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Purpose: In response to resistance exercise training, it has been shown that individuals with a previous training history acquire muscle volume at an accelerated rate. This phenomenon may be attributed, in part, to the myonuclear enrichment resulting from the proliferation of muscle progenitor cells, which promotes essential protein synthesis following subsequent muscle training. As a highly energy demand tissue, the successful hypertrophy of muscle fiber depends on mitochondrial biogenic progression. Moreover, the majority of genes that encode mitochondrial proteins are within nuclear genome. Therefore, in this study, we investigated the effect of increased number of myonuclei in response to the previous resistance exercise preconditioning on mitochondrial adaptations to subsequent resistance training. Our central hypothesis was that pre-trained muscles would show an accelerated acquisition of training-induced mitochondrial function leading to a greater skeletal muscle hypertrophy compared to previously non-trained muscles and this may be associated with increased number of myonuclei in the pre trained muscles. Methods: Thirty-two Sprague-Dawley rats were randomly assigned to four groups (n=8 per group) which include control, pre-training, training, and retraining group. Resistance exercise training was carried out by ladder climbing with weights attached to the tail at ages of either 8- (pre-training) and 36-week-old (training), or both (retraining). Each training session consisted of 3 sets of 5 repetitions, and the training protocol was performed every third day for 8 weeks. At 44 weeks of age, specific muscle groups were carefully collected and stored at -80 °C until further analyses. 4', 6-Diamidino-2-phenylindole staining, hematoxylin & eosin staining, cytochrome c oxidase and succinate dehydrogenase staining were performed. Western blotting and immunohistochemstry were performed to assess the abundance of mitochondrial regulatory proteins and the mitochondrial content. In complementary in vitro studies, confluent L6 myoblast cells were further grown in differentiation media for 4 days with or without insulin-like growth factor 1 (50 ng/ml) supplementation. Mitochondrial gene expression levels and mitochondrial respiratory function were assessed after 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR, 1 mM), a 5' AMP-activated protein kinase activator, treatment. Results: Myonuclei numbers were higher in training and retraining groups than control group (all, p < 0.05), suggesting that ladder climbing training protocol increased myonuclei number. There was a significantly higher level of myonuclei number in pretraining group compared to the control group indicating that the acquired myonuclei during exercise preconditioning were retained over the 20-week detraining period. Muscle cross-sectional area, mitochondrial content and mitochondrial enzymatic activities (COX and SDH) were significantly greater in retraining group compared to training group (p < 0.01, p < 0.01 and p < 0.05, respectively). In in vitro study, L6 myotubes preconditioned with IGF-1 showed increased myonuclei numbers within each myotube and presented a higher level of mitochondrial gene expression and oxygen consumption rate under AICAR treatment condition. Conclusions: These data provide physiological evidence that pre-trained muscle with more myonuclei make the muscles more responsive to subsequent training in terms of muscle hypertrophy and mitochondrial remodeling. Furthermore, this study provides a proof-of-concept of biological processes underlying potential nuclear-mitochondrial interplay during muscle hypertrophy. These findings warrant future studies to identify a novel target for mitochondrial medicine to treat muscle atrophy. / Kinesiology

Cardiovascular disease (CVD) is the leading global cause of death. Coronary Artery Disease (CAD) is a grouping of the most common cardiovascular diseases and is the current leading cause of death in developed countries. Treatments for CAD include pharmaceuticals as well as surgical interventions such as percutaneous coronary intervention (PCI) and coronary artery bypass grafting. However, these treatments do not completely remove the risk of adverse outcomes. Endothelial dysfunction is the underlying cause of CAD and is initiated by the chronic inflammation of the vasculature due to increased oxidative stress and production of reactive oxygen species (ROS). Previous studies have shown that the deletion of caveolin, a signaling molecules abundant within endothelial cells, can enhance inflammatory responses and lead to increased oxidative stress and ROS production. Mitochondrial ROS created from dysfunctional mitochondrial dynamics has also been shown to contribute to the inflammation of the endothelium. We hypothesize that due to the link between caveolin and endothelial dysfunction, and the link between mitochondria and endothelial dysfunction, caveolin has an important function in mitochondrial dynamics and that the loss of caveolin increases the mitochondrial fission via a Drp1-dependent pathway. Our data shows that adenoviral silencing of caveolin-1 in rat aortic endothelial cells increases Drp1 expression but does not significantly alter mitochondrial morphology. Overexpression of caveolin-1 via an adenoviral construct in these cells produces a decrease in Drp1 expression without altering mitochondrial morphology. This data provides insight into the pathophysiology of CAD and could provide us with new therapeutic targets in the future. / Biomedical Sciences

