Biology

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This is the collection for the University of Waterloo's Department of Biology.

Research outputs are organized by type (eg. Master Thesis, Article, Conference Paper).

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Effects of Trastuzumab on Alternative Splicing in HER2+ BT474 Breast Cancer Cells
(University of Waterloo, 2024-09-12) Piticaru, Benjamin
Alternative splicing (AS) is a fundamental process that enhances transcriptomic diversity and protein isoform complexity in eukaryotic cells. Deregulation of AS plays a critical role in various diseases, particularly cancer, where erroneous splicing patterns contribute to oncogenesis, tumor progression, and therapeutic resistance. Human epidermal growth factor receptor (HER)2, overexpressed in more than 20% of breast cancers, has clinical relevance in AS. The humanized monoclonal antibody (mAb) Trastuzumab has been used for decades to fight HER2+ breast cancer with positive results. However, there are many unknowns surrounding the mechanism of this drug and the effects it has on alternative splicing. Next generation long read sequencing technologies like Oxford Nanopore allow researchers to sequence full length transcripts. The ability to sequence long intron/exon spans and repeating regions enables long read sequencing technology to provide new insights into the AS patterns of genes. Identifying AS events is important for understanding changes in function and pathways affected by differing conditions (e.g., drug treatment, diseased cells). Software tools tailored to long read data like Long-read Isoform Quantification and Analysis (LIQA), Full-Length Alternative Isoform Analysis of RNA (FLAIR), FLAME, and Nanosplicer enhance data analysis abilities to detect AS events. They analyze sequencing data by mapping reads to genomes, identifying splice junctions, and clustering AS events. These tools then compare AS events between conditions (e.g., treated vs. untreated cells) to pinpoint significant AS variations. Accurate transcriptome sequencing is vital for research in drug development and diagnostics. The optimization of sequencing methods is an ongoing endeavour that requires continuous development. A chapter of this thesis compares the impact of omitting polyadenylation (poly(A)) enrichment in Oxford Nanopore Technologies (ONT)’s direct complementary deoxyribonucleic acid (cDNA) sequencing protocol to improve library prep efficiency and sequencing accuracy. Analysis indicated that excluding poly(A) selection does not negatively affect sequencing metrics but enhances read length and enables the sequencing of histone messenger ribonucleic acid (mRNA). Further, significant differences in poly(A) tail lengths between selected and unselected samples were found, suggesting a bias against shorter tails with enrichment. Gene composition and unique gene identification remain consistent across conditions. These findings support the potential advantages of omitting poly(A) enrichment in transcriptome sequencing while advocating for further validation. With optimized methods, the impact of Trastuzumab on AS profiles in BT474 and SKBR3 cell lines was tested. Using ONT long-read sequencing, significant AS events were identified in response to Trastuzumab treatment. The findings reveal differential isoform expression in genes involved in cellular signaling, RNA processing, and stress response pathways. Notably, nuclear paraspeckle assembly transcript 1 (NEAT1) and pre-mRNA processing factor 38B (PRPF38B) exhibited distinct AS patterns across multiple cell lines, suggesting HER2-mediated regulatory mechanisms. This study underscores the utility of ONT sequencing and high throughput data processing as an effective and efficient means for explaining complex AS landscapes affected by therapeutic treatments.
Structural and Functional Characterization of a Modular Immunoglobulin A Protease from Thomasclavelia ramosa
(University of Waterloo, 2024-08-29) Tran, Norman
Immunoglobulin A proteases (IgAPs) are a diverse group of enzymes secreted from a wide range of mucosal bacteria. These enzymes have convergently evolved to cleave immunoglobulin A (IgA), the main antibody found on the mucosa, as a means of modulating the bacterium’s relationship with their host tissues. Due to the various biological functions and biochemical properties of these enzymes, the study of IgAPs has provided multifaceted insight into aspects of mucosal immunity, enzyme structure and function, and the structural basis for substrate specificity. Only two of three known IgAP enzyme families have been investigated using an in-depth structural and functional approach. This thesis thus aimed to carry out these analyses on the IgAP from Thomasclavelia ramosa as a representative member of this last poorly characterized family. X-ray crystallographic, small-angle X-ray scattering, and gel-based kinetic techniques were used to reveal that, unlike the other two IgAP families, the T. ramosa IgAP has a truly modular protein architecture that can be split into and produced as distinct minimized domains that retain function. The crystal and solution-scattering structures of various domain constructs were also used to generate a working model for how the T. ramosa IgAP recognizes and has high specificity for IgA. This thesis provides the first in-depth biochemical account of this IgAP family and pave the way for advances in clinically relevant IgAP-related research and our understanding of IgAPs as a whole.
Wetland hydrology and the impacts of beaver dams in the Upper Columbia River floodplain wetlands
(University of Waterloo, 2024-08-28) Leven, Catriona
The Upper Columbia River floodplain wetlands are the last remaining undammed stretch of floodplain wetlands along the Columbia River and continue to experience a natural flood pulse. This flood pulse interacts with natural levees and beaver dams across the floodplain and habitat heterogeneity results, with individual wetlands within the Columbia Wetlands having different hydrographs. I conducted research in 38 wetlands from 2020 to 2022 and aimed to determine if differing wetland hydrology allowed for wetland groups to be determined, and if those groups could be attributed to gaps in natural levees and beaver dams. Hydrograph attributes can be used to differentiate wetland groups, with three or four groups being identified depending on year. Random Forest models based on measurements of the levees, levee gaps, and beaver dams had an Out-Of-Box Error Estimate of between 36% and 53% across all groups depending on year, indicating correct classification of between 64% and 47%. Combining hydrograph attributes and levee gap and beaver dam metrics, we can describe these groups on a gradient of connectivity to the Columbia River, being Most Connected, with large open levee gaps, Partially Connected, with levees without gaps or with gaps dammed by beaver dams that are smaller, and Least Connected, with levees without gaps or gaps dammed by beaver dams that are bigger. This demonstrates the large impacts of beavers on shaping wetland systems and has implications for the differing impacts of climate change on these different wetland groups.
