La connaissance du métabolisme demeure incomplète mais sa compréhension reste un but majeur tant pour la recherche fondamentale qu’appliquée. Environ 40% des gènes des organismes procaryotes n’ont pas de fonction précise proposée. En conséquence, de nombreuses voies métaboliques restent incomplètes, rendant très difficile la prédiction du métabolisme d’un organisme. Les recherches et les avancées dans la compréhension de la cascade des « omiques » incluant la génomique, la transcriptomique, la protéomique et la métabolomique jouent aujourd’hui un rôle central dans la connaissance du métabolisme. En tant qu’intermédiaires des réactions biochimiques, les métabolites connectent les différentes voies métaboliques d’un organisme. La quantité et la nature de ces molécules étant la résultante de l’expression des gènes, la composition en métabolites de la cellule est donc liée de très près à son phénotype. La métabolomique, qui étudie l’ensemble de ces petites molécules présentes dans un échantillon biologique, constitue une source d’informations importante nécessaire à la compréhension du métabolisme. En ce sens, le travail présenté dans ce manuscrit décrit la mise en place d’une approche métabolomique au sein du Genoscope pour l’étude du métabolisme de la bactérie modèle Acinetobacter baylyi (ADP1) en utilisant la spectrométrie de masse à haute résolution (LTQ-Orbitrap) couplée à la chromatographie liquide (LC/MS).Nous avons suivi la réorientation du métabolisme d’ADP1 à la suite d’une perturbation environnementale, induite par un changement de source de carbone. Nous avons comparé les métabolomes d’ADP1 cultivée sur succinate (source de carbone de référence) et sur quinate (source de carbone alternative). Environ 450 métabolites potentiels ont été détectés et plus d’une centaine ont pu être identifiés. Plusieurs phénomènes ont pu être observés. Premièrement, l’utilisation du quinate comme source de carbone engendre une réponse spécifique attendue, liée à sa dégradation (intermédiaires cataboliques détectés et gènes associés surexprimés). Toutefois, alors que le métabolisme central est peu affecté par ce changement biotique, la concentration d’environ la moitié des métabolites détectés est significativement modifiée. Ce résultat inattendu est en accord avec les expériences de transcriptomique qui indiquent, qu’avec ce changement de source de carbone, 12% des gènes sont différentiellement exprimés. Nos résultats montrent que la perturbation du métabolisme dans ces conditions s’étend bien au-delà de la voie de dégradation du quinate ; elle engendre un bouleversement global du métabolisme (Stuani, Lechaplais et al. 2014).Cette étude a également permis de détecter de nouveaux métabolites, produits dans des cellules utilisant du quinate. L’élucidation structurale de l’un d’entre eux est décrite dans ce manuscrit. Il s’agit d’une tyrosine substituée en position benzylique par un groupement méthylamine (BMAT). Son identification aura nécessité des expériences de CID-MSn séquentielles avec détection à haute résolution et d’échanges H/D. Des études structurales effectuées sur deux autres molécules suggèrent des similarités structurales entre elles et avec le BMAT. Nous pourrions donc être en présence d’une famille de nouveaux métabolites secondaires, impliqués dans une même voie métabolique. C’est donc une approche métabolomique qui a permis à un simple changement de source de carbone de nous orienter vers la découverte de nouvelles voies métaboliques et d’appréhender une part cachée du métabolisme d’ADP1. / While incomplete, the knowledge of metabolism remains a priority for both basic and applied research. With approximately 40 % of genes of prokaryotic genomes with no accurate function, a number of metabolic pathways remain incomplete, which impedes the accurate prediction of the metabolism of a cell.Recent progress and breakthroughs in the understanding of the Omics Cascade that includes genomics, transcriptomics, proteomics and metabolomics play an important role in the knowledge of metabolism. As intermediaries of biochemical reactions, metabolites connect the different metabolic pathways of the cell. The amount and nature of these molecules being the result of gene expression, the metabolite content of a cell is thus closely related to its phenotype. Metabolomics, which aims at studying the whole set of metabolites present in a biological sample, is an invaluable source of information for the understanding of metabolism.This is why the work presented in this thesis reports the setup of a metabolomic approach at Genoscope for the study of metabolism of the model bacterium Acinetobacter baylyi ADP1 (ADP1) through high-resolution mass spectrometry (LTQ-Orbitrap) coupled to liquid chromatography (LC/MS).We investigated the metabolism of ADP1 after an environmental perturbation induced by a change of carbon source. We compared the metabolomes of ADP1 grown on succinate (reference carbon source) and on quinate (alternative source). About 450 potential metabolites could be detected and more than a hundred were identified. Many observations were made. First, quinate as a carbon source triggered an expected specific metabolic response in relation with its dissimilation (quinate catabolites were detected and the corresponding genes were up-regulated). However, while central metabolism is not impacted by this biotic change, about half of the detected metabolites had their concentration changed. This unexpected result is consistent with transcriptomic experiments that revealed that with the change of carbon source, 12% of the total number of genes are differentially expressed. Our results show that in these conditions, metabolic alteration goes far beyond the quinate degradation pathway and causes a global overhaul of the metabolism of ADP1 (Stuani, Lechaplais et al. 2014).This study also led to the detection of novel metabolites that were exclusively produced in quinate-grown cells. The structural elucidation of one of them is reported in this manuscript. It is a tyrosine substituted in the benzylic position by an aminomethyl group (BMAT). Its identification required sequential CID-MSn experiments with high-resolution detection and isotopic H/D exchange experiments. Structural studies conducted on two other molecules suggest structural similarities with BMAT. We thus could be in the presence of a family of novel secondary metabolites, involved in the same metabolic pathway.In conclusion, a metabolomic approach allowed a mere change of carbon source to guide us toward the discovery of novel metabolic pathways and gain insight into the hidden part of ADP1 metabolism.