<p> Three pieces of work are contained in this thesis. OGRe is a relational database that stores mitochondrial genomes of animals. The database has been operational for approximately five years and the number of genomes in the database has expanded to over 1000 in this period. However, sometimes, new genomes can not be added to the database because of small errors in the source ffies. Several improvements to the update method and the organizational structure of OGRe have been done, which are presented in the first part of this thesis. </p> <p> The second part of this thesis is a study on codon usage in mitochondrial genomes of mammals and fish. Codon usage bias can be caused by mutation and translational selection. In this study, we use some statistical tests and likelihood-based tests to determine which factors are most important in causing codon bias in mitochondrial genomes of mammals and fish. It is found that codon usage patterns seem to be determined principally by complex context-dependent mutational effects. </p> <p> The third part of this thesis is a phylogenetic study of 159 avian species obtained using mitochondrial rRNA sequences that were provided by Dr. van Tuinen. In this study, two methods are used: one considers sites of sequences as independently evolving; the other includes the secondary structure of rRNAs. Unfortunately, the amount of information in the rRNA sequences seems to be insufficient to determine the whole phylogeny of birds. However, our results make it clear that several traditionally defined orders are polyphyletic and therefore need to be redefined. </p> / Thesis / Master of Science (MSc)

The Yellow Warbler (Dendroica petechia) is a highly polytypic bird species with a vast breeding range in the Americas. To assess the level of population structuring within the northern part of its range, I surveyed the nucleotide variation present in a 344bp segment of the mitochondrial DNA (mtDNA) control region I (CR-I) from 155 breeding individuals. These birds were caught at seven locations in Canada and Alaska. Fifty-nine haplotypes were observed in this sample, with pairwise distances between haplotypes ranging from 0.29 to 4.35%. The number of nucleotide sites with multiple hits indicates a high rate of evolution in this region. A homologue to the CR-I was also identified and likely originated from a paralogous duplication event, as suggested by the comparison of sequences from the two regions. Significant population structuring in Yellow Warblers across North America was revealed by analyses of nucleotide diversity and molecular variance, which demonstrated the existence of a major subdivision between eastern (Manitoba to Newfoundland) and western (Alaska and British Columbia) warbler populations. This finding provides evidence for very low levels of gene flow between these two groups. Fifteen out of 21 pairs of populations differ significantly in their genetic composition, indicating further structuring at a smaller geographic scale. Within the eastern group of populations, both the high mutation rate of the CR-1 and an isolation-by-distance process seem to be responsible for differences between locations. Phylogenetic analyses indicate that western birds form a monophyletic group whereas eastern birds are paraphyletic with respect to the western ones. However this conclusion remains hypothetical because of a lack of statistical support for the monophyly of western haplotypes. Nevertheless, this situation is consistent with a historical splitting of warbler populations by a vicariant event, possibly of Pleistocene origin, and provides intraspecific support for vicariance as a mechanism leading to isolation and speciation of western warbler taxa, as hypothesized by Mengel (1964). However, other scenarios, such as a founder event in the west from an eastern stock, cannot be excluded, although they are less likely based on mtDNA data. The absence of phylogeographic structure in the East suggests a recent expansion of Yellow Warbler populations from a restricted geographic range. These findings demonstrate that populations of continentally distributed North American passerine species can show high level of population structuring when assayed with an hypervariable molecular marker such as the mtDNA control region I. / Thesis / Master of Science (MSc)

N/A / Thesis / Master of Science (MS)

Mitochondria are intracellular organelles responsible for oxidative phosphorylation. They contain their own DNA which encodes some components involved in oxidative phosphorylation. Mitochondrial DNA is very susceptible to mutations. Mitochondrial abnormalities have been observed in several disorders of muscle and brain. Alzheimer's disease is a form of dementia characterized by the formation of numerous neuritic plaques and neurofibrillary tangles. There is evidence suggesting a possible role for mitochondrial abnormalities in Alzheimer's disease. The goal of this project was to determine if there were quantitative changes in mitochondrial DNA content in large neurons from Alzheimer's disease patients, compared to age-matched control patients. The relative mitochondrial DNA content per unit area was assessed in brain sections from Alzheimer's disease subjects and age-matched control subjects using in situ hybridization to mitochondrial DNA. The results were not conclusive due to technical concerns with the in situ hybridization technique which are discussed. / Thesis / Master of Science (MS)

