The recognition of small organic compounds and metabolites is essential for living systems, enabling the cell to sense environmental stimuli and respond appropriately. Developing quantitative models of living systems which can incorporate these environmental stimuli would accordingly benefit from comprehensive mapping of interactions between proteins and small molecules of interest. While high-throughput experimental methods provide a wealth of interaction data, the scale of chemical space currently precludes comprehensive enumeration of protein-compound interaction space. Computational methods can help to bridge this gap by inferring proteome-scale protein-compound interactomes, elucidating structural features within protein families which mediate specificity of binding to specific small molecules, and inferring the affinity of binding for specific protein-compound interactions. In this thesis, we attempt to use, and in some cases develop, methods to study protein-compound interactions at these three scales. First, we describe recent work in extending our structure-based algorithm for predicting protein-compound interactions throughout the proteome to include a wider array of small molecules. We demonstrate that this method performs comparably to existing methods and describe an online database storing the results of this analysis. We also report several case studies illustrating how this database can be used along with cautionary vignettes indicating areas where the method fails and directions for future improvement. We subsequently analyze druggable pockets occurring within protein-protein interfaces (PPIs) to assess whether they are less structurally conserved than analogous pockets of conventional drug sites. We find that PPI interfacial pockets are associated with fewer expected off-targets than conventional drug sites, however that this finding is specific to individual protein families, rather than a general feature of interfacial PPI pockets. Finally, we use Free Energy Perturbation to predict the binding affinity of an array of small volatile odorants with an olfactory receptor from the jumping bristletail, Machilis hrabei, as well as attempt to further optimize the system in order to study the effects of mutating receptor binding site residues on binding affinity to its active ligands.

Phytate is the major storage form of phosphorus in seeds of soybeans. Because phytate chelates mineral cations including calcium, iron, and zinc, these mixed salts are often excreted by non-ruminant animals such as humans, swine, poultry, and fish. While this causes iron and zinc deficiencies, phytate is also considered a water pollutant due to the excess phosphorus excreted in animal waste. These negative environmental and nutritional effects, create a need for low phytate soybeans. While several low phytate soybean lines have been developed, a major drawback is the reduced seedling emergence of these lines resulting in low yields. Therefore, understanding the genetic and molecular bases of the low emergence trait in relation to seed phytate content in major crops such as soybean is of great economic importance. This PhD project worked towards the long term goal of developing low phytate soybean cultivars with good seedling emergence and high-yield. This dissertation focused on metabolomic differences between low and normal phytate lines and how these could relate to the low emergence phenotype. The genetic materials used here include four near isogenic lines that differ in mutations in two multi drug resistance-associated proteins (MRPs). Only the line with both mutations was low phytate. The phytate levels, field- and lab-based emergence rates were determined for these lines, their parents and a control line through replicated field experiments for three consecutive years. The emergence rates of the low phytate lines were not always reduced. This showed that the environment the seeds were produced in is highly important, especially when breeding and commercially growing low phytate lines. A protocol was developed for successful metabolomic discrimination of these closely related soybean lines. The polar and non-polar metabolite profiles were determined using ultra performance liquid chromatography mass spectrometry and metabolomic differences between the low and normal phytate lines were identified. The low phytate double mutant did not contain C22 glucose terminated Group A soyasaponins and almost exclusively contained C22 xylose terminated Group A soyasaponins (A4, A5 and A6). Compared to the normal phytate lines, the low phytate soybean line showed a higher concentration of storage lipids (triacylglycerols and diacylglycerols) and certain phospholipids. / Ph. D.

Systematic fractionations of crude extracts from various strains of Aspergillus versicolor were carried out by a combination of techniques, including liquid-liquid partition, column chromatography and thin-layer chromatography. As a result of these fractionations a total of eleven metabolites were obtained in pure form. Ten of these were identified as the known compounds, sterigmatocystin, de-0-methylsterigmatocystin, 5-methoxysterigmatocystin, versicolorin A, versicolorin C, aversin, averufin, griseofulvin, dechlorogriseofulvin and 3,8- dihydroxy-6 -methoxy-1-methylxanthone. A new metabolite was isolated and identified as 6,8-di-O-methylnidurufin. Two high-performance liquid chromatography systems were developed for the analysis of these metabolites, using microparticle silica gel and microparticle polar bonded phase columns. Studies were also carried out on the reduction of the hemi-acetal derivatives of sterigmatocystin and versicolorin A. Reduction with a limited amount of sodium borohydride yielded in each case a mixture of two major products. The fully reduced products were identified as diols, while the partially reduced products were found to be new hemiacetals. The structure elucidation of these compounds was carried out with the aid of carbon magnetic resonance spectroscopy and of chemical conversions. A modified pathway for the biosynthesis of versicolorin A from averufin is proposed that incorporates nidurufin as an intermediate. / Ph. D.

