Objectives: The objectives of this study were to a) examine the distribution of chondrocalcinosis (CC), b) determine the risk factors of CC, and c) examine the radiographic phenotype of osteoarthritis (OA) associated with CC. Methods: Data from the Genetics of Osteoarthritis and Lifestyle (GOAL) study were used to describe the radiographic distribution of CC, and to conduct a case-control study in which cases with CC were compared with controls without CC. All participants had already completed a detailed questionnaire, been examined by a research metrologist, had radiographs of knees, hands, and pelvis, and had given urine and blood samples. All radiographs had been scored for structural radiographic changes of OA, and for the presence of CC. Frontal plane knee alignment was measured on all knee radiographs. The prevalence (95% confidence interval (CI)) of CC was calculated. The odds ratio (OR) and 95% CI were calculated for risk factors of CC, and for structural changes associated with CC in joints with OA. This was adjusted for age, gender, body mass index (BMI), and OA as appropriate, using logistic regression. Results: 3170 participants were included in this study. There were 431 cases with CC. The overall prevalence (95%CI) of CC in the GOAL population was 13.7% (12.5% - 14.9%). In the GOAL population, knee was the commonest site of CC. However, 42% of participants with CC did not have any knee involvement. There was evidence for a generalized predisposition to CC. For example, CC at one joint associated with CC at distant joints. Joints with CC clustered together more than would be expected by chance alone. At knees, wrists and hips, bilateral CC was more likely to associate with CC at distant joints than unilateral CC – also supporting the existence of a systemic predisposition to CC. After adjusting for confounding factors, there was an association between CC and increasing age, lower current BMI, and OA. The association between OA at one joint and CC at the same joint was present for all joints except for the hip. There was no association between CC and gender, diuretic intake, and selected single nucleotide polymorphisms in enzymes involved in pyrophosphate (PPi) metabolism. CC associated with peri-articular calcification, vascular calcification, low cortical bone mineral density (BMD) but not with low cancellous BMD. Self-reported arthroscopy, meniscectomy, knee injury, occupational knee joint loading and knee mal-alignment in the 3rd decade of life associated with knee CC. However, after adjusting for confounding factors including OA, there was no association between either self-reported or radiographically assessed current knee mal-alignment and knee CC. In joints with OA, the additional presence of CC at the same joint associated with a different radiographic phenotype of structural arthropathy. For example, in knees with OA, knee CC associated with attrition. In hips with OA, hip CC associated negatively with osteophytes, joint space narrowing, and sclerosis at the right hip but not at the left. Similarly, in wrists with OA, wrist CC associated with sclerosis in the right but not in the left wrist; in scapho-trapezioid joints (STJs) with OA wrist CC associated with sclerosis on both sides; in metacarpophalangeal joints with OA, wrist CC associated with cysts in the right but not in the left hand; and in 1st carpometacarpal joint with OA, wrist CC associated with cysts in the left but not in the right hand. In knees with OA, the additional presence of CC at distant joints associated with knee attrition. Those with knee CC + OA were excluded from this analysis to remove any local effects of CC. CC at distant joints did not associate with a distinct structural OA phenotype in other joints examined. Conclusion: These findings suggest that CC results form a systemic predisposition, and that it commonly occurs at other joints in the absence of knee involvement. Established risk factors of CC such as age, OA, and previous arthroscopy and/or meniscectomy were validated in this study. Several novel risk factors of CC e.g. low current BMI, low cortical BMD, and vascular calcification were identified. Several novel associations of knee CC i.e. early life knee malalignment, self-reported knee injury, and occupational knee loading were also recognised. There was convincing evidence to suggest that in joints with OA, the additional presence of CC modifies the OA phenotype, and that this varies from joint to joint.

