Emery, Neil

Amyloid fibrils are fibrous protein aggregates that arise from misfolding and self-assembly processes, collectively referred to as amyloidosis. These aggregates are strongly associated with incurable neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease, and Amyotrophic Lateral Sclerosis (ALS). Elevated levels of Reactive Oxygen Species (ROS) and dysregulated metal-ion homeostasis often impaired by environmental and lifestyle factors can induce oxidative stress that undermines cellular antioxidant defenses, which cause the amyloid formation and toxicity. This thesis investigates multiple amyloidosis models, emphasizing the contribution of metal ions and ROS to aggregation pathways, and evaluates the potential inhibitory or protective roles of cytokinin (CK) plant hormone.Chapter 2 focuses on Gelsolin amyloidosis, a hereditary condition driven by point mutations that promote aberrant amyloid formation. Using microscopic and spectroscopic approaches, this work characterizes the aggregation behavior of peptides derived from domain 2 of plasma gelsolin and secreted by muscle cells. Three peptides were studied: the wild-type(WT) sequence and two clinically relevant mutants, K184N and N187Y. Each variant exhibited distinct aggregation rates, reflecting mutation-dependent effects on self-assembly. Furthermore, two CKs Kinetin (Kin) and trans-Zeatin (tZ) were shown to modulate gelsolin aggregation, suggesting their potential as anti-aggregation molecules. Chapter 3 revolves on the aggregation properties of TDP-43 peptides associated with ALS pathology. Within the RRM I domain, two cysteine residues serve as key redox-active sites susceptible to oxidation. ESI-MS and spectroscopic methods were used to analyze three peptide variants: WT, a mutant (MT) in which cysteine were substituted with alanine, and WT-S, a disulfide-linked dimer. All variants displayed higher aggregation under mildly acidic conditions. CKs, Kin and isopentenyl-adenine (iP) showed antioxidant capacity and their influence on peptide stability. Chapter 4 investigates the effects of copper(II)-induced oxidative stress in C2C12 muscle cells and evaluates cellular responses to various CK forms. ESI-MS profiling identified 20 CKs in copper-treated samples and revealed 24 untargeted metabolites with significant level changes, indicating their possible involvement in metal-induced oxidative pathways. In conclusion, this thesis highlights the multifaceted roles of CKs in biological systems, particularly their potential to mitigate ROS overproduction, counteract metal-driven amyloidgenesis, promote fibril destabilization, and lessen oxidative stress.

Author Keywords: Amyloid, Anti aggregation, cytokinins, inhibition, Peptide aggregation, Protein aggregation

Fungi are a diverse group of organisms that play crucial roles in various ecological processes and have immense economic importance. Understanding the intricate mechanisms underlying fungal growth and development is fundamental to harnessing their potential and exploring their applications in different fields. Signalling molecules, such as hormones, have been identified as key regulators in fungal physiology, orchestrating intricate processes and modulating biological responses to the environment. Phytohormones, commonly associated with plants, have been proposed as potential myco-hormones due to their production in a wide variety of fungi. Metabolomic analyses were performed via LC-MS/MS to investigate the role of phytohormones, specifically cytokinins (CKs) and indole-3-acetic acid (IAA), along with lipids and energetic metabolites such as organic acids during the growth and development of the model fungus Sordaria macrospora. The results revealed a clear switch between CK ribosides and CK free bases during the ascosporegenesis stage, with increased levels of cZ and iP and decreased levels of iPR and cZR. A similar pattern was observed in the mutant strain smgpi1 but demonstrating higher levels of CK free bases and increased fruiting body formation compared to the wild type. These findings provide insights into the regulation of phytohormones especially during fungal fruiting body development. In terms of IAA, the levels increased during the transition to sexual development in all strains, with the per5 mutant, unable to produce lipids via the cytosol, demonstrating a higher concentration than the wild type. The interplay between energetic metabolites and IAA suggests a potential role in the transition to sexual development. Additionally, the dose-dependent effects of exogenous CK application were investigated, showing the potential of low concentrations, from 1 to 10 μM, of CKs in promoting biomass accumulation or sexual development. Furthermore, gene editing in S. macrospora was proposed as a future direction to explore the functions of CKs and other metabolites during fungal development. Overall, this research contributes to our understanding of phytohormone-mediated processes in fungi and opens avenues for future investigations in fungal biology.

