Background Opioids are the most potent analgesics available and their treatment is well established in cancer and acute pain. However, their long-term use in non-cancer pain is worrisome due to insufficient evidence on long-term effectiveness and safety. Population-based research examining the utilisation of opioids in non-cancer pain patients in UK primary care remains limited and little information exists on patients receiving long-term therapy. Aims This thesis aimed to examine the demographics, clinical traits, prescribing patterns and healthcare use of non-cancer pain patients prescribed strong opioids in primary care, with a focus on long-term strong opioid use. Methods The thesis was a retrospective observational study using the Clinical Practice Research Datalink. Prescription data of four strong opioids (morphine, buprenorphine, fentanyl and oxycodone) issued between 2000-2010 to adults (aged ≥18 years) without cancer diagnosis 12 months within the date of first prescription use were included. The annual prevalence and incidence of non-cancer pain patients prescribed strong opioids over the 11 years were initially assessed using joinpoint regression analysis, and the users' demographic and clinical characteristics were explored descriptively. The numbers of prescriptions per patients, total number of days of drug supply, and total oral morphine equivalent daily dose (OMED) were subsequently calculated annually and compared between long-term (>90 days/year) and short-term (≤90 days/year) users. Multivariate linear regression modelling with generalized estimating equations was consecutively used to identify baseline and time-varying covariates linked to long-term strong opioid use. Lastly, long-term strong opioid users' primary and secondary healthcare use was quantified using CPRDs' data link with Hospital Episode Statistics (HES), and the nature of hospital admissions and patient-level factors of influence were determined by multivariate regression data analysis. Results A total of 135,941 non-cancer pain patients (63.34% female, mean age 66.34±17.98 years) were prescribed strong opioids and were included in the analysis. The incidence of long-term prescribing (>90 days) continued to increase by 16.96% per year (95%CI: 13.70%, 20.30%, p < 0.001) from 3.60 (95%CI: 3.38, 3.82) per 10,000 person-years in 2001 to 12.75 (95%CI: 12.41, 13.10) per 10,000 person-years in 2010. The mean number of pain diagnoses amongst long-term users was 3.00±2.16, with back pain, abdominal pain and osteoarthritis presenting the most common diagnostic disorders, and the mean number of co-morbidities was 2.04±1.70. Depression and anxiety were prevalent in 50.02% and 22.13% of long-term users, respectively. The mean daily oral morphine equivalent dose (OMED) of long-term users (95.88±109.03 mg/day/year) was almost twice that of short-term users (54.80±54.55mg/day/year). Over a quarter (26.01%) of long-term users received high oral morphine equivalent daily dose and their mean annual OMED was 221.19±148.07mg/day and mean annual days' supply was 277.05±84.55 days. Forty-one percent of long-term users had one or more all cause in-patient admissions during the study period, and a rising trend of admission spells was noted annually by 28.16% (95% CI: 26.40, 29.90, p < 0.001). Musculoskeletal pain disorders were the main cause of hospital admissions and re-admissions. Factors associated with hospitalisation included; greater GP visits per year (>3 consultations/year), >120mg morphine equivalent daily dose (aRR: 1.37; 95%CI: 1.27, 1.49) and co-prescriptions of psychoactive medications, including antidepressants (aRR: 1.07; 95%CI: 1.00, 1.14), benzodiazepines (aRR: 1.17; 95%CI: 1.09, 1.26) and non-benzodiazepines (aRR: 1.15; 95%CI: 1.05, 1.27). Conclusions Prescribing of strong opioids over the 11 years was characterized by a shift towards long-term prescribing. Primary care non-cancer pain patients exposed to strong opioids long-term were typified by multiple pain and co-morbid illnesses that included common psychiatric disorders, which suggest complex, and vulnerable, high-risk patients that are susceptible to negative health consequences. The thesis has identified several covariates linked to strong opioid-utilisation and healthcare use, which exemplifies key findings that can be used to inform clinical decision-making, targeted management interventions and monitoring of non-cancer pain patients treated with strong opioids long-term.