Characterization of darter (Etheostoma spp.) interspecific energetic responses to climate change induced temperature changes
(University of Waterloo, 2024-08-27) Weber, Allison
Shallow freshwater ecosystems are predicted to experience increases in temperature variability as the occurrence and severity of heat waves continues to rise. Ectothermic organisms like fish are especially vulnerable to these acute temperature increases as their physiological functioning is directly regulated by environmental conditions. Thus, understanding their capabilities of responding to thermal stress is critical to predict how these species will be affected by climate change. Here, we characterized the elevated temperature responses of three closely related darter species: Fantail (Etheostoma flabellare; FTD), Rainbow (Etheostoma caeruleum: RBD), and Johnny darter (Etheostoma nigrum; JD) native to the Grand River of Southern Ontario, via three experiments: Experiment #1: assessment of thermal tolerance limits and energetic enzymatic activity, Experiment #2: determination of thermal preference, and Experiment #3: characterization of metabolic responses to elevated temperatures. Specifically, Experiment #1 determined each darter’s CTmax and quantified activity and gene expression of enzymes involved in glycolysis, the Krebs cycle, and the electron transport chain in brain and heart tissue at 15C baseline and at thermal tolerance limits. Experiment #2 determined differences in thermal preference and mobility between species, and Experiment #3 compared darter aerobic scope (AS) during exposure to four different heat ramp exposures designed to mimic previously recorded heat waves. Significant differences were observed in the thermal tolerance limits of each species. For brain tissue, FTD had higher baseline enzymatic activity compared to JD and RBD, however at CTmax, this difference was lost, as both JD and FTD had similarly high enzyme activity relative to RBD. Intraspecifically, JD demonstrated a superior plastic ability, often having a significantly higher enzyme activity at CTmax compared to its baseline counterparts, while RBD activity declined at CTmax with respect to its baseline levels. Heart tissue exhibited no interspecific differences in activity levels at baseline. At CTmax, however, JD had greater activity than RBD for all heart enzymes, although neither JD or RBD were different from FTD. Similar intraspecific trends as brain were observed, with FTD and JD increasing activity at CTmax, and RBD decreasing. No differences were observed in thermal preference between species, although FTD demonstrated significantly higher mobility than JD. Metabolically, FTD AS was significantly greater than JD and RBD at both 25C and 30C, however no differences were observed at 15 or 20C. These results suggest that FTD may be the best equipped at responding to temperature-induced increased metabolic demands due to their higher baseline enzymatic activity and broader aerobic scope. This FTD advantage, and the interspecific differences observed throughout this study, may a be a result of prior adaptation and acclimatization to each species’ respective microhabitat conditions, as it is expected that FTD reside in warm, high flow, and thermally variable regions, JD in warm, moderately thermally variable, low flow environments, and RBD in cold, fast flow, and thermally stable habitats. Exposure to warm and fluctuating habitats, and high mobility levels, have been shown to broaden metabolic function, potentially explaining the high enzyme activity and aerobic scope seen in FTD, defining their superior CTmax. Collectively, these findings provide insight to predict how climate change will affect local species, and may have conservation applications for determining which species may be most at risk with increased occurrence of extreme heat events.
Enhanced Precision of Aerial Herbicide Application for Invasive Species Management
(University of Waterloo, 2024-08-23) Lew-Kowal, Grace
Invasive common reed (Phragmites australis ssp. australis) has established and dominated in Ontario wetlands for decades. The detrimental effects of P. australis invasions on wetland habitats have demanded intervention through aggressive suppression efforts. However, constraints in available control methods to suppress P. australis have led to persistence. To improve P. australis management in wetlands, we investigated remotely piloted aircraft systems (RPASs) as a precision tool for herbicide application. We applied Habitat® Aqua with a spray-equipped RPAS at selected pilot sites, marking the first-ever application of its kind in Canada. We evaluated the suppression efficacy of RPAS-based herbicide application to P. australis and examined the plant community changes one-year after the initial herbicide application. We found a > 99% reduction in live P. australis stems, along with reductions in species richness (33%), Shannon-Weiner diversity (73%), Simpson’s reciprocal diversity (50%), and Pielou’s evenness (73%) in the year following herbicide application. We performed an in-field application experiment to quantify the herbicide deposition of an RPAS-based application through the vertical profile of a P. australis canopy, comparing medium and coarse droplet sizes. We determined that both droplet sizes achieved similar coverage and vertical penetration of herbicide within the P. australis canopy. Lateral droplet drift occurred at least 8 m away from the application area with medium droplets diminishing at a faster rate than the coarse droplets. We used remotely-sensed images taken before and after the RPAS-based herbicide application to determine the application accuracy and post-treatment effects. We determined that RPAS-based herbicide application to P. australis was on-target 91% of the time. The herbicide drift footprint extended up to 20 m away from the application area, representing a distance that is 96% lower than the label-recommended buffer distance for Habitat® Aqua application with a helicopter.
Seasonal temperature induced heart-collagen remodeling response in the rainbow darter (𝘌𝘵𝘩𝘦𝘰𝘴𝘵𝘰𝘮𝘢 𝘤𝘢𝘦𝘳𝘶𝘭𝘦𝘶𝘮)
(University of Waterloo, 2024-08-22) Hamel, Michael
Acclimation to temperature changes in fish has been shown to prompt a cardiac remodeling response, with collagen protein playing a key role, although the mechanism of this response remains unclear. Currently, it is believed to be a seasonal adaptation to shifting temperatures, with studies indicating that microRNA-29b (miR-29b), an epigenetic non-coding RNA, targets collagen mRNA in the heart. To further explore these questions, this thesis characterizes the remodeling response in a wild population of rainbow darters (Etheostoma caeruleum) to examine seasonal effects in a natural environment, which moves beyond the current lab-only studies. Rainbow darter heart tissue was collected on-site at three season timepoints (Spring, Summer, Fall 2023) from the Grand River, Grand Valley, ON. Water temperature loggers were deployed to monitor the fluctuating river temperatures throughout the study. Gene expression of miR-29b, and the three collagen type I protein transcripts (col1a1, col1a2, & col1a3) was measured in heart tissue through RT-qPCR. Hearts were embedded for histological analysis to visualize heart morphology and quantify the collagen protein content through picro-sirius red staining. Additionally, western blot analysis was performed to measure collagen type I abundance for each season. Temperature loggers revealed substantial daily water temperature fluctuations, with differing fluctuation profiles dependent on season. Results from RT-qPCR revealed seasonal differences in expression of col1a1, and col1a2, and western blot revealed a season effect driving a trend in collagen type I protein differences, suggesting the presence of this remodeling response in a non-model species. Although only a sex-specific difference was found in miR-29b expression and no significant regression was found between miR-29b and any of the collagen type I mRNA, further investigation into its role in this remodeling in vivo will be required. The lack of a compact myocardium layer was determined from cardiac tissue sections, which previously had not been investigated in the rainbow darter and evidence of cardiac hypertrophy at acclimatization to colder temperatures was demonstrated by higher relative heart mass compared to body mass in spring and fall, compared to summer. Understanding the impacts of temperature fluctuations and extreme weather events on local fish populations is increasingly crucial. With limited studies of cardiac collagen remodeling in natural environments, it is important that there be continued focus on bringing elements of natural seasonality into the context of lab studies to further investigate this remodeling response as seasonal plasticity and flexibility. This thesis contributes to a more comprehensive understanding of seasonal effects in a natural environment.
