Stock, Naomi

The detection of ciguatoxins (CTXs) in biological samples is challenging due to their low concentrations, the presence of various congeners, and the absence of standardized methods. This study uses high resolution mass spectrometry (HRMS) with P-CTX-3B as a reference standard. The protonated molecules ([M+H]+) were most prevalent, especially when acetonitrile/water was utilized, providing enhanced sensitivity. Optimized collision energies of 15 eV for protonated molecules and flow rates of 10 µl/min enhance sensitivity and peak intensities, respectively. Acetonitrile/water (ACN/H2O) is recommended as the primary solvent for HRMS method, an aspect underexplored in existing literature. The detection of CTX-3B in fish tissue samples proved to be challenging, caused by variations in ion peak intensities and matrix effects, requiring a deeper exploration of the impact of complex matrices on CTX detection. The study emphasizes the need for a reliable internal standard to mitigate these effects and highlights the ongoing challenge of developing a rapid, simple, and sensitive detection method. The study's specific focus on the P-CTX-3B analogue significantly contributes to methodology development for this congener, serving as a foundational step in understanding and detecting CTX. Despite notable progress, the study acknowledges the absence of an ideal assay, outlining key challenges for future research on ciguatera analysis. It underscores the continuous necessity for method reevaluation, testing, and the broader goal of establishing a more clarified and rugged method for the identification of CTX in fish.

Author Keywords: Analytical Chemistry, Ciguatera Fish Poisoning, Ciguatoxin, High-Resolution Mass Spectrometry, Optimization, P-CTX-3B

Access to human blood for forensic research and training in bloodstain pattern analysis (BPA) can be difficult due to many ethical, safety and cost concerns. Mammalian blood alternatives can be sourced, especially from local and willing abattoirs, but some concerns remain, and the added difficulties of high variation and species-specific differences in cellular components pose other issues. Therefore, synthetic alternatives to human blood provide practical options for the BPA community. This thesis explores the use of alginate hydrogels as a base material for forensic blood substitute (FBS) development. Hydrogels are first explored as a suitable environment for DNA stability and functionality and compared to other polymer systems. The ability of DNA to remain intact while undergoing electrospray ionization (ESI) is also investigated. The FBS design focuses on mimicking the fluid properties and genetic capabilities of whole human blood – a material not developed in FBSs previously. ESI was used to develop microparticles (MPs) that serve as cellular components of human blood (the red blood cells – RBCs, and white blood cells – WBCs). The microparticles were ionically crosslinked using calcium to provide small MPs (RBCs) or covalently crosslinked with functional DNA to provide larger WBC-like functional particles. The integration of these novel MPs into alginate-based materials is optimized and their use in BPA scenarios is explored. The FBS is tested in BPA scenarios of dripping experiments, impact patterns, and the ability to extract and amplify the contained DNA. In addition, the stability (or shelf-life) of the FBS was also assessed. The FBS exhibited similar spreading ratios to blood and demonstrated feasibility in use for impact angle (a) determination and impact pattern creation. Importantly, the DNA contained within the FBS could be processed with analogous protocols used in DNA evidence processing, enhancing its applicability to BPA research and training.

Author Keywords: Alginate hydrogels, Bloodstain pattern analysis, Electrospray ionization, Forensic blood substitutes, Forensic materials, Synthetic DNA design

Phenolic compounds are used in industry, such as agriculture and biotechnology, and inevitably end up in our environment. These compounds may serve as a phenolic precursor to produce raw materials for a wide range of applications. Chemical oxidation has been the common synthetic pathway to oxidize phenols and related compounds. However, traditional chemical approaches suffer from use of harsh chemicals, waste generation, and lack of reaction selectivity. Electrochemical synthesis has emerged as an alternative method to mitigate common challenges associated with organic synthesis. Herein, electrochemical oxidation of 2,6-diphenylphenol (DPP) and 2,2-dihydroxybiphenol (DHBP) was carried out and compared to traditional chemical oxidation. Contrasted with chemical oxidation, cyclic voltammetry of DPP resulted in a range of products based on the specific potential ranges used, whereas chemical oxidation of DHBP yield a dark-coloured polymeric product. The electrooxidation and chemical oxidation of DPP and DHBP resulted in a solution colour change, indicative of the formation of new, but different products monitored by UV-vis, and characterized by nuclear magnetic spectroscopy (NMR), X-ray single crystal diffraction, IR spectroscopy, transmission electron microscopy (TEM), and gas chromatography-mass spectrometry (GC-MS). The data indicate that the synthetic outcomes are dependent on the synthetic methodology employed, and that electrooxidation and chemical oxidation can form products unique to the pathway utilized.