Background: Fibromyalgia (FM) and chronic widespread pain (CWP) are highly prevalent chronic painful conditions that have substantial impact on patients, health care systems, and society. Diagnosis is complex and management strategies are associated with various levels of evidence for effectiveness and cost-effectiveness. Pain management services (PMSs) have been shown to be effective in some settings and are therefore recommended by clinical practice guidelines as a rational treatment. As these services are resource intensive, evidence is needed to demonstrate their cost-effectiveness. Quality-adjusted life-years (QALYs) are commonly used as an outcome for the economic evaluation of health interventions. To perform an economic evaluation, mapping from a disease-specific measure onto a generic preference-based measure is essential when utility values of health states are not available in the same sample. In the field of FM/CWP, mapping studies that have estimated the EuroQoL 5-dimensional 3-level instrument (EQ-5D-3L) from the Revised Fibromyalgia Impact Questionnaire (FIQR) are lacking. Aims: The aim of the research was to conduct a programme of feasibility work in preparation for the conduct of a definitive randomised controlled trial (RCT) of PMSs in managing people with fibromyalgia or chronic widespread pain, compared with standard care (SC). The programme included a systematic review, a feasibility observational study, an economic modelling, and a statistical mapping of FIQR to EQ-5D-3L. Methods: The study followed the Medical Research Council (MRC) recommendations for evaluation of complex interventions through an iterative process of observation work before evaluation by large scale studies. Therefore, this work comprised four phases. Firstly, a systematic review was performed, using standard methods of systematic review along with quality and reporting criteria for published economic evaluation studies of PMSs in people with FM/CWP. An electronic search was performed in clinical and economic databases, from their inception to April 2016. Full economic evaluations undertaken from any perspective were included. The Cochrane Risk of Bias tool and the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) checklist were used to assess the methodological quality. Secondly, a prospective observational multicentre study, using patient-level data was conducted among newly referred adults with FM/CWP (≥18 years-old) at two community-based PMSs in Nottingham. The feasibility of the study and recruitment, retention and follow-up rates were assessed. Participants provided details of clinical outcomes and health resource-use, using a postal questionnaire and diaries, at baseline and then three and six months after recruitment. Outcome measures were the FIQR, the Brief Pain Inventory (BPI), and the EQ-5D-3L. Thirdly, data from the observational study, systematic review and secondary data sources were then integrated in a de novo Markov decision analytic model to assess the cost-effectiveness of PMSs compared with SC. Data included transition probabilities, utilities, healthcare resource use and their costing tariff. Costs per extra QALY were calculated from the perspective of National Health Services (NHS) England, using a lifetime horizon. For validating the decision analytic model, the Assessment of the Validation Status of Health-Economic decision models (AdViSHE) criteria were followed. Finally, access to an anonymised dataset of 160 Spanish adults (≥18 years-old) with a confirmed diagnosis of FM, according to the American College of Rheumatology (ACR) 1990 criteria, was obtained from a researcher, to perform the mapping from the FIQR to the EQ-5D-3L. The econometric models investigated were ordinary least square (OLS), censored least absolute deviations (CLAD) and multinomial logistic (MNL) models. The choice of model was based on the ‘Best’ performance, which was defined as: the lowest absolute difference (AD), mean absolute error (MAE), and root mean square error (RMSE), and the highest R2 statistics. Results: In the systematic review, six economic evaluation studies of PMSs in people with FM/CWP were identified. However, due to methodological weakness and variability, the review was unable to draw a firm conclusion about the cost-effectiveness. In addition, all identified studies were performed alongside randomised controlled studies (RCTs) with a short follow-up period. As FM/CWP are chronic diseases, the effectiveness and cost-effectiveness over longer periods of time need further investigation, and it is likely that this will require the use of economic evaluation modelling approaches. Recruitment to