Department of Chemistry Graduate Theses

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    The Liquid Microjunction-Surface Sampling Probe as an Emerging Tool for Medicinal Mass Spectrometry
    McKeown, Mark J.; Chemistry; Oleschuk, Richard
    Ambient ionization mass spectrometry (AIMS) is an attractive technique for providing supplementary information that medical professionals can use to guide clinical decisions. The group of techniques includes ionization methods, such as desorption electrospray ionization (DESI) and rapid evaporative ionization mass spectrometry (REIMS) that, when combined with mass spectrometry (MS) analyze thousands of compounds (e.g., highly polar/non-volatile, non-polar/volatile analytes) within resected tissues. From these compound profiles, one can distinguish between healthy and diseased states on a cellular level. Tissue analysis by MS is difficult to perform as it is challenging to obtain the required data from these specimens while minimizing the loss of information from time-dependent metabolomic changes. Despite technological advances, many AIMS techniques are limited in processing speed, analyte extraction, and sample restraints for widespread clinical utility. Herein, the liquid microjunction- surface sampling probe (LMJ-SSP) is investigated in profiling and imaging MS to directly interrogate fresh tissue surrogates of various sizes/shapes/thicknesses. A previously developed conductive feedback system is enhanced and used with the probe to produce four-dimensional (4D) images that describe the topography and chemistry of uneven surfaces using the LMJ-SSP. The platform enables the analysis of these tissue specimens with absolute height variations of up to centimeters, and the ability to monitor the dynamics of changing surfaces. Secondly, the versatility of the LMJ-SSP is explored to profile underwater tissue surrogates. To this end, normal saline solutions (water and 0.9% sodium chloride, weight by volume [w/v]) are typically used to temporarily maintain tissue integrity before analysis. The two described workflows use a low miscible solvent to directly interrogate these specimens immersed in saline solutions or extract compounds from saline microenvironments. Submerged analytes are still identifiable for their molecular weights at high salt concentrations (0 - 3.5% NaCl, w/v). Underwater analysis of an insoluble compound reveals the effects of salt on electrospray ionization (ESI). The LMJ-SSP provides the speed of information needed to revolutionize patient care and outcomes.
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    The Study of the Activation of Primary Terminal C-H Bonds by Rieske Oxygenase Enzymes and the Identification of a Serine Protease with Anti-nociceptive Activity
    Ramachandra, Manasa; Chemistry; Ross, Avena
    The first project in this thesis focused on the study of the activity of Rieske oxygenase enzymes, TamC and PtTamC. Tambjamines are methoxy-bipyrrole alkaloids isolated from marine invertebrates and bacteria, and possess several biologically useful properties. The first cyclic analogue, tambjamine MYP1, was isolated from a marine bacterium, Pseudoalteromonas citrea (also proposed to exist in Pseudoalteromonas tunicata). TamC and PtTamC, both Rieske oxygenase enzymes (ROs), are proposed to catalyse the cyclisation of tambjamine YP1, a linear analogue, to tambjamine MYP1. The macrocyclisation in MYP1 would involve a C-C bond formation at a primary carbon atom (terminal methyl) of the alkyl chain in YP1. This cyclisation activity was successfully demonstrated by the heterologous co-expression of TamC or PtTamC along with the identified redox partner enzymes of TamC, PcFdx and PcRed, in Escherichia coli, followed by substrate-feeding assays with synthesised tambjamine YP1 and tambjamine BE-18591 as substrates. These are the first reported C-C bond formations being initiated at the terminal methyl group of an alkyl chain in oxidative cyclisations and therefore, the first successful activation of an unreactive terminal C(sp3)-H bond in an oxidative carbocyclisation reaction catalysed by the RO enzymes, TamC and PtTamC. TamC, PtTamC, and PcRed were also successfully heterologously expressed and purified. The goal of the second project was the identification of serine proteases with anti-nociceptive activity from Faecalibacterium prausnitzii. Inflammatory bowel disease (IBD) induces hyperexcitability in dorsal root ganglion (DRG) nociceptors contributing to abdominal pain. Proteases modulate nociceptive signals in abdominal pain via activation of cell-surface receptors present on sensory neurons. Recent findings indicate that serine proteases from F. prausnitzii, a gut commensal bacterium, can reduce DRG neuron excitability, thereby suppressing abdominal pain. Genome mining and structure simulation tools led to the heterologous expression and purification of three proteases with potential anti-nociceptive activity. Patch clamp experiments conducted by our collaborators at Queen’s University led to the identification of a single protease with anti-nociceptive activity. The successful identification, expression, and purification of the anti-nociceptive serine protease, Clp-fp, from F. prausnitzii is the first step in the microbial modulation of IBD-related abdominal pain.
