Chemokines are central to the migration of leukocytes around the body, during both inflammatory and homeostatic conditions. Chemokines mediate their effects by binding to chemokine receptors found on the migrating cell’s surface. Chemokine binding to the chemokine receptor results in signaling, which allows the cell to migrate towards the epicenter of chemokine production. In addition to ‘classical’ chemokine receptors which are involved in leukocyte migration, a discrete family of chemokine receptors exist which are considered to be ‘atypical’, as binding to their cognate ligands does not result in classical signaling as detected by calcium flux assays. One of these atypical chemokine receptors is the chemokinescavenging receptor D6, which can bind to and internalize at least 14 inflammatory CC chemokines in vitro. In addition, an analysis of D6 function in vivo has shown that D6 is important for the resolution of the inflammatory response. D6 KO mice treated with phorbol ester to the shaved dorsal skin developed an inflammatory skin pathology that resembled the human condition psoriasis in many respects. In contrast, WT mice treated with phorbol ester developed a very mild inflammatory response, which quickly resolved. These data suggested that a loss of D6 expression ‘primed’ the mouse to develop a psoriasisform pathology, requiring only minor irritation/trauma to develop the pathology. Similarly, histologically normal (uninvolved) skin from a psoriatic patient has a propensity to developing inflammatory lesions upon minor trauma, and could also be suggested to be ‘primed’ for lesion development. Collectively, these data led us to the following hypothesis, ‘A loss of D6 expression in uninvolved psoriatic skin is associated with the development of psoriatic lesions’. To test this hypothesis, D6 expression in clinical samples from psoriasis patients was analysed. Full thickness biopsies from psoriasis patients were taken from a histologically normal site (uninvolved psoriatic skin), in addition to an elliptical biopsy covering the skin directly adjacent to the psoriatic lesion (peri-lesional psoriatic skin), in addition to the lesion itself (lesional psoriatic skin). D6 expression was analysed in these biopsies by QPCR and immuno-staining. It was observed that D6 expression was significantly elevated in psoriatic skin compared to healthy control skin. In particular, in uninvolved psoriatic skin D6 was significantly increased compared to healthy control skin, or peri-lesional 3 psoriatic skin or lesional psoriatic skin. The increase in D6 expression in uninvolved psoriatic skin localised to the epidermis and the LVs. A significant increase in PBMC-D6 expression was also noted in psoriatic patients compared to healthy control PBMCs. These data suggest that at sites not directly involved in the pathology, D6 is elevated in an attempt to limit inflammation-induced damage. Further immuno-staining showed the inflammatory CC chemokines CCL2 and CCL5 (both high affinity D6-binding ligands) were detected in uninvolved psoriatic epidermis, but were apparently unable to mediate their function due to the lack of significant leukocyte infiltration into the tissue. These data gave rise to the idea that D6 in uninvolved psoriatic skin was significantly elevated in an attempt to block the release of inflammatory CC chemokines into the dermis, and subsequent migration of inflammatory leukocytes into the tissue, and the onset of lesion formation. Interestingly, D6 expression on the epidermis was strongest towards the lower layers of the epidermis, which suggested a role for epidermal- D6 in ‘barrier function’, preventing the uncontrolled release of inflammatory CC chemokines into the dermis. In addition to inflammatory CC chemokines, a variety of inflammatory cytokines have been previously detected in uninvolved psoriatic skin. Several of these cytokines were shown to increase D6 expression in vitro in this study. Therefore, it is possible the significant increase in D6 expression in uninvolved psoriatic skin is partly mediated by cytokine stimulation. A loss of D6 expression was observed when comparing uninvolved psoriatic skin and perilesional psoriatic skin. These data suggested that a loss of D6 expression occurs directly before the onset of lesion formation. It was also shown in this study that a loss of D6 expression could occur after micro-trauma to uninvolved psoriatic skin, which suggests a possible mechanism of how D6 expression is lost in peri-lesional psoriatic skin. To analyze whether the increase in D6 expression in psoriatic skin was disease specific, or a generic response to cutaneous inflammation, D6 expression in eczema skin was studied. It was found that D6 expression in eczema skin is elevated compared to healthy control skin, but less so compared to psoriatic skin. Collectively these data suggest that increased D6 expression may be a feature of inflammatory skin diseases.

