Focal Adhesion Kinase (FAK) is an intracellular kinase known to mediate integrin signalling following cell adhesion to the extracellular matrix. It is now emerging as a promising therapeutic target in many tumour types due to its overexpression in tumour cells and is associated with various cellular processes involved in cancer progression. Given that existing literature demonstrating that FAK plays a key role in the transduction of HER2 signalling in HER2+ cells and that the levels of FAK expression strongly correlated with HER2 overexpression in clinical samples, we explored the potential for improvement of current therapies for HER2+ breast cancer by combination treatment strategies with the small molecule FAK inhibitor, PF878. FAK activity was assessed in a panel of cell lines reflecting HER2- (MCF7, T47D) and HER2+ (BT474, MDA-361, SKBr3) disease by Western blotting. FAK activity was relatively increased in HER2+ versus HER2- cell lines with HER2+ cells demonstrating greatest sensitivity to PF878 with respect to suppression of FAK phosphorylation at Y397. The effects of PF878 on cell proliferation as a monotherapy and in combination with Herceptin were assessed using MTT and direct coulter cell counting and by Ki67 immuno-staining. Whilst PF878 did not affect the proliferation as a monotherapy, treatment of HER2+ cells with PF878 and Herceptin combined resulted in synergistic inhibitory action on cell proliferation with an associated suppression in AKT pathway activity. This combination treatment strategy produced the greatest effects in MDA-361 cells which were intrinsically insensitive to Herceptin-monotherapy. Inhibition of FAK activity also suppressed HER2+ cell migration in response to the (1) exogenous ligand Heregulin and (2) conditioned-media derived from fibroblasts (FCM), as assessed in Boyden Chamber migration assays. In this latter context, our data suggests that FAK may act through a STAT3-dependent mechanism to regulate fibroblast-stimulated migratory and invasive responses. Collectively, these data support a role for FAK in HER2+ breast cancer where its targeting has the potential to improve Herceptin response as well as suppress stromal-induced signalling that can contribute to disease progression and spread.

In plant ecology, understanding which species live where and why is fundamental for ensuring successful conservation management with the aim of maintaining biodiversity and ecosystem services. Biodiversity patterns in space can be empirically linked to spatially scaled environmental processes, providing a greater understanding to how plant species assemblages change along differing environmental gradients. In the same way, biodiversity patterns in time can be linked to temporal trends in the environment, empirically linking changes in vegetation to changes in the external environment. This furthers understanding of how plant communities are likely to respond to future scenarios of environmental change, providing an insight into future shifts in biodiversity patterns and, in turn, how his may influence valuable ecosystem functions and services upon which humans ultimately depend. Trends in human land use have been shown to be the single most influential driver of current global biodiversity change to date. This, coupled with a changing climate, suggests vegetation communities on a global scale are under increasing pressure to adapt to multiple dynamically changing environmental constraints. This thesis focuses on a globally-rare semi-natural, coastal grassland habitat termed ‘Machair’, renowned for its high biodiversity and cultural heritage importance. Confined solely to the north-western fringe of Europe, the Machairs have formed through unique combinations of geo-physiological and climatic conditions, and even more importantly, century-long associations with human land use through which the low-intensity intermediate disturbance is considered to be vital in maintaining its biodiversity value. However, change in the management of the Machairs alongside other environmental drivers of change, particularly climate, provides major concerns for the biodiversity value of this habitat. In this thesis, spatial and temporal biodiversity patterns of Machair vegetation are investigated to assess the major drivers of change and identify regions which may require future conservation efforts to restore and/or maintain the future biodiversity value of this globally-rare habitat. To achieve this, a spatio-temporal dataset of Scottish Machair vegetation first collected in 1976-77 and then re-surveyed in 2009-10 was used. Analyses found both climate and land use management to influence spatial and temporal vegetation patterns of Scotland’s Machair and Machair grassland. Climate was shown to operate at relatively broad scales (>50km), while the influence of different measurable components of climate was also found to significantly affect temporal turnover patterns of Machair grassland assemblages. Land use management, predominantly at a relatively fine scale (<1.5km), is shown to be the single most influential driver of spatial turnover patterns among both the national extent of Scotland’s soft coast habitats (i.e. low lying coastal areas composed of sand, shingle or mud) and Machair. This indicates the relative importance of land use management in