Epstein Barr Virus (EBV) is a major risk factor in Multiple Sclerosis (MS), via as yet unclear mechanisms. Several hypotheses have been proposed to explain how EBV infection could cause MS and the aim of this thesis was to better understand the mechanisms of action of EBV in the context of MS studying a) the role of EBV in myelin antigen presentation by B cells and b) the association of HERVs with MS. In a non-human primate experimental autoimmune encephalomyelitis (EAE) model, an EBV-related lymphocryptovirus enables B cells to protect a proteolysis-sensitive immunodominant myelin oligodendrocyte glycoprotein (MOG) peptide (residues 35- 55) against destructive processing. This facilitates its cross-presentation to autoaggressive cytotoxic MHC-E-restricted cytotoxic T cells. The present study extends these observations to human B cells and identifies a key role of autophagy. EBV infection upregulated antigen presentation-related markers on B cells and activated the cross-presentation machinery. Although human MOG protein was degraded less in EBV-immortalized B-lymphoblastoid cell lines (LCL) than in uninfected B cells, induction of cathepsin G activity by EBV led to total degradation of the immunodominant peptides. Inhibition of cathepsin G or citrullination of the arginine residue within a LC3-interacting regions (LIR) motif of immunodominant MOG peptides abrogated their degradation. Internalized MOG co-localized with autophagosomes, which may protect it from destructive processing. Thus, EBV infection switched MOG processing in B cells from destructive to productive possibly facilitating cross-presentation of disease-relevant epitopes to CD8+ T cells. This mechanism could facilitate presentation of myelin autoantigens that may be involved in MS induction and progression. The first part of this thesis shows a possible EBV-mediated mechanism involved in MS pathogenesis, but it is likely that different mechanisms act alternatively or cumulatively in different individuals based on environmental and genetic differences. A further mode of action of EBV is through the activation of Human Endogenous Retroviruses (HERVs). In normal conditions HERVs are silenced or expressed at low levels, but in some pathological cases, like MS, their expression is higher than in the healthy population. We performed a systematic review and meta-analysis of the literature on the association between HERVs and MS. The systematic review suggested a strong association between HERV expression and MS, in particular with the HERV-W family. The meta-analysis showed odds ratios of 22, 44, and 6 for the expression of MSRVpol in serum/plasma, MSRVenv in PBMC and MSRVpol in CSF respectively. Furthermore, we confirmed the association experimentally. An increased expression of MSRV/HERV-Wenv and TLR4 RNA was detected in blood of MS patients compared with control groups and the viral protein Env was expressed mainly by B cells and monocytes, but not by T cells. Our finding that EBV infection can induce the expression of MSRV/HERV-Wenv is consistent with previous reports in the literature. We also established that such increased expression was not due to a repression of retroviral restriction factors in LCL. A further connection between HERVs and MS is supported by the observation that people infected by HIV may have a lower risk of developing MS than the HIV non- infected, healthy population. We found that the expression of MSRV/HERV-Wenv RNA in HIV-infected people was lower than in MS patients and similar to healthy controls. Nevertheless, there was no difference in MSRV/HERV-Wenv expression between antiretroviral drug -treated and -untreated HIV patients. The expression of MSRV/HERV-Wenv was also detected in vitro in LCL treated with different classes of antiretroviral treatments (ART) and only Efavirenz (NNRI) reduced MSRV/HERV- Wenv expression. In conclusion, taking in consideration the multifactorial aetiology of MS, it is likely that EBV infection and increased expression of MSRV/HERV-W are significant contributing factors in genetically predisposed individuals. This thesis helps to better understand the mechanisms of action of EBV and HERVs in the context of MS.

