Edited Abstract: Mechanotransduction is the process by which cells convert mechanical stimuli into an adaptive gene- and protein-level response, via signalling cascades or direct physical effects of the cytoskeleton on the nucleus, and appropriate mechanosignalling is crucial for tissue development and function. Most techniques currently used to study cellular mechanoresponses are relatively damaging to the cells. In contrast, topographically structured substrates, such as microgrooves, have great potential for use as non-invasive mechanostimuli. In this study, quartz microgrooved substrata (2 μm depth x 25 μm pitch) were used as platforms for the confinement and alignment of cells. A multi-layered approach was adopted to begin to integrate the changes induced by the topographical mechanostimulus at the chromosome, small RNA, transcript, protein and structural levels. Together, the results provide insight into multiple facets of topography-induced mechanotransduction, which should contribute to understanding of mechanotransduction and cell-material interactions.

Background: Acceptance and Commitment Therapy (ACT) aims to help individuals live a life congruent to their values by cultivating psychological flexibility (PF); the ability to respond to experiences with acceptance and creativity. Concurrently, Compassion Focused Therapy (CFT) addresses the role of self-attacking cognitions on psychological difficulties. Recent work suggests that integrating aspects of CFT into an ACT approach (i.e. developing a person’s PF to self-attacking thoughts through self-compassion) may offer additional therapeutic value. There remains no assessment of this specific therapeutic process. Aims: The project aimed to develop and validate a new scale to assess flexibility of responses to self-critical thoughts (FoReST). Methods: Factor Analysis was used to explore factor structure of the FoReST in a convenience sample of 253 adults. Construct validity was explored by comparing FoReST with measures of similar constructs (PF, self-compassion, self-criticism) and potentially related outcomes (anxiety, depression, quality of life). Findings: Alternative 2-factor (‘unworkable action’ and ‘avoidance’) and 1-factor (‘unworkable action’) versions of the FoReST showed high concurrent validity with similar measures, good predictive validity for mental health and wellbeing outcomes and good internal consistency. The relative strengths and weaknesses of both versions are discussed. Recommendations: Findings indicate that the FoReST may offer a useful clinical and research tool for emerging forms of ACT for people high in self-criticism. Future research will be required to confirm the factor structure of the FoReST, confirm concurrent, predictive validity, test-retest reliability, and validate the scale in relevant clinical populations.

Nerve repair is still a major challenge in surgery, regenerative medicine and tissue engineering even if progress has been made over the last 30 years. Functional recovery after severe lesions to a nerve is often incomplete and rarely totally successful. In this thesis I present a multi-disciplinary approach to improve the regenerative potential of “nerve repair tubes” that aim to reconnect wounded nerves and refine or replace autologous nerve graft, the clinical current gold standard. The efficacy of such tubes has already been shown in the clinic especially for small gap injuries, but the outcomes are still limited, and ought to be improved by e.g. micro/nano-topography, growth factor delivery systems, supportive cells or active features such as electrical stimulation, which have individually been shown to enhance nerve regeneration. In this study organotypic cultures of dorsal root ganglions (DRG) isolated from neonatal rats were used throughout as an in vitro model of nerve regeneration. Here I tested different devices in combination with growth factors to contribute to the fundamental and technical knowledge necessary to improve the regenerative potential of such tubes. I investigated the interaction between surface features and growth factors in their joined influence on regenerating DRGs. For this polydimethylsiloxane (PDMS), a polymer with adjustable elasticity was used together with photolithography to build devices of different stiffness with different surface microgrooves, on which DRG could be grown. To optimise the use of nerve growth factor (NGF) in conjunction with these devices, and to show how NGF interacts with stiffness and topography the reaction of the DRG was tested. To ease the making of three-dimensional internally microstructured tubes I have developed up a novel, timesaving, fabrication technique for polycaprolactone (PCL) “Swiss roll” nerve repair tubes. This technique improves the reproducibility of the scaffold, and using DRG its potential for nerve regeneration is being demonstrated. The influence of time-variant, balanced, pulsed electric stimulation is a potentially useful means to influence nerve regeneration. To narrow down the parameter space the effect of various electric fields was tested in their effect on DRG regeneration using commercially available devices. In collaboration with Christopher Martin from the School of Engineering, novel custom-made devices that allowed us to quantify the directional response of the regenerating axons were developed, and the guidance effect of pulsed alternating current (AC) electrical fields on regenerating DRGs axons was investigated in vitro. This approach allows in principle to transfer the use of this nerve guiding strategy to potentially improve nerve repair tubes.

