Osteoarthritis (OA) is a highly prevalent, debilitating disease affecting many joints including the knee. Despite the involvement of several tissues, it is believed that the articular cartilage is the primary site of pathogenesis in humans. Within this study, a new scoring system of OA was devised, incorporating the articular cartilage and underlying bone, aimed at providing a more comprehensive means of grading the severity of tissue damage. We examined changes progressively from mild to severe and were able to deduce from the scoring system that bone changes may precede those of the overlying cartilage. Immunohistochemistry was used to assess stem cell marker expression, proliferation and progressive changes within the extracellular matrix of sectioned osteochondral plugs, however no distinct pattern of change could be extrapolated, highlighting the variable nature of this taxing disease. Previous studies have demonstrated the presence of a sub-population of chondroprogenitor cells present in normal hyaline cartilage. We demonstrated in this study that a similar group of cells reside in osteoarthritic articular cartilage. We were able to isolate and expand clonally derived primary cell lines to beyond 50 population doublings whilst maintaining a chondrogenic phenotype, and demonstrated the tri-lineage potential of these cells. That said, a significant amount of variation was observed and it was, therefore, postulated that there may be a smaller cohort of viable cells within this sub-population isolated from osteoarthritic cartilage. A preliminary study was also carried out comparing chondroprogenitors from normal articular cartilage to those isolated from OA tissue. Heterogeneity was again encountered, suggesting that there was a group of OA chondroprogenitors with similar characteristics to the normal cells, which differed from the other less metabolically active cells. This finding was agreeable with the aforementioned postulation. Data from our preliminary integration study was promising as we demonstrated the potential for using these chondroprogenitor cells in combination with other cells whilst achieving successful integration. However, further work is necessary to distinguish between the cell lines with the potential for integration from those that lacked this ability, thereby eliminating the heterogeneity. The presence of viable chondroprogenitor cells in OA tissue challenges the dogma that the tissue is irrecoverable, and opens the scope for regenerative medicine using resident progenitor cells. This is an exciting prospect that could significantly contribute to articular cartilage repair.

Flavonoids and phenolic compounds (aka polyphenols) are phytochemicals, thought to participate in plant development and defence mechanisms. Polyphenols are ubiquitous plant secondary metabolites, and are usually found conjugated as glycosides or esters. These compounds have been of particular interest as part of the human diet, and have been the focus os many studies in nutrition research. Many epidemological studies have found a correlation between flavonoid intake and protection against certain chronic diseases such as cancer and cardiovascular events. The mechanisms underlying such benefits, however, remain to be unveiled, as judged by the number of in vitro and animal model intervention studies. The primary property attributed to polyphenolic and phenolic compounds relates to their antioxidant activities. Outcome from in vitro studies have established the ability of polyphenols to scavenge radical species and bind to metal ions, thus preventing the damage caused by oxidative stress. Recent progress in the field has broadened the knowledge of how polyphenols exert their beneficial effects, which appears to depend on more than simply antioxidant activity. Indeed, polyphenols are thought to actively participate in the modulatory effects involved in signal transduction in cells, responsible for the regulation of genes, apoptosis and cell proliferation.

