The work described in this thesis concerns the use and quality of primary care service use by people living beyond a diagnosis of breast, colorectal and prostate cancer and long-term risks associated with cancer. Firstly, the thesis provides a background for this work, with a definition of long-term survivors as those living at least five years past cancer, and the role of primary care in the care of this population. The second section describes use of the General Practice Research Database amongst a cohort of cancer survivors compared to a control population. Breast and colorectal cancer survivors consult more frequently than controls up to 10 years post-diagnosis, while prostate survivors continue to see their GP up to 3 times more for at least 15 years. Most survivors receive adequate preventative care and chronic disease monitoring, excepting mammography for long-term breast cancer survivors. Cancer survivors receive more prescriptions for pain relief, anti-depressants and erectile dysfunction, suggesting higher rates of pain, depression and sexual dysfunction. Breast cancer survivors have an elevated risk of incident heart failure, coronary artery disease and hypothyroidism, while colorectal survivors experience increased risk of dementia and diabetes. All three groups of cancer survivors had higher risks of osteoporosis and second cancers, all-cause, non-cancer and cancer mortality compared to controls. The third section describes a qualitative study of the primary care usage and unmet needs of 40 long-term survivors of breast, colorectal and prostate cancer. Most respondents did not need active GP involvement. Others had ongoing information and psychological service needs. Some felt that their GPs did not have the right expertise for cancer related issues or were too busy, while others had concerns about continuity of GP care. Overall, this thesis provides a background on how the increasing numbers of cancer survivors use and experience primary care in the UK, areas of good practice, and areas where care can be improved in the future.

Replication defective adenoviruses have emerged as promising vectors for delivery of vaccine antigens. The development of new vaccine vectors has recently focused on serotypes t, which the human population is less exposed in order to circumvent pre-existing anti vector immunity. This thesis describes the construction and optimisation of ChAdOX1, a new vector based on chimpanzee adenovirus Y2S, which has recently been manufactured to clinical grade for a Phase 1 human trial. Comparative immunogenicity studies between vectors of different serotype were performed in mice, with careful consideration of the infectious titer of vector preparations, since this parameter can confound studies based solely on viral particle estimation. Aft intramuscular administration, HAdV-S (Human adenovirus C) based vectors elicited superior transgene product specific T cell and antibody responses compared to a selection of chimpanzee adenovirus vectors (from Human adenovirus EJ including ChAdOX1. The construction of ChAdOXl in a bacterial artificial chromosome (BA C), enabled precise, and flexible modification of the genome by recombmation mediated genetic engineering. (recombmeering). Reverse genetics was performed to identify vector determinants of immunogenicity. Chimeric ChAdOXl vectors were created by replacement of native virus associated RNA (VA-RNA) and fiber sequences with the corresponding sequences from HAdV-5 Using these chimeric vectors, the importance of innate immunity and vector transduction in determining vector immunogenicity was investigated. Though the mechanisms responsible ultimately remain unclear, superior transgene product specific immune responses with HAdV-5 correlated with higher levels of transgene expression in vivo after vector administration. The current study has conclusively demonstrated that neither VA-RNA sequences, nor the fiber protein, are responsible for differences in immunogenicity between vectors, contrary to hypotheses based on previous studies.

The absence of invariant Natural Killer T cells (iNKT cells) in mice infected with Influenza A virus (flu) has previously been shown to augment the expansion of Myeloid-derived suppressor cells (MDSCs), a bone marrow derived population that powerfully suppress the development of viral and tumor immune responses. Moreover, iNKT cell adoptive transfer into flu-infected mice has been shown to abolish the expansion and flu-induced suppressive activity of the MDSCs in a CD1d- and CD40-dependent manner. However, the mechanisms by which this relatively small subset of T cells influence myelopoiesis and MDSC differentiation remain largely unknown. In this manuscript we firstly better define the MDSCs found in flu-infection as IL-10-secreting neutrophils that can suppress T cell proliferation. We then go further to show that the flu-induced ability to suppress T cells is acquired as early as the level of the Granulocyte-Macrophage Progenitors (GMPs) in the bone marrow and that iNKT cells can not only abrogate the suppressive activity of the IL-10-secreting neutrophils in the periphery but also that of the GMPs by a direct CD1d-dependent GCSF-mediated crosstalk. MDSC expansion has previously been shown to be associated with the expression of the myeloid-related protein S100A9, and the mechanism of action of granulocytic-MDSCs shown to be ARG1-dependent. We built upon both these findings to show that iNKT cells influence the expansion and function of the MDSCs in part by regulating S100A9 and ARG1 expression. Following this we then showed for the first time that the acute phase protein Serum Amyloid A (SAA), shown to increase during flu-infection, has a dual reciprocal role: having the ability to up-regulate S100A9 and ARG1 in myeloid cells and differentiate IL-10-secreting suppressive neutrophils, while simultaneously facilitating the ability of the MDSCs to crosstalk with iNKT cells in a CD1d-dependent GCSF-mediated manner to abrogate the SAA-induced suppressive activity. All together the data highlights the complexity of the immune response and the role iNKT cells play in influencing the differentiation of MDSCs during demand-driven myelopoiesis. More importantly however, it further affirms that research into harnessing the immunomodulatory capacity of iNKT cells remains an exciting prospect in bolstering future vaccination strategies and should continue to be pursued.

