input in leaf and wood breakdown. Breakdown rate and pattern of microbial colonization of leaves and wood were typical of results reported for surface streams. Unlike in surface streams, CPOM input did not influence breakdown rate or microbial colonization on leaves and wood, apparently because nutrients are not limiting in cave streams. Nutrient addition had little effect on microbial films on wood in either stream type. Gammarus minus is an important shredder in Organ Cave streams and G. minus colonization accelerated leaf breakdown rates. Leaf and wood transport rates were low and, when combined with breakdown rates, suggest that CPOM will be retained and transformed to fine particles near its entry point to the subsurface. In chapter 3 I examine cave stream food web structure and the role various organic matter sources in stream trophic dynamics. I used stable isotope (13C and 15N) natural abundance analysis and a 13C-acetate tracer release to establish feeding relationships and to trace the use and importance of bacterial carbon in cave streams with and without CPOM input. Cave streams contained three trophic levels consisting of organic matter sources, primary consumers, and predators. Patterns of 13C labeling in the stream were similar to that in similar studies of surface streams. 13C acetate was incorporated into epilithic biofilms and fine benthic organic matter (FBOM). Some primary consumers, Fontigens tartarea, Gyraulus parvus, and Physa were highly labeled and showed a longitudinal labeling pattern consistent with the consumption of epilithic biofilms. An epigean caddisfly, Dolophilodes, was highly labeled and probably feeds on suspended organic matter. Other primary consumers, Gammarus minus and Caecidotea holsingeri, feed on FBOM and epilithon. Two amphipods, Stygobromus emarginatus and S. spinatus, and a planarian, Macrocotyla hoffmasteri, are predators in the streams. Leaves and wood were not major energy sources directly used by stream animals. Dissolved organic matter (DOM) originating from soils appears to be the primary energy source for stream food webs by fueling bacterial production that is then used by higher trophic levels. Because epilithon C turnover times were relatively long (12.7 - 17 days), DOM can be immobilized in cave stream biofilms, enhancing the efficiency with which the microbial loop may transfer energy to higher trophic levels. / Ph. D.