Magnetic resonance imaging (MRI) is a powerful technique for the in vivo study of experimental disease models. The application of MRI to animal models requires the development of specialized methods which can provide insight into anatomy, function, physiology and specific pathology. This thesis research focused around the development of MRI methods for imaging the mouse heart and other body organs. In this work, single slice arterial spin labelling (ASL) was implemented and optimized for the in vivo measurement of perfusion in the mouse heart. A fast ECG-gated Look-Locker sequence was used for T1 mapping with data logger recordings for the assessment of respiration corruption and additional prospective gating. A variability and repeatability study was performed to assess the applicability of the technique in vivo. This technique was then extended to have multi-slice capabilities through the implementation of a multi-slice cardiac T1 mapping sequence. In order to apply the multi-slice sequence in vivo, a new method of perfusion quantification was developed to compensate for the input function of the blood magnetization. Amyloidosis is a severe condition where amyloidotic fibrils of mis-folded proteins accumulate in the extracellular space. With the development of new therapies, there is an urgent need for sensitive imaging markers for the monitoring of amyloidosis. In this research, the extracellular volume fraction, as measured using equilibrium contrast MRI with primed infusions of gadolinium, was assessed as a marker for the detection of amyloidosis and for the monitoring of amyloid depletion during therapy. Finally, in order to remove spike noise in MRI data sets, a post-processing algorithm was implemented and validated for the removal of RF spikes in k-space Overall, this thesis research presents methodological developments of cardiac and body MRI for the in vivo study of experimental models of disease.

This thesis describes the development and evaluation of a stochastic mortality model that is intended to support shared decision-making between health care professionals and patients; health care planning; and actuarial analysis for life insurance, health insurance and pensions. The model uses risk factors and cause of death to calculate all-cause mortality in a modularised Markov Chain Monte Carlo approach. This modularisation allows the substitution of different cause of death models and hazard ratios, enabling customisation, calibration to different populations, and further research. The cause-specific sub-models used in this evaluation incorporate a method of auto-calibration to different populations using baseline population mortality rates, risk factor distributions, and individual risk factor values. The model can investigate the impact of changing trends in risk factors and population mortality rates. Trends in lifestyle factors such as smoking cessation, medical interventions that adjust risk factors such as blood pressure or serum cholesterol reduction, or interventions that reduce mortality from specific causes, can be examined. Non-specific, observed trends like the select transition effect seen in life insurance portfolios, or cohort effects relating to year of birth, can also be applied. A thorough internal and external evaluation of the model is presented, including its performance in simulating three randomised controlled trials (two of the treatment of hypertension, and one of cholesterol reduction in subjects experiencing a cardiovascular event), as well as three simulations of historical prospective cohort data projected over at least 16 years. A demonstration of the application of the model in the assessment of the impact of changing trends in obesity rates in the UK over the next 50 years suggests that rising obesity makes a modest negative contribution to mortality improvement, but not enough to reverse current improvement rates.

Background: Evidence of a relationship between maternal postnatal depression and child development is consistently growing, but there has been no distinction between depression that is clearly diagnosed and treated, and depression that is never identified by health professionals. Previous models assessing the cost-effectiveness of screening for postnatal depression have been unable to account for child outcomes and the effects of undiagnosed maternal depression due to a lack of research in this area. Without these outcomes, screening for maternal postnatal depression is not currently considered to be cost-effective. Methods: Longitudinal survey data from the Millennium Cohort Study is used to explore the differential effects of undiagnosed and diagnosed maternal depression on child cognitive and behavioural development over time, and to re-examine whether screening for postnatal depression could be considered cost-effective once longer term child outcomes are included. Results: Depression that is undiagnosed has a substantial effect on the behavioural development of children. Children of mothers who are depressed but not diagnosed at 9 months are at least equally likely as those of mothers with diagnosed and treated depression to have behavioural problems later on in childhood. Identifying and treating maternal depression showed some short-term beneficial effect for child behavioural development up to age 5, but this was not maintained at age 7. Higher levels of persistent depression were identified in women who were diagnosed and treated for depression and this persistency was found to have an additive effect on child outcomes, with longer-term maternal mental health problems much more strongly associated with child outcomes than postnatal depression alone. Conclusions: This research highlights the limited success of current treatments for maternal depression, both in benefiting child development and providing long-term symptom remediation for mothers. As current treatments lack benefit for children over the longer-term, the recommendation that screening for postnatal depression appears not to be cost-effective remains unchanged.