Strategies to reduce infarct size in ischaemia-reperfusion (IR) syndromes such as acute myocardial infarction are of high clinical and scientific interest. Remote ischaemic preconditioning (rIPC) is one such strategy but its mechanisms remain incompletely understood. Multiple lines of evidence from animal studies suggest that the endogenous purine nucleoside adenosine is a key mediator of preconditioning pathways but no evidence exists as to adenosine’s role in the more complex physiology of humans. The work in this thesis aims to elucidate the role of endogenous adenosine in the physiological phenomenon of rIPC and to examine the role of exogenous adenosine in triggering preconditioning-like states. In a randomised, placebo controlled study using healthy volunteers and the human forearm model of ischaemia-reperfusion injury, I demonstrate that delivery of the adenosine receptor antagonist caffeine, prior to the initiation of a rIPC stimulus abrogates the protective effect of rIPC on IR. By then selectively infusing caffeine to achieve high local but low systemic concentrations, I also demonstrate that adenosine receptor activation is important in the ‘trigger’ phase of rIPC rather than in the ‘effector’ phase and that blockade of the trigger phase effectively inhibits the release of a circulating humoral protective factor. These studies provide evidence of the crucial role of adenosine receptor activation in human rIPC, demonstrating their sites of action and illuminating their potential mechanism of action. To study whether exogenously delivered adenosine can recapitulate preconditioning-like states, in initial studies in a large mammal model of acute myocardial infarction, I demonstrate that adenosine, given after the onset of ischaemia, but prior to reperfusion, significantly reduces myocardial infarct size. In a subsequent study, translating these findings to humans with coronary disease, I demonstrate that the delivery of adenosine in a range of concentrations is able to illicit the release of a circulating preconditioning factor which is transferrable across species and can reduce infarct size in a murine model of myocardial IR.

The history of peoples has always evoked a great deal of both academic and popular interest, and the peoples of Britain, with its island position and semi-mythic serial invasions, have evoked as much as any. As most of the period during which Britain has been inhabited by modern humans lies in prehistory, archaeology has long been the best method for elucidating the past. In recent years, however, genetics has come to complement the reconstruction of peoples' pasts, with its ability to trace lineal human biology instead of transferable human culture. The purpose of this thesis is to assess population genetics systems of Britain against the backdrop of archaeologically determined history, informed for later periods by linguistics, and attempt to ascertain any marked congruities or incongruities between this history and modern genetic data. The genetic datasets included in this work are the People of the British Isles Project collection, and some ancillary cohorts from surrounding countries. The genetic systems assessed include mitochondrial DNA, classical marker genes, lactase, pigmentation genes and some phenotypes, and finally a suite of candidate genes for determining normal facial variation. In a self-contained section, the principle of relating population genetic data to population histories is illustrated by a study focusing on Central Asia (a larger area), but using fewer genetic markers. The chosen markers systems overall reveal modest amounts of genetic differentiation among different groups in Britain, but consistently highlight Wales and Orkney especially as relatively distanced from the rest of Britain. This is in keeping with the historically quite isolated state of the former, and the comparatively recent heavy influx of Norse Vikings in the latter. Further details are observable from subsets of this study: all are discussed in the context of archaeological and linguistic evidence. These findings provide support and foundation for a forthcoming study from the People of the British Isles Project, using a genome-wide SNP approach rather than selected markers, which will likely increase the nuance of this initial picture and contribute further to answering specific questions regarding Britain's past.

