Multiple myeloma (MM) is a malignancy of clonal plasma cells (PC) which develops as a consequence of a multistep process of transformation from a normal PC to an asymptomatic stage known as monoclonal gammopathy of undetermined significance (MGUS) to MM to the more aggressive plasma cell leukaemia (PCL). MGUS is the most common PC disorder and the majority of cases never progress to MM requiring treatment, as progression only occurs in ~1% of patients per year. From a genetic point of view, specific abnormalities represent initiating events (i.e. IgH translocations or hyperdiploidy) of this multistep process while others occur at later stages. In this study, interphase-FISH showed that initiating events are present in MGUS (n=187) and asymptomatic MM (SMM, n=128) at similar frequencies as found in MM (n=400) (the only exception was t(4;14)) and showed that these abnormalities alone, regardless of their biological impact in MM, cannot drive progression to overt disease. The time of occurrence of deletion/monosomy of chromosome 13 (13) was found to depend on the presence of specific concurrent abnormalities. 13 was extremely rare in MGUS and SMM with translocations directly involving CCND1 and CCND3 suggesting a possible role of 13 in the progression of disease specifically in these genetic sub-groups. However, it was clear that, excluding 13 in these sub-groups, standard interphase-FISH abnormalities are insufficient to predict progression of MGUS and SMM. High resolution array CGH showed an increasing level of genomic complexity from MGUS (n=25) to SMM (n=15) to MM (n=47) to PCL (n=11). In MGUS, the number of copy number changes per case was highly associated with progression (P=0.003). The simplest profiles belonged to MGUS cases with t(11;14) and t(14;20); surprisingly, none of these patients had progressed to MM by the end of this study (median follow-up=72 months). The integration of results from interphase-FISH, array CGH and metaphase analysis suggested that there were various abnormalities (corresponding to distinct molecular pathways) responsible for disease progression. A number of chromosomal changes were found to be strongly associated with progression (del(1)(p22.3-p23); del(6)(q25), MYC changes, del(12)(p13), 13 in t(11;14), abnormalities involving members of the NF-B pathway, del(17)(p13)). Such associations were not only suggested by the fact that these abnormalities were rare in MGUS/SMM compared to MM, but also by the observation that all pre-malignant patients positive for these changes progressed to overt disease. However, among patients who did progress and carried the same abnormalities, time to progression was found to be highly variable from case to case. This suggested that other factors (genetic or otherwise) must be interacting with chromosomal abnormalities in order to lead to progression. Other changes, e.g. 1q21 gain, despite being rare in pre-malignant cases compared to MM and despite some being associated with a dismal prognosis in MM, did not appear to be linked to rapid progression. This study has made significant progress towards understanding the progression from pre-malignant disease to MM, which will provide information towards potential novel targets for therapy to prevent progression or prolong the pre-malignant phase of a highly aggressive disease

The impact of blood pressure changes on cerebral blood flow is an important area of investigation. The cerebral autoregulation mechanism acts to maintain blood supply to the brain, despite changes in blood pressure. Blood flow alterations are closely linked to neuronal activation, and this activity can be visualised using blood oxygenation level dependent magnetic resonance imaging (BOLD MRI) – functional MRI. The aim of this project is to investigate the effect of dynamic blood pressure stimuli on the BOLD MRI signal in the brain. Two blood pressure stimuli were employed; thigh cuff deflation and the Valsalva manoeuvre. BOLD MRI signal changes were measured throughout both challenges. Arterial and venous blood pressure and tympanic membrane displacement (TMD) measurements were also made during these challenges. Blood pressure data was used to drive two linked models. The first model represented cerebral vascular physiology (Ursino) and this fed into a second model (Buxton), which predicted the resulting BOLD signal changes. This allowed comparison with experimental BOLD data. TMD data was also compared to intracranial pressure changes predicted by the Ursino model. The experimental BOLD data was found to agree reasonably well with the BOLD signal changes predicted by the modelling. BOLD signal changes are most influenced by deoxyhaemoglobin changes, predominantly as a result of blood flow alterations during the blood pressure challenges, which are not immediately compensated for by the autoregulation mechanism. TMD changes did not reflect intracranial pressure changes predicted by the modelling. In conclusion, if such blood pressure changes do occur during a functional MRI experiment, they may cause changes in the BOLD signal that are not due to neuronal activation. These signal changes may be employed to investigate the cerebral autoregulation mechanism across the brain, or to correct for inaccuracies in functional MRI data in patients with impaired cerebral autoregulation

