Medicine - Doctoral Theses
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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.Item Fall prevention in older people in long-term care facilities: a mixed-methods study investigating effective staff education to prevent falls in older residents(University College Cork, 2024) Albasha, Neah Mohammad A.; Timmons, Suzanne; Cornally, Nicola; McCullagh, Ruth; Mchugh, Sheena; Princess Nourah Bint Abdulrahman UniversityBackground: More than 50% of older residents in long-term care facilities(LTCFs)fall each year, associated with adverse psychological and physical consequences. In intervention studies, some fall prevention activities reduced morbidity, but challenges remain in clinical practice. Fall prevention can be enhanced by LTCF staff knowledge and skills and by identifying barriers and facilitators to success. This thesis aimed to explore LTCF staff education needs (i.e. knowledge gaps) and selfidentified learning needs, and to identify the most common implementation strategies used in fall prevention studies. Methods: To synthesise the evidence on implementation strategies and outcomes(categorised using the Expert Recommendations for Implementing Change(ERIC)Taxonomy and Implementation Outcomes Framework), and on clinical outcomes of fall prevention interventions, a systematic search of six electronic and eight grey literature databases from 2001 to 2021 was conducted, updated to 2023. Using an exploratory, parallel, mixed methods design, staff in 14 LCTFs(varying in size and provider type) in southwest Ireland were invited to complete a survey, a hybrid of two previously developed questionnaires. Staff knowledge, attitudes, and confidence in fall prevention, along with previous training and future education preferences, were evaluated. Suggestions were sought on how to prioritise fall prevention activities and current practices regarding fall incidents. Staff in five of the LTCFs were invited to participate in online interviews, using a semi-structured guide to explore barriers and facilitators of fall prevention activities, and staff perceptions of their learning needs. Survey analysis included descriptive statistics and content analysis, mapping categories to the refined theoretical domains framework(TDF) and an existing fall prevention guideline, and reflective thematic analysis using Braun and Clarke's method. The two data sets were then triangulated to compare/contrast findings and determine whether educational needs reflected inadequate training and/or their perceived learning needs. Results: The SR identified 39 implementation strategies for fall prevention used in 27 studies but highlighted poor reporting, limited assessment and no clear pattern in implementation strategies within positive studies, requiring improvement. In total, there were 155 survey participants across 13 LTCFs, predominantly HCAs and nurses, with a response rate of 15%. Overall, 60% of LTCF staff, mainly senior nurses, had high levels of knowledge about fall prevention, but role-specific knowledge gaps involved exercise, medication review, antipsychotic drug usage and toileting issues. Respondents had high confidence in delivering fall prevention interventions (90%); greater experience in healthcare was associated with greater knowledge and confidence. The most common fall prevention activities suggested were environmental reviews and modifications, staff education, resident monitoring, using alarm/call systems and protective equipment, while fall prevention activities targeting resident-related risk factors (e.g., assessing risk factors) were less recognised. Staff also used a standardised approach to respond to falls, implementing comprehensive post-fall control measures. Concurrently, 17 LTCF staff members from 4 sites participated in online 1:1 interviews (n=7) or small group interviews (n=10). Analysis identified six main themes, leading to the conclusion that integrating fall prevention into daily routines is feasible and practical through rapid-cycle audits, falls champions, informal communication, and staff collaboration. Half of participants had received fall prevention training, but many welcomed further education, especially in-service,multidisciplinary training tailored to staff roles and local contexts. Triangulation of the data revealed that there were both knowledge gaps and selfidentified learning needs in topics such as fall risk assessment, manual handling techniques, medication review, while practice- based learning was preferred. Conclusion:The most common strategy used in falls prevention studies is education, but LTCF staff have both educational and learning needs. This thesis provided insights into fall prevention education, and identified required curriculum elements, and emphasised the importance of tailoring multidisciplinary fall prevention education to staff preferences and educational needs. Future fall prevention education programmes should consider the greatest knowledge gaps, as well as self-identified learning needs and staff preferences for delivery, along with the nursing home’s culture, to most effectively integrate fall prevention activities into practice.Item Evaluation of biomarkers for the diagnosis, management and follow-up of women with gestational trophoblastic disease(University College Cork, 2024) Joyce, Caroline Martha; O'Donoghue, Keelin; McCarthy, Tommie V.; Coulter, John; Irish Research CouncilGestational Trophoblastic Disease (GTD) describes a spectrum of disorders arising from the abnormal proliferation of trophoblastic tissue. Human chorionic gonadotrophin (hCG) is an excellent biomarker for most forms of this disease as its concentration accurately reflects trophoblastic activity. Hydatidiform mole (HM) or molar pregnancy is the