the observational study was a significant challenge. Despite modifications to the study protocol to enhance recruitment and retention, only 14 participants were identified, of whom 10 were recruited and eight completed the study (July 2015-September 2016). Hence, the effectiveness and healthcare resource use data were affected by the small sample size Following the integration of data from the feasibility work and published studies, PMS generated a mean of 15.2 (2.5% percentile -1.7; 97.5% percentile 33) more QALYs per patient, at a mean reduced cost of -£10453 and -£11148.8 (2.5% percentile -£31420.2; 97.5% percentile £3494.5) for deterministic and probabilistic analysis, respectively. PMS has a 97% probability of cost-effectiveness compared with SC at a willingness-to-pay of £20000 per QALY. Sensitivity analysis demonstrated that targeting each disease severity with PMS has a probability over 0.96 of cost-effectiveness compared with SC, at a willingness-to-pay of £20000 per QALY. In the mapping sample, it was possible to map from the FIQR to the EQ-5D-3L. The predicted mean utilities in all OLS models were identical (up to four decimals) to the observed means. Among the ten specifications used, the best performing model was an OLS model, which included the FIQR (collapsed items in a discrete form), age, and educational level as independent variables in the regression. This model produced lowest errors (AD = 0.0000; MAE = 0.1279; MSE = 0.1576; R2= 0.4675) compared with the more advanced CLAD and MNL models. Conclusions: The programme of feasibility work performed in this thesis found that due to the low sample size in the feasibility observational study, it has been concluded that it is not feasible to perform a future RCT to evaluate the cost-effectiveness of PMS for people with FM/CWP. However, this study has highlighted key methodological issues which should be taken into consideration in future studies. Moreover, the systematic review highlighted has key design areas that researchers need to consider when conducting economic evaluations, including following standard design guidelines. The indicative economic evaluation of PMS for people with FM/CWP suggested that PMS can lead to a significant health gain and cost savings to the NHS. Finally, it was found that mapping from the FIQR to the EQ-5D-3L is possible. The OLS was the best performing model, which predicted the mean tariff score of the EQ-5D-3L with an accuracy of up to four decimal places. Findings from the statistical mapping will enable future researchers to perform economic evaluations in the absence of EQ-5D-3L data from RCTs of FM/CWP.

In this thesis I have explored different components of the pathogenesis of several related EBV associated cancers. In the first part of the thesis I focus on the microenvironment of two of these cancers, nasopharyngeal carcinoma (NPC) and diffuse large B cell lymphoma (DLBCL). Our group has developed a therapeutic vaccine against EBV which has already been shown to be safe in patients with NPC. Therefore, in the first results chapter (chapter 3), I present a description of the phenotyping of expression of the immune microenvironment including immune checkpoint (ICP) genes and MHC class I and class II genes in NPC tissues. I showed for the first time in NPC tissue samples, two types of PD-L1 expressing tumours: diffuse and marginal. In re-analysis of published data, I found co-expression of immune checkpoint genes and their receptors in EBV positive NPC samples; information which is likely to inform the design of combination immunotherapy in NPC patients. I have shown in my re-analysis of EBV positive NPC that PD-L1 is not up-regulated by LMP-1. In chapter 4, I show the results of studies of the expression of collagen and collagen receptors in DLBCL in which I have identified the over-expression of a potentially novel immune checkpoint receptor, LAIR-1, on the macrophages infiltrating this tumour. In the second part of the thesis I switch my line of inquiry to the tumour cells of EBV-associated cancers, this time focussing on Hodgkin lymphoma (HL), another EBV-associated lymphoma. During the course of the work presented in this chapter I was able to define a role for aberrant sphingosine-1-phosphate (S1P) signalling in driving PI3-K activation mediated through up-regulation of S1PR1 and downregulation of S1PR2 receptors in HL. I also showed that in turn, PI3-K signalling increases the expression of potentially oncogenic downstream transcription factors, such as BATF3 which I have shown to be overexpressed in HL. These data suggest that antagonists of S1P could be considered for the treatment of patients with HL.