Author Keywords: fungi, LC-MS, metabolomics, myco-hormone, phytohormone, sexual development

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive loss in function of motor neurons. Elevated levels of biologically important metal ions, such as copper (II) (Cu(II)), zinc (II) (Zn(II)) or iron (III) (Fe(III)), may contribute to the disease. Moreover, Cu(II) interactions with other proteins associated with ALS have been investigated; however, the effects of metallation on TAR DNA-binding protein of 43 kDa (TDP-43) are less known. The aim of the thesis was to evaluate interactions between full-length TDP-43 and metal ions, and gain insight into the mechanisms of these interactions. In Chapter 2, electrochemical methods were used to evaluate the coordination of Cu(II) ions to immobilized TDP-43. Cu(II)-TDP-43 binding was favourable at a neutral pH. Surface characterization confirmed protein immobilization and Cu(II)-TDP-43 coordination. Competitive Zn(II) ion binding studies revealed Zn(II) increases Cu(II) binding. In Chapter 3, Fe(III) ion binding studies revealed that Fe(III) reduces Cu(II) binding when co-exposed to the TDP-43-Au surface. Data shows significant uptake of Cu(II) by TDP-43 protein which may have important implications in normal and diseased states of TDP-43, indicating surface bioelectrochemistry is a viable tool for fundamental exploration of proteins and metals, and their interactions, as they inform disease mechanisms, disease detection and drug screening.

Author Keywords: Amyotrophic Lateral Sclerosis, bioelectrochemistry, electrochemistry, metalloprotein, surface characterization, TDP-43

Heated leaf extracts of Euphorbia neriifolia (sweet aloes) are used traditionally in Guyana for asthma therapy, but the phytoconstituents have not been studied and phytohormones are generally unexplored in Euphorbia species. Phytoconstituents in asthma-effective traditional heated extracts were analyzed and compared with unheated leaves and latex extracts as screens to identify candidate compounds for asthma treatment. Analysis employed untargeted and targeted mass spectrometry-based metabolomics. The untargeted approach revealed thousands of features in samples with higher abundance in heated extracts and confirmation of 33 metabolite identities (confidence levels 2 and 3) which are known to have antioxidant and anti-inflammatory properties. Highly accumulated and unique features were detected in latex, and those characteristic compounds may reflect the expanded therapeutic uses of E. neriifolia globally. A targeted metabolomic analysis identified and quantified several cytokinins, including aromatic kinetin, and acidic phytohormones. Traditional heating improved the content of several cytokinins with known therapeutic potential, indicating they may be candidates for asthma management.

Author Keywords: asthma, cytokinins, Euphorbia neriifolia, metabolomics, sweet aloes, traditional remedies

Fungi are a diverse group of organisms that play crucial roles in various ecological processes and have immense economic importance. Understanding the intricate mechanisms underlying fungal growth and development is fundamental to harnessing their potential and exploring their applications in different fields. Signalling molecules, such as hormones, have been identified as key regulators in fungal physiology, orchestrating intricate processes and modulating biological responses to the environment. Phytohormones, commonly associated with plants, have been proposed as potential myco-hormones due to their production in a wide variety of fungi. Metabolomic analyses were performed via LC-MS/MS to investigate the role of phytohormones, specifically cytokinins (CKs) and indole-3-acetic acid (IAA), along with lipids and energetic metabolites such as organic acids during the growth and development of the model fungus Sordaria macrospora. The results revealed a clear switch between CK ribosides and CK free bases during the ascosporegenesis stage, with increased levels of cZ and iP and decreased levels of iPR and cZR. A similar pattern was observed in the mutant strain smgpi1 but demonstrating higher levels of CK free bases and increased fruiting body formation compared to the wild type. These findings provide insights into the regulation of phytohormones especially during fungal fruiting body development. In terms of IAA, the levels increased during the transition to sexual development in all strains, with the per5 mutant, unable to produce lipids via the cytosol, demonstrating a higher concentration than the wild type. The interplay between energetic metabolites and IAA suggests a potential role in the transition to sexual development. Additionally, the dose-dependent effects of exogenous CK application were investigated, showing the potential of low concentrations, from 1 to 10 μM, of CKs in promoting biomass accumulation or sexual development. Furthermore, gene editing in S. macrospora was proposed as a future direction to explore the functions of CKs and other metabolites during fungal development. Overall, this research contributes to our understanding of phytohormone-mediated processes in fungi and opens avenues for future investigations in fungal biology.