Normal mechanical loading potently induces bone formation mediated by osteocyte effects on osteoblasts. Current in vitro bone models do not reflect these cellular interactions, either focusing on mechanical loading of osteoblasts in monolayers or in 3D and therefore not elucidating the osteocyte-osteoblast interactions that regulate mechanically-induced bone formation. Adenosine, calcium-sensing and glutamate signalling have been shown to influence bone biology, with both adenosine precursors and glutamate having been implicated in mechanotransduction. The aims were to develop a novel in vitro 3D co-culture model of bone to investigate mechanically-induced signalling, and to determine the expression of adenosine, calcium-sensing and glutamate signalling components within the 3D model and their contribution to the regulation of mechanically-induced bone formation markers. A 3D model was developed as a two-phase culture system where MLO-Y4 osteocytes were embedded within type I collagen gels and MC3T3-E1osteoblasts were layered on top. In this model, cells were viable over 7 days (100 % osteoblasts, 87 % osteocytes), maintained appropriate morphology and contacted neighbouring cells through CX43 labelled projections. RT-qPCR revealed Runx2, OCN and E11 mRNA expression in both osteoblasts and osteocytes. COL1A1 mRNA expression was significantly higher in the osteoblasts (P=0.0001), whereas ALP mRNA was higher in the osteocytes (P=0.001). RT-PCR revealed expression of adenosine receptors A2A and A2B and glutamate transporter GLAST1 in osteoblasts and osteocytes, as well as glutamate receptors AMPAR2 and KA1 in osteocytes. Immunostaining confirmed expression of A2A, GLAST1 and KA1, and revealed expression of CaSR, in both osteoblasts and osteocytes. A novel mechanical loading device was developed which was used to apply osteogenic loads (5 min, 10 Hz, 2.5 N) to 3D osteocyte mono-cultures and 3D osteocyte-osteoblast co-cultures. A minimum of 48 hr pre-load time was required for a reliable load response. 3D osteocyte mono-cultures cultured for 48-72 hr or 7 days pre-load, remained viable, significantly increased PGE2 0.5 hr after load (48-72 hr: P=0.0249, 7 days: P=0.041) and decreased their IL-6 synthesis. RT-qPCR revealed a load-induced decrease in E11 (P=0.018) and RANKL (P=0.0486) mRNA, in 48-72 hr cultures. In 7 day cultures, E11 mRNA (P=0.041) increased as a result of loading. Preliminary data showed that the same loading conditions increased PINP synthesis, a bone formation marker, in 3D co-cultures (P=0.022). The AMPA/KA receptors antagonist NBQX increased PINP synthesis by 2-fold over 5 days, similar levels induced by loading in untreated cultures, suggesting that NBQX has similar anabolic effects as mechanical stimuli. Similarly, the A2A receptor antagonist SCH 442416 increased osteoblast ALP mRNA expression by 3.5-fold at day 1 post-load and increased PINP synthesis by 1.9-fold, in co-cultures after 5 days. This 3D osteocyte-osteoblast co-culture model represents a useful in vitro model for the investigation of the osteocyte-osteoblast interactions that lead to mechanically-induced signalling and regulation of bone formation markers. Adenosine, calcium-sensing and glutamate signalling components are expressed within the model, facilitating future investigations of their roles in mechanically-induced signalling. Preliminary experiments indicated that adenosine and glutamate signalling may each contribute individually to the regulation of mechanically-induced bone formation markers.