Legionella pneumophila was first recognized as a cause of human pneumonia in 1976 . Since then, much has been learned about the microbiology, pathophysiology and epidemiology of this organism. The features which permit one strain but not another to invade human lung tissue and produce disease remain incompletely understood. This study e valuated several attributes of a virulent and an avirulent strain of L. pneumophila in an attempt to identify characteristics which would distinguish the two. Evaluation of a new medium, buffered egg yolk agar, showed that virulence was maintained after 26 passages, which was the same as the buffered charcoal yeast extract agar used for comparison. However, growth appeared earlier and was heavier on the charcoalcontaining medium. Morphologically, the avirulent strain produced greater numbers of filamentous forms and was found to be encapsulated more frequently. Treatment with polymixin B produced morphologic changes similar to those previously reported but failed to alter the virulence of either strain. No plasmids were found in either strain nor were consistent differences in protein content demonstrated using sodium dodecylsulfate polyacrylamide gel electrophoresis. Both strains reacted less intensely as cultures aged with a monoclonal but not a polyclonal antibody in a direct fluorescent antibody assay . This change was more pronounced when the virulent organism was tested. Chemotactic assays showed similar tendencies when human or guinea pig mononuclear cells were compared to two estuarine species (hogchoker and spot) and one freshwater species (golden shiner minnow) of fish. In vivo results were also similar when two of the three species of fish were tested, suggesting that either the spot or the minnow may be used in evaluation of certain characteristics of L. pneumophila. Organisms were isolated from apparently healthy fish up to 15 days after inoculation in some instances. This suggests that fish may be a possible additional environmental reservoir for Legionella pneumophila.

Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) is a major obstacle in the success of head and neck cancer therapy. Despite efforts by several groups to understand the mechanisms of resistance to tyrosine kinase inhibitors such as erlotinib, there has been little success in improving the patient survival. Given that there are a number of ongoing clinical trials testing the efficacy of erlotinib in head and neck cancer, it is essential to investigate the novel mechanisms of erlotinib resistance to improve its efficacy and patient survival. This dissertation addresses this issue of erlotinib resistance in head and neck cancer, underscoring the role of inflammatory cytokine signaling. Chapter 1 introduces the problem of erlotinib resistance and discusses the potential link between inflammatory signaling and cancer progression and erlotinib resistance in head and neck squamous cell carcinoma. Chapter 2 discusses the role of the cytokine interleukin-6 signaling in acquired resistance to erlotinib in head and neck squamous cell carcinoma. Chapter 3 describes the role of IL-1 signaling in acquired resistance to erlotinib in head and neck squamous cell carcinoma. Chapter 4 discusses the specific role of IL-1α (an agonistic ligand for IL-1 signaling) in acquired resistance to erlotinib in head and neck squamous cell carcinoma. Chapter 5 discusses ideas to test for future work in this field. Altogether, this dissertation endeavors to emphasize the contributory role of inflammatory cytokine signaling in erlotinib resistance in head and neck squamous cell carcinoma so that it helps in the development of effective anti-cancer therapies and biomarkers of resistance and/or response in HNSCC.

No description available.

In the eastern U. S. and other wheat growing areas of the world, powdery mildew is a major constraint to the production of wheat. Currently, the most effective and economical means of control is to use resistant cultivars. Unfortunately, the diversity of the pathogen population enables the pathogen to overcome new resistance genes, often within several seasons of commercial deployment. Surveys to determine the virulence of the population to a set of resistance genes have been performed in many areas of the world. Often, differential frequencies of virulence at varying geographic areas are extended to infer a putative population structure. Due to selective pressure, virulence frequencies are poorly suited to high-resolution analysis of population structure and results may reflect regional cultivar choices and not underlying population phylogeny. A collection of 206 single-ascospore derived powdery mildew isoaltes were utilized to determine virulence frequencies at varying locations in the eastern U. S. Primers were developed to amplify coding sequences in order to assess single nucleotide polymorphisms within this population, which allowed allowed analyses of the distribution of presumably neutral genetic variation. Dendrograms based on Nei?s standard genetic distance (Gst) indicated clustering of virulence frequencies into northern and southern subpopulations, with North Carolina as the putative boundary between groups. DNA sequence based AMOVA analysis using groups derived from Hudson?s sequence based subdivision test (Snn) support this conclusion. Tests for population richness indicate greater haplotype diversity in Virginia and Delaware; a result with several possible explanations based on the known history of North American wheat cultivation.