Author Keywords: fungi, LC-MS, metabolomics, myco-hormone, phytohormone, sexual development

Increased plant size is expected to have negative consequences for mating by increasing pollen transfer among the same plant. However, recent theoretical studies have demonstrated that this may not be true for clonal plants. Instead, clonal expansion could enhance outcrossing opportunities without increasing selfing by reducing distances to potential mates. I investigated how the spatial structure of clones influences patterns of pollen dispersal, selfing rates and siring success in a natural population of Sagittaria latifolia. I found that pollen dispersal distances typically exceeded the spatial extent of clones and there was a positive association between clone size and the likelihood that clones were intermingled. Together, this resulted in a weak positive association between clone size and selfing rates, and a strong positive association between clone size and outcross siring success. This is the first empirical support for the theoretical expectation that any negative effects of selfing in large clones might be offset by increased siring success.

Author Keywords: clonal growth, fitness gain curve, geitonogamy, plant mating, plant reproductive ecology, sex allocation theory

In Ontario, the dominant cash crop rotations consist of soybean (SB), which is a leguminous crop grown in rotation with maize (MZ) and winter wheat (WW). In addition to these crops, some farmers integrate cover crops (CC) into crop rotation, especially during the fallow period and winter seasons, to reduce nitrogen (N) losses via nitrate (NO3-) leaching and emission of N2 and the greenhouse gas nitrous oxide (N2O). This thesis focused on understanding the impact of crop phases in a MZ-(SB-WW)-CC rotation on the abundance of N-cycling bacterial communities that mediate nitrification and denitrification pathways. In addition, the influence of CCs on soil cytokinin (CK) profiles, which are plant growth-promoting hormones, were studied in a greenhouse trial to assess their potential impacts when integrating CCs into crop rotations. In particular, the relationship between traditional soil health parameters and the soil CK profiles was studied to understand how CKs might reflect biotic interactions and soil vitality. Results indicate N fertilizer application mono ammonium phosphate (MAP) and starter N:P: K (24:6:24) during WW planting in fall largely supported nitrifying bacterial communities (amoA) and potentially contributed to NO3- leaching. Management of MZ, which included spring-applied MAP resulted in larger denitrifying (nirK) bacterial communities, increasing the potential risk of N-loss via emission of dinitrogen gas (N2) and greenhouse gas N2O. However, CC soils had significantly lower nirK than MZ, reflecting the importance of strong and deep root systems of CCs, which have a higher ability to scavenge the substrates for denitrifying communities (NO3-). This highlights the importance of growing CCs in reducing the potential risk for N-loss via leaching and denitrification. Additionally, in the greenhouse trial, the ability of CCs to affect CK was detected, highlighting the importance of integrating CC in crop rotations. This is particularly noteworthy, given that total CK profiles showed strong associations with traditional soil health parameters such as labile or active carbon and soil microbial community diversity. It was concluded that total soil CK can be used as a novel and dynamic soil health measure. Future research on quantifying N2O fluxes and levels of NO3- in leachates would provide a more precise understanding of the impact of different crop rotation phases on N-dynamics in these fields. Further studies on single or combined measures of soil CKs are warranted to develop its potential as a practical and effective soil health parameter.