Expanding global populations, unequal food distribution and disease pressure suggest food poverty is increasing. Consequently, much attention is focussed on alternative natural methods in which to increase agricultural yield. Previously, it was observed that Trichoderma hamatum strain GD12 and its respective N-acetyl-β-D-Glucosamine mutant ∆Thnag:hph promoted plant biomass and fitness that, as a result, may provide a credible natural alternative to synthetic fertilisers. However, on a molecular level, the manner in which this is achieved has not been fully elucidated. In this thesis, I report the biofertiliser effect of GD12 and mutant ∆Thnag::hph once applied to autoclaved peat microcosms as sole applications. Furthermore, I demonstrate the biocontrol ability of GD12 when co-inoculated with Sclerotinia sclerotiorum or Rhizoctonia solani and reveal, that once mycelium co-inoculation has occurred, GD12 increase plant biomass and provide protection; whilst ∆Thnag::hph does not. Consequently, I challenged the biocontrol effects of Trichoderma metabolite extract where I validate that both Trichoderma wild type GD12 and mutant ∆Thnag::hph are incapable of suppressing pathogen growth. Subsequently, I characterised the up-regulated signatures associated with GD12 and ∆Thnag::hph using LC-MS techniques where unique compounds were discovered from each strain of Trichoderma. In conclusion, I provide evidence that N-acetyl-β-D-Glucosamine mutation bring about metabolomic changes that affect the fungal secretome which, in turn, alters plant phenotype, fitness and germination. Furthermore, I have shown that these effects are species specific and depend upon pathogen, plant and fungal properties. However, further investigations are needed to fully elucidate the compound(s) responsible for biocontrol and biofertilisation; especially plant-specific effects that take place as a consequence of fungal activity.

No description available.

Notre travail de thèse avait pour objet l’étude du potentiel anti-diabétique des urolithines A, B, C et D, métabolites de polyphénols formés par le microbiote colique à partir des tanins de l’acide ellagique (présents notamment dans la grenade et les noix).La première partie, bibliographique, constitue un rappel :• de la régulation de l’équilibre glycémique et le rôle de la sécrétion d’insuline dans cette régulation ; •de l ‘épidémiologie et la physiopathologie du diabète type 2 (DT2) ; •des polyphénols et leurs métabolites, ainsi que de leurs effets antidiabétiques potentiels.La seconde partie décrit les effets des urolithines sur différents modèles expérimentaux : •Sur un modèle de cellules β insulino-sécrétrices (lignée INS-1), les urolithines induisent une amplification concentration-dépendante de la sécrétion d’insuline induite par le glucose, mais également par d’autres sécrétagogues comme un analogue du GLP-1 ou une sulfonylurée (médicaments utilisés dans le diabète). Les urolithines préviennent également l’altération sécrétoire induite par un stress oxydant. •L’effet insulino-sécrétoire des urolithines a été confirmé sur îlots de Langerhans isolés. •L’urolithine C étant apparu comme le composé le plus prometteur, nous avons poursuivi la caractérisation de son activité sur un modèle ex vivo mimant la situation physiologique, le pancréas isolé perfusé. Alors que l’effet sécrétoire de l’urolithine C n’apparaît pas en présence de 5mM de glucose, l’urolithine C (20µM) a stimulé la sécrétion d’insuline dans des conditions de stimulation modérée de la sécrétion d’insuline par le glucose (8.3mM). Cet effet est strictement dépendant du glucose, la sécrétion d’insuline retournant immédiatement à son niveau basal lors du passage de 8,3 à 5mM de glucose en présence d’urolithine C. •Des études de pharmacocinétique ont permis de mettre au point une méthodologie de dosage plasmatique de l’urolithine C dans le plasma de rat par chromatographie liquide / ionisation electrospray /spectrographie de masse en tandem. Cette méthodologie a été appliquée à une première étude pharmacocinétique chez le rat après injection de 10mg/kg d’urolithine C par voie intra-péritonéale. Cette étude montre notamment que le profil pharmacocinétique suit un modèle à 3 compartiments et suggère un stockage tissulaire du composé.D’autres résultats (confidentiels) ne peuvent être évoqués dans ce résumé mais confirment l’intérêt potentiel de l’urolithine C dans le traitement du diabète de type 2 en tant que médicament insulinotrope dépendant du glucose. / The objective of our thesis was to study the anti-diabetic potential of metabolites of ellagic acid tanins, present notably in pomegranate and nuts, that are formed by the colon microbiote. The metabolites are urolithins A, B, C and D.The first part of thesis is bibliographic and reviews: •The control of glycemic plasma levels, and in particular the role of insulin secretion in this process; • The pathophysiology of Type 2 Diabetes (T2D); •The various polyphenols and their metabolites, along with their potential anti-diabetic activity.The second part describes the effects of urolithins on various experimental models: •On a model of insulin secreting beta cells (the INS-1cell line), urolithins concentration-dependently amplified insulin secretion induced by glucose, but also by insulinotropic drugs used in the treatment of T2D such as a GLP-1 analogue or a sulfonylurea. In addition, urolithins were able to induce insulin secretion on cells rendered unresponsive to glucose by oxidative stress. • The insulinotropic effect of urolithins was also confirmed on isolated rat islets of Langerhans. •As urolithin C appeared to be the most promising antidiabetic compound, we further characterized its activity on an ex vivo model mimicking the physiological situation, the isolated infused pancreas. While urolithin C (20µM) had no effect in the presence of 5 mM glucose concentration, it amplified the stimulation of insulin secretion in the presence of 8.3mM glucose. The effect of urolithin C was also strictly glucose-dependent, as insulin secretion immediately returned to basal level when glucose concentration was switched from 8.3 to 5mM glucose in the presence of urolithin C. •We also conducted studies aiming at designing a validated methodology for rat plasma urolithin C determination using a liquid chromatography-electrospray ionization-tandem mass spectrometry method. The applicability of this assay was demonstrated in a preclinical pharmacokinetic study carried out in rats receiving intraperitoneal administration of urolithin C (10mg/kg). We found that the urolithin C followed a three-compartment model, suggesting a long-term tissue storage of urolithin C.Some other (confidential) results, not described in this abstract, confirmed urolithin C as a potential glucose-dependent insulinotropic treatment for type 2 diabetes.

