The amount of pain we feel is not always directly related to the amount of damage our body suffers. In fact, research over the last decade has shown that the experience of pain is strongly linked with how we feel about our body, including its shape and how much we like it. Chronic pain is associated with a distorted mental representation of the body, which can have a significant impact on everyday live. Evidence suggests that multisensory illusions can modulate pain and can lead to changes in body perception. However, the factors that may contribute to previously observed analgesic effects remain unclear. This thesis aimed to systematically asses the effects of these illusions on pain and body representation in both healthy individuals and individuals with chronic pain. First, this thesis aimed to investigate the effects of multisensory illusions on body representation, sense of body ownership, and pain in healthy individuals. Chapter 2 found that multisensory illusions can alter perceived body shape, body satisfaction, without losing ownership. Chapter 3 examined whether distorting the size and shape of the virtual hand could modulate pain in healthy participants. No evidence was found for this, which could indicate that pain is not necessarily affected by virtual body ownership over a distorted hand. Having established that multisensory illusions can alter body perception and the affective experience of the body, the second aim of this thesis was to investigate the effect of multisensory illusions on body representation, body ownership, and pain in individuals with hand osteoarthritis (HOA). Chapter 4 investigated whether people with HOA performed differently on a hand laterality motor task to investigate whether people with HOA present with a disruption of the working body schema. Though no evidence was found for a general impairment on this task, the findings suggested that performance on the task was mediated by the presence of pain. Chapter 5 investigated effects of multisensory illusions on pain and pain sensitivity in individuals with HOA. The results demonstrated that viewing the body could modulate pain; and affected body representation, body ownership, and agency. Chapter 6 examined the effects of multisensory illusions on subjective and objective aspects of body perception in people with HOA of the hands, compared to healthy controls. Though no evidence was found for an analgesic effect of the illusion, results showed that participants with HOA have a disturbed experience of the size of their hand compared to healthy controls. Furthermore, the results suggest that individuals with HOA may have an abnormally high body dissatisfaction that cannot readily be altered by multisensory illusions. This thesis found mixed support for the analgesic effects of multisensory illusions on pain in HOA and concludes that the specific context in which pain occurs is important. It also highlights the many perceptual and cognitive factors that may contribute to the modulation of pain. The findings imply that future work should focus on interventions that are more portable and accessible for home use, and focus on developing research around the effects of repeated and prolonged exposure to multisensory illusions on pain, body image disturbances and body dissatisfaction.

Background: The chronic joint disease osteoarthritis (OA) represents a significant global problem, not only at the present time, but also for the future. Characterised by articular cartilage degeneration, inflammation of the synovium, and subchondral bone changes, it is the chronic pain associated with OA which presents the most serious consequences. There is a clear need to understand the mechanisms under lying chronic joint pain, and to identify novel therapeutic targets. Two targets which may have therapeutic potential are BDNF and cordycepin. Objectives: The objectives of this thesis are two-fold; firstly to establish a slow progressing murine model of OA that is representative of post-traumatic OA, and secondly to investigate peripheral targets which may modulate chronic OA pain. Methods: Surgical destabilisation of the medial meniscus (DMM) was carried out in adult C57BL/6 mice. Weight bearing asymmetry and hindpaw withdrawal threshold were measured up to 16 weeks post-surgery. Joint pathology was then assessed post-mortem at 16 weeks post-surgery. Another cohort of adult C57BL/6 mice underwent a modified surgical destabilization of the medial meniscus. Pain behaviour and joint pathology outcomes were measured 16 weeks and 20 weeks post-surgery. Osteoarthritis was induced in adult male Sprague Dawley rats via intra-articular injection of monosodium iodoacetate (MIA) or vehicle (50ul 0.9 % saline. A second cohort of male Sprague Dawley rats underwent either meniscal transection (MNX) or sham surgery. Rats then received intra-articular injections of either trkB-fc or human IgG. Pain behaviour was tested up to 3 hours post injection. Adult C57BL/6 mice underwent DMM or sham surgery. Pain behaviour was measured up to 16 weeks post-surgery. From 14 weeks post-surgery mice were orally dosed with either cordycepin or vehicle every two days for two weeks. Joint pathology was then assessed post-mortem at 16 weeks post-surgery. Results:There was a significant