The work undertaken explored the potential for discovery of new bioactive metabolites from soil fungi. The research developed selective mycological isolation techniques and maximised metabolite production from active selected fungi by application of the OSMAC approach and concept of hormesis. Novel active compounds were discovered and many other known compounds characterised.

The work undertaken explored the potential for discovery of new bioactive metabolites from soil fungi. The research developed selective mycological isolation techniques and maximised metabolite production from active selected fungi by application of the OSMAC approach and concept of hormesis. Novel active compounds were discovered and many other known compounds characterised.

PART I. Asymmetric syntheses of both natural (+)- and nonnatural (-)- byssochlamic acid via a [2+2] photoaddition-cycloreversion strategy are described. X-ray crystallographic analysis of the cyclohexylamine salt 99 showed that the structure of the monomethyl ester 100 from esterase hydrolysis of 44 was originally misassigned as 56. The enantiomeric relationship of the two diolides 106 and 70 permitted syntheses of nonnatural byssochlamic acid (-)-3 and natural byssochiamic acid (+)-3 from enantiopure alcohol (+)-64 and from its enantiomer (-)-110, respectively. Through the use of (��)-103 to reach both enantiomers of byssochlamic acid (3) and subsequent epimerization of the npropyl chain, it was proved that the cis configuration of the two alkyl substituents is strongly preferred in the natural product. PART II. An asymmetric approach towards the nootropic agent huperzine A is described. Formation of cyclobutane 122 with the desired stereochemistry was accomplished using intramolecular [2+2] photoaddition of the enantiopure enone 121. Attempts to prepare the methoxypyridine system via an azadiene Diels- Alder reaction were unsuccessful. However, intramolecular Michael addition of 181 produced silyl ether 182 which was converted into the pyridone 187 by treatment with hydrogen fluoride followed by selenoxide elimination. Attempts to effect the key sigmatropic rearrangement of ketone 197 into a direct precursor of huperzine A were unsuccessful. / Graduation date: 2001

M.Sc. / Plants have the ability to continuously respond to various stimuli which alter their physiology, morphology and development. These stimuli may be abiotic or biotic and range from essential to toxic in their effects. One of these stimuli is a steroid from fungal membranes, ergosterol (C28H44O), which does not occur in plants. Ergosterol acts as a pathogen-associated molecular pattern molecule and triggers defence mechanisms in plants, characterised by highly regulated and interrelated events that include the elicitation of the oxidative burst and expression of a number of defencerelated genes. However, the ergosterol-induced global cellular reprogramming of the host has not been fully investigated in all aspects. No metabolomic study has previously been conducted to elucidate, for instance, the effect of ergosterol on plant metabolism. A clear and broader understanding of the molecular mechanisms involved in plant : ergosterol interactions is of paramount importance, for it would open up possibilities of developing novel, more effective and sustainable strategies to control or eradicate fungal diseases in plants. In plants, the metabolome is a compilation of all primary and secondary metabolites. The latter are the final recipients of genetic information, and their levels can influence gene expression and protein stability. Metabolite patterns reveal the actual cellular dynamic environment. Hence, qualitative and quantitative measurements of extra- and intracellular metabolites yield insights into the cellular processes that control the biochemical phenotype of the cell, tissue or whole organism. Metabolomics, the most recent of the ‘omics’ approaches, is the holistic analysis of metabolites present within a biological system under specific physiological conditions. In the present study a metabolomic approach was used to elucidate and analyse changes in the metabolism of tobacco (Nicotiana tabacum) cells following ergosterol treatment. Special attention is given to sesquiterpenoids since the antimicrobial compounds (phytoalexins) isolated from plants within the Solanaceae are mostly bicyclic sesquiterpenoids. Suspension of tobacco cells were treated with different concentrations (0 - 1000 nM) of ergosterol and incubated for different time periods (0 - 24 h). A viability assay, based on the ability of viable cells to reduce 2,3,5- triphenyltetrazolium chloride (TTC), was used to determine whether cell death occurred due to ergosterol treatment. No loss of cell viability was observed over the concentration range and time periods used in this study, indicating that the observed responses were due to the treatment alone and possible secondary responses due to cell death could be excluded. Intracellular metabolites were extracted with two methods: a selective dispersive liquid-liquid micro extraction and a general methanol extraction. Chromatographic techniques (TLC/HPTLC, GC-FID, GC-MS, GC×GC-TOF-MS, UPLC-MS) and 1H NMR spectroscopy were used for quantitative and qualitative analyses. Multivariate data analyses (PCA and OPLS-DA models) were used to extract interpretable information from the multidimensional data generated from the aforementioned techniques.