The Effect of Acute Heat Stress on the Chinook Salmon Immune System and on Ability to Combat 𝘝𝘪𝘣𝘳𝘪𝘰 𝘢𝘯𝘨𝘶𝘪𝘭𝘭𝘢𝘳𝘶𝘮 Infection
(University of Waterloo, 2024-08-13) McKenzie, Emily
The frequency, duration, and intensity of heatwaves in western Canada is expected to rise in the coming years. As a result, shallow rivers and streams have also experienced drastic changes, and water temperature may fluctuate by up to 13 °C throughout the duration of a single day. This poses a problem for salmon species, as they travel from marine to freshwater environments to spawn. Additionally, salmon are poikilothermic, meaning their physiological functions are influenced by their surrounding water temperature. Encountering water that is outside of their optimal growth temperature of 11-17 °C may result in behavioural and metabolic changes and, in extreme cases, rapid death. This may also result in increased susceptibility to infectious disease, as warming waters may render them temporarily immunocompromised and increase the virulence factors of some pathogens. Salmon have several cellular mechanisms to survive these stressful events, including heat shock proteins, immune responses, and secretion of glucocorticoids. Interestingly, the concept of thermal preconditioning is emerging as a method to aid salmonids’ ability to handle increasing water temperatures – exposure to a controlled, short term stimulus triggers a physiological response that prepares them for future, more extreme stress exposure. However, thermal preconditioning has never been tested before exposure to a pathogen. Further, the immune effects of heat waves have scarcely been investigated in salmon. In this study, we performed an environmentally applicable mock heat shock on juvenile Chinook salmon (Oncorhynchus tshawytscha) and measured their immunological responses up to 14 days afterwards. Chinook salmon also received an injection of live Vibrio anguillarum after heat shock to determine if heat deterred their ability to fight a systemic bacterial infection. The transcripts of IL-1β, IL-8, TNF-α, IL-10, TGF-β, IFN-γ, IL-2, cathelicidin, hepcidin, MHC1α, tapasin, MHC2α, MHC2β, HSP47, HSP70, and HSP90 were quantified by qPCR in the spleen, gills, and hindgut, as well as assessment of the stress response by measuring glucose, lactate, cortisol, and HSP47 levels in the plasma. Heat shock did not affect mortality rates due to vibriosis compared to salmon that received V. anguillarum alone. Additionally, heat shock mitigated the pro-inflammatory and corresponding anti-inflammatory responses needed to combat infection by initially upregulating il1b, tnfa, il8, and il10, then returning to normal levels by three days post-infection. The antimicrobial peptides cathelicidin and hepcidin played a significant role in combatting infection in both V. anguillarum treatment groups. Transcript levels of hsp47 and hsp90 were upregulated in response to both heat shock and bacterial infection; however, hsp70 expression was surprisingly low through the duration of the trial. HSP47 proteins measured by ELISA in the serum did not differ between any treatment groups. Heat shock caused a significant increase in plasma and cortisol lactate concentrations that both returned to basal levels 6-hours post-heat shock. Altogether, these data indicate that the experimental heat shock had a positive preconditioning effect on Chinook salmon and provides new insights on the interactions between the host, environment, and pathogen over a 14-day period. This research also provides a first step in understand how increasing river temperatures affect the salmon immune and stress responses and can help inform decision making in policy and sustainability initiatives to protect Pacific salmon populations in the coming years.
Early life exposure to diel thermal variation alters microRNA expression and performance in zebrafish.
(University of Waterloo, 2024-08-12) Gavarikar, Sana Manish
Freshwater ecosystems are characterized by large thermal variations, especially in the summer months. However, this variability is rarely accounted for in laboratory studies examining physiological processes of fish inhabiting these environments, thereby producing results which lack ecological realism. Understanding how animals respond to and cope with rapid changes in temperature is more crucial than ever as the range of thermal fluctuations in these habitats is expected to increase dramatically due to increases in the frequency and severity of heat waves. Recently, it was posited that epigenetic mechanisms could buffer fish against such thermal fluctuations as they act on a more rapid timescale than genetic adaptation. Thus, the aim of this thesis was to understand how chronic exposure to thermal variability impacts the physiology of freshwater fish, and if these effects are associated with changes in epigenetic modulators called microRNAs (miRNAs) which act by repressing the translation of target genes. This was achieved by subjecting zebrafish (Danio rerio) to realistic diel thermal fluctuations (FLUX; 28 ± 5°C) throughout the embryonic and larval stages to evaluate the effects on physiological processes like metabolism and survival, and on the expression of seven thermosensitive miRNAs and three heat shock proteins (HSPs). These fish were compared to those kept at constant control (CTRL; 28°C) and constant elevated (HEAT; 33°C) conditions to allow for comparisons with fish reared under optimal lab conditions and in traditional climate change experiments, respectively. After the developmental stages, the fish from CTRL and FLUX treatments were reared under common control conditions until adulthood to understand if developmental exposure to fluctuations altered miRNA expression profiles in the brain and the upper thermal tolerance (CTmax). This study revealed that while the HEAT conditions reduced survival throughout development, the FLUX thermal regime had no impact on this. Additionally, both FLUX and HEAT conditions significantly altered body weight as well as the miRNA expression profiles during early life stages. Although juveniles from both FLUX and HEAT conditions were able to metabolically compensate to their respective thermal regimes, the degree of compensation was greater in FLUX fish. I found that miR-181a-5p, which regulates pathways associated with mitochondrial biogenesis and respiration, was significantly upregulated in the juveniles from these groups, suggesting that changes in this miRNA could modulate the observed metabolic impacts. Notably, this miRNA remained elevated in the brains of longitudinal adults with FLUX developmental histories, though their CTmax was unaffected. Besides its role in modulating mitochondrial function, insights from mammalian models suggest that this miRNA can also enhance neuronal injury/damage in vertebrate brains. This could indicate that FLUX fish have an altered capacity for recovery following acute thermal stress. Collectively, the findings presented in this thesis demonstrate that developmental plasticity may be regulated by changes in epigenetic processes and underscore the need to incorporate variability into our experiments, as it produces a robust and long-lasting impact on the physiology of fish that is distinct from static temperature exposures. Ultimately, ecologically realistic conditions and the plasticity potential of populations need to be accounted for in investigations to accurately predict how fish will respond to the multiple stressors caused by climate change.
