Aeromonas hydrophila ML09-119 is an important fish pathogen that severely affects channel catfish aquaculture. To better understand this strain’s virulence factors, outer membrane vesicles (OMVs) were isolated, and their proteome was assessed. Using transmission electron microscopy and dynamic light scattering, OMVs were shown to be monodispersed particles with an average diameter of 120.33 nm. OMV proteins were identified using mass spectrometry, and analysis of the resulting proteome of 74 proteins revealed that many originated from the cytoplasm, but there was an enrichment of outer membrane, periplasmic, and extracellular proteins compared to the total proteome. The majority of the functional classifications were associated with bacterial metabolism. Of the predicted virulence factors, several had a putative function in adherence, and there were type III secretions system proteins as well as three secreted exotoxins. Overall, our data reveal new insights into A. hydrophila OMVs and their potential roles in physiology and virulence.

Breast cancer (BC) is the second most commonly occurring malignant disease in women and one of the leading causes of cancer-related death worldwide, globally accounting for almost half-a-million deaths per year. In Canada, BC is the second leading cause of death in women preceded only by lung cancer. Invasion and metastasis are the most common causes of mortality in patients with BC. Studies show that extracellular vesicles (EVs) play an important role in immune system evasion, invasion and metastasis. Studies have shown a significant elevation of EVs in the serum of cancer patients compared to healthy subjects. Furthermore, elevated secretion of EVs has been correlated with cancer malignancy. Therefore, it has been suggested that EVs may be an important non-invasive diagnostic and prognostic tool for cancer. Herein our in vitro studies show that ER-α is secreted via EVs from MCF-7 cells. Furthermore, our mass spectrometry (MS)-based proteomic study showed that the proteomic profile of EVs from the plasma of BC patients differs from that of healthy subjects. In addition, we have also shown that vesicular abundance of proteins associated with tumour malignancy, such as tissue factor (TF), plasminogen activator inhibitor (PAI-1), a disintegrin and metalloproteinase 12 (ADAM12) and β-Catenin is different between primary tumour and metastatic disease. / Thesis / Master of Science (MSc)

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No description available.

Atherosclerosis, the pathophysiology of many cardiovascular diseases (CVD), is a chronic inflammatory process caused by the sustained accumulation of cholesterol, followed by endothelial dysfunction, and the resulting vascular inflammation. The established treatment for atherosclerosis, to date, involves the use of statins. These medications are hydroxymethylglutaryl coenzyme A reductase (HMG-CoA) inhibitors and lower the levels of by inhibiting HMG-CoA, a rate limiting step in the biosynthesis of cholesterol. Statin therapy varies in effectiveness based on dosage and individual differences, making effective treatment of patients challenging. More recently, extracellular vesicles (EVs) have emerged as a promising field in cardiovascular research. Once thought of as “platelet dust,” EVs are now recognized for their potential as therapeutic targets and tools. In this review, a comprehensive characterization of EVs is provided to explain how EVs are involved in normal physiological function and pathological processes of atherosclerosis. Evidence supports a model where EVs participate in the initiation and progression of atherosclerosis and may also be used as a delivery tool in disease therapy. Currently, cell-derived EVs can be therapeutic agents in animal models, an effective tool in gene therapy, or a drug delivery vehicle. Future experiments enhancing the therapeutic potential of EVs promise to deepen our understanding of EV-based therapy for atherosclerosis precision medicine.

Doctor of Philosophy / Department of Food, Nutrition, Dietetics and Health / Tonatiuh Melgarejo / Mark Haub / Human umbilical cord-derived mesenchymal stromal cells (HUC-MSCs) have an enormous therapeutic potential because of their immunomodulatory and anti-inflammatory properties. However, there are limitations for their therapeutic use due to low cell survival after implantation, the risk of culture-borne pathogens, and the risk of embolism and thrombosis after intravenous infusion. Exosomes, on the other hand, constitute an important part of the MSCs secretome and may play a role in their therapeutic effects. Here, it was demonstrated that HUC-MSC-derived exosomes accumulate in human and mouse osteosarcoma cell lines in vitro and reduce their proliferation. The distribution of HUC-MSCs exosomes was shown in osteosarcoma tumor- bearing mice. Exosome distribution in vivo was observed using Magnetic Resonance Imaging (MRI) of gadolinium-labeled exosomes and by fluorescent imaging after infusion of near infrared dye-labeled exosomes. HUC-MSC exosomes accumulated in the tumor throughout the 48 hours ours post-injection period. In contrast, synthetic lipid nanoparticle accumulate in tumor only for the first 3ours post-injection. These results suggest that labeling with gadolinium or near-infrared dye may affect exosome accumulation within the spleen. In summary, this study showed that HUC-MSCs exosomes can accumulate to osteosarcoma cells in vitro and in vivo, and thus they may be useful for detecting cancer metastasis.

