McKay, Hayley Elizabeth
29 July 2019
No description available.
Exploring the Role of Human Endogenous Retroviral Gag in the Formation and Content of Extracellular VesiclesMcCulloch, Danielle 30 August 2018 (has links)
Human Endogenous Retroviruses (HERV) are derived from exogenous retroviruses that infected inheritable germline tissues millions of years ago and account for 8% of the human genome. Like other retroviruses HERVs encode Gag, Pol and sometimes Env proteins. During a retroviral infection, retroviral Gag recruits the hosts Endosomal Sorting Complex Required for Transport (ESCRT) and associated proteins (ALIX and TSG101) to produce precisely sized viruses from endosomes or the plasma membrane. The ESCRT machinery is also involved in cytokinesis and control growth factor receptor signalling. HERV-K is the most recent HERV family to insert into the genome and is still able to produce mostly intact transcripts, including Gag. When expressed, Gag causes cells to release Virus-Like Particles (VLP) that lack HERV genomes. These retroviral VLP are remarkably similar to a sub-category of extracellular vesicles (EVs) called exosomes. Exosomes require ALIX, TSG101 and the ESCRT machinery for their production. It is possible that HERV-K Gag is required for exosome production or that HERV VLPs are a major contaminant of exosome preparations that account for many of the functions attributed to exosomes. Our data shows that HERV-K Gag over-expression or knockdown did not change the number of EVs released per cell in two cell lines. As well there was no difference in the amount of ALIX and TSG101 in the EVs in these conditions. The most intriguing observation made was the increase of cell number with expression of HERV-K Gag and decrease when HERV-K Gag was knocked down in HEK293T. We are currently unable to conclude the role of HERV-K Gag on EV production and content. We speculate that HERV-K Gag might affect cells through controlling cell proliferation or death, for example by competing with ESCRT machinery to impact signalling through growth factor receptors. This study begins to outline the potential effects HERV-K Gag might have on EV release and cell proliferation.
email@example.com / 1 / Adedoyin Johnson
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)
Individuals with diabetes have an increased mortality due to the macro- and microvascular complications, which are commonly preceded by endothelial dysfunction. We have shown that endothelial microparticles (eMPs) are markers and mediators of vascular injury and pathology. However, their utility as a biomarker of hyperglycemia-induced endothelial damage and their influence on the vasculature remains unclear. We hypothesized that high glucose (HG) exposure alters eMPs protein composition, making them reflective of active signalling processes characteristic of a hyperglycemic environment. In addition, HG alters eMPs bioactivity, making them more potent inducers of oxidative stress, thrombosis and endothelial damage. Therefore, we assessed the exclusive effects of HG on eMPs formation, composition, and signalling. Results: Exposure of endothelial cells to high glucose for 24 hours caused a 3-fold increase in eMPs formation, increased mean vesicle size and their absolute electronegativity. Proteomic analysis of eMPs identified 1,212 independent proteins, with 68 exclusive to HG and associated with signalling processes related to metabolic processes, oxidation-reduction reactions, hemostasis and thrombosis and cellular interactions at the vascular wall. Compared to eMPs formed under normal conditions, eMPs formed in response to HG possess a ~3-fold greater procoagulant activity, induced a greater production of cellular ROS and were more potent inhibitors of endothelial-dependent relaxation. Conclusions/Interpretation: Taken together our results indicate HG alters the composition of eMPs, making them more potent mediators of endothelial damage. With similar changes in bioactivity being evident in the protein composition and the associated enriched biological processes, eMPs protein content may provide insight into the pathophysiological status of the cells in a hyperglycemic environment and provide use clinically, to identify dysregulated pathways for therapeutic targeting.
Effect of extracellular vesicles on cancer cell lines in vitro and biodistribution in an ectopic osteosarcoma mouse modelJavier, Abello January 1900 (has links)
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.
13 June 2019
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.
Comprehensive Proteomic Analysis and Characterization of Human Bone Marrow Mesenchymal Stem/Stromal Derived Extracellular VesiclesMunshi, Afnan M N Alam 23 August 2019 (has links)
No description available.
Exploration of a novel non-lytic viral transmission mechanism utilized by a non-enveloped positive-sense RNA virusYang, Jie Eune 12 June 2018 (has links)
While enteroviruses, including poliovirus, are conventionally released upon cell lysis, recent studies show that phosphatidylserine-enriched infectious extracellular vesicles (IEVs) shed by infected cells can transport clusters of enteroviruses from cell to cell, resulting in increased infectivity. Combining structural and biochemical analyses, we focused on IEVs shed from poliovirus-infected cells, a classical prototype for studying enteroviruses. Transmission cryo-electron microscopy, cryo-electron tomography and computational reconstruction, present the first three-dimensional structures of well-preserved IEVs and purified exosomes. We observed that single-membraned IEVs present a wide size range in diameter. Clusters of virions can be either densely packed within a protein-coated irregularly shaped IEV, or concentrated at one or both ends of an IEV, forming a polar structure. In addition to virions, IEVs often contain internal vesicles, “ramen-noodle”-like structures with strong density, and partially assembled virion-like structures. Viral replication complex components, including viral proteins polymerase 3D, 3CD, 3A, 3AB, 2BC, 2C and (+) and (-) stranded RNAs were detected in IEVs. Furthermore, (-) stranded RNA templates are protected by the IEVs, not packed in viral capsids. The transported viral replication components (viral proteins and RNAs) and virions within IEVs initiate a stronger and faster viral replication in recipient cells than free virions. Both cryo-electron tomographic and mass spectrometry data also showed that virions and “ramen-noodle”-like structures were also observed in purified CD9 positive exosomes from poliovirus-infected cells. Viral protein 3AB, detected on the membrane of IEVs, can invaginate membranous structures to engulf large proteins into a closed lumen. Our study demonstrates that IEVs can transport viral replication complex components to initiate a rapid onset of viral replication, as part of a novel viral transmission mechanism. Viral protein 3AB may contribute to forming IEVs throughout the infection. / 2019-06-12T00:00:00Z
04 September 2020
Amyotrophic lateral sclerosis is a relentlessly progressive and fatal neuromuscular disease with no effective biomarkers, treatments or cure. In the early stages of ALS, it can be difficult to provide a diagnosis as patients do not meet diagnostic criteria until they become symptomatic, a sign of neuron degeneration. Early detection is therefore crucial to provide access to therapeutics prior to significant neuron loss. Extracellular vesicles are an ideal source of biomarkers as they contain a mix of proteins and nucleic acids reflective of the physiological state and are released from all cell types. We identified valosin-containing protein, integrin-beta 1 and gelsolin as potential biomarkers for ALS14 through proteomic analysis of EVs isolated from cell lines carrying the ALS-associated VCP-R155H mutation. My results indicate that EVs may serve as a valuable source of protein biomarkers in diagnostic, prognostic and predictive applications.
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