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Actinobacteria, primarily the genus Streptomyces, have led to the development of a number of antibiotics, which result from their secondary metabolites or modified derivatives. Secondary metabolite production can result from competition with neighboring microbes in an effort to disrupt growth, aiding in the competition for vital nutrients in impoverished conditions. Such secondary metabolites have the potential to affect a plethora of cellular functions in target cells, including, cell wall development, protein synthesis, protein function and fatty acid synthesis/metabolism. Due to the pandemic spread of antibiotic resistant bacteria, it is imperative to continue the search for new therapeutic agents targeting these deadly organisms. As such, our group explored soil and marine samples from Tampa Bay’s surrounding farmlands and waterways for secondary metabolite producing microbes using culture methods specific to Actinobacteria. Through these efforts we isolated over 750 bacterial species, of which almost half are confirmed Actinobacteria. In an attempt to derive new and novel chemistry from these organisms, we used our novel collection, and developed techniques for epigenetic modification to un-silence dormant and cryptic metabolic pathways. Our work reveals that a number of these Actinobacteria produce secondary metabolites that are effective against the ESKAPE pathogens, some at very low concentrations. Although the bioactivity from secondary metabolites is a well-known source for antibiotic drug discovery, our epigenetic methods suggest a potential to isolate previously overlooked compounds that have a very real possibility for use as antibacterial therapeutics.

En plus de leurs propriétés structurales comme constituants majeurs des membranes biologiques des cellules, les lipides jouent de nombreux rôles dans la signalisation cellulaire, le stockage d’énergie et le transport de protéines. Leurs importances biologiques ont mené à une augmentation accrue des méthodes analytiques pour la caractérisation d’espèces moléculaires uniques. De récents progrès en spectrométrie de masse ont amené à la caractérisation et à la quantification des espèces moléculaires des lipides dans des extraits lipidiques bruts (Han and Gross, 2005; Murphy et al., 2001). Par exemple, les espèces moléculaires de phospholipides peuvent être identifiées spécifiquement par leur tête polaire, la nature de leurs chaînes d’acide gras et leur positionnement au niveau du squelette glycérol. / In addition to their structural properties as main constituents of biological membranes, lipids play a multitude of roles such as in cell signalling, energy storage, and protein transport. Their biological importance has led to an increasing focus on analytical methods for the characterisation of their individual molecular species. Improvements in mass spectrometric technology has provided a great advantage for the characterisation and quantification of molecular lipid species in total lipid extracts (Han and Gross, 2005; Murphy et al., 2001). For instance, phospholipid molecular species can be identified on the basis of a characteristic fragment of the lipid class, the nature of the acyl chains and their positions on the glycerol backbone.A method allowing the quantitative profiling of the yeast lipidome was developed in a recent study using automated shotgun infusion strategy (Ejsing et al., 2009). We applied this method to characterise several lysophospholipid acyltransferase yeast mutants produced using reverse-genetics. These enzymes are involved in essential biological processes like de novo synthesis or remodelling of the phospholipid membrane component (Testet et al., 2005; Le Guedard et al., 2009). The comparative analysis of phospholipid molecular species from the wild-type strain and the corresponding deletion mutants has allowed us to identify lipid compositional changes, and has given us significant indications about the in vivo function of the encoded lysophospholipid acyltransferases.