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.

Harnessing the actions of the resolvin pathways has the potential for the treatment of a wide-range of conditions associated with overt inflammatory signalling. Aspirin-triggered resolvin D1 (AT-RvD1) is a potent anti-inflammatory lipid derived from docosahexaenoic acid (DHA). In rodent, the biological effects of AT-RvD1 are mainly mediated by its specific G-protein coupled receptor, formyl peptide receptor 2 (ALX/FPR2). AT-RvD1 has been demonstrated potent anti-inflammatory and tissue resolution promoting (pro-resolving) activities in preclinical model of inflammatory diseases. Continuous peripheral activity of nociceptive fibres induces neuroinflammatory responses and alters the excitability neurones in both peripheral and central pain pathways. This thesis focused on spinal neuroinflammation which plays important roles in the sensitisation of nociceptive processing of the spinal dorsal horn neurones and contributes to the nociceptive hypersensitivity of the central pain pathway in acute and chronic pain. Since AT-RvD1 possess a potent anti-inflammatory activity, therefore, enhancing AT-RvD1 signalling in the spinal cord may attenuate the spinal nociceptive sensitisation and provide analgesia. AT-RvD1 also has robust antinociceptive effects in behavioural models of pain, however the potential underlying spinal neurophysiological mechanisms contributing to these inhibitory effects in vivo are yet to be determined. The purposes of this thesis were to investigate the differential effects of spinal AT-RvD1 on evoked responses of spinal neurones in vivo in well characterised pain models include carrageenan-induced acute inflammatory pain, monosodium iodoacetate (MIA)-induced OA pain and paclitaxel (PCX)-induced peripheral neuropathic pain and to investigate the alterations in the expression of spinal genes relevant to the resolvin system and neuroinflammation that may underlie the differential effects of AT-RvD1 among these pain models. Following model induction, pain behaviour was assessed and then rats were prepared for single unit extracellular recordings of dorsal horn wide dynamic range (WDR) neurones on the following day post carrageenan or day 28-32 post MIA or PCX induction. At the time-matched the spinal recording, ipsilateral dorsal quadrants of spinal cord form separate groups of rats were collected for gene expression quantification using TaqMan Low Density Arrays (TLDA). AT-RvD1 clearly demonstrated differential inhibition on evoked responses of WDR neurones in the pain models of interest. Low dose AT-RvD1 (15 ng/50ul) selectively produced a robust inhibition of electrical-evoked responses (Adelta-, C-fibre, post-discharge, input, wind-up) of WDR neurones in carrageenan rats but not in control rats. These effects were abolished by spinal pre-administration of a FPR2/ALX antagonist, butoxy carbonyl-Phe-Leu-Phe-Leu-Phe (BOC-2) (50 ug/50ul). In MIA rats, AT-RvD1 given at a high dose (150 ng/50ul) produced only mild inhibition of electrical evoked Adelta responses but not at a low dose (15 ng/50ul). AT-RvD1 (15 and 150 ng/50ul) had no significant effects on electrical evoked responses of PCX rat WDR neurones. Interestingly, AT-RvD1 produced a dose dependent and selective inhibition of low intensity mechanical evoked responses PCX rats whereas it had no effects in control rats. At high dose (150 ng/50ul), the magnitude of inhibition of 8g mechanical evoked responses was comparable to morphine (1 ug/50ul) applied at the end of the experiments. AT-RvD1 produced a dose dependent inhibition of acetone-evoked responses in PCX rats, however WDR neurones in the control rats were also inhibited to a similar degree. TLDA revealed the distinct alteration of resolvin genes in spinal cord underpinning the differential inhibition of AT-TvD1. Upregulated 5-lipoxygenase activating protein (FLAP) gene, encoding protein determining endogenous resolvin synthesis, in carrageenan and PCX rats may underlie the robust inhibition of AT-RvD1 on WDR neurones in these two models. The minimal effects AT-RvD1 of MIA rats were associated with upregulated 15-hydroxyprostaglandin