Medicine - Doctoral Theses

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Identification of bacteria-regulated mechanisms for the pathogenesis of inflammatory bowel diseases (IBD) and development of upconverting nanoparticle (UCNP) luminescence imaging for the monitoring of gut bacteria
(University College Cork, 2022) Singh, Raminder; Melgar Villeda, Silvia; Andersson-Engels, Stefan; Shanahan, Fergus; Science Foundation Ireland; APC Microbiome Institute
Inflammatory bowel disease (IBD) is a chronic inflammatory state of the gastrointestinal tract, including two inflammatory conditions Crohn’s disease (CD) and ulcerative colitis (UC). The environment, the gut microbiota, the genetic make-up and the immune response are believed to contribute to the aetiology of the disease. Non-steroidal anti-inflammatory drugs (NSAIDs) are believed to exacerbate inflammation in patients with IBD. However, the literature demonstrates no consensus on the association between NSAID use and IBD, with some studies reporting that only high dose NSAID treatment are more likely for IBD exacerbation or relapse. In this study, using the piroxicam-accelerated model of colitis in interlukin-10 deficient (IL-10-/-) mice, we showed that mice fed 100 ppm piroxicam in food for 9 days (high dose) followed by 5 days of regular chow, develop colitis. The colitic phenotype was associated with activation of caspase-8, NLRP3 inflammasome and apoptosis and independent of initial gut microbiota. However, 5 days exposure (low dose) to 100 ppm piroxicam did not lead to colitis development. Adherent-invasive Escherichia coli (AIEC) is widely prevalent and heterogeneously present in the mucosa of IBD patients, particularly in Crohn’s disease. We hypothesise that the presence of AIEC in the gut of IBD patients might explain the NSAID-induced inflammation in IBD under low dose treatment (5 days piroxicam treatment). Indeed, 5 days piroxicam exposure was sufficient to induce colitis in AIEC-precolonised animals, indicating a synergism between AIEC colonisation and piroxicam treatment. Inhibition of NLR family pyrin domain containing 3 (NLRP3) or Caspase-8 activity ameliorated colitis. This synergism was lost under high dose piroxicam (9 days exposure) treatment. Our data indicate that under low dose NSAID treatment, AIEC can potentiate NSAID-induced inflammation in IL-10-/- mice by regulating the intestinal epithelial function and the immune response, highlighting its potential role in NSAID-induced inflammation in IBD patients exposed to low dose NSAIDs. Although, under high dose, NSAID itself is sufficient to induce colitis. This data suggests that the lack of consistency in the association between NSAID use and IBD could be explained by the NSAID dose and the presence of AIEC in the patients with IBD. Future studies should consider both these factors while studying association between NSAID use and symptomatic worsening in IBD. The role of microbes in IBD is supported by various studies performed in animal models where germ free mice are protected from intestinal inflammation. However, the precise interaction(s) between microbes and the host are not well understood. In vivo imaging techniques using custom designed fluorescent probes and bioluminescence have been classically used to track gut microbes. Several disadvantages while using fluorescent probes include autofluorescence, photobleaching and photodamage. To overcome these limitations, we propose to develop upconverting nanoparticles (UCNPs) luminescence imaging for monitoring of gut bacteria. Their unique property of photon upconversion enables them to convert low energy near-infrared (NIR) light into higher energy visible/NIR light offering greater tissue penetrance and signal-to-noise ratio. Here, we investigated the possibility of using UCNP 1) to image a single gut bacterium (e.g., AIEC) using UCNP-conjugated anti-E. coli antibody, and 2) to image the endogenous gut bacteria using metabolic labelling of bacterial peptidoglycan with azido-D-amino acids and strained-cyclooctynes such as Dibenzocyclooctyne (DBCO) -functionalised UCNPs. We showed a proof of principle for both these approaches using a fluorescent dye, but we were not able to replicate it with UCNP. Preliminary data suggests that the size difference between the UCNPs and the fluorescent dyes may be one of the potential reasons for lack of labelling. Future experiments should consider using smaller UCNPs and/or use a polyethylene glycol (PEG) linker between DBCO and UCNPs to increase the length of DBCO-UCNP construct and reduce the steric hinderance for better stability of UCNP-bacteria conjugation.