maintaining the high habitat heterogeneity and a high proportion of edge habitat believed to be vital for maintaining the high biodiversity value typical of these habitats. Considerable change in Machair biodiversity for many of Scotland’s regions was observed, much of which could be reasonably well linked to reported shifts in land use, particularly shifts towards increased use of inorganic fertilisers and either increased or decreased agricultural intensification. Not all change was deleterious, many regions which were once included in government incentivised schemes specific to the protection and biodiversity maintenance of Machair grassland displayed higher biodiversity, including species of conservation importance. This highlights the importance of financial incentives to encourage environmentally sensitive land use management more inline with traditional practices, which are increasingly becoming socially and economically unviable. The analyses used were deliberately targeted to identify the community assembly processes by which the Machair communities are governed at local, regional and national scales. It is evident that these communities are governed by environmental filtering at relatively broad regional scales principally through climatic constraints, and similarly at relatively fine local scales through constraints imposed by land use management. It is also evident that community assembly processes are not mutually exclusive and operate simultaneously, at hierarchical scales. Biotic processes at extremely fine scales certainly operate to govern the vegetation communities of Machair grasslands, evident through functional trait divergence. Furthermore, shifts in land use management are shown to impact on these assembly processes, displaying for several regions, an increase in constraints through environmental filtering and reduced resilience through reduced niche differentiation. This study makes a valuable contribution to knowledge of how land use and climate impact on spatial and temporal biodiversity patterns among Machair vegetation, identifying the relative importance of climate and land use determinants, the scale at which they operate, and, how current trends are influencing the biodiversity value of the Machairs at national and regional scales. Furthermore this research demonstrates the utility of different methodological advances and techniques in investigating patterns of biodiversity change for a better understanding of the patterns and processes that govern plant communities.

Age related macular degeneration (AMD) is the leading cause of blindness among the elderly population. It occurs in two forms, a nonexudative "dry" form which can lead to geographic atrophy of the retinal pigment epithelial (RPE) cells in the region of the macula and an exudative "wet" form which involves neovascularisation from the choroid, through Bruch's membrane and the RPE layer and into the photoreceptor layer causing significant damage. Cystatin C is a cysteine protease inhibitor known to inhibit cathepsin activity. It is a highly abundant transcript in the RPE and is known to be processed through the secretory pathway . A variant form of this protein with an amino acid substitution in its signal sequence results in the protein being retained intracellularly and is found localised with the mitochondria. This variant form has been associated with increased risk of developing exudative AMD. In this study a number of key cellular processes were examined to elucidate the effect the variant B protein has on the RPE. These processes were respiration, apoptosis, autophagy and oxidative stress; all of which have been implicated in AMD pathology or ageing. In addition the protein-protein interactions of variant B cystatin C were assessed through mass spectrometry analysis of pulled-down cystatin C protein from transfected cell lysates. No effect on respiration, apoptosis or autophagy was identified. However a statistically significant difference in oxidative stress was identified as a result of overexpression of either wild type cystatin C or variant B cystatin C. Mass spectrometry analysis resulted in two highly promising proteins that were found interacting with variant B cystatin C at a statistically significant level, prohibitin and voltage-dependent anion-selective channel protein 1 (VDAC1). Although oxidative stress was found for both proteins given the nature of the expression (driven by a CMV promoter) it seems likely that the oxidative stress response is due to a high level of intracellular cystatin C. This suggests that the retention of variant B protein within the cell would lead to increased oxidative stress levels. It can be speculated that this response could be a contributory factor to development of AMD. VDAC1 and prohibitin may offer an explanation of what happens to the variant B protein within RPE cells. VDAC1 is the major pore-forming protein in the mitochondrial membrane, disruption of its functioning by the binding of variant B protein might be expected to have detrimental effects. Prohibitin is an even more promising target as it has been associated with oxidative stress. In addition it is known to translocate between the nucleus and the mitochondria, offering the tantalising possibility of a complete explanation for the mitochondrial mislocalisation.