Background Community acquired pneumonia (CAP) is a common illness in patients admitted to hospital, accounting for nearly 10% of acute medical admissions. Despite widespread use of antimicrobial therapy, morbidity and mortality from this disease remains high. In recent years in the UK, there have been significant developments in both preventative and treatment strategies for this illness. To understand the impact of these changes and direct future management strategies, it is important that the epidemiology of this disease is fully understood. This thesis investigates changes in epidemiology and outcomes in adult patients admitted to UK hospitals with a primary diagnosis of CAP, in recent years and with reference to the herd protection effect of the conjugate Streptococcus pneumoniae vaccine. Methods There are 3 study populations presented in this thesis. Data were derived from (a) the British Thoracic Society national CAP audit database, (b) a longitudinal cohort study of adults hospitalised with CAP, within the Greater Nottingham area, and (c) an observational study of adults admitted to four hospitals within the East Midlands area with a diagnosis of CAP. The specific methods used for the identification of study participants, laboratory and statistical analysis are described in detail in ensuing chapters. Results Across the UK, there was a significant reduction in 30-day mortality between 2009 and 2014; this improvement in outcome may be attributable in some part to improved processes of care. Whilst data derived from coding databases have previously been used to report CAP related mortality trends, this thesis has demonstrated significant variation in coding accuracy across UK institutions and that miscoded cases of pneumonia had lower odds of 30 day mortality compared to those individuals with CAP. Consequent to herd effects from national infant vaccination programmes and changes in nasopharyngeal carriage of S pneumoniae, this thesis shows that (a) school holiday periods were associated with increased incidence of pneumococcal CAP in hospitalised adults, (b) older adults at high risk of pneumococcal disease were less likely to be hospitalised with vaccine serotype CAP compared to non-vaccine-serotype pneumococcal CAP, and (c) there was a decrease in the overall burden of vaccine-serotype pneumococcal CAP compared to non-vaccine-serotype pneumococcal CAP. Conclusion Important changes in the epidemiology of adult CAP in the UK over recent years are reported in this thesis. This includes temporal decreases in mortality rates of all cause CAP, as well as a significant ongoing burden of non-vaccine serotype pneumococcal CAP. This data on the current burden of disease and associated outcomes should help inform future CAP management strategies.

Background Malaria and pneumonia are the leading causes of under-five mortality in sub-Saharan Africa especially in Nigeria. The Integrated Management of Childhood Illness (IMCI) guidelines were developed by the World Health Organisation (WHO)/United Nations Children’s Fund (UNICEF) as a strategy to reduce the burden of these and other preventable childhood diseases. However, there appears to be a paucity of evidence on the diagnostic performance of the revised IMCI guidelines and whether they offer an advantage over lay diagnosis (caregiver) for malaria and pneumonia management in Nigeria. Aim and specific objectives This study evaluates early diagnostic approaches (IMCI guidelines and lay diagnosis) for malaria and pneumonia in children under-five at the primary healthcare level. To address the overarching aim of the study, the following four specific objectives were studied: I. To assess the diagnostic accuracy of the IMCI guidelines and lay diagnosis (caregiver) for malaria and pneumonia in comparison to reference diagnostic approaches (microscopy and chest X-ray for malaria and pneumonia respectively). The extent of agreement between caregivers’ and health workers’ diagnosis of malaria and pneumonia is also assessed. II. To estimate the burden of malaria and pneumonia among children under-five presenting to study primary healthcare centres (PHCs) according to various diagnostic approaches. III. To determine the clinical outcomes in children diagnosed with malaria and pneumonia according to the IMCI guidelines and risk factors for severe outcomes. IV. To qualitatively explore caregivers’ and health professionals’ perspectives on lay diagnosis and IMCI guidelines as diagnostic approaches for childhood malaria and pneumonia. Methods A mixed methods approach was used for this study. The quantitative component used a consecutive sampling approach to recruit 903 children aged 2–59 months who met study eligibility criteria for malaria and pneumonia assessment according to the IMCI guidelines at presentation to five study PHCs in Benin City, Nigeria. Caregivers of these children were also asked what they thought the diagnosis was (lay diagnosis). Diagnostic accuracy was assessed in terms of sensitivity, specificity, positive and negative predictive values, Area under the Receiver Operating Characteristic Curves (AUROC) values and 95% Confidence Intervals (C.I). The extent of agreement was assessed in terms of Cohen’s kappa statistic (k) and 95% CI. The estimated burden of malaria and pneumonia during the study period was assessed using proportions and 95% C.I. Clinical outcomes in children diagnosed