272 research outputs found
Environmental DNA as an ecological monitoring tool for the Canadian Arctic
No full textEnvironmental DNA is a method where DNA shed by organisms into the environment is captured and analysed to give insights into various aspects of the ecosystem. Since detection of organisms is based on capturing theoretically as little as a single strand of shed DNA, the method is highly sensitive, cost-effective, and can be applied to any target organism or groups of organisms. These advantages, among others, have led eDNA methods to become a popular tool in environmental monitoring programs. Recent advances in DNA sequencing technologies have lowered the cost of analysis and allowed for many different applications of eDNA to become viable. Despite its widespread use, there are still a limited number of studies that have been conducted in remote regions such as the Canadian Arctic. This project aims to adapt eDNA methods for use in the Canadian Arctic, specifically in the monitoring of several lakes in the vicinity of established and developing mining sites through metabarcoding. Six of the eight target fish species were detected throughout the project, with the addition of one unanticipated species. The established mine site showed little change, while the developing site showed indications of fish movement that were consistent with the change in affected water bodies. Additional work supported the use of eDNA methods in frozen environments where sampling is required through ice. Overall, eDNA sampling was successfully employed in monitoring for fish presence near an active and developing mine in the Canadian Arctic.October 202
Kynurenine to tryptophan ratio as a biomarker of acute stress in fish
No full textThe aim of this study was to determine the kynurenine (KYN) to tryptophan (TRP) ratio in fish liver and brain tissue to assess its usefulness as a biomarker of acute stress exposure. The activity of the IDO enzyme in the kynurenine pathway is known to be activated during periods of stress, infection, inflammation, and immunomodulation. The enzyme activity was measured indirectly using the ratio of TRP and its primary metabolite KYN. Laboratory held rainbow trout (Oncorhynchus mykiss) were subjected to an acute physical stressor and kynurenine, TRP, serotonin (5-HT), and cortisol were measured in liver and brain tissue extracts sampled at 4- and 48-hrs post-stress exposure. The analytical method used to extract and detect our analytes was based on lyophilization; liquid-solid extraction followed by isotope dilution high-performance liquid chromatography positive ion electrospray tandem mass spectrometry. The [KYN]/[TRP] ratio (KTR) was greater in fish liver and brain in the 48-hrs post-stress exposure group (n=8) relative to controls (n=8, p<0.05); a similar increase was not observed in fish in the 4-hr post-stress exposure group. The levels of TRP in the liver and brain samples did not decrease substantially and the increase in the KTR can be attributed to the increase in KYN levels. Hepatic and brain cortisol levels were also elevated in fish from both stress-induced groups relative to their respective controls implying that cortisol responded more quickly to the stressful stimulus than KYN, TRP, and 5-HT. My results suggest that (i) cortisol responds more quickly to an induced stressor than KYN and TRP and (ii) KTR is a promising acute stress diagnostic biomarker in fish (iii) KTR increases is due to KYN increasing and TRP slightly decreasing. The broad scope of TRP metabolism in organisms suggests that the KTR may also be a useful biomarker for stress in different species. Efforts are ongoing to assess whether the KTR can be used as a biomarker for chronic stress in fish exposed to aquatic contaminants and other environmental stressors and if similar assessments can be made on tissues collected via non-lethal approaches.March 202
Evaluating the chromatographic behaviour of peptides carrying post-translational modifications for proteomics applications
No full textAmong all types of biomolecules, proteins make up a large portion of the core function of cellular processes. Studying the proteins in a cell is called proteomics, and all the proteins which make up this system, the proteome. Proteomics relies on robust and sophisticated liquid chromatography mass spectrometry (LC-MS) instrumentation with highly developed protocols to achieve confident results in peptide and protein identifications. For such applications, it is useful for the chromatographic and mass spectrometric properties of peptides to be well characterized and consistent across all forms which a peptide can be found. These forms are either native, where peptides are found as simple strings of amino acids or be altered with what are called post-translational modifications (PTMs), where a functional group has been added to the protein endogenously (in the cell) or by chemical treatment. These modifications can have drastic ramifications on the analytical properties of peptides and can even affect the protein identification output by artificially altering the observed abundance of a protein within a sample if the diminished intensities on LC-MS are not taken into consideration. Moreover, endogenous modifications often represent a feature of a protein which is being used to communicate within a signalling pathway or alter the function of that protein in the cell, thereby making PTMs an important choice for biological investigations.
