Background: Several studies have documented differences between individuals from different ethnic groups in terms of the way in which medications are viewed. These views can potentially impact on medication adherence. However, comparisons of adherence between rheumatoid arthritis (RA) patients from other minority ethnic groups and South Asians have not been reported. Method: Two studies were undertaken in patients with RA: a quantitative survey (180 patients) to investigate the relation between demographic, diseases related and psychological variables and a qualitative study explored the reasons for poor adherence to medications used in RA. Results: The quantitative survey undertaken for this thesis demonstrated that South Asian RA patients exhibited more negative beliefs about medicines and lower levels of adherence to RA medicines than did their White British counterparts and were more dissatisfied with the information they received about their medicines. Results from the interviews showed that four interlinking themes influenced adherence in both South Asian and White British ethnic groups. Conclusion: This thesis has demonstrated that some beliefs about medicines and illness perceptions differ between the South Asian and White British ethnic groups; these beliefs and perceptions are important in understanding differences in adherence between these two groups.

Dr James A. Thomson reached a milestone discovery in 1998, as he managed to isolate and in vitro expand embryonic stem cells originating from human blastocysts. Since then, human embryonic stem (hES) cells have served as excellent tools for the understanding of a plethora of events that take place during embryogenesis. A full and comprehensive analysis of the molecular mechanisms that regulate both pluripotency and differentiation procedures will ultimately allow these cells to be utilised for therapeutic purposes. The first part of the present thesis is dedicated to investigating the implication of ADP-Ribosylation Factor 6 (ARF6) in TGFβ signalling. ARF6 is a low molecular weight GTPase involved in various cellular functions. Our preliminary data indicate that ARF6 interacts with SMAD4. Building on that, we uncover novel interactions of ARF6 with proteins SMAD2/3 and the interconnection between nucleotide status and downstream signalling events. The connection between ARF6 and TGFβ signalling led us to hypothesize a role for the GTPase in hES cells. In that system, we characterise the effects of ARF6 activation or knockout on both Activin A and BMP4 signalling. In addition, we uncover a novel role for the GTPase during mesendoderm specification. In the last part of the thesis, we utilise a broad transcriptomic approach to reveal novel candidates that are implicated in early differentiation of hES cells to mesendoderm. The assay has been carried out using a novel culture system, based on the ability of Activin A to preserve pluripotency and BMP4 to initiate differentiation.

Clinical trials are prospective studies in volunteers to test the safety and efficacy of a drug or intervention in a well-defined, controlled experiment. Pharmaceutical companies spend billions of dollars each year on clinical trials. Yet, despite the rising levels of chronic diseases and evidence suggesting that black patients may respond to treatments differently than their white counterparts, Sub-Saharan Africa is still represented in very few industry-sponsored trials. In addition to any immediate potential therapeutic benefit and the ability to grant patients greater access to drugs that they might not normally be able to obtain, clinical trials may also bring collateral benefits, such as investments in infrastructure and resources. To this end, clinical trials may be useful in helping to address the rising levels of chronic disease in the Sub-Saharan region of Africa. Additionally, it may not always be appropriate to extrapolate data from trials conducted in patients in the West and apply them to patients in other regions of the world, as the literature demonstrates that for certain medicines, treatment effects may differ due to genetic variations between ethnic groups. Aim: The aim of the study was to better understand stakeholder perceptions of the issues associated with the conduct of pharmaceutical industry-sponsored clinical trials in chronic diseases in Sub-Saharan Africa. A further goal was understanding what benefit, if any, conducting such trials could confer to the population and region. Methods: A multi-methods approach was adopted. The first part of the study focused on the use of semi-structured qualitative interviews with various stakeholders to identify the themes most relevant to the research objectives. The contents of the interview transcripts were thematically analysed, and a quantitative online questionnaire was created on the basis of the themes that emerged from the interviews. This questionnaire was then administered to a larger number of similar stakeholders to corroborate the findings from the first part of the study. Results: The interviews identified five main overarching themes. Those themes were as follows: (1) ethical, (2) commercial, (3) medical/scientific, (4) educational, and (5) practical. All five themes are closely related and oftentimes impact one another. The ethical issues largely related to the provision and availability of medicines post-trial and informed consent, as well as to the potential for corruption and fraud by both investigators and pharmaceutical companies operating outside the scope of tightly regulated Western competent authorities and ethics committees. The commercial considerations that were raised primarily