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.

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.

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

Proximale Spinale Muskel Atrophie (SMA) ist eine autosomal rezessive Krankheit, charakterisiert durch eine Degeneration des zweiten Motorneurons und einer progressiven Paralyse und Atrophie proximaler Muskeln. Nach Zystischer Fibrose, ist SMA die häufigste autosomal rezessive Erkrankung bei Menschen und der häufigste genetische Grund für Säuglingssterblichkeit. SMA, monogenetisch im Ursprung, ist verursacht durch eine Mutation in einem einzelnen Gen, dem Survival Motor Neuron 1 (SMN1) Gen, was zu einer reduzierten Menge an Survival Motor Neuron (SMN) protein führt. SMN ist ein ubiquitär expremiertes Protein mit house-keeping Funktion in snRNP Biogenese und pre-mRNA splicen. Dennoch, eine reduzierte Menge an SMN beeinträchtigt vor allem Motor Neurone und Muskeln aus bisher unverständlichen Gründen. Es wurde demonstriert, dass SMN mit ß-actin mRNA interagiert und an dessen Transport entlang des Axons beteiligt ist. Funktionelle Studien an der Neuromuskulären Synapse (NMJ) haben gezeigt, dass Evozierte Neurotransmitterfreisetzung um 55 % reduziert war in den meist betroffenen Muskelgruppen, dies indiziert, dass eine verringerte Menge an Vesikeln fusioniert, währenddessen asynchrone Transmitterfreisetzung um 300 % erhöht ist aufgrund von einer abnormalen Akkumulation von Calcium in der Nervenendigung in SMA Mäusen. Eine Mögliche Erklärung für diese Calcium Erhöhung ist eine herabgesetzte Calcium Aufnahme durch Mitochondrien während Serien von Aktions Potenzialen. Diese Studie präsentiert eine umfassende Analyse mit einem Fluoreszens Konfokal Mikroskop über die Organisation und Fülle Synaptischer Vesikel (SVs), Mitochonrien und Aktin in Nervenendigungen von SMA Mäusen ( Smn -/-; SMN2; SMNdelta7). Wir visualisierten Synaptische Vesikel mit einem Antikörper gegen den Acetylcholin ( VACht) und konnten zeigen, dass im Transversus Abdominis (TVA) Muskel SV Klusters während des Reifungsprozesse klein verbleiben mit einer Reduzierung von 50% der totalen Fläche die von SVs bedeckt ist. Diese schwere Reduktion von SVs wurde auch im der kaudalen Muskelstrang des Levator auris longus (LAL) Muskel gefunden, obwohl nur leichte Veränderungen in der Postsynapse dieses Muskels festzustellen sind. Diese Ergebniss von Präsynaptischer Pathologie, neben fast normalen postsynaptischen Status, verstärkt die Hypothese dass SMN-induzierte Veränderungen im Muskel nicht auschließlich eine reine Konsequenz von Motor Neuron Degeneration sein können. Als Nächstes, haben wir Mitochondria mit Mitotracker angefärbt und haben gefunden, dass die Fläche,die von Mitochondrien in Mutanten Mäusen bedeckt ist, etwa nur die Hälfte der Fläche im Wild typ beträgt. Überraschenderweise waren SVs und Mitochondrien stak kolokalisiert. In vielen Fällen war ein Kern von Mitochondrien deutlich umgeben von einem Ring aus SVs. Diese Verteilung war unbeeinträchtig in der Mutanten Maus und könnte eine mehr generelle Bedeutung in Nervenendigungen haben. Phalloidin gefärbtes Aktin zeigte das F-aktin ringförmige Strukturen um SV Klusters formt. Diese Strukturen und der Prozentuale Anteil der Nervenendigung der von Aktin bedeckt ist, war geringer in SMA Mutanten Mäusen. Aktin ist an multiblen Schritten des Vesikel Zyklus beteiligt. Kurz Strecken-Transport von Vesikeln und Organellen, wie Mitochondrien in Wachstunskegeln und Nervendigungen ist vor allem vom aktin-myosin-basierten Transport abhängig. Weitere Arbeit ist notwendig um zu klären ob die Charakteristiken des SMA Phänotyps wie abnormales SV Klustering, Reduktion von Mitochondrien, unabhängig auftreten oder eine gemeinsame Konsequenz von einer Dysfunktion des Aktin Zytoskeleton sind, was Aktin eine Schlüsselrolle in der SMA Pathogenese verleihen würde.

