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- PublicationExtracellular Vesicles in Cardiovascular Disease – Modulation of Circulating Extracellular Vesicle Signatures by Rivaroxaban Therapy(University College Dublin. School of Biomolecular and Biomedical Science, 2021)Thrombosis remains a major cause of mortality and morbidity worldwide. Common risk factors include pro-inflammatory conditions, endothelial dysfunction, and aberrant platelet activation, all augmenting the patient’s risk of developing a thrombus. Intriguingly, these risk factors also manifest in increased levels of circulating extracellular vesicles (EVs). EVs are a heterogenous group of membrane vesicles released from all cells. Due to the expression of tissue factor and phospholipids on their surface, EVs can accelerate thrombus formation. To circumvent adverse events in thrombotic disease, pharmacological thromboprophylaxis is frequently indicated. Rivaroxaban, a direct FXa inhibitor, mediates anti-inflammatory and cardiovascular-protective effects besides its well-established anticoagulant properties, however, these remain poorly characterized. Given the prevalent role of EVs in thrombotic diseases, we hypothesized that Rivaroxaban’s anti-inflammatory properties are reflected upon differential molecular profiles of circulating EVs. Here, we used single vesicle analysis and comparative proteomics to, for the first time, characterise small (<200nm) and large (200-1000nm) plasma EVs from patients with non-valvular atrial fibrillation (AF), venous thromboembolism (VTE) and stable cardiovascular disease (CVD) treated with Rivaroxaban compared with cohort-specific controls. We identified profound changes in circulating EV profiles and proteomic signatures that may contribute to Rivaroxaban’s pleiotropic effects. Circulating EV profiles were fundamentally altered across the cohorts analysed. While AF patients displayed profoundly reduced levels of total small EVs, we identified a decrease in the proportion of large EVs between 400-700 nm in VTE patients. Patients with stable CVD, on the other hand, indicated increased small EV mode size with a concomitant increase in the proportion of large EVs between 600-800 nm. Comparative proteomic characterisation of enriched EV fractions revealed differential expression of proteins involved in the regulation of complement activation, vascular integrity, and inflammation, collectively mirroring the reported anti-inflammatory and cardioprotective characteristics associated with Rivaroxaban therapy. In summary, we have determined that circulating EV profiles are powerful surrogate markers of the Rivaroxaban-mediated pleiotropic effects. Regardless of the underlying thrombotic disease, Rivaroxaban appears to confer vascular protective effects in both venous and arterial thrombotic conditions. While several mechanisms, including inhibition of protease activated receptor or NF-¿B signalling, have been proposed to contribute to these vascular protective characteristics mediated by Rivaroxaban, we here provide original evidence that the modulation of circulating EV signatures may pose a distinct mechanism through which Rivaroxaban therapy may elicit such pleiotropic effects, an exciting finding that may be leveraged when planning future studies exploring personalized management strategies, such as identification of those patients most likely to benefit from this therapy, particularly in jurisdictions where access to therapy may be restricted. Alleviating the underlying pro-inflammatory state of patients with thrombosis poses a promising future therapeutic target to avert major adverse thrombotic events. Further investigations into Rivaroxaban-mediated changes in circulating EV signatures may provide a future avenue to open Rivaroxaban’s use for extended patient cohorts, such as rheumatoid arthritis.
- PublicationValorisation of dairy side streams using processing technology and biotechnology(University College Dublin. School of Biomolecular and Biomedical Science, 2022)Dairy side streams, namely, whey permeate (WP) and delactosed permeate (DLP), are a key challenge for dairy processing. Excess WP and DLP volumes can be a sustainable bottleneck for the expansion of dairy industries and finding a sustainable disposal route with environmental and economic incentive for these dairy side streams can be challenging. This thesis explores a second-generation (2G) biotechnological process for the bioconversion of dairy side streams into bio-based L-lactic acid. WP and DLP are supersaturated in fermentable lactose that can be converted biochemically into biodegradable bio-based monomers such as lactic acid (LA). The bioconversion of whey side streams into LA and subsequent polymerization into biodegradable polymers such as polylactic acid creates new value chains in the dairy industry but also the opportunity to reduce the environmental and economic burden associated with WP and DLP processing. However, WP and to a greater extent, DLP, have a high mineral content which can affect microbial fermentation performance and increase the cost and complexity of the downstream purification process, thus, requires a pre-treatment demineralisation step. At pilot-scale, the pre-treatment of dairy side streams into a suitable fermentation substrate was investigated using two different pre-treatment methods, NaOH and Ca(OH)2 pre-treatment, applying different dairy side stream feedstock types. Furthermore, microbial L-LA fermentation performance was then investigated at pilot-scale (100 L and 3000 L) using both NaOH and Ca(OH)2 pre-treated dairy side streams as the fermentation substrate. Analytical data was compiled and assessed after completing all stages of the 2G LA biotechnological process, namely, pre-treatment demineralisation, microbial L-LA fermentation, and the downstream purification of the lactate salt into high-quality L-LA. The generated data was then used to develop a techno-economic assessment to validate if the valorisation of dairy side streams into LA was economically feasible at industrial scale production. To sum up, this thesis underpins a feasible bioprocessing valorisation route for WP and DLP to produce L-LA. A 2G LA valorisation platform solves the issue of finding a sustainable solution for excess dairy side stream volumes, creates a new value chain, and supports the transition of dairy industries into a circular dairy bioeconomy.
