Heidelberg, Univ., Diss., 2000.

University, Diss., 2005--Heidelberg.

Universiẗat, Diss.--Köln, 2006.

No description available.

Chemical species carry information in two dimensions, in their concentrations and their isotopic signatures. The concentrations of metabolites or synthetic compounds describe the composition of a chemical or biological system, while isotopic signatures describe processes in the system by their reaction pathways, regulation, and responses to external stimuli. Stable isotopes are unique tracers of these processes because their natural abundances are modulated by isotope effects occurring in physical processes as well as in chemical reactions. Nuclear magnetic resonance (NMR) spectroscopy is a prime technique not only for identification and quantification of small molecules in complex systems but also for measuring intramolecular distribution of stable isotopes in metabolites and other small molecules. In this thesis, we use quantitative NMR in three fields: in food science, environmental pollutant tracing, and plant-climate science. The phospholipid (PL) composition of food samples is of high interest because of their nutritional value and technological properties. However, the analysis of PLs is difficult as they constitute only a small fraction of the total lipid contents in foods. Here, we developed a method to identify PLs and determine their composition in food samples, by combining a liquid-liquid extraction approach for enriching PLs, with specialized 31P,1H-COSY NMR experiments to identify and quantify PLs. Wide-spread pollution with synthetic compounds threatens the environment and human health. However, the fate of pollutants in the environment is often poorly understood. Using quantitative deuterium NMR spectroscopy, we showed for the nitrosamine NDMA and the pesticide DDT how intramolecular distributions (isotopomer patterns) of the heavy hydrogen isotope deuterium reveal mechanistic insight into transformation pathways of pollutants and organic compounds in general. Intramolecular isotope distributions can be used to trace a pollutant’s origin, to understand its environmental transformation pathways and to evaluate remediation approaches. The atmospheric CO2 concentration ([CO2]) is currently rising at an unprecedented rate and plant responses to this increase in [CO2] influence the global carbon cycle and will determine future plant productivity. To investigate long-term plant responses, we developed a method to elucidate metabolic fluxes from intramolecular deuterium distributions of metabolites that can be extracted from historic plant material. We show that the intramolecular deuterium distribution of plant glucose depends on growth [CO2] and reflects the magnitude of photorespiration, an important side reaction of photosynthesis. In historic plant samples, we observe that photorespiration decreased in annual crop plants and natural vegetation over the past century, with no observable acclimation, implying that photosynthesis increased. In tree-ring samples from all continents covering the past 60 – 700 years, we detected a significantly smaller decrease in photorespiration than expected. We conclude that the expected “CO2 fertilization” has occurred but was significantly less pronounced in trees, due to opposing effects. The presented applications show that intramolecular isotope distributions not only provide information about the origin and turnover of compounds but also about metabolic regulation. By extracting isotope distributions from archives of plant material, metabolic information can be obtained retrospectively, which allows studies over decades to millennia, timescales that are inaccessible with manipulation experiments.

Ce travail décrit l’analyse de la distribution des flux de carbones au sein de deux souches de Streptomyces coelicolor A3(2) : la souche sauvage nommée M145 et son mutant M1146 incapable de produire les antibiotiques actinorhodine, undecylprodigiosine, et l’antibiotique dépendant du calcium. Metabolite Balance Analysis et Isotopomer Balance Analysis sont mis en œuvre pour proposer un modèle de distribution des flux de carbones de S. coelicolor en phase exponentielle de croissance. Les souches M145 et M1146 sont cultivées dans un milieu minimum limitant en azote et leurs comportements métaboliques sont comparés. Dans la souche non productrice M1146, un taux de croissance plus élévé, un flux plus important dans la voie des pentoses phosphates, un flux plus faible au niveau du cycle de Krebs ainsi qu’une activité respiratoire plus faible sont mis en évidence. Cela traduit le coût énergétique important associé à la production d’actinorhodine par M145. De plus, ce travail propose un rôle important de la nicotinamide nucléotide transhydrogénase pour le maintien de l’homéostasie du NADPH lors de la production d’actinorhodine par M145. Comme il existe de bonnes corrélations entre les données expérimentales et celles issues de la modélisation au niveau des bilans carbones, des bilans de pouvoir réducteur et des échanges gazeux, il sera intéressant d’utiliser cette modélisation avec la technique de Flux Balance Analysis pour prédire les variations de la distribution des flux de carbones dans des mutants de S. coelicolor pour lesquels des gènes auraient été sur-exprimés ou délétés. / This work describes an analysis of carbon flux distribution in two strains of Streptomyces coelicolor A3(2), namely the wild type strain M145 and its derivative M1146 that is no longer able to produce the antibiotics actinorhodin, undecylprodigiosin and the calcium dependent antibiotic. Metabolite Balance Analysis and Isotopomer Balance Analysis were used to propose a model for carbon flux distribution in S. coelicolor during the exponential phase of growth. Strains M145 and M1146 were grown under nitrogen limitation in minimal medium and their metabolic behaviour were compared. In the non-producing strain M1146, a higher growth rate, a higher flux via the pentose phosphate pathway, a decreased flux through the TCA cycle and a decreased respiratory activity were evidenced. This highlighted the high energetic cost for actinorhodin production in M145. In this paper, we also propose a key role for the nicotinamide nucleotide transhydrogenase in NADPH homeostasis in M145 during actinorhodin production. As there are good correlations between experimental data and the model in terms of carbon balance, reducing power balance and gas exchanges, this model will be of great interest for Flux Balance Analysis to predict carbon-flux distribution changes in S. coelicolor strains in which gene are deleted or overexpressed.