Sialic acid (SA) is a known major virulence factor of Haemophilus influenza (Hi). This study aims to analyse the functions of some candidate sialometabolism genes, and to further our current understanding on the Hi sialometabolism gene regulation. Two candidate sialometabolism genes (HI0227 and HI0148) and their adjacent ORFs (HI0228, HI0148.1 and HI0149) were studied. HI0148.1 and HI0149 are transcribed as a single gene in screened NTHi strains, and we refer to the combined ORF as NTHI0236 (the designation in strain 86-028NP). Across Hi strains screened, the sequences of HI0227, HI0148 and NTHI0236 are conserved. However, the sequence of HI0228 is heterogeneous. Mutants that lack the functions of HI0227, HI0228, HI0148 and NTHI0236 were compared to their respective wild type parent strains for ability to grow on SA (in aerobic and microaerophilic conditions), their ability to sialylate LPS and their ability to resist complement mediated killing. The mutants did not exhibit major differences in the tested aspects of sialometabolism compared to their respective wild type strains. Changes observed in some of the mutants in serum bactericidal assays and LPS profiles were due to the effect of phase variable genes. The sialometabolism functions of HI0227, HI0148, and NTHI0236 remain obscure, and we postulate that HI0228 is a pseudogene. Investigation of Hi sialometabolism gene regulation was conducted using mutants that lack different steps of the Neu5Ac catabolism pathway and the Neu5Ac activation pathway. The expression of nanE and siaP, respectively representing the Neu5Ac catabolism and transport operons, were assessed using RT-PCR and qPCR. We investigated a temporal/concentration effect of Neu5Ac on the expression of sialometabolism operons, which highlights the importance of studying the Hi sialometabolism gene regulation as a dynamic process. We further demonstrated that GlcN-6P, a Neu5Ac intermediate from the catabolism pathway, is likely the SIS sugar that interacts with SiaR, the repressor protein of the Hi sialometabolism operons. We postulate that upon binding of GlcN-6P to SiaR, the SiaR-mediated repression on the Hi sialometabolism operons is relieved, resulting in the induction of the expression of Neu5Ac catabolism and transport genes.

The focus of this thesis is on practice changing clinical studies which impact upon the day to day treatment of patients with pleural infection, answering specific questions on several aspects of patient management. Specific areas of assessment in this thesis include: Assessment of the current evidence for optimal drain size choice in patients with pleural infection; Analysis and statistical modelling of a previous cohort of patients with pleural infection, in order to assess optimal drain size choice in pleural infection; The design, conduct and analysis of a 2 x 2 factorial multi-centre randomised, placebo controlled trial to assess the efficacy of two novel intrapleural agents (tPA and DNase) in aiding drainage in patients with pleural infection (The 2nd Multi-centre Intrapleural Sepsis Trial, referred to from here on as MIST2); Validation work informing the primary outcome measure of MIST2, assessing the relationship between chest radiograph imaging of infected pleural effusion and CT measured volume of pleural fluid using novel digital measurement strategies.

Stroke or cerebral vascular accident (CVA) is the 4th leading cause of mortality and the principle cause of long-term disability in the United States. Unfortunately, current reperfusion-based treatments (e.g. thrombolysis, tPA) cannot be administered to the majority of patients presenting with ischemic stroke. Accordingly, new treatments for ischemic stroke are desperately needed. Reactive astrocytes perform key roles in tissue repair and remodeling following stroke such as preservation and repair of the blood-brain barrier, modulation of immune cell invasion, glutamate uptake and neuroprotection, and glial scar formation. The proliferative subpopulation of reactive astrocytes found immediately adjacent to the infarct core after stroke (known as the peri-infarct area) is particularly important for protecting the brain parenchyma from ischemic damage and inflammation. Defining the signaling network that controls reactive astrocyte formation and function has potential to provide new treatment strategies for patients ineligible for reperfusion therapy. Notch1 signaling is required for the proliferation of peri-infarct reactive astrocytes after stroke. To identify downstream targets and potential functional effectors of Notch1 signaling in reactive astrocytes, we developed an ex vivo forward signaling screen. To generate large quantities of adult reactive astrocytes, we employed adult Reactive astrocyte-derived Neural Stem Cells (Rad-NSCs) isolated from the peri-infarct area of mice after stroke. Astrocytes re-differentiated from Rad-NSCs (AstroRad-NSC) were then exposed to immobilized Jagged-1, a Notch1 ligand. In response to Jagged-1, many genes involved in reactive astrocyte-mediated tissue protection, metabolic regulation, angiogenesis and glial scar formation were up-regulated. Of special interest, several genes for proteins that regulate with glutamate uptake and metabolism were increased by Jagged-1/Notch signaling, including the glial-specific GLutamate-ASpartate Transporter (GLAST). With loss-of-function experiments, we determined that deletion of Notch1 decreased GLAST transcript and protein levels in cultured AstroRad-NSC. Furthermore, we isolated reactive astrocytes directly from cerebral cortex after stroke and confirmed the effects of Notch1 on GLAST in vivo. Our results suggest that treatments designed to stimulate Notch1 signaling after stroke may promote glutamate uptake, thereby decreasing excitotoxicity and neuronal cell death. Binding of Endothelin peptides to the type B Endothelin receptor (ETBR) has been shown to alter cell proliferation. Investigating a possible relationship between Jagged-1/Notch1 and Endothelin signaling in reactive astrocytes, we determined that Notch1 signaling regulated ETBR indirectly, by activating STAT3, an unidentified transcriptional activator of ETBR. Using inducible transgenic astrocyte-specific conditional knockout (cKO) mice (GFAP-ETBR-cKO), we found that specific deletion of ETBR in reactive astrocytes phenocopied the defect in reactive astrocyte proliferation observed in our previous work with GFAP-Notch1-cKO mice. Notably, the Notch1-STAT3-ETBR axis we identified is likely to control reactive astrocyte proliferation in most, if not all, forms of CNS injury. The experimental results presented in this doctoral dissertation provide novel insight into signaling mechanisms that may someday be exploited to improve care for patients with stroke and other forms of CNS injury or disease.