Introduction : La présence des moisissures et de leurs mycotoxines dans les matrices complexes de papiers est peu étudiée. Notre travail a porté sur la recherche de mycotoxines sur les papiers patrimoniaux altérés par le foxing et les papiers peints moisis issus de logements dont les habitants ont été diagnostiqués comme porteurs de symptômes allergiques et du syndrome des bâtiments malsains. Objectifs : Identifier les espèces fongiques de ces deux types de supports papiers et y déterminer la production de métabolites fongiques. Matériels et Méthodes : Le foxing a été caractérisé par des techniques pluridisciplinaires (physiques, biologiques, bioanalytiques, tests de cytotoxicité). Les métabolites fongiques dans les extraits hydro-organiques de ces papiers ont été recherchés et identifiés par spectrométrie de masse afin d'évaluer s'ils pouvaient être reliés aux espèces fongiques détectées par microbiologie. Puis le risque toxique de ces extraits de papiers a été évalué sur un modèle cellulaire in vitro. Résultats : Pour le foxing, nous avons pu en exclure une origine métallique et montrer que les micro-organismes présents dans celui-ci sont essentiellement des espèces fongiques. Pour les papiers peints, des pics relatifs à des métabolites fongiques ont été retrouvés. Grâce à l'ensemencement individuel d'espèces fongiques sur papier peint et à l'aide de la MS2, nous avons pu relier les composés de m/z 401 et 487 à S. chartarum. Les tests de cytoxicité ont montré une augmentation significative de la cytotoxicité des cellules A549 avec certains papiers peints moisis par rapport au papier peint témoin. Les cellules A549 ont montré une surexpression du TNF-α, de l'IL-8 et du CYP 1A1, après contact avec ces mêmes papiers peints. Discussion-Conclusion : Nous n'avons détecté aucune mycotoxine dans le foxing excluant ainsi d'éventuels liens entre inhalation de mycotoxines émanant de vieux manuscrits et symptomatologie respiratoire. Pour les habitants des logements et leurs symptômes respiratoires, il est difficile de les relier à une espèce fongique donnée ou à un métabolite donné, bien que S. chartarum ait pu être mis en cause. Ces premiers résultats doivent être confirmés par des études ultérieures / Introduction: Little study has been carried out on the presence of fungi and their mycotoxins in complex paper matrices. Our work has focused on finding mycotoxins on heritage paper altered by the foxing and wallpapers from moldy homes whose residents have been diagnosed with symptoms of allergies and sick building syndrome. Objectives: To identify the fungal species of these two types of paper and to determine the production of fungal metabolites. Materials and methods: The foxing has been characterized using multi-disciplinary technics (physical, biological, bioanalytical, cytotoxicity assays). The fungal metabolites in the hydro-organic extracts of these papers were sought and identified by mass spectrometry to assess whether they could be related to fungal species detected by microbiology. The toxicological risk of wallpaper extracts was then evaluated on model in vitro cells. Results: For the foxing, we could exclude a metal origine and we showed that the microorganisms present are mainly fungal species. For wallpapers, fungal metabolites were found. By seeding individual fungal species on wallpaper and using MS2, we were able to link the compounds of m/z 401 and 487 to S. chartarum. Cytotoxicity tests showed a significant increase in cytotoxicity of A549 cells with moldy wallpaper in comparison with the control wallpaper. A549 cells showed an overexpression of TNF-α after contact with these wallpapers as well as a significant overexpression of IL-8 and CYP 1A1.Discussion-Conclusion: We detected no mycotoxin in foxing, thus excluding possible links between inhalation of mycotoxins from old manuscripts and respiratory symptoms. For people in their homes and respiratory symptoms, it is difficult to draw any relationships to a given fungal species or a metabolite although S. chartarum has been implicated. These initial results should be confirmed by further study

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Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (p. 161-186).

Thesis (M.Sc.) --Memorial University of Newfoundland, 1987. / Typescript. Bibliography: leaves 113-122. Also available online.

Thesis (Ph.D.)--University of Melbourne, School of Chemistry, Bio21 Molecular Science and Biotechnology Institute, 2010. / Typescript. Includes bibliographical references (p. 183-213)