The skin is the most extensive and accessible organ in the human body. It efficiently provides a barrier to an external hostile environment whilst maintaining and regulating fundamental physiological functions. The sophisticated and complex nature of this natural barrier requires continued analytical advancement to offer further insight into both its biological mechanisms and how to target the delivery of compounds through it. This work presents the use of a recently emerging technique in the field of skin research, time of flight - secondary ion mass spectrometry (ToF-SIMS), to investigate the presence of both native and exogenous compounds in skin tissue from samples collected both in vivo and ex vivo. Subtle changes to the stratum corneum lipid composition have been shown to exert significant effects on the barrier properties of the skin and are associated with numerous skin disorders. The analysis of these lipid species and factors affecting their composition, both internal and external, is therefore a vital area of research. Using ToF-SIMS, this work has conducted an examination of changes to this lipid composition that have resulted from aging of the skin. This has been achieved by undertaking extensive development of a recently proposed surface analysis method to analyse sequential tape strips of stratum corneum. This study was unprecedented in demonstrating that ToF-SIMS could obtain information on human skin from samples collected in vivo. Changes in the levels of both cholesterol sulfate and long chain fatty acids were observed as a consequence of both intrinsic and extrinsic aging, offering confirmatory evidence to previously theorised skin aging mechanisms. Research relating to the effective permeation of compounds across this skin barrier is also of upmost importance, both to the pharmaceutical and cosmetic industries, to enable the design of new topical formulations for skin delivery. Currently employed methods to assess the permeation of a compound are heavily focused on dermal delivery, with limited information obtained on the effectiveness of a compound to permeate into the upper layers of the skin. This research has therefore pioneered a dynamic SIMS method to conduct depth profile analysis of ex vivo porcine skin tissue, enabling the permeation of exogenous compounds to be monitored as a function of skin depth. This work is novel in successfully producing 3D spatially resolved chemical profiles of exogenous compounds within biological tissue using ToF-SIMS. The permeation of four different vitamin C related compounds, popular ingredients in anti-aging cosmetic formulations, were assessed using this method, highlighting a significant difference in permeation efficiency between them. An investigation into the delivery of ascorbic acid to the skin from various different formulations was also achieved, highlighting a permeation enhancing effect from delivery via a novel supramolecular gel formulation. The method developed for surface analysis was also successfully applied to monitor the permeation of ascorbic acid through human stratum corneum following in vivo application of an ‘off-the-shelf’ cosmetic product.

The ability of pathogens and their surrogate host cells to exchange molecules is at the core of all host-pathogen interaction processes. Small chemical molecule signals provide a rich vocabulary for cellular communication, and pathogens have evolved the capacity to modulate, mimic or hijack these signals in a defensive, exponentially increasing the scope and complexity of this chemical language between host cells and pathogens. The objective of this work is to use Raman microscopy (RMS) to analyze molecular changes during infection of human cells by intra- (Toxoplasma gondii) and extra-cellular (Acanthamoeba castellanii) protozoan parasites. The work is divided into two parts. Firstly, we studied host-pathogen biomolecular interaction by using RMS. Secondly, we employed stable isotopes substitution technique to induce spectral changes that are specific to the labelled molecules. In this approach mammalian cells are selectively grown in culture medium in which only certain molecules are substituted with stable-isotope counterparts, and the exchange of these molecules are monitored between individual mammalian cells and parasites in real-time. Our results show that Raman spectroscopy can be a great tool to understand the molecular processes that mediate the interaction between the pathogens and host cell by providing quantitative spatially- and temporally-resolved information regarding molecular trafficking and exchanges. A better understanding of these complex interactions will advance our understanding of microbial pathogenesis and potentially identify new therapeutic targets.

