La consommation d'aliments riches en ellagitannins (ETs) pourrait être associée principalement à la prévention des maladies cardiovasculaires et la régulation des cancers hormono-dépendants. Néanmoins, les ETs ne sont pas biodisponibles en tant que tel et, après avoir été partiellement transformés en acide ellagique (EA) dans le tractus gastro-intestinal (GI) supérieur, ils sont métabolisés dans le côlon par la flore intestinale en urolithines, un groupe de molécules plus biodisponibles et bioactives qui peuvent persister jusqu'à 4 jours à des concentrations relativement élevées dans le plasma et l'urine. La variabilité de l'excrétion des urolithines dans l'urine est importante et à partir d'un échantillon de population de 26 volontaires sains, trois groupes principaux d'individus ont pu être distingués : "faible ou non-excréteur d'urolithin », « Excréteur prédominant d'UA et dérivés» et « Excréteur prédominant d'UB et dérivés»". Ces groupes ont également été observés en considérant la cinétique totale d'excrétion sur une période de 4 jours après ingestion du jus et à des périodes différentes tout au long d'une année. Bien que les variabilités inter-et intra-individuelles soient relativement élevées, les individus conservent leur statut au cours des différentes périodes d'intervention même en modifiant les quantités d'ETs ingérées. L'analyse par UPLC-PDA/ESI-Q-TOF/MS2 a permis d'attribuer hypothétiquement une identité à 15 autres métabolites d'ETs dans l'urine, mais le profilage métabolomique n'a pas permis de discriminer d'autres composés exceptés les dérivés d'UA ou d'UB. La fermentation in-vitro des ETs et EA, par les matières fécales a montré une voie métabolique spécifique qui débouche sur la production d'UA. Néanmoins, les métabolites excrétés in vivo sont beaucoup plus complexes ce qui met en évidence de fortes interactions entre le système excréteur de l'hôte et la composition du microbiote intestinal. La recirculation hépatique suivie par une re-conversion des métabolites de phase II dans le côlon permettrait d'expliquer l'excrétion d'UB chez certains volontaires. L'écologie spécifique de la flore intestinale évaluée par la méthode des empreintes PCR-DGGE a permis d'identifier quelques microorganismes associés à une plus grande capacité de bioconversion des ETs en urolithins / Consumption of dietary ellagitannins (ETs) could be associated mainly with prevention of cardiovascular diseases and regulation of hormone-dependent cancers. Nonetheless, ETs are not bioavailable as such; therefore, after being partially converted into ellagic acid (EA) in the upper gastrointestinal (GI) tract, they undergo sequential bioconversion in the colon by gut microbiota into urolithins, a more bioavailable and bioactive group of molecules that persist up to 4 days at relatively high concentrations in urine. Variability of urolithin excretion in urine is high and three main groups, “no or low urolithin excreters,” “predominantly UA derivatives excreters” and “predominantly UB derivatives excreters,” were observed on a cohort of 26 healthy volunteers. These categories were also unambiguously observed following the total excretion of main ETs' metabolites over a 4 day period after ingesting one shot of juice, and at different periods of time along one year. Although relatively high inter- and intra-individual variabilities were observed, individuals preserved their status during various intervention periods with different amounts of ETs ingested. UPLC-PDA and ESI-Q-TOF/MS1 and MS2 allowed the tentative assignment of an identity to 15 other ETs metabolites in urine, but this profiling did not allow the discrimination of any other compounds aside from UA or UB derivatives. In-vitro fermentation of ETs and EA with fecal stools showed a specific metabolic pathway ending in the production of UA. Nonetheless, metabolites excreted in-vivo are much more complex, highlighting strong interactions between host excretory system and composition of gut microbiota. Hepatic recirculation and additional bioconversion of Phase II metabolites in the colon may explain predominant excretion of UB in some volunteers. Microbiota ecology assessed by PCR-Denaturing Gradient Gel Electrophoresis (DGGE) fingerprint method allowed the association of some microorganism species to higher capacity of bioconversion of dietary ETs into urolithins.Key words: Ellagitannins, blackberry, urolithin, colonic metabolites, ETs degradation patterns, gut microbiota, gastrointestinal tract,

