Natural products as sources of novel therapeutic agents experienced a steady increase from around the turn of the twentieth century until it peaked in the 1970s and 1980s. However since this time pharmaceutical research in natural products has experienced a decline. Despite this trend the natural products industry now seems to be experiencing a revival of sorts. This thesis represents a continuation of the work on the isolation and structure elucidation of potential drug leads from terrestrial fungal sources that the natural products group at the University of Canterbury is engaged in. The known compound, pseurotin A (2.7) and two novel diastereomers, pseurotin A2 (2.8) and pseurotin A3 (2.9) were isolated from the extract of a Penicillium sp. of fungus collected from the foreshore of a beach in Vancouver, Canada. The absolute stereochemistry of pseurotin A2 and proposed absolute stereochemistry for A3 were elucidated using a combination of X-ray crystallography (A2 only), circular dichrosim, oxidative cleavage reactions, and J2-resoved 2D NMR experiments. The extract of an as yet unidentified endophytic fungus has yielded eight novel compounds related to the spirobisnaphthalene class of compounds. These eight compounds fall into to distinct groupings. The spiro-mamakones, distinguished by a structurally unprecedented oxygenated spiro-nonene skeleton, comprise five compounds, spiro-mamakones A-E (3.11, 3.15-3.18). In addition to these naturally occurring compounds, the semi-synthetic compounds, 4-oxo-spiro-mamakone A (3.12) and O-acetyl-spiro-mamakone A (3.21), were also synthesised. spiro-Mamakone A was found to be racemic, while X-ray crystallography and optical rotation revealed spiro-mamakone C (3.15) to be present as an enantiomeric mixture (4S*, 5S*, 9R*). Unfortunately the enantiomeric excess was unable to be elucidated. NOE experiments revealed spiro-mamakone B (3.16) to have the relative stereochemistry 4S*, 5S*, 9S*. The relative stereochemistry of spiro-mamakones D (3.17) (4S*, 5S*, 8S*, 9S*) and E (3.18) (4S*, 5S*, 8S*, 9R*) was proposed from comparison of coupling constant calculations from energy-minimised models with those of the experimentally determined values. The second group, comprising three novel compounds named the mamakunoic acids, mamakunoic acid A-C (3.8, 3.7, 3.10), are characterised by their acid substituted dihydro benzofuran system. The low yield obtained of these compounds, unfortunately prevented their stereochemical elucidation. In addition to structure elucidation, biosynthetic studies on spiro-mamakone A and mamakunoic acid B were also carried out. Analysis of the NMR spectra derived from spiro-mamakone A, labelled with isotopic acetate, revealed a situation complicated by the presence of isotopomers and racemisation, resulting in NMR spectra that were somewhat anomalous in appearance. These irregularities however, were resolved leading to the proposal that spiro-mamakone A was derived from a dihydroxynaphthalene (DHN) intermediate, which proceeds through to spiro-mamakone via an epoxide intermediate. Despite problems with purity and low yields of isotopically labelled mamakunoic acid B, it was proposed that like spiro-mamakone A, it proceeded via a DHN intermediate. The extract derived from a Malaysian Scleroderma sp. was found to contain a new dichlorinated pulvinic acid derivative, methyl-3',5'-dichloro-4,4'-di-O-methylatromentate (4.14), the structure of which was confirmed by X-ray crystallography. In addition three previously reported compounds, 4,4'-dimethoxyvulpinic acid (4.11), methyl-3'-chloro-4,4'-di-O-methylatromentate (4.12) and methyl-4,4'-dimethoxyvulpinate (4.13), were also isolated. The extract of another, as yet unidentified endophytic fungus was found to contain the new acetogenin, 1,5-dihydroxy-6-(2-hydroxyethyl)-3-methoxyacetophenone (5.7), differing from the known compound, 2,4-dihydroxy-6-(2-hydroxyethyl)-3-methoxyacetophenone (5.8) only by virtue of the substitution pattern. The structure of 5.7 was confirmed by X-ray crystallography. The implementation of efficient dereplication procedures is paramount for those working in the field of natural products. The recent advances that have been made in the dereplication process in the natural products group at the University of Canterbury are given using examples from this research and where necessary from other group members.