Three root-colonizing fungi, binucleate Rhizoctonia (BNR) isolate BNR621, BNR isolate P9023, and Trichoderma hamatum 382 (T382) were studied for suppression of Botrytis blight in geranium and demonstration of induced systemic resistance (ISR) as a mechanism of biocontrol. Root isolations of the BNR fungi confirmed that BNR621 and P9023 colonized the geranium root system. Root colonization is considered a requirement for biocontrol. Induction of resistance to Botrytis blight was observed in geraniums transplanted into potting mix amended with formulations of P9023 and T382 2 weeks prior to inoculation with B. cinerea when grown under environments either highly or less conducive to disease development. In the less disease-conducive environment, P9023 and T382 provided protection equivalent (P<0.0001) to a weekly rotation of fenhexamid and chlorothalonil fungicides. The effects of cellular and biochemical changes of ISR on germination of conidia of B. cinerea and Botrytis blight lesion area and expansion rates were tested in vitro. No differences in conidia germination were observed between treatments at any sampling time, with a mean germination rate of 80% across all treatments. The size of the leaf lesion area was dependent upon the length of time between topdressing of the geranium plants with the biocontrol agents and detachment of the leaves for inoculation. In geranium leaves detached and inoculated 7 days after topdressing with a Pesta formulation of either BNR621 or P9023, AUDPC calculated from lesion area was reduced (P<0.0001). In contrast, leaves detached and inoculated 14 days after topdressing with a formulation of T382 had a smaller (P<0.0001) AUDPC from lesion area than plants treated with a Pesta formulation of BNR621. Based on the overall suppression of Botrytis blight in geranium and the reduction in lesion size observed in the detached leaf assays, a role for restriction of lesion development is suspected in the control of B. cinerea in geranium. In a comparison study, induction of resistance to Rhizoctonia stem rot in geranium cuttings taken from stock plants treated with formulations of BNR621, P9023, or T382 was evaluated. No consistent control of Rhizoctonia stem rot in geranium cuttings by a biocontrol treatment was observed. However, cuttings taken from geraniums topdressed with a Pesta formulation of BNR621 14 days prior to propagation and inoculation had a greater probability (P=0.03) of rooting and growing out to healthy plants as compared to inoculated cuttings taken from untreated stock plants.