Investigation of the atypical type III secretion system in Pseudomonas syringae strains using long read sequencing technology
(University of Waterloo, 2024-08-12) Lorv, Janet
Ubiquitous across many environments, Pseudomonas syringae is a phytopathogen that is spread through the water cycle. With a broad host range, Pseudomonas syringae has been reported to cause outbreaks across many agricultural crops including potato, mango, and kiwifruits. Although this species is highly studied, interest in agricultural impacts has meant that sequencing efforts have focused primarily on virulent strains isolated from diseased plants. In this work, we explored the genetic differences of a recently diverged, closely related and putatively non-virulent (i.e., not visibly detected by host plant) subphylogroup of P. syringae strains, subphylogroup 2c. Strains in this monophyletic group are a part of the larger virulent phylogroup 2 and more broadly grouped with other agriculturally virulent strains in phylogroups 1 and 3. Virulence of this bacterial species complex has been attributed to the genetic mobility of genes encoding type III effector proteins that are secreted by the type III secretion system. Gain or loss of these genes can cause P. syringae strains to switch between virulent and avirulent (i.e., detected by host plant and rendered not virulent) phenotypes. Many other niche adaption genes such as phytotoxin biosynthesis genes can also contribute to a virulence phenotype. We propose that the mechanisms contributing to the putative non-virulence phenotype of subphylogroup 2c may be elucidated via identification and examination of the divergent gene clusters within P. syringae strains. To capture the full complement of genes, we sequenced and assembled complete genomes of two subphylogroup 2c strains, a biocontrol strain P. syringae pv. syringae 508, and a strain isolated from a healthy leaf, P. syringae TLP2. These strains were sequenced using Oxford Nanopore Technology's MinION long read sequencer to generate complete genomes without additional sequencing. Using one strain per R9.4 flowcell, we were able to generate >300x coverage for each strain with maximum read lengths exceeding 500kb and 250kb respectively allowing easy assembly of the full genome lengths. However, the ease-of-use of nanopore sequencing is hampered somewhat by the relatively high sequencing error rate of the reads, requiring extensive testing and optimization of recently developed software to maximize assembly quality. Despite optimization, many genes in the initial assembly remained fragmented. To address this issue, we developed the software tool Kastor that identifies and corrects draft assembly errors through comparisons to reference genomes and/or protein sequence information. Users can reference related genomes for more precise error identification, or use reference proteins for a more generalized identification method that doesn't rely on data from closely related species. For both the Pss508 and PsyTLP2 sequenced genomes, fewer than 3000 errors (comprising <0.05% of the assemblies) were corrected, however assembly completeness was increased from <85% to >99.8% as benchmarked using single copy orthologs. The completed assemblies were then annotated using NCBI's PGAP. The annotated gene sets of the two P. syringae strains were then compared to 428 P. syringae strains across the species complex. We identified several divergent gene clusters in both sequenced and subphylogroup 2c genomes, with the most prominent diverging gene cluster set encoding the atypical variant of the type III secretion system (T3SS). Using PIRATE, we identified a related gene cluster in the distantly related phylogroup 13. With the analysis of both gene sequence and gene content, we propose that the atypical variant is a result of horizontal transfer and not divergence from the canonical variant in closely related strains. The horizontal acquisition of this T3SS variant in the most recent common ancestor of subgroup 2c likely triggered the divergence of the subgroup from group 2, the loss of common type III effector genes, and a likely niche/virulence change, resulting in putatively weakly-virulent strains.
Wastewater Surveillance of Influenza (A, B) and Respiratory Syncytial Virus (RSV) in Southern Ontario
(University of Waterloo, 2024-07-16) Abu Farah, Joud
Wastewater-based surveillance (WBS) of SARS-CoV-2 has been highly effective at tracking trends of COVID-19 infections across the globe. This success led to the question of the possible applicability of WBS in monitoring other respiratory viruses that are also posing a concern for healthcare systems. Traditional influenza and RSV surveillance consists of monitoring hospital admissions, clinical testing data and outpatient visits. Even when clinical surveillance and hospitalization admissions are used to monitor the spread of these infections, they are often underestimated, with a lag between detection and community spread. In this study, wastewater surveillance of influenza A, influenza B and respiratory syncytial virus (RSV) was investigated, by quantifying the viral RNA of these viruses in wastewater and comparing these trends to clinical metrices. The solid–liquid partitioning behaviors of influenza A, influenza B, RSV and SARS-CoV-2 in wastewater were examined to understand the behavior of the viruses in wastewater. Analyzing whether each virus is present in the liquid or solid fraction of wastewater may affect the data interpretation and inform further method development. The viral RNA in the liquid and solid fractions were separated and enriched by undergoing different centrifugation settings, overnight polyethylene glycol (PEG) precipitation followed by centrifugation, or ultrafiltration using a Centricon Plus-70 device. The influenza A, influenza B and RSV viral RNA concentrations were found to almost exclusively partition in the solids fraction of wastewater, which increased with an increase in centrifugal settings, unlike SARS-CoV-2 that had a more even split in signal between the solids and liquid fractions. The effectiveness of normalization using the endogenous pepper mild mottled virus (PMMoV) was also examined, by comparing the normalized influenza signal with the raw signal. Normalization using the PMMoV biomarker did not increase or hinder the correlation with clinical testing data relative to the raw influenza signal in wastewater. Wastewater samples were collected once weekly from two wastewater treatment plants in the Region of Waterloo, Canada, from September 15, 2022, to June 21, 2023. Wastewater was treated overnight with polyethylene glycol (PEG), centrifuged (12,000g 1.5 h), then extracted (pellet) with Qiagen RNeasy PowerMicrobiome Kits and quantified using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). The RNA concentrations of influenza A, influenza B and RSV in wastewater strongly correlated with the cases reported by episode date and hospitalizations, in the cities of Kitchener and Waterloo (Spearman’s Rank correlation coefficient rho ranging from 0.41-0.85). Wastewater surveillance can be a very effective additional surveillance tool to support public health officials in monitoring the trends of respiratory viruses in communities. Further development of WBS for respiratory virus monitoring will help public health to better prepare for these and other emerging pathogens in the future.