increase in weight bearing asymmetry from 13 weeks post DMM surgery in C57BL/6 mice, but no changes in hindpaw withdrawal thresholds. There were also significant increases in chondropathy and synovitis at 16 weeks post-surgery. When the surgical induction of the DMM model was modified there were still significant changes in joint pathology, but no significant changes in pain behaviour. Intra-articular injection of TrkB/fc chimera in rats with established MIA induced joint pain was found to acutely reduce weight bearing asymmetry and increase ipsilateral hindpaw withdrawal thresholds. There was also a significant reduction in pain behaviour in rats with MNX established joint pain when TrkB/fc chimera was injected into the knee joint. Following systemic administration of cordycepin in mice with DMM induced joint pain there was a significant reduction in weightbearing asymmetry when compared to vehicle treated mice. There was also a significant reduction in DMM induced chondropathy, subchondral bone thickening, and osteophytosis in mice treated with cordycepin compared to vehicle treated mice. Conclusions: The changes in pain behaviour outcomes between the traditional and modified DMM, despite similar joint pathology outcomes, suggests a role for meniscal damage as a peripheral driver of OA pain. Localised injection of TrkB/fc chimera into the knee joint of rats with both MIA and MNX induced joint pain was found to acutely reverse joint pain. This implies that peripheral BDNF may be involved in mediating OA joint pain. Oral administration of cordycepin was found to reduce both pain behaviour and joint pathology changes in the DMM model in mice. These results suggest a role for local protein translation underlying both OA chronic pain and joint damage.

Uveal melanoma (UM) is the most common intraocular malignancy in adults. Despite successful ocular treatment, about 50% of patients succumb to metastatic dissemination, which occurs haematogenously and mainly affects the liver. On the basis of clinical, histopathological and genetic features of the primary tumour it is possible to predict if the individual patient is at high risk (HR) or low risk (LR) of developing metastases. However, the mechanisms responsible for the development of metastatic disease in UM are still largely unknown; therefore no adjuvant treatment is currently offered to HR patients to prevent development of fatal disease. As the time to discovery of clinically detectable metastases can range from months to decades, a secreted biomarker(s) that could be routinely tested in blood is much needed. The scope of the work presented in this thesis was to use proteomics as a tool to identify potential novel, UM-specific biomarkers. Moreover, the proteomic data acquired would complement genomic and transcriptomic information already generated by the Liverpool Ocular Oncology Research Group, with the ultimate aim of increasing our understanding of UM development and dissemination. The aim of Chapter 2’s project was to compare the proteome of UM tissue samples at HR versus LR of developing metastatic disease using isobaric tags for relative and absolute quantitation (iTRAQ) labelling and mass spectrometry (MS). The quantification of proteins in our samples, proteomic analysis and further validation by immunohistochemistry has led to the identification of two novel prognostic and potentially therapeutic target, S100A6 and the tumour suppressor PDCD4. In Chapter 3 we focused on proteins released in the conditioned medium (secretome) of short-term cultures of HR and LR UM cells, as well as normal melanocytes. Using a label-free quantitative proteomic approach, almost 2000 proteins were identified and quantified, with more than 30% of these identified as secreted and/or previously described in exosomes. Using these data, an 18-protein signature able to discriminate between HR and LR UM was identified. Further validation will be necessary in secretome samples and in the peripheral blood of UM patients, but this has the potential of being translated into a clinically useful assay to detect early development of metastatic disease. As reported in Chapter 4, we also conducted a pilot clinical study on circulating tumour cells (CTC) in UM, using the CellSearch® platform with the novel melanoma kit to enumerate CTC in the peripheral blood of UM patients at LR, HR or with overt metastatic disease. CTC were detected in metastatic and HR tumours and were not present in LR UM, however, the number of CTC detected varied widely, calling into question the clinical value of using this platform in UM patients. The research detailed in Chapter 5 had a direct clinical value, as it addressed the procedures undertaken during the acquisition and processing of prognostic biopsies from UM tumours treated conservatively. The modifications introduced led to a significant improvement of the success rate of such prognostic biopsies for risk stratification, which is essential for clinical management, follow-up and research purposes. In conclusion, the work conducted throughout this PhD has provided further insight into the molecular characteristics that can differentiate between HR and LR UM, identifying novel potential biomarkers that will need validation in the clinical setting.

