Allogeneic bone marrow transplantation (BMT) is an important and commonly used treatment for a variety of haematological malignancies. Central to the therapeutic benefits of allogeneic BMT is the role of donor T cells, which mediate a potent anti-neoplastic effect against tumours in the recipient. However, the presence of donor T cells is also tightly associated with the development of the life-threatening condition graft-versus-host-disease (GVHD), a disorder where alloreactive donor T cells recognise the recipient as ‘non-self’ and elicit a potent anti-host immune response. Antigen presenting cells (APC) are critical to the induction of acute GVHD. However, recent studies have questioned whether any particular subset of APC has a non-redundant role in the priming of an alloreactive donor T cell response. One of the most commonly affected organs in acute GVHD is the skin, which contains a diverse array of professional APC subsets capable of initiating potent immune responses. One such subset is Langerhans cells (LC), a population of radioresistant dendritic cells (DC) that form a dense interlacing network within the epidermis. Using an in vivo mouse model of acute GVHD where host LC can be conditionally depleted, LC were found to play an important role in the development of acute GVHD in the skin. The depletion of host LC significantly reduced the severity of cutaneous GVHD that developed following allogeneic BMT. Host LC were required in situ in the epidermis for both the accumulation and function of alloreactive donor CD8 T cells in the skin. A role for host Langerin+ dDC, a population shown to be critical for CD8-mediated T cell responses in the skin, was also ruled out. These findings describe a novel role for host LC in the regulation of cutaneous GVHD in situ, which could identify a novel target for organ specific immunosuppression following allogeneic BMT.

The autoinflammatory syndromes are an emerging group of diseases, some with defined genetic cause, characterised by seemingly unprovoked inflammation which derives from a disruption of innate immunity. Novel as yet undefined syndromes are increasingly recognised in consanguineous families who may have normal parents and both affected and unaffected offspring. This type of family is ideal for genetic mapping as both copies of the (presumed recessive) disease causing alleles are likely to have originated in a recent shared common ancestor, and may be linked with markers which will be homozygous in the affected children. In this thesis affected and unaffected offspring and parents in three first-cousin Pakistani families were genotyped with Illumina 610 SNP arrays. This data was used for homozygosity mapping and parametric multipoint linkage analysis. For the first family, a 5Mb region was identified from this mapping. Candidate genes were chosen by members of an expert panel and the exons of these genes were Sanger sequenced. DNA from the entire homozygous region linked to the disease locus (5Mb) was captured using a custom designed 385k array from Nimblegen and then resequenced using the Illumina Genome Analyser II, revealing over 50 coding change variants. These entered a filtering process involving: the selection of rare variants and screening of extended family members, ethnically matched controls and disease controls, and the exclusion of unlikely candidates. This ultimately identified two missense variants of interest in two genes in family A, a novel variant in MEFV the gene affected in Familial Mediterranean Fever, and a variant in TNF Receptor Associated Protein 1 gene TRAP1. The potential contribution of disrupting of the function of these genes to the pathogenesis was assessed using siRNA knockdown in HeLa and THP1 cells. Outcomes assessed include levels of reactive oxygen species (ROS), which are central to many inflammatory processes, and cytokine production. This suggested that TRAP1 may be involved through elevated ROS and TNFα secretion.

The thymus provides a unique microenvironment with all cytokines, cell surface ligands and extra-cellular matrix components necessary T cells to develop. The cellular programme of this development is well characterized, whereby CD4-CD8-double negative (DN) cells require pre-TCR induced signals to give rise to the C04+CD8+ double positive (DP) cells. The OP population undergoes selection to form self-MHC restricted and self-tolerant C04 or CD8 single positive T cells. Extensive work has helped to identify few genes that are important in the pre-TCR induced ON to DP transition. However, our understanding of the temporal regUlation of the genome-wide molecular events underpinning the developmental programme remains restricted. The work done in this thesis focusses on addressing this gap in the understanding of T cell development. Using time course microarrays on thymocytes sorted from foetal thymic organ cultures (FTOCs), transcriptomes of developing T cells were measured through time during the ON to OP transition. A transcriptional modelling strategy, based on the principle that gene expression observed at a particular moment is the balance of synthesis and degradation of its mRNA, was applied. This allowed dissection of the transcriptional signature of the pre-TCR by temporally integrating all the molecular mediators under the pre-TCR signal into defined transcriptional groups. The study revealed previously unknown regulatory factors regulating this phase of T cell development, a few of which, such as the transcriptional repressor Bcl6, were tested in vitro. I describe here, a role for Bcl6 in promoting preTCR induced ON to OP differentiation, and I identify the genes repressed by it, in developing thymocytes. Among them, were members of the Fragilis protein family that are also targets of morphogens including Hedgehog (Hh) proteins, which regulate T cell development in the thymus and differentiation in the periphery. Fragilis proteins were found to influence multiple stages of thymocyte development and function. Genome wide analysis revealed a possible mechanism upstream of Stat1 and the IL27 receptor. Finally, the molecular mediators regulated by Hh signalling at the ON to OP stage are examined, using time course microarrays on foetal thymocytes from a transgenic Gli2 mouse model. This identified a group of transcriptional targets including Bmp4, which can signal directly to thymocytes and attenuate the pre-TCR induced ON to OP differentiation.