Rethinking stillbirth through behaviour change
(University College Cork, 2022) Escañuela Sánchez, Tamara; O'Donoghue, Keelin; Matvienko-Sikar, Karen; Meaney, Sarah; Byrne, Molly; Science Foundation Ireland
Background Worldwide, two million babies are stillborn every year. While the majority of stillbirths occur in low and middle-income countries, stillbirth is still one of the most common adverse pregnancy outcomes in high-income countries. In Ireland, the latest National Perinatal Mortality Clinical Audit report states a stillbirth rate of 4.20 per 1000 births for the year 2020, showing an increase compared to previous years. The belief that reduced stillbirth rates in high-income countries cannot be achieved is refuted by differences in stillbirth rates across different countries. Although not all stillbirths are preventable, there has been a call made in high-income countries to focus on risk factors for stillbirth, in order to reduce stillbirth rates. These risk factors include sociodemographic factors, medical factors, obstetric history-related factors, placental and fetal-related factors as well as behavioural and lifestyle-related factors. Some of these factors are modifiable through medical management or through behaviour change modification. This Thesis focuses on risk factors that have the potential to be modified through maternal behaviour change interventions: substance use (smoking, alcohol, and illicit drug use), high BMI, sleep position, and attendance at antenatal care. Strategies have been successfully implemented internationally to reduce stillbirth rates by designing and implementing care bundles that, amongst other elements, take into consideration the modifiable/behavioural risk factors for stillbirth. However, in Ireland, no such initiatives have been developed, although recommendations have been made that support their development. For behaviour change interventions or public health initiatives to have the best possible success in reducing the rates of stillbirth, they need to be designed with a solid evidence base. Hence, the overall objective of this Thesis was to build the evidence base to enhance the understanding of the modifiable behavioural risk factors for stillbirth and pregnancy. Further, this evidence base is needed to inform the future development of a behaviour change intervention that could be part of a care bundle with the objective of reducing stillbirth rates in Ireland. Methodology To address the Thesis´s aims, both qualitative and quantitative methods were utilised. Applying multiple methods to explore a phenomenon provides flexibility to analyse different aspects of it in the different studies. Initially, a non-systematic review of the literature was conducted to identify the target behavioural risk factors that this project was going to focus on (Chapter 2). A website quantitative content analysis was conducted to assess the availability of information related to stillbirth and behavioural risk factors for stillbirth in Irish and UK websites (Chapter 3). For this study, descriptive and inferential statistics were utilised. Further, three systematic qualitative meta-synthesis were conducted to identify facilitators and barriers to modify identified behavioural risk factors according to the pregnant women’s experience (Chapters 4-6). A meta-ethnographic approach as described by Noblit and Hare was adopted to conduct these qualitative meta-syntheses. Reflexive Thematic Analysis as described by Braun and Clarke, with a constructivist approach, was used to conduct a qualitative semi-structured interview study with postpartum women about their experiences of stillbirth information provision and behaviour change during their antenatal care (Chapter 7). Finally, a systematic review of interventions designed in the context of stillbirth prevention that targeted behavioural risk factors was conducted (Chapter 8). This systematic review had the objective of identifying which behaviour change techniques (BCTs) have been used to date. Results The findings of the literature review (Chapter 2) showed that the modifiable behavioural risk factors with the strongest evidence of associations with stillbirth were substance use, smoking, heavy drinking and illicit drug use, lack of attendance and compliance with antenatal care, weight-related risks, and sleep position. The quantitative content analysis of websites (Chapter 3) revealed that information about stillbirth and behavioural risk factors for stillbirths was scarce on websites directed at the pregnant population, with only one website containing all the information sought. Five main areas of concern were identified across the three meta-synthesis of qualitative research of facilitators and barriers influencing women’s prenatal health behaviours (Chapters 4-6), regardless of the behaviour explored: 1) health literacy, awareness of risks and benefits; 2) insufficient and overwhelming sources of information; 3) lack of opportunities and healthcare professionals attitudes interfering with communication & discussion; 4) social influence of environment, and 5) social judgement, stigmatisation of women and silence around stillbirth. Further, the qualitative study with postpartum women (Chapter 7) revealed that women perceived behaviour change during pregnancy as easy and natural, as they were focused on obtaining the best outcomes for their babies. Although women had high levels of awareness regarding health advice, their awareness about stillbirth was very limited. Women reported a lack of discussion about stillbirth and behavioural risk factors during their antenatal care; however, most women showed a positive disposition towards receiving this information because “knowledge is key”, as long as it is done in a “sensible manner”. The systematic review of interventions designed in the context of stillbirth prevention identified nine relevant interventions. From the BCT coding, it was established that the most common BCT used was “information about health consequences”, followed by “adding objects to the environment” (Chapter 8). Conclusion This research makes a valuable contribution to the understanding of the maternal behaviours associated with an increased risk of stillbirth, and it provides a necessary evidence-base to inform future prevention strategies to reduce rates of stillbirth in Ireland and in similar healthcare settings. This research sought to incorporate women’s voices and use research methods to produce high-quality results that meet the research objectives. The findings from the studies in this Thesis support four overarching topics and highlight issues related to 1) health literacy and sources of information, 2) relationships with healthcare professionals (HCPs), 3) healthcare systems and structural barriers, and 4) interpersonal, social and structural factors. In response to the research findings, several recommendations are made in relation to policy, practice and research which are grounded on women’s experiences during pregnancy. Regarding policy, these recommendations include improving education and information sources for women and HCPs, providing pregnancy-specific supports, utilising community services to support women with behaviour change, and developing a care bundle to tackle the behavioural risk factors for stillbirth. Furthermore, the work practice recommendations made include developing clinical guidelines to support HCPs in providing care to pregnant women, and prioritising health promotion during antenatal care. These priorities might also serve to help funders and researchers to design and conduct policy-relevant research. The key future research areas identified by this Thesis are in relation to the involvement of PPI representatives, the assessment of the quality of the available sources of information and the further exploration of potential facilitators and barriers to modifying pregnant women’s sleeping position from a qualitative perspective. In addition, this Thesis proposes a detailed process to continue building on the work set out in the different studies to develop a pregnancy-specific behaviour change intervention for the modifiable behavioural risk factors for stillbirth in the future.