Breast carcinoma is the most frequent type of cancer affecting women. Among the recently described molecular and phenotypic classes of breast cancer, human epidermal growth factor receptor 2 (HER2)-positive tumours are associated with a poor prognosis. HER2 status is currently assessed in routine breast cancer reporting using immunohistochemistry (IHC) in addition to in situ hybridisation (ISH) in borderline cases. The ability of HER2 gene status to predict response to targeted therapy (Trastuzumab) is well documented. However, prognostic information provided by IHC expression categories and prognostic value added by using ISH in borderline cases remains unclear. HER2 plays an important role in cancer progression being targeted to provide predictive and prognostic information. Moreover, HER2 is related to cancer resistance against a variety of therapies; however, trastuzumab has proved successful in treatment of this subgroup. Nevertheless, patients may acquire resistance to this drug after a period of treatment, which indicates that other molecular mechanisms might influence success of this therapy. Dimerisation between members of the HER family may contribute to resistance against treatments due to different combinations that trigger different downstream pathways. This is promoted by ligands, which are expressed as transmembrane precursor protein molecules and have a conserved epidermal growth factor-like domain. Through resistance to trastuzumab, other drugs are being developed to interact in different domains of HER2 protein. The study of interaction between receptors/ligands will characterise specifically their signalling pathway and understand which strategy to acquire. The main aim of this thesis was to assesses the status of HER2 protein (IHC), HER2 gene (chromogenic ISH) and HER heterodimers (in situ proximity ligation assay (PLA)), including HER2/EGFR, HER2/HER3 and HER2/HER4, in two BC series prepared in tissue microarray format; a series of consecutive primary operable BC cases (n = 1858) including HER2+ trastuzumab naïve cases (TrN, n = 221), the second series of HER2+ trastuzumab adjuvant treated cases (TrT, n = 143). Therefore determining the biological characterisation of these biomarkers by associating against clinicopathological parameters, survival outcome and understand the trastuzumab therapy value. There was excellent overall concordance between HercepTest negative (scores 0/1+) and positive (3+) with CISH positive/negative (defined as HER2/Chr17 copy number ratio of ≥ 2; p < 0.001). Kaplan-Meier analysis for breast cancer specific survival (BCSS) and disease free interval (DFI) revealed statistically significant differences between HER2 positive and negative cases detected by HercepTest and CISH (p < 0.001). Interestingly, it was identified that HercepTest 2+ non-amplified cases were not significantly different with those amplified 2+ or 3+ cases with respect to their behaviour (BCSS and DFI). The results revealed an inverse association between the HER heterodimerisation status and hormone receptor status (p < 0.001), and a significantly worse outcome amongst cases revealing high levels of all heterodimers (p < 0.001). Among ER+ cases, the heterodimers high levels were significantly associated with worse prognosis (p < 0.001) overall. However amongst the two HER2+ populations dimerisation status did not show an association with patient outcome. The overall concordance between HercepTest and CISH analysis for HER2 status was excellent. All HercepTest 2+ cases identified were observed to have poor outcomes similar to those HercepTest 3+ cases regardless of gene amplification status. In the current clinical environment cases exhibiting IHC 2+ and non-amplified gene HER2 status will not be offered targeted HER2 therapy but do exhibit aggressive clinical behavioural characteristics. Even though those patients with high levels of the HER2 truncated form, p95HER2, have shown poor outcome, this biomarker does not reveal any extra findings comparing with the HER2 expression results. Beside HER2, EGFR is the only monomer that reveals prognostic value amongst the breast cancer patients. Tumours exhibiting high levels of HER heterodimerisation have an adverse prognosis, however in the context of HER2+ breast cancer no association with clinical outcome was observed regardless of use of trastuzumab treatment. HER2/HER3 heterodimerisation status assessed in multivariate analyses has shown that this protein-protein interaction is associated with a poor prognostic outcome, which needs further investigation and assessment of clinical utility to use in the future in breast cancer treatment decision. Further quantification analysis of dimer/ligand complex using PLA of other HER family members may be useful to identify subset of patients associated with distinct outcome, response to treatment and relationships with HER signalling related biomarkers.

PCOS is one of the leading causes of infertility worldwide affecting 1 in 10 women of a reproductive age. One of the fundamental abnormalities in women with PCOS can be seen within hormonal irregularities, which may include hyperandrogenemia hyperinsulinemia and hyper secretion of luteinising hormone (LH); and it is hypothesised that a defect in steroid secreting ovarian theca cells is involved due to their contribution in non-PCOS hormonal synthesis. Hyperinsulinemia has been associated with hyper-androgenemia through in vitro studies of cultured PCOS theca, where it has been suggested that insulin increases progesterone and androstenedione secretion when compared to normal theca cells. Furthermore the augmented effects of LH and insulin have been seen to increase ovarian androgen synthesis in non-PCOS theca cultures whilst also increasing the expression of steroidogenic enzymes specific to the PI3-K pathway. Many theories exist toward the etiology of hyper androgenemia within PCOS. Very few approaches however, consider dysfunction in multiple tissue types that may contribute to hormonal imbalances. It is well established that an association between obesity and PCOS exists and it is