Midwifery practice in Ireland has emerged from a system of care dominated by the biomedical model of childbirth. The aim of this study was to explore the experiences of labour ward midwives who are potentially complicit with this approach. This study reveals how midwives’ environment impacts on their construction of childbirth. The opening of a large new maternity hospital afforded the opportunity to see if the move to this setting would influence midwifery practice. A hermeneutic phenomenological approach was used based on the work of Heidegger and Gadamer. The study was undertaken in two phases, the first involved interviewing six labour ward midwives working in a busy obstetric led labour ward which was due to close. The findings revealed that midwives complied with the norms for the unit and did not take responsibility for the biomedical approach to care. The second phase was undertaken twelve months after the opening of the hospital which was an amalgamation of three maternity units. Seventeen midwives were interviewed for this phase of the study. The move to the larger unit revealed a paradox for midwifery autonomy and enabled midwives to practice in new ways. The maternity service was delivered through a system that values detachment and an attempt at equal (not individualised) care under conditions of limited resources and constraints. This had resonance with Lipsky’s and Foucault’s work. A contrasting situation occurred within the individual labour rooms as the midwives worked in relative isolation, away from the general activity of the unit. This phase of data was framed in terms of Merleau-Ponty’s four existentials. Midwives had opportunities to enact ‘real midwifery’ and normalise birth for women using a range of strategies rather than resorting to interventionist therapies. Midwives shared in the joy of achievement when positive births occurred. The paradox of this mega maternity unit enabled authentic midwifery to emerge. The study provides an insight into the experience of labour ward midwives and how midwifery identities are revealed by the narratives they relate. It also highlights the complexity of contemporary maternity care in large centralised maternity units.

Tobacco use is the leading preventable cause of death worldwide. In order to address this epidemic, it is important that we have a thorough understanding of the aetiology of tobacco use and dependence. Twin and adoption studies have consistently demonstrated the importance of genetic factors in smoking behaviours. The advent of genome-wide technologies has greatly facilitated the search to determine which specific genetic factors contribute to tobacco use phenotypes. A locus within the nicotinic acetylcholine receptor gene cluster CHRNA5-A3-B4 has generated particular interest – that marked by variants rs16969968 in CHRNA5 and rs1051730 in CHRNA3. The primary aim of this thesis was to determine the role played by this locus in smoking-related behaviours, with an emphasis on phenotype refinement. A number of different approaches were utilised to address this objective, namely systematic review and meta-analysis, genetic epidemiology (including detailed phenotyping of smoking behaviour in adolescence), laboratory-based techniques, and genome-wide meta-analysis. Compelling evidence for a small, robust association was observed between the rs1051730/rs16966968 variants and daily cigarette consumption, equivalent to a per allele effect of approximately one cigarette per day. This effect was consistent across population sub-groups. Compelling evidence for an association between this locus and level of tobacco exposure was further illustrated through genome-wide meta-analysis of cotinine levels in current smokers. No association was observed between this locus and smoking initiation however, as examined in a prospectively assessed cohort using precisely defined phenotypes. An association between rs1051730/rs16969968 and smoking topography has yet to be explored. However, a full protocol was developed and piloted to investigate this. In addition, this research has also illustrated the importance of precise, objective, phenotype definition, an observation which has important implications for the fields of molecular genetics and epidemiology.

A potential benefit of the use of artificial media for the suspension of platelets as concentrates is a reduction of the morphological, functional and metabolic changes observed in platelets during storage and collectively referred to as the platelet storage lesion (PSL). A better understanding of the nature of the PSL may suggest strategies for manipulation of the storage environment to improve platelet viability and efficacy posttransfusion. In this context, two principal considerations formed the basis for the study: · The hypothesis that apoptosis is a central mechanism responsible for the changes observed in the PSL. · The investigation of this hypothesis within the applied setting of improving the storage environment of platelet concentrates. The study investigated the role on the PSL of plasma protein (in the form of albumin), acetate and glucose in leucoreduced platelet concentrates suspended in a medium with minimal plasma. A 14-day storage study on platelet concentrates in either plasma or a 70:30 ratio of a commercial additive solution (SSP+Ô) and plasma provided an overview of platelet in vitro characteristics under standard storage conditions. The work led to targeted investigations into the nature of the cell death mechanism in platelet concentrates. Results suggested that in storage media with adequate energy stores, a Bcl-2 proteinmediated mechanism of cell death was viable, though possibly storage-time dependent and limited by pre-existing levels of anti-apoptotic Bcl-2 proteins in the platelets. Further studies would be required to determine if this mechanism is akin to caspasedependent apoptosis. In media lacking glucose, a mechanism more reminiscent of necrosis was observed, associated with decreased ATP levels, accelerated mitochondrial dysfunction, elevated intracellular free calcium and culminating in platelet disruption.

