Human African trypanosomiasis (HAT) is a disease that is in desperate need of new pharmacological agents active against the causative parasite, the flagellated protozoan Trypanosoma brucei. In this thesis, new metabolomics techniques have been developed to study pathways in response to drug action with the aim of defining the mode of action of current and future drugs. Eflornithine, a polyamine pathway inhibitor, was used as a proof of principle, revealing both expected changes that correlate well with the literature and unexpected changes that lead to pathways and metabolites not previously described in bloodstream form trypanosomes. One metabolite not previously described in trypanosomes is acetylornithine, whose levels correlate well with ornithine and whose production comes directly from ornithine transported from the medium. Nifurtimox and the nifurtimox- eflornithine combination therapy were assayed for changes to their metabolomes revealing changes in nifurtimox treatment that included alterations to sugar and purine levels. The combination therapy had reduced changes to some metabolites compared to each drug in isolation suggesting reasons for the combination‟s lack of synergy. Isotopically labelled metabolites were also of use in determining flux through the pathways identified as being affected by drug perturbation. These techniques, along with other biochemical techniques, were used to show arginase activity is absent in bloodstream form trypanosomes and that ornithine is not made from arginine when ornithine is present in the medium. Arginine can, however, be used to produce ornithine through an arginase-independent mechanism when exogenous ornithine is lacking. Evidence is also provided that parts of the pentose phosphate pathway, not thought to be active in bloodstream form trypanosomes, may still be active in in vitro grown cells. A mechanism of resistance to eflornithine involving the deletion of an amino acid transporter that is able to transport eflornithine is also described. It is hoped that simple PCR-based tests for this resistance mechanism will be of use in resistant foci in prescribing appropriate drugs to HAT patients.

Sample complexity is one of the key challenges facing contemporary proteomic analysis. A variety of methods is commonly employed to reduce this complexity, both at an intact protein and digested peptide level. For complex lysates containing many thousands of proteins, orthogonal (mutually independent), multidimensional separation methods must be employed to provide sufficient resolution to characterize the appropriate number of different species. The most common of these methods are two dimensional gel electrophoresis (2DGE) of proteins and multidimensional liquid chromatographic separation (MuDPIT) of peptides, which rely on isoelectric focusing followed by mass separation in the former, and ion exchange followed by reversed phase separation in the latter. These methods have significant drawbacks in terms of sample bias, sample preparation and reproducibility, and therefore a new methodology that combines the positive aspects of both separation technologies in an automatable, reproducible form is highly desirable. New developments in column technology have allowed rapid improved-resolution separation of intact proteins in complex samples, coupled to improved methodology for peptide and protein identification. The separation of complex protein mixtures using both on- and off-line 2D liquid chromatography using derivitized polystyrene-divinylbenzene (PS-DVB) pellicular ion-exchange resins and PS-DVB monolithic reversed-phase columns is described. Proteolytic digestion of the fractions followed by rapid liquid chromatography - tandem mass spectrometry was used to complete the analysis. An alternative methodology, relying on direct analysis of the second dimension eluents by top-down (mass spectrometric analysis of intact proteins) methodology, using an Apex IV 12 T Fourier-transform ion cyclotron resonance mass spectrometer (Bruker Daltonics) and an HCT ion trap (Bruker Daltonics) equipped with electron transfer dissociation has allowed in-depth analysis of intact proteins. Sample types investigated to establish the utility of the methodology include bacterial lysates (Bordetella parapertusis, and Escherichia coli), a eukaryotic parasite (Leishmania donovani), and transformed human cell lines. These developments lead to a multidimensional intact protein separation methodology suitable for small-sample proteomics (minimum effective protein load of 200 μg) and good reproducibility (1.5% variation in the ion exchange dimension, 0.5% variation in the reversed phase dimension). Analysis of the digested fractions gave good coverage of the proteome as well as a high predicted dynamic range, capable of detecting proteins with codon adaptation index scores ranging from 0.22 to 0.99, out of a logarithmic scale from 0 to 1. Proteins representing low (8 kDa) and high (500 kDa) molecular mass and extremes of predicted pI were identified, as well as a number of membrane proteins. Resolution of the overall protein separation was such that single protein species often occurred in one or two fractions for both the ion-exchange and reversed phase separations, with the fractions varying in complexity. Separation of modified proteins in the ion-exchange dimension demonstrated separation of isoforms. Most analyses coupled anion-exchange chromatography in the first dimension to monolithic reversed phase separation in the second dimension. To provide alternative first dimension methodologies for specific sample types, external gradient chromatofocussing (based on separation by isoelectric point) and high-pH ion-pair reversed phase were evaluated as additional techniques. In particular the pISEP (CryoBioPhysica) technique for chromatofocussing constituted an effective orthogonal separation methodology for separation, and its use in a proteomics context was compared to that of the original anion-exchange, reversed phase technology. To rapidly analyse the large number of fractions