Assessment of the immunomodulatory effects of raw/farm milk and probiotic bacteria

The overall aims of this study were to investigate the differences between raw/farm milk and pasteurised milk with respect to potential immune modifying effects following consumption and investigate the bacterial composition of raw milk compared to pasteurised milk. Furthermore, in this thesis, panels of potential probiotic bacteria from the Bifidobacterium and Lactobacillus genera were investigated. The overall bacterial composition of raw milk was compared with pasteurised milk using samples obtained from commercial milk producers around Ireland using next generation sequencing technology (454 pyrosequencing). Here the presence of previously unrecognised and diverse bacterial populations in unpasteurised cow’s milk was identified. Futhermore the bacterial content of pasteurised milk was found to be more diverse than previously thought. The global response of the adenocarcinoma cell line HT-29 to raw milk and pasteurised milk exposures were also characterised using whole genome microarray technology. Over one thousand differentially expressed genes were identified which were found to be involved in a plethora of cellular functions. Interestingly a reduction in immune related activity (e.g. Major histocompatability complex class II signalling and T and B cell proliferation) was identified in cells exposed to pasteurised milk compared with raw milk exposures. Further studies comparing human cell response to raw versus pasteurised milk was performed using peripheral blood mononuclear cells (PBMC) from healthy donors. A reduction in CD14 was identified following raw milk exposures compared with pasteurised milk and the pattern of cytokine production may indicate that gram positive bacteria in the raw milk were contributing to the differences in the cellular response to raw versus pasteurised milk. Panels of potentially probiotic bacteria (comprising of lactobacilli and bifidobacteria) were further assessed for immunomodulatory capabilities using cell culture based models. Gene expression and cytokine production were used to evaluate stimulated and unstimulated (LPS) cellular responses as well as interaction mechanisms

The ELDERMET biobank: Isolation and characterization of the intestinal microbiota from elderly Irish subjects

The human gastrointestinal (GI) tract is colonized by a dense and diverse bacterial community, the commensal microbiota, which plays an important role in the overall health of individuals. This microbiota is relatively stable throughout adult life, but may fluctuate over time with aging and disease. The adaptation of the gut microbiota to our changing life-style is probably the reason for the large inter-individual variation observed among different people. Since the gut microbiota plays an essential role in interactions with host metabolism, it is of utmost importance to explore this relationship. The elderly intestinal microbiota has been the subject of a number of studies in recent years. The results presented in this thesis have further contributed to the expansion of knowledge related to gut microbiota research highlighting the combined effect of culture based and molecular methods as powerful tools for understanding the true impact of microbes. The degree of correlation between measurements from both methods suggested that a single method is capable of profiling intestinal Bifidobacterium spp., Lactobacillus spp. and Enterobacteriaceae populations. Bacteriocins have shown great promise as alternatives to traditional antibiotics. In this respect, the isolation and characterisation of bacteriocinogenic strains are important due to growing evidence indicating bacteriocin production as a potential probiotic trait by virtue of strain dominance and/or pathogen inhibition in the mammalian intestine. The selection pressure applied on the bacterial population during antibiotic usage is the driving force for the emergence of antibiotic resistant bacteria. Identification of antibiotic resistant isolates opens up the possibility of using such probiotics to offset the problems caused by antibiotics to the gut microbiota and to improve the intestinal microbial environment. Future work is required to explore the culture collection housing thousands of bacterial isolates as a valuable source of potential probiotics for use for the elderly Irish community.

Continuous non-destructive monitoring of cell health using impedance based interdigitated electrode structured sensors

This article examines some preliminary tests which were performed in order to evaluate the best electrode configuration (width and spacing) for cell culture analyses. Biochips packaged with indium tin oxide (ITO) interdigitated electrodes (IDEs) were used to perform impedance measurements on A549 cells cultured on the surface of the biochip. Several tests were carried out using a 10 mM solution of Sodium Chloride (NaCl), cell medium and the cell culture itself to characterize some of the configurations already fabricated in the facilities at Tyndall National Institute.

Pretty good governance: balancing policy drivers and stakeholder interests in developing fisheries ecosystem plans

