This thesis describes the study of the three-dimensional structure and dynamics of the hepatitis B virus (HBV) encapsidation signal, epsilon, by means of nuclear magnetic resonance (NMR) and mutational data. HBV replicates by reverse transcription of an RNA pregenome into the viral DNA genome, which becomes enclosed in viral particles (encapsidation). Epsilon is a stem-loop structure within the RNA pregenome and both the primary sequence and secondary structure of epsilon are strongly conserved, in agreement with its essential function of propagating HBV. Epsilon is therefore a potential target for drug design. Studying the structure of epsilon requires development of new methods in the field of structural biology, as it is such a large RNA. Knowing the structure of epsilon will help to better understand the encapsidation mechanism and priming step of reverse transcription. This will help us in the search for antiviral drugs that block epsilon and prevent the viral reverse transcriptase from binding. NMR spectroscopy is a method that provides detailed structural and dynamical data in solution under natural conditions. However, the size of the molecules that can be studied with NMR is limited. NMR spectra become more and more difficult to interpret as the size of the molecule increases. To circumvent this problem, large RNA molecules can be divided into smaller parts and only the parts essential for NMR studies are selected. The information obtained from these smaller fragments can then be used to determine the structure of the larger molecule. Furthermore, a new method of enzymatically synthesizing nucleoside triphosphates with isotopes suitable for NMR has made it possible to specifically label the RNA molecules. Using this method it is possible to derive highly detailed molecular structures of RNA up to a size of 150 nucleotides. The method of selective isotope labelling was applied to different parts of HBV epsilon. Three RNA fragments of 27 (apical loop), 36 (internal bulge) and 61 (whole epsilon) nucleotides (nt) were synthesized in the unlabelled form. The 27-nt and 36-nt RNAs were also synthesized with (13C, 15N, 1', 3', 4', 5', 5"-2H5)-labelled uridines. The 61-nt sequence was (13C, 15N)-guanidine labelled. This labelling allowed unambiguous assignment of otherwise inaccessible parameters. The unlabelled and labelled RNA sequences provided the necessary data for structure derivation of the whole epsilon. The apical loop of epsilon forms a pseudo-triloop motif. There is only one conformation of the loop that fulfils all the restraints, including experimental chemical shifts. However, the loop adopts several structures that fulfil the experimental distance, torsion angle and residual dipolar coupling restraints. This may reflect true flexibility. Indeed, relaxation studies on the unlabelled and labelled 27-nt sequences show that the residues that show multiple conformations are flexible. This can be an important feature for the recognition and subsequent binding of epsilon to the viral polymerase. The information gained on the HBV encapsidation signal is useful in our understanding of the initiation of replication of the virus. This can in turn contribute to the search for drugs against HBV.

No description available.