centred on the fact that pharmaceutical companies are businesses, many of which have obligations to shareholders, and on the fact that drug development is tremendously expensive. The majority of the profit generated by pharmaceutical companies comes from their sales in the West, which is why their focus remains on that part of the world. The medical and scientific issues were primarily related to the evolution of Sub-Saharan Africa’s disease landscape and pharmaceutical companies’ responsibility to their global patients to ensure a robust understanding of how their drugs affect patients of varying ethnic backgrounds in different parts of the world. The educational issues were mainly linked to public awareness regarding what clinical trials are, as well as to the education of investigators, research staff, and ethics committee members. The final theme to emerge was practical issues raised in relation to a lack of infrastructure and oversight. The results of the questionnaire mostly echoed the findings of the interviews. Through their questionnaire responses, participants indicated that they felt that the pharmaceutical industry does have an ethical and scientific responsibility to do more to ensure that its drugs are tested in developing parts of the world, such as Sub-Saharan Africa. However, respondents indicated that pharmaceutical companies should not conduct trials in regions where they have no intention of selling their products and that the three largest barriers precluding the conduct of clinical trials in that part of the world are a lack of adequate infrastructure, a lack of commercial attractiveness, and concerns around unethical behaviour. Discussion: Although there are inherent risks and disadvantages associated with participating in clinical trials, the benefits are well known and understood for participants in the West. Therefore, most respondents across the stakeholder groups could see the potential benefits of research for Sub-Saharan Africa. However, many within the pharmaceutical stakeholder group exhibited unfamiliarity with the evolving disease landscape and level of infrastructure within Sub-Saharan Africa. The ethical issues and associated practicalities of conducting trials in that part of the world were likewise not well understood. The results of the study suggest that respondents across all stakeholder groups feel that the pharmaceutical industry needs to do more to make drugs available to patients in developing countries, both commercially and through research. As a justification, they pointed to the industry’s ethical and scientific responsibilities to do so. The commercial benefits that the industry could gain from conducting an increased number of clinical trials in Sub-Saharan Africa did not appear to be well understood by the research participants. The results also illustrated that the respondents did not think that chronic diseases should be prioritised over infectious diseases, or vice versa. By carrying out this research, important questions were raised regarding the capabilities of countries within Sub-Saharan Africa, and topics associated with the increasing prevalence of chronic diseases in that region were explored. All stakeholder groups agreed that pharmaceutical companies can play a role in addressing levels of rising chronic disease through the conduct of clinical trials. The findings of this research led to several recommendations, including allowing countries in the region to participate in bridging studies as a starting point, establishing national databases, and revisiting the restrictive wording in certain current ethical regulations.

This thesis is concerned with the synthesis, characterisation, and purification of 19 organoiridium(III) complexes, seventeen of which are novel. The complexes are of the general structure [CpX Ir(azopyridine)Z]A, where the iridium centre is coordinated to either a pentamethyl-cyclopentadienyl (Cp*) ligand, a tetramethyl(phenyl)-cyclopentadienyl Cpxph ligand, or a tetramethyl(biphenyl)-cyclopentadienyl Cpxbiph ligand. The azopyridine acts as an N,N-chelated bidentate ligand with a variety of substituents, the chemical and biological e↵ects of which are investigated. Z represents a monodentate halido ligand. In this work, complexes with chlorido and iodido ligands in this position are investigated. A represents the counterion to the cationic organoiridium complex. In this work, complexes bearing the hexafluorophosphate (PF6 ), Cl , and I anions are investigated. X-ray crystal structures of eight of the complexes are determined, confirming that the complexes adopt the expected ’piano-stool’ configuration. The anticancer properties of these complexes are thoroughly investigated in multiple cancer cell lines, revealing that several are more potent than many clinically-utilised chemotherapeutics including cisplatin (CDDP), as well as many previously reported metal-based anticancer complexes. The mechanism of action (MoA) of this family of complexes has been investigated, revealing an MoA based on the generation of reactive oxygen species (ROS) and superoxide (SO) in addition to mitochondrial membrane depolarisation. Drugs with this MoA hold the potential to selectively kill cancer cells over normal ones as cancer cells have higher levels of basal ROS and are therefore more sensitive to perturbation of their ROS balance. The charge, solubility, hydrophobicity, hydrolytic behaviour, and mechanism of action (MoA) of these complexes can all be modified with small synthetically trivial adjustments, resulting in highly potent complexes. This demonstrates this family of complexes as an effective and versatile platform for drug design.