It has been proposed that intermittent exercise can differentially affect mitochondrial responses to training, with training volume being more important than intensity for increasing skeletal muscle mitochondrial content and with intensity playing a greater role in mitochondrial respiration. To test this hypothesis, we examined markers of skeletal muscle mitochondrial content and respiration in response to two different interval training protocols performed using single-leg cycling, which permitted a within-subjects design. Ten healthy active adults [6 males / 4 females, 22±4 y, peak oxygen uptake (VO2peak) = 42±4 ml/kg/min] were recruited. Each leg was randomized to either a HIIT [4 × (5 min at 65% Wpeak and 2.5 min at 20% Wpeak)] or SIT [4 x (30-s “all-out” sprints and 4 min active recovery)] protocol and completed three exercise sessions/wk over 4 wk for a total of 12 exercise sessions/leg. The mean work performed during each session was 133±32 and 44±8.0 kJ for HIIT and SIT respectively, and the average workload during intervals was 95±25 W and 322±77 W for HIIT and SIT respectively. Citrate synthase (CS) maximal activity increased compared to baseline after training interventions, with the change being greater after SIT vs HIIT (42±25% vs 16±13%, interaction p=0.01). COXIV protein content and succinate-supported state 3 were unchanged. Single-leg VO2peak and time to exhaustion (TTE) increased to a similar extent in both HIIT and SIT (main effect of time, p<0.05). These data suggest that, in contrast to what has been proposed by others, training intensity is more important than volume for increasing mitochondrial content during short-term interval training in human skeletal muscle. / Thesis / Master of Science in Kinesiology / Mitochondria are an important component of cells that use oxygen to convert fuels such as sugars and fats into energy. One of the factors that determines the amount of mitochondria in skeletal muscle is physical activity. Aerobic exercise training can be performed over a range of intensities, from relatively easy to very hard, and in an intermittent or continuous manner. This thesis examined the effect of short-term, intermittent exercise training performed at two different intensities on the content of mitochondria in human skeletal muscle. It found that both high- intensity interval training (HIIT) and sprint interval training (SIT) increased mitochondrial content. The increase was greater after SIT compared to HIIT, even though the total “dose” or amount of exercise was lower in the former compared to the latter. These results suggest that intensity is an important determinant of skeletal muscle remodelling induced by intermittent exercise in humans.

Background: Infertility has become a growing concern across the world as cases continue to increase each year. Research has now shifted to identifying novel biomarkers to predict male fertility. While mtDNAcn has recently been found to show promising results as potential biomarker, its regulation remains unclear. Method: Triplex probe-based PCR was used to quantify mtDNA levels, while 850K Array was used to measure methylation levels. A-clustering algorithm followed by generalized estimating equations (GEE) lead to clustering of individual CpG sites, containing a minimum of 2 CpGs within 1000 base pairs of each other. These clusters were used for analysis of the association between mtDNAcn and DNA methylation within sperm. Metascape1 was used to annotate gene ontology terms. Result: Generalized estimating equation model analysis produced 6,038 FDR significant (q Conclusion: Thus, we show that sperm mtDNAcn is strongly associated with sperm DNA methylation and the associated implicates mtDNAcn as an influence on infertility.

The relationship among oxidative stress, mitochondrial DNA integrity, and longevity continues to be without a general consensus. Here, we hypothesize that short- and long-lived birds, including the budgerigar (Melopsittacus undulatus), guineafowl (Numida meleagris), quail (Corturnix japonica), and turkey (Meleagris gallopavo) differ in oxidative stress measured by blood markers and that this difference correlates with mitochondrial genomic integrity both within and among species. In preliminary studies and to establish a reference and standard for the search for single nucleotide polymorphisms (SNPs), we used a combination of experimental and in silico tools for genome analysis to screen selected regions of the chicken (Gallus gallus) mitochondrial genome (mtGenome) for SNPs. A total of 113 SNPs was identified which formed 17 haplotypes. The length of the turkey mtGenome sequence developed was 16,967 bp in length, while that of the budgie was 18,193 bp. Annotation of both sequences revealed a total of 13 genes and 24 RNA (22 tRNA and 2 rRNA). Within the budgie mtGenome sequence, a duplicated control region was observed, and there was an additional nucleotide in the NADH dehydrogenase subunit 3 sequence of both the turkey and budgie. The total number of SNPs within the D-loop and 16S rRNA in each of the four species ranged from zero in the quail to 22 in the budgie. The new mtGenome sequences revealed that the turkey was most closely related to the chicken and quail, and the budgie was closest to kakabo (Strigops habroptilus). Oxidative stress, estimated by biomarkers thiobarbiturate acid reacting substance (TBARS), plasma uric acid (PUA), and glutathione (GSH) and at 10, 30, 55, and 80 wks-of-age within each species, was not consistent. The level of GSH was highest in guineafowl, but lowest in budgie. While PUA, an antioxidant, exhibited a significantly (P<0.05) decrease as birds grew order, TBARS, a lipid peroxidation index, increased with age. In general, oxidant and antioxidant status appeared to vary among species and to be significantly affected by age, unlike mutations in the mtDNA which remained the same in younger and older birds. This primary findings and discoveries of this dissertation research include the large scale SNP discovery in previously described and novel avian mtGenomes including the chicken and turkey, the two main poultry species, and the determination that oxidative stress in birds appears to vary with age but that this does not affect mitochondrial DNA variation. Recent evidence of work in mice appears to support results described in this dissertation that mitochondrial DNA mutations do not increase with age, the central paradigm of the "Free Radical Theory of Aging". The dissertation also described resources and data that will be a foundation for the use of birds, especially the budgie, as a model for testing this theory that remains of interest to both agricultural and biomedical sciences. / Ph. D.