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Sarcopenia is the age-related progressive loss of skeletal muscle (SkM) mass, function, and strength. It has been well elucidated that resistance exercise can attenuate the development of sarcopenia. A population of extracellular vesicles, termed ‘exosomes’ (EXO), can contain microRNA and facilitates intercellular communication, including within SkM, though the response to prolonged training is not well understood. Given the potential role of SkM-derived exosomes in the response to exercise, we examined older adults (n = 30, OLD) before (PRE) and after a 12-week (POST), resistance training program. Healthy, young controls (n = 12, YNG) were used for comparison of baseline measures. Exosomes were isolated from platelet-free plasma using size exclusion chromatography in combination with ultracentrifugation (SEC-UC) and characterized via western blotting, nanoparticle tracking analysis and electron microscopy. To assess exosome biogenesis and miRNA synthesis in skeletal muscle, biopsies were taken from the vastus lateralis. Circulating EXO-enclosed and SkM miRNA expression was measured using RT-PCR. In SEC-UC isolates, EXO-markers CD81 and CD9 were significantly lower in PRE compared to YNG (p<0.05) but did not change with training. At baseline, ALIX, TSG101 and CD63 (markers of exosomes) were not altered with aging as compared to YNG; however, their expression significantly increased with training (p<0.05) Circulating EXO-derived mir-1, -133, -23 and -27a were significantly lower in expression of OLD participants as compared to YNG. Following resistance training, their expression significantly increased (p<0.05), returning to a YNG phenotype. Next, we aimed to investigate the contribution of skeletal muscle in the exosome responses. Our data indicate that a small fraction of circulatory exosomes may originate from skeletal muscle. In addition, in biopsy-derived SkM tissue, expression of proteins involved in EXO and miRNA biogenesis (Alix, XPO-5, DICER) were significantly higher in PRE compared to YNG (p<0.05), and further increased with resistance training (POST, p<0.05). Expression of Rab27a, a marker of exosome trafficking, was significantly higher in PRE (p<0.05) but did not respond to training. In conclusion, here we show alterations in circulating EXO content and cargo with age and resistance training partially restores the values to a younger phenotype. / Thesis / Master of Science in Medical Sciences (MSMS) / Aging is the slow and time-dependent process that our organs, down to the cellular level, deteriorate in function reducing the biological fitness of our bodies. Aging specific to skeletal muscle, or sarcopenia, is especially important because skeletal muscle makes up 40% of our weight, is essential for posture, balance, locomotion and breathing. Sarcopenic individuals have low muscle mass, strength, and function and as a result are associated with low independence in activities of daily living and increased risks of falls and fractures. Exercise, and in particular resistance training, has been shown to be beneficial and cost-effective in treating sarcopenia and delaying aging throughout the body. Part of the underlying mechanism regarding how exercise affects us in a multi-systemic manner is not well understood. We know that skeletal muscle releases a multitude of molecular factors during exercise. Amongst them, extracellular vesicles and specifically exosomes are worth investigating because they have been shown to function in intercellular communication by delivering molecular signals, called microRNAs, from origin cells to recipient cells throughout the body. In this thesis project, we investigate exosomes in circulation of older individuals before and after a 12-week resistance training program. We found that aging alters the exosome pool in circulation as well as their miRNA content. After resistance training, many of miRNAs altered with age, return to levels comparable to young. In addition, we showed that at the skeletal muscle level, aging and resistance training affect exosome biogenesis and miRNA expressions. In conclusion, we provide evidence that aging significantly alters circulatory exosomes and miRNA and show that resistance training normalizes the miRNA profile to levels seen in exosomes derived from young plasma. How exosomes and their molecular signals change with aging and how exercise affects them gives us an insight on how exercise elicits multi-systemic benefits against aging and sarcopenia.

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Breast cancer (BC) is the second most diagnosed cancer in Canadian women. Early detection of this cancer is critical to improve patient survival and prognoses. Exosomes are proposed to be involved in tumor proliferation through the transfer of diverse biomolecules, including metabolites. The use of exosomes as biomarkers for early diagnosis of BC has recently garnered interest due to them having unique biomolecules in diseased cohorts. Hence, an untargeted metabolomic analysis of BC exosomes was performed using nano high-performance liquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS) for BC diagnostic biomarker discovery. A total of 9 independent metabolite samples from non-tumorogenic MCF10A and highly metastatic MDA-MB-231 cell lines were analyzed. Bioinformatic analysis revealed 27 potential metabolite candidates unique to MDA-MB-231. Amongst 4 metabolites tested, one, N-Acetyl-L-Phenylalanine, was successfully validated. Overall, this study reveals that exosomes possess metabolites that can be candidates for early BC diagnosis.