Randomised controlled trials are considered the gold standard study design, as random treatment assignment provides balance in prognosis between treatment arms and protects against selection bias. When trials are subject to departures from randomised treatment, however, simple but naïve statistical methods that purport to estimate treatment efficacy, such as per protocol or as treated analyses, fail to respect this randomisation balance and typically introduce selection bias. This bias occurs because departure from randomised treatment is often clinically indicated, resulting in systematic differences between patients who do and do not adhere to their assigned intervention. There exist more appropriate statistical methods to adjust for departure from randomised treatment but, as demonstrated by a review of published trials, these are rarely employed, primarily due to their complexity and unfamiliarity. The focus of this research has been to explore, explain, demonstrate and compare the use of causal methodologies in the analysis of trials, in order to increase the accessibility and comprehensibility by non-specialist analysts of the available, but somewhat technical, statistical methods to adjust for treatment deviations. An overview of such methods is presented, intended as an aid to researchers new to the field of causal inference, with an emphasis on practical considerations necessary to ensure appropriate implementation of techniques, and complemented by a number of guidance tools summarising the necessary clinical and statistical considerations when carrying out such analyses. Practical demonstrations of causal analysis techniques are then presented, with existing methods extended and adapted to allow for complexities arising from the trial scenarios. A particular application from epilepsy demonstrates the impact of various statistical factors when adjusting for skewed time-varying confounders and different reasons for treatment changes on a complicated time to event outcome, including choice of model (pooled logistic regression versus Cox models for inverse probability of censoring weighting methods, compared with a rank-preserving structural failure time model), time interval (for creating panel data for time-varying confounders and outcome), confidence interval estimation method (standard versus bootstrapped) and the considerations regarding use of spline variables to estimate underlying risk in pooled logistic regression. In this example, the structural failure time model is severely limited by its restriction on the types of treatment changes that can be adjusted for; as such, the majority of treatment changes are necessarily censored, introducing bias similar to that in a per protocol analysis. With inverse probability weighting adjustment, as more treatment changes and confounders are accounted for, treatment effects are observed to move further away from the null. Generally, Cox models seemed to be more susceptible to changes in modelling factors (confidence interval estimation, time interval and confounder adjustment) and displayed greater fluctuations in treatment effect than corresponding pooled logistic regression models. This apparent greater stability of logistic regression, even when subject to severe overfitting, represents a major advantage over Cox modelling in this context, countering the inherent complications relating to the fitting of spline variables. This novel application of complex methods in a complicated trial scenario provides a useful example for discussion of typical analysis issues and limitations, as it addresses challenges that are likely to be common in trials featuring problems with nonadherence. Recommendations are provided for analysts when considering which of these analysis methods should be applied in a given trial setting.

All DNA transactions involving overtwisting or undertwisting of the double helix alter the topological state of DNA, yielding distortions known as DNA supercoiling or DNA torsional stress. Under conditions of negative supercoiling susceptible sequences within B-form DNA may be transformed to stable, non-B-DNA structures such as G-quadruplex. G-quadruplex DNA has the potential to create or destroy transcription factor binding sites or influence nucleosome positioning and so may contribute to gene regulation, making it an attractive therapeutic target. Although in vitro and in silico studies support the notion that G-quadruplex is regulatory, a compelling in vivo demonstration of G-quadruplex function is lacking. Using computational and experimental approaches previously developed to identify DNA segments that actually adopt non-B DNA conformations in vivo, I have identified sequences with a high probability of secondary structure formation. Identified sequences are located in the promoters of three genes central to cancer biology where much of the work in G-Quadruplex has been focused. I have expanded this focus into the field of neuroscience by identifying a further three sequences in the promoters of genes implicated in a range of neurological disorders. Preliminary biochemical analysis of these regions assessed methylation status and transcription factor binding profiles corresponding to a dynamic model of G-quadruplex formation at the well characterised CT element in the c-MYC promoter. In order to provide definitive structure function analysis of G-quadruplex I designed a gene editing strategy to reengineer the well characterized CT element of c-MYC. The initial strategy used AAV- targeting vectors to introduce strategic mutations that destabilize G-quadruplex DNA conformations in the CT element of c-MYC in the HCT-116 cell line via homologous recombination. In parallel to these structure-destabilizing mutations, neutral base changes were targeted to the 5´-UTRs of the targeted alleles, distinguishing them from their respective unmodified homologous partners. qPCR and ChIP analysis of the relative expression and factor binding profiles of targeted and wild-type alleles will enable a definitive structure function analysis of basal or induced expression; determining whether these secondary structures have a functional role in regulating gene expression and the factors involved. Low targeting efficiencies of the AAV gene editing strategy prompted me combine the recent technology of the CRISPR-Cas system with traditional AAV targeting to produce a highly efficient, high fidelity and streamlined approach to gene editing.