Unfortunately, despite the importance in studying PTMs, their chromatographic characteristics are rarely reported. In this thesis, the chemical characteristics of various PTMs, general and specific, were uncovered and reported, permitting explanations for standard and ‘anomalous’ retention behaviours for peptides carrying PTMs. The general features across all PTMs studied in our lab over the last 10 years were summarized, while more specific behaviours based on peptide primary structure and the position of the modified amino acid were elucidated for peptides carrying deamidations and HexNAc glycosylations. Moreover, one mode which largely showed promise in investigating PTMs, electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), was studied for its retention properties. This led to the development of a retention time prediction algorithm which explained the sequence-specific behaviour of peptides and general features of PTMs in ERLIC.February 202
Biophysical analysis of the interaction of laminin-related protein Netrin with its receptor Neogenin
No full textNetrins are axon guidance cue molecules belonging to the laminin related protein. These molecules play a significant role in neuronal migration, by releasing chemotrophic cues and allowing either chemoattraction or chemorepulsion. Netrin holds capability to bind multiple different receptors including Deleted in Colorectal Cancer (DCC), Uncoordinated 5 (UNC5) and Neogenin (Neo). In order to investigate the binding of Netrin with its receptor Neogenin, two different constructs of Neogenin, Neogenin 3-5 Long and Neogenin 3-5 Short were investigated with its binding partner Netrin in a biophysical approach followed by crystallization screening. Netrin and the two constructs of Neogenin alone and in complex were analysed by gel electrophoresis, Size exclusion chromatography, dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). The findings from the low-resolution models reveal that the models for the complex appear larger and more compact than the individual models of the chNet△C, Neogenin Long 3-5 and Neogenin Short 3-5, which appear elongated. This analysis provides some insight into the interaction of Netrin with Neogenin and identifies 47% w/v 2-methyl-2,4- pentanediol 100mM HEPES, pH 7.5 and 35% w/v 2-methyl-2,4- pentanediol 100mM Imidazole, pH 8.0 as preliminary conditions for crystallization of the complex.October 201
Polycyclic aromatic compounds in Canadian Arctic seabirds: spatiotemporal trends and bioaccumulation patterns
No full textPolycyclic aromatic compounds (PACs) are a complex class of organic contaminants associated with petroleum. Many PACs have been identified as a concern due to their persistence in the environment, their potential to bioaccumulate, and their toxicity to living organisms. In the Canadian Arctic, emissions from natural seeps and increasing industrial activities, particularly shipping and mining, pose significant threats to the region’s vulnerable marine ecosystems. While numerous contaminants have been reported in this region, targeted monitoring of PACs remains limited. Seabirds serve as established bioindicators of marine ecosystem health and are commonly used for environmental monitoring. This thesis presents two complementary studies investigating PAC contamination in Arctic seabirds using gas chromatography-tandem mass spectrometry. The first study was a spatial and temporal analysis of PACs in seabird eggs collected from high Arctic and low Arctic colonies between 1993 and 2024. Higher PAC concentrations were detected in eggs from seabirds breeding in the high Arctic, with considerable interannual variation at both sites and many of the higher PAC concentrations occurring in the past decade. The second study focused on species- and tissue-specific PAC accumulation in three species of Arctic seabirds. Substantial variation in PAC concentrations and chemical profiles was observed between tissue types and among species. This variability reflects species-specific differences in PAC exposure, accumulation, and metabolism, driven by a combination of foraging ecology, breeding phenology, and physiological factors. As activity in the Arctic and interest in Canada’s northern shipping routes continue to grow, the need for monitoring oil and gas related contaminants in the Arctic region is essential to detect emerging trends, identify potential sources and support informed decision-making to ensure the protection of Canada’s Arctic wildlife.October 202
Acute effects of quinoline and 2-methylquinoline on electrical activity of great pond snail (Lymnaea stagnalis) neurons