often the first therapeutic target for re-establishing reproductive function in obese PCOS patients. Furthermore PCOS patients tend to show distinct gynoid body fat distribution, which is reported to aggravate PCOS symptoms. It was therefore valid to examine the involvement in adipocyte function and its contribution to androgen levels within peos. This is further supported through the link between metabolic disorders such as insulin resistance and hyperinsulinemia, and their associations to obesity. Our study employed isolated preadipocyte and thecal cultures with close regulation of the influential factors LH and insulin. In doing so, we analysed androgen synthesis through activation and expression of steroidogenic enzymes CYP17 within both normal and polycystic ovaries. This allowed us to examine whether protein/hormonal concentrations vary across non-PCOS and peos cultures. This also allowed us to examine the possibility of a novel pathway leading to localised adipocyte synthesis as well as pinpointing whether dysfunction existed within the insulin-signalling pathway of thecal androgen steroidogenesis. The work in this thesis shows that adipocytes derived from non-PCOS and PCOS women, maintained in vitro differ on the basis of their morphology, rates of differentiation and proliferation. Furthermore, they reacted differently under conditions designed to mimic PCOS in vitro (increased insulin and LH), with reduced non-PCOS proliferation, and increased non-PCOS androgen secretion on insulin treatment. We also found increased steroidogenic CYP 17 expression in PCOS cultures under insulin stimulation. However PCOS adipocytes androstenedione secretion remained unaffected by insulin stimulation and secreted constant levels of androstenedione similar to that seen by insulin stimulated non-PCOS adipocytes. Our examination of non-PCOS and PCOS primary thecal cultures showed CYP17 expression is increased in pcas theca under basal conditions and that increases in insulin and LH leads to increases in in vitro theca proliferation. These conditions were also seen to lead to significant increases in androstenedione secretion over non-PCOS thecal cultures, and the results suggest it to be acting through the PI3-K pathway. These results therefore point to a specific area of dysfunction that should be further targeted for examination. Furthermore, they suggest that an adipocyte dysfunction exists within PCOS patients that may significantly contribute to hyperandrogenemia through localized synthesis of androgens.

Progesterone has been shown to be neuroprotective in a number of central nervous system injury models, including cerebral ischaemia. There is still a lack of understanding behind progesterone’s neuropotective properties, and the purpose of this project is to clarify some of these issues. Osmotic mini-pump infusion was hypothesised to more effective in delivering progesterone to the target organ of the brain, when compared to a bolus intraperitoneal injection. Progesterone pharmacokinetic profiles were compared between different dosing regimes. Intraperitoneal progesterone injection had a short half-life in both plasma and brain, while osmotic mini-pumps delivered higher concentrations of progesterone in plasma and particularly in brain, over a longer period, which supports the hypothesis. It was hypothesised that progesterone will reduce NO production and cell death in in vitro. Progesterone reduced nitric oxide production after challenging microglia with LPS, which supports our hypothesis and the nuclear progesterone receptor was found not to have a major role in nitric oxide attenuation. Neither of the microglial cell lines, BV-2 and HAPI cells produced elevations in NO formation in ischaemic conditions. The in vitro oxygen and glucose deprived model of ischaemia, reduced viability in both microglial and neuronal cells. Also, high pharmacological concentrations of progesterone exacerbated ischaemic injury, which does not support the hypothesis of progesterone in reducing cell death. Progesterone administration, via osmotic mini-pump infusion, was hypothesised to have a better outcome compared to vehicle treatment. After the onset of experimental stroke, progesterone delivery via osmotic mini-pump with loading dose was found to be beneficial in terms of neurological deficit score in adult male mice, which supports the hypothesis. Also, we hypothesise that co-morbidity can affect the efficacy of progesterone treatment in outcomes. Aged animals have an increased sensitivity to experimentally induce stroke and did not display, in the outcomes measured, any benefit from progesterone treatment. NOD/ShiLtJ mice had severe symptoms, resulting in high mortality after surgery and are not recommended as a model of diabetes for experimental stroke. Hypertensive BPH/2 mice are a potential hypertensive model and had better functional outcomes after treatment with intraperitoneally administered progesterone, compared to non-treated hypertensive animals in our small preliminary study. This supports our hypothesis that co-morbidity can affect the efficacy of progesterone treatment in outcomes. The gold-standard for assessing intervention effects across studies within and between subgroups is to use meta-analysis based on individual animal data. We hypothesise meta-analysis would reveal progesterone to reduce lesion volume, but also discover other effects in different subgroups of animals. Progesterone significantly reduced lesion volume, it also appeared to increase the incidence of death following experimental stroke. Furthermore, this negative effect appears to be particularly apparent in young ovariectomised female animals. These findings support the hypothesis that progesterone reduces lesion volume and progesterone having other effects in different subgroups. This investigation has clarified some issues and expanded our understanding on the neuroprotective properties of progesterone. However, these findings indicate further investigation is still required before progesterone can be considered for use in clinical trials as a neuroprotectant in stroke.