College of Medicine and Health - Masters by Research Theses
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Item The development of a health communication passport for stroke(University College Cork, 2024) O'Leary, Norma; Kelly, Helen; O'Toole, CiaraIntroduction: The Irish National Stroke Strategy (2022-2027) (HSE, 2022) has recommended the introduction of a Stroke Passport. However, the perspectives of stakeholders on the purpose, design, format, and content of a stroke passport are not yet known. Moreover, given that a Stroke Passport can be categorised as a complex intervention, characterised by multiple interacting components and the need to modify established practices, it is crucial to examine current practices related to information provision across the stroke care continuum. Consequently, this study also seeks to investigate the methods currently employed by Healthcare Professionals (HCPs) to provide information to stroke patients and their families or caregivers. Additionally, it aims to identify the barriers and facilitators influencing information provision throughout the stroke care continuum from the perspective of HCPs. Methods: The study took place over two phases. A cross-sectional online survey initially explored the perceptions of HCPs working in stroke care in Ireland and UK. Focus groups interviews were then conducted with HCPs who worked in an acute stroke ward and/or Early Stroke Discharge (ESD) team. Phase one quantitative data was analysed using descriptive and inferential statistics. Qualitative survey data was analysed using content analysis mapped onto the Theoretical Domains Framework (TDF). Phase two interview data were analysed using Braun & Clark’s (2022) six-step Thematic Analysis framework. Results: The survey was completed by 111 HCPs (64% Ireland:36% UK). Wide variation in methods and terminology was noted. Differences across countries was observed relating to opinions about patient and family satisfaction with current information provision. There was consensus that information be provided at multiple care pathway timepoints and be available in both digital and paper formats. Phase two consisted of 22 HCPs who took part in one of the four focus groups (acute stroke ward n=19, ESD n=3). Participants included: Allied Health Professionals (n=10), Nursing staff (n=6), Medical doctors (n=3), Pharmacy (n=1), Psychology (n=1) and Medical Social Work (n=1). Findings suggest that a Stroke Passport, as a central source of information, has the potential to empower patients and family/caregivers. Participants offered suggestions regarding the ideal content, format, and timing of the provision of information. There was a general agreement that a Stroke Passport should be digital rather than paper based. They suggested that issues relating to data protection, longevity of the Stroke Passport, and user buy-in are more challenging with a paper version. Key factors were identified that pose potential obstacles to implementation. This includes a lack of digital infrastructure and the ability of Healthcare Information Systems (HIS) to communicate across systems (interoperability). In addition, the identification of the HCP(s) who would take responsibility for updating the Stroke Passport beyond acute care was considered a necessary component to successful implementation. Conclusion: The provision of a resource such as a Stroke Passport was considered an important asset to patient care. Our findings emphasise the critical role of co-design in its development to better address the complex needs of stroke patients, improving outcomes, satisfaction, and engagement. However, greater attention to addressing current gaps in our stroke healthcare system, particularly digital infrastructure, and interoperability, during the transition from hospital to home is required prior to implementation of a Stroke Passport.Item Engineering therapeutic messenger RNA for triple-negative breast cancer(University College Cork, 2024) Leahy, Mark; Krajewska, Malgorzata; Kowalski, Piotr; O'Connor, RosemaryBackground: Targeted therapies are revolutionising the landscape of cancer treatment, providing better therapeutic responses and minimising the adverse effects of conventional treatments such as radiation and chemotherapy. Human epidermal growth factor receptor 2 (HER2) is overexpressed in multiple cancer types and remains one of the therapeutic flagship targets in oncology. The HER2-targeting monoclonal antibody trastuzumab has dramatically improved the survival of patients with HER2-positive breast cancer. More recently developed antibody-drug conjugates, such as trastuzumab deruxtecan (T-DXd), have demonstrated unprecedented efficacy in HER2-overexpressing cancers. However, only a small subset of patients can benefit from HER2-targeted treatments (15-30% of breast cancers). Triple-negative breast cancer (TNBC) is characterised by the absence of HER2 and hormone receptors. TNBC is an aggressive subset of breast cancers for which few targeted therapies are available. It has recently been shown that artificial overexpression of HER2 could sensitise TNBC xenograft models to trastuzumab, however the clinical relevance of this strategy was limited by the use of viral vectors. Messenger RNA (mRNA) technology has been cemented as a safe and effective therapeutic modality, as evidenced by its success in SARS CoV-2 vaccination. Its potential to revolutionise cancer therapy is now becoming increasingly evident. We hypothesised that mRNA technology could be used to transiently overexpress HER2 in TNBCs to sensitise them to anti-HER2 therapeutics. Methods: Molecular cloning techniques were used to engineer a plasmid vector for in vitro transcription (IVT) of a truncated variant of HER2 (TrHER2), lacking the intracellular domain (ICD) to eliminate the potential for proliferative signalling. Modified nucleosides were incorporated into IVT reactions and mRNA was enzymatically capped and poly(A) tailed. TrHER2 and control (Luciferase and WtHER2) mRNA were delivered to TNBC cells using conventional transfection reagents. TrHER2 protein expression and HER2 