The inflammatory response is critical to healing outcome after cutaneous injury. Our current understanding of the response to injury has been compiled from targeted studies on components expected to play a role. It was hypothesised that an unbiased approach to characterisation of soluble mediators within the injured tissue might identify additional components, thereby increasing our understanding of the cellular processes involved. The aim of this research was to develop and characterise a model of injury with the potential to identify novel mediators of the early response. Microdialysis was chosen as both the sampling method and the means by which the tissue was injured as probe insertion causes a single injury that can be sampled continuously without further damaging the tissue. Early injury responses were initially characterised in terms of changes in blood flow and known markers of the inflammatory response, using Laser Doppler Imaging and a bead-based cytokine flow cytometric assay, respectively. A shotgun proteomic analysis was then undertaken to characterise of the protein content of the fluid obtained using microdialysis, dialysate. The phosphorylation status of proteins was also characterised following the implementation and optimisation of a recently reported method that uses dendrimer conjugation chemistry to capture phosphopeptides. Blood flow and cytokine measurements in the dialysate confirmed the occurrence of a reproducible inflammatory response to the microdialysis injury. Proteomic analyses of dialysate suggests that it has a relatively simple protein composition and is dominated by highly abundant components. The identified proteins originate from both intra- and extracellular locations and play a range of roles, including regulation of coagulation, cellular ii communication, and the immune response. Several are likely to undergo post-translational phosphorylation and hence their phosphorylation status was also investigated. Using the phosphopeptide capture method, potentially novel phosphorylation sites were identified in two abundant proteins, albumin and apolipoprotein L1 at positions S603 and S314, respectively. Collectively, the data obtained in this investigation increase our knowledge of the proteins and processes involved in responses to injury, and suggest that microdialysis may be of some use for studies in this area. Further, analysis of protein phosphorylation in dialysate suggests that this is an informative approach that could shed light on extracellular signalling events that occur during the progression of the response to injury.

The myeloproliferative disorders (MPDs) are a group of haematopoietic stem cell diseases, characterised by proliferation of one or more cells of the myeloid lineage. Several lines of evidence have highlighted the importance of aberrant tyrosine kinase signalling in the pathogenesis of these disorders. Cloning of rare chromosomal translocations and point mutation analysis in the MPDs has identified diverse deregulated tyrosine kinase genes, notably PDGFRA, PDGFRB, FGFR1 and JAK2. However the majority of atypical MPDs still remain to be characterised and identification of patients harbouring fusions, particularly those involving the PDGF receptors is of increasing importance, as they are likely to be responsive to targeted therapy with imatinib. I am investigating MPD patients for abnormalities affecting tyrosine kinase signalling, and have used three approaches, translocation cloning, expression analysis and SNP array analysis to detect regions of loss of heterozygosity (LOH). Thus far, by translocation cloning I have identified a previously undescribed partner gene fused to PDGFRB and two new PDGFRA fusion genes. I have also designed two reverse transcriptase PCR (RT-PCR) assays and a cDNA MLPA assay to detect over-expression of specific tyrosine kinases screening approximately 200 patients. Each assay identified all patients previously diagnosed with known fusions. Additionally, two patients identified with overexpression of PDGFRB have been found to have cryptic ETV6-PDGFRB fusions and overexpression of PDGFRA in one patient lead to the discovery of a previously undescribed fusion involving a novel partner gene (KIF5B). Recent evidence has indicated that acquired isodisomy is a novel mechanism by which mutations in cancer may be reduced to homozygosity. Typically, acquired isodisomy is associated with oncogenic changes rather than tumour suppressor genes, eg. the activating JAK2 V617F mutation and 9p aUPD. I have undertaken a screen using Affymetrix 50K SNP arrays for regions of acquired isodisomy as a means to identify genomic regions that may harbour novel oncogenes in different subgroups of MPD patients. Large tracts of homozygosity (defined as >20Mb running to a telomere), strongly suggesting acquired isodisomy, were seen in 40% aMPD patients. The homozygous tracts encompassed diverse genomic regions in aMPD, but two common regions (3 cases for each region) were identified at 7q and 11q. Mutations in the CBL ubiquitin ligase gene were discovered in all three aCML patients with 11q aUPD as well as in an additional 23 MPD patients following further screening.