with malaria and pneumonia by the IMCI guidelines were described in terms of frequency and percentages, while the potential risk factors associated with clinical outcomes were assessed using odds ratios (ORs) and 95% C.I. For the qualitative component, health stakeholders (17 health professionals and 13 caregivers) who met the study eligibility criteria were purposively recruited and interviewed using semi-structured interviews. An inductive approach to thematic analysis was used for data analysis. Results Compared to microscopy, the diagnosis of malaria by health workers using the IMCI guidelines was poorly accurate with an AUROC value of 0.57 (with sensitivity and specificity of 51.8% and 61.3% respectively). Similarly, caregivers’ diagnosis of malaria was poor with an AUROC value of 0.55 (with sensitivity and specificity of 31.1% and 79.5% respectively) as compared to microscopy. Using the IMCI guidelines as the reference diagnostic test, caregivers’ diagnosis of malaria was more accurate (AUROC 0.60) in comparison to that of pneumonia (AUROC 0.54). There was a slight or minimal level of agreement (k=0.14; 95% CI: 0.09-0.19) between caregivers and health workers in the diagnosis of malaria and pneumonia. The estimated burden of malaria and pneumonia was relatively low, varying by the study local government areas, PHCs and seasonality, irrespective of the diagnostic approach. Where follow-up data were available, approximately 57% (172/304) and 78% (81/104) of the children diagnosed with malaria and pneumonia, respectively, recovered without complications within 30 days. Self-medication prior to presenting to study PHCs and use of preventive measures against malaria were independently and significantly associated with improved clinical outcomes. In contrast, exposure to solid fuels increased the odds of severe illness following malaria or pneumonia diagnosis. The qualitative component of the study found that caregivers rely on lay diagnosis despite the awareness of its limitations. The perceptions of malaria and pneumonia appeared to influence caregivers’ home management practices and health seeking behaviours. Caregivers showed willingness to be trained in the IMCI guidelines for improved home-based management of malaria and pneumonia. Health professionals believed that the IMCI guidelines were useful for managing both malaria and pneumonia. However, there are some recurring challenges to the wide-scale and sustainable implementation of the IMCI strategy in Nigeria. These include inaccurate diagnosis of malaria and inadequate funding. Conclusion The IMCI guidelines are crucial in the effective management (diagnosis and treatment) of malaria and pneumonia at the primary healthcare level in Nigeria. Although not perfect, lay diagnosis has an important contribution in the early detection and management of malaria and pneumonia at the community level in Nigeria. However, there is need for further investment in the training of both health professionals and caregivers in the IMCI guidelines for better health outcomes in under-five population. The training of caregivers in the IMCI guidelines and potential for a scale-up will benefit from careful design, piloting, implementation, and monitoring.

Background Papular pruritic eruption (PPE) of human immunodeficiency virus (HIV) infection is common HIV-infected populations who live in tropical and subtropical regions. It is characterized by chronic and intensely itchy papules that are usually more highly concentrated on the extremities, adversely impacting on quality of life. Its aetiology has been postulated to be an altered and exaggerated immunological response to insect bites or stings. It has been reported to diminish in severity or resolve with antiretroviral therapy (ART). Its presence after at least six months of ART has been proposed as one of several clinical markers of failure of antiretroviral treatment. Objectives 1. To systematically summarise the evidence of interventions for PPE 2. To translate, culturally adapt, and test oral administration of a Runyankore-version of Skindex-16 for use in dermatology research in Mbarara, Uganda 3. To determine factors associated with PPE in HIV-infected Ugandan adults receiving ART for at least 15 months 4. To describe the natural history of PPE in HIV-infected Ugandan adults over two years from the time of ART initiation and explore the association between recurrent or persistent PPE and antiretroviral treatment failure Methods Systematic review of interventions for PPE Electronic searches of Medical Literature Analysis And Retrieval System Online (Medline), Excerpta Medica Database (Embase), Cumulative Index To Nursing And Allied Health Literature (CINAHL), Global Health Library, Cochrane Library, World Health Organization (WHO) International Clinical trials registry and National Library Of Medicine (NLM) gateway were carried out from January 1980 to July 2014. Studies of any design were included. The primary outcome measure for this review was resolution of skin disease. The quality of evidence was assessed using the Newcastle-Ottawa quality assessment scale and Grading Of Recommendation, Assessment, Development And Evaluation (GRADE) approach, where appropriate. Two authors carried out data extraction and quality assessment of studies independently. Runyankore-version of Skindex-16 for oral administration in Mbarara, Uganda Skindex-16 