No full textPolycyclic aromatic compounds (PAC) are organic compounds found ubiquitously in the environment and originate naturally or anthropogenically. They are persistent, bioaccumulative, and toxic (PBT). PACs have been found to modulate voltage gated ion channels in fish cardiomyocytes. Many studies have examined PACs effects, however, there is growing concern about a less studied class of PACs: hetero-polycyclic aromatic compounds (HPACs). Quinoline and its derivative 2-methylquinoline are two-ringed HPACs found in high concentrations in sediment and tissues of fishes from the Great Lakes. Studies investigating synthetic quinoline derivatives used in the pharmaceutical industry have shown HPACs modulate voltage gated sodium, calcium and potassium ion channels in cardiomyocytes of fishes and mollusk neurons. We therefore designed a study investigating the acute neurobiological effects of quinoline and 2-methylquinoline on Great Pond Snail (Lymnaea stagnalis) neurons, a model with a large easily accessible central nervous system and well conserved ion channels. Electrophysiological characteristics were measured by performing suction electrode experiments on the right internal
nerve of the right parietal Lymnaea stagnalis ganglion. Extracellular recordings before and after application of various quinoline or 2-methylquinoline concentrations were used to determine the frequency of right parietal nerve activity which we normalized as percent of control. The extracellular recordings revealed that quinoline affects right parietal nerve activity in a dose dependent manner. Quinoline caused a statistically significant effect on right parietal nerve activity at 1000 nM, but not 100 nM, and 10 nM, with mean percent of control values being 89.2 ± 4.3%, 103.9 ± 4.6% and 99.9 ± 3.5% respectively. 2-Methylquinoline had no statistically significant effect on right parietal nerve activity at 1000 nM. These experiments highlight the importance of understanding the neurobiological effect of PBT environmental contaminants quinoline and 2-methylquinoline, as well as demonstrate the utility of suction electrode recordings as an electrophysiology technique to evaluate acute neurotoxicity of environmental contaminants
New approaches to the analysis of polycyclic aromatic compounds in bird serum, blood and feathers
No full textPolycyclic aromatic compounds (PACs) are a structurally diverse class of environmental contaminants many of which are persistent (P), bioaccumulative (B) and inherently toxic (T). They can be formed from natural activities (such as volcanic activities and wildfires) or human activities (incomplete combustion and power plants). Due to their PB&T properties, PACs are considered an ecological threat, and there is great interest in measuring PACs in environmental compartments especially in biological samples. Because of their ability to accumulate contaminants, birds are an invaluable bioindicator of environmental health, and many countries use them in their environmental monitoring programs. The focus of my research was to develop and validate new approaches to the analysis of PACs in bird matrices and to evaluate if bird feathers can be used as a non-invasive matrix for the determination of PACs. Extraction of PACs from serum and blood, was achieved using microbead beating homogenization, and for feathers, I used pressurized fluid extraction. Detection and quantitation of PACs were based gas chromatography-tandem mass spectrometry. The performance characteristics of my methods were done in strict accordance with the EURACHEM guide - The Fitness for the purpose of Analytical Methods. The importance of my research is the development of more accurate, less time-consuming and more cost-effective approaches to the analysis of PACs in these matrices. Finally, my research is a reasonable first step toward the use of feathers as a non-invasive approach for monitoring the concentration of PACs in the environment.February 202
Dietary accumulation of hexabromocyclododecane diastereoisomers in juvenile rainbow trout (Oncorhynchus mykiss): bioaccumulation/depuration parameters and evidence of bioisomerization
No full textThe major objectives of this research were to examine the bioaccumulation parameters [depuration rates (kd), half life (t1/2) and biomagnification factor (BMF)] of individual isomers of hexabromocyclododecane (HBCD, C12H18Br6) in fish and to test the hypothesis of in vivo bioisomerization. This was done by exposing three groups of juvenile rainbow trout (Oncorhynchus Mykiss) to food fortified with known concentrations of an individual diastereoisomer (α, β, γ) for 56 days (uptake phase) followed by 112 days (depuration phase) of unfortified food. A fourth group of fish were exposed to unfortified food for the duration of the experiment. Fish (n=4) from all four aquaria were sacrificed on days 0, 7, 14, 56, 63, 70, 112 and 168 and muscle tissue was extracted and analyzed for diastereoisomer concentrations by high performance liquid chromatography tandem mass spectrometry (LC/MS/MS).