Gangliosides, complex sialic acid-containing glycolipids, and other glycosphingolipids are active physiological membrane components with an array of functions in development and disease. Altered profiles are found in many disorders including those of neurological and cancerous aetiologies. Glycosphingolipids are also important for lateral membrane organization, cell communication and as binding sites for extra-cellular components. These lipids have long been implicated as targets in autoimmune diseases such as Guillain-Barré Syndrome (GBS) and Multifocal Motor Neuropathy. In GBS, auto-antibodies bind native membrane gangliosides signalling immune-mediated breakdown of nerves causing acute flaccid paralysis. While the fundamental pathology is understood, differences in clinical presentation, and preference for motor over sensory nerves, have yet to be explained. Understanding the precise nature of native gangliosides, including low abundance species and modifications, is an important first step. Meanwhile genetically engineered mouse models are under development that should increase our understanding of disease pathogenesis. To be truly functional it is essential these models contain a full range of complex and simple glycosphingolipids in the neurological tissue. Mass spectrometry has recently been applied with great effect to lipidomics; the comprehensive profiling of all lipids involved in a system. However, heavily glycosylated, low abundance and chemically unusual lipids such as the gangliosides tend to be neglected in otherwise thorough lipidomic studies. It was the aim here to optimise separation and mass spectrometry methodologies for ganglioside analysis. Workflows were developed for high performance thin layer chromatography (HPTLC) combined with direct imaging mass spectrometry (IMS) detection and identification, and for high performance liquid chromatography (HPLC) with online high resolution mass spectrometry detection and identification with dissociation to confirm structures (MSMS). A range of lipid standards were analysed using this second method to build a database of characteristic ionization behaviour, retention times, and product ion spectra to aid the analysis of unknowns in complex mixtures. Methods were then applied to molecular phenotyping in novel mouse models of GBS, and to glycosphingolipidomics in peripheral sensory and motor nerves. Finally the recently developed technique of imaging mass spectrometry, using matrix assisted laser desorption ionisation (MALDI) and secondary ion mass spectrometry (SIMS) ion sources, was investigated for its capability for direct ganglioside analysis in brain and spinal cord tissue sections. Results are presented below demonstrating the significant benefits of the mass spectrometry-based workflows over more conventional profiling methods as well as comparing and contrasting the two techniques developed here. Limitations and potential areas for future development are debated. Findings from profiling knockout and rescue mouse models and from single nerve glycosphingolipidomics are discussed along with further experiments and directions for these studies. The discovery of a full range of complex gangliosides in neurological tissue from rescue mice, albeit at low levels compared to the wild type, confirmed their molecular usefulness for modelling neurological autoimmune diseases. The sensitivity and reproducibility of the mass spectrometry technique enabled relative quantitation, revealing details into the abundance of different ganglioside species and inclusion of ceramide structures in each mouse type. The ability to detect very low abundance lipids with an additional dimension of structural description also suggested that O-acetylation of the second sialic acid on native disialylated lipids is more prevalent than previously thought. Finally imaging mass spectrometry results are presented. Although sensitivity was limited, both simple and complex gangliosides were detected in spinal cord sections; the first known IMS detection of these lipids outside of the brain. Results also demonstrate the abundance of parallel lipidomic information that can be obtained using these methods. Possible solutions to increasing the sensitivity limit are discussed that may increase IMS usefulness to glycosphingolipid studies in future.

Rheumatoid Arthritis (RA) is a chronic systemic autoimmune disease characterised by extensive synovitis resulting in cartilage and bone erosions. Both the innate and adaptive immune pathways contribute to the initiation and the maintenance of the disease. Understanding the role of these pathways is central to develop new therapeutics. We have developed a murine model of RA where ovalbumin (OVA) specific Th1 cells induced a breach of self-tolerance and a transient monoarthritis. This thesis aimed firstly to create a model of chronic autoimmune polyarthritis and then to investigate the contribution of B cells and innate inflammation to the induction of arthritis. Relapse of arthritis was associated with the nature of the antigen (OVA) employed and the route of administration. The analysis of collagen specific B cell response revealed that anti-type II collagen antibodies titres rise during the induction of the relapse of arthritis and that they were directed against the epitope U1. Although typical RA autoantibodies were detected in OVA-mediated arthritis, a mild arthritis could be elicited in absence of antigen presenting B cells and in complete absence of mature B cells. B cells were not necessary in the induction of pathology even though their presence was associated with a higher joint histology score. Finally, this thesis describes that an innate inflammatory stimulus, such as LPS, elicited joint pathology but was insufficient to breach B and T self-tolerance. On the contrary, antigen-specific T cell activation led to arthritis and the production of several autoantibodies typical of RA. The relapse and spread of arthritis developed in this thesis provides a useful tool to investigate the contribution of the innate and adaptive immune pathways in the development of autoreactive responses. A better understanding of these mechanisms will hopefully help to design new therapeutic intervention aiming to re-establish immunological tolerance.