Background. The growing population of migrants (including sub-Saharan Africans) in the United Kingdom poses challenges to British general practice. First, migrants tend to seek health care at times of crisis rather than for preventive measures. This is despite being at increased risk of certain chronic conditions compared with the indigenous population. For sub-Saharan Africans this includes hypertension-related diseases and some cancers. Little has been published about Africans’ awareness of this risk or their knowledge of associated causative factors. Second, discordant health beliefs and healthcare expectations between migrants and doctors in the UK have been found to undermine trust during consultations with general practitioners and to lead to poor patient satisfaction. Little is known about the health behaviours of African migrants whose expectations are not met by primary care in the UK. A related area where health beliefs and practices differ between African migrants and their GPs is in the use of traditional medicines. A final challenge lies in considering the wider issues that GPs must address when consulting with migrant patients, including time pressures, organisational factors and the complex nature of problems presented by migrant patients. These issues are the focus of this study. Aims. To examine African migrants’ perceptions of chronic disease and their experience of seeking primary health care in the UK. To explore the impact upon GPs of caring for migrants. Objectives. To explore: 1) perceptions of chronic disease risk facing African migrants and their underlying explanatory models; 2) experiences of consultations about antibiotic prescriptions; 3) traditional African medicine use in the UK; and (4) to consider the effect of workload and work patterns on GP consultations with migrants. Design. In-depth interviews were conducted with 19 Africans from French- or Swahili- speaking countries, one African key informant and 13 GPs working with migrants. African participant recruitment was from community organisations and GPs were approached via an informal network of doctors. Interviews were transcribed and ten were translated by the principal investigator (three Swahili and seven French). Data analysis was undertaken following the approach of applied thematic analysis using the Nvivo software package. Data collection and analyses were underpinned by the following theoretical frameworks: Kleinman’s explanatory models of illness and of cultural health care systems and Lipsky’s street-level bureaucracy. Results. Narratives suggested low awareness of chronic disease risk amongst Africans. Infectious diseases were considered the dominant health threat for African migrants, mainly HIV but also tuberculosis and ‘flu’. Chronic diseases were sometimes described by Africans as contagious. Explanatory models of chronic disease included bodily/dietary imbalance, stress/exertion, heredity/predisposition and food contamination. Cancer was feared but not considered a major threat. Cancer was considered more common in Europe than in Africa and was attributed by Africans to chemical contamination from fertilizers, food preservatives and industrial pollution. Evidence cited for these chemicals was rapid livestock/vegetable production, large size of farmed products (e.g. fish), softness of meat and flavourless food. Chemicals were reported to circulate silently inside the body and cancer to develop in the part where they deposit, sometimes years later. Africans’ belief in infective explanations of disease extended to minor illnesses and was manifested in an expectation of antibiotics from GPs for problems such as a sore throat. This arose from participants’ experience in Africa, witnessing life-threatening infectious diseases and experience of unregulated access to antibiotics. Africans described various alternative measures to fulfil their unmet expectations, including approaching other National Health Service doctors, importing medication, and using private healthcare services in London, francophone Europe and east Africa. A further option was the use of traditional African medicine, reported by one quarter of African participants. Traditional African herbal medicine use was based upon a perception of its purity and natural origin in African soil and a deep belief in its efficacy. Consulting traditional African healers in the UK was reported to be undertaken in secret. Some GPs and Africans described consultations in terms of pressure, processing and conflict. Migrants were reported to present with complex health problems that were frequently compounded by language barriers. GPs described a need to remain in control of consultations and this included some use of personal discretion to render their tasks easier to complete. The most common example was accepting patients’ family and friends as informal interpreters – a choice that ran contrary to formal policy of only using professional interpreters. Burnout was reported to be one consequence of excessive workload for patient-centred GPs working with vulnerable groups like asylum seekers. Conclusions. There is a need to improve health literacy amongst African migrants in order to promote preventive behaviours for chronic disease and alternatives to antibiotics for minor illnesses. As part of this, further research is required into the use and properties of traditional African medicine. Interventions should be built upon participants’ existing knowledge of disease causation, their self-reliance in the pursuit of a healthy lifestyle and desire to retain cultural practices. One challenge to improving migrant health lies in the service dilemmas facing GPs, including excessive workload, the complex nature of migrants’ presenting problems and professional dilemmas. GPs who act as advocates for vulnerable migrant patients may be at increased risk of burnout and greater consideration should be given to providing them with appropriate support.