- PublicationEffects of Pharmacological Modulation of Nrf2 Signalling on Chemical Carcinogenesis in Renal Epithelial Cells(University College Dublin. School of Biomolecular and Biomedical Science, 2022)Cancer is a major cause of morbidity and mortality globally. There were 23.6 million cancer cases and 10 million cancer deaths globally in 2019 and cases are estimated to reach 28.5 million in 2040. Around 30-50 % of cancer cases can be prevented by avoiding risk factors and implementation of prevention strategies. Exposure to chemical carcinogens can increase a person’s risk of developing cancer in their lifetime. Chemical carcinogens are substances which directly induce malignant tumour formation, increase tumour incidence, or decrease the time taken for a tumour to form at an increased rate compared to background. Carcinogens Aristolochic acid (ARAI) and Ochratoxin A (OTA) are major contaminants of herbal medicines and foodstuffs globally. The kidneys are one of the principal organs affected by carcinogens due to their function in maintaining tissue homeostasis. Renal cell carcinoma arises from the epithelium of the nephrons of the kidney which are disproportionately affected by chemical carcinogens due to their role in filtering plasma for removal of waste products and xenobiotics. In most eukaryotic organisms, oxygen is essential for normal cellular functions. During cellular respiration and metabolism, by-products such as reactive nitrogen species and reactive oxygen species (ROS) are produced. Antioxidants remove free radicals and help restore cellular balance. Oxidative stress occurs when excess free radicals are produced in cells which overwhelms the normal antioxidant capacity. This can be caused by both intrinsic (e.g., metabolism) and extrinsic (e.g., xenobiotics) factors. Excess ROS can damage DNA, proteins and lipids and is associated with many pathophysiological processes including carcinogenesis. The Nuclear Factor Erythroid 2-related factor (Nrf2) pathway is an intrinsic, inducible stress mitigation response which regulates antioxidant response genes to reduce oxidative stress. Bardoxolone methyl (CDDO-Me), a synthetic triterpenoid induces Nrf2 through inhibition of the endogenous repressor Keap-1. Several human carcinogens elicit tumourigenic effects by modulating associated oxidative stress pathways including the Nrf2 pathway. For this research the normal (RPTEC/TERT1) cell line was exposed to carcinogens ARAI and OTA, pre-treated with CDDO-Me. Several effects associated with cellular transformation were characterised including loss of junctional protein expression and barrier function, lipid peroxidation and effects on the primary cilium. Pre-treatment with CDDO-Me affected these endpoints and elicited a spectrum of effects dependent on cell type and the carcinogen in question. To further characterise and contextualise the effects observed, whole cell proteome analysis and systems biology modelling was performed. In summary, the current research suggests that the beneficial effects of Nrf2 induction are highly dependent on the nature of the carcinogenic insult applied. In some cases, Nrf2 enhancement abrogated some of the deleterious effects whilst in others, it appeared to potentiate the cellular damage caused.
- PublicationUncovering novel drugs that restore vision by combining biological and computational drug discovery processes(University College Dublin. School of Biomolecular and Biomedical Science, 2022)Retinal degeneration is the leading cause of blindness in the industrialised world1 and is characterised by progressive loss of the light sensing cells, photoreceptors, in the retina. Retinal degeneration occurs in both inherited retinal degenerations (IRD) and age-related macular degeneration (AMD). Limited therapies are available for both conditions and there is a pressing need to uncover novel therapies to rescue/preserve vision. With the advent of computational technology and new screening techniques, novel compounds restoring vision can be uncovered by combining computational and phenotypic drug discovery methodologies. Here, I present two complementary workflows to identify compounds rescuing vision. Firstly, ligand-based virtual screening was used to uncover 3D analogues of 7,8-DHF using Cresset Ltd, Blaze’s software. The second workflow utilised orthogonal pooling to screen 720 compounds from the Chembridge DIVERSet™ compound library for hit compounds. Screening for both workflows was conducted using the optokinetic response assay in the atp6voe1-/- zebrafish model of inherited blindness. Three hit compounds were discovered to rescue vision. Compounds UCD-OPGG-A2 and UCD-OPGG-B15 were identified as 3D analogues of 7,8-DHF restoring vision. Compound UCD-OPGG-3E was uncovered during the randomised library screen. RT-qPCR of 3E and A2 did not confirm alterations in inflammatory or oxidative stress related genes in atp6voe1-/- after drug treatment. Light microscopy analysis suggests 3E may reduce cell death within the ciliary marginal zone of atp6voe1-/- larvae. Tolerability studies performed in collaboration with Experimentica Ltd, indicate that intravitreal injections of A2 and 3E are tolerated in mice. In conclusion, I helped develop two complementary workflows to efficiently detect compounds rescuing vision. Although additional experimentation is needed, compounds A2 and 3E are promising starting points for the discovery of novel compounds to restore vision.