Prostate cancer has high cancer incidence in males and is the second highest cause of male cancer related mortality. Currently the main stay therapy for localised and metastatic disease is maximum androgen blockade (MAB). This aims to inhibit androgen production or action, thereby reducing stimulation of the androgen receptor (AR). This in turn prevents the activation of androgen-regulated genes, which normally result in on-going growth and survival. Inhibition of testicular androgen production may be achieved surgically (bilateral orchidectomy) or chemically, using gonadotropin-releasing hormone (GnRH) agonists. The latter induces castrate levels of testosterone by down-regulating pituitary GnRH receptors (and therefore gonadotropin hormone production) through constant stimulation. The action of androgen may be blocked at a peripheral level using anti androgens, which inhibit ligand binding to AR and subsequent activation. Although this approach has initial response rates of over 80% the majority of men relapse with castrate resistant prostate cancer (CRPC) and this is the cause of significant morbidity and mortality. To overcome this and to improve patients treatment options the mechanisms of castrate resistance need to be addressed. The PI3K/Akt cascade regulates several cellular processes such as proliferation and apoptosis. Akt activation results in phosphorylation of multiple substrates and has been implicated in prostate carcinogenesis and castration resistance. Research has suggested that Akt interacts with signallingcascades implemented in carcinogenesis, in particular the NFkB cascade and AR signalling. The current study investigated the hypothesis that the expression and activation of PI3K/Akt cascade influences the progression to castrate resistant disease using clinical prostate cancer tumours. Fluorescent insitu hybridisation and Immunohistochemistry revealed that PTEN deletion was a common event in castrate resistant prostate cancer and low PTEN protein expression was significantly associated with a poor outcome. PTEN negatively regulates PI3K signalling. Consequently increased levels of PI3K and activated Akt (pAkt ser 308 and pAkt ser 473) were significantly associated with a shorter time to biochemical relapse and shorter disease specific survival. Inhibition of PI3K resulted in a significant reduction in cellular proliferation and Akt phosphorylation. The downstream affects of Akt activation were also investigated. Akt has been reported to directly activate the NFkB signallingcascade both directly and indirectly but no correlations between Akt and NFkB were observed in the current study. Using an immunohistochemical approach NFkB, IкBα and MMP-9 expression were observed to be significantly associated with shorter time to death from relapse and disease specific death. MMP-9 and IкBα expression were also significantly associated with metastases at relapse. Using paired hormone naive and castrate resistant LNCaP cells lines allowed the functional consequences of NFkB inhibition to be investigated. Reduced NFkB activation significantly inhibited cellular proliferation and induced apoptosis in both cell lines. Having shown a significant link between expression and activation of the PI3K cascade and progression to castrate resistant disease, the interaction between Akt and the AR was investigated in both clinical prostate tumours and cell lines. The phosphorylation of AR at the Akt consensus site serine 213 (pARser213) was significantly associated with disease progression. Patients with high expression pARser213 had a significantly shorter time to death from relapse and disease specific survival. Additionally 42% of patients displayed an increase in pARser213 expression, these patients also had a significantly shorter time to death from relapse and disease specific survival. Inhibition of PI3K resulted in a reduction of pARser213 expression in both cell lines and using siRNA knockdown to target PI3K p85 regulatory subunit reduced pARser213 expression. This research highlights the impact of both the PI3K/Akt and NFkB signallingcascades on prostate cancer progression and development of castrate resistant disease. In particular this study highlights the impact of Akt phosphorylation in castrate resistant prostate cancer patients. Therefore phosphorylation of AR at serine 213 may serve as a diagnostic tool to predict patient outcome in response to maximum androgen blockade and inhibition of AR 213 phosphorylation via the Akt cascade may be an effective therapeutic avenue to investigate for treatment of prostate cancer.