The extracellular calcium-sensing receptor (CaSR) is a G-protein coupled receptor central to systemic Ca2+ homeostasis in mammals. We have previously shown that CaSR is expressed in primary bovine aortic vascular smooth muscle cells (VSMCs). Furthermore, dominant-negative adenoviral knockdown of this receptor in bovine VSMCs in vitro causes enhanced calcification in the presence of culture conditions which mimic vascular calcification in vivo. Using mineralising conditions in vitro, we have also previously demonstrated that positive pharmacological allosteric activation of CaSR using the calcimimetic, R-568, significantly reduces calcification of bovine VSMCs. These findings now implicate the CaSR, not just in systemic Ca2+ homeostasis, but also in potential protection against vascular calcification; a condition associated with increased blood pressure, left ventricular hypertrophy, chronic kidney disease and cardiovascular morbidity. Using a specific targeted deletion strategy, we have developed CaSR-specific knockout in VSMCs driven by the SM22α promoter. Using in vitro, ex vivo and in vivo approaches, here, I have characterised the cardiovascular phenotype of this novel transgenic mouse model. In vivo data demonstrate that ablation of CaSR from VSMCs causes hyperkalaemia at 3 months of age and hypercalcaemia throughout life. 3 month old CaSR-VSMC Knockout (KO) mice also exhibit reduced bone mineral integrity and increased heart weight at the age of 18 months. Ex vivo analysis implicates the VSMC-CaSR as a modulator of blood vessel tone. CaSR-VSMC KO mice exhibit reduced luminal diameters of the aorta and mesenteric arteries. CaSR-VSMC KO also reduces contractile tone in addition to evoking Ca2+-dependent relaxation only, compared to CaSR-wild-type (WT) mice which exhibit both Ca2+-dependent contraction and relaxation of the aorta. In vitro analyses confirm that CaSR expression and activation reduces Ca2+-dependent proliferation and mineralisation. In conclusion, the VSMC CaSR is a modulator of vascular tone ex vivo and of VSMC proliferation and calcification in vitro.

PDIp is a close homolog of the well known protein folding catalyst PDI. Unlike PDI however, PDIp exhibits restricted protein expression and is found predominantly in the exocrine pancreas. Currently, the physiological function of PDIp is unknown but previous work has shown a clear specificity for substrates containing a hydroxyaryl group. Again this is in contrast to PDI which exhibits more general specificity. This work to investigate the in vitro activities of PDIp, was stimulated by the hypothesis that PDIp has an essential role in folding a specific subset of secretory proteins. The identity of these proteins is currently unknown. In this work, the redox-mediated conformational changes of PDIp have been studied and compared using far UV CD, dynamic light scattering and limited proteolysis. Compared to PDI, these changes are conservative in PDIp. Also, unlike PDI for which b’xa’c is the minimal redox-active cassette, the PDIp a and b domains seem to be involved in modulating these conformational changes. This may indicate that PDIp has a unique substrate binding mechanism that may work synergistically with its restricted substrate specificity. Using the insulin reduction assay, PDIp was shown to have ~50% of the oxidoreductase activity of PDI and this was not due to the aberrant threonine residue in the a’ domain active site motif (CTHC). Further investigation by stopped flow kinetic studies showed that the low activity could be due to the abnormally high pKa for the PDIp a domain N-terminal catalytic cysteine. This result was unexpected because PDI and PDIp a share the same catalytic active site motif (CGHC) indicating that nearby residues may act as mediators of activity. Future work to clarify this will be essential. This is the first study of the structure of PDIp and its molecular basis for activity. Through investigation of these two areas, it is hoped that the general understanding of the role of PDIp and its contribution to oxidative folding in secretory tissues will be improved.