Although mitochondrial disorders are the most common inherited form of neuromuscular disease, there are currently limited effective treatments that directly improve mitochondrial function – either by modulation of the effects of mutated genes or by increasing the proportion of healthy mitochondria. In this work, two different approaches were employed to develop treatments for mitochondrial diseases: the design of mitochondrially targeted anti-sense oligonucleotides and the development of a high throughput screen of a unique library of bacterial extracts. The heteroplasmic nature of mitochondrial DNA (mtDNA) enables the use of anti-genomic strategies to specifically prevent the replication, transcription or translation of mutated molecules of mtDNA or mitochondrial mRNA in patients with heteroplasmic mtDNA mutations. In conjunction with an industrial partner (Ugichem GmbH), a mitochondrial targeting oligonucleotide was developed using cell membrane crossing oligomers (CMCOs) – a new class of oligonucleotide with the ability to enter and accumulate within the cytoplasm. By conjugating “mitochondrial targeting” molecules to the CMCOs, translocation to mitochondria was shown, potentially enabling the use of anti-sense therapies in the treatment of mtDNA diseases. To complement the mutation specific approach of anti-sense oligonucleotide therapies a large scale screen was carried out to discover compounds that could cause a general improvement in mitochondrial function. A library of unique bacterial extracts, provided by Demuris Ltd, was screened for effects on mitochondrial biogenesis in HeLa cells. To that end, a high throughput assay, which used fluorescent markers to detect changes in relative mitochondrial mass, was designed and validated using mitochondrially active control compounds. The screen of bacterial extracts discovered several one extract which caused mitochondrial mass to increase 2 fold after 48 hours incubation.

Salivary agglutinin, encoded by the gene DMBT1, is a multifunctional high molecular mass glycoprotein (340 kDa) that acts as a pattern recognition receptor (PRRs) in innate immunity and mediates epithelial differentiation. The central region of the protein contains 13 tandemly-repeated scavenger receptor cysteine-rich (SRCR) domains that are copy number variable and bind to bacteria and viruses. The paralogue ratio test (PRT) was used to estimate the exact copy number of two distinct CNV (1 & 2) regions of DMBT1 gene and results were compared with other CNV estimation assays. Both CNV1 and CNV2 at DMBT1 were multiple allelic CNVs and diploid copy number varied in different populations. The de novo mutation rate at CNV1 and CNV2 of DMBT1 was estimated using a segregation study of 520 samples from 40 multigenerational CEPH families; a high mutation rate was found at both loci of DMBT1 (CNV1 - 1.4% and CNV2 - 3.3% per generation). The evolutionary basis of CNV at DMBT1 was examined using 971 samples from 52 populations from the Human Genome Diversity Panel (HGDP-CEPH). The study found that the subsistence history of human populations affected the frequency distribution of both CNVs at DMBT1. The increase in dental caries following the development of agriculture, and the likely causative role played by an increase in Streptococcus mutans following transition to a starch-rich diet, the present study suggests that this has favoured CNV1 and CNV2 alleles at DMBT1 with more S. mutans-binding SRCR domains in agricultural populations. Due to the functional importance of DMBT1, the study analysed association of DMBT1 copy number in different disease cohorts. The study found no evidence of the association between DMBT1 copy number with Crohn’s disease (n=2900), Urinary tract infection (UTI; n=405), vesicoureteral reflux (VUR; n=625), Chronic obstructive pulmonary disease (COPD; n=241) and Asthma cohorts (n=850). A significant association was found between CNV2 copy number and base-line HIV (n=987) viral load just before anti-retroviral therapy.