dehydrogenase (15-HPGD) gene, encoding a major enzyme responsible for D-series resolvin inactivation. Data presented in this thesis provide for the first time the differential inhibition of AT-RvD1 on spinal nociceptive processing in different types of pain and the evidence of heterogeneous spinal alterations of the resolvin signalling potentially underlie such inhibition. This thesis strongly supports further investigation of AT-RvD1 as a novel analgesic. Another part of this thesis demonstrated changes in the responses of spinal WDR neurones in PCX rats. Spinal WDR neurones from PCX rats displayed characteristics representing pain sensitisation including: reduced the thresholds for Abeta and C-fibre responses; increased proportion of neurones exhibiting spontaneous activity, acetone responsiveness and post-discharge following low intensity mechanical stimuli and a more convergence in stimulus processing. A remarkable upregulation of multiple genes in proinflammatory signallings, toll-liked receptor 4 (Tlr4), interleukin-1 (Nlrp1a) and tumour necrosis factor-alpha receptors (Tnfrsf1a and Tnfrsf1b) and chemokines (Cxcl6, Cxcr1, Cxcr5, Ccr1, Cx3cr1), was found in the spinal cord of PCX rats. This suggests that a high inflammatory component in the spinal cord could contribute to the changes in the responses of spinal WDR neurones in the PCX rats. This thesis supports further investigation of targeting neuroinflammation as a promising approach for the treatment of PCX-induced neuropathic pain.

I have explored the infection of human cells by the gammaretroviruses XMLV (xenotropic murine leukaemia virus) and FeLV (feline leukaemia virus). For XMLV the main aim was to assess the risks and consequences of contamination of human-mouse xenografts. For FeLV, the aim was to explore the susceptibility of human cells in vitro to assess the risks and identify the barriers to zoonotic infection in vivo. Xenografting of human cells to mice is used commonly in many disciplines of biomedical science. Infection of xenograft-derived cell lines has been reported as a common observation but it was unclear how many of these lines were infected due to in vitro cross-contamination rather than de novo infection in vivo. I conducted a prospective study and demonstrated that more than 40% xenografts passaged through BALB/c nude mice acquired XMLV. Xenografts passaged through NSG, another commonly used mouse strain for engraftment studies, did not yield replication-competent XMLV, although there was some evidence of activation of related replication-defective viruses. The source of XMLV in BALB/c mice appeared to be Bxv1, a locus encoding a replication competent virus which I showed was absent from NSG mice. I also showed that de novo isolated XMLV replicates to high copy number in MCF7 breast cancer and induces subtle changes in growth properties. Transcriptome analysis suggested that up regulation of EGR1 (early growth response gene 1) may be responsible for these growth effects. I also analysed susceptibility to XMLV infection in human cells and showed that while primary PBMCs are highly resistant, many cell lines can be infected. The Raji Burkitt’s lymphoma cell line was found to be highly susceptible to XMLV and displayed a much larger transcriptional response compared to MCF7, with marked up regulation of a series of markers of innate immunity. I examined the susceptibility of human cells to infection with FeLV B, the viral subtype which appears to be the most likely zoonotic agent. Cell lines of human origin were found to vary in susceptibility with no clear relationship to cell lineage. While some cell lines were completely susceptible for FeLV B, most showed limited virus replication and G to A hypermutation that correlated with the expression of APOBEC family members. Primary PBMCs and some leukaemia cell lines showed profound resistance to FeLV B infection at an early stage of replication and accumulated proviral DNA with only a few mutations. The mediator of this early block has not yet been identified but appears likely to be important in preventing cross-species spread of gammaretroviruses to the human population.