Secoisolariciresinol (SECO) is the major lignan present in flaxseed, but unlike the structurally related lignan nordihydroguaiaretic acid, it is not associated with toxicity. The major phase I metabolite of SECO is lariciresinol, likely formed as a result of para-quinone methide (p-QM) formation followed by an intramolecular cyclization, thereby minimizing any toxicity associated with the p-QM. Four analogues of SECO were used to investigate substituent effects on lignan metabolism and formation of reactive quinones. HPLC methods were developed for analysis of SECO analogues and their metabolites. The stability of SECO analogues (1 mM) in a 50 mM Na2HPO4 buffer at pH 6.0 and 7.4 were quantified. Enzymatic oxidation experiments using mushroom tyrosinase and microsomes harvested from male Sprague-Dawley rats were performed with and without a GSH trapping system. Mass spectrometry and LC-MS were used to identify metabolites. Life Technologies was contracted to perform IC50 inhibition assays on SECO and the SECO analogues against CYP3A4, CYP3A5, CYP2C9 and CYP2C19 cytochrome P450 isoforms. All SECO analogues were stable at pH 6.0. SECO-2 was stable at pH 7.4 but SECO-1, -3 and -4 were unstable at pH 7.4. Autoxidation of SECO -1, -3 and -4 were 1st order reactions with t1/2 of 9.0 h, 1.7 h and 7.0 h respectively. Mushroom tyrosinase oxidations were performed to generate ortho-quinone standards. SECO-1 -3 and -4 were oxidized by mushroom tyrosinase but SECO-2 was not. Trapping with GSH produces aromatic ring conjugates for SECO-1, -3, -4. Results from microsomal oxidations for SECO-1, -3 and -4 are consistent with these standards. SECO-2 was metabolized by a microsomal system to produce a benzyl GSH adduct. Dealkylation products were also observed. All SECO analogues formed quinones but interestingly, GSH conjugation was competitive with intramolecular cyclization. All cytochrome P450 isoforms were inhibited by every analogue tested to varying degrees, a potential cause of toxicity concerns. Quinones are known to cause toxicity in vivo, including cytotoxicity, immunotoxicity, and carcinogenesis. Our results suggest that since the phenol and catechol lignans form GSH adducts in addition to intramolecular cyclization products, this class of lignans have the potential to cause toxicity.

No description available.

This thesis focuses on studies investigating the structure of intermediates involved in fluorometabolite biosynthesis, and the potential applications of the fluorinase enzyme in positron emission tomography (PET). Chapter 1 introduces the rare natural occurrence of fluorinated compounds. The bacterium Streptomyces cattleya is known to biosynthesise two fluorinated secondary metabolites: the toxin fluoroacetate (FAc) and the antibiotic 4-fluorothreonine (4-FT). The enzymes and intermediates identified on this fluorometabolite biosynthetic pathway in S. cattleya, prior to this research, are discussed in detail. Chapter 2 presents studies towards the unambiguous structural identification of (3R,4S)-5- deoxy-5-fluoro-D-ribulose-1-phosphate (5-FRulP) as the third fluorinated intermediate on the biosynthetic pathway to fluoroacetate and 4-fluorothreonine in S. cattleya. Chapter 3 describes the synthetic routes to key molecules, necessary as reference compounds and substrates, to underpin the subsequent studies in this thesis. In particular, synthetic routes to 5'-deoxy-5'-fluoroadenosine (5'-FDA), 5'-deoxy-5'-fluoroinosine (5'-FDI), 5-deoxy-5-fluoro-D-ribose (5-FDR) and 5-deoxy-5-fluoro-D-xylose (5-FDX) are described. Chapter 4 describes the use of the fluorinase enzyme from S. cattleya as a tool for the synthesis of new [¹⁸F]-labelled sugars with potential application in positron emission tomography (PET). A new route to 5-deoxy-5-[¹⁸F]fluoro-D-ribose ([¹⁸F]FDR) is developed in a two-step enzymatic synthesis. A total of three potential radiotracers ([¹⁸F]FDA, [¹⁸F]FDR and [¹⁸F]FDI) are synthesised using fluorinase-coupled enzyme reactions. In addition, in vitro studies are reported with these [¹⁸F]-labelled sugars to investigate their uptake and potential as PET radiotracers in cancer cells. A preliminary rat imaging study with [¹⁸F]FDA is reported. Chapter 5 details the experimental procedures for the compounds synthesised in this research and the biological procedures for chemo-enzymatic syntheses and protein purification.

No description available.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Obesity is the most prevalent nutritional disorder encountered in small animal medicine. Problems related with obesity are the higher incidence of morbidity and mortality. Nutritional and physical activity interventions have been common strategies employed; however, they have shown low compliance rates. Because of it more attention has been given to the nutrient composition of diets. Using the canine model, three experiments were conducted to examine the effect of fish oil or barley on protein and lipid metabolism, as well as postprandial glycemia, and nutrient digestibility in mature and in young adult dogs. In Exp. 1, seven female dogs were randomly assigned to one of two isonitrogenous and isocaloric diets, control (CO) or fish oil (FO), in a crossover design. Animals fed the FO diet tended to be more sensitive to glucose, showing a lower glucose half life. Cholesterol and HDL decreased (p<0.05) on the FO treatment. Overall, the supplementation of fish oil may improve glucose clearance rate and is effective in decreasing cholesterol in mature overweight dogs. In Exp. 2, eight female Beagles were randomly assigned to one of two isonitrogenous and isocaloric diets, control (CO) or fish oil (FO), in a crossover design. Overall, feeding a FO containing diet showed a protective effect against the rise of plasma CHOL and it increased plasma ghrelin levels. However, it did not appear to improve protein metabolism or postprandial glycemia in adult lean dogs. In Exp. 3, sixteen female dogs were randomly assigned to four experimental diets; control (40% corn) or three levels of barley (10, 20, 40%). The data suggest that inclusion of barley up to 40% in diets for adult dogs is well tolerated and does not negatively impact nutrient digestibility of the diets. However, inclusion of barley did not improve aspects related to fecal odor, postprandial glycemia, or plasma cholesterol. Overall, the research presented herein suggests that different nutritional strategies - dietary lipid or carbohydrate manipulation - may be beneficial in ameliorating health issues (e.g., hyperlipidemia) or in improving the health status of dogs (e.g., gut health by increased SCFA production).