<p> The purpose of this study is to provide a commentary on part of Seneca's lengthy treatise on benefits. An attempt is made to provide an understanding of the meaning of the text. This involves, at times, consideration of the continuity of thought, of textual problems and lacunae (although by no means all difficulties of that order have been discussed). The statements which Seneca makes in his other philosophical works, whether in agreement or contradictory, are adduced for purposes of elucidation. It is apparent that some of the expressions and statements which seem innocuous in their context are sometimes coloured by their appearing elsewhere in doctrines of considerable complexity. Some of the works of Cicero, another valuable source of Roman philosophy, particularly Stoicism, are introduced for purposes of comparison. The ethical works of Aristotle likewise prove to be a valuable source of comparative statements. In general the background provided illustrates that Seneca did not provide the reader with much original thought but that he presented his material with the skill of an effective instructor.</p> / Thesis / Doctor of Philosophy (PhD)

In this thesis, synthetic chemistry was used as a tool in the exploration of various aspects of natural products discovered by the natural products research group at the University of Canterbury. Work on the constituent amino acids and connectivity of the pteratides, a potently cytotoxic series of cyclodepsipeptides, had been completed before the beginning of this work (carried out by Miss C. Chen). The elucidation of the stereochemistry of the constituent amino acids was undertaken in this present work. The synthesis of all stereochemical entities of a number of unusual amino acids, which were either not available commercially or were expensive, was carried out, providing reference materials for comparison to the natural products. The synthesis of the diastereoisomers of one of these amino acids, 4-methylproline, was carried out by modification of literature procedures, which led to the development of an improved, concise and stereoselective synthesis. The hydrolysis of the natural products, derivatisation of the resultant hydrolysates, synthetic and commercial reference amino acids and HPLC analysis allowed the full stereochemical assignment of the pteratide series. The total synthesis of spiro-mamakone A, a cytotoxic polyketide isolated by Dr S. van der Sar, was undertaken. The synthesis was not successfully completed due to difficulties in the late-stage formation of a crucial enedione motif. However, very advanced intermediates were successfully synthesised. These synthetic analogues of the natural product were analysed for biological activity, allowing valuable insight into the structure-activity relationship, for example, demonstrating the importance of the enedione moiety to biological activity.