Degradation of Polyethylene Terephthalate (PET) and Polyamide (PA)
(University of Waterloo, 2024-07-16) Griffiths, Erin
Microplastics have become an increasing concern to humans and ecosystems as plastic production continues to soar, due to their prevalence in the environment and lifespan. Plastic is cheap and durable making it an ideal industrial and commercial material. However, because of this popularity, it resides in most places on earth, including in human blood, and is difficult to remove due to its small size. These plastics can enter the environment through numerous methods, from landfills and dumps to washing machines and sinks. In recent years, there has been significant investigation in reducing plastic pollution. This a difficult task attributed to the varying size, shape, polymer type, chemical properties and location plastic can be found. It’s critical to understand the rate of degradation and the factors that influence it for two main reasons; it provides accurate timelines of degradation and techniques that may increase degradation need a starting point. In Chapter 2, I investigate the degradation rate of laboratory grade polyethylene terephthalate (PET) using a model enzyme (Huimcola insolens cutinase) to hydrolyze the plastic. This research aims to characterize the polymers used such that results can be compared and identify the analyses which capture degradation and characterize the polymer best. Environmental factors controlling enzymatic plastic degradation are not well studied and this experiment aimed to study the effect of incubation temperature, exposure to freeze-thaw cycles (FTCs) and extreme temperatures on the degradability of laboratory-grade PET. In addition, we also assessed the degradability of consumer-grade PET, sourced from plastic bottles, for comparison to the laboratory-grade PET. The first test was under variable temperatures, where plastic was incubated at 25 ˚C, 40 ˚C and 55 ˚C. The results show increased temperatures increase the rate of polymer degradation. The second set of tests were conducted under different pretreatments; treatments the plastic would undergo before incubation at 40 ˚C. Plastic was exposed to a series of freeze-thaw cycles (FTCs) or extreme temperatures (-70 ˚C or +55 ˚C). It was found any type of pretreatment increased the rate of degradation compared to plastics that did not undergo any pretreatment. The final condition tested was plastic type, where PET water bottles were obtained and incubated at 55 ˚C to determine the differences in degradability between laboratory-grade PET and consumer-grade PET. Consumer-grade PET was found to not have any significant degradation after 10 weeks of enzyme exposure, raising serious concerns regarding its degradability and lifespan. This result suggests that modifications to the consumer-grade PET during the fabrication process, such as heat treatments, are altering its chemistry and its degradation kinetics. Analyses for degradation and characterizing the polymers included: Fourier-transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and tensile strength measurements. Analysis of the crystallinity, tensile strength, SEM images and FTIR spectra measured indicate that PET’s physical and chemical properties were modified when degraded. Overall, the PET’s tensile strength decreased and the crystallinity increased with increasing hydrolytic degradation. FTIR spectral changes were seen early on, with peaks of interest at 1237 cm-1, 1016 cm-1 and 1087 cm-1, and finally at 1716 cm-1, and the flattening of these peaks increased with increasing hydrolytic degradation. The results highlight that enzymatic degradation rates can be highly variable due to differences in environmental conditions. It also highlights the large difference in the degradability of consumer-grade versus laboratory-grade PET, which has significant implications for in situ environmental degradation rates. In Chapter 3, I investigated the rate of laboratory-grade PET and polyamide (PA) degradation in stormwater pond sediment over a 16 month period in a stormwater pond in Kitchener, Ontario. Microplastic accumulation in the environment, especially in bodies of water and sediment is a well-known problem. Stormwater ponds act as a microplastic sink and draw pollutants from urban and industrial wastewater before it enters oceans or lakes. This results in high levels of microplastics remaining in stormwater pond sediment. Stormwater ponds are an excellent site to determine realistic plastic degradation in the environment, in a contained area where high concentrations of plastic is known to be present. To date, no long-term polymer degradation studies have been conducted in a stormwater pond despite the rising popularity of these ponds. For this study, 8 pore water samplers (peepers) were packed with pond sediment and plastic pieces were inserted into each cell of the peeper. An additional 8 peepers filled with water, such that pore water chemistry could be collected. The peepers were inserted into the pond sediment and sacrificed periodically over the course of 16 months. For the first 8 months both PET and PA plastic increased in mass as they absorbed water. After 16 months of field incubation, PA had degraded by 0.42% and PET was still net positive (higher mass than before the incubation) however it was close to its original weight. The obtained results highlight the lack of degradation to plastics in stormwater pond sediment and suggest lifespans are longer than previously estimated. Based on previous degradation studies under sediment conditions, this study suggests that stormwater pond sediment is the least effective at degradation polymers, which may be attributed to the pond water chemistry and microbial communities present. Microplastics are known to accumulate in stormwater pond sediment but they are found to degrade at slower rates than other sediment profiles. The laboratory experiment results in Chapter 2 show under ideal conditions laboratory-grade PET degrades minimally at low temperatures. Additionally, the lack of degradation seen with the consumer-grade PET in Chapter 2 suggests that under environmental conditions, the polymer would take even longer than the laboratory-grade polymers to degrade. The combination of Chapter 2 and 3 demonstrate the difference between ideal and environmental conditions for polymer degradation. This research provides evidence to strongly advocate for the removal of microplastics before they enter the environment as I have proven they take considerable lengths of time to degrade under various conditions. I encourage this research to be used by any future researchers who hope to develop methods for plastic pollution reductions.
Patterns in stream biofilm communities and organic matter processing in an agricultural stream network: A multi-scale assessment of the influence of groundwater
(University of Waterloo, 2024-06-03) Banks, Lauren
Benthic stream biofilm communities support stream ecosystem structure and function by mediating nutrient and carbon cycling. Understanding how environmental factors shape biofilm communities in stream ecosystems is therefore essential. Biofilm communities have been shown to be strongly influenced by nutrient availability and temperature, factors that can be modified by groundwater input at multiple spatial scales. However, in enriched streams, groundwater input as a driver of heterogeneity in surface water environmental conditions has not been well-explored among stream reaches (kilometer scale), habitat types (meter scale), and patches (centimeter scale), nor has the seasonal consistency of these relationships been studied. To investigate the association of groundwater input to biofilm communities, I conducted three interconnected field studies in Kintore Creek, a nutrient-rich agricultural stream network in Ontario, Canada. First, I assessed if variability in groundwater input altered patterns of biofilm communities and cellulose decomposition among reaches over four temperate seasons (Chapter 2). Next, I compared habitats (i.e., riffles and runs) in reaches with high, moderate, and low groundwater inputs to determine if habitat type modified the effects of groundwater input on stream biofilm communities and cellulose decomposition by varying environmental conditions (Chapter 3). Lastly, I assessed the response of stream biofilm communities and cellulose decomposition to a gradient of groundwater upwelling at the patch scale and tested whether small scale variations in environmental conditions are associated with biofilm communities and cellulose decomposition (Chapter 4). The results of Chapter 2 showed no within season association of groundwater input to biofilm communities, with vii seasonality driving heterogeneity in biofilm communities. Findings in Chapter 3 demonstrated that habitat type modified effects of groundwater input on biofilm communities. Groundwater influence was expressed by greater primary production and decomposition in runs in reaches with groundwater input compared to runs in the reach with no groundwater input. At the patch scale (Chapter 4), groundwater upwelling did not appear to generate substantial variation in surface water conditions, and variability stream velocity was the primary driver of heterogeneity in stream biofilm communities. The findings of this this thesis are in contrast to past work that found effects of groundwater on stream biofilm communities in nutrient-poor streams. These results may be due to cumulative effects of groundwater input throughout the stream network, thereby limiting the ability to detect environmental drivers of groundwater influence at small spatial (i.e., habitat, patch) scales. Therefore, additional studies comparing catchments with differing levels of groundwater are needed to fully understand the influence of groundwater on stream biofilm communities in differing landscape contexts. A major challenge across spatial scales was the ability to represent the impact of groundwater inputs through environmental measures and biofilm communities, suggesting further investigations at the stream water – biofilm interface is required to disentangle the environmental drivers associated to heterogeneity in biofilm communities. The results of this thesis suggest that the influence of groundwater input on stream biofilm communities and processes depends on the context of stream ecosystem, therefore understanding effects of groundwater input requires future research across a diverse range of stream ecosystems.
