Thesis (P.D.)--University of Wisconsin--Madison, 1984. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 248-258).

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.

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.

archives@tulane.edu / Gram-negative bacteria secrete outer membrane vesicles (OMVs) that play critical roles in intraspecies, interspecies, and bacteria-environment interactions. Some OMVs, such as those produced by Pseudomonas aeruginosa, have previously been shown to possess antimicrobial activity against competitor species. In the current work, we demonstrate that OMVs from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi and exhibit antibiofilm activity against methicillin-resistant S. aureus (MRSA) and Streptococcus mutans. We show that a number of compounds, including peptidoglycan hydrolases, 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline (HMNQ) and long-chain rhamnolipid present in B. thailandensis OMVs exert antimicrobial activity. Furthermore, we demonstrate that HMNQ and rhamnolipid possess antimicrobial and antibiofilm properties against various microbes. Rhamnolipid is superior at reducing the integrity of biofilms while HMNQ displays greater bactericidal activity. We attempted to use HMNQ and rhamnolipid to combat MRSA and promote wound healing in a murine full-thickness wound model. However, further optimization of the model and characterization of the molecules in antimicrobial efficacy, wound healing, and host immune responses are required. Overall, this work indicates that B. thailandensis secretes antimicrobial OMVs that may impart a survival advantage by eliminating competition. In addition, bacterial OMVs may represent an untapped resource of novel therapeutics effective against biofilm-forming and multidrug-resistant organisms. / 1 / Yihui Wang

Breast cancer (BC) is the second leading cause of death in Canadian women. Detection of the disease at an early stage greatly increases the average 5-year survival rate, however non-invasive early detection methods are not available to-date. Cells release various types of extracellular vesicles (EVs) to mediate intercellular communication by transferring signals in the form of bioactive molecules such as proteins, metabolites, and nucleic acids. Understanding the composition of these biomolecules may shed light on the physiological state of the cell of origin. Therefore, EVs are a promising source of biomarkers for non-invasive detection of BC. However, the surface proteome of EVs is not yet understood well enough to propose BC biomarkers that could be detected directly from biofluids. In this study, small EVs (sEVs) and medium EVs (mEVs) were isolated by differential ultracentrifugation from breast cancer MDA-MB-231 and MCF7, and non-cancerous breast epithelial MCF10A cell lines and analyzed by nano-liquid chromatography coupled to tandem mass spectrometry. EV proteins were analyzed by two approaches: (1) global proteomic analysis and (2) enrichment of EV surface proteins by labelling surface-accessible proteins with a Sulfo-NHS-SS-Biotin reagent. Potential BC biomarkers were obtained from the first approach (1) by identifying the presence of cell line specific sEV proteins, filtering for membrane/surface proteins using UniProt annotations, and predicting the co-localization of proteins on sEVs with known EV marker proteins (CD63, CD9, CD81) by correlation analysis. This resulted in 11 potential BC sEV biomarkers (C8A, AXL, ST14, FAM20B, PROM2, CLDN3, ITGA7, MEGF10, SHISA2, GJC1, IFNGR1); the presence of ST14, CLDN3 and ITGA7 was validated by Western blot analysis. The surface labelling approach (2) enriched proteins previously not identified using the first approach (1). Potential general BC biomarkers were selected from surface proteins commonly identified from MDA-MB-231 and MCF7, but not identified in MCF10A EVs. Annotation with known BC disease associations from DisGeNET yielded 9 and 2 potential surface proteins on sEVs and mEVs, respectively. This study demonstrates the emerging role of EVs as a rich source of known and novel biomarkers which may be used for non-invasive detection of BC.