Natural products as sources of novel therapeutic agents experienced a steady increase from around the turn of the twentieth century until it peaked in the 1970s and 1980s. However since this time pharmaceutical research in natural products has experienced a decline. Despite this trend the natural products industry now seems to be experiencing a revival of sorts. This thesis represents a continuation of the work on the isolation and structure elucidation of potential drug leads from terrestrial fungal sources that the natural products group at the University of Canterbury is engaged in. The known compound, pseurotin A (2.7) and two novel diastereomers, pseurotin A2 (2.8) and pseurotin A3 (2.9) were isolated from the extract of a Penicillium sp. of fungus collected from the foreshore of a beach in Vancouver, Canada. The absolute stereochemistry of pseurotin A2 and proposed absolute stereochemistry for A3 were elucidated using a combination of X-ray crystallography (A2 only), circular dichrosim, oxidative cleavage reactions, and J2-resoved 2D NMR experiments. The extract of an as yet unidentified endophytic fungus has yielded eight novel compounds related to the spirobisnaphthalene class of compounds. These eight compounds fall into to distinct groupings. The spiro-mamakones, distinguished by a structurally unprecedented oxygenated spiro-nonene skeleton, comprise five compounds, spiro-mamakones A-E (3.11, 3.15-3.18). In addition to these naturally occurring compounds, the semi-synthetic compounds, 4-oxo-spiro-mamakone A (3.12) and O-acetyl-spiro-mamakone A (3.21), were also synthesised. spiro-Mamakone A was found to be racemic, while X-ray crystallography and optical rotation revealed spiro-mamakone C (3.15) to be present as an enantiomeric mixture (4S*, 5S*, 9R*). Unfortunately the enantiomeric excess was unable to be elucidated. NOE experiments revealed spiro-mamakone B (3.16) to have the relative stereochemistry 4S*, 5S*, 9S*. The relative stereochemistry of spiro-mamakones D (3.17) (4S*, 5S*, 8S*, 9S*) and E (3.18) (4S*, 5S*, 8S*, 9R*) was proposed from comparison of coupling constant calculations from energy-minimised models with those of the experimentally determined values. The second group, comprising three novel compounds named the mamakunoic acids, mamakunoic acid A-C (3.8, 3.7, 3.10), are characterised by their acid substituted dihydro benzofuran system. The low yield obtained of these compounds, unfortunately prevented their stereochemical elucidation. In addition to structure elucidation, biosynthetic studies on spiro-mamakone A and mamakunoic acid B were also carried out. Analysis of the NMR spectra derived from spiro-mamakone A, labelled with isotopic acetate, revealed a situation complicated by the presence of isotopomers and racemisation, resulting in NMR spectra that were somewhat anomalous in appearance. These irregularities however, were resolved leading to the proposal that spiro-mamakone A was derived from a dihydroxynaphthalene (DHN) intermediate, which proceeds through to spiro-mamakone via an epoxide intermediate. Despite problems with purity and low yields of isotopically labelled mamakunoic acid B, it was proposed that like spiro-mamakone A, it proceeded via a DHN intermediate. The extract derived from a Malaysian Scleroderma sp. was found to contain a new dichlorinated pulvinic acid derivative, methyl-3',5'-dichloro-4,4'-di-O-methylatromentate (4.14), the structure of which was confirmed by X-ray crystallography. In addition three previously reported compounds, 4,4'-dimethoxyvulpinic acid (4.11), methyl-3'-chloro-4,4'-di-O-methylatromentate (4.12) and methyl-4,4'-dimethoxyvulpinate (4.13), were also isolated. The extract of another, as yet unidentified endophytic fungus was found to contain the new acetogenin, 1,5-dihydroxy-6-(2-hydroxyethyl)-3-methoxyacetophenone (5.7), differing from the known compound, 2,4-dihydroxy-6-(2-hydroxyethyl)-3-methoxyacetophenone (5.8) only by virtue of the substitution pattern. The structure of 5.7 was confirmed by X-ray crystallography. The implementation of efficient dereplication procedures is paramount for those working in the field of natural products. The recent advances that have been made in the dereplication process in the natural products group at the University of Canterbury are given using examples from this research and where necessary from other group members.