signalling were measured by means of western immunoblot. TrHER2 localisation was assessed by immunofluorescent microscopy and flow cytometry. The impact of TrHER2 expression on migration and clonogenicity was measured by scratch and colony formation assays. Trypan blue and CellTiter-Fluor assays were used to assess the impact of TrHER2 expression on the efficacy of T-DXd and trastuzumab. Induction of apoptosis was determined by flow cytometry with Annexin V and propidium iodide stain. The effect of TrHER2 mRNA on trastuzumab function was determined by co-culture of TNBC cells with human immune cells. Results: TrHER2 plasmids were engineered and validated by sanger sequencing. IVT reactions with these plasmids produced pure, high yield TrHER2 mRNA. TrHER2 protein was shown to be approximately 100 kDa, lacking expression of the HER2 ICD. TrHER2 mRNA expression demonstrated cell surface localisation with a >110-fold increase in expression, exceeding that of WtHER2 mRNA (50-fold) at an equal dose. TrHER2 mRNA delivery did not active the HER2 signalling pathway, instead reducing cell migration and clonogenicity compared to WtHER2 mRNA. TrHER2 mRNA expression significantly increased the efficacy of T-DXd, causing an 80% reduction in viability (p<0.0001) by inducing DNA damage and apoptosis. TrHER2 mRNA also sensitised TNBC cells to antibody-dependent cell-based cytotoxicity by trastuzumab, causing an 37% reduction in viability compared to cells transfected with reporter mRNA (p<0.001). Significance: These findings demonstrate proof-of-concept for utilising mRNA-based overexpression as a strategy to enhance the sensitivity of TNBC cells to anti-HER2 therapies. Additionally, the results validate the safety profile of the engineered TrHER2 mRNA. The observed high efficacy suggests promising in vivo effectiveness. When paired with a suitable delivery system, this approach could have the potential for clinical translation.Item Socioeconomic deprivation as a risk factor for stillbirth: a case-control study(University College Cork, 2024) Keane, Jessica; Leitao, Sara; Corcoran, Paul; Greene, Richard A.Introduction: Stillbirth (SB) is a devastating outcome for families. Identifying and addressing risk factors is of crucial importance. Level of deprivation has been linked to adverse perinatal outcomes. This thesis aims to comprehensively examine the relationship between area-level deprivation and the risk of SB by conducting a scoping review of international literature and a case-control study using an Irish based cohort. Methods: Guided by the Joanna Briggs Institute methodology and PRISMA-ScR framework, this scoping review focused on studies examining SB in upper-middle and high-income countries using composite geographical indices of deprivation. A systematic search across six scientific databases was performed. The screening process, involving title, abstract, and full text reviews, followed specified inclusion criteria. Data extraction and synthesis were carried out and presented in a narrative summary. An observational case-control study was conducted, matching cases of SB (n=127) with a control cohort of live births (n=266). A ratio of 2 controls to 1 case was identified. Retrospective data over four years (2018-2021) was collected from a tertiary university maternity unit in the Republic of Ireland. The study employed the Pobal HP Deprivation Index to categorise small areas into levels of deprivation, ranging from affluence to deprivation. Variables, including maternal age, parity, BMI, booking visit gestation were identified as confounding factors. Statistical analysis was conducted using SPSS and included; frequency tables to present the distribution of certain variables, Chi-squared tests to identify associations between deprivation and the confounding factors with risk of SB, T-tests and logistic regression for crude and multivariate analysis on the relevant confounding factors, including odds ratio calculations, to identify any significant differences in the risk of SB across the deprivation levels and categories of the confounding factors. Results: A total of 29 studies were included in the scoping review, from 9 countries (the majority UK-based; n=20, followed by the Netherlands; n=2 and Brazil; n=2). A range of deprivation indices were utilised internationally (n=18), the UK’s Index of Multiple Deprivation (IMD) was the most commonly used (n=8), followed by the Townsend and Jarman indices (n=6 and n=3, respectively). Income, employment, education and access to services were some of the most common factors included in the indices. Results show an association between SB rates and areas identified as being more deprived, with 22 of the 29 studies (75.9%) showing positive correlations. The results of the case-control study demonstrated no statistically significant correlation between level of deprivation and risk of experiencing SB within our sample (p-value 0.288). When readjusted into quintiles of deprivation, a slightly higher representation of SB was noted in the more deprived levels, though not significant. When examined by cause of death, there was a significant association between deprivation and placental causes of death (p-value 0.045). BMI was consistently associated with risk of SB, while booking visit gestation and maternal age also showed associations. Conclusion: This comprehensive investigation into the relationship between area-level deprivation and SB risk provides important insights. The complex nature of measuring deprivation and of SB aetiology is demonstrated. The scoping review suggests that countries and regions should consider assessing deprivation when evaluating their SB rates, as this could offer valuable information for care programme development to impact deprived areas. While the case control study demonstrates the need for further national research to understand the relationship between deprivation and adverse perinatal outcome in the Irish context. Ultimately, using the evidence to guide and inform local policy and resource management will aid in tackling the inequalities in healthcare provision, thus encouraging