Cancer pain remains poorly managed despite technological developments worldwide. The reason for this problem is linked to numerous barriers related to the healthcare system, healthcare professionals and patients themselves. A range of studies that have utilised educational programmes and cognitive behavioural programmes to improve cancer pain management have shown unclear effects. This study reported in this thesis aimed to evaluate the experiences of Jordanian cancer patients who were exposed to a multidisciplinary pain management programme (PMP) in Jordan. The programme is the first of its kind in Jordan and was developed for people with a range of cancer diagnoses experiencing cancer-related pain at the King Hussein Cancer Centre (KHCC). The programme consisted of different services including doctors' consultations, pain medication, physiotherapy and psychotherapy on cancer pain experience. In order to evaluate the experiences of Jordanian cancer patients who were exposed to the PMP, the study adopted a mixed-methods approach based on a concurrent design in which both quantitative and qualitative data were collected simultaneously from 58 participants. Data were collected at baseline and at three time points over six months (July 2010 to January 2011). Data was collected using the Barrier Questionnaire (BQ) to capture patients' bel iefs and attitudes toward cancer pain management and the Brief Pain Inventory (BP!) to investigate pain experience during the PMP. Qualitative data were collected through face-to-face interviews with a subset of the PMP participants (n= 21) to capture their views about the impact of the programme on pain experience. The findings suggest that there is a significant change in cancer pain experience during the PMP, evinced by pain scores measured via the BP!. All pain levels (worst (P =.00), average (P =.00), and current pain (P =.03) were significantly decreased after TO. The analysis of the BQ revealed no change in beliefs and attitudes (P= 0.20). The results also showed substantial early improvement in total pain experience, as well as total pain interference at T 1 (P < .05). However, the pain remained constant at T2 and T3, as no further decline in pain intensity occurred after Tl. The major theme from the interviews was the patient-healthcare professionals relationship, which was found to have a major influence on patients' behaviours and was a modifier for barriers that influence effective cancer pain management. This theme consists of four main sub-themes, including "being the focus of healthcare professionals," "gained knowledge," "trust" and "satisfaction." The majority of interviewees reported good pain relief related to a successful relationship with healthcare professionals, but a small subset of participants were unsatisfied with the PMP's effects as the programme it did not help them to manage their pain.

Neuropathic pain, a debilitating condition, occurs as a consequence of nerve injury with symptoms such as spontaneous pain, hyperalgesia, and allodynia commonly reported. In animal models of peripheral nerve injury, there is accumulation and proliferation of microglial cells in the injured spinal cord and macrophages within the injured nerve and dorsal root ganglion (DRG), contributing to the development of pain-like behaviours. The pro-inflammatory cytokine interleukin-1β (IL-1β) plays a key role in acute and chronic inflammation. It causes potent mechanical and thermal hyperalgesia when injected into peripheral tissues, and increased expression of IL-1β in the spinal cord, DRG, and injured nerve is seen in several animal models of inflammatory and neuropathic pain. A key step in the release of active IL-1β is the cleavage of pro-IL-1β by active caspase-1, generating mature IL-1β. Activation of this enzyme requires assembly of the inflammasome, a multi-protein complex. The active complex contains a central scaffold protein (eg nod-like receptor-1 (NLRP1), NLRP3), the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and pro-caspase-1. It is known to assemble in response to a variety of exogenous and endogenous danger signals (eg adenosine-5’-triphosphate (ATP), monosodium urate (MSU) crystals). Data presented in this thesis demonstrates that, following L5 Spinal Nerve Transection (SNT), an animal model of neuropathic pain, inflammasome components NLRP3, ASC, and caspase-1 are upregulated in the injured DRG and the ipsilateral lumbar dorsal horn. Immunohistochemical analysis reveals that ASC and caspase-1 are both highly co-localised with macrophage and microglial marker ionised calcium-binding adaptor molecule-1 (IBA1), but not with astrocytic marker glial fibrillary acid protein (GFAP) or neuronal marker NeuN. While daily intrathecal administration of Ac-YVAD-CMK, a cell-permeant caspase-1 inhibitor, significantly attenuated established behavioural hypersensitivity following L5 SNT, ASC-/- and NLRP3-/- mice developed behavioural hypersensitivity normally in the Partial Sciatic Nerve Ligation (PSNL) model of neuropathic pain, indicating an inflammasome-independent role of caspase-1 in neuropathic pain. In a model of central inflammatory pain, pharmacological inhibition of caspase-1 prevented the development of hindpaw mechanical hypersensitivity following intrathecal administration of lipopolysaccharide (LPS). ASC-/- mice had a significantly reduced mechanical hindpaw hypersensitivity following intrathecal administration of LPS. However NLRP3-/- mice developed hindpaw hypersensitivity normally following intrathecal administration of LPS, indicating a NLRP3-independent role for ASC and caspase-1 activation in this animal model of central inflammatory pain. Finally, although ASC appears to contribute to the development of hypersensitivity in a model of central inflammatory pain, ASC-/- mice developed behavioural hypersensitivity normally following intraplantar administration of complete Freund’s adjuvant (CFA) in an animal model of persistent peripheral inflammatory pain. Thus data presented in this thesis reveal a complex role for the inflammasome in animal models of pain, with differential contribution of inflammasome components to behavioural hypersensitivity in animal models of neuropathic and central and peripheral inflammatory pain.