Effective immunity is fundamental to life on a dirty planet. Appropriate immune responses control infections and protect against cancer. Inappropriate immune responses lead to autoimmunity and allergy. A fine balance between aggression and tolerance is therefore central to effective immune function at the system level. This is a particular problem for T cells, which recognize peptide antigens bound to host major histocompatibility complex (MHC) molecules. Faced with a composite antigenic structure, the distinction between “foreign and dangerous” and “self and harmless” becomes both difficult and imperative, especially when the antigen persists. In this thesis, antigen-specific T cell responses were investigated under conditions of chronic antigenic stimulation to inform our understanding of this process. In T cell receptor transgenic mice, continuous antigenic stimulation without adjuvant lead to increased in vivo turnover of antigen-specific CD4+ T cells but “aborted” immune activation, characterized by depletion of these cells from the circulation and spleen. Full immune activation and expansion of antigen-specific memory/effector CD4+ T cells required the presence of adjuvant, in this case IL-1β, which induces an inflammatory environment. Further isotope labelling studies in human immunodeficiency virus-infected subjects suggested that the surface marker CD57 demarcates a “steady state” within the CD8+ T cell memory compartment, whereby CD57+ cells have lower in vivo turnover rates compared to their CD57- counterparts. These observations provide a potential mechanistic explanation for the preferential accumulation of CD57+CD8+ cells under conditions of chronic antigenic stimulation. Another persistent pathogen, cytomegalovirus (CMV), expresses a viral interleukin (IL)-10 homologue. Memory T cell inflation and antiviral cytokine production in murine CMV(MCMV)-infected mice were suppressed by IL-10. Conversely, IL-10 blockade or deficiency lead to the inflation of certain antigenspecific T cell populations and reduced viral load, most likely as a consequence of the enhanced immune response. Reactivation of human CMV was also apparent in subjects with dasatinib-associated large granular lymphocyte expansions. Consistent with a causative association, the expanded T cell and NK cell populations in these subjects were oligoclonal and exhibited a late differentiated (CD27-CD57+) phenotype, indicative of chronic antigenic stimulation. In addition, CD8high and CD8low T cells were observed within both the total and CMV-specific CD8+ T cell compartments, consistent with CMVdriven activation. In summary, these data show that antigen alone is not sufficient to induce full immune activation, even under conditions of chronic stimulation. Additional signals, such as those provided by an inflammatory environment, are required to trigger full T cell activation and expansion. Persistent viruses attempt to undermine this process, for example by the expression of homologues that mimic host immune regulators. Even in the presence of viral reactivation and immune system perturbations, however, the T cell compartment can demonstrate remarkable resilience in its ability to generate fully differentiated and functional expansions. The persistence of certain memory T cell subsets under such conditions appears to play an important role in the immune response to chronic “dangerous” antigens.

