Medicine - Doctoral Theses
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Item Bi-directional tumour-host interaction in a murine model(University College Cork, 2001) O'Brien, Mike; Shanahan, FergusTumours evoke immunologic responses yet they continue to grow and develop. The majority of people who develop tumours are immunocompetant, evoke immune responses and yet metastases occur in lymphnodes, potentially the most hostile immune environment. This is the paradox that we are attempting to unravel. It is hoped that this work would eventually lead to improved immunotherapeutic strategies. The primary objective of the work was to study the mechanisms by which tumours of differing origins and antigenic spectra circumvent the anti-tumour immune response to grow and develop. Responses were characterised for two histologically distinct murine tumours grown in immunocompetant, syngeneic mice. Immunological response profiles were defined in terms of lymphocyte proliferation, cytotoxic function and lymphocyte subsets in naive non-tumour bearing mice and mice with increasing tumour burden. The effect of this antitumour immune response on the developing tumour was examined in a model of mixed tumour antigenicity established in our laboratory. The influence of tumour derived immunosuppressive factors on the immune response was examined. These factors were partially characterised and compared to determine if the differing response profiles could be attributed to differing immunosuppressive factors. The possible effect of tumour antigens shed into and processed by the gut associated lymphoid tissue with consequent down regulation of the systemic immune response (Oral Tolerance) to a developing tumour was examined.Item Real-world Orkambi Cork (ROCK) study - a prospective 12 months analysis addressing the impact of CFTR modulation in patients with cystic fibrosis homozygous for F508del CFTR variant(University College Cork, 2024) Arooj, Parniya; Plant, Barry; Eustace, JoeCystic fibrosis (CF) stands as one of modern medicine's success stories, with significant and sustained improvements in survival rates, transforming it from a childhood fatal condition to one of adult survival. Historically, CF management has centered on addressing the consequences of CFTR dysfunction. The introduction of Lumacaftor-Ivacaftor marks a pivotal shift, allowing researchers to assess the impact of CFTR function restoration on both pulmonary disease and extrapulmonary manifestations. Globally, approximately 82% of individuals with CF are homozygous for the F508del mutation, whereas this prevalence is 53% at the Cork CF centre. This study examines the effects of CFTR modulation on clinical outcomes, patient-reported measures, systemic and airway inflammation, and lung microbiota composition.Item Gut-heart axis in a large animal model of metabolic syndrome and heart failure(University College Cork, 2024) Cluzel, Gaston; Caplice, Noel M.; Stanton, Catherine; SFI ManufacturingBackground The metabolic syndrome (MetS) is a pathological condition diagnosed as the combination of obesity with either hypertension, dyslipidaemia, or hyperglycaemia. MetS constitutes a deadly cocktail of cardiovascular risk factors that greatly increases patient mortality. Among the cardiovascular complications of MetS, heart failure with preserved ejection fraction (HFpEF) represents one of the greatest unmet clinical needs of the 21st century. Indeed, as HFpEF prevalence increases along with soaring MetS cases, current therapeutic strategies fail to prevent disease complications. Therefore, novel approaches are required. MetS and HFpEF are accompanied by a low-grade inflammation (LGI) state. LGI is characterised as a steady but lingering increase in circulating inflammatory factors. Inflammatory signalling is known for promoting structural and functional changes in the myocardium that may contribute to HFpEF. Thus, decreasing LGI may reduce HFpEF progression. While the precise origin of LGI is uncertain, the gut microbiome has recently emerged as a hidden organ with critical immune regulatory functions. Crucially, the gut microbiome is tightly connected to the intestinal barrier. In MetS and HFpEF, patients show alteration of the gut microbiome and of the intestinal barrier, a phenomenon called gut permeability. Gut permeability results in the translocation of bacterial antigens from the gut lumen to circulation. Circulating bacterial antigens are pro-inflammatory, that contributes to LGI and, indirectly, to cardiac structural changes and HFpEF. Therefore, therapeutic strategies aimed at the gut microbiome may effectively prevent HFpEF via reducing gut permeability and LGI. This concept is described as the “gut-heart” axis. The gut-heart axis constitutes a novel field of investigation in cardiometabolic disorders and may answer the urgent need for novel therapeutic strategies directed against HFpEF. However, more research is needed to characterise the mechanisms involved in gut-heart signalling. Aims of the project This thesis aimed at characterising the cardiac pathological mechanisms involved in gut-heart signalling, and determining whether they can be modulated by a microbiota-targeted treatment. Methodology In this project, gut-heart axis pathological signalling was characterised using a porcine model of MetS and HFpEF induced by Western diet (WD) and hypertensive corticosteroid salts (desoxycorticosterone acetate, DOCA). Then, to investigate the effects of a gut microbiome-targeted