Otolin-1 is a collagenous, C1q domain-containing extracellular matrix protein that has been identified and characterized in the inner ear. Recently, mass spectrometry analysis of left ventricular cardiac tissue detected a peptide of Otolin-1. Experimental analysis confirms Otolin-1 is an insoluble protein present in the left ventricular extracellular matrix whose expression decreases dramatically post-myocardial infarction beginning at day 5 post-MI. The protein is localized to the gap junctions of cardiac myocytes, and depletion of protein levels in the infarcted region of the left ventricle shows strong association with ventricular dimensions, observed via echocardiography.

Endothelial dysfunction, present in most cardiovascular disease, results in up-regulation of inflammatory adhesion molecules/cytokines, increases in vascular permeability, and decreased vasoprotective factors leading to vascular dysfunction. A novel means of communication between almost all cells are small vesicles containing biologically active proteins, nucleic acids, and lipids known as extracellular vesicles. Despite the advances in cardiovascular biology, the role of extracellular vesicles between endothelial cells and cells of vascular wall are underexplored. Therefore, we hypothesized that endothelial activation results in the release of pro-inflammatory vesicles that initiate inflammatory remodeling of vascular smooth muscle cells of the aorta. Extracellular vesicles were released from both endothelial cells and vascular smooth muscle cells with characteristic size, shape, and content. However, serum-free collection in endothelial cells resulted in endothelial activation of cell in culture and resulted in altered function in vascular smooth muscle cells, characterized by increased monocyte adhesion, altered protein synthesis/signal transduction, and signs of pro-senescent features. These effects were not recapitulated in any combination of endothelial-vascular smooth muscle cell extracellular vesicle communication. Unbiased mass spectroscopy of vascular smooth muscle cell treated with serum-free endothelial vesicles identified several proteins significantly up- regulated, including high mobility group box 1 and 2. Pharmacologic and genetic inhibition of these molecules significantly attenuated NF-kB activation, VCAM-1 expression, and monocyte adhesion. In summation, we suggest a new axis through which endothelial activation releases vesicles that skew the function of vascular smooth muscle cells to phenotype characterized by inflammatory properties through up-regulation of high mobility group box proteins 1 and 2. This highlights the importance of extracellular vesicles as a novel communication method between cells of the vasculature and how alterations in the host cell function may change the function of these vesicles. / Biomedical Sciences

The purpose of this study was to relate femoral venous plasma potassium concentrations ([K⁺]) following a fatiguing submaximal isometric quadriceps contraction, to the excitability of the muscle cell membrane as assessed by the compound muscle action potential (M-Wave) . Ten healthy male volunteers (22. 0 ± . 5 yrs) performed a unilateral 3 minute (min) sustained isometric quadriceps contraction at 30% of their maximum voluntary contraction (MVC) . M-Waves, peak evoked twitch torque, plasma lactate concentration ([La⁻]), and plasma potassium concentration ([K⁺]) were measured before, and at predetermined times over the course of a 15 min recovery period following the fatigue paradigm. Immediately post-exercise, twitch torque decreased to 58% of baseline, femoral venous [La⁻] had risen to 10 ± 0.8 mmol/1, and [K⁺] was significantly increased from 4.0 ± 0.1 mmol/1 to 5.9 ± 0.2 mmol/1. M-Wave amplitude illustrated a trend for potentiation increasing 9.5%-from 13.9 ± 2.4 mV pre-exercise, to 15.3 ± 2.8 mV at 1 min 20 seconds post-exercise. M-Wave area exhibited a similar trend from baseline, but values showed no statistical significance during this time. These results suggest that in spite of increased extracellular [K⁺] following this type of fatiguing exercise, muscle membrane excitability is maintained, which is probably due to the electrogenic nature of the highly active Na⁺ /K⁺ pump. This study was supported by the Natural Sciences and Engineering Research Council of Canada. / Thesis / Master of Science (MSc)