Chronic conditions have an impact on the quality of life (QoL) of families as well as patients themselves, and the two are often linked; the greater the effect on the patient, the more the QoL of the family members is reduced. Research into family QoL exists in several medical specialties, but studies have usually been focused on carers or families of patients with one specific disease. Currently, there is no generic instrument that can be used to measure the impact of illnesses on the partner or family members of patients. This study describes the development of the Family Reported Outcome Measure (FROM-16)©. The aims of this study were to investigate the impact of disease on family members of patients over a wide range of specialties, identify key impact areas and develop a generic family quality of life measure. Semi-structured interviews were carried out with 133 family members of patients from 26 medical specialties. Family members were invited to discuss all the areas of their lives that had been affected by having an unwell relative. Thematic analysis was carried out using NVivo9© software. A preliminary 31-item measure was developed from the content of the interviews with family members. Content validity was assessed using qualitative and quantitative data from expert panels involving clinicians and family members. A separate cohort of 240 family members was recruited for both Rasch analysis and factor analysis to reduce items. A further 120 family members completed the final version of the FROM-16 for full psychometric testing including construct validity and reliability. Most family members interviewed were female (61%), the partner or spouse of the patient (56%) or the parent (22%). The mean age was 56.1 years (range= 21-85) and the mean duration of the patient’s disease was 8.9 years (range= one month to 60 years). 10 key themes of family quality of life were identified from interviews. The median number of themes reported by family members was 6 (range= 1-10). The key themes included: emotional impact (mentioned by 92% of subjects), daily activities (91%), family relationships (69%), sleep and health (67%), holidays (62%), support and medical care (61%), work and study (52%), financial impact (51%), social life (37%), and time planning (14%). Relationships between the themes were identified. A 31-item generic family quality of life instrument, the Family Reported Outcome Measure (FROM)©, with a 5-point Likert response scale was developed. The content validity panel's ratings of each item on a 4-point scale for the four attributes showed either "strongly agreed" or "agreed" (88%), with an ICC value of 0.98 (CI=0.97-0.99) suggesting a high agreement between the panel members' responses. Collapsing response categories, removing misfitting items and combining items with residual correlations produced a good fit to the Rasch model (n=240, Total χ2 = 56.6, df = 48, p = III 0.18). Factor analysis produced a 16-item measure with two factors. The FROM showed high internal consistency (n=120, Cronbach’s α= 0.91), high reproducibility (n=51, ICC=0.93) and a mean completion time of two minutes. Construct validity was proven through the correlation between the FROM and the WHOQOL-BREF total scores (n=119, r=-0.55, p<0.001), and the correlation between the FROM and the patient’s overall health score (n=120, r=-0.51, p<0.001). This large scale multi-specialty study has demonstrated the great, yet similar impact that illness can have on the quality of life of family members of patients. Family quality of life is a previously neglected area of healthcare which needs to be addressed in order to provide better support for the patient and for the family unit. The FROM is both reliable and valid for use in family members of patients. It has a potential for wide use, including clinical (all medical specialties), industrial and social sciences.

Diabetes mellitus (DM) is a major health problem currently affecting over 225 million people worldwide. It is often described as a major metabolic disorder, which can result in numerous long-term complications including retinopathy, nephropathy, neuropathy and cardiomyopathy. DM is due to a deficiency of insulin or insulin resistance. Of the 225 million diabetic patients, around 5-10% suffer from type 1 DM (T1DM) and the remaining 90-95% suffer from type 2 diabetes (T2DM). T1DM is due to insulin deficiency whereas T2DM is due to either a reduction in insulin secretion or insulin resistance. Patients with both T1DM and T2DM normally require insulin and hypoglycaemic drugs, respectively. Changes in life style habits, regular exercise and healthy diets can also help to control blood glucose in T2DM patients. Mediators that can help to increase the health of pancreatic islets to synthesize and secrete insulin will be of tremendous benefit to diabetic patients. This study investigated the beneficial effects of incretins, substances such as glucagon-like peptide-1 (GLP-1) and its synthetic agonist, exenatide on the diabetic rat pancreas compared to healthy, agematched controls. These incretins exert their beneficial effects by repairing the pancreatic islets. Thus, increasing pancreatic beta (β) cell mass and in turn it will help to synthesize and secrete insulin into the circulation. The rationale of this study was to find out how these two incretins can improve insulin secretion both in vivo and in vitro employing the rat model of T1DM following injection with streptozotocin (STZ). The project