Worldwide, more than 1 million infants die as a result of premature birth. In the United States, where 1 in 10 births occurs preterm, premature birth is the leading cause of infant mortality. Premature infants have high rates of mortality and morbidity, with the highest rates seen in those infants born extremely preterm -- prior to 30 weeks gestation. Severe morbidity in these infants often contributes to life-long health problems. Maternal hypertension (HTN) is one contributor to preterm birth and also contributes to fetal growth restriction, resulting in birth weights which are small for gestational age (SGA, and generally within the lowest 10th percentile). Within this high risk population, SGA infants have increased risk of mortality compared to appropriate for gestational age infants. Therefore the impact of maternal HTN on neonatal outcome might be presumed to be negative. Previous studies however, have been contradictory, with both higher and lower rates of infant mortality reported in infants born to mothers with HTN, as well as differing reports analyzing the relationship between serious morbidity and maternal HTN. Utilizing the Vermont Oxford Network Very Low Birth Weight database, a collaborative database of Level III Neonatal Intensive Care Units across the world, 88,275 North American infants born between 22+0 and 29+6 weeks gestational age between 2008 and 2011 were identified. This dissertation explores the relationship between maternal HTN and gestational age at time of birth within this population, and the reported rates of morbidity and mortality in infants born prior to 30 weeks gestation. The independent contributions of maternal HTN with neonatal morbidity and mortality in our population were estimated using logistic regression and adjusting for factors previously known to be associated with risk, including birth weight, antenatal steroid exposure, infant sex, maternal race/ethnicity, prenatal care, inborn/outborn status, and birth year. We hypothesized that mortality rates would be lower for infants born to mothers with HTN compared to those born due to other factors, when corrected for the noted confounding variables and surviving infants would have better prognoses, as evidenced by lower rates of severe morbidity, including bronchopulmonary dysplasia, intraventricular hemorrhage, periventricular leukomalacia, necrotizing enterocolitis, and infection. Within the higher-risk SGA population, we hypothesized that mortality rates would be higher than observed in appropriately grown infants, but decreased in those born to mothers with HTN, despite the association between maternal HTN and SGA. This dissertation begins with an explanation of current knowledge about preterm birth, maternal HTN, and their associations. Chapter 2 focuses on the relationship between maternal HTN and infant mortality in extremely preterm infants. Chapter 3 examines the risk associated with severe morbidities in surviving infants. In addition, we also use a combined morbidity risk assessment score which has previously been used to determine future risk of long term disability. In Chapter 4, SGA infants are separately evaluated for their risk of mortality and the association with maternal HTN. These analyses support the high mortality and morbidity rates seen in extremely preterm infants. Maternal HTN, after adjustment, results in reduced risk of both mortality and severe morbidities in infants compared to infants born to mothers with other underlying contributors to preterm birth. This suggests that clinical practices and parental counseling should reflect differing risk profiles in sub-populations of extremely preterm infants.

Background and Purpose: More than one-third of American adults are obese. The prevalence of extreme obesity is rapidly rising. Nine medications are currently approved for weight loss yet they remain under utilized with the focus primarily on lifestyle modifications. The study's objective was to determine current prescribing patterns and attitudes of weight loss medications in the management of obesity among primary care providers (PCPs). Methods: PCPs were surveyed to determine practice patterns, attitudes, barriers, and facilitators for prescribing weight loss medications. Conclusions: A total of 105 surveys were completed. 76% of all PCPs did not prescribe weight loss medications for long-term weight loss therapy and 58% of PCPs had negative perceptions of pharmacotherapy as a treatment. Significant differences existed between prescribing patterns and attitudes of advanced practice clinicians and physicians. Safety concerns were identified as the greatest barrier. Having 2+ comorbidities and severe obesity were identified as facilitators for prescribing weight loss medications. Under utilization of pharmacotherapy suggests that PCPs may not have sufficient knowledge about medication safety profiles and efficacy. Delaying treatment until patients have reached a high level of morbidity may be less efficacious than earlier treatment. Implications for Practice: Education regarding effectiveness and risks of weight loss medications for obesity management is needed and earlier interventions with pharmacotherapy may prevent significant morbidity and mortality.