in English was translated to Runyankore, and then back-translated to English. The original and back-translated versions of Skindex-16 were compared for fidelity of translation. The Runyankore-version was administered orally to 47 dermatology patients and 47 random hospital visitors. Study participants were also asked about the characteristics of their skin condition including its duration, presence of skin colour change and ease or difficulty of concealment as well as an open question on how their skin condition has affected them. Case control study examining factors associated with PPE in the ART era This is a case–control study nested within a 515-person cohort receiving care at the HIV clinic of a teaching hospital in Mbarara, Uganda. Forty-five cases and 90 controls were enrolled. Cases had received ART for ≥15 months, fulfilled the clinical case definition of PPE and had skin biopsy findings consistent with PPE. Each case was individually matched with two controls for age, sex and ART duration. Cohort study describing the natural history of PPE over two years from ART initiation This is a cohort study of HIV-infected Uganda adults initiating ART and receiving care at the HIV clinic of a teaching hospital in Mbarara, Uganda who fulfilled the clinical case definition of PPE and had skin biopsy findings consistent with PPE. Standardised interviews, clinical photography, HIV viral load, CD4 counts and CD8+ T-cell activation markers were measured at three-month intervals for two years. Results Systematic review of interventions for PPE No randomised controlled trials were identified. Thirteen studies with a total 188 participants were included. ART was associated with resolution of PPE in a prospective observational study that had high loss to follow-up rates. Two observational studies reported positive responses of PPE to oral antihistamines (promethazine and cetirizine). Pentoxifylline was associated with diminished signs and symptoms of PPE in an uncontrolled open trial and superior to dapsone and a combination of antihistamine and topical corticosteroids in a parallel group non-randomised trial. Resolution of PPE was reported with a combination of topical corticosteroids and oral antihistamines in a case report. The efficacy of ultraviolet B (UVB) phototherapy was reported in an observational study with eight participants and three case studies with a total of five participants. Runyankore-version of Skindex-16 for oral administration in Mbarara, Uganda Oral delivery was feasible, taking ≤10 minutes per subject. High Cronbach α values (0.86, 0.88 and 0.85 for Symptoms, Emotions and Functioning subscales, respectively) demonstrated internal consistency reliability. As hypothesised, subjects with reported skin problems, dyspigmentation and difficulty in concealment had higher mean Skindex-16 scores, indicating construct validity. A large proportion (72.4%) of responses to the open-ended question were addressed in Skindex-16, indicating content validity. Case control study examining factors associated with PPE in the ART era Twenty-five of 45 cases (56%) had histological findings consistent with PPE (known as PPE cases). At skin examination and biopsy (study enrolment), a similar proportion of PPE cases and their matched controls had plasma HIV RNA <400 copies/ml (96% vs. 85%, p=0·31). The odds of having PPE increased four-fold with every log increase in viral load at ART initiation (p=0.02) but not at study enrolment. CD4 counts at ART initiation and study enrolment, and CD4 gains and CD8 T-cell activation measured 6 and 12 months after ART commencement were not associated with the presence of PPE. Study participants who reported daily insect bites had greater odds of being cases [odds ratio (OR) 8.3, p<0.001] or PPE cases (OR 8.6, p=0.01). Cohort study of natural history of PPE over 2 years from ART initiation Seventeen (15 female and 2 male) participants with a median age of 29.8 years were enrolled. Median CD4 count and HIV viral load at ART commencement was 108 cells/mm3 and 114,442 copies/ml, respectively. Resolution of PPE occurred in 13 of 17 (76%) study participants at a median time of 8.5 months after ART initiation, although PPE recurrence was observed at seven participants during the study period. Two participants had persistent PPE. Virological failure was not detected in any study participant. HIV RNA was less than 400 copies/ml at a median time of three months from ART initiation in all study participants. Conclusions 1. The evidence base of interventions for PPE is of low quality. There is some evidence of the efficacy of ART in the management of PPE. Pentoxifylline and phototherapy may have a role in its management but are unlikely to be available in resource-limited settings. Oral antihistamines and topical corticosteroids may be helpful in some individuals affected by PPE. 2. The orally administered Runyankore-version of Skindex-16 is reliable, with construct and content validity, and feasible for use in dermatology research in Mbarara, Uganda. 3. PPE in HIV-infected Ugandan adults receiving ART for at least 15 months was associated with reported daily insect bites and greater HIV viraemia at ART commencement, independent of CD4 count. 4. Recurrent or persistent PPE in HIV-infected Ugandan adults observed over two years from initiation of ART was not associated with virological failure in participants of this study.