Aedes aegypti is the main vector of dengue worldwide. A highly anthropophilic and endophilic mosquito, its behaviour is a major influence on dengue epidemiology and a major challenge to vector control, which is the only dengue prevention method available. A series of studies into host-seeking and resting behaviour were carried out in the laboratory. In Penang, Malaysia, the efficacy of standard and a novel modified form of indoor residual spraying was evaluated in a field trial and the insecticide susceptibility of local vector populations was determined. Arrival patterns of female Ae. aegypti were investigated at a seated human-bait protected by an adhesive-coated net. Mosquitoes preferentially landed on the top and nearest upper vertical surfaces of the net, clustering in a region above the volunteer’s head. Although not previously reported in Ae. aegypti, this behaviour supported the proposition that a plume of potential host attractants rises from the human host. Resting preferences of unfed female Ae. aegypti were investigated using simple two-dimensional panel targets and resting boxes. Exploring the influence of colour, texture, adhesive and target height, the highest resting rates were found on black targets in a vertical configuration at 90 cm above ground. Target texture and adhesive factor did not influence target attractiveness. Data also indicated that female Ae. aegypti were randomly distributed on the panels. In laboratory tests, significantly higher numbers of mosquitoes were captured in resting boxes by raising internal humidity to over 65%. However, a field test in Malaysia did not capture any Aedes sp., although Cx. quinquefasciatus were caught. A randomised-controlled trial was conducted in Penang, Malaysia to investigate the effect of indoor residual spraying (IRS) on Ae. aegypti and Ae. albopictus populations. Two insecticides (lambda-cyhalothrin and pirimiphos-methyl) were delivered either by standard (entire interior surface sprayed) or selective IRS (upper walls and ceilings sprayed) methods. Throughout the three-month study, entomological indices fluctuated considerably and, while there was some evidence of an overall effect throughout the study area, due to a number of confounders comparison between treatments was not possible and the outcome was ultimately inconclusive. At the trial study site in Penang, the insecticide susceptibility status of local populations of Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus were investigated. All were found to be resistant to lambda-cyhalothrin. For pirimiphos-methyl, Ae. aegypti and Ae. albopictus remained susceptible but Cx. quinquefasciatus was classed as ‘suspected resistance’ and potential resistance management strategies are discussed. The study has demonstrated the potential to improve traps or targets for Ae. aegypti by simple alterations to their design. The potential of IRS in the control of dengue vectors remains to be confirmed. The data on emerging insecticide resistance in the mosquito vector populations is timely and provides an evidence base for local authorities to reconsider management strategies that are currently in place for the control of dengue vectors in Malaysia.

Extracellular signal-regulated kinase 5 (ERK5) is the newest member of the mitogen- activated protein kinase (MAPK) family following the discovery of ERK1/2, JNK and p38 MAPK. ERK5 consists of an N-terminal kinase domain, similar to the other MAPK members, and a large C-terminal domain, which is unique in structure and function from the other MAPK members. ERK5 is activated in response to a multitude of extracellular stimuli, including pro-angiogenic factors in endothelial cells. The physiological importance of ERK5 was highlighted following Erk5 gene ablation in mice, where a severe disruption to cardiovascular development and loss of vascular integrity was observed, resulting in embryonic lethality. This study investigated the differences in ERK5 activation between vascular endothelial growth factor (VEGF) stimulation of primary human dermal microvascular endothelial cells (HDMECs) compared to epidermal growth factor (EGF) stimulation of HeLa (immortalised epithelial cervical cancer cell line) cells. It was discovered that in contrast to other growth factors, VEGF appeared unique in its ability to induce ERK5 phosphorylation in HDMECs, stimulating ERK5 activity via a VEGFR-2/PLCγ-dependent pathway. Utilisation of the innovative Phos-tagTM reagent in SDS-PAGE facilitated the novel discovery that VEGF was only able to induce phosphorylation of the threonine (Thr)218/tyrosine (Tyr)220 residues present within the activation loop of the kinase domain. In contrast, EGF stimulation of HeLa cells resulted in phosphorylation of ERK5 on Thr218/Tyr220 as well as additional C- terminal residues such as Thr732. It was further demonstrated that contrary to EGF, VEGF stimulation of HDMECs did not evoke a nuclear translocation of ERK5, instead, ERK5 appeared to localise to the cytoplasm and plasma membrane. The analysis of intracellular signalling pathways following treatment with small-molecule inhibitors against MAPK/ERK kinase 5 (MEK5) and ERK5 kinase activity, revealed that VEGF-mediated ERK5 activation regulated phosphorylation of AKT in HDMECs. Furthermore, with an apparent co- localisation of ERK5 and AKT in the cytoplasm and at the plasma membrane of HDMECs, it was hypothesised that the two proteins were interacting partners. Adenoviral-mediated expression of FLAG-tagged ERK5 revealed that VEGFR-2, ERK5 and AKT co- immunoprecipitated in HDMECs, suggesting the possibility of a complex between these proteins at the cell periphery following VEGF stimulation and VEGFR-2 internalisation. Taken together, this study shows that VEGF appears to induce a unique activation of ERK5 in endothelial cells, which facilitates its interaction with AKT and subsequent regulation of AKT phosphorylation, ultimately regulating endothelial cell survival.