intervention on MetS and HFpEF, this model was supplemented with a synbiotic product combining soluble corn fibre and Lactobacillus mucosae. Inflammatory signalling associated with HFpEF structural changes was investigated in the four cardiac chambers. In particular, the project focused on the roles of tumour necrosis factor (TNF)-α, lipopolysaccharide (LPS) and NOD-like receptor family, pyrin domain containing 3 (NLRP3). These central inflammatory pathways may be key in transducing gut-originating LGI into cardiac pathological signalling in HFpEF. Results Upon WD and DOCA challenge, the porcine model constituted a clinically-relevant reproduction of MetS and HFpEF. MetS was characterised by increased body weight, severe hypertension, hypercholesterolemia, and hypertriglyceridemia. HFpEF was characterised by left atrium enlargement (LAE) and left ventricle hypertrophy (LVH). LAE was associated with tissue apoptosis, and LVH was accompanied by cardiomyocyte hypertrophy. Left atrium (LA) and left ventricle (LV) also had increased inflammatory activity with cardiac macrophage (Mφ) expansion, and activation of TNF receptor 1 (TNFR1), toll-like receptor 4 (TLR4), and NLRP3 pathways. Moreover, the increase in TNFR1, TLR4, and NLRP3 activity was colocalised with cardiac Mφ, microvascular endothelial cells, and cardiomyocytes. While no structural or pressure-induced changes were observed in right heart chambers, the right atrium and the right ventricle also exhibited prominent inflammatory signalling. Data not reported in this thesis indicated that the model exhibited LGI and features of gut permeability. Overall, the porcine model of MetS and HFpEF was characterised by inflammatory cardiac changes along with systemic and intestinal alterations. Synbiotic treatment of MetS pigs reduced LAE, LA cardiomyocyte apoptosis, and LVH, but did not affect MetS core parameters. These improvements in cardiac structural changes were associated with a reduction in cardiac Mφ expansion and in TNFR1, TLR4, and NLRP3 activity in all four cardiac chambers. Reductions in TNFR1, TLR4, and NLRP3 activity were colocalised within the cardiac Mφ, microvascular endothelial cells, and cardiomyocytes populations. Data not reported in this thesis also indicated that synbiotic treatment reduced LGI and gut permeability. Therefore, synbiotic treatment targeted at the gut microbiome reduced pathological signalling along the gut-heart axis, and effectively reduced cardiac structural changes associated with HFpEF. Discussion The porcine model of MetS and HFpEF stood out as a robust model for investigating gut-heart axis inflammatory signalling. The study also highlighted the central role of TNFR1, TLR4, and NLRP3 in driving structural changes in HFpEF through pro-apoptotic and pro-hypertrophic signalling. Crucially, synbiotic treatment targeted at the gut microbiota effectively reduced HFpEF-associated structural changes via reducing cardiac inflammatory signalling. Finally, while exempt of structural changes, the right heart reflected accurately and dynamically the systemic changes in gut-heart axis pathology and treatment. Conclusions Synbiotic targeting of the gut microbiome resulted in cardiac structural improvements in a clinically-relevant porcine model of MetS and HFpEF. This study demonstrates the critical role of gut-heart inflammatory signalling cardiometabolic disease progression.Item Evaluation of the role of MxA in oesophageal cancer(University College Cork, 2024) Hayes, Robert Michael; McKenna, Sharon L.; O'Donovan, Tracey; Breakthrough Cancer Research; Health Research BoardOesophageal cancer is the seventh most common cancer worldwide and the sixth leading cause of cancer death. The incidence of oesophageal cancer is predicted to increase by 30% by the year 2040, however five-year survival remains at 20% in Ireland. There are two main subtypes, oesophageal squamous cell carcinoma and oesophageal adenocarcinoma. Both are aggressive cancers which frequently develop resistance to chemotherapeutics. Thus, novel therapeutic options are needed. Myxovirus resistance A (MxA) is an interferon inducible antiviral protein that is frequently upregulated in oesophageal cancer. It is currently unknown whether MxA plays a role in the development and progression of oesophageal cancer. We investigated the relationship between MxA expression and the response to chemotherapeutics (5-fluorouracil and oxaliplatin) in oesophageal cancer cell lines. MxA was differentially expressed in five oesophageal cancer cell lines. KYSE450 and KYSE140 cells did not express MxA and were apoptosis incompetent. FLO-1, KYSE270 and OE21 cells expressed MxA and were apoptosis competent. MxA was artificially overexpressed in cell lines without endogenous expression. This increased the resistance of KYSE450 but not KYSE140 cells. Both cell lines remained apoptosis incompetent. MxA expression was depleted in FLO-1 cells using siRNA, and in OE21 cells using CRISPR knockout. Knockdown of MxA significantly increased drug sensitivity and caspase-3 activation in FLO-1 cells. OE21-MX1KO cells were also more drug-sensitive, but caspase-3 activation was reduced. Therefore, these data indicate MxA can influence drug sensitivity, but this is not always related to apoptosis. We examined the impact of MxA on autophagy. Loss of MxA led to significant elevation of basal and drug-induced autophagy in all cell lines. Limited colocalisation was observed between MxA and the autophagosome marker LC3, suggesting MxA’s negative regulatory effect on autophagy is unlikely to involve direct interaction with autophagosomes. MxA was distributed in variably sized, discrete structures which were more numerous and peripherally located following treatment with a proteasome inhibitor or oxaliplatin. We also found that MxA is a significant component of the secretome of oesophageal cancer cells. This secretome included the mitochondrial protein TOM20. MxA knockout diminished the secretion of TOM20 from cells, in conjunction with the autophagy marker LC3. Therefore, we have established that MxA plays a role in the secretion of mitochondria and may act at the intersection between autophagic degradation and export of cellular material in oesophageal cancer cells. We evaluated the effects of MxA knockout on gene expression by RNAseq analysis. 