Author Keywords: Cover crops, Crop rotations, Cytokinin hormone, Nitrogen Cycle, qPCR, Soil health

During the COVID-19 pandemic, nucleic acid and antibody-based testing methods were heavily relied upon, but can be costly, time-consuming and exhibit high false -negative and -positive rates. Thus, alternative strategies are needed. Viral antigens such as the SARS-CoV-2 spike (S) glycoprotein are critical in the function of the virus and useful as diagnostic biomarkers for viral infections. For biosensing applications, aptamers are suitable high-affinity and cost-effective binding partners for their specific targets. Using localized surface plasmon resonance (LSPR), real-time, rapid acquisition of results can be achieved, essential for improving the efficacy of a sensor. Herein, LSPR aptamer sensors were fabricated for the detection of the SARS-CoV-2 protein. Data indicate that the best performing aptasensor was the streptavidin-biotin sensor, while the current gold aptasensor exhibited lower sensitivity and the fabrication of the carboxyl aptasensor was unsuccessful. The S1 aptamer selectively bound the S1 protein with high binding affinity. Excellent shelf-life stability, reusability, and high recovery in complex matrices was also maintained. Additionally, a receptor binding domain (RBD) functionalized sensor was fabricated to examine the interactions with angiotensin converting enzyme 2 (ACE2), for future assessment of inhibitors used in drug therapies. Overall, LSPR has been demonstrated as a viable tool for measuring SARS-CoV-2 related aptamer-protein and protein-protein interactions, and this strategy may be applied to other viral or non-viral antigen targets.

Author Keywords: Antigen-based Detection, Coronavirus, COVID-19, Inhibition, Localized Surface Plasmon Resonance, SARS-CoV-2

Heavy metal pollution threatens human and ecosystem health. E. gracilis was investigated for its potential use in bioremediation due to its tolerance for heavy metals and ability to sequester them from the environment. E. gracilis can remove metals by producing metal binding compounds enriched in sulfur and nitrogen. In this thesis, E. gracilis cultures that were pretreated with elevated levels of sulfur or nitrogen had increased tolerance to CdCl2 compared to non-pretreated cultures. RNA-sequencing revealed that both pretreatments led to transcript level changes and that exposure to CdCl2 led to further transcript level changes. Gene ontology (GO) enrichment analysis reflected changes in nitrogen and sulfur metabolism as well as physiological processes related to metal binding. The data from this thesis revealed important transcription level changes that occur when E. gracilis is challenged with CdCl2 and helps us understand how organisms adapt to heavy metal pollution in the environment.

Author Keywords: bioremediation, Cadmium, Euglena gracilis, GO-enrichment, metal-binding, RNA-Sequencing

Euglena gracilis is an enigmatic and adaptable organism that has great bioremediationpotential and is best known for its metabolic flexibility. The research done in this dissertation addresses (1) how growth conditions impact cellular composition, and (2) how chemometric approaches (such as statistical design of experiments and artificial neural networks) are viable alternatives to the conventional biosorption models for process optimization. Using high-resolution mass spectrometry for biosorbent characterization is a powerful way to assess the chemical characteristics of lyophilized and fractionated cells with high precision, especially to screen for compound classes that may have potentiality for rare earth element removal. Growth conditions impacted cellular composition and separated size fractions of cells yielded different molecular/chemical properties as described by compositional abundances, thus different biosorptive potential. Untargeted analysis demonstrated that exponential dark-grown cells with glucose supplementation were abundant in polyphenolic- and carbohydrate-like compounds, molecular species highly involved in rare earth element binding. Light grown cells had more heterogeneity and the highest molecular weighted fractions from light grown cells (fraction D) had the most abundances of polyphenolic- and protein-like structures. Chemometric modeling used identified the best and worst conditions for iii dysprosium sorption and showed that pH had the most significant influence on bioremoval. Bioremoval ranged from 37% at pH 8 to 91% at pH 3 at Dy concentration ranging from 1 to 100 μg L-1. The work presented in the PhD dissertation will aid in understanding the chemical characteristics of biosorbents by using a Van krevelen analysis of elemental ratios whether algal cells are grown in different environmental growth conditions, or when algal cell are size fractionated. This is especially applied for the screening for metal binding potentiality to Dysprosium. Chemometric methods provide an alternative method for the investigating factors for bioremoval, and applications for process optimization and for real-world applications. This dissertation will aid in understanding chemical characteristics when a biosorbent is grown in a given condition and which factors are important for rare earth element (REE) bioremoval. The significance of this work aims to look for alternate ways to screen biosorbents and using a more efficient experimental design for REE bioremoval.

Author Keywords: bioremoval, biosorption, chemometrics, dysprosium, euglena, mass spectrometry