Macrophages are central players during atherosclerosis. Especially, macrophage cholesterol efflux, which promote the removal of free cholesterol from foam cells, are crucial to prevent lipid accumulation and reverse atherogenesis. microRNAs (miRNAs) are important regulators of various pathways involved in atherosclerosis. During inflammation, macrophages secrete extracellular vesicles (EVs) carrying miRNAs to communicate signals to nearby cells. However, the role of macrophage-derived EVs in atherogenesis is not known. In the first study, we find that EVs derived from cholesterol-loaded macrophages can inhibit macrophage migration in vitro and in vivo. This effect appears to be mediated by the transfer of several miRNAs, including miR-146a, to recipient macrophages where they repress the expression of specific pro-migratory target genes Igf2bp1 and HuR. Our studies suggest that EV-derived miRNAs secreted from atherogenic macrophages may accelerate the development of atherosclerosis by decreasing cell migration and promoting macrophage entrapment in the vessel wall. Understanding macrophage communication via EVs provided the rationale for the design of nanoparticles (NPs) that mimic macrophage EVs to deliver beneficial miRNAs to the atherosclerotic plaque. While cationic lipid/polymer-based NPs have been employed as systemic delivery vehicles of siRNA, none of these have been used to deliver miRNAs to macrophages in vivo. In the second study, we developed a chitosan NP platform for effective delivery of miRNAs to alter macrophage function in vivo. We showed that our NPs made using a cross-linked chitosan polymer can protect as well as transfer miR-33 to naïve macrophages and regulate the expression of its target gene (Abca1) as well as cholesterol efflux in vitro and in vivo. Finally, almost all miRNAs that have been characterized are efflux-repressing miRNA, thereby accelerating atherosclerosis. miR-223 is one of a few miRNAs whose overexpression can promote cholesterol efflux, modulate the inflammatory response, and thus, be antiatherogenic. However, its contribution to the pathogenesis of atherosclerosis in vivo and the mechanism underlying its effects has not been thoroughly characterized. We herein find that miR-223 is capable of suppressing plaque development via modulating cholesterol efflux and inflammatory responses, thus may serve as a potential therapeutic to reduce atherosclerosis. These effects of miR-223 appear to be dependent on the inhibition of its target gene, the transcription factor Sp3. Overall, this thesis highlights the importance of both endogenous and extracellular miRNAs in controlling different aspects of atherogenic response.

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

Physical activity (PA) reduces the risk for deleterious outcomes in both mother and fetus during pregnancy and improves health across the lifespan. How these benefits are bestowed remains poorly understood but may involve the placenta, the critical interface responsible for fetal growth and survival during pregnancy. This thesis first aims to determine whether small extracellular vesicles (sEVs), potential biological mediators of cell-to-cell communication, are released into circulation after acute exercise during pregnancy and how this compares in the non-pregnant state. Pregnant women were found to have greater circulating sEVs levels compared to non-pregnant controls after a moderate-intensity treadmill walk. Since exercise-associated sEVs are proposed to mediate tissue cross talk in response to exercise, exercise-associated sEVs were examined for their ability to influence trophoblasts (specialized placental cells) in vitro using the BeWo choriocarcinoma cell line. Exercise-associated sEVs from pregnant and non-pregnant women interacted with trophoblast-like cells but did not alter their proliferation, gene expression of angiogenic growth factors, or production of the pregnancy hormone, human chorionic gonadotropin. Finally, the relationship between differing intensities of maternal PA and fetoplacental vascular density in a cohort of healthy pregnant women followed prospectively from 24 weeks of gestation until term delivery. Using traditional histopathological point-counting techniques, there was no difference in the fetoplacental vascular density of individuals meeting or exceeding recommended 150 min of moderate-to-vigorous intensity PA. However, the analysis revealed unexpected associations between fetoplacental vascular density and lower intensities of PA, and sedentary time. Together, the work presented in this thesis highlight the potential for exercise-associated sEVs to communicate the benefits of PA to mother and fetus and the need to investigate the effects of varying PA intensities on placental vascular development.

In this work, the properties of the liposomes, the artificially created vesicles by various methods, are explored by a resistive pulse method using micropipettes. The fact that vesicles are fundamental in the wide range of functionalities they fulfill as organelles strengthen the desire of understanding the properties of them. The motivation of this work comes from the significant roles that liposomes play in the development of targeted drug delivery systems. Among other significant variables, the size of liposomes is found to be one of the dominating parameters in liposome based drug delivery, and the correlation between liposome size and delivery efficiency is discussed. To help improving the size evaluation ability, a few mainstream methods for liposome size detection and measurements are reviewed. As a reliable and accessible alternative method for liposomes detection, the resistive pulse method is introduced and the measurement on liposomes size change upon pH gradient was performed using this method. With our current liposome composition, we found the size increases as environmental pH increases. Further investigation is performed with vesicular pH=6, 7, and 8, respectively. Lastly, the stability of the small unilamellar vesicles (SUV) was studied via resistive pulse method, by monitoring the size change of 50nm liposomes as function of time. A significant size change in freshly prepared 50nm liposomes is recorded. This information will provide invaluable knowledge for targeting tumor with tight tissues, where small size liposomes are needed.

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