employed six groups of rats, with three groups serving as age-matched, healthy controls and the other three groups rendered diabetic. One set of rats from each group was untreated while the rats from the other four groups were given either GLP-1(50 nmol/kg body weight) or exenatide (1 μg/kg body weight) over 10 weeks. The project measured body weight, levels of blood glucose and insulin. The plasma levels of liver and kidney markers were also determined. The in vitro study measured insulin secretion from pancreatic fragments, the distribution of insulin- and glucagon-positive cells in pancreatic islets, granules, co-localization of different peptides in the islets, biochemical, and molecular biological changes, which may occur in the pancreas during the experimental period. For the in vivo study, the results have shown mild gain in body weight and no change in blood glucose levels in both treated and untreated age-matched normal control rats. Furthermore, the results show a significant reduction in blood glucose levels in diabetic rats treated with either GLP-1 or exenatide, but the beneficial effect was more pronounced following GLP-1 treatment. The results also show no changes in glucose handling between normal treated or normal untreated rats following blood glucose tolerance test (GTT). However, in diabetic rats, the results show that the GTT reveals a better glucose tolerance in these diabetic animals treated with either GLP-1 or exenatide but the effect was more significant with GLP-1 compared with untreated diabetic rats. The present study shows that diabetic rats secreted significantly less insulin in the blood than normal healthy rats and a significant increase in serum insulin was detected in both normal and diabetic rats treated with either GLP-1 or exenatide compared to untreated controls. The results also show significant reductions in the liver enzymes, aspartate transferase and alanine transferase in the diabetic rats. A similar beneficial effect on kidney function was obtained owing to a small reduction in blood urea nitrogen, serum creatinine and serum uric acids in both normal and diabetic rats treated with either GLP-1 or exenatide. In the lipid profile study, the results show a mild reduction in serum cholesterol and a marked reduction in serum triglyceride in both normal and diabetic rats treated with either GLP-1 or exenatide. The results from the in vitro study show that either GLP-1 or exenatide can evoke marked dose-dependent release (secretion) of insulin from pancreatic tissue fragments of normal and diabetic rats, indicating that there is a clear role for either GLP-1 or exenatide in inducing insulin secretion. In this study, an attempt was also made to investigate both the number and distribution of endocrine cells in the control and diabetic rat pancreas using immnohistochemistry. The results show a significant increase in the number of cells containing either insulin or GLP-1 in both normal and diabetic treated rats. However, in the case of exenatide, catalase and glutathione reductase-positive cells were only significantly increased in diabetic rats, but the increase was not significant in normal rats treated with either GLP- 1 or exenatide. These results show that the significant increase in number of catalase and glutathione-positive cells in diabetic rats treated with either GLP-1 or exenatide reveal the beneficial antioxidant effect of both GLP-1 and exenatide in treatment of oxidative stress, which usually occurs in DM. On the other hand, there was a significant decrease in glucagon-positive cells in both normal and diabetic rats treated with either GLP-1 or exenatide. The immunohistochemical and immunofluorescent studies also revealed that insulinpositive cells were distributed both in the central and peripheral portions of the islets of Langerhans in normal pancreas. In contrast, glucagon-positive cells were located in the peripheral part of the islets of Langerhans. After the onset of diabetes, the number of insulin-positive cells was reduced significantly. In contrast, the number of glucagonpositive cells increased significantly with abnormal pattern of distribution compared to normal pancreas. The pattern of distribution of both GLP-1 and exenatide has indicated co-localization not only with insulin, but also with glucagon. Furthermore, catalase and glutathione reductase-positive cells were distributed homogenously all over the islet of Langerhans with no specific co-localization with specific type of endocrine cell. In the gene expression study, the results show significant increases in the levels of mRNA of pancreatic duodenal hoeobox-1, heat shock protein-70, glutathione peroxidase, insulin receptor and glucagon like peptide-1 receptor in both normal and diabetic rats treated with either GLP-1 or exenatide. However, the increase was not significant in mRNA gene expression of either insulin receptor or glucagon like peptide-1 receptor in normal rats treated with GLP-1. On the other hand, the gene expression results show that glucagon mRNA level was significantly decreased in both normal and diabetic rats treated with either GLP-1 or exenatide. In conclusion, the results of this study have clearly demonstrated that both GLP-1 and exenatide have marked beneficial effects on pancreatic islet cells, especially β- and α- cells, which produce insulin and glucagon, respectively. The two incretins seem to repair the diabetic pancreas, which in turn secretes more insulin and less glucagon.