In this thesis, synthetic chemistry was used as a tool in the exploration of various aspects of natural products discovered by the natural products research group at the University of Canterbury. Work on the constituent amino acids and connectivity of the pteratides, a potently cytotoxic series of cyclodepsipeptides, had been completed before the beginning of this work (carried out by Miss C. Chen). The elucidation of the stereochemistry of the constituent amino acids was undertaken in this present work. The synthesis of all stereochemical entities of a number of unusual amino acids, which were either not available commercially or were expensive, was carried out, providing reference materials for comparison to the natural products. The synthesis of the diastereoisomers of one of these amino acids, 4-methylproline, was carried out by modification of literature procedures, which led to the development of an improved, concise and stereoselective synthesis. The hydrolysis of the natural products, derivatisation of the resultant hydrolysates, synthetic and commercial reference amino acids and HPLC analysis allowed the full stereochemical assignment of the pteratide series. The total synthesis of spiro-mamakone A, a cytotoxic polyketide isolated by Dr S. van der Sar, was undertaken. The synthesis was not successfully completed due to difficulties in the late-stage formation of a crucial enedione motif. However, very advanced intermediates were successfully synthesised. These synthetic analogues of the natural product were analysed for biological activity, allowing valuable insight into the structure-activity relationship, for example, demonstrating the importance of the enedione moiety to biological activity.

No description available.

The development of novel bioremediation systems has widespread benefits for human health and natural ecosystems. Optimization of such systems is only possible with a thorough understanding of the processes that drive bioremediation. This thesis developed novel understanding of carbon sources and cycling relationships for microbial communities that are integral in controlling contaminant fate in two contaminated environments. In the first case (Chapter 2), biodegradation in the soil microbial community was determined to be the primary pathway for recalcitrant petroleum pollutant removal. Microbial uptake and metabolism of petroleum hydrocarbons was conclusively demonstrated via 14C analysis of their PLFA biomarkers. This microbial community was the most 14C depleted bacterial system detected in an environmental system to date. In addition, complete mineralization of petroleum carbon was demonstrated with 14C analysis of soil COz. The second paper (Chapter 3) identified unique Phospholipid Fatty Acid (PLFA) biomarkers and stable carbon isotopic fractionation patterns for heterotrophic and autotrophic bacterial communities of an acid mine drainage (AMD) system. The characteristic isotopic fractionations observed during biosynthesis of PLF A biomarkers in autotrophic versus heterotrophic metabolic pathways provided the basis for a model capable of elucidating the relative roles of these members of the microbial community in the environment. The major implications of the knowledge developed in this thesis, are two new methods to identify microbial carbon cycling pathways and processes in contaminated environments. These advances may lead to new methods for mitigating the effects of contamination in environmental systems through better understanding of the microbial processes at the contaminated sites. / Thesis / Master of Science (MSc)

Unambiguous structure assignment of standards is essential for metabolome characterization of infected plants. Complete structure elucidation of eleven natural polyphenols, resveratrol (1), (-)-epicatechin (2), pelargonidin chloride (3), cyanidin chloride (4), cyanin chloride (5), keracyanin chloride (6), caftaric acid (7), quinic acid (8), procyanidin B1 (9), procyanidin B2 (10), and procyanidin C1 (11) by 1H-, 13C, COSY-, TOCSY-, ROESY-, and HMBC-NMR is described. The sinusoidal modulation frequency of 1H-13C-cross-peaks (J-HMBC) was fitted iteratively to sin(πJH,Ctvar) and yielded 2,3J-coupling values for 1H-13C-correlations in the natural polyphenols. Satisfactory fit to standard Karplus-equations was achieved for glycosides directly attached to the aromatic core in cyanin chloride. Molecular dynamics simulation data in vacuum at the AM1-level of theory were shown to approximate the NMR-solution data reasonably well. Analysis of these standards enables the characterization of unknown plant metabolites produced by bacterially stressed Thompson grape plants provided by the USDA. Initial steps for structure elucidation of 72 unknown fractions is discussed. In addition, selective HCl-catalyzed H/D-exchange was observed for aromatic protons H6 and H8 in flavonoid structures containing a 5,7-meta-disubstituted chromelynium core with free OH-groups. The exchange took place readily in compounds 3, 4, and 6, whereas 1, 2, and 5 did not exchange.

No / This article reports the structural elucidation of the Alangium alkaloid, tubulosine (1) on the basis of systematic 2D-NMR analyses (DEPT, COSY, TOCSY, NOESY, ROESY, HMQC and HMBC). The data obtained allowed the unambiguous assignment of all proton and carbon signals in 1 for the first time.

No description available.

No description available.