Dendritic cells (DCs) are potent antigen presenting cells and are crucially involved in the induction of the adaptive immune response. Multiple DC subsets exist in the body at rest and during inflammation with unique tissue origins and development. The main function of DCs is the uptake of antigen from the peripheral tissue by endocytosis, and the transfer of this antigen to T cells within the lymph nodes (LNs) to generate an immune response against the antigen. Given the potency of the potential T cell response, DCs are an attractive cell type for therapeutic use in contexts such as cancer. Over 20 years of clinical trials have developed DCs for this purpose: although significant progress has been made, DC clinical trials still show subclinical and variable T cell responses in patients. Numerous strategies have been tested to improve this, including use of specific DC subsets, ex vivo activation and maturation of the cells using cytokines, different antigen loading strategies and different routes of injection. These, however, do not sufficiently take into consideration the migration capability of these injected cells, which severely limits the potential cell function. CCR7 is the chemokine receptor crucially involved in DC homing to LNs and is a marker of DC maturity, but it has been shown that ex vivo-generated cells do not consistently express this receptor. Using a novel sorting methodology to isolate DCs expressing CCR7, it is possible to improve the maturity and function of the injected cells, as well as in vivo migration. CCR7 expressing cells are more capable of generating mature T cell responses in vitro due to the expression of co-stimulatory molecules such as CD80 and CD86 and the production of T cell-attracting chemokines. The B16F10 mouse melanoma was used to assess the potential improvement in therapeutic DC use following CCR7-sorting. In the subcutaneous model after a single injection, or multiple prophylactic injections, of CCR7- sorted DCs there was significant control of the tumour growth and this resulted in a longer survival duration. This was attributed to the increased T cell response induced by DCs reaching the LN, as more antigen-specific T cells were present in the CCR7-expressing DC-receiving animals and that the T cell phenotype was more mature by surface marker expression. In the metastatic model, however, there was no difference in the overall tumour burden between groups despite having a significantly improved antigen-specific T cell response following injection of the CCR7-sorted DCs. Finally, it was shown that this CCR7-sorting methodology is directly translatable to clinical use using the clinical grade MACSQuant Tyto cell sorter, and that CCR7-sorted human monocyte-derived DCs were also more potent activators of T cells in vitro.

The contribution of store-operated calcium entry (SOCE) in the response of human sperm to progesterone, a steroid secreted from the cumulus cells surrounding the oocyte, has not yet been elucidated. The aim of this study was to investigate the presence of SOCE proteins in human sperm and examine the effects of pharmacological modulation of SOCE on the progesterone-induced biphasic intracellular calcium concentration ([Ca\(^{2+}\)]i) response. STIM (stromal interacting molecule) and Orai, proteins of the SOCE system were detected in human sperm in a similar location to intracellular Ca\(^{2+}\) stores. 2-aminoethyldiphenyl borate (2-APB; SOCE modulator) altered SOCE in human sperm in a bimodal manner as seen in other cell types. Furthermore, 5\(\mu\)M 2-APB potentiated the initial progesterone-induced [Ca\(^{2+}\)]i transient within the neck and midpiece, but not in the flagellum. In the sustained phase of the progesterone-induced [Ca\(^{2+}\)]i response both 5\(\mu\)M 2-APB and 10\(\mu\)M loperamide (another modulator of SOCE) potentiated the [Ca\(^{2+}\)]i response. Higher doses of 2-APB (50-200\(\mu\)M) didn’t potentiate the transient [Ca\(^{2+}\)]i and inhibited the sustained response consistent with reported actions on SOCE. Ryanodine receptors were localised to the neck/midpiece region which suggested that they may mobilise intracellular Ca\(^{2+}\) stores in response to progesterone, leading to activation of STIM/Orai and initiating SOCE.