most common form of GTD which may be suspected on ultrasound but requires pathological confirmation for diagnosis. Prognosis is generally excellent however close surveillance with hCG monitoring is imperative to detect cases progressing to malignant disease, referred to as gestational trophoblastic neoplasia (GTN). Clinical management and treatment of women with GTN in specialist centres achieves cure rates approaching 100%, emphasising the importance of accurate hCG monitoring for early detection of disease persistence to ensure optimal outcome. This thesis aims to evaluate pathological, genetic, and biochemical biomarkers used in the diagnosis, monitoring, and follow-up of women with GTD. In this thesis, a scoping review was performed to generate evidence and identify gaps in the GTD knowledge base to inform areas for future research. Key findings were the need to standardise hCG immunoassays for effective GTD surveillance, to improve diagnostic reporting pathways and to establish more accurate incidence rates. In order to identify patient priorities and inform initiatives to enhance the quality of care, a service evaluation was performed with women enrolled in the National GTD Registry invited to participate. Feedback revealed a knowledge gap regarding GTD amongst healthcare professionals outside GTD centres. This study also revealed shortcomings in psychological support and bereavement counselling offered to women after a molar pregnancy. To enhance the diagnostic accuracy of HM reporting, an in-situ hybridisation ploidy assay was developed with a customised scoring system to aid partial hydatidiform mole (PHM) diagnosis. This innovative technique provides a reliable adjunct to morphological assessment for PHM classification. The accuracy of this ploidy technique was confirmed by evaluation with molecular short tandem repeat genotyping. A national pathology questionnaire was performed to gather information on GTD diagnostic rates. Data collected was cross-referenced with cases documented in the National GTD Registry. This revealed a concerning under-enrolment of women with GTD (42%) onto the National GTD Registry by their clinicians. An audit of HM diagnostic rates over a 3-year period was performed following the Implementation of the new ploidy technique to aid diagnosis . This revealed a higher local PHM incidence rate (1 in 296 births) than reported nationally in the pathology audit (1 in 484 births). An electronic questionnaire was distributed to all European laboratories offering hCG for GTD management to gather insights on 5 key areas: hCG methodology, quality parameters, reporting and operational procedures, and use of hCG for non-GTD testing. This revealed considerable inter- and intra-laboratory variability in practice, emphasising the need to promote standardisation and harmonisation of immunoassays for GTD-derived hCG. A review of five challenging cases managed at the Irish GTD centre was conducted to evaluate the role of serum hCG and molecular genotyping in guiding management decisions, treatment strategies, and risk stratification for women with molar pregnancy, GTN and GTD mimics. These complex cases confirmed the pivotal role of serum hCG and molecular genotyping in monitoring disease persistence, prognostic stratification, and clinical management Novel strategies to measure hCG were pursued to address the lack of centralised hCG testing for GTD in Ireland. A proof-of-concept study for remote capillary blood collection was performed to evaluate the clinical efficacy and user acceptability of this method of collection for hCG monitoring in early pregnancy. The study confirmed the equivalence of capillary and venous blood hCG testing, demonstrated complete clinical concordance, thereby offering an alternative to venepuncture for hCG measurement. To explore the accessibility of hCG testing in the community, a point-of-care testing (POCT) device was evaluated for its suitability to triage women with pregnancy of unknown location (PUL) in the early pregnancy unit. The Abbott iSTAT®1 POCT instrument was chosen for this purpose and was clinically concordant with central laboratory hCG results facilitating the use of clinical decision thresholds for PUL. The research presented in this thesis provides valuable clinical insights through a series of nine research studies and has broadened the knowledge base in GTD. The findings offer an opportunity to positively impact diagnostic practices. The knowledge gap regarding GTD amongst healthcare professionals revealed in the patient survey points to a need for targeted educational initiatives in this area. Widespread integration of the HER2 ploidy technique to support morphology and standardise HM reporting could improve diagnostic accuracy, addressing a suspected underdiagnosis of PHM nationally and possibly internationally. It also suggests the need to re-evaluate incidence rates following the introduction of advanced diagnostic techniques such as ploidy assessment and molecular genotyping. This thesis identified an under-registration of women with GTD in Ireland supporting the need for initiatives such as mandatory registration or advocacy by professional organisations to increase registration rates. The inter and intra-laboratory variation in hCG immunoassays revealed in the European laboratory survey merits renewed harmonisation efforts through pilot external quality assurance and sample exchange programmes. The use of novel methods to enhance the accessibility of hCG monitoring for GTD in the community, through capillary blood collection or use of point-of-care analysis, warrants further study. The positive patient feedback indicated a preference for this approach, suggesting better compliance with serial test monitoring, a priority for GTD management.