equal and quality maternity care. Tailored initiatives have the potential to enhance healthcare services, clinical practice, and ultimately improve maternal and perinatal outcomes.Item Development of an end-to-end monoclonal antibody production process(University College Cork, 2024) Pradeep, Rithwik; Allen, Evin; Tangney, MarkMonoclonal antibodies (mAbs) represent the dominant modality of biological therapeutics currently marketed. The manufacture of monoclonal antibodies consists of a series of processing steps including upstream cell culture, downstream purification and formulation and a final step of drug product manufacture. In this thesis, a small-scale end-to-end production process was developed for two monoclonal antibodies namely an anti-TNF Alpha mAb and cNISTmAb whose cell lines were sourced from commercial and public bodies respectively. A cell culture process for both cell lines was successfully developed including the creation of working cell banks (WCB). Titers were optimised, for both cell lines through a series of media, bioreactor and cell density modifications. In addition, the critical quality attributes of potency and purity were assessed via cell-based assays and SDS-PAGE respectively, that were designed and developed as part of this thesis. The potency of anti-TNF Alpha mAbs produced was assessed through two distinct in-vitro cell-based assays developed using HEK and L929 reporter cell lines and determined to be functional and more potent than a commercially available anti-TNF Alpha antibody The stability of cNISTmAb was also analysed across a range of temperature conditions from -20°C to 40°C after 50 days and also post-lyophilisation, with stability maintained at room temperature. Additional modifications of cNISTmAb were performed in an attempt to yield antibody fragments through pepsin digestion with partial success through pepsin digestion to yield F(ab’)2 fragments. This work demonstrates the successful production of two monoclonal antibodies and fragments but also sets the stage for further process improvement and enhanced characterisation to fully define and analyse an endto-end monoclonal antibody production systemItem Neurobiological effects of food fermentation-derived metabolites for metabolic and mental health(University College Cork, 2023) Carey, Nathan; Schellekens, Harriet; O'Mahony, Siobhain M.Nutrition and diet are becoming increasingly popular therapeutic interventions as we discover more about the complex roles the foods we consume play in maintaining our health status. It is now clear that foods we ingest daily and their metabolites interact with systems both within and outside the gastrointestinal tract including the gut microbiome, the nervous system, immune system and hormonal system. Each of these play essential roles in the bi-directional communication pathway of the microbiota-gut-brain axis. Interactions between our food and this axis can potentially influence centrally mediated processes such as cognition, mood and even appetite. While several foods have been identified as being beneficial to our health, there is one food group that remains under investigated and holds promise as a reservoir of both beneficial bacteria and bioactive compounds – fermented foods. Fermented foods are created through the controlled enzymatic conversion of foods to simpler organics substances by microorganisms. Common examples include foods like kimchi (a fermented cabbage product), kefir (a fermented milk product) and kombucha (a fermented sweet tea beverage). While human studies on fermented foods remain sparce, one recent finding recorded lower perceived stress in human adults who underwent a dietary intervention that included fermented food intake. Recent findings in rodents suggest that fermented foods can alter social behaviour, reduce body weight and lead to reduced anxiety in animals. The mechanism by which fermented foods act is still unknown but it likely due to a number of factors such as their probiotic bacteria content, their metabolite content including short chain fatty acids (SCFA), and the ability to breakdown their starter compounds into simpler molecules and increase their bioavailability such as phenolic compounds in fruits and vegetables. The research conducted in this thesis aims to investigate the ability of food-fermentation derived metabolites, with a specific focus on SCFAs and polyphenols, to alter the neurobiological functions associated with central appetite regulation (hypothalamus) and cognition (hippocampus). Using in vitro assays, we tested the selected panel of metabolites shown to be found in fermented foods, and capable of crossing the blood brain barrier, on both immortal cell lines (hypothalamic and hippocampal) and on primary neurosphere cultures (hippocampal). A panel of SCFA were administered to hippocampal and hypothalamic cell lines and were capable of altering brain-derived neurotrophic factor (BDNF) gene expression. These metabolites were also tested on primary hippocampal cells using a neurosphere assay of proliferation. Positive trends were observed across many of the treatments, however these trends were not significant. Perhaps most interesting were our findings when submitting the same hippocampal neurosphere assay to a panel of phenolic compounds. Apigenin and kaempferol (both flavonoids) significantly increased hippocampal cell proliferation. Moreover, to enhance the efficiency of data analysis, a semi-automatic quantification pipeline was developed for high-throughput screening of primary neurosphere cultures. This pipeline offers a systematic and reliable method for evaluating neurosphere proliferation, providing a valuable tool for future studies in the field. Our results highlight the modulatory effects of SCFA and phenolic compounds on hypothalamic and hippocampal cells in vitro, emphasising the potential role of dietary metabolites and fermented foods as a whole on brain function related to metabolic and mental health. These findings also highlight the need for more in depth analysis of fermented foods and their neuromodulator effects both in vivo.