Author Keywords: chemoselectivity, electrochemistry, phenols, radical, synthesis

Perfluorinated carboxylic acids (PFCAs) are anthropogenic environmentally ubiquitous surfactants that tend to concentrate on water surfaces. This investigation looked at the effect of simulated rain on the atmospheric concentration of a suite of PFCAs (C2 - C12) above the bulk water system. Increased air concentrations of all PFCAs were detected during simulated rain events. Long chain PFCAs (>C8) were found to be much more concentrated in the air above the bulk water system than their short chain counter parts (

Author Keywords: aqueous aerosols, perfluorinated carboxylic acids, surfactants environmental fate, water to air chemical transfer

Dissolved organic matter (DOM) in oceans provides nutrients and ultraviolet radiation protection to microbes. Some DOM compounds can chelate with metals, including copper, controlling their transport and bioavailability in marine systems. As copper functions as both a nutrient and toxicant, studies into the chemical structures of Cu-ligands is important, however currently limited. In this thesis, the chemical composition of total and Cu-binding DOM is investigated using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) in the Canadian Arctic and North Pacific. Chapter 2 reveals chemical differences in DOM composition between the southern and northern Canada Basin, revealing the influence of terrestrial and biological sources. Chapter 3 shows the uniqueness of Cu-binding ligands found in the Canadian Arctic and North Pacific Ocean. Studying the composition of DOM gives insight into the chemical diversity of marine DOM, helping to predict the effects of a changing climate on marine ecosystems.

Author Keywords: biological, dissolved organic matter, fluorescence, immobilized metal-ion affinity chromatography, mass spectrometry, terrestrial

Cis-isomers of the cytokinin plant hormone family are thought to have low activity or impact on plant growth and development. Mutants with independent silencing of the pathway leading to cis-CK (cis-cytokinin) were investigated at the phenotype and metabolite levels. Phenotypic deviations were noted in trichome development, fresh weight, rosette diameter, number of non-rosette leaves, shoot height, delayed flowering, flower number, and carotenoids. Exploratory metabolomic analysis detected a number of metabolite features that have been associated with CK, auxin, and ABA (abscisic acid) activity. Evidence from both phenotype and metabolomic analysis support the hypothesis that cis-CK production is biologically important for plant growth and development.

Author Keywords: arabidopsis, cytokinin, IPT, metabolmics

Giardia lamblia is an intestinal parasite found globally in freshwater systems that is responsible for endemic outbreaks of infectious diarrhea. As a unicellular parasite that lacks mitochondria, a respiratory chain and lives in the anaerobic environment of its host's intestine, Giardia was assumed for decades to lack heme proteins. However, its genome encodes several putative heme proteins, including three with sequence similarity to the cytochrome b5 family, referred to as Giardia cytochromes b5 (gCYTb5). Recombinant expression of one of these genes (gCYTb5-I), results in a protein (17-kDa) that is isolated with noncovalently bound heme. Resonance Raman and UV-visible spectra of gCYTb5-I in oxidized and reduced states resemble those of microsomal cytochrome b5, while sequence alignment and homology modelling supports a structure in which a pair of invariant histidine residues act as axial ligands to the heme iron. The reduction potential of gCYTb5-I measured by cyclic voltammetry is -165 mV vs the standard hydrogen electrode and is relatively low compared to those of other family members. The amino- and carboxy-terminal sequences that flank the central heme-binding core of the gCYTb5 are highly charged and do not occur in other family members. An 11-kDa core gCYTb5-I variant lacking these flanking sequences was also able to bind heme; however, we observe very poor expression of this truncated protein as compared to the full-length protein.

Author Keywords: b-type cytochrome, cytochrome b5, electron transfer protein, Giardia intestinalis, heme/heam protein, spectroelectrochemistry