Bioaccumulation of the γ diastereoisomer was linear during the uptake phase while the α and β diastereoisomers were found to increase exponentially with respective doubling times of 14.1 and 20.5 days. Both the β and γ diastereoisomers followed first order depuration kinetics with calculated t1/2’s of 94 ± 25 and 84 ± 51 (± 1 × standard error) days, respectively. The BMF for the α diastereoisomer (BMF = 4.1) was one and a half times greater than the β-diastereoisomer (BMF = 2.6) and about one fifth larger than the γ-diastereoisomer (BMF = 3.6). The large BMF for the α diastereoisomer is consistent with this diastereoisomer dominating higher trophic level organisms in wildlife. Although the BMF of the β diastereoisomer suggests that it will biomagnify, because it is present in small quantities in commercial mixtures it is rarely detected in environmental samples. Results from these studies also provide evidence of bioisomerization of the β and γ diastereoisomers. Most importantly, the α diastereoisomer which was recalcitrant to bioisomerization by juvenile rainbow trout in this study and known to be the dominant diastereosiomer in fish, was bioformed from both the β and γ diastereoisomers. To our knowledge, this is the first report of bioisomerization of a halogenated organic pollutant in biota.May 200
Towards the development of a MALDI/TOF-MS Fingerprint Library for the Identification and Differentiation of Cannabis Extracts
No full textThe legalization of recreational cannabis in Canada has resulted in an increased interest for research on the plant within the scientific community. Currently in Canada, there is no requirement for the confirmation of cultivar/strain identity for cannabis that is sold in recreational distributors. Strains of cannabis differ through their chemical make-up which result in different pharmacological effects. The aim of this research is to develop a method using sequential extraction and matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and Brukers BiotyperTM software that uses cannabis extracts to fingerprint and distinguish strains purchased from the recreational market.
Cannabis was first extracted sequentially using methanol, acetonitrile and hexane to obtain a range of biomolecules from cannabis. Using a methanol extract, BiotyperTM software was optimized to generate peak lists that would include the maximum number of distinguishable peaks in the desired mass range. Then, using generated score values and dendrograms, MALDI mass spectra of strain extracts were compared.
It was found that the spectra of the methanol and acetonitrile extracts contained previously observed peaks that are consistent with cannabinoids. Also, some consistent patterns could be found using the methanol and acetonitrile extracts from cannabis. The use of hexane as an extracting solvent was less practical as it is not miscible with the optimized matrix solution. Biotyper software was sometimes consistent but overall showed discrepancies for how peaks were matched. Further work for this research includes the use of tandem mass spectrometry (MS/MS) to determine if matched pairs of peaks are truly identical. Another avenue for future work is method exploiting high
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molecular weight (MW) cannabis proteins for fingerprinting instead of low MW compounds as described in this work.October 202
Improving vitrification of sulfur in P-doped borosilicate glasses: a solid-state nuclear magnetic resonance spectroscopy study
No full textVitrification is a widely accepted method to immobilize hazardous heterogeneous high-level radioactive waste. Along with their favorable mechanical and thermal properties, the capacity of borosilicate glasses to accommodate a wide range of chemical species makes them excellent candidates for nuclear waste forms. However, the presence of high-valent species such as S6+ in the waste, which resist incorporation into the glass, demands improvements in glass composition to prevent the devitrification of sulfates at target waste-loading levels. A phase-separated sulfate layer can be an environmental threat, as it sequesters radioactive species such as 90Sr2+ and 135,137Cs+, and could contaminate ground-water resources during long-term geological disposal. Based on reports of improved sulfate incorporation in phosphate glasses, I have investigated doping borosilicate glasses with phosphate to evaluate its potential to enhance sulfur loading without compromising chemical durability. I have characterized a series of borosilicate glasses with various S6+ and P5+ contents using x-ray diffraction (XRD) and solid-state nuclear magnetic resonance (NMR) spectroscopy to gain insight into the identities of the devitrified products and to better understand the short-range structure of the glasses. 23Na NMR is complementary to XRD for determining the sulfate phases, whereas 31P NMR provides information about phosphate speciation in the glass. 29Si, 11B and 27Al NMR are used to determine the glassy short- range structure and polyhedral connectivity. Together, these results indicate that the preparation of homogeneous sulfur-bearing glasses requires the balance of the high field-strength cations, P5+ and S6+.February 202
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