Recombinant protein production by mammalian cells is a core component of today’s multi-billion dollar biopharmaceutical industry. Transcriptome and proteome technologies have been used to probe for cellular components that correlate with higher cell-specific productivity, but have yet to yield results that can be translated into practical metabolic engineering strategies. The recognition of cellular complexity has led to an increasing adoption of systems biology, a holistic investigation approach that aims to bring together different omics technologies and to analyze the resulting datasets under a unifying context. Fluxomics is chosen as the platform context to investigate cell metabolism because it captures the integrated effects of gene expression, enzyme activity, metabolite availability and regulation, thereby providing a global picture of the cell’s metabolic phenotype. At present, the routine quantification of cell metabolism revolves around very basic cellular parameters: growth, substrate utilization and product formation. For a systems approach, however, just measuring gross metabolic features is insufficient; we are compelled to perform high-resolution, large-scale fluxomics in order to match the scale of other omics datasets. The challenges of performing large-scale fluxomics come from two opposing fronts. Metabolic flux analysis (MFA) is the estimation of intracellular fluxes from experimental data using a stoichiometric model, a process very much susceptible to modelling biases. The in silico challenge is to construct the most comprehensive model to represent the metabolism of a specific cell, while the in vivo challenge is to resolve as many fluxes as possible using experimental measurements or constraints. A compromise needs to be established between maximizing the resolution of the MFA model and working within technical limitations of the flux experiment. Conventional MFA models assembled from textbook pathways have been available for animal cell culture for the past 15 years. A state-of-the-art model was developed and used to analyse continuous hybridoma culture and batch CHO cell culture data (Chapter 3). Reasonable metabolic assumptions combined with constraint based analysis exploiting irreversibility constraints enabled the resolution of most fluxes in central carbon metabolism. However, while the results appear consistent, there is insufficient information in conventional measurement of uptake, secretion and growth data to assess the completeness of the model and validity of all assumptions. 13C metabolic flux analysis (13C MFA) can potentially resolve fluxes in the central carbon metabolism using flux constraints generated from 13C enrichment patterns of metabolites, but the multitude of substrate uptakes (glucose and amino acids) seen in mammalian cells, in addition to the lack of 13C enrichment data from proteinogenic amino acids, makes it very difficult to anticipate how a labelling experiment should be carried out. The challenges above have led to the development of a systematic workflow to perform large-scale MFA for mammalian cells. A genome-scale model (GeMs), an accurate compilation of gene-protein-reaction-metabolite associations, is the starting basis to perform whole-cell fluxomics. A semi-automated method was developed in order to rapidly extract a prototype of GeM from KEGG and UniProtKB databases (Chapter 4). Core metabolic pathways in the mouse GeM are mostly complete, suggesting that these databases are comprehensive and sufficient. The rapid prototyping system takes advantage of this, making long term maintenance of an accurate and up-to-date GeM by an individual possible. A large number of under-determined pathways in the mouse GeM cannot be resolved by 13C MFA because they do not produce any distinctive 13C enrichment patterns among the carbon metabolites. This has led to the development of SLIPs (short linearly independent pathways) for visualizing these under-determined metabolic pathways contained in large-scale GeMs (Chapter 5). Certain SLIPs are subsequently removed based on careful consideration of their pathway functions and the implications of their removal. A majority of SLIPs have a cyclic configuration, sharing similar redox or energy co-metabolites; very few represent true conversion of substrates to products. Of the 266 under-determined SLIPs generated from the mouse GeM, only 27 SLIPs were incorporated into the final working model under the criterion that they are significant pathways and are potentially resolvable by tracer experiments. Most of these SLIPs are degradation pathways of essential amino acids and inter-conversion of non-essential amino acids (Chapter 8). In parallel, OpenFLUX was developed to perform large-scale isotopic 13C MFA (Chapter 6). This software was built to accept multiple labelled substrates, and no restriction has been placed on the model type or enrichment data. These are necessary features to support large-scale flux analysis for mammalian cells. This was followed by the development of a design strategy that uses analytical gradients of isotopomer measurements to predict resolvability of free fluxes, from which the effectiveness of various 13C experimental scenarios using different combinations of input substrates and isotopomer measurements can be evaluated (Chapter 7). Hypothetical and experimental results have confirmed the predictions that, when glucose and glutamate/glutamine are simultaneously consumed, two separate experiments using [U-13C]- and [1-13C]-glucose, respectively, should be performed. If there is a restriction to a single experiment, then the 80:20 mixture of [U-13C]- and [1-13C]-glucose can provide a better resolution than other labelled glucose mixtures (Chapter 7 and Chapter 8). The tools and framework developed in this thesis brings us within reach of performing large-scale, high-resolution fluxomics for animal cells and hence realising systems-level investigation of mammalian metabolism. Moreover, with the establishment of a more rigorous, systematic modelling approach and higher functioning computational tools, we are now at a position to validate mammalian cell culture flux experiments performed 15 years ago.