Aim: To assess the use of the Quantitative Light-induced Fluorescence-Digital BiluminatorTM (QLF-DTM) as an oral hygiene evaluation tool to detect demineralisation and plaque during orthodontics Design: Randomised clinical trial Settings: Liverpool University Dental Hospital Subjects: 33 patients (21 females, 12 males) currently undergoing upper and lower fixed orthodontic appliance treatment were recruited. The median age of patients was 14.6 years with a range from 11.0 to 37.4 years. Methods: The patients were randomly allocated, stratified by the presence of demineralisation at baseline (T0), to receiving oral hygiene reinforcement (OHR) at four consecutive appointments (T1-T4) using the White light (WL) or Quantitative Light-induced Fluorescence (QLF) images, taken with the QLF-DTM device (Inspektor Research Systems BV, Amsterdam, The Netherlands), as visual aids. The standard of oral hygiene was assessed on the QLF images using customised software to provide quantitative scoring of fluorescence loss (ΔF) and plaque coverage (ΔR30) at each appointment. Inter-examiner reliability assessments were conducted by 4 examiners using QLF and WL images from 7 patients. One examiner assessed the images on a second occasion two weeks later to ascertain the intra-examiner reliability. A debriefing questionnaire, distributed on completion of the study, was used to ascertain the patients’ perspectives of the QLF-DTM images. Results: There were no significant differences in demineralisation (ΔF: P=0.56) or plaque accumulation (ΔR30; P=0.95) between the WL and QLF groups from T0 to T4. There were no significant reductions in ΔF in the WL or the QLF group from T0-T4 (P > 0.05), however there was a significant reduction in ΔR30 (P < 0.05). The inter-examiner reliability of QLF image assessment, using ICC, was 0.994 and 0.998 for ΔF and ΔR30 respectively. The inter-examiner reliability of WL image assessment, using kappa, ranged from 0.504 to 0.785. The intra-examiner reliability scores were additionally high with an ICC of 0.988 and 1.0 for ΔF and ΔR30 respectively on the QLF images. The kappa score of demineralisation assessment on the WL images was 1.0. All of the participants found being shown the images helpful and were able to see areas of demineralisation and plaque accumulation. 100% of the QLF group thought it would be useful to be given OHR for the full duration of orthodontic treatment compared to 81% of the WL group (OR 2.3, 95% CI: 1.5-3.5). Conclusion: QLF-DTM can be used to detect and monitor demineralisation and plaque during orthodontics. The image analysis demonstrated high levels of inter- and intra-examiner reliability. OHR at consecutive appointments using the WL or QLF images as visual aids is effective in reducing plaque coverage. Whilst there was no apparent statistical benefit in terms of reducing levels of demineralisation or plaque of using QLF images over WL images, patients reported that they were more informative.