Frontline healthcare workers (HCWs) in the UK have been prioritised for free occupational immunisation against seasonal influenza since 1999. During the 2009-10 influenza pandemic, they were identified as a priority group to receive the strain-specific vaccine. Nevertheless, take-up rates among HCWs have rarely exceeded 50%, even during the pandemic. Most attempts to change this situation have been predicated on the assumption that these low rates are the result of reluctance or resistance by individual HCWs, who must be persuaded or coerced to comply with employer directives. To gain a novel understanding of this immunisation programme, an actor-network theory approach is adopted to trace the journeys of vaccines through two Local Health Boards in Wales during the 2009-10 H1N1 influenza pandemic and in the following winter influenza season (i.e. during 2010-11). The research reported shows that low uptake is largely the result of complex social, organizational and cultural processes. Only when these have been changed will it be appropriate to frame the remaining problem as reluctance or resistance by individual HCWs. The study reveals that this immunisation programme is inherently unstable and subject to ambivalence from actors at all levels. Suggestions for practical improvement are given.

The Seamless Stressometer® roll (Seamless STR) is used to measure the flatness of aluminum and steel strip when there is an extreme demand on the surface finish. To protect the roll and strip, the roll is coated with two layers deposited by high velocity oxygen fueled spraying (HVOF), Cr-Ni(Si,B) closest to the roll and WC-Co on top. This solution has several disadvantages; high cost and complicated logistics, corrosion sensitivity and high residual stresses creates the need for two coatings which in turn complicates the process. Cobalt is, in addition, sensitive to low pH coolants and environmentally unfriendly. These problems have given rise to the idea of switching both the method and material of the coating. In the first part of this work, high velocity air fueled spraying (HVAF) was evaluated as an alternative deposition method. Three materials, Cr3C2-NiCr, WC-Co and WC-CrC-Ni were deposited on steel coupons with varying chamber pressure, powder feed rate and distance from the nozzle, in order to evaluate if HVAF can be a valid technique for use in this application and to optimize the spraying recipe. The objectives were to get a sufficiently high thickness per sweep, to be able to make the depositions in a manageable number of sweeps, and to get low porosity, since the coatings need to be dense to be hard and possible to polish smooth. The tests showed that all three materials can be sprayed with the high settings on the parameters to obtain coatings that exceeded the set limits of the objectives. In the second part of this work, the recipe obtained from the first part was used to deposit samples for further analysis. The coatings were compared regarding cost, hardness, friction, wear and pick-up properties to evaluate if a switch in material from WC-Co was possible. The coatings showed both similarities and differences. The friction was very similar for the three materials. Cr3C2-NiCr was substantially cheaper than the other two, had lower hardness and higher porosity, but still probably acceptable values, and was satisfactory regarding wear and pick-up. WC-Co and WC-CrC-Ni were very similar to each other regarding cost, hardness and porosity but WC-Co was the best regarding wear and pick-up, where WC-CrC-Ni was the worst. The only clear advantage of WC-CrC-Ni over WC-Co is the lack of cobalt. Taking everything into consideration, including the fact that the wear and pick-up tests in this work was quite exaggerated, Cr3C2-NiCr is an interesting option for this application due to its low cost and acceptable test results, WC-Co had the best results but is expensive and contains cobalt and WC-CrC-Ni had as good results as WC-Co except for the wear and pick-up tests and does not contain cobalt.