The Development and Evaluation of Next-Generation Metallic Nanomedicines for Oncology
(University of Waterloo, 2024-05-27) Youden, Brian
Nanoparticles (NPs) are ultrasmall objects with profound applications in research, industry, and medicine. Next-generation nanomedicines, such as gold, hafnium, iron, and copper nanoparticles, are particularly interesting due to their excellent physical, chemical, and quantum properties that can be exploited for cancer diagnosis and therapy. However, despite their demonstrated preclinical effectiveness, the potential of these inorganic nanomedicines, both in oncology and the broader medical field, is hampered by mechanistic uncertainty and a lack of detailed regulatory guidance. Together, these factors have resulted in many failed clinical trials and unexpected and sometimes severe side effects for approved formulations. The therapeutic efficacy and toxicity of nanomedicines are controlled by an extremely complex interplay of nanoparticle physicochemical properties and individual patient biology, where many confounding factors exist. This makes designing and evaluating nanomedicines a challenging task. To progress metal-based nanomedicines to the clinic and for them to be considered safe, even in the life-or-death circumstances of cancer, a deep understanding of nano-bio interactions is necessary across different stakeholders. This includes physicians, academia, industry, and government. By understanding and utilizing these in vivo behaviors, powerful nanomedicines and novel treatments can be applied to oncology. This thesis begins with a summary of the fundamental concepts relating to nanotechnology and the origins, properties, and treatment of cancer. Chapter 2 expands this discussion for a comprehensive analysis of cancer nanomedicines and their structure-activity relationships (SARs) in the body, which are central to both treatment efficacy and safety. Fundamentally, SARs describe the interactions between NP properties and the biological systems that ultimately produce their effects. To assist in the communication of this information, identified SARs were integrated into a simple, adaptable, and guiding framework composed of a parameter space, a pathway model, and various evaluation metrics. By resolving the complexity of nanomedicine into three parts, representing the interactions of NPs with 1) whole organs, 2) individual cells, and 3) fundamental biochemical pathways, this framework provides a clear illustration of how to fine-tune nanomedicines via pathway analysis. This framework and SARs were then used to guide the design, application, and evaluation of next-generation nanomedicines containing gold and copper. Gold nanoparticles (GNPs) have long been proposed as promising agents for cancer phototherapy and image-guided radiation therapy (IGRT) due to their strong absorption of near-infrared (NIR) light and X-rays. GNPs are also among the most studied NPs owing to their general biocompatibility and easy synthesis. Despite this, only one GNP has been approved for clinical use owing to long-term safety concerns. Among various SARs, those related to size are often the most critical parameters for both efficacy and safety. This stems from both the nanoparticles themselves and the size-restrictive nature of kidney, liver, and tumor filtration of blood. To optimize the use of GNPs for enhanced IGRT, drug delivery, and photothermal therapy (PTT), drug-loadable lipid NPs were utilized as a scaffold for GNP assembly, forming a versatile nanocomposite (Lipogold). Overall, this allows small NPs to function collectively as one larger nanoshell with plasmonic properties. Over time, this shell will degrade as the soft liposome core is stressed and deformed, resulting in renal-clearable NPs that can be cleared by the body following treatment. This thin shell of gold also optimizes the Auger process for RT and enables PTT, while the hollow core allows for encapsulation and delivery of drugs and molecular contrast agents. Thus, Lipogold nanocomposites demonstrate the advantages of both large and small NPs while adding multifunctionality. In this work (Chapter 3), medical radiation sources and cellular models were used to test their ability to sensitize cancer cells to megavoltage X-ray radiation therapy, provide contrast for computed tomographic (CT) imaging, deliver drugs, and engage in NIR-based PTT. In addition to GNPs, plasmonic copper sulfide (Cu2-XS or just CuS) NPs are also emerging as increasingly popular nanomedicine candidates due to their photothermal properties, biodegradability, an ability to convert less-toxic H2O2 into more potent reactive oxygen species (ROS) for chemodynamic therapy (CDT). However, this approach in cancer therapy is fundamentally limited by several factors, principally the low concentration of H2O2 in the body. To overcome this issue, the properties of the tumor microenvironment (TME) were exploited for nanomedicine design, where CuS NPs were combined with the enzyme glucose oxidase (Gox) for a synergistic combination of starvation therapy, CDT, and PTT. Gox was used to convert glucose, which is upregulated in the TME, into H2O2 and acid, starving the cancerous cells and activating the Fenton-like reactivity of the CuS NPs. Deep-penetrating NIR could then be used for PTT and to enhance reaction kinetics specifically at the tumor site. The fundamental reaction mechanism was also investigated, highlighting the accelerative effect of chloride ions on the copper-Fenton reaction, which are present at high concentrations within skin and individual cells. In Chapter 4, the therapeutic efficacy and biocompatibility of the Gox@CuS nanocomposite were demonstrated using in vitro and in vivo melanoma models. To further improve the safety profile of the Gox@CuS nanocomposite, the emerging technology of microneedle patches were explored as a transdermal drug delivery approach. Since conventional injections can lead to off-target uptake and toxicity, transdermal delivery may improve both efficacy and safety by maximizing local delivery and limiting blood exposure. This approach was extensively reviewed (Chapter 5) to determine the viability, design considerations, and fabrication methods of MNs containing light-responsive NPs such as Gox@CuS. Applications outside oncology were also reviewed to fully understand the advantages and limitations of this delivery system. Gox@CuS were then integrated into dissolvable polymeric microneedle (DPMN) patches and compared to hypodermic injection using another mouse melanoma model. In this study (Chapter 6), the microneedle patches were demonstrated to deliver a higher amount of Gox@CuS to the tumor site and reduce the risk of systemic toxicity. Further mechanistic insight into the catalytic behavior of CuS NPs was also collected, specifically identifying the effect of chloride ions on the generation of both hydroxyl radicals and singlet oxygen. Overall, this thesis contributes to our overall understanding of cancer nanomedicine and demonstrates several novel next-generation treatment strategies using metal-containing NPs. The framework proposed in this work is an adaptable and potentially valuable resource for researchers and regulators to understand SARs. Additionally, the pathway modelling used by this framework can assist in the development and integration of machine learning models that will increasingly play a role in the regulatory and industrial development of nanomedicine formulations.
Differential Gene Expression Analysis using Resampling
(University of Waterloo, 2024-05-09) Yang, Yifan
The primary objective in high-throughput sequencing is to identify differentially expressed genes, which provides substantial information of gene expression and regulation. A persistent challenge in this field is the bias caused by limited replicates. To address this, we have developed a novel Bootstrapping method. This approach enhances the power of DEG detection by augmenting the sample data points. New data points are generated through weighted geometric combinations of bootstrap samples and a pooled distribution. The pooled distribution consists of sample data from genes with similar expression levels. Through simulation tests and evaluations on real-world data, our proposed Bootstrapping method exhibited competitive performance compared to common DEA tools (edgeR, DESeq2, Limma-Voom). A key advantage of the Bootstrapping method is its independence from any assumptions about the sample distribution. This independence avoids the bias raised from inaccurate assumptions, offering the potential for broad application across various areas of genomic research.