The niche in which stem cells reside is involved in the regulation of stem cell fate, such as differentiation and self-renewal, by providing ECM proteins, growth factors, cell-cell interactions and balancing chemical factors such as the level of oxygen and pH. ECM proteins are involved in maintaining stemness of stem cells and in regulating differentiation via integrin-mediated signalling. Following the interaction of ECM proteins with integrins, integrins cluster and interact with large complexes of signalling proteins. These adhesion complexes have been reported to contain at least 150 proteins, which have been termed the adhesome. Adhesion complex proteins interact with the actin cytoskeleton and signalling pathways to play an essential role in stem cell fate. The hypothesis in this study was that the interaction of stem cells via integrin receptors with ECM proteins, lead to changes in the abundance or composition of adhesion complexes, which potentially activates signalling pathways involved in either maintaining or differentiation of stem cells. In this study, three principal advances have been made:First, a method was developed using ligand-coated magnetic beads for the isolation of integrin-associated complexes from pluripotent human embryonic stem cells (hESCs). The isolated integrin-associated complexes from hESCs were analysed by proteomic methods, which led to the detection of key integrin-associated adhesion proteins such as talin, vinculin, alpha actinin 4, filamin B, filamin C and zyxin. Second, isolation of integrin-associated complexes from multipotent MSCs was performed using a method based on “de-roofing” MSCs from FN or PDL coated plastic dishes, leading to the detection of key adhesome components by mass spectrometry. Ontological analysis of proteins enriched on FN demonstrated the enrichment of adhesion complexes. Third, following the induction of multipotent MSCs into early adipogenic MSCs and the isolation of integrin-associated complexes from early adipogenic MSCs and undifferentiated MSCs, core adhesome components were identified in induced and non-induced MSCs, with induction hypothesised to cause putative changes in the FN-induced adhesome network. Also, the level of adhesion complexes increased significantly in MSCs on FN upon induction into adipocytes compared to non-induced MSCs on FN and versus the control as shown by bioinformatics analysis. This data led to the hypothesis that upon induction of MSCs into adipocytes the abundance of proteins in integrin-associated complexes or the number of adhesion complexes increases.In conclusion, in this study two biochemical affinity methods were developed for the isolation of integrin-associated complexes from hESCs and MSCs, using ligand coated magnetic beads and ligand-coated plastic dishes. The development of these methods led to the isolation of adhesion-related proteins from pluripotent hESCs and differentiated MSCs and the detection of a pattern of changes in the abundance of adhesion related proteins in differentiated MSCs incubated on FN. The development of methods for the isolation of adhesion related complexes from stem cells can lead to a better understanding of the role of adhesion in differentiation and maintenance of pluripotency in stem cells. A better understanding of adhesion could have future implications in obtaining pure populations of undifferentiated stem cells for cell-based therapies and differentiated cells for the use in tissue engineering and repair.

See Abstract on Page 2 / Dissertation (MSc. (Chemistry))--University of Pretoria, 2007. / Chemistry / unrestricted

Existing instrumental techniques must be adaptable to the analysis of novel explosives if science is to keep up with the practices of terrorists and criminals. The focus of this work has been the development of analytical techniques for the analysis of two types of novel explosives: ascorbic acid-based propellants, and improvised mixtures of concentrated hydrogen peroxide/fuel. In recent years, the use of these explosives in improvised explosive devices (IEDs) has increased. It is therefore important to develop methods which permit the identification of the nature of the original explosive from post-blast residues. Ascorbic acid-based propellants are low explosives which employ an ascorbic acid fuel source with a nitrate/perchlorate oxidizer. A method which utilized ion chromatography with indirect photometric detection was optimized for the analysis of intact propellants. Post-burn and post-blast residues if these propellants were analyzed. It was determined that the ascorbic acid fuel and nitrate oxidizer could be detected in intact propellants, as well as in the post-burn and post-blast residues. Degradation products of the nitrate and perchlorate oxidizers were also detected. With a quadrupole time-of-flight mass spectrometer (QToFMS), exact mass measurements are possible. When an HPLC instrument is coupled to a QToFMS, the combination of retention time with accurate mass measurements, mass spectral fragmentation information, and isotopic abundance patterns allows for the unequivocal identification of a target analyte. An optimized HPLC-ESI-QToFMS method was applied to the analysis of ascorbic acid-based propellants. Exact mass measurements were collected for the fuel and oxidizer anions, and their degradation products. Ascorbic acid was detected in the intact samples and half of the propellants subjected to open burning; the intact fuel molecule was not detected in any of the post-blast residue. Two methods were optimized for the analysis of trace levels of hydrogen peroxide: HPLC with fluorescence detection (HPLC-FD), and HPLC with electrochemical detection (HPLC-ED). Both techniques were extremely selective for hydrogen peroxide. Both methods were applied to the analysis of post-blast debris from improvised mixtures of concentrated hydrogen peroxide/fuel; hydrogen peroxide was detected on variety of substrates. Hydrogen peroxide was detected in the post-blast residues of the improvised explosives TATP and HMTD.