Yes / One of the main obstacles to therapeutic success in colorectal cancer (CRC) is the development of acquired resistance to treatment with drugs such as 5-fluorouracil (5-FU). Whilst some resistance mechanisms are well known, it is clear from the stasis in therapy success rate that much is still unknown. Here, a proteomics approach is taken towards identification of candidate proteins using 5-FU-resistant sublines of human CRC cell lines generated in house. Using a multiplexed stable isotope labelling with amino acids in cell culture (SILAC) strategy, 5-FU-resistant and equivalently passaged sensitive cell lines were compared to parent cell lines by growing in Heavy medium with 2D liquid chromatography and Orbitrap Fusion™ Tribrid™ Mass Spectrometry analysis. Among 3003 commonly quantified proteins, six (CD44, APP, NAGLU, CORO7, AGR2, PLSCR1) were found up-regulated, and six (VPS45, RBMS2, RIOK1, RAP1GDS1, POLR3D, CD55) down-regulated. A total of 11 of the 12 proteins have a known association with drug resistance mechanisms or role in CRC oncogenesis. Validation through immunodetection techniques confirmed high expression of CD44 and CD63, two known drug resistance mediators with elevated proteomics expression results. The information revealed by the sensitivity of this method warrants it as an important tool for elaborating the complexity of acquired drug resistance in CRC. / Sadr ul-Shaheed and the University of Bradford Proteomics Facility were supported by Yorkshire Cancer Research, UK (Cancer Medicine Discovery II, grant B381PA).

La compréhension de la fonction des protéines et des systèmes biologiques passe par une connaissance fine des mécanismes moléculaires sous-jacents. La cristallographie et la résonance magnétique nucléaire permettent d'appréhender ces mécanismes au niveau atomique en fournissant des informations sur la structure et sur la dynamique des macromolécules biologiques. Nous nous sommes ainsi intéressés à deux protéines, la lipase lip2 de la levure Yarrowia lipolytica et la protéine membranaire OmpA de la bactérie Klebsiella pneumoniae. Nous avons recherché des conditions d'expression de la protéine lip2 marquée uniformément ou spécifiquement sur une boucle (appelée " lid ") afin d'en étudier la dynamique. Des conditions de marquage uniforme à l'azote 15 de lip2 recombinante dans Yarrowia lipolytica ont été mises au point, mais le marquage acide aminé spécifique n'a pu être réalisé à cause de phénomènes de dilution isotopique trop importants dans cette levure. Nous avons résolu par cristallographie aux rayons X la structure du domaine C-terminal de la protéine OmpA et étudié sa dynamique en solution par RMN (techniques de relaxation 15N). Nous avons caractérisé la dynamique de son domaine N-terminal membranaire reconstitué en liposomes par RMN du solide : en utilisant la rotation à l'angle magique à 60kHz et à la détection 1H sur un spectromètre 1 GHz, nous avons pu attribuer une majorité des résonances du tonneau ? et établir un profil de paramètre d'ordre des vecteurs NH. Des expériences de protéolyse ménagée ont révélé par ailleurs un site de coupure unique à la trypsine au sein de la boucle extracellulaire L3. Enfin, une première caractérisation de la protéine complète exprimée dans la membrane externe d'Escherichia coli a été entreprise par RMN du solide sur membranes externes natives. / Understanding the function of proteins and biological systems requires an accurate knowledge of the underlying molecular mechanisms. Crystallography and nuclear magnetic resonance provide a detailed description of these mechanisms, with an atomic resolution, by providing data on both structures and motions. We investigated two proteins, the lip2 lipase from the yeast Yarrowia lipolytica and the membrane protein OmpA from the bacteria Klebsiella pneumoniae. We tried to produce lip2 with uniform and amino-acid specific stable isotope labelling on its functional loop (the lid) for NMR experiments. The homologous recombinant expression in Yarrowia lipolytica turned out to be the most efficient for uniform labelling but failed for specific labelling due to extensive isotope scrambling. We solved the structure of OmpA C-terminal domain by X-ray crystallography, and analyzed its dynamics in solution by NMR (15N relaxation techniques). We characterized its transmembrane N-terminal domain in proteoliposomes by solid state NMR: using state of the art ultra-fast MAS (60 kHz), 1H detection and a 1 GHz spectrometer, we could assign most ?-barrel resonances and establish a NH order parameter profile. In a complementary approach, we used proteolysis to reveal a unique trypsin cleavage site on the extracellular loop 3. Finally, a first characterization of the full-length protein expressed in the outer membrane of Escherichia coli was initiated by solid state NMR on intact outer membranes.