The ghrelin receptor (GRLN-R) was cloned in 1996 after the discovery that a series of synthetic growth hormone-releasing compounds (the growth hormone secretagogues) acted through a receptor distinct from the growth hormone-releasing hormone receptor. In 1999 the endogenous ligand of the receptor, ghrelin, was discovered. As well as stimulating growth hormone release, ghrelin has been shown to be involved in many other processes such as appetite stimulation and the regulation of energy homeostasis, making the ghrelin/GRLN-R system an attractive pharmaceutical target for the treatment of disorders such as growth hormone deficiency, cachexia and obesity. The GRLN-R displays a high level of ligand-independent (constitutive) activity and has been suggested to couple to Gaq/11, Gai/o, Gas and Ga12/13 G protein pathways, although little is known about the signalling of ghrelin and the growth hormone secretagogues in all but the Gaq/11 pathway. Two of the growth hormone secretagogues, GHRP-6 and L-692,429, have been described as ‘ago-allosteric modulators’ of the GRLN-R as, when co-administered with ghrelin, GHRP-6 and L-692,429 were reported to act both as co-agonists (increasing the efficacy of the ghrelin response) and as negative or positive (respectively) regulators of the potency of ghrelin. This study sought to investigate the pharmacology of the GRLN-R through the Gai/o pathway. [35S]GTPyS binding assays were used to measure activation of the Gai/o pathway, demonstrating that GHRP-6, L-692,585 (a commercially available analogue of L-692,429) and a third growth hormone secretagogues, MK-677, acted with higher efficacy than ghrelin. At least in the system tested, upon co-administration with ghrelin each of the growth hormone secretagogues acted in a simple competitive fashion with ghrelin. Radioligand binding experiments showed that the dissociation kinetics of [His[125I]]- ghrelin from the GRLN-R were not altered by co-administration of the growth hormone secretagogues. Fitting data to a modified operational model of allosterism demonstrated that GHRP-6, L-692,585 and MK-677 were not ago-allosteric modulators of the GRLN-R but simple orthosteric agonists. In order to further examine the receptor-specific effects of ghrelin and the growth hormone secretagogues, a Flp-In™ T-REx™ HEK293 cell line expressing the GRLN-R was constructed. [35S]GTPyS binding assays confirmed that the GRLN-R was constitutively active through both the Gaq/11 and Gai/o pathways, demonstrated by an increase in [35S]GTPyS loading upon receptor expression which could be reduced by the administration of the GRLN-R inverse agonist SPA. Upon expression of the GRLN-R a considerable level of cell detachment was observed with the remaining cells appearing rounded compared to parental HEK293 cells, an affect that appeared to be mediated by the constitutive activity of the receptor. In contrast to the [35S]GTPyS assays used to measure activation of Gai/o, [35S]GTPyS assays with a Gaq-immunoprecipitation step demonstrated that the growth hormone secretagogues acted with equal efficacies to that of ghrelin, demonstrating functional selectivity at the GRLN-R. Intact cell assays were also used to measure Gaq/11 and Gai/o responses, however, a Gai/o-mediated response could not be measured in cAMP accumulation assays, suggesting that the GRLN-R could signal via activation of Gao but not Gai1-3. Although the activation of the Gas pathway by the GRLN-R remains controversial, in this study ghrelin and the growth secretagogues could evoke a Gas-mediated cAMP response (although L-692,585 acted with a lower efficacy than ghrelin). Finally, two naturally occurring missense mutations of the GRLN-R (A204E in the second extracellular loop and I134T in the third transmembrane helix) were analysed to investigate whether these mutations led to retention of the receptor within the endoplasmic reticulum and to investigate whether the mutations affected the ability of the GRLN-R to signal to the growth hormone secretagogues. The A204E mutation caused partial retention of the GRLN-R within the endoplasmic reticulum, whilst receptor that was transported to the plasma membrane did not display any measurable constitutive activity. In contrast, the I134T mutation did not alter receptor localisation, nor did it have any effect on the constitutive or ligand-induced activation of the GRLN-R, however, it appeared to lower the efficacy of the inverse agonist SPA.

The circadian clock is an internal mechanism found in most organisms generating a 24h rhythm, evolved to anticipate predictable environmental changes thus making best use of resources. Intensely studied in the model plant Arabidopsis thaliana (L.) Heynh, the circadian clock was found to control a large number of physiological traits and the expression of more than a third of the plant genes. Clocks therefore play a central role in the life of organisms, in fact plants lacking clock functionality lose their ability to anticipate the dawn showing a reduced fitness. In a perspective of raising global temperatures, impact on crops production of plants with a non optimised clock could be of major importance. The present study therefore investigated the importance for Arabidopsis plants of having a functional clock at high temperatures. Arrhythmic plants over-expressing the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene were compared to wild-type Col-0 plants: while at 17°C wild-type plants showed more leaf area, biomass and chlorophyll content than CCA1ox, at 27°C the difference was even greater. This increased difference at high temperature was also confirmed in successive transcript and metabolite profiling of the two lines. Not only a functional but also an accurate clock was previously found to be important for a good plant performance. For this reason investigation on the impact of high temperature was extended to plants with an internal clock period not matched to the external light/dark cycle. Period mutant lines ztl (30h) and toc1 (20h) along with their wild-types (24h) were grown in 12LD, 15LD or 10LD cycles. ztl line showed no difference from Col-0 at 17°C but a marked difference at 27°C, where the lines with a period matching the external light/dark cycle performed better. Similarly the toc1-1 line performed better at high temperature when its clock period matched the environment. Another key feature of circadian clocks is temperature compensation, a mechanism able to maintain an accurate and robust rhythm with a period close to 24h over a broad range of temperatures. To quantify the importance of temperature compensation at high temperatures, the mutant line gi-11, defective for the temperature compensation mechanism, was used. Little differences were found at both 17°C and 27°C between the gi-11line and its wild-type WS. Finally, a screen was performed to identify new components of the clock specifically required for function at high temperatures. At the end of the screen pipeline, four putative circadian mutants were identified, which will need to be further characterised to confirm their altered rhythmicity and eventual position in the current clock model.