Abstract Tungsten carbide-cobalt powder is pressed before sintering into a compacted form using punches and a die cavity. After the powder has been pressed to a specific shape, it is sintered and shrinks a certain amount to a final size. To accommodate this shrinkage, the pressing tools are designed to a certain “shrinkage percentage” and thus the pressed component or compact is larger than the sintered component by that percentage amount. During the pressing process, there is a large amount of friction between the powder being compacted and the die cavity wall. To counter pressing friction, a lubricant is pre-mixed with the tungsten carbide powder. In the past at Powder Industries, the powder was mixed with wax and all of the tools were designed to a 20% shrinkage. In recent times, the wax in the powder has been replaced by PEG (polyethylene glycol) by most manufacturers as this increases the quality of the final product and is easier to remove in the furnaces. As a result of the new PEG lubricant, the tool wear rate at Powder Industries increased and because a higher pressure had been necessary to achieve powder pressing to the same shape and form, often the pressed components exhibited cracks or were not pressed ideally. On account of the problems introduced by PEG, correct tool design for the shrinkage was obtained by a ‘trial & error’ process. This project has been motivated by the need of establishing pressing and/or design ‘rules’ that would do away with trial and error when designing compaction tooling. The project has consisted of investigating the physical properties of 23 grades of WC-Co powder (with or without TiC and TaC) and of performing a series of pressing tests for each grade. A relationship between the apparent density of a powder and the ideal green density of the green compact pressed from the same powder has been found. Using this relationship, an equation has been derived between ideal shrinkage, powder apparent density, component sintered density and powder volatile content. Since the last three parameters are known to the tool designer, this equation can be used to calculate the ideal shrinkage when designing new compaction tooling. This method of calculating shrinkage is now in general use at Powder Industries and many successful sets of compaction tooling have already been manufactured

The growth of TiN on cemented carbide, deposited by chemical vapour deposition (CVD), was studied. Today TiN is used as a seeding layer between the cemented carbide and the following layer. Previous experiments have shown that the coverage is uneven on the cemented carbide surface showing pits with a different growth than the main part of the surface. These pits most likely occur on some of the WC surfaces. Therefore the growth and orientation relation between the two phases were examined. Cemented carbide specimens were deposited with TiN under two different pressures and with different deposition time to give a layer as thick as the seeding layer used in the production and one about ten times thicker to study growth after the whole surface had been covered. Two pre-treatments where used on the specimens; one which were polishing with diamond and the other where the specimens were boiled in acid to remove the binder phase and expose the WC surfaces. The specimens were studied using XRD and a SEM equipped with EDS and EBSD detectors. The pictures taken with SEM showed that initial growth occurred at grain boundaries and polishing scratches. It also showed that growth occurred on all surfaces, which were confirmed by EDS. Both processes showed about the same appearance in the thinner layers but very different appearance in the thicker. This was confirmed by XRD were the thinner layers showed about the same result while the thicker ones differ from one another. Thus further growth is dependent on the parameters of the CVD process and not the surface beneath. EBSD showed an orientation relation between TiN and WC crystals in both processes. The process at lower pressure gave much finer grains which were difficult to index with EBSD, giving results in only three points. The process at higher pressure gave coarser grains which were more easily indexed. The relations WC{0001}-TiN{110}, giving WC{101̅0}-TiN{100}, and WC{0001}-TiN{111}, giving WC{101̅0}-TiN{211}, could be seen in more than one point. These results are consistent with previous studies of the growth of TiC on WC. This comparison between TiC and TiN makes it possible to draw the conclusion that also Ti(C,N) should behave the same. It also shows an orientation relation that is consistent no matter what WC plane is on the surface. The poor growth and the pits depend on the growth orientation of TiN on each specific WC surface.

Hepatitis C Virus currently affects up to 3% of the world’s population. There is no effective vaccine yet available and the natural immune response to infection is largely inefficient. Progress has been made in isolating several broad-acting neutralizing antibodies that target the viral envelope protein E2. However, a dominant element of the epitopes targeted is an overlap with the highly conserved CD81 binding sites. Various E2 constructs were investigated as possible targets to be used in phage display panning of a combinatorial library of the phagemid vector pComb3H. HVR2 deletion showed optimal exposure of the CD81 binding sites and D535A disrupted known CD81 epitopes. A selection technique was designed to improve exposure of conserved sites on an E2 construct target molecule that disrupts CD81 epitopes while remaining conformationally correct. Optimisation of the screening methodology was used to assess the quality of enrichment of the library panning along with more efficient selection of specific clones. The approach adopted in this project isolated Fab clones specifically reactive to the protein target, one of which also showed preferential binding in acidic environments. Taken together, the information gathered on E2 and the implementation of the phage display method described will contribute to more effective ways of isolating novel antibodies.

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