Tamoxifen is an effective and well-tolerated treatment for early disease and/or pre-menopausal patients with breast cancer (BC); although many women go on to develop resistance. Currently the five-year survival rate following Tamoxifen resistance (TR) is < 20%; hence the mechanisms need to be better understood. Recent research has focussed on specific pathways, however additional mechanisms are involved and we investigated these using cell line models of BC (MCF7) and TR using a variety of proteomic approaches. Differential expression and phosphorylation of proteins between the MCF7 and TR cell lines were detected by antibody arrays; which detected changes in Mitogen Activated Protein Kinases and Receptor Tyrosine Kinases family members, and in apoptosis related proteins. There were 21 novel proteins found to be altered in TR. 262 quantifiable proteins were found using SILAC; 29% over expressed in resistance and 25% down regulated. 5 were subsequently picked for validation by Western blot and 2 of these (IQGAP1 and cortactin) were chosen for further investigation with siRNA and functional assays. IQGAP1 was found to play a role in TR; as decreasing expression of IQGAP1 using SiRNA decreased the proliferation of TR cells and significantly modulated the TR cells ability to invade matrigel.

The interest in the development of anticancer metal complexes for cancer therapy is growing spurred by the encouraging successful stories of platinum drugs. Osmium arene chlorido complexes had been found to show anticancer activity in vitro. In this thesis, the osmium arene iodido complexes were mainly explored and investigated. It is found that iodido OsII arene complexes with a general structure: [Os(η6- arene)(XY)I]PF6 (XY = p-hydroxy or p-dimethylamino phenylazopyridine, arene = p-cymene or biphenyl) are potently cytotoxic at nanomolar concentrations toward a panel of human cancer cell lines. In contrast to the chlorido osmium arene anticancer complexes, the iodido complexes are stable and inert toward aquation. More than thirty half sandwich azopyridine OsII arene complexes [Os(η6- arene)(azopyridine)X]+ (where X is chloride or iodide, the arene is p-cymene or biphenyl and the pyridine ring of azopyridine ligand bearing a variety of substituents) were synthesized and characterized. A preliminary structure activity relationships (SARs) were built up based on the anticancer activity towards A2780 human ovarian cancer cell line. In general, the introduction of an electronwithdrawing group (e.g. F, Cl, Br or I) at specific positions on the pyridine ring significantly increases cytotoxic activity and aqueous solubility. Changing the arene from p-cymene to biphenyl or the monodentate ligand (X) from chloride to iodide resulted in a significant increase in the anticancer activity. Studies in A2780 human ovarian cancer cells suggested that cellular uptake and targeting to cellular organelles play important roles in determining the anticancer activity. According to the 60 cancer cell lines screening results from National Cancer Institute (NCI), the anticancer activity achieved by the most potent OsII arene azopyridine complex is 100 times more than cisplatin; 1000 times activity was found in some cell lines. The mechanism of action may involve the inhibition of tubulin polymerization. One iodido osmium complex was selected for anticancer efficiency evaluation in vivo: [Os(η6-p-cym)(Azpy-NMe2)I]PF6 (FY026). This complex delayed the growth of HCT116 human colon cancer xenografts in mice, with negligible toxicity. It is the first example of in vivo antitumour activity for an organometallic osmium arene complex. Its activity appears to involve redox mechanisms. Its potency towards A2780 ovarian and A549 lung cancer cells is increased significantly when used in combination with L-buthionine-sulfoximine (L-BSO) indicating that L-BSO can be a good candidate for combination therapy treatment with iodido osmium complexes. Further study on the bioisosteres of FY026 was carried out by changing the azo bond (N=N) to imine bond (CH=N). Sixteen osmium arene iminopyridine complexes were synthesized, well characterized and showed good anticancer activity. Different structure-activity relationships comparing iminopyridine complexes with azopyridine complexes were identified which suggested a different anticancer mechanism. In contrast to FY026, [Os(η6-p-cym)(Impy- NMe2)I]PF6 (6) and [Os(η6-p-cym)(Impy-NMe2)Cl]PF6 (14) were found to undergo hydrolysis and the binding was observed between their hydrolyzed product (14A) and 9-ethylguanine. Moreover, a hydride transfer from NADH to form an osmium hydride intermediate which is involved in a catalytic process resulting in the formation of NAD+ was discovered. This process might be involved in the anticancer mechanism of action. A dual mechanism of action was proposed based in the interaction of these compounds with DNA nucleobase and catalytic oxidation of NADH.