Countries are increasingly implementing pay for performance (P4P) as a way to improve health services. The evidence base is conflicting and difficult to interpret. It is necessary to more systematically explore evaluations of P4P schemes in order to synthesize more useful evidence to inform the use of P4P schemes in health care. This thesis starts with a literature review, which shows that the results of evaluations of P4P schemes are heterogeneous, which may possibly be explained by differences in programme design, context, implementation, and evaluation study design. I sought to find ways to better analyse and make sense of these evaluations using two approaches. A quantitative approach was used to systematically explore the heterogeneity. I developed and tested a theoretical typology to categorise P4P schemes by their design features. This typology considers who receives the incentive, type of incentive, size of incentive, and perceived risk of not earning the incentive. I then used the typology to quantitatively explore the influence of P4P design features and evaluation designs on it effectiveness using meta-regression and multilevel logistic regression analyses. I also undertook a formative evaluation of a pilot P4P scheme in Nigeria (a case study). This used semi-structured in-depth interviews with 36 purposively sampled health workers to explore how contextual and implementation factors (e.g. delay in incentive payment) influenced the impact of the scheme. This research presents three notable and novel contributions to knowledge about P4P in healthcare. First a useful typology was developed, which can be used to help categorize, think about, structure and report P4P schemes in a standardized and theoretically informed way. Second, I show that P4P schemes with design features such as payment to groups, large incentive size (>5% of salary or usual budget), and low perceived risk of not earning the incentive are more likely to be effective compared to schemes characterized by payment to individuals, small incentives, and high perceived risk of not earning the incentive. In addition, I demonstrate that P4P evaluations without adequate controls over-estimate the effectiveness of P4P. Third, I show that contextual factors such as incentive payment delays, poor health worker understanding of the P4P scheme, and poor infrastructure affect the effectiveness of the Nigerian P4P scheme and need to be addressed in its future development.

In this thesis I investigated whether an intensive computerised, home-based therapy programme could improve phonological discrimination ability in 19 patients with chronic post-stroke aphasia. One skill specifically targeted by the treatment demonstrated an improvement due to the therapy. However, this improvement did not generalise to untreated items, and was only effective for participants without a lesion involving the frontal lobe, indicating a potentially important role for this region in determining outcome of aphasia therapy. Complementary functional imaging studies investigated activity in domain-general and domain-specific networks in both patients and healthy volunteers during listening and repeating simple sentences. One important consideration when comparing a patient group with a healthy population is the difference in task difficulty encountered by the two groups. Increased cognitive effort can be expected to increase activity in domain-general networks. I minimised the effect of this confound by manipulating task difficulty for the healthy volunteers to reduce their behavioural performance so that it was comparable to that of the patients. By this means I demonstrated that the activation patterns in domain-general regions were very similar in the two groups. Region-of-interest analysis demonstrated that activity within a domain-general network, the salience network, predicted residual language function in the patients with aphasia, even after accounting for lesion volume and their chronological age. I drew two broad conclusions from these studies. First, that computer-based rehabilitation can improve disordered phonological discrimination in chronic aphasia, but that lesion distribution may influence the response to this training. Second, that the ability to activate domain-general cognitive control regions influences outcome in aphasia. This allows me to propose that in future work, therapeutic strategies, pharmacological or behavioural, targeting domain-general brain systems, may benefit aphasic stroke rehabilitation.