Photorhabdus are motile Gram-negative bacteria that have a mutualistic association with entomopathogenic Heterorhabditis nematodes. Nematodes vector the bacteria from one insect host to another, while the bacterial symbiont produces toxins and secondary metabolites that kill that the insect host. In this study, we characterize the bacterial symbiont of Heterorhabditis sonorensis, recently discovered in the Sonoran desert. Biochemical and molecular methods including sequence data from five genes: 16s rDNA, gyrB, recA, gltX, dnaN were considered. Evolutionary relationships of this new Photorhabdus subsp. were inferred considering maximum parsimony and Bayesian analyses. We also surveyed for secondary metabolites (SM) produced by this microorganism, considering HPLC and mass spectrometry analyses. SM crude extracts showed activity against the corn ear worm Helicoverpa zea, the root-knot nematode (Meloidogyne incognita), the bacterium Pseudomonas syringae, and the fungus Fusarium oxysporum; and were more toxic that those produced by related species. Results from these studies showed that Photorhabdus l. sonorensis' secondary metabolites have potent antagonistic activity against these plant pathogens.

Chamuvarinin (22) is a unique annoanceaeous acetogenin isolated from the roots of Senegalese medicinal plant Uvaria chamae by Laurens and co-workers in 2004. It displays highly potent cytotoxicity towards the cervical cancer cell lines (KB 3-1, IC₅₀= 0.8 nM). Structurally, chamuvarinin is the first reported acetogenin to contain an adjacently linked bis-THF-THP ring system spanning the C15-C28 carbon backbone. However, initial efforts to assign the relative and absolute configuration within this stereochemical array, on the basis of ¹H and ¹³C NMR analysis, provided only partial information pertaining to the relative configuration of C15-C19 region. As a consequence, 32 diastereomeric structural possibilities exist for the highly unusual structure of chamuvarinin; an unrealistic target for total synthesis. The synthesis of the central core tricyclic (BCD) intermediate represents the most challenging aspect in the entire synthesis, which in turn will aid ultimate structural proof. At the outset of the project the stereochemical configuration of C15-C28 (BCD) of chamuvarinin was uncertain and a library approach was proposed to enable structure elucidation (Scheme A-1). Chapter 2 and Chapter 3 detail the synthesis of possible diastereomers of the C9-C21 (51) and C22-C34 fragments (52). Chapter 4 details the intial strategy to couple the key diastereomeric fragments in a series of model studies. Chapter 5 describes the successful coupling strategy via an revised synthetic approach to reach the advanced C9-C34 intermediate 251 (Scheme A-2).

The success of biocontrol bacteria is often restrained due to their low persistence in the rhizosphere and fluctuations in expression of antagonistic compounds. In the first part of this thesis the ability of the biocontrol agents (BCAs) Pseudomonas brassicacearum DF41 and Pseudomonas chlororaphis PA23 to resist grazing by the bacterivorous nematode Caenorhabditis elegans was investigated. We found that both BCAs are capable of killing the nematodes through exposure to toxic metabolites. We discovered that in addition to HCN, pyrrolnitrin (PRN) is a potent nematicide produced by PA23. Unique for a pseudomonad, DF41 was also found to kill the nematodes by forming biofilms on the nematode anterior, causing starvation. Biofilm formation was dependent upon the Gac two-component system and NaCl concentration of the media. Co-culturing these BCAs in the presence of nematodes increased expression of a number of genes associated with biocontrol. We observed elevated exoproduct formation, consistent with our gene expression analysis. The nematicidal activity exhibited by DF41 and PA23 towards C. elegans bodes well for their persistence in the environment. In the second part of this thesis the role of hydrogen cyanide (HCN) and the anaerobic regulator ANR in PA23 biocontrol was explored. An hcn mutant was created and in vitro antifungal (AF) assays revealed the involvement of HCN in Sclerotinia sclerotiorum suppression. Addition of glycine promoted both AF activity and HCN production. In addition, HCN was found to be positively regulated by quorum sensing (QS). Besides a phz box, an anr box was identified in the hcnA promoter, suggesting a role for ANR in regulating hcnA. An anr mutant was generated and phenotypic characterization revealed that ANR is a key regulator governing PA23 secondary metabolite production. Through gene expression analysis, ANR was shown to positively regulate phzI/phzR and PhzR negatively regulate anr. Furthermore, expressing anr in trans partially complemented the QS-deficient phenotype with respect to several biocontrol genes and exoproducts. Overall, the global regulator ANR is vital for PA23-mediated biocontrol and a significant overlap exists between the QS and ANR regulons. / October 2016