Peronospora tabacina Adam. is the causal agent of blue mold or downy mildew of tobacco. The pathogen is a fungus-like organism and is a member of the Oomycota. P. tabacina is an obligate parasite restricted to the genus Nicotiana spp. Identification of the pathogen is difficult since symptoms and signs generally occur 6-12 days post inoculation. The spread of the pathogen occurs through aerial long distance dispersal of inoculum and severe epidemics occur yearly in tobacco growing areas of the world. One objective of this work was to develop a real-time Taq Man assay for the detection and quantification of P. tabacina. Optimization of the assay was established at a final concentration of 450nM of primers and 125nM of probe. The assay was useful for detection of the pathogen down to a lower limit of 1fg of DNA. The pathogen could be detected after 4 days post inoculation. The real-time PCR assay was useful for the specific detection of P. tabacina in field samples, artificially inoculated leaves, roots, and systemically infected tobacco seedlings and could be used as a tool for regulatory agencies interested in the detection of the pathogen. A second objective was to examine the genetic structure of the pathogen in North America, Central America, the Caribbean and Europe and determine the direction of migration of the pathogen. The intergenic spacer Igs2 region of the nuclear ribosomal DNA (rDNA) and the Ras-related protein (Ypt1) gene, and the mitochondrial cytochrome c oxidase subunit 2 (cox2 gene) were used. Populations of P. tabacina were characterized by high nuclear diversity, low population division and a possible mixed sexual and asexual reproductive system. Large population size, the mechanism of dispersal, mutation rate and genetic diversity indicate that this pathogen is a high evolutionary risk plant pathogen. An isolation with migration (IM) model was used to study genetic diversity in the U.S./Central America and the Caribbean (CCAM) and the European subpopulations. Results support migration from the CCAM region, Florida and Texas into the northern part of the U.S. including North Carolina. These data validate previous migration reports of the pathogen by the North American Plant Disease Forecasting Center at NCSU. In Europe estimates for the migration of the pathogen from North Central to Western Europe and both these regions to Lebanon support migration reports for the first introductions of the pathogen into Europe. Mitochondrial sequences of P tabacina and Hyaloperonspora parasitica genome were generated using bioinforrmatics approaches and PCR methodology. One quarter of the mitochondrial genome of P. tabacina has been annotated and compared with that of Phytophthora infestans and Hyaloperonospora parasitica. Similarities in direction, arrangement and number of genes and regions have been found. Results from this research will be useful in understanding the evolutionary history of the Oomycetes as well as future population genetics research to investigate how these organisms adapt, survive and attack plants in a changing environment.

Cyst nematodes in the genus Heterodera are sedentary endoparasites that induce elaborate feeding cells within host roots by secreting proteins produced within nematode esophageal glands into plant cells. Functional analyses of selected cyst nematode parasitism genes that encode such secreted proteins was the objective of this dissertation. Homologs of four parasitism genes initially isolated from Heterodera glycines, including Hg4F01 (annexin-like protein), HgSYV46 (CLAVATA3-like plant peptide mimic), Hg4E02 and Hg5D08 (novel proteins with putative host nuclear localization) were isolated from Heterodera schachtii, which can infect Arabidopsis thaliana. Greater than 90% nucleotide and predicted amino acid identity existed between the four parasitism genes homologs of H. glycines and H. schachtii. mRNA in situ hybridization and immunolocalization confirmed the expression of each gene product exclusively within the nematode esophageal gland cells. Since eukaryotic annexins affect many cellular processes involving calcium-dependent membrane association, the potential function of the Hs4F01 secreted into plant cells was analyzed. Similar to annexin mutants in Arabidopsis, transgenic Arabidopsis expressing Hs4F01 produced no observable plant phenotype, but were more susceptible to nematode infection. Hypersensitivity to osmotic stress in an Arabidopsis annAt1 annexin mutant was reduced (complemented) in mutants that expressed Hs4F01, suggesting a functional similarity of nematode and plant annexins within plant cells. Host derived RNA interference (RNAi) to silence Hs4F01 transcripts significantly reduced the number of H. schachtii females developing on roots that express dsRNA to Hs4F01. Expression of Hs4E02 and Hs5D08 in Arabidopsis produced no observable plant phenotype and susceptibility to H. schachtii was not altered in plants that expressed Hs4E02. Silencing of HsSYV46 using host-derived RNAi demonstrated a significant reduction in the development of nematode females on Arabidopsis roots that expressed double-stranded RNA to HsSYV46. Expression of dsRNA to Hs4E02 and Hs5D08 in Arabidopsis roots did not affect nematode susceptibility. In summary, parasitism gene products confirmed to have cellular functions similar to their plant homologs, including Hs4F01 (annexin-like protein) and HsSYV46 (CLAVATA/ESR-like peptide) were demonstrated by RNAi to have a significant biological role in cyst nematode parasitism of host plant roots.