With intensive and global usage, flame retardants (FRs) have played critical roles in the prevention of fires for decades. However, there are increasing concerns about the potential adverse effects of these chemicals due to the well documented environmental and human exposures to FRs. To date, relatively little is known about the molecular mechanisms of the potential toxic effects of human exposure to FRs. In this study, microarray-based transcriptomics and direct injection mass spectrometry based metabolomics were employed to investigate the molecular responses of human lung cancer cells (A549) and human hepatoma cells (HepG2/C3A) exposed to a range of sub-lethal concentrations of hexabromocyclododecane (HBCD), tris (1, 3-dichloro-2-propyl) phosphate (TDCIPP) and a mixture of FRs at equivalent concentrations to those found in typical household dust. Combined with the quantification of FRs levels in cells after exposure, this work using the non-targeted capabilities of multi-omics approaches has revealed that at the concentrations investigated, and which are relevant to human exposures, significant molecular perturbations are not induced by exposure to the FRs under study. The results from this thesis are beneficial for both understanding the potential mechanisms of effects of human exposure to FRs and for future risk assessment of these chemicals.

Glucocorticoids are a class of steroid hormones which are highly relevant in human health and disease as they are involved in the regulation of carbohydrate, protein and fatty acid metabolism and are instrumental in the onset or progression of various diseases including those associated with glucocorticoid deficiency or excess. As an example, cortisol and insulin are involved in diurnal metabolic processes but their effects and interaction on healthy subjects are not completely elucidated yet. My PhD programme had the objectives to (1) assess and validate computational methodologies for application in untargeted metabolomic studies of healthy humans and those diagnosed with glucocorticoid-related diseases and (2) to investigate the global and tissue-specific metabolic changes induced by glucocorticoid excess and deficiency and their integrated effects with the relevant hormone insulin. A study of data pre-processing methods applied for untargeted metabolomics including different normalisation, missing value imputation, transformation and scaling methods were investigated on an in-silica modified dataset. The results showed that different combinations of data pre-processing methods influenced the results and different data pre-processing methods should be applied for univariate and multivariate analysis. Untargeted metabolomic studies ofbiotluids were applied to investigate in-vivo global effects of glucocorticoids. The study of the separate and integrated effects of cortisol and insulin showed that insulin may have negating effects on the influence of cortisol and treatment with cortisol should be timed appropriately during the day to minimize the effect of insulin on the therapeutic effect of cortisol. The studies reported here have shown the influence of the interactions between glucocorticoids and insulin across the metabolic network.