Abstract available in pdf file.

Abstract available in pdf file.

Thesis (M.Phil.) - University of Queensland, 2006. / Includes bibliography.

New drugs, particularly antibiotics, are urgently required to combat the increasing problem of antibiotic resistant human pathogens. Due to the scarcity of products available today, the pharmaceutical industry is now under pressure to reassess compounds derived from plants, soil and marine organisms. Pharmaceutical companies are showing renewed interest in marine biotechnology as the oceans represent a rich source of both biological and chemical diversity of novel molecular structures with anti-cancer, anti-inflammatory and antibiotic properties. Formerly unexplored locations, such as deep ocean sediments, show great potential as a source of genetically novel microorganisms producing structurally unique secondary metabolites. In this research, a metabolite producing marine Pseudoalteromonas strain, known as AP5, was initially used to develop methods for the detection, optimisation of production and extraction of bioactive metabolites from other potentially novel marine isolates. Two hundred and seventy six (276) marine isolates from water and sediment samples from the Antarctic Ocean and Marion Island were isolated. Ten visually different isolates were screened for bioactivity against Gram-positive and -negative bacteria, fungi and yeast. Three out of the 10 isolates, WL61 , WL 114 and WL 136, appeared to be novel Streptomyces spp. showing activity against different test organisms. Many of these marine microorganisms are difficult to culture in the laboratory, particularly when they are cultivated continuously in shake flasks as they can stop producing bioactive compounds. The cultivation of marine isolates in bioreactors may be a more beneficial process for the optimisation of metabolite production compared to conventional liquid fermentation techniques whereby the solid-liquid-air interface of membrane bioreactors can imitate the natural environment of microbes. The membrane bioreactor system is a stable growth environment with low shear that supports steady-state biofilm growth consisting of a high cell density due to a high mass transfer of nutrients and oxygen to the cells. This approach was employed and isolates WL61, WL114 and WL136 were immobilised onto ceramic membranes using Quorus single fibre bioreactors (SFR). The SFRs were used to establish the most suitable growth medium for continuous secondary metabolite production. The best growth conditions were applied to the Quorus multifibre bioreactor (MFR) for scale up of biologically active metabolites, highlighting the potential of bioreactor technology for use in bioprospecting for isolating and screening novel and known organisms for new and interesting natural products. Furthermore, the Quorus MFR was shown to be suitable for the production of high yields of antimicrobial metabolites and is an efficient new fermentation production system. Purification by HPLC fractionation was used to characterise four major compounds from isolate WL 114 extracts. NMR structure elucidation identified one of the two primary compounds as Bisphenol A. The complete chemical structure for the second potent bioactive compound could not be determined due to the low concentration and volume of material. / KMBT_363 / Adobe Acrobat 9.54 Paper Capture Plug-in