The aim of this study was to investigate the potential use of ELISA and LC-MS-MS in combination and as individual techniques, for the detection of drugs of abuse in biological matrices. Overall the LC-MS-MS method showed good correlation results for opiates compared to the GC-MS method. 6-MAM was however detected in more root segments and segments excluding roots by LC-MS-MS. Morphine was detected in a greater number of root segments by LC-MS-MS compared to GC-MS. However, morphine was detected in a greater number of segments excluding roots by GC-MS. Codeine and dihydrocodeine were also detected in a greater number of root segments and segments excluding roots by GC-MS. The cocaine results showed excellent qualitative correlation between the LC-MS-MS and GC-MS methods for cocaine and benzoylecgonine. The GC-MS method did not however extract greater concentrations of cocaine and its metabolites compared to LC-MS-MS due to the higher recovery of the drug group specific GC-MS method. Cocaethylene and EME were detected in some samples by LC-MS-MS method for opiates and cocaine and its metabolites compared to the GC-MS method; there may be some cases where the GC-MS method would detect the analytes where the LC-MS-MS method would not. This has been demonstrated in 3 samples for morphine and in 6 samples for codeine. The LC-MS-MS method analysed for and detected amphetamines in samples that were not tested for amphetamines by GC-MS. In one sample that was tested by both methods, amphetamine was detected in the root sample by LC-MS-MS where GC-MS failed to detect it. Also a greater concentration of amphetamine was extracted using the LC-MS-MS method in the segment without roots. The LC-MS-MS method was useful for the analysis of 17 drugs of abuse in post-mortem hair samples in forensic toxicology cases. Using this method, it is possible to obtain maximum information from one hair sample which is extremely useful when the sample weight is limited. The ability of the LC-MS-MS method to extract and analyse a greater number of drug groups from one hair sample highlights the advantages of using this method over GC-MS which targets individual drug groups and requires splitting of the sample. This method is particularly applicable for implementation in the forensic toxicology laboratory at the University of Glasgow where currently GC-MS methods that target individual drug groups are used for routine hair screening and confirmation.

Multiple sclerosis (MS) is a chronic disease of the human central nervous system (CNS) in which repeated episodes of inflammatory demyelination result in formation of persistently demyelinated plaques of gliotic scar tissue associated with varying degrees of axonal loss. MS is now considered a “complex trait” that is triggered in genetically susceptible individuals by environmental factors. The disease is also considered to contain an autoimmune component where both the adaptive and innate immune systems have been implicated in disease pathogenesis. There has been a steady accumulation of circumstantial evidence from both clinical and experimental studies that implicate a role for autoantibody dependent mechanisms. However this issue remains controversial in the absence of formal evidence that patients actually develop a pathogenic autoantibody response. The aim of this thesis was to resolve this question. To do this we developed an in vitro bioassay based on a dissociated myelinating culture system from embryonic rat spinal cord. We demonstrated that this in vitro system could reproduce many features of in vivo myelinated axons. To validate this model as a viable screening assay characterised complement mediated autoantibody responses using a series of monoclonal antibodies and anti-sera. Due to their significance in the literature we focussed in particular on the MOG specific and Nfasc specific responses and comprehensively demonstrated that our bioassay offered a robust screening strategy in which to detect pathogenic antibody responses in the presence and absence of exogenous complement. To determine whether we could use our model to detect pathogenic autoantibody responses in MS patients, we purified the IgG fraction from a cohort of MS patients (n=20), OND (n=10) and healthy controls (n=13). Using this patient purified IgG we demonstrated a MS specific demyelinating activity, which was present in ~50% of samples screened. However in 10% of patients demyelination occurred secondary to pronounced axonal injury. These effects were dependent on exogenous complement and were unique to the MS cohort. Pathogenic antibody responses tended to be most prevalent in those patients with an aggressive disease course. In addition to complement mediated CNS injury we also demonstrated that this pathogenic MS IgG could disrupt myelin formation in developing myelinating cultures. Attempts to define the specificity revealed that this was heterogeneous, however in one MS patient we discovered that Nfasc155 provided a dominant antigen for pathogenic autoantibody responses. Together these data provide formal demonstration that MS is associated with pathogenic autoantibody responses. This has significant long term consequences for the clinical management of the disease