Three year outcomes in a cohort of patients with newly-diagnosed epilepsy
(University College Cork, 2022) Stack, Jessica; Costello, Daniel; O'Reilly, Eilis
Background: Epilepsy is a common and internationally recognised neurological disorder with often life-long implications for its sufferers and their families. While a number of studies have assessed the outcomes of epilepsy, there have been none carried out on a well-defined ‘all-comers’ cohort of patients identified in an epilepsy incidence study. Our primary aim was to evaluate the early history and mortality of newly diagnosed epilepsy and the impact of diagnosis on overall quality of life three years after diagnosis. In addition, the incidence of psychogenic non-epileptic seizures arising from the same original cohort was investigated both at the time of initial data collection and at a three-year follow-up point. Methods: The studied cohort had been previously identified and consisted of patients of all ages (excluding neonates) diagnosed with epilepsy and seizure mimics during 2017 among a population of 542,868 in Cork City and County, using already-published methods. Follow-up involved prospective and retrospective collection of data including phone interviews and interrogation of patient’s charts, clinic letters and electronic hospital databases for up-to-date information on seizure outcomes, quality of life indicators and deaths. Incidence rates for PNES were calculated for the initial time of data collection and at the three-year follow-up point to adjust for any changed diagnoses within the epilepsy and PNES cohorts. Data was collected in a pseudonymised format and stored in an encrypted excel file on a secure storage device. Data was analysed using SPSS software. Results: There were 334 definite and probable patients with newly diagnosed epilepsy defined in the 2017 cohort and 40 diagnosed with psychogenic non-epileptic seizures. At three-years, 75.85% of the epilepsy cohort were taking a single ASM with a large proportion (69.92%) reporting seizure freedom of over one year and a smaller number becoming drug-refractory (9.75%). A minority of participants reported negative impact of an epilepsy diagnosis on social outcomes such as driving, relationships, education, employment and mental health. Seventy-two people (21.5%) had died during the three years since diagnosis with a standardised mortality ratio (SMR) of 2.01. A further 13 had died by five years. Those aged 65 and over represented the age group with the highest number of deaths. The majority of deaths were secondary to infections and malignancy with a small number of direct seizure-related deaths. The initial crude incidence rate of possible PNES cases in the 2017 study was 7.37 per 100, 000 people which was recalculated as 7.55 per 100, 000 people at three years to include changed diagnoses within the PNES and epilepsy cohorts. Conclusion: The outcomes of this study are encouraging and indicate that the majority of newly diagnosed epilepsy patients will have good outcomes, with minimal numbers of injuries, hospitalisations, neuropsychiatric complications or other negative impacts on quality of life and a high level of seizure freedom. However, despite this generally favourable prognosis in epilepsy, premature mortality within the newly diagnosed cohort remains a significant concern, particularly for the already vulnerable older population. The incidence of PNES in this study was higher than previously quoted which may reflect the wide-ranging ascertainment methods, providing a more comprehensive assessment of the true incidence rates in Ireland.