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    The Development of the Controlled-Atmosphere Flame Fusion Methodology for the Growth of Nickel Single Crystals and the Interplay between the Oxidation of Nickel and the Hydrogen Oxidation Reaction in Alkaline Media
    Esau, Derek M.; Chemistry; Jerkiewicz, Gregory
    Nickel (Ni) and Ni-based materials have become promising alternatives to the platinum group metals that are currently used in sustainable energy technologies. To improve the fundamental knowledge of Ni electrochemistry, Ni single crystal electrodes that have highly ordered surface arrangements of atoms were used. The development of the controlled atmosphere flame fusion methodology as part of this thesis work has allowed for bench top growth of poly-oriented spherical single crystals (POSSCs) of non-noble metals. Ni POSSCs were oriented, cut, and polished to produce hemispherical monocrystalline electrodes (Ni(hkl)), namely, Ni(111), Ni(100) and Ni(110). The surfaces were then characterised using cyclic voltammetry (CV) in solutions outgassed with nitrogen gas (N2(g)) in 0.10 M aqueous NaOH solution at a potential scan rate (s) of 50.0 mV s-1 and temperature (T) of 295 K. Each Ni(hkl) had a unique electrochemical response for the formation and reduction of α‒Ni(OH)2 and β‒NiOOH. Due to the importance of inductive heating for the pre-treatment of Ni(hkl), the technique was described in detail, including the relevant theory of electromagnetism, heat conduction and radiation emission with temperature. This was followed by a discussion of practical, experimental considerations to best utilize inductive heating and examples of the electrochemical response of a Ni(111) with varying degrees of surface oxidation. Lastly, the influence of s on the formation and reduction of α‒Ni(OH)2, and the hydrogen oxidation reaction (HOR) in 0.10 aqueous NaOH at T = 298 K was studied. The CV response in electrolyte that was outgassed with N2(g) and saturated with hydrogen gas (H2(sat.)) with s = 1.00, 2.00, 5.00, 10.0, 20.0, 50.0 and 100 mV s-1 in the potential range of ‒0.20 V ≤ E ≤ 0.50 V was studied. In solution outgassed with N2(g), the analysis suggests that Ni is likely partially covered by α‒Ni(OH)2 with sections of metallic Ni at the peak. In H2(sat.) electrolyte, the HOR occurs after the formation α‒Ni(OH)2 and allowed us to propose the “bifunctional mechanism” for the HOR. This work has provided valuable experimental methodology to produce Ni(hkl) cheaply and effectively, but also valuable fundamental information about the electrochemistry of monocrystalline Ni electrodes.
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    Preparation, Characterization, and Application of Palladium and Nickel Hydroxide Nanomaterials for Electrochemical Energy Storage and Generation
    Fujita, Sho; Chemistry; Jerkiewicz, Gregory
    Nanostructured materials are of great importance to the advancement of energy storage and generation systems. Particularly, palladium (Pd) and nickel hydroxide (Ni(OH)2) nanomaterials have attracted great attention due to their remarkable stability and electrocatalytic activity. However, there is a lack of a comprehensive investigation elucidating (i) the interfacial electrochemistry unique to their nanostructures and (ii) the key factors that determine their electrocatalytic activity and stability. Filling this knowledge gap is crucial to develop ideal material/electrocatalyst designs using Pd and Ni(OH)2 nanomaterials to further improve the performance of energy storage and generation systems. Therefore, in this research, synthesis, characterization, and comprehensive analyses on various Pd and Ni(OH)2 nanomaterials were conducted to address these challenges. In the first part of this research, the electrochemical property and stability of shape-controlled Pd NPs were investigated in aqueous alkaline solution. It was envisaged that this nanomaterial would find application as an anode material in miniaturized nickel-metal hydride (Ni-MH) batteries. The results indicated that repetitive H absorption and desorption induced structural changes to the Pd NPs, which were attributed to the absorbed H being trapped in the subsurface site of the Pd NPs. The cathode in the miniaturized Ni-MH batteries also requires a nanomaterial due to the limited volume of the batteries. Thus, the second part of this research focused on the analyses of electrochemical and chargedischarge behaviour of carbon-supported β-Ni(OH)2 nanosheets. The results indicated that the β-Ni(OH)2 nanosheets gradually became aggregated upon repetitive charge-discharge cycling in aqueous alkaline solution, leading to the decrease in the specific capacity of the nanomaterial due to the modification of the electrochemical and material properties after the charge-discharge cycling. In the third part of this research, the synthetic approach of the β-Ni(OH)2 nanosheets was modified to the preparation of Pd NPs supported by β-Ni(OH)2/C composite material for the envisaged use in alkaline-exchange membrane fuel cells (AEMFCs). This electrocatalyst possessed enhanced activity towards hydrogen oxidation reaction (HOR) in aqueous alkaline solution and remarkable stability upon accelerated durability test (ADT). The novel results in this study provide a new insight into the design of unique anode electrocatalysts for the advancement of AEMFCs.
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    Biosynthesis of Alterochromide Natural Products in Pseudoalteromonas piscicida JCM 20779
    Li, Longyu; Chemistry; Ross, Avena
    The alterochromides are a family of structurally related lipopeptides (many of which are brominated) produced by several species of Pseudoalteromonas including Pseudoalteromonas piscicida, Pseudoalteromonas flavipulchra, and Pseudoalteromonas maricaloris. Previous investigations identified the alt locus in P. piscicida as the biosynthetic gene cluster responsible for production of alterochomides and particularly the gene product of altN as being responsible for the bromination. AltN corresponds to a flavin-dependent halogenase that appears to selectively generate brominated alterochromides. It is not, however, known the timing of the bromination within the biosynthesis of alterochromides. However, it was hypothesized that AltN would carry out the bromination reaction once coumaric acid has been covalently linked to an acyl carrier protein towards the beginning of the fatty acid synthesis. The objective of this thesis is to determine at which step of the biosynthesis procedure does AltN act to brominate the natural product moiety by conducting in vitro enzymatic assays to reconstruct the brominated natural product moiety. Enzymes native to the P. piscicida JCM 20779 biosynthetic pathway, AltA, AltC, and AltN were successfully heterologously expressed and purified. The carrier protein AltB, however, could not be successfully expressed. Accessory enzymes including the 4′-phosphopantetheinyl transferase Sfp, NAD(P)H-dependent FMN reductase SsuE, and NAD-dependent phosphite dehydrogenase PtdH were successfully heterologously expressed and purified. Enzymatic activities of AltA were examined using its native substrate L-tyrosine, and the kinetic parameters of AltA were determined using steady state kinetic experiments. Substrate promiscuity tests were further conducted, and results showed that AltA was highly selective towards L-tyrosine compared to L-, D-phenylalanine, L-, D-tryptophan, and L-, D- histidine.