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Skeletal stem cell based therapies offer tremendous potential for regeneration of a patient's bone. With the demo graphics of an ageing population, the demand for skeletal reconstruction to replace lost or damaged bone is expanding dramatically. Novel bone tissue engineering techniques offer the opportunity to push the boundaries of bone regeneration, yet few strategies have been translated to clinical practice. This thesis aims to explore novel bone regeneration strategies in vitro and in vivo, and details the clinical application of those techniques. The effects of skeletal stem cells, growth factors and material properties on osteogenesis of bone tissue engineering constructs were explored: • Skeletal stem cells and human fibronectin were shown to augment the biomechanical characteristics of impacted allograft. • Alteration of porosity in a synthetic ceramic scaffold had an effect on osteogenesis. • Innovative technology for enriching the skeletal stem cell fraction from aspirated bone marrow was successfully trialled on bone marrow from an elderly COhOli of . patients, reaching a therapeutic cellular concentration. • A pathological role for osteogenic cells was demonstrated in unicameral bone cysts, with up-regulation of RANI<L related cytokines and stimulation of osteoclastic activity. • Retrieval of tissue from an early translated tissue engineering case provided the oppOliunity for ex vivo analysis, with discussion on lessons that can be learned for future translation. • A novel tissue engineering strategy, to augment the biological and mechanical characteristics of impacted allograft, was subsequently translated to a case series of four patients with avascular necrosis of the femoral head. Surgical technique, clinical follow-up and analysis of retrieval tissue is described. This study has shown the efficacy of skeletal stem cells for bone regeneration in vitro and in vivo, and explored techniques to further augment their osteogenic capacity on bone graft extenders. The translational potential of this bone tissue engineering technology has been realised from the bench to the clinic.

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Invariant NKT cells (iNKT cells) have been increasingly recognised as an important cell type in regulating certain immune responses. Advances in the understanding of iNKT biology have provided opportunities for manipulating their functions for combating malignant, infective and autoimmune diseases. During the tenure of my thesis I have explored aspects of iNKT function in healthy individuals, assessed the activity of novel iNKT compounds in an ex vivo human system and evaluated this important conserved T cell subset in a human disease setting. iNKT cells were initially evaluated in healthy individuals to explore whether there were differences in their function and development that co-associated with an enumeration that extended over a three log range. We performed phenotypic and functional assessments of ex vivo, polyclonal iNKT cells in 47 healthy donors. We established that those individuals with low numbers of iNKT cells (<200/105 T cells) displayed a different iNKT phenotype and functional profile to those with a high numbers of iNKT cells (>500/105 T cells). Following the establishment of a robust screening platform for human iNKT responses a series of 14 novel iNKT cell ligands were designed and analysed for functional distinctiveness from the prototypic !-GC ligand. We identified ligands that had been modified in the acyl and sphingosine chains, which both dissociated iNKT proliferation from cytokine production and skewed effector responses in a Th1, Th2 or Th17 direction. We similarly identified novel compounds that were able to reduce iNKT anergy after initial activation. Finally we showed that these functional differences were modified by CD1d affinity and the nature of the antigen presenting cell, co existent cytokines and donor phenotype. Lastly, we applied our iNKT investigative tools to a clinical disease where the link between iNKT cells and B cell help for antibody production was a possibility. We showed that in the commonest antibody deficiency syndrome, common variable immunodeficiency, iNKT were absent or functionally impaired in the majority of cases and that their number was correlated with presence of memory B cells

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