In the new era of drug abuse, the proliferation of new psychoactive substances (NPS), commonly referred to as designer drugs or legal highs, has been a global concern. These substances are produced to circumvent current legislation for controlled substances with minor modifications in their chemical structure. Although many efforts have been made previously, the characterization of such substances are still challenging because of (1) the continual emergence of newly identified substances, (2) the lack of a universal screening test for NPS that are structurally similar to each other, and (3) the complex and time-consuming chromatographic techniques currently used. Therefore, it is necessary to develop novel analytical methods that can be readily adapted by forensic laboratories to overcome these challenges. In this dissertation, various analytical techniques have been evaluated for qualitative analysis of these emerging NPS. For rapid screening purposes, a commercial ion mobility spectrometry with a 63Ni ion source (63Ni-IMS) and also direct analysis in real time coupled to a quadrupole time-of-flight mass spectrometer (DART-QTOF-MS) were investigated first. The results showed that rapid detection by 63Ni-IMS and identification by DART-QTOF-MS can be achieved with sub-nanogram detection capability and high speed total analysis time less than two minutes. In recent developments of gas chromatography mass spectrometry (GC-MS), gas chromatography (GC) has been coupled to state-of-the-art mass spectrometers, including triple quadrupole (MS/MS) and quadrupole time-of-flight (QTOF). It was revealed that the application of GC-MS/MS and GC-QTOF facilitates the unambiguous identification of emerging NPS with a chemical ionization (CI) source. In addition, constitutional isomers of NPS were differentiated with the capabilities of product ion scan and multiple reaction monitoring (MRM) modes. Finally, the coupling of IMS with a mass spectrometer (IMS-MS) was investigated as an alternative confirmatory technique. With the development of an optimal solvent system in the electrospray ionization (ESI) process, the rapid analysis and identification of synthetic cathinone was successfully achieved less than five minutes. As a proof-of-concept, seized drugs samples provided by a local forensic laboratory were analyzed using these developed methods by various analytical techniques. The results from these seized samples are also presented in this evaluation.

(i) Hydrogen Bonding Studies - The nature of hydrogen bonding in solutions of alcohols, ROH, in various solvents has been studied using nuclear resonance techniques. Data obtained from dilution-shift curves for the OH protons in alcohols have been combined with information derived from infra-red investigations of the OH stretching regions in these compounds. The information obtained has been interpreted in terms of three effects, (i) The electronic effects of the group R. (ii) The steric effects of the group R. (iii) Effects due to other forms of molecular association. On this basis, deductions have been made concerning the degree and type of association in these compounds. The relative hydrogen bonding strengths have been predicted in some instances. The importance of steric inhibition of hydrogen bonding by bulky substituent groups has also been demonstrated. Some dilution-shift studies of acrylic acid in various solvents have been carried out. Studies of the effect of temperature on the OH proton resonances of alcohols have led, amongst other things, to a value for the average hydrogen bond energy in a methanol / carbon tetrachloride solution. The temperature-shift curves for a variety of ortho-substituted phenols have also been obtained and discussed in the light of existing infra-red spectral evidence concerning the nature of hydrogen bonding in these substances. The relation between the association shifts and the integrated absorption intensities of alcohols has been discussed. A correlation between these two quantities was found for alcohols of a similar type. (ii) Studies of Unstable Positive Ions. (a) Triphenyl Carbonium Ions. The NMR spectra of a variety of substituted triphenyl carbonium ions in a trifluoroacetic acid trifluoroacetic anhydride solvent have been obtained at 40 Mc and 60 Mc. No unequivocal evidence as to the structures of these compounds has been obtained, i.e. no distinction between 'symmetrical propeller’ and assymetric forms was possible, due to the presence of exchange effects. The data have given information about the changes in electron density in the aromatic rings due to the various substituent groups. Partial assignments of the aromatic proton spectra have been given. The importance of hyperconjugative electron release by aliphatic substituents is indicated. Some preliminary investigations of the protonated form of 1,1-Di-p-anisylethylene have also been carried out. (b) The l⁺ Ion. The NMR spectra of solutions of iodine in oleum have been investigated to try and shed light on the possibility of the existence of the l⁺ ion in such systems. The measured broadenings and shifts of the oleum proton resonances at various iodine concentrations have been interpreted in terms of the presence of this species, which should be paramagnetic. A value for the magnetic moment of this ion has been obtained. Other evidence for the existence of the l⁺ ion has been fully discussed. / Science, Faculty of / Chemistry, Department of / Graduate