868 genes were differentially expressed, including interconnected networks of genes involved in cell motility, immune/inflammatory signalling, metabolism, and cell differentiation. 59 differentially expressed genes overlapped with genes affected by inhibition of HDAC6, a central mediator of cancer associated pathways. We examined a potential interaction between MxA and HDAC6. Immunofluorescence analysis of OE21 and FLO-1 cells identified colocalisation between MxA and HDAC6 in particulate structures resembling stress granules. Colocalisation was increased in response to treatment with a chemotherapeutic agent or proteasomal inhibitor. Co-immunoprecipitation analysis indicated a specific interaction between MxA and HDAC6. We then compared migration in OE21 and OE21-MX1KO cells. A wound-healing assay demonstrated that migration was significantly reduced in MxA KO cells. Therefore, our data indicates a diverse role for MxA in oesophageal cancer biology. Depletion of MxA expression increased sensitivity to chemotherapeutics. MxA knockout resulted in differential expression of interconnected networks of genes which are associated with cancer progression. MxA negatively regulated autophagy and promoted secretion. A novel interaction between MxA and HDAC6 was identified, which may have significant consequences for the activity of both proteins. Importantly, as depletion of MxA can increase drug sensitivity and reduce migration, targeting this protein may be beneficial for the treatment of oesophageal cancer in future.Item Identifying dietary additive regulated mechanisms impacting intestinal epithelial cell responses and inflammatory processes(University College Cork, 2024) Saiz-Gonzalo, Gonzalo; Melgar Villeda, Silvia; Joyce, Susan; Irish Research Council for Science, Engineering and TechnologyIn recent years, research has increasingly identified diet as a crucial factor in the development and management of gastrointestinal diseases like Inflammatory Bowel Disease (IBD). Dietary influences are now considered significant in determining an individual's susceptibility to IBD, yet much remains unknown about the long-term effects of various dietary components. The main objective of my thesis is to explore the mechanisms through which dietary emulsifiers and sweeteners influence gut epithelial cell health, with a focus on their potential role in exacerbating gastrointestinal diseases. The first part of the study focuses on the effects of food emulsifiers, specifically lecithin, carboxyethyl cellulose (CMC), and polysorbate 80 (p80), on human intestinal epithelial cells and murine organoids. While lecithin and CMC showed no significant harmful effects, p80 was found to induce cell death at concentrations below the FDA-approved level. Through RNA sequencing and a series of assays including cell viability, cell death, lipid peroxidation autophagy, Seahorse, transmission electron microscopy, western blotting to name a few, the study reveals that p80 triggers ferroptosis—a type of cell death associated with iron-dependent lipid peroxidation—causing mitochondrial damage. Interestingly, p80 did not seem to activate the inflammatory response in the cells. The second part of the thesis explores the role of the lipid peroxidation and lipid accumulation prompted by p80 and ferroptosis. Using lipidomic analysis and inhibitors targeting lipid formation enzymes, the research identifies specific lipid changes and profiles, especially in the polyunsaturated fatty acids, associated with ferroptosis. The study highlights how lipid metabolism is intricately linked to cell death mechanisms, suggesting potential therapeutic approaches to mitigate emulsifier-induced lipotoxicity by using lipid and ferroptosis inhibitors. In the final part, the study examines the effects of rare sugars and sweeteners on human intestinal epithelial cells under both steady-state and inflammatory conditions such as IBD. Among the sweeteners tested—Psicose, Lactitol, Maltitol, Saccharin, and Tagatose—saccharin shows a significant impact on gene expression, particularly under inflammatory conditions. Saccharin activates JAK/STAT pathway related to inflammation by increasing cytokine secretion and cell death, indicating a potential role of saccharin in potentiating intestinal inflammation. Collectively, this thesis provides comprehensive insights into how specific dietary additives influence intestinal cell health. By unravelling the complex interactions between diet and cellular processes, the study contributes to a better understanding of potential dietary risks and offers guidance for future research aimed at improving dietary management in IBD and GI-related disorders.