Glycosaminoglycans (GAGs), such as Heparan Sulfate (HS), are vital components, of all multicellular organisms, with confirmed roles in development, cell-cell communication and diseases such as dementia and cancer. As such, the interaction of this important group of molecules with proteins is an important process to comprehend and improvements to current techniques are sought. To achieve this HS and its structural analogue heparin, were investigated in various biological systems to simultaneously improve our collective knowledge of the biological systems employed and to improve techniques available to study them. Hen Egg White Lysozyme (HEWL), a model amyloid forming protein, is found to be destabilised with a complex of HS in the zinc (HS(Zn)) from 67.7 °C to 57.6 °C compared to 65.3 °C in the presence of HS in the Na form (as measured by differential scanning fluorimetry). These complexes were studied structurally using synchrotron radiation circular dichroism (SRCD) and found to contain extremely similar secondary structure. To differentiate these similar structures principal component analysis (PCA) of thermal and UV degradations of the complexes using CD were analysed. The HS and HS(Zn) ligands were hypothesised to interact with the two tryptophan residues (Trp) in the active site of HEWL. To confirm this, magnetic circular dichroism (MCD) was also developed as a technique to observe direct Trp:ligand interactions. MCD demonstrated an interaction between HS/HS(Zn) and Trp in HEWL using a signal at the novel position of 286 nm. Antithrombin (AT) was studied in relation to the active pentasaccharide drug and inactive shrimp heparinoids (SH) that contain the important 3-O-sulfate, thought to be key for AT activity against factor Xa. These active and inactive complexes were found to be structurally similar again when studied with SRCD so were subjected to UV degradation. The active and non-active compounds were finally differentiated via their induced stability in AT to thermal degradation. This important correlation challenges the current dogma, which relates activity with a particular pentasaccharide sequence. 2D generalised correlation techniques were applied to the data obtained in this thesis increasing the resolution of the data significantly, allowing for <1nm resolution in some examples. A separate method utilizing the same analysis techniques reveals subtle features within a spectrum, thereby increasing the information available and will be useful for future developments of algorithms assuring improved reliability. A new method of data handling is also proposed for Raman optical activity data, which allows for the correction of baseline drifts, enabling a series of data to be directly compared.

Alkaptonuria (AKU) is an ultra rare autosomal recessive disorder resulting from a deficiency of the homogentisate 1,2-dioxygenase (Hgd) enzyme and is characterized by accumulation of homogentisic acid (HGA) in plasma. The disorder has three distinct stages of disease beginning with the excretion of large quantities of HGA in the urine, followed by deposition of HGA as a polymerized pigment in collagenous tissues principally in the cartilages of loaded joints (termed ochronosis), and finally the early onset of severe and devastating osteoarthropathy. There is currently no effective treatment available to AKU patients. Studying the extreme osteoarthritis (OA) phenotype seen in AKU is helping to increase understanding of more common OA, and may help elucidate the mechanisms behind the initiation and progression of OA. Although a murine model of AKU has previously been reported, published studies reported that Hgd-/- mice did not develop ochronosis. The aim of this thesis was to make a comprehensive survey of Hgd-/- mice to identify if ochronosis was present, determine the pathogenesis of the disorder, and to establish whether a potential treatment could prevent pigment deposition in tissues. Through studying a large number of Hgd-/- mice, of a wide variety of ages, this thesis has provided novel findings in relation to the presence of ochronosis in these mice. Using a modified version of Schmorl’s stain, which can specifically identify ochronotic pigment, ochronosis was observed in Hgd-/- mice for the first time. The identification of the earliest stages of ochronosis in Hgd-/- mice provided an opportunity to follow the pathogenesis of the disease throughout their lifespan. Pigmentation was initially identified in the pericellular matrix (PCM) of chondrons in the articular calcified cartilage (ACC), before progressing intracellularly. Examination of aged mice revealed widespread pigmentation throughout all areas of the tibio-femoral joint. Quantification of the pigmented chondrons demonstrated a progressive, linear increase in pigmentation with increasing age. Similar to ochronosis observed in AKU patients, Hgd-/- mice exhibited signs of ochronotic osteoarthropathy which became progressively worse with age. The early identification of ochronosis and its associated osteoarthropathy in Hgd-/- mice is helping to investigate the biochemical and pathological changes associated with AKU in humans. Following the identification of ochronosis Hgd-/- mice were treated with nitisinone, which had been identified as a possible therapeutic for AKU. Administration of nitisinone throughout the lifespan completely prevented deposition of ochronotic pigment. When given mid-life, nitisinone stopped any further pigment deposition but was unable to reverse the effects of ochronosis which had already taken place. The results showed nitisinone to be an effective treatment against the initiation and progression of AKU. During the course of investigation to identify ochronotic pigment at the ultrastructural level, high resolution transmission electron microscopy revealed the presence of previously undescribed microanatomical concentric lamellae in the ACC of Hgd-/- and wild type mice. Although the pathogenesis of these structures is still undetermined they may play a role in OA development as they appear to be associated with tidemark advancement and increased cartilage mineralization. In summary the studies reported in this thesis present novel findings on the identification of pigmentation, and on the initiation, progression and mechanism of ochronosis which leads to ochronotic osteoarthropathy in Hgd-/- mice. The prevention of ochronotic pigmentation, using the drug nitisinone, was also reported for the first time.