The formation, phase ordering, and evolution has been studied in lipid and liquid crystal (LC) colloidal aqueous mixtures as a function of LC concentration and thermal history. The lipid used was 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) while the liquid crystal was pentylcyanobiphenyl (5CB). POPC is a naturally occurring lipid in eukaryotic cell membranes and mimics many of the properties of human cell walls. 5CB is a polar liquid crystal that exhibits a thermodynamically stable orientationally ordered (nematic) state at room temperature. Colloidal dispersions were made at various 5CB and POPC concentrations in water and studied via optical microscopy (phase contrast, confocal, florescence, and cross-polarizing) to probe phase order and evolution as well as by calorimetry to study phase transformations. Very large vesicles (larger than 100 micrometers) were observed to form that appear to use the phase separated 5CB droplets as scaffolds. Also, there appears a unique promotion of dye (used to image the lipid bilayers) crystallization within liquid crystal domains well above room temperature.

Bacteroides fragilis is an important, anaerobic commensal of the human gastro-intestinal tract. As a Gram-negative bacterium, B. fragilis produces a large number of outer membrane vesicles (OMV), spherical globules consisting of outer membrane and periplasmic material, which have a range of potential functions and which are known to be able to deliver their cargo to host dendritic cells (DCs). One of the proteins believed to be packaged into the OMV of B. fragilis is BfUbb (encoded by the ubb gene) which shares 63% homology with human ubiquitin. Ubiquitin is a small, common, eukaryotic protein modifier, which is conjugated to target proteins via a series of activating, conjugating and ligating enzymes, and which has known roles in a wide range of eukaryotic cell processes. Due to key differences between the two proteins, BfUbb has the potential to act as a suicide substrate mimic of ubiquitin. BfUbb was therefore assayed for its ability to interact with ubiquitin E2 conjugating enzymes of the ubiquitylation cascade in vitro, and was found to covalently bind the majority of available enzymes in a DTT-sensitive manner. BfUbb showed a preference for three specific E2 enzymes, all of which are involved in the degradation of mitotic check point proteins, suggesting a role for BfUbb in the inhibition of cell cycle progression and, consequently, tumorigenesis. No binding partners of BfUbb were identified outside of the ubiquitylation cascade, however BfUbb was found to form spontaneous multimers in vitro, the biological function of which is unknown. This study also describes the construction of two sets of plasmids. The first set will allow the expression of untagged and fluorescently tagged forms of BfUbb for purification and use in biochemical assays. The second set will allow the expression of his-tagged and fluorescently tagged forms of BfUbb in mammalian cells, so that the effects of BfUbb on the host epithelial cells may be studied. The proteome of the OMV of B. fragilis was solved using LTQ-Orbitrap mass spectrometry. The identified proteins indicated several putative roles for B. fragilis OMV, including nutrient acquisition and protease inhibition. The suitability of techniques used during the isolation and proteomic analysis of OMV in different studies is discussed. BfUbb-carrying B. fragilis OMV were able to inhibit growth of Salmonella enterica Typhimurium, thus indicating a role for BfUbb in the inhibition of competing, pathogenic bacteria in the gastro-intestinal tract. The conclusions of this study are that the putative roles of both BfUbb and the OMV of B. fragilis may promote both survival of the bacterium and the gastro-intestinal health of the host.

Thesis (Ph. D.)--Cornell University, January, 2007. / Vita. Includes bibliographical references (leaves 100-109).

Thesis (M. Phil.)--University of Hong Kong, 1980. / Typescript.