Doctor of Philosophy / Department of Biochemistry / John M. Tomich / Peptide-based delivery systems show great potential as safer drug delivery vehicles. They overcome problems associated with lipid-based or viral delivery systems, vis-a-vis stability, specificity, inflammation, antigenicity, and tune-ability. We have designed and synthesized a set of 15 and 23-residue branched, amphiphilic peptides that mimic phosphoglycerides in molecular architecture. They undergo supramolecular self-assembly and form solvent-filled, bilayer delineated spheres with 50-150 nm diameters (confirmed by TEM and DLS). Whereas weak hydrophobic forces drive and sustain lipid bilayer assemblies, these structures are further stabilized by β-sheet hydrogen bonding and are stable at very low concentrations and even in the presence of SDS, urea and trypsin as confirmed by circular dichroism spectroscopy. Given sufficient time, they fuse together to form larger assemblies and trap compounds of different sizes within the enclosed space. They are prepared using a protocol that is similar to preparing lipid vesicles. We have shown that different concentrations of the fluorescent dye, 5(6)-Carboxyfluorescein can be encapsulated in these assemblies and delivered into human lens epithelial cells and MCF-7 cells grown on coverslips. Besides fluorescent dyes, we have delivered the plasmid (EGFP-N3, 4.7kb) into N/N 1003A lens epithelial cells and observed expression of EGFP (in the presence and absence of a selection media). In the case of large molecules like DNA, these assemblies act as nanoparticles and offer some protection to DNA against certain nucleases. Linear peptides that lacked a branching point and other branched peptides with their sequences randomized did not show any of the lipid-like properties exhibited by the branched peptides. The peptides can be chemically decorated with target specific sequences for use as DDS for targeted delivery.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

The goal of my doctoral work is to understand how proteins involved in vesicle trafficking contribute to proper animal development. To understand aspects of this process, I studied how two vesicle trafficking proteins, Liquid facets(Lqf)/epsin1 and D-Epsin-Related, affect Drosophila eye development. I determined that Lqf, an endocytosis protein, together with Fat facets (Faf), a deubiquitinating enzyme, regulate the Notch and Delta signaling in the developing Drosophila eye. Notch signaling pathway is used in most developmental processes and is dependent on its ligand Delta. Faf deubiquitinates Lqf in the signaling cells, thereby increasing Lqf protein levels and also levels of Delta endocytosis. This event is necessary for Notch activation in neighboring cells. Lqf probably works in concert with the E3 ubiquitin ligase Neuralized (Neur), which ubiquitinates Delta. These conclusions are consistent with a relatively new model describing an obligate role for endocytosis in the signaling cells to effect activation in neighboring cells. To understand how Lqf functions mechanistically in this process, I performed a structure/function analysis of the Lqf protein. Lqf proteins with strategic deletions of certain functional domains were tested for their ability to function in vivo. The major result of these experiments is that the N-terminal ENTH domain of Lqf and a protein without the ENTH domain each retain significant activity. This suggests that Lqf has two functions: the ENTH domain function and the ENTH-less function. These data are in contrast with the most popular model suggesting that ENTH-less epsins are non-functional proteins. I present possible models for how ENTH-less epsins may retain function. The final part of my thesis focuses on D-Epsin-Related (D-Epsin-R) protein. I showed that D-Epsin-R is a Golgi protein, like its homologs. Surprisingly, D-Epsin-R ENTH domain is not required for function because an ENTH-less D-Epsin-R can substitute for endogenous D-Epsin-R. Also, D-Epsin-R has essential and probably specific developmental roles in the eye as D-Epsin-R mutants exhibit impaired cell growth. This work suggests that epsins are specific components of certain developmental pathways. / text

No description available.

Parkinson's disease is a neurodegenerative disease characterized by the buildup of aggregated and spread of misfolded alpha-synuclein. How the misfolded alpha-synuclein contributing to the toxicity and death of neuronal cells has been the focal point of research. The spread of alpha-synuclein has been attributed to many mechanisms, one of which is via cell-derived vesicles called exosomes. This project aims to examine the controllability of exosome release. SH-SY5Y, MCF-7 and CHO-K1 cells were transfected with dopamine receptor 3-green fluorescent protein, G-protein receptor 143 or green fluorescent protein and treated with either dopamine or L-DOPA. Medium was harvested and subjected to ultracentrifugation and a silver stain and western blot were performed. There was no significant difference in the total protein in the exosome fraction lanes between the treatment groups or within them. Another aim was to test the specificity of exosomes. Exosomes isolated from SH-SY5Y or MCF-7 were labeled with Exo-Red dye and introduced to wells containing SH-SY5Y, MCF-7 and CHO-K1 cells at room temperature and -4C. At room temperature, exosomes were observed intercellular in all of the cell lines, however, they did not deliver their content. At -4C exosome uptake was halted and they remained on the surface of the cells. Exo-Red labeled SH-SY5Y exosomes were treated with proteinase K and were introduced to CHO-K1 cells at -4C and room temperature. CHO-K1 did not take up exosomes, suggesting exosomes contain one or more necessary proteins needed to interact with the cellular membrane to initiate internalization. CHO-K1 cells were treated with versene to examine the involvement of integrin proteins. Exo-Red labeled SH-SY5Y exosomes were trapped on the surface of CHO-K1 after versene treatment. Lastly, Exo-Red labeled SH-SY5Y exosomes were biotinylated and magnetically captured then introduced to SH-SY5Y and MCF-7 cells and a silver stain and a biotinylated blot were performed. MCF-7 bound more Exo-Red labeled SH-SY5Y exosomes.

Não consta resumo na publicação / Abstract not available