The measurement of oxygen saturation SO2 is one of the vital signs relied on by the medical profession. Pulse oximeters are widely used in many branches of medicine; and are the most widely used method of assessing oxygen saturation. However they can only be applied to an extremity (usually a finger or toe), need calibration, and are known to be inaccurate under certain conditions. The object of this research was to develop an oximeter, that does not require a pulsatile signal, (and so can be used anywhere on the body); can be used in either transmission of reflective mode; does not require calibration; and does not suffer from the known problems of pulse oximeters. The instrument must work with reflected light, and so the first step was to develop a Monte Carlo simulation of the Attenuation spectra, for visible light, from a scattering media (tissue). A mathematical model of the attenuation surface had then to be found, and its effect on the absorbtion spectra of oxyhemoglobin HbO2 and de-oxyhemoglobin Hb understood. Then the oxygen saturation the ratio of HbO2 to total haemoglobin could be recovered. Methods of computing oxygen saturation from the raw reflectance spectra were devised and then tested with single reflection spectra, the results indicate that a low cost instrument could be developed. The technique was applied to images from a hyper-spectral camera, this instrument takes a full spectrum at each pixel of an image, and enabled an oxygen saturation map for large areas of the body to be produced. The technique is being used with AstraZenca Ltd as a bio marker skin for irritation studies.

Forensic science is growing rapidly in the world today. During the past decade, medico-legal investigations have been highly expanded to include all areas of forensic science. The present study investigated three important aspects of forensic biology. First, this present project investigated, a total number of 5,703 reported medico-legal cases diagnosed as un-natural deaths by The Forensic Department in Kuwait, during the year 2003-2009. The results show that accidental, homicidal and suicidal deaths accounted for 86%, 8% and 6%, respectively. The results showed that most people who died of unnatural deaths were more predominant in the age group 20-29 years (third decade). Road Traffic Accidents accounted for 65% of accidental deaths, and 4% out of them were related to alcohol consumption. The results also illustrated that the highest rate of homicide in Kuwait was due to stab wound injuries (38%) compared to the lower rate of homicidal pattern for infanticides (3%). Similarly, the study showed that the most common method of suicide in Kuwait was death by hanging and this accounted for (60%). This study further demonstrated the effectiveness of virtual autopsy technique as a new tool in forensic investigations to determine various un-natural death causes. A total of thirty (30) male forensic cadavers were employed in this project. The cases were RTA (11), firearm injuries (10), drowning (4), head injuries (3) and lastly strangulation (2). All these cases were compared to the findings of traditional autopsy. The results show similar findings for virtopsy compared to traditional autopsy. This study clearly revealed that virtopsy could be an effective alternative in certain situation, being noninvasive and rapid. The present project also investigated 28 samples of human blood, saliva or semen. The experiments were done at four different temperatures (55°C, 37°C, 24°C and 4°C) and four different humidity ranges (41%, 55%, 58% and 61%), respectively. The results showed that, DNA quantity in blood, saliva and semen samples remained more or less the same at temperatures of 4°C, 24°C and 37°C compared to values for day one with all other days. In contrast, when the temperature was raised to 55 °C, the DNA started to degrade with time until it reached zero at day 12 for saliva and day 15 for blood, but not for semen. The results clearly show that DNA in saliva and blood samples is extremely sensitive to heat compared to semen. In conclusion, the study reveals the different causes of unnatural deaths, the value of virtual autopsy and the need for early DNA measurement in Kuwait.