Metatranscriptomic analysis of pediatric acute sinusitis: pathogen detection and host response profiling
(University of Waterloo, 2024-05-08) Abu Mazen, Nooran
Acute sinusitis (AS) is the fifth leading cause of antibiotic prescriptions in children. Distinguishing bacterial AS from common viral upper respiratory infections in children is crucial to prevent unnecessary antibiotic use but is challenging with current diagnostic methods. Despite its speed and cost, untargeted RNA sequencing (RNA-seq) of clinical samples from children with suspected AS has the potential to overcome several limitations of other methods. However, the utility of sequencing based approaches in analysis of AS has not been fully explored. Here, we performed RNA-seq of nasopharyngeal samples from 221 children with clinically diagnosed AS to characterize their pathogen and host-response profiles. Results from RNA-seq were compared with those obtained using culture for three common bacterial pathogens and qRT-PCR for 12 respiratory viruses. Metatranscriptomic pathogen detection showed high concordance with culture or qRT-PCR, showing 87%/81% sensitivity (sens) / specificity (spec) for detecting bacteria, and 86%/92% (sens/spec) for viruses, respectively. 22 additional pathogens not tested for in the clinical panel were detected, and plausible pathogens were identified in 11/19 (58%) of cases where no organism was detected by culture or qRT-PCR. 205 viruses were assembled across the samples including novel strains of coronaviruses, respiratory syncytial virus (RSV), and enterovirus D68. By analyzing host gene expression, host-response signatures were identified that distinguished bacterial and viral infections and correlated with pathogen abundance. Ultimately, this study demonstrates the potential of untargeted metatranscriptomics for in depth analysis of the etiology of AS, comprehensive host-response profiling, and using these together to work towards optimized patient care.
Metabolic engineering strategies for biomanufacturing of chemicals using Yarrowia lipolytica and Escherichia coli
(University of Waterloo, 2024-04-24) Kefale, Teshager Bitew
This dissertation advances metabolic engineering by optimizing the genetic and metabolic capabilities of Yarrowia lipolytica and Escherichia coli to enhance their applications in biotechnology. It focuses on improving Y. lipolytica's mannitol and amino acids production by varying fermentation temperatures and employing techniques like shake flask fermentation, HPLC, and NMR. Notably, mannitol production was enhanced through targeted modifications of FBP1 gene at elevated temperatures. RNAseq analyses highlighted shifts in metabolic pathways under thermal stress, markedly in lipid, sugar and amino acids metabolism. Additionally, a dual-gRNA CRISPR-Cas9 system was integrated within the pCRISPRYL2 plasmid, noticeably improving genetic editing precision by overcoming the constraints of the non-homologous end joining (NHEJ) pathway. Furthermore, the study pioneered a Cell-Free Metabolic Engineering (CFME) strategy to synthesize 5-Aminolevulinic Acid (5-ALA) utilizing optimized enzymatic reactions and operational conditions, presenting a scalable and eco-friendly alternative to conventional whole-cell systems. In parallel, engineered E. coli demonstrated robust heme production capabilities in both whole-cell and cell-free systems. Heme derivatives, including valuable pigments like biliverdin, Phycocyanobilin (PCB) and Phycoerythrobilin (PEB) were also produced at a 1L bioreactor scale utilizing E. coli engineered with unexplored enzymes. Overall, this work not only expands the scope of metabolic engineering but also sets a foundational work for future innovations in biomanufacturing.
Structural and Biochemical Insights into Previously Uncharacterized Activity of Nucleotide-Dependent Phosphoenolpyruvate Carboxykinases
(University of Waterloo, 2024-04-08) Barwell, Sarah
Phosphoenolpyruvate carboxykinase (PEPCK) enzymes are central to glucose metabolism, with their main role in catalyzing one of the first steps of gluconeogenesis. In addition to its primary role, PEPCK is also involved, whether directly or indirectly, in glyceroneogenesis, amino acid metabolism, and lipogenesis. The PEPCK enzyme is responsible for the conversion of oxaloacetic acid to phosphoenolpyruvate using a triphosphate nucleotide as a phosphoryl donor. This dissertation focuses on human cytosolic PEPCK and Escherichia coli PEPCK as representative enzymes of the PEPCK family. The hcPEPCK enzyme is well characterized and extensively studied. Recent research suggests a new role for this enzyme, and potentially other GTP-dependent PEPCKs, as a protein kinase. hcPEPCK is thought to phosphorylate the ER protein INSIG, resulting in a cascade effect leading to increased lipogenesis. Expanding upon the information in the literature, phosphomimetic mutants were used to study the effects of phosphorylation on hcPEPCK, and its subsequent interactions with the INSIG protein. The ATP-dependent PEPCKs are less studied than their GTP counterparts, and some of the research in the literature is not up to date. Historically, the bulk of the research has involved GTP-dependent PEPCK. Using E. coli PEPCK as a proxy for the ATP-PEPCK class, a full kinetic and structural characterization was performed to further our understanding on these enzymes and compare differences and similarities between the classes. In addition, a new 31P-NMR assay was designed to address the hypothesis that ATP-dependent PEPCK can catalyze two additional reactions: the conversion of phosphoenolpyruvate to pyruvate (pyruvate kinase-like activity), and the nucleotide-dependent catalysis of phosphoglycolate.
Environmental DNA barcoding as a method of amphibian species detection compared to conventional monitoring techniques in southern Ontario vernal pools
(University of Waterloo, 2024-03-06) Zamora, Cailyn
Ongoing monitoring is vital for the conservation of amphibian species and is conducted through conventional auditory and visual surveys. A molecular method, termed environmental DNA (eDNA) barcoding, may offer a more sensitive method of species detection that negates the need for direct species observation. The research aims of this thesis were to conduct a comparative analysis of eDNA barcoding versus conventional (audio/visual) species detection methods for six amphibian species in southern Ontario. I hypothesized that eDNA barcoding would offer equal or greater species presence detections compared to the conventional methods. Conventional surveys and eDNA collections were conducted in three vernal pools from April-July 2019 in collaboration with rare Charitable Research Reserve (Cambridge, ON). Conventional methods included collection of daily audio files from acoustic song meters and weekly/biweekly visual encounter surveys. Audio data was analyzed using Kaleidoscope Pro. Alongside conventional surveys, duplicate water samples containing eDNA were collected at multiple sampling locations around three vernal pools. After water collection, eDNA was concentrated by filtration, extracted, and quality controlled. eDNA samples were processed using optimized eDNA barcoding assays using quantitative PCR. Comparative analysis between conventional methods and eDNA barcoding contradicts a one-size-fits-all model of amphibian monitoring. eDNA barcoding offered a reliable and effective method of species detection for five of the target amphibians especially for obligate vernal pool breeding species, however this method failed to accurately detect the spring peeper despite detections by passive acoustic surveys. I propose using eDNA barcoding alongside a conventional method of species detection to optimize detections across a spatiotemporal scale, however, this should be catered to the target species of interest. Future studies could implement a multi-year study as well as a comparison of eDNA barcoding to metabarcoding for Ontario amphibian species. eDNA barcoding offers a new method of species detection that could aid in ongoing amphibian monitoring and therefore conservation efforts of the declining taxa.