Characterisation of the effect of electroporation and electrochemotherapy on cancer cells and immune cells in the tumour microenvironment
(University College Cork, 2022-10) Bendix, Maura; Brint, Elizabeth K.; Houston, Aileen M.; Amu, Sylvie; Forde, Patrick; Health Research Board; Breakthrough Cancer Research
Lung cancer is the leading cause of cancer-related death worldwide, with the lung cancer incidence rate expected to rise further. Despite recently developed novel therapy options, 5-year survival rates for lung cancer patients remains below 20% generally and below 5% for late-stage diagnosis, thus additional therapy options are still needed. Electrochemotherapy (ECT), the application of an electric pulse to deliver chemotherapy drugs into cells, could be a new treatment option for lung cancer patients. ECT is a locally very effective treatment, with local tumour reduction of up to 85%, while the systemic effects are more varied. For clinical application ECT treatment modalities have been standardized since 2006, after the ESOPE study, which optimized ECT parameters to 8 pulses at 1000V/cm with 100µs pulse length at 1Hz frequency and either bleomycin or cisplatin as the drug of choice. To evaluate whether ECT could be a potential treatment option for lung cancer patients’ ECT parameters, the needed electric field strength and the needed drug and drug concentration, were optimized for in vitro lung cancer research. In our study, we initially developed a standard operating protocol (SOP) to determine the optimal electric field strength for a given cancer cell line in vitro, while keeping the other ESOPE parameters constant. The developed SOP combined short-, medium-, and long-term assays to fully visualize the impact treatment, at a given field strength, has on the tested cancer cell line. This evaluation showed that human lung cancer cell lines (A549, H460 and SK-MES 1) and the human pancreatic Pan02 cell line have an optimal electric field strength of 800V/cm, the melanoma A375 (human) and B16F10 (murine) cell lines as well as the murine pancreatic Mia-PACA2 cell line have an optimal electric field strength of 700V/cm, while the murine Lewis Lung carcinoma (LLC) cell line has an optimal electric field strength of 1300V/cm. In addition, our study findings demonstrate that cisplatin at 11µM would be the drug of choice when using ECT for lung cancer treatments. In recent years while the importance of the immune system in lung cancer development and treatment results has become increasingly clear, little is known about how ECT treatments impact immune cells. Therefore, the impact of ECT on murine T cells, dendritic cells (DCs) and macrophages was evaluated in vitro. Our data indicates that while T cells are able to tolerate electric field strengths of up to 1400V/cm, DCs and macrophages are significantly negatively impacted by electric field strengths exceeding 800V/cm. Further investigation on the impact of ECT on dendritic cells demonstrated that DCs die via necrosis following ECT treatment, while ECT at electric field strength exceeding 1000V/cm leads to DC maturation and activation of the surviving cells. In addition, DCs remain partially functional following ECT treatment in a stimuli and treatment dependent manner with distinctively different sets of genes upregulated 4-hours post treatment at 800V/cm compared to treatment at 1000V/cm and 1300V/cm. Taken together, our data indicates that it is worthwhile to further investigate ECT as a potential therapy option for lung cancer patients, while more attention needs to be paid to the impact ECT has on immune cells in order to maximize treatment results.
A population-based epidemiologic study of adult neuromuscular disease in the Republic of Ireland
(University College Cork, 2014) Lefter, Stela; Ryan, Aisling; Hardiman, Orla
This research project generates for the first time prevalence rates (PR) for all adult neuromuscular diseases (NMD) as well as the incidence rates (IR) for Guillain-Barre syndrome (GBS) in the Republic of Ireland (ROI). Multiple case ascertainment sources were used to achieve as complete as possible case ascertainment to accurately estimate the country-wide prevalence. Acquired demyelinating polyneuropathies represented the biggest cohort (26.0%) followed by muscular dystrophies (22.5%) and myasthenia gravis (19.7%). For GBS, in the 20 year period (1992 to 2012), comparable with other international studies, incidence rates of 0.3-1.3 per 100,000 person-years were attained. Higher PR figures for chronic inflammatory demyelinating polyneuropathy and sporadic inclusion body myositis were found (5.87 per 100,000 and 11.7 per 100,000 in those older than 50 year, respectively) compared with previous studies. The PR for myasthenia gravis in ROI is comparable with countries such as Australia, Italy and Norway but is lower than in Northern Ireland. The PR for Charcot-Marie-Tooth disease and limb girdle muscular dystrophies were similar with United Kingdom PR; however, a definite diagnosis was attained in only one third of Irish patients. Given the fact that paediatric cases were excluded from our study, lower PR figures were obtained for dystophinopathies. Interestingly, the PR for myotonic dystrophy type 1 and facioscapulohumeral muscular dystrophy (FSHD) in the ROI was lower than in United Kingdom but not considerably. A high PR for periodic paralysis (PP) was found in our study, nearly ten times higher for Hyper PP and as twice as high for Hypo PP when compared with figures from England; however, the ROI figures were derived from large families suggesting a founder effect. Of all the inherited NMD, 46% of cases had a definite histopathology and/or genetic diagnosis. The combined PR for inherited and acquired NMD was high, 62.6 per 100,000, when compared with other chronic neurological diseases in ROI. During the course of the study, a definite genetic diagnosis was achieved in families with rare previously unreported NMD in ROI, such as Laing distal myopathy, Andersen Tawil syndrome and FSHD2. This research defines the burden of NMD in the ROI, raises awareness of these conditions, will facilitate earlier intervention where appropriate and will provide a strong argument for service provision for these patients. This research project will serve as a useful originator for future research studies, especially with regard to obtaining genetic confirmation of currently undefined cases.

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