Dynamics of carbon and phosphorus flows in arbuscular mycorrhizal symbiosis Mgr. Tereza Konvalinková (doctoral thesis) Abstract Arbuscular mycorrhizal fungi (AMF) are widespread and highly specialized root symbionts, which gain all of their carbon (C) from the hosts, supplying plants with mineral nutrients (particularly with phosphorus, P) in return. This thesis focuses on the size and flexibility of C and P flows in arbuscular mycorrhiza in relation to environmental conditions, in particular to light and P availability. The indications that the symbiotic flows are regulated actively by both partners are discussed. The main findings are presented as a compilation of separate scientific works (two research articles, one review and one book section). A glasshouse experiment has shown that both mycorrhizal benefits and mycorrhizal colonization of medic (Medicago truncatula) by an AMF species (R. irregularis) decline along the gradient of decreasing light intensity. Interestingly, morphological adaptation of medic to the long-term light deprivation was boosted by mycorrhiza, probably because of C demand of AMF and due to the improved nutrition of the mycorrhizal plants. On the other hand, sudden 6-day shading caused rapid decline of shoot P content of mycorrhizal plants, accompanied with the accumulation of P...

The aim of this thesis was to develop stable-isotope steady-state metabolic flux analysis (MFA) based on ¹³C labeling to quantify intracellular fluxes of central carbon metabolism in plants. The experiments focus on the analysis of a heterotrophic cell suspension culture of Arabidopsis thaliana (L) Heynh. (ecotype Landsberg erecta). The first objective was to develop a robust methodology based on combining high quality steady-state stable labeling data, metabolic modeling and computational analysis. A comprehensive analysis of the factors that influence the outcome of MFA was undertaken and best practice established. This allowed a critical analysis of the subcellular compartmentation of carbohydrate oxidation in the cell culture. The second objective was to apply the methodology to nutritional perturbations of the cell suspension. A comparison of growth on different nitrogen sources revealed that transfer to an ammonium-free medium: (i) increased flux through the oxidative pentose phosphate pathway (oxPPP) by 10% relative to glucose utilisation; (ii) caused a substantial decrease in entry of carbon into the tricarboxylic acid cycle (TCA); and (iii) increased the carbon conversion efficiency from 55% to 69%. Although growth on nitrate alone might be expected to increase the demand for reductant, the cells responded by decreasing the assimilation of inorganic N. Cells were also grown in media containing different levels of inorganic phosphate (Pi). Comparison of the flux maps showed that decreasing Pi availability: (i) decreased flux through the oxPPP; (ii) increased the proportion of substrate fully oxidised by the TCA cycle; and (iii) decreased carbon conversion efficiency. These changes are consistent with redirection of metabolism away from biosynthesis towards cell maintenance as Pi is depleted. Although published genome-wide transcriptomic and metabolomic studies suggest that Pi starvation leads to the restructuring of carbon and nitrogen metabolism, the current analysis suggests that the impact on metabolic organisation is much less extreme.