The Organisation for Economic Cooperation and Development (OECD) established standardized tests to predict the chemical behaviour in the environment. Difficulties exist in the extrapolation of data from laboratory OECD tests into the environment, and prediction of possible scenarios regarding environmental pollution is not accurate. The current project aimed to increase the realism of OECD tests and to investigate the microbial populations involved in the biodegradation of paranitrophenol (PNP). River water, sediment and biofilms were sampled from a stretch of River Dene with an effluent discharge point (Wellesbourne WWTP), and were used as microbial inocula in biodegradation studies. The effect of light, PNP concentration and inoculum preparation on PNP biodegradation was determined. Culture dependent techniques were used for isolation of PNP-degrading bacteria while culture independent techniques; including 16S rRNA Terminal Restriction Fragment Length Polymorphism, QPCR, and high throughput sequencing targeting the PNP functional genes (pnpA and mar), enabled detection and characterization of PNP-degrading bacteria. Light incubation lead to increased river water pH which inhibited PNP degradation. A threshold PNP concentration was determined around 42 μg/L. Application of biofilm inocula improved the reproducibility of PNP biodegradation at a concentration of 2 mg/L, and increased the amount of microbial biomass in test systems. Pseudomonas syringae was identified as a key PNPdegrader. Additional groups of PNP-degrading bacteria were detected based on the analysis of pnpA and mar functional markers. It was shown that location of sampling site for inoculum collection had no impact on biodegradation test outcome but variation of microbial inocula between sampling dates may affect the biodegradation of PNP. Core and satellite taxa analysis demonstrated that ‘biodegradation lottery’ is not the major process which determines the successful chemical biodegradation, and that probably microbial interactions within inoculum affect proliferation of PNPdegraders, and therefore impact on test results. This project revealed the lack of consideration of microbial inocula in the OECD biodegradation test guidelines. Further experimental work was suggested to expand current studies to different chemicals and rivers, and to develop more predictable approaches for better chemical risk management by Industry and Regulatory bodies.

The role of CRH and CRH related peptides in mediating HPA axis and various pathophysiological processes is well known. Over the last decade the role of CRH in mediating cancer has been widely studied and considered to exert variable functions. This study was undertaken to study the biological role of CRH in ER(+) and ER(-) breast cancer cell lines. Since estrogen is related to normal as well as the neoplastic growth, the CRH effects were studied with and without estrogen in ER (+) MCF7 and ER (-) SKBR3 cell lines. The role of the stress hormone CRH in breast cancer is complex, and its abundance and biological activity may be modulated by estrogen. The signaling mechanisms were investigated by employing phosphokinase assay to identify protein kinases activated by CRH in ER(+) MCF7 cells. CRH activated numerous kinases and downstream effectors, at least some of which were mediated by the CRH receptor type 1 (CRH-R1). MAPK, GSK3β and Akt were further investigated to study downstream effects. The analysis of the spatiotemporal characteristics of MAPK activation suggested that CRH mediated p38 response is strong compared to ERK1/2 which is inhibited by the CRH in MCF7 cells. UCN II also showed a similar response, but the extent of p38 response is not as strong as with CRH. The MAPK effects were studied in SKBR3 cells and interestingly, the CRH and UCN II mediated effect were inhibited by 24 hrs of estrogen treatment. CRH also increased the transcription of many genes that encode effectors, transcriptional targets, or regulators associated with estrogen signaling. CRH mediates its effects by activating two types of CRH receptors, i.e R1 and R2. The tissue sensitivity to agonists is determined by the presence of receptors in the plasma membrane and signal activation. This project was undertaken to investigate cellular expression of CRH-R and CRH-R1 splicing variants (α,β,c,d) and their internalization characteristics. CRH-R has been confirmed in both the cell lines. One of the goals of this project was to identify the expression of CRH-R1 splicing variants in both the cell lines with and without estrogen. CRH-R1α and R1d expression was confirmed in the ER (+) cells. However the CRH-R1d expression was lacking in SKBR3 cells. The CRH mediated splicing of CRH-R1 receptor was dose dependent. This splicing event is regulated by the splicing factors, thus in silico analysis was performed to identify splicing factor, with high affinity binding at exon 12 which is missing in CRH-R1d. It was hypothesized that Estrogen regulated these effect downregulating serine/arginine-rich splicing factor 55 (SRp55) expression resulting in generation of CRH-R1d. The results show that E2 is a driving factor influencing CRH-R1 gene exon 12 splicing and causing an increase in the expression of CRH-R1d. Immunofluorescence demonstrated that CRH treatment initiates the internalization of receptor inside the cytoplasm but this effect is lost when cells were treated for 24 hrs with E2, probably due to generation of CRH-R1d which loses internalization properties. This effect is lost in the absence of E2 receptors (SKBR3 cells).