Some people report a number of negative psychosocial outcomes associated with living with a visible difference. There is, however, little research into whether such effects are also experienced by those living with a genetic condition that affects appearance, and no research into the associated ethical issues. This interdisciplinary study accordingly investigates the ethical and psychosocial aspects of living with a visible difference associated with a specific genetic condition, Ectodermal Dysplasia (ED). The enquiry employed a critical applied ethics approach integrating empirical exploration with theory. Semi-structured interviews were conducted with thirteen people with ED and twelve clinicians in the field of ED, and analysed using inductive thematic analysis. Three key concepts emerged from the data: wellbeing and identity, which were related to living with a visible difference that consequently influenced reproductive choice. Appearance was found to be an indirect factor influencing reproductive choice through its association with wellbeing and identity. These findings were then synthesised with the existing psycho-social and ethical literature. The synthesis of the findings with the existing literature led to theories of wellbeing, identity and reproductive choice that reflected the multi-faceted nature ofthese concepts. Therefore, a relational and virtue ethics account was most appropriate for reproductive choice, subjective and objective (hedonic and eudaimonic) components were required in a comprehensive theory of wellbeing, alongside a combined theory of identity that incorporated embodiment, narrative identity, and symbolic interaction ism. This thesis therefore presents evidence from a previously under-reported subject matter, providing the reader with a novel overview of the experience of living with ED. Possible future research lines are identified, and the data and arguments provided here offer a platform for future practice and policy, by informing clinicians, policy makers, academics, those who have ED and their associates about the challenges that people with ED may face.

Inhealthy physiological systems all organs act to maintain relative constancy or homeostasis through a highly complex and integrated communication control network of feedback loops. This constant interaction results in a fluctuation in the beat to beat value of the nominal heart rate. Heart rate variability (HRV) is used to describe the variation in the intervals between consecutive heartbeats or the intervals between consecutive R peaks of the QRS complex obtained from an electrocardiogram (ECG). The hypothesis that illness and injury is associated with a reduction in heart rate variability has been tested in various clinical settings and continues to be evaluated. However standard measures of heart rate variability using power spectral analysis have not always been conclusive and the interpretation of results is still being debated. Newer methods based on ideas from nonlinear mathematics are controversial. Inparticular, the method of approximate entropy (ApEn) may give misleading results. In this study a novel normalized entropy measure L was developed for assessing HRV, using theoretical methods and data from an acute hypovolaemic shock model for validation. Electrocardiogram data from the shock model experiments was processed to . obtain the R-R intervals for analysis and used to validate the measure L. The results were statistically significant in showing the differences between the baseline state and the post shock state and between the baseline and the post resuscitation state after allowance was made for the effects of changes in heart rate. From theoretical analysis and from experimental data the method was shown to be valid in both the time and frequency domain. Theoretical predictions of the inconsistencies of approximate entropy were confirmed by experimental results and in particular the method did not give statistically significant results for the experimental data using the accepted range of tolerance values and number of elements being compared. The standard measures of power spectral analysis were not statistically significant. However, by modifying the analysis to that of amplitude rather than power a statistically significant reduction was shown in the total amplitude and high frequency amplitude, after allowance was made for changes in heart rate, at the same states as the measure L.

Pattern recognition receptors (PRR) detect microbial products and induce cytokines which shape the immunological response. Interleukin-12 (IL-12) is a proinflammatory cytokine important for the differentiation of T helper 1 (Th1) cells which produce IFN-γ to activate macrophages and eradicate intracellular pathogens. In contrast, interleukin-10 (IL-10) is an immunosuppressive cytokine that minimises immune-driven host pathology, but can also lead to defective pathogen clearance. The regulation of IL-10 and IL-12 is therefore of interest due to their central roles in the orchestration of an effective but regulated immune response. C57BL/6 and BALB/c mice differ significantly in their resistance to several pathogens. We observed that macrophages generated from these mice produce reciprocal levels of IL-10 and IL-12 in response to the bacterial ligands LPS and Pam3CSK4, which activate TLR4 and TLR2 respectively, and heat-killed Burkholderia pseudomallei, a Gram-negative bacterium which activates TLR2 and TLR4. We have investigated this differential cytokine production in order to further dissect the molecular mechanisms underlying the regulation of IL-10 and IL-12. Detailed analyses of protein production, signal transduction and transcriptional kinetics have identified a type I IFN dependent, but IL-27 independent mechanism for the differential production of IL-10 in LPS and heat-killed B.pseudomallei stimulated C57BL/6 and BALB/c macrophages. Microarray analysis of LPS stimulated C57BL/6 and BALB/c macrophages further revealed potential regulatory networks that may differ between these mouse strains. These findings highlight key pathways responsible for the regulation of IL-10 and IL-12, and may provide valuable information on factors contributing to the ability of C57BL/6 and BALB/c mice to clear bacterial infections.