Apoptosis is an important process in normal mammary gland physiology and evasion of apoptosis has also been identified as a hallmark of cancer. In breast cancer cells apoptotic resistance is an acquired feature that can promote tumour growth and progression. Induction of apoptosis by the extrinsic death ligand TRAIL has been shown to be a promising clinical therapy targeting a number of different cancer cells whilst sparing normal cells. Unfortunately most breast cancers are inherently resistant to TRAIL treatment. Herein it is shown that by reducing the expression of the downstream TRAIL inhibitor c-FLIP, a range of different breast cancer subtypes can be sensitised to TRAIL treatment resulting in significant cancer cell death. Significantly, suppression of c-FLIP in combination with TRAIL (FLIPi/TRAIL) ablated the tumour-initiating breast cancer stem cell (bCSC) subset, as defined by mammosphere formation assay, within cell lines. This selective killing of bCSCs translated to reduced tumour initiation and metastasis in animal transplant models. However, continued culture of FLIPi/TRAIL treated cell lines in adherent conditions resulted in bCSC re-acquisition suggesting a phenotypic plasticity of non-bCSC cells. Re-acquired bCSCs also demonstrated sensitivity to repeated FLIPi/TRAIL treatment and maintaining reduced c-FLIP expression prevented bCSC re-acquisition. These results substantiate the importance of resistance to apoptosis in tumour initiation and metastasis and identify the targeting of c-FLIP proteins as a promising anti-cancer therapeutic approach. Acquired resistance to existing mainstay therapies such as antiestrogens (AEs) (tamoxifen and Faslodex) and aromatase inhibitors (AIs) is an ongoing obstacle in treatment of a large number of breast cancer patients. AE-resistant models of breast cancer and a multiple endocrine-resistant patient sample demonstrated hypersensitivity to TRAIL. This sensitivity was observed in both in vitro and in vivo models of AE resistance and cell death was prevalent in both bulk tumour cells and bCSCs. Sensitisation was not attributed to combination AE/TRAIL treatment suggesting cellular changes during the acquisition of AE resistance are responsible for TRAIL sensitivity in these models. Further investigation suggested that the mechanism of AE-resistant cell sensitivity to TRAIL was not dependant on functional estrogen receptor signalling and is most likely dependant on the AE agent that the cancer cells have acquired resistance to. Interestingly tamoxifen-resistant MCF-7 cells were shown to have reduced c-FLIP protein expression compared to parental cells, further supporting c-FLIP’s potential in cancer therapy. Recent success in the use non-MHC-restricted γδ T cells as a targeted immunotherapy in clinical trials has identified this therapeutic methodology as desirable. Here it is shown that TRAIL is readily expressed by this subset of T cells that also demonstrate cytotoxicity to breast cancer cell lines. Neither the secretion of TRAIL or surface expression of TRAIL appeared to contribute significantly towards γδ T cell cytotoxicity and the majority of breast cancer cell death induced by γδ T cells would seem to be perforin-mediated. The suppression of c-FLIP in target cells increased γδ T cell cytotoxicity but again not via TRAIL. Preliminary results also indicated that the bCSCs of some cell lines were exquisitely sensitive to γδ T cell treatment. In summary these results indicate that targeting c-FLIP and TRAIL can be therapeutically beneficial in a range of different breast cancer subtypes by certain therapeutic strategies.