generated by the technique, bottom-up protocols required improvements in the throughput of peptide separations. PS-DVB monoliths were employed for rapid peptide separations and conditions for analysis were evaluated and optimized. The implementation of a parallel 200 μm monolith system for tryptic peptide separations ensured minimal sample loss and improved sample throughput with little loss of sensitivity. For simple mixtures, reversed-phase separation times could be reduced to a few minutes without significantly affecting data content, although rapid scanning capability is essential due to the very narrow peak widths. Quantitation is of paramount importance to any proteomic technique, and liquid chromatographic separation of intact proteins provides flexibility for differential analysis of complex samples. Three categories of quantitative analysis were evaluated for two dimensional intact protein separation by liquid chromatography: ultraviolet-absorbance maps, isotopic labeling and label-free computational analyses. UV-absorbance maps of wild-type and pentamidine resistant Leishmania donovani were compared using standard two dimensional gel electrophoresis analysis software and resulted in the identification of a small number of quantitiative differences. Modifications to the isotope coded affinity tag (ICAT) and isotope tags for relative and absolute quantitation (ITRAQ) protocols were developed to allow protein labeling prior to separation, and the related methodologies ExacTag, isotope coded protein label (ICPL) and stable isotope labels for amino acids in culture (SILAC) were evaluated. Finally, label-free techniques have been employed for protein quantitation by liquid chromatography (LC)/Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). A particular niche for liquid chromatographic separation of intact proteins is in top-down analysis, in which mass spectrometric analysis of intact proteins is performed. The separation technology was directly coupled to high-resolution FT-ICR MS for analysis of standards, mixtures, and cellular lysates, leading to the identification of an intact Leishmania protein and post-translational modification (PTM) mapping of histone H4. In addition, the recently developed electron transfer dissociation (ETD) fragmentation methods coupled with proton transfer reaction (PTR) for charge reduction allowed analysis of standards and isotopically labeled cellular lysates using an ion trap instrument. In summary, we have developed a general proteomic methodology with unique flexibility as well as applicability to top-down analysis. It has been applied to multiple complex samples in a variety of analysis conditions including quantitation methodologies and state-of-the-art mass spectrometric techniques. In general, the method equals other separation methods in terms of protein identification rates, is substantially more reproducible and automated, but is more time-consuming. Currently, two dimensional intact protein separation by liquid chromatography using polystyrene-divinylbenzene-based columns is particularly applicable to top-down analysis of heavily modified proteins. However, there is considerable remaining room for optimization and improvement of methodologies, and further development will enhance the technique for general use.

No description available.

A fruit and vegetable-rich diet has been associated with decreased risk of developing chronic diseases such as cardiovascular disease and cancer in humans. These protective effects have been attributed in part, to the presence of phytochemicals in fruit and vegetables, in particular flavonoids and phenolic compounds. Some plants have been used in traditional medicine for healing, ritual ceremonies and as health tonics or food supplements. Recent interest in the health-promoting properties of Malaysian traditional vegetables has been based on claims about their uses in health and medicine. However, scientific information to support these claims is largely unexplored. The overall objectives of the present study were to investigate, determine and quantify the phytochemicals, particularly phenolic compounds, in the seven samples from five species of selected Malaysian traditional vegetables (Anacardium occidentale, Centella asiatica, Colubrina asiatica, Pluchea indica and Premna cordifolia) and to evaluate their activities in vitro, including antioxidant and antibacterial activities of extracts of these plants and individual phytochemicals. In the first section of this project, discussed in Chapter 3, Malaysian traditional vegetable extracts were screened for phenolic compounds using several complimentary techniques, namely high performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry and the total phenolic content determined using the Folin-Ciocalteu assay. Flavonol glycosides were predominant in most of the species, particularly A. occidentale with levels ranging from 6434 to 12420 µg/g fresh weight. Chlorogenic acids were the main components identified and quantified in C. asiatica and P. indica. The total phenolic content of the vegetables were between 100 ± 7.8 and 415 ± 20 mg/ kg gallic acid equivalent (GAE) in batch 1 but lower in batch 2 ranging from 62 ± 2.5 to 386 ± 41 mg/ kg GAE. The total phenolic content of plant extracts was positively correlated with total antioxidant capacity, determined by 2, 2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and ferric reducing antioxidant potential (FRAP) assays. A. occidentale exhibited the highest total phenolic content and total antioxidant activity, whereas Colubrina asiatica, which had the lowest total phenolic content, also had low antioxidant activity in vitro. Phenolic content and antioxidant activity were significantly (p<0.05) influenced by environmental factors, as in this study, plant materials in batch 1 which was harvested in rainy season, had a higher total phenolic and antioxidant content than batch 2, which was harvested in the dry season. Based on the hypothesis that other components in addition to phenolics also contributed to