Recombinant protein production by mammalian cells is a core component of today’s multi-billion dollar biopharmaceutical industry. Transcriptome and proteome technologies have been used to probe for cellular components that correlate with higher cell-specific productivity, but have yet to yield results that can be translated into practical metabolic engineering strategies. The recognition of cellular complexity has led to an increasing adoption of systems biology, a holistic investigation approach that aims to bring together different omics technologies and to analyze the resulting datasets under a unifying context. Fluxomics is chosen as the platform context to investigate cell metabolism because it captures the integrated effects of gene expression, enzyme activity, metabolite availability and regulation, thereby providing a global picture of the cell’s metabolic phenotype. At present, the routine quantification of cell metabolism revolves around very basic cellular parameters: growth, substrate utilization and product formation. For a systems approach, however, just measuring gross metabolic features is insufficient; we are compelled to perform high-resolution, large-scale fluxomics in order to match the scale of other omics datasets. The challenges of performing large-scale fluxomics come from two opposing fronts. Metabolic flux analysis (MFA) is the estimation of intracellular fluxes from experimental data using a stoichiometric model, a process very much susceptible to modelling biases. The in silico challenge is to construct the most comprehensive model to represent the metabolism of a specific cell, while the in vivo challenge is to resolve as many fluxes as possible using experimental measurements or constraints. A compromise needs to be established between maximizing the resolution of the MFA model and working within technical limitations of the flux experiment. Conventional MFA models assembled from textbook pathways have been available for animal cell culture for the past 15 years. A state-of-the-art model was developed and used to analyse continuous hybridoma culture and batch CHO cell culture data (Chapter 3). Reasonable metabolic assumptions combined with constraint based analysis exploiting irreversibility constraints enabled the resolution of most fluxes in central carbon metabolism. However, while the results appear consistent, there is insufficient information in conventional measurement of uptake, secretion and growth data to assess the completeness of the model and validity of all assumptions. 13C metabolic flux analysis (13C MFA) can potentially resolve fluxes in the central carbon metabolism using flux constraints generated from 13C enrichment patterns of metabolites, but the multitude of substrate uptakes (glucose and amino acids) seen in mammalian cells, in addition to the lack of 13C enrichment data from proteinogenic amino acids, makes it very difficult to anticipate how a labelling experiment should be carried out. The challenges above have led to the development of a systematic workflow to perform large-scale MFA for mammalian cells. A genome-scale model (GeMs), an accurate compilation of gene-protein-reaction-metabolite associations, is the starting basis to perform whole-cell fluxomics. A semi-automated method was developed in order to rapidly extract a prototype of GeM from KEGG and UniProtKB databases (Chapter 4). Core metabolic pathways in the mouse GeM are mostly complete, suggesting that these databases are comprehensive and sufficient. The rapid prototyping system takes advantage of this, making long term maintenance of an accurate and up-to-date GeM by an individual possible. A large number of under-determined pathways in the mouse GeM cannot be resolved by 13C MFA because they do not produce any distinctive 13C enrichment patterns among the carbon metabolites. This has led to the development of SLIPs (short linearly independent pathways) for visualizing these under-determined metabolic pathways contained in large-scale GeMs (Chapter 5). Certain SLIPs are subsequently removed based on careful consideration of their pathway functions and the implications of their removal. A majority of SLIPs have a cyclic configuration, sharing similar redox or energy co-metabolites; very few represent true conversion of substrates to products. Of the 266 under-determined SLIPs generated from the mouse GeM, only 27 SLIPs were incorporated into the final working model under the criterion that they are significant pathways and are potentially resolvable by tracer experiments. Most of these SLIPs are degradation pathways of essential amino acids and inter-conversion of non-essential amino acids (Chapter 8). In parallel, OpenFLUX was developed to perform large-scale isotopic 13C MFA (Chapter 6). This software was built to accept multiple labelled substrates, and no restriction has been placed on the model type or enrichment data. These are necessary features to support large-scale flux analysis for mammalian cells. This was followed by the development of a design strategy that uses analytical gradients of isotopomer measurements to predict resolvability of free fluxes, from which the effectiveness of various 13C experimental scenarios using different combinations of input substrates and isotopomer measurements can be evaluated (Chapter 7). Hypothetical and experimental results have confirmed the predictions that, when glucose and glutamate/glutamine are simultaneously consumed, two separate experiments using [U-13C]- and [1-13C]-glucose, respectively, should be performed. If there is a restriction to a single experiment, then the 80:20 mixture of [U-13C]- and [1-13C]-glucose can provide a better resolution than other labelled glucose mixtures (Chapter 7 and Chapter 8). The tools and framework developed in this thesis brings us within reach of performing large-scale, high-resolution fluxomics for animal cells and hence realising systems-level investigation of mammalian metabolism. Moreover, with the establishment of a more rigorous, systematic modelling approach and higher functioning computational tools, we are now at a position to validate mammalian cell culture flux experiments performed 15 years ago.

The topic of this thesis is the study of nitrogen functionalities in nitrogen-doped reduced graphene oxide using Raman spectroscopy. Specifically, the project set out to investigate if the Raman active nitrogen-related vibrational modes of graphene can be identified via isotope labelling. Previous studies have used Raman spectroscopy to characterise nitrogen doped graphene, but none has employed the method of isotope labelling to do so. The study was conducted by producing undoped, nitrogen-doped and nitrogen-15-doped reduced graphene oxide and comparing the differences in the first-order Raman spectrum of the samples. Results of this study are inconclusive. However, some indications linking the I band to nitrogen functionalities are found. Also, a hypothetical Raman band denoted I* possibly related to \spt{3} hybridised carbon is introduced in the same spectral area as I. This indication of a separation of the I band into two bands, each dependent on one of these factors could bring clarity to this poorly understood spectral area. As the results of this study are highly speculative, further research is needed to confirm them and the work presented here serves as a preliminary investigation.

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.

No description available.

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.

No description available.

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).