This thesis aimed to evaluate the contribution of several pneumococcal two-component systems to virulence by analysis of null mutants in a murine model of infection. In addition, a putative zinc metalloprotease, ZmpB, located immediately downstream of one of the TCS, was analysed for its role in virulence. Data indicated that one of the systems studied, TCS08, does not contribute significantly to virulence in serotype 2 pneumococcus, but may have a slightly more important role in a serotype 3 background. A second two-component system, TCS09, was found to be essential for virulence in a serotype 2. Despite the completely avirulent phenotype of the mutant, no difference in expression of many of the previously identified pneumococcal virulence-associated genes was detected in the mutant compared to its isogenic parental strain. Microarray analysis indicated that in serotype 2, TCS09 may be involved in nutrient perception in nutrient perception. TCS09 was found to be required for full virulence in a serotype 3 strain. In this strain, mutants appeared to be impaired in their ability to disseminate from the lungs to the blood in a pneumonia model of infection, but were not attenuated in virulence following direct inoculation into the systemic circulation. These data provide evidence that virulence determinants can behave differently based on the genetic background of the parental strain. ZmpB was found to contribute significantly to pneumococcal virulence in a serotype 3 strain. Further analysis of the contribution of this protein to infection found that ZmpB appears to have a role in promoting inflammation. Thus this work has identified ZmpB as being a novel pneumococcal virulence factor. The role of this protein in inflammation is being investigated further. This thesis has thus identified several genes important in the virulence of S. pneumoniae and work is currently ongoing to assess the potential of these genes as future vaccine or drug candidates. Data presented within this work also provides evidence that virulence determinants can behave differently based on the genetic background of the parent bacterial strain. This important observation could have significant implication for the future characterisation of pneumococcal virulence factors and may apply to other bacterial pathogens.

Background: Malnutrition and the cachexia syndrome are common in people with cancer. A combination of reduced nutritional intake and abnormal metabolism can lead to physical and psychological disturbances which may impair quality of life and reduce survival. Improved patient outcomes are more likely if treatments and nutritional support can be initiated before significant weight loss has occurred. Methods: A three phase, mixed methods study was undertaken. The primary aim was to gain a greater understanding of the complex factors that have an effect on, and can predict, weight loss in people with cancer. Phases I and II involved the psychometric testing of the Cancer Appetite and Symptom Questionnaire (CASQ). The instrument was tested for reliability among patients receiving radiotherapy (n=34). Predictive validity of the CASQ, using ROC curve analysis, was determined in patients with lung or upper GI cancer (n=185). Total CASQ scores (possible range, 0 to 48) were assessed at baseline, together with percentage weight change after 3 months. An exploratory qualitative study, following the principles of grounded theory, was conducted to explore the causes and influencing factors on weight change. Results: When tested for reliability, the intra-class correlation coefficient of the CASQ was 0.80 (95% CI 0.68 to 0.92) and the difference between total CASQ scores at the two time points was -0.20 (95% CI -1.21 to 0.80). The optimum cut point of the total CASQ score to predict >5% weight loss was 31/32 (C statistic = 0.64; sensitivity 65%, specificity 62%, PPV 33%, NPV 86%), and to predict >10% weight loss was 29/30 (C statistic = 0.75; sensitivity 71%, specificity 66%, PPV 19%, NPV 95%). Exploratory modelling using multiple linear regression techniques suggested that BMI, MUST score, age and the CASQ items of enjoyment of food and pain, were most predictive of weight loss. Nine patients with lung or upper GI cancer and three carers participated in semi-structured interviews. Analysis of the data confirmed the vulnerability of this patient group in terms of symptom burden and nutritional risk. From the findings, a conceptual model that explains the influences on weight change in people with cancer was proposed. Conclusions: Patients with lung and upper GI cancer are at high risk of malnutrition. Psychometric testing of the CASQ suggests that the instrument can predict weight loss in this patient group. Due to the low PPV, further refinements are needed before the instrument is able to be used in clinical practice. A conceptual model which explains the complex process of influences on weight change in people with cancer can improve knowledge and understanding, ultimately informing healthcare practice.