The central research question that this thesis addresses is whether there is a significant gap between fishery stakeholder values and the principles and policy goals implicit in an Ecosystem Approach to Fisheries Management (EAFM). The implications of such a gap for fisheries governance are explored. Furthermore an assessment is made of what may be practically achievable in the implementation of an EAFM in fisheries in general and in a case study fishery in particular. The research was mainly focused on a particular case study, the Celtic Sea Herring fishery and its management committee, the Celtic Sea Herring Management Advisory Committee (CSHMAC). The Celtic Sea Herring fishery exhibits many aspects of an EAFM and the fish stock has successfully recovered to healthy levels in the past 5 years. However there are increasing levels of governance related conflict within the fishery which threaten the future sustainability of the stock. Previous research on EAFM governance has tended to focus either on higher levels of EAFM governance or on individual behaviour but very little research has attempted to link the two spheres or explore the relationship between them. Two main themes within this study aimed to address this gap. The first was what role governance could play in facilitating EAFM implementation. The second theme concerned the degree of convergence between high-level EAFM goals and stakeholder values. The first method applied was governance benchmarking to analyse systemic risks to EAFM implementation. This found that there are no real EU or national level policies which provide stakeholders or managers with clear targets for EAFM implementation. The second method applied was the use of cognitive mapping to explore stakeholders understandings of the main ecological, economic and institutional driving forces in the Celtic Sea Herring fishery. The main finding from this was that a long-term outlook can and has been incentivised through a combination of policy drivers and participatory management. However the fundamental principle of EAFM, accounting for ecosystem linkages rather than target stocks was not reflected in stakeholders cognitive maps. This was confirmed in a prioritisation of stakeholders management priorities using Analytic Hierarchy Process which found that the overriding concern is for protection of target stock status but that wider ecosystem health was not a priority for most management participants. The conclusion reached is that moving to sustainable fisheries may be a more complex process than envisioned in much of the literature and may consist of two phases. The first phase is a transition to a long-term but still target stock focused approach. This achievable transition is mainly a strategic change, which can be incentivised by policies and supported by stakeholders. In the Celtic Sea Herring fishery, and an increasing number of global and European fisheries, such transitions have contributed to successful stock recoveries. The second phase however, implementation of an ecosystem approach, may present a greater challenge in terms of governability, as this research highlights some fundamental conflicts between stakeholder perceptions and values and those inherent in an EAFM. This phase may involve the setting aside of fish for non-valued ecosystem elements and will require either a pronounced mind-set and value change or some strong top-down policy incentives in order to succeed. Fisheries governance frameworks will need to carefully explore the most effective balance between such endogenous and exogenous solutions. This finding of low prioritisation of wider ecosystem elements has implications for rights based management within an ecosystem approach, regardless of whether those rights are individual or collective.

Supporting therapist engagement in evidence based practice: Developing a continuing education programme through participatory action research

Therapists find it challenging to integrate research evidence into their clinical decision-making because it may involve modifying their existing practices. Although continuing education (CE) programmes for evidence-based practice (EBP) have employed various approaches to increase individual practitioner’s knowledge and skills, these have been shown to have little impact in changing customary behaviours. To date, there has been little attempt to actively engage therapists as collaborators in developing educational processes concerning EBP. The researcher collaborated with seven clinical therapists (one occupational therapist, four physiotherapists and two speech and language therapists) enrolled in a new post-qualification Implementing Evidence in Therapy Practice (IETP) MSc module to monitor and adapt the learning programme over ten weeks. The participating therapists actively engaged in participatory action research (PAR) iterative cycles of reflecting→ planning→ acting→ observing→ reflecting with the researcher. Mixed methods were used to evaluate the IETP module and its influence on therapists’ subsequent engagement in EBP activities. Data were gathered immediately on completion of the module and five months later. Immediate post-module findings revealed four components as being important to the therapists: 1) characteristics of the learning environment; 2) acquisition of relevant EBP skills; 3) nature of the learning process; and 4) acquiring confidence. The two themes and sub-themes which emerged from individual interviews conducted five months post-module expanded on the four components already identified. Theme 1: Experiencing the learning (sub-themes: module organisation; learning is relational; improving the module); and theme 2: Enacting the learning through a new way of being (sub-themes: criticality and reflection; self agency; modelling EBP behaviours; positioning self in an EB work culture). The therapists’ perspectives had by then shifted from that of a learner to that of a clinician constructing a new sense of self as an evidence-based practitioner. Findings from this study underline the importance of the process of socially constructed knowledge and of empowering learners through collaboratively designed continuing education programmes. In the student-driven learning environment, therapists chose repetitive skill-building and authentic problem-solving activities which reflected the complexity of the environments to which they were expected to transfer their learning. These findings have implications for educators designing EBP continuing education programmes, during which students develop professional ways of being.

Ex vivo culture of patient tissue and examination of gene delivery

Gene therapy has emerged as a realistic prospect for the treatment of cancer due to its potential for selective tumour cell targeting. The greatest challenge gene delivery vectors face is the ability to safely and efficiently deliver genes into target cells. The overall objectives of this thesis are to evaluate the efficacy of various gene delivery methods in a clinically relevant tumour model and to also investigate potential strategies for tumour selective delivery. We began with the development of a tumour slice model system using patient waste tissue. This model involves the use of fresh human tumour tissue, cut into thin slices and maintained ex vivo and is universally applicable to gene delivery methods, using a real-time luminescence detection method to assess gene delivery. The nature of the ex vivo culture system permitted examination of specific physiological variables, the influence of intratumoural factors and tissue specific effects on vector expression. Adenoviral vectors under the control of the human CXCR4 promoter demonstrated a 'tumour on' and 'normal off' expression profile when compared with the ubiquitously active CMV promoter when tested in patient tumour tissue. In addition, we developed an ex vivo system of changing oxygenation using the hypoxia inducer, cobalt, to mimic the transient hypoxic conditions found in solid tumours. We found that Adenoviral transgene expression was robust in the cycling hypoxic conditions relevant to solid tumours and re-oxygenation of chronically hypoxic tissue enhanced transgene expression. Finally, we demonstrated an AAV-based tumour targeting strategy using a tumour-selective promoter allowing for the efficient targeting of AAV vectors to cancer cells and the sparing of normal tissue in both murine metastatic liver tumours models and patient tissue. The thesis highlights the importance of indepth preclinical assessment of novel therapeutics and may serve as a platform for further testing of novel gene delivery approaches.