In this work tumour cells expressing well-defined antigens were generated to study the importance of interferon <IFV> modulation of T cell mediated immune responses to foreign antigens. The antigens used were those of a murine leukaemia virus (XLV). DIA clones of this virus were manipulated using standard molecular biology techniques to generate replication-defective viruses expressing individual XLV antigens (env or gag) when transfected into tumour cells. The tumour cell line used in this study (C3H201) was produced in our laboratory by the transformation of murine fibroblasts (C3H10TX) with the Kirsten murine sarcoma virus (MSV) which expresses the ras oncogene. Initially cells transfected with the MLV genes were selected by neomycin resistance and preliminary investigations using these cells suggested that gag is the main T-cell target in this system; the gag expressing cells were more susceptible to MSV/XLV-specific Tc lysis and were less tumourigenic than the env transfected cell lines. However it was found that the cells expressing the neomycin resistance <neo*> gene alone also elicited an Immune response (increased susceptibility to Tc lysis and less tumourigenic than the untransfected C3H201 cells) thus it was suggested that this selection system was not suitable for cells to be used in immunological studies and an alternative selection system was needed. To overcome the problems observed with neomycin resistance a novel selection system using the major histocompatibility (MHC) antigen D* was developed. The H-2D* gene was transfected into the H-2D* negative cell line C3H201 < H-2* > with the DIA of interest and then the cells expressing the D* antigen were sorted on the FACS following IFI-f treatment and indirect immunofluorescence staining. Experiments to determine the Immunological importance of the antigen of interest were then performed in the FI progeny from a cross between C3H/Ha (H-2*) and C57BL6 (H-2*) mice which recognise the endogenous and acquired KHC antigens of the transfected cell line as self. This selection system thus enabled the examination of the immune response to the proteins of interest without actually interfering with the response they elicit. This system was used to produce gag expressing cells and also a cell line that expresses all the XLV proteins without producing the virus itself (designed to facilitate an examination of the effect of virus replication and subsequent infection on the response to XLV antigens by comparison with a XLV expressing cell line). These cell lines were found to be susceptible to lysis by Xo-MSV/XLV-specific Tc and this lysis was increased by IFI-V treatment (cells transfected with D* alone, DC3H201, showed no such lysis). It is now known that Tc usually recognise antigen in association with class I KHC antigen. Furthermore IFV-y treatment of C3H201 cells has been shown to increases class I KHC antigen expression on these cells. Thus it is proposed that the increased susceptibility to lysis observed for these IFI-t treated cells is due to increased class I KHC antigen expression. These cells also show reduced tumourigenicity in mice in comparison with DC3K201 and C3H201 thus confirming the recognition of gag by the immune system (in the case of DK-gagl and DX- gag2) and furthermore the correlation between the tumourigenicity and susceptibility to Tc lysis of all these cells supports the importance of the Tc element in the immune response to these tumour cells.

Tumour development is a complex process involving susceptibility loci (oncogenes) and the accumulation of spontaneous somatic mutations in tumour suppressor genes that interact to result in cancer. Tumours may require the accumulation of a variety of mutations before developing but the general principle is that the mutations result in an imbalance between the oncogene and tumour suppressor gene function (Berger et al., 2011). This multi-hit process can be tested by subjecting strains of mice susceptible to certain types of tumours to chemical mutagens, thereby inducing further mutations. This technique is used in this study to identify loci resulting in the susceptibility to thymic lymphoma in the C57BL/6J strain in order to further our understanding of the processes involved in the development of such tumours.

This thesis aims at examining the contribution of Vocational Education and Training (VET) to advancing Human Development (HD) and reducing inequality, using the example of Palestine as case-study rich in multi-layered inequalities, some of which are experienced in the region and worldwide, while others are specific to adverse conditions. The case of Palestine provides fertile ground for understanding inequality and human development, and for echoing the developed knowledge through to the understanding of VET and HD globally. The thesis contributes to the previous work on VET and HD introduced by McGrath (2012c) and shaped by many scholars, including McGrath and Powell, through the Capability Approach (CA). It uses Powell’s (2014) work on “aspiration” and capability lists (CL) and relates VET to human development within the Palestinian context. The thesis provides empirical evidence of the VET Learners’ and graduates’ capabilities and functions achievements through VET in the adverse conditions present in the Palestinian context. The thesis validates and develops the VET CL, integrating inequality elements and linking it to political economy theory. The thesis utilises Gender and Development Theory (GAD), complemented by the intersectionality approach. The research borrows Kabeer’s (1999) “empowerment framework”, based on the notion of “choice” in addressing disempowerment, to analyse how VET can contribute to reducing inequality. The thesis also engages with Political Economy arguments and specifically the work of Phillip Brown, Andy Green and Hugh Lauder (2001) on the need for “social