The aim of thesis was to establish a methodology for 210Pb measurements by Accelerator Mass Spectrometry (AMS). The potential application is to measure 210Pb in people who have been exposed to radon. This will better our understanding of radon toxicity, which is not possible now with current radiometric and mass spectrometry techniques. The determination of 210Pb by AMS was done in two major studies 1) Studying Pb chemistry in a Cs+ sputter source used in AMS and 2) Evaluating 204,205 & 208Pb spikes for the quantification of 210Pb by isotope dilution. Pb chemistry was investigated using an 834 SIMS-type and a SO-110 Cs+ sputter source at the IsoTrace Laboratory and A.E Lalonde AMS facility, respectively. Different molecular anions of Pb were studied with the 834 SIMS-type Cs+ sputter source and the strongest molecular anion current of Pb and thus greatest ionization efficiency was achieved form the superhalogen PbF3-. The average 208PbF3- current was unaffected by varying the ratio of the fluorinating compounds (AgF2 and CsF) packed into a target. The average current of 208PbF3- was reproducibly increased by chemically mixing the targets of AgF2, CsF and PbF2 in concentrated HF rather than mechanically mixing them the powders with a stir rod. The count rate of 210Pb reproducibly increased by a factor of 20 when μg quantities of PbF2 were present in mg AMS targets compared to AMS targets that had pg quantities of PbF2. The average current of 208PbF3- for pure PbF2 targets in an SO-100 Cs+ sputter source was reproducibly increased when the Cs+ flux was decreased by a factor of 10. This phase of my work maximized the overall efficiency of PbF3-, to a value of 1.8x10-10 ±8x10-11s-, which was a key first step in the measurement by AMS. Then isotope dilution was tested to quantify 210Pb and the next stage of my work evaluated the use of 204,205 & 208Pb spikes. 210Pb was measured in the +3 charge state by isotope dilution assays using 204,205 & 208Pb spikes. 204Pb+3 reproducibly suffered from the molecular interference from 68Zn3+3, which could not be easily removed without negatively impacting the detection limit for 210Pb. 205Pb+3 continually suffered from 205Tl+3 interference which could be readily be removed but not without negatively affecting the II detection limit for 210Pb. 208Pb+3 suffers from no molecular interferences but if a large amount of 208Pb is needed to swamp the Pb in a sample, this could limit the detection limit for 210Pb as the abundance sensitivity is 210Pb/208Pb=1.3×10-12. A calibration curve is required when 208Pb is used as a spike due to a difference in collection efficiency of a Faraday cup, where 208Pb+3 is detected and the gas ionization chamber, where 210Pb+3 is detected. The quantification of 210Pb with 208Pb as a spike yielded a detection limit of 4.4mBq at the IsoTrace facility. A theoretical detection limit of ≤0.11mBq is expected at the A.E Lalonde AMS facility. The expected detection limit at the A.E Lalonde AMS facility is on par with α-spectroscopy but AMS samples can be counted in less than 1 hour whereas alpha spectrometry samples must be counted for about 1 day.