Natural killer (NK) cells are a key component of innate immunity and have been implicated in determining the outcome of HCV infection in both genetic and functional studies. The last two decades have seen significant advances in the understanding of NK cell regulation with the discovery of a multitude of activating and inhibitory receptors. CD94:NKG2A operates in tandem with the polymorphic killer cell immunoglobulin-like receptors (KIR) and Ly49 systems to inhibit NK cells, however it is not clear as to the benefits of having two distinct inhibitory receptor:ligand systems. Down regulation or modification of MHC class I expression is a key feature of NK cell recognition of virus infected cells. However, viruses can subvert this mechanism of NK cell surveillance by encoding peptides that can bind to MHC class I. The aim of this thesis is to further our understanding of the interaction between viral and host derived MHC class I binding peptides and their effect on NK cell inhibition. By using an MHC deficient cell line, we have shown that HCV core35-44 peptide is capable of enhancing cell surface expression of MHC class I (HLA-C and HLA-E). Although this peptide stabilises HLA-E, the HLA-E:HCV core35-44 complex alone is insufficient to inhibit at NKG2A positive NK cells. However, in the presence of HLA-E binding MHC class I signal peptides, HCV core35-44 has a synergistic effect in suppressing the NKG2A+ NK cell population. Peptides derived from other viruses such as EBV and HIV, and the stress related peptide derived from heat shock protein 60 also augment inhibition of NKG2A+ NK cells, but only when in the presence of MHC class I signal peptides. This augmentation is caused by recruitment of the non-signalling CD94 molecule to the immune synapse in the absence of its inhibitory signalling partner NKG2A. Thus CD94 can function independently as an enhancer of inhibition. The augmentation of inhibition of CD94:NKG2A by non-inhibitory peptides, contrasts with antagonism of inhibition of KIR by low affinity peptide:MHC complexes. We also show that KIR+ and NKG2A+ NK cells respond with differing stoichiometries to MHC class I down-regulation. Thus peptide selectivity and MHC class I sensitivity of natural killer cell receptors provides a rationale for the evolution of two distinct inhibitory systems for MHC class I.

Aberrantly expressed carbohydrates occur on cancer cells and are antigenic. This thesis is focused on studies to elucidate two elusive hybridoma-defined, cancer-associated carbohydrate antigens: a prostate cancer-associated antigen F77 and a broadly distributed epithelial cancer-associated antigen AE3. The key experiments performed are microarray analyses with mucin-type glycoproteins and generation of designer arrays, multidimensional chromatographies and mass spectrometry of O-glycomes. Antigen-positive sequences assigned are corroborated with focused arrays of natural and chemically synthetized oligosaccharides or glycolipids and products of glycosidase treatments. The F77 antigen is assigned as blood group H type 2 on a 6-linked branch of a poly-N-acetyllactosamine backbone (structure a). This sequence is shown to occur on O-glycans and on glycolipids. The F77 antibody can bind, with lower intensities, to the blood group A and B analogues of structure a. The minimum F77 antigenic sequence is shown to be a pentasaccharide (underlined in structure a). The close association of F77 antigen with prostate cancer is proposed to be a consequence of up-regulation of branching enzymes together with persistent expression of H antigen. This may account for the prevalence of F77 antigen in prostate cancers irrespective of the patient's ABO blood group status. There appear to be two distinct forms of AE3 antigen as evidenced by the ability of AE3 antibody to bind: (i) the O-glycan structure b, known as T antigen, which is joined by α-linkage to serine on mucin-type glycoproteins, and (ii) a sulphated glycolipid known as SM1a (structure c) in which 'T' sequence is joined by β-linkage to galactose. An O-glycan analogue of structure c (structure d), has not been reported so far. Chemo-enzymatic synthesis of structure d will shortly be attempted for antigenic analysis. With knowledge of details of these two antigens, the biosynthetic pathways, the biological functions and clinical applications can now be rationally pursued.

The normal adult mammalian lung has a robust capacity to regenerate following injury, and evidence for alveolar regeneration was recently demonstrated in adult man. In contrast, mounting evidence suggests COPD/emphysema represents a failure of regeneration. COPD represents an enormous worldwide clinical and social burden, with currently no cure besides lung transplantation. An appealing therapeutic option is induction of endogenous lung regeneration using retinoic acid (RA), demonstrated to stimulate alveolar regeneration in animal models of emphysema. However, clinical trials investigating retinoids for chronic lung diseases have been disappointing. Thus, there is a profound stimulus to understand how the regeneration-inducing effects of RA in animal models translate to man. The molecular regulation of RA signalling in emphysema has not been investigated hitherto, and the role of RA in repair of specific human alveolar cell types, alveolar type 2 cells and lung microvascular endothelial cells, is unknown. Work in this thesis was conducted to address these questions. We demonstrated that CYP26A1, which breaks down RA, is enriched on an mRNA and protein level in emphysematous lung tissue. We also demonstrated using in vitro cell culture assays that RA is unlikely to directly regulate alveolar epithelial wound healing. In contrast, RA stimulated lung microvascular endothelial angiogenesis, likely via retinoic acid receptor alpha, and was associated with induction of angiogenic genes. Further work presented herein involved development of an ex vivo model of lung regeneration using precision cut lung slices (PCLS) derived from adult human distal lung tissue. We demonstrated that human PCLS retain architecture and viability through slicing, that 10% serum supplementation is inappropriate for long-term PCLS culture, and that human PCLS remain viable for at least 4 days in culture, suggesting they are amenable to development of an injury/repair model within this time frame.