the total antioxidant activities in the plants, the next objective, which was presented in Chapter 4, was to investigate the occurrence of phytochemicals such as triterpenes, carotenoids, α-tocopherol and vitamin C. The level of total triterpenes, biomarkers of C. asiatica was not significantly different between batches. The main component was madecassoside with 91 ± 4.8 µg/g fresh weight in batch 1 and 77 ± 3.4 µg/g fresh weight in batch 2. The level of carotenoids and vitamin C were low compared to previous reports. This was almost certainly due to dried samples being used in the present study, as some of the compounds would have broken down during drying process. This would have particularly affected the levels of vitamin C, which contributed only 0.9 to 5.5% to the total antioxidant activity of the plants under study. Total antioxidant activities of plant essential oils were determined using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and the result was in agreement to the total antioxidant activities of plant extracts, which A. occidentale having the highest amount. The highest antioxidant activity exhibited by A. occidentale oil was attributed to the presence of high amounts of γ-terpinene (28%) and terpinen-4-ol (4.2%), both of which were shown to have strong radical scavenging activity. The high phenolic content, antioxidant activity and occurrence of volatile components exhibited by A. occidentale has led to the final objective of this study, which is presented in Chapter 5. This was to screen for antimicrobial activities of A. occidentale extracts and essential oil against selection of Gram-positive (Enterococcus faecalis, Staphylococcus aureus, Meticillin-resistance Staphylococcus aureus (MRSA), coagulase negative Staphylococci (CoNS) and Lactobacillus acidophilus), Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) and fungi (Candida albican) using disc diffusion and minimum inhibitory concentration (MIC) methods. Investigation of the modes of action was determined using growth inhibition curve, scanning (SEM) and transmission (TEM) electron microscopy. A. occidentale was shown to have promising effects at 25 mg/ml with regard to inhibiting the growth of Gram-positive bacteria including MRSA. The essential oil and its major component, γ-terpinene at only 2.5% (v/v) inhibited the growth of all Gram-positive and Gram-negative bacteria. None of the A. occidentale extracts or oil exhibited antibacterial activities against Lactobacillus acidophilus, an important strain of bacteria found in the human gut. This indicates selective effects of A. occidentale. A. occidentale extract and oil inhibited the growth of S. aureus cells within a 2-hour incubation observed in time-kill experiments. SEM and TEM examination revealed that the oil and its component, γ-terpinene, inhibited the bacteria through bacteriostatic and bactericidal effects which damaged the bacterial cell wall. Testing the oil and γ-terpinene against epidemic-MRSA (EMRSA) biofilms indicated an anti-adhesive effect, which disrupted the bacterial colonies in the biofilms to produce more extracellular polysaccharides (EPS). The effects of A. occidentale oil were comparable with tea tree oil, a widely used topical antiseptic. All the Malaysian traditional vegetables under study are claimed to have medicinal properties and health effects. The results in the present study have provided some information on phytochemical and nutritional properties of Malaysian traditional vegetables, and as a consequence provide a sound scientific base for promoting their consumption particularly in Malaysia.

The mechanism of strand exchange by Tn3 resolvase was studied using FRET based methods. For this purpose Cy3 dye and a Cy5 dye were attached to modified thymines within Tn3 res site I, the DNA substrate of Tn3 resolvase. The dyes formed a FRET pair and followed the movements of the substrates, resulting in changes of the distance between both dyes and therefore changes in the FRET efficiency of the FRET pair which were monitored using fluorescence spectroscopy. A library of short, double-stranded substrates containing one copy of Tn3 res site I and Cy3 and/or Cy5 dyes attached to at different positions within site I was generated. Fluorescent substrates which do not interfere with the formation of synapses and with the recombination process were selected using gel-based assays. Fluorescent dyes attached at positions about five bases from the centre of site I were found to allow uninhibited recombination. Short double-stranded substrates with dyes attached at the selected positions within site I were studied in ensemble FRET experiments. Recombination of substrates containing a FRET pair with an excess of non-fluorescent substrates resulted in a strong decrease of the FRET efficiency due to the formation of recombinant products containing one dye each. This observation suggested that recombination and strand exchange could indeed be studied using FRET-based methods. The ensemble FRET experiment and FRET based experiments showed that the short substrates are usually recombined in both parallel and anti-parallel orientation. The lack of control over the orientation of the substrates in the synapses motivated the development of U-shaped substrates which consisted of two double-stranded arms, each containing one copy of site I, and a single stranded linker which connected both arms. In gel based assays, the U-shaped substrates were found to prefer the intramolecular recombination of both sites in one defined orientation. A U-shaped substrate containing a Cy5 and a Cy3 dye was studied in a single-molecule FRET experiment. In the presence of an activated Tn3 resolvase mutant, several specific FRET states and transitions between FRET states had been observed. The FRET states and transitions differed in the presence and absence of MgCl2 allowing the identification of a FRET state transition potentially corresponding to the conformational changes during ligation and several transitions corresponding to intermediate steps during strand exchange.