Seasonal and environmental effects on physiological tolerances and gill adaptations of imperilled Eastern Sand Darter (Ammocrypta pellucida)
(University of Waterloo, 2024-02-15) Firth, Britney
In the absence of accurate biological information, conservation and recovery strategies carry the risk of failing to meet their objectives. Understanding species-specific physiological tolerances is important for several aspects of conservation, such as better understanding habitat requirements, determining the significance of future environmental change, and evaluating the suitability of recovery activities such as reintroduction. Currently, there is relatively little scientific information on the physiological tolerances of Eastern Sand Darter (Ammocrypta pellucida), a small benthic fish listed as threatened under the Species at Risk Act in Canada. The goal of this thesis was to define temperature and oxygen tolerances of Eastern Sand Darter and determine the role of temperature and turbidity, two common environmental stressors, on these tolerances. I also aimed to determine the response of three co-occurring darter species to the same environmental stressors. All objectives were conducted to evaluate physiological thresholds, thereby improving our understanding of species habitat, which allows us to identify restoration priorities and evaluate the suitability of candidate sites for reintroduction. To achieve this goal, field trials were conducted between June and November 2019 in the Grand River, Ontario, to encompass a range of ambient water temperatures (7-25℃) and August 2020 in the comparatively more turbid Thames River, Ontario. Critical thermal maximum (CTmax), agitation temperature (Tag), hypoxia tolerance (loss of equilibrium, LOE; and critical oxygen tension), and metabolic rate were measured to test the effect of seasonal temperature change and turbidity. Gills were collected from incidental mortalities of Eastern Sand Darter, and from three co-occurring darter species (Blackside Darter, Percina maculata; Greenside Darter, Etheostoma blennioides; and Johnny Darter, Etheostoma nigrum) to assess gill morphometrics. Chapter 2 demonstrated that Tag and CTmax significantly increased linearly with water temperature but there was no significant influence of turbidity. However, there was a significant interaction between turbidity, mass, and CTmax, indicating that turbidity has a hypothesized, indirect effect on CTmax by reducing growth. Overall, study results better define the sensitivity of Eastern Sand Darter to temperature and turbidity fluctuations and provide a better understanding of suitable thermal habitat. In Chapter 3, I demonstrated that temperature alone had no impact on metabolism but significantly and positively impacted hypoxia tolerance metrics (Loss of equilibrium and critical oxygen tensions). However, temperature explained very little of the variability seen in the response of metabolism and both hypoxia tolerance metrics across seasons, while environmental and fish-specific factors (reproduction and condition) explained more of the variation. Overall, temperature may be a weak predictor for Eastern Sand Darter metabolism and hypoxia tolerance, but the identification of a relatively high hypoxia tolerance helps to explain why the species persists in sand substrates that may have low availability of oxygen. In Chapter 4, I determined that turbidity significantly increased gill interlamellar space, filament width, and loss of equilibrium (i.e. decreased hypoxia tolerance) but had no significant influence on any other gill morphometric measurements, or on species metabolism. The findings suggest that turbidity has limited impact on Eastern Sand Darter gill physiology and that current turbidity levels do not surpass most physiological thresholds of Eastern Sand Darter. Chapter 5 demonstrated that temperature and turbidity have limited impact on the gill morphology of three common darter species, only impacting ionocyte number, lamellae width, and hematocrit. Findings suggest that the three darter species have limited plasticity in gill morphology under the range of temperature and turbidity tested. Having data on other darter species lets one put Eastern Sand Darter in context of other members of the ecosystem that are phylogenetically related. Overall, my thesis provides information on the physiological tolerances of Eastern Sand Darter that can be used to inform species recovery under Canada’s Species at Risk Act, such as refining the description of critical habitat, identifying habitat restoration objectives, and evaluating the suitability of candidate sites for reintroduction. Additionally, the discovery that current turbidity levels have limited impact on Eastern Sand Darter physiology allows for a refined understanding of how siltation impacts the species survival.
Transcriptional regulation of North American wood frog (Rana sylvatica) dorsal skin during development and in response to chronic exposure to a microplastics mixture
(University of Waterloo, 2024-02-09) Thompson, John Drew
Amphibian populations have been experiencing declines globally for decades due to factors such as habitat loss, invasive species, climate change, overexploitation, pollution, and disease. Skin is an important immune organ in amphibians, employing physical, chemical, microbiological, and immunological defences to prevent infection. Amphibian skin also undergoes dramatic morphological changes during metamorphosis. Therefore, it is important to gain a deeper understanding of the biological changes that amphibian skin undergoes during metamorphosis and whether these tightly regulated processes can be disrupted by environmental contaminants. Microplastics are a persistent, ubiquitous environmental contaminant of increasing concern with a wide range of effects on exposed organisms. Previous studies have reported mortality, reduced growth, behavioural changes, and hepatotoxicity in amphibians exposed to microplastics. However, few studies have evaluated sub-organismal effects of microplastic exposure on amphibians, particularly in relation to the immune system. Furthermore, few studies have chronically exposed amphibians to an environmentally realistic microplastics mixture. To address these gaps, an outdoor mesocosm experiment was performed where developing North American wood frogs (Rana sylvatica) were exposed from embryos throughout metamorphosis to a microplastics mixture consisting of an equal number of particles by count of polypropylene, polystyrene, and polyethylene terephthalate microplastics. Developing wood frogs were exposed to one of three concentrations: a 0× negative control of 0 g/L, a 1× treatment of 0.069 g/L, or a 10× treatment of 0.691 g/L of the microplastics mixture. This thesis reports on the transcriptomic analysis of dorsal skin of wood frogs from three different developmental timepoints: (1) after 50 days of exposure, average Gosner stage (GS) 34; (2) after 74 days of exposure, average GS 42; and (3) GS 45, 74 – 82 days of exposure. The first analysis performed compared the dorsal skin transcriptome in the 0× unexposed wood frogs at different timepoints to evaluate changes in dorsal skin gene expression during development. Trends corresponding to known developmental phenomena such as keratinization of the skin and active ion transport nearing the end of metamorphosis were observed in the data. Downregulation of immune genes was also seen in the skin after the first timepoint, and is in line with the systemic immunosuppression metamorphosing amphibians undergo leading up to and during metamorphic climax. This result demonstrates that the systemic immunosuppression is paired with suppression of immune function in skin tissues. The second analysis evaluated the effects of microplastic exposure on the dorsal skin transcriptome at each of the three selected developmental stages. Concentration- and timepoint-dependent effects were observed, with the greatest number of differentially expressed genes found in the 1× treatment at the first timepoint (GS ~34). Clusters of differentially expressed genes involved in mitochondrial function, protein synthesis, transmembrane transport, and immunity were found in at least one timepoint/treatment group. Developmental plasticity, morphological and behavioural changes during development, and a potential nonmonotonic dose response to microplastics in the wood frog are ideas proposed to account for the differences in response seen to different microplastic concentrations and at different developmental timepoints. Downregulation of immune genes was seen at the first developmental timepoint in response to the 1× microplastic treatment, suggesting that microplastics could cause developmental stage-specific immunosuppression in developing wood frogs. Because amphibian populations are already experiencing declines due to infectious diseases, it is important for future studies to continue to evaluate the potential for microplastics to act as a stressor and predispose amphibians to infection.

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