Epstein-Barr virus (EBV) establishes lifelong latent infections in humans. EBV isolates worldwide are classified as type 1 or type 2 based on their EBNA-2 gene sequence. Type 1 EBV is more efficient at B cell transformation, a property previously mapped to the EBNA-2 locus. Previous work using EREB2.5 cells in a trans-complementation assay showed that the superior ability of type 1 EBNA-2 to sustain B cell proliferation is mostly determined by its C-terminal region. In this study, conversion of a single amino acid in the transactivation domain (TAD), from serine in type 2 EBNA-2 to the aspartate residue in the type 1 protein (S442D), was remarkably found to confer the type 1 growth-promoting phenotype in the EREB2.5 growth assay. The mechanism of the greater transformation efficiency of type 1 EBV appears to involve differential regulation of EBNA-2 target genes. The superior growth properties of type 1 EBNA-2 correlate with the greater induction and activation of viral LMP-1 and cellular CXCR7, compared to type 2 EBNA-2. 5' RACE was used to identify the transcription start site (TSS) of the CXCR7 promoter transcribed in response to EBNA-2. In chromatin immunoprecipitation (ChIP) assays, type 1 EBNA-2 was found to associate more strongly than the type 2 protein with EBNA-2 binding sites near the LMP-1 and CXCR7 genes. D442 was shown to increase binding of type 2 EBNA-2 to some sites at differentially regulated genes. Unbiased motif searching identified an ETS-interferon regulatory factor (IRF) composite element (EICE) that closely resembles the sequence known to mediate EBNA-2 regulation of the LMP-1 promoter. This element may therefore confer the differential effects of type 1 and type 2 EBNA-2 on both LMP-1 and cell gene activation resulting in superior immortalisation by type 1 EBV.

Minimally invasive surgery (MIS) in children is epitomised by delicate and complex reconstructive procedures undertaken on diminutive anatomy within constrained operative workspaces. Conventional MIS equipment imposes restrictions on a surgeon's ability that may limit the eligibility, safety and effectiveness of keyhole surgery in paediatric settings. Enhancements provided by medical robotics may restore and augment operative capabilities to overcome these limitations. This thesis represents a detailed healthcare technology assessment of robotic technologies in paediatric MIS. Systematic review of the first decade of clinical adoption identifies a progressive diffusion pattern. Fundoplication and pyeloplasty procedures stand out as dominant target applications. Meta analyses of comparative effectiveness literature for these procedures identified outcomes to be either comparable or marginally better, but at higher financial cost. An international survey was conducted to understand perceptions of paediatric surgeons towards robotic technologies. To address a deficiency in dedicated education resources, an inaugural European workshop was hosted. Large prospective fundoplication and pyeloplasty series were then analysed to determine multi-dimensional learning curves. Other features of robots in paediatric surgery were explored in further detail, specifically dextrous 'wristed' instruments and haptic feedback loss. In a pre-clinical randomised study of robotic versus non-robotic instruments a threshold workspace size of <200cm3 was identified for which existing larger robotic instruments are less well suited for advanced bimanual tasks. Investigation of in vivo suture damage revealed that experience-related factors compensate for haptic loss. A force-sensing paediatric laparoscopy simulator was developed and validated to provide a platform for compensatory haptic skills to be acquired. Overall, first-generation robotic systems can safely be used to perform all paediatric procedures currently undertaken with conventional MIS techniques. However, cost-effectiveness is not sustainable in most healthcare settings. If genuinely disruptive clinical improvements are to be achieved, creative new procedural approaches need to be considered that are facilitated by purpose-designed technology. To pursue this agenda, the application of two novel single-shaft flexible robotic system prototypes are investigated for future roles in advancing surgical care in children.