Climate is changing and it is certain that this change is due to human activities. Atmospheric greenhouse gases have been rising in an unprecedented way during the last two centuries, although the land biosphere has dampened their increase by absorbing CO2 emitted by anthropogenic activities. However, it is unclear if this will continue in the future. This uncertainty makes it difficult to predict future climate changes and to determine how much greenhouse gas emissions must be reduced to protect climate. To understand the future role of plants in limiting the atmospheric CO2 level, the effect of increasing CO2 on plant photosynthesis and productivity has been studied. However, studies on trees showed contradictory results, which depended on the duration of the experiment. This revealed that an initial strong CO2 fertilization may be a transient response that disappears after a few years. Because climate changes over centuries, we must explore the response of vegetation to increasing CO2 on this time scale. Studying tree rings is a good alternative to impractical decade-long experiments, because trees have experienced the CO2 increase during the last 200 years and may already have responded to it. This thesis shows that the intramolecular distribution of the stable hydrogen isotope deuterium (deuterium isotopomer distribution, DID) of tree rings is a reliable tool to study long-term plant-climate adaptations. The premise for this is that the deuterium abundance in tree rings depends on environmental as well as physiological factors. Using newly developed methodology for DID measurements, the influences of both factors can be separated. Applied to tree rings, separating both factors opens a strategy for simultaneous reconstruction of climate and of physiological responses. The results presented show that DIDs are influenced by kinetic isotope effects of enzymes, allowing studies of metabolic regulation. We show that the abundances of specific D isotopomers in tree-ring cellulose indeed allow identifying environmental and physiological factors. For example, the D2 isotopomer is mostly influenced by environment, its abundance should allow better reconstruction of past temperature. On the other hand, the abundance ratio of two isotopomers (D6R and D6S) depends on atmospheric CO2, and might serve as a measure of the efficiency of photosynthesis (ratio of photorespiration to assimilation). The presence of this dependence in all species tested and in tree-ring cellulose allows studying adaptations of plants to increasing CO2 on long time scales, using tree-ring series or other remnant plant material. / Klimatet förändras och det är numera allmänt vedertaget att detta beror på människans aktiviteter. Halten av växthusgaser har stigit onormalt mycket under de senaste två århundradena och detta beror i största del på människans användning av fossila bränslen. Landbiosfären har hittills haft en buffrande effekt på klimatförändringen eftersom den tar upp och lagrar mycket av växthusgasen CO2. Det är dock osäkert om, och i så fall hur länge, denna effekt kvarstår. Detta gör det mycket svårt att förutsäga framtida klimatförändringar, och därmed hur mycket utsläppen av växthusgaser måste reduceras för att skydda klimatet. För att förstå växternas framtida förmåga att begränsa halten atmosfäriskt CO2 har man studerat effekten av förhöjda halter av CO2 på växters fotosyntes och produktivitet. Resultaten av dessa försök varierar i stor omfattning. Studier på träd odlade under höga halter CO2 indikerar att den initiala ökningen av en trädets produktivitet kan vara en temporär effekt som försvinner redan efter några år. Eftersom klimatförändringen sker under århundraden, måste även växternas anpassningar på förhöjd CO2 halt utforskas på denna tidsskala, men experiment som skulle ta tiotals år är opraktiska att utföra. Trädringar är ett bra sätt att studera sådana anpassningar, eftersom träd redan har upplevt de senaste två hundra årens ökning av koldioxid och dess trädringar därför kan innehålla information om en respons som redan skett. Denna avhandling visar att den intramolekylära fördelningen av den stabila väteisotopen deuterium i trädringar är en tillförlitlig metod för att studera växters anpassningsförmåga till långsiktiga klimatförändringar. Antagandet bakom denna strategi är att isotopfördelningen i trädringar beror på faktorer både från miljön och växtens fysiologi. Om båda faktorerna skulle kunna utvinnas från trädringar, skulle detta öppna en helt ny väg för parallell rekonstruktion av klimatet och växters anpassning till det. Avhandlingen presenterar den första tekniken för att mäta isotopfördelningen av deuterium i växtglukos. Resultaten visar att deuteriumfördelningen hos växtglukos påverkas av enzymers isotopeffekter, vilket möjliggör att regleringen av växternas metabolism kan kartläggas. I avhandlingen bevisas att halten deuterium i skilda intramolekylära positioner (isotopomerer) av glukos från trädringcellulosa bestäms av miljöfaktorer respektive trädets fysiologi. T.ex. påverkas deuteriumhalten i position 2 (D2 isotopomer) av glukosmolekylen huvudsakligen av miljön, vilket kan användas för att förbättra temperaturrekonstruktioner från trädringar. Å andra sidan är kvoten deuterium mellan två andra positioner (D6R och D6S) relaterat till halten atmosfäriskt koldioxid, och kvoten skulle kunna användas som mått för fotosyntesens effektivitet, dvs. förhållandet mellan fotorespiration och fotosyntes. Närvaron av denna relation i trädringar och annat växtmaterial i alla de växter vi hittills studerat, öppnar en helt ny möjlighet att studera växters anpassning till den ökande mängden CO2 i atmosfären under århundraden.