- PublicationEvaluation of the disease relevance and therapeutic potential of cysteinyl leukotriene receptors in uveal melanoma(University College Dublin. School of Biomolecular and Biomedical Science, 2022)Uveal melanoma (UM) is a rare, ocular cancer that arises from melanocytes within the uveal tract. This cancer imposes the threat of visual impairment, ocular pain, enucleation, and in half of all cases, death from metastatic disease. Approximately 50% of UM patients will develop metastases, which occur most frequently in the liver. Despite advances in control of the primary tumour, there is currently no approved therapy that can prevent or halt the growth of UM metastases. The prognosis for metastatic UM patients is extremely poor; the median overall survival is approximately 13.4 months, with as few as 8% of patients surviving beyond 2 years. Recent reports suggest that Ireland has one of the highest incidence rates in the world, with an average of 45 new cases diagnosed annually. The development of therapies that can prolong the life of UM patients is an area of urgent unmet need. This research evaluates the clinical relevance and therapeutic potential of cysteinyl leukotriene receptors (CysLT1 and CysLT2) in UM. The cysteinyl leukotrienes (CysLTs) are a group of inflammatory, lipid mediators that signal through G-protein couple receptors, CysLT1 and CysLT2. The role of CysLT receptor expression, and signalling, in several cancers has recently emerged. This, coupled with the identification of an oncogenic mutation in CYSLTR2 in a subset of UM patients, led us to hypothesise that these receptors could be targeted therapeutically in the disease. We have shown that high expression of CYSLTR1 and CYSLTR2 are significantly associated with reduced disease-free survival and reduced overall survival in UM patients. The expression of both receptors was further investigated by IHC. In two, independent patient cohorts we validated that high expression of CysLT1 is significantly associated with reduced overall survival. These findings solidified the importance of CysLT receptor expression in UM and its link to patient outcomes. As CysLT receptors are druggable targets, we hypothesised that pharmacological antagonists may attenuate cancer phenotypes including viability, proliferation, angiogenesis, inflammation, and metabolism, of UM cells in culture. We found that CysLT1 antagonists, but not CysLT2 antagonist, HAMI 3379, produced significant anti-cancer effects in primary and metastatic UM cell lines through the inhibition of cell survival and cell proliferation. Novel CysLT1 antagonists, quininib and 1,4-dihydroxy quininib, produce cell line-dependent effects on the cancer secretome of UM cell lines. In terms of metabolism, CysLT1 antagonists significantly reduce oxidative phosphorylation in primary and metastatic UM cells. We sought to validate our findings in in vivo and ex vivo preclinical models of UM. In zebrafish cell line-derived xenograft models, CysLT1 antagonists significantly inhibit the growth of primary and metastatic cell lines in vivo and have a greater effect in zebrafish ocular orthoxenograft models. In a cell line-derived orthotopic rodent xenograft model of metastatic UM, treatment with CysLT1 antagonist, 1,4-dihydroxy quininib, did not significantly decrease tumour weight versus vehicle control but did decrease tumour weight and expression of Ki-67, a marker of proliferation, versus standard-of-care, dacarbazine. 1,4-dihydroxy quininib significantly decreases ATP5B, a marker of oxidative phosphorylation, versus vehicle, mimicking our in vitro data. Treatment of UM ex vivo explants derived from primary UM with 1,4-dihydroxy quininib significantly alters the secretion of inflammatory mediators in the tumour microenvironment. The secretion of IL-13, IL-2 and TNF-a was significantly increased following treatment of primary UM tumours for 72 hours. These preclinical data strengthen the importance of CysLT signalling in UM. Our findings suggest that high expression of CysLT1 in UM could act as a biomarker and that antagonism of CysLT1 may be of therapeutic interest in the treatment of UM.