Robichaud, Karyn2026-04-132026-04-132026-04-132026-04-07https://hdl.handle.net/10012/22998Aquatic and terrestrial environments are dynamic due to natural and anthropogenic sources, including pollutants, thermal variability, and hypoxia. These stressors or perturbations can result in changes to energetic demands of animals resulting in metabolic stress. As a major energy transduction site within cells, mitochondria can respond to alterations in environmental conditions and metabolic stress by changing their physiology. Specifically, mitochondrial oxidative phosphorylation may change during stress, resulting in altered oxygen consumption rates and efficiency of energy transduction. These plastic responses of mitochondria can be regulated by post translational modifications to proteins; however post-transcriptional regulation of genes may also alter mitochondrial physiology. Genes can be post-transcriptionally regulated by microRNA; microRNAs are small, non-coding RNA molecules that canonically suppress mRNA expression, and thus protein expression. MicroRNAs regulate nuclear encoded genes in response to a variety of physiological stressors, however microRNA can also redistribute subcellularly into mitochondria. Mitochondria contain their own genome which encodes proteins involved in oxidative phosphorylation, therefore mitochondrial microRNAs (mitomiRs) can also regulate mitochondrial gene expression in response to stress. The overall goal of this thesis was to investigate mitomiRs in animals and predict their potential role in regulating mitochondrial function in response to metabolic stress. Most research on mitomiRs prior to this thesis was conducted in mammals, and with respect to disease. Therefore, this thesis aimed to compare mitomiRs across species, identify whether mitomiRs change in abundance with exposure to different stressors, and predict mitomiR mRNA targets. It was hypothesized that mitomiR abundances differ based on environmental changes (stressor type, stressor duration) and inherent differences (species, sex), resulting in changes to mitochondrial function during metabolic stress. Chapter 2 investigated the mitomiR profiles of zebrafish (Danio rerio) brains under control conditions and during acute exposure to two known metabolic stressors (hypoxia and elevated temperature). Exposure to each stressor resulted in distinct mitomiR profiles, where two mitomiRs were differentially abundant during hypoxia, and another mitomiR had altered abundance during thermal stress. The predicted nuclear targets of these mitomiRs were mainly involved in metabolic pathways, with many distinct predicted targets. Furthermore, brain mitochondrial respiration was only altered during thermal stress, and results indicated a potential decrease in ATP synthesis efficiency. Overall, brain mitochondrial respiration and mitomiR abundances had stressor-specific effects. Chapter 3 investigated whether venlafaxine, an antidepressant commonly found in wastewater effluent, altered zebrafish brain mitochondrial respiration and mitomiR abundances. In vitro, this study first confirmed that venlafaxine suppressed brain mitochondrial respiration. Then, an acute time-course exposure was conducted using zebrafish. In vivo, venlafaxine had minimal effects to brain mitochondrial respiration, however, three mitomiRs were differentially abundant based on exposure, sex, and time sampled. Changes to mitomiR abundance may have been due to their host gene expression, circadian rhythm, and venlafaxine exposure. Chapter 4 utilized three species of wild fish (Etheostoma spp.), to determine if they responded similarly to chronic exposure to wastewater effluent outfall in the Grand River, Waterloo, with respect to mitomiR abundances and mitochondrial function. Wild rainbow (Etheostoma caeruleum), fantail (E. flabellare), and Johnny darters (E. nigrum) were collected from up and down stream of the Waterloo wastewater treatment plant, then liver mitomiR abundances and mitochondrial cytochrome c oxidase activities were measured, and species-specific differences were detected. Results indicated that these darter species had species-specific changes to mitomiR abundances and mitochondrial enzyme activity when living downstream of the Waterloo wastewater treatment plant. Chapter 5 provided a comparative study between vertebrate taxa using a mammal that experiences drastic changes in mitochondrial respiration during torpor and interbout euthermia hibernation states. During torpor, mitochondrial respiration is suppressed and returns to summer values during interbout euthermia. This study profiled mitomiRs in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) during summer and hibernation. Changes in mitomiR abundances were detected during hibernation, and mitomiRs had predicted effects that may indicate their involvement in regulating changes to mitochondrial respiration and function during hibernation. The major finding of this thesis was that mitomiRs are differentially abundant based on stressor, sex, and species, despite some conservation of mitomiRs among species studied. This thesis also predicted targets for differentially abundant mitomiRs within mitochondrial and nuclear genomes, and measured changes in mitochondrial enzyme activities and respiration in fish to provide context for how mitomiRs may aid in regulating mitochondrial function. Overall, this thesis contributed to knowledge of mitomiRs and how they show distinct abundance patterns using a variety of comparative approaches.enDoctoral Thesis