No description available.

Lipid metabolism is critically important to the normal function of individual cells and for signaling between tissues of the human body. There is a great need to quantify changes in lipid metabolism because of its implications in both normal healthy conditions and during disease development. In our first study, we developed a liquid chromatography mass spectrometry based workflow to assess in vivo metabolism of murine brain lipids. This involved sample preparation, data acquisition, and analysis software development and improvements. Regarding sample preparation, we maintained the mice at 5% body water for the duration of the experiment. As mouse metabolism proceeds, enzymes can add deuterium atoms (D) from D2O into lipid C-H bonds. These newly synthesized D-labeled lipids display shifts in their isotopic envelopes. To observe these shifts, we used mass spectrometry acquisitions to measure the mass spectra of isotopic envelopes. We calculated changes in these isotopic envelope shifts to deduce several metabolic metrics. We used these metabolic metrics to make inferences about metabolism changes. These metrics include n-value, fraction new, rate, and asymptote for each lipid. We deduced n-value by replacing one hydrogen with a deuterium at a time in the lipid's theoretical chemical formula. The number of deuterium atoms in the theoretical D-labeled chemical formula that agrees best with its respective empirical spectrum is the n-value. A large part of this effort was assessing the reproducibility and quality control of n-values that were derived from empirical spectra. We compared these n-values to two sets of ground truth n-values that we generated. We generated one set of ground truth n-values by referencing biochemical pathways and published n-values. We used a linear algebra approach to deduce the other set of ground truth n-values. We compared both sets of ground truth n-values to n-values derived from empirical D-labeled lipid spectra. We found that both sets of ground truth n-values correlate well with n-values from empirical spectra. Using these n-values, we calculated fraction new for each lipid. This fraction new indicates what percentage of a lipid's pool is newly synthesized at a given time. For a given lipid, we calculated the fraction new for each time point and biological replicate. We plotted these fraction values together against time in days. From this fraction new vs time plot for each lipid, we deduced its asymptote and rate constant. In our second study, we added the additional dimension of drift time to the data acquisition and analysis using ion mobility spectrometry. We added this additional dimension so that we could further separate lipid isomers and prevent spectral convolution. Preliminary results suggest that lipid isomers may have distinct metabolic regulation.

In der vorliegenden Diplomarbeit wurden sekundäre Isotopeneffekte auf NMR-chemische Verschiebungen mit ab-initio quantenchemischen Methoden bestimmt. Um die zu erwartenden Fehler verschiedener Methoden abschätzen zu können, wurden die sekundären Isotopeneffekte von Fluor(2-2H1)ethan auf HF, MP2 und CCSD(T)-Niveau mit tripel- und quadrupel-Zeta Basissätzen berechnet. Anhand der Ergebnisse dieser Rechnungen lässt sich zeigen, dass gauche und antiperiplanare Konformere bereits mithilfe von sekundären Isotopenverschiebungen unterschieden werden können, die auf HF/tz2p-Niveau berechnet wurden. Weiterhin wurde untersucht, ob es eine Abhängingkeit des Isotopeneffektes von den Diederwinkeln zwischen Deuterium- und Fluorsubstituenten gibt. Die sekundären Isotopeneffekte auf 19F-NMR-chemische Verschiebungen von exo- und endo-2-Fluornorbornanen mit Deuterium Substituenten in den endo-3, exo-3, syn-7 oder anti-7 Positionen wurden auf HF/tz2p-Niveau berechnet. Es wird gezeigt, dass die berechneten Isotopeneffekte an 2-Fluornorbornanen eine Identifizierung der verschiedenen Stereoisomere erlauben. / In the present Diploma thesis secondary isotope effects on NMR chemical shieldings were determined using ab-initio quantum chemical methods. In order to estimate errors of various methods, secondary isotope effects on fluoro(2-2H1)ethane were calculated at the HF, MP2 and CCSD(T) level of theory using triple- and quadruple zeta basis sets. On the basis of these calculations it can be shown that gauche and antiperiplanar conformers can already be distinguished by their secondary isotope shifts calculated at the HF/tz2p level of theory. Furthermore it was investigated, whether a dependency of the isotope effects on dihedral angles between the deuterium and the fluorine substituent exists. The secondary isotope effects on 19F chemical shifts of exo- and endo-2-fluoronorbornanes with deuterium subsituents in the endo-3, exo-3, syn-7 or anti-7 positions were calculated at the HF/tz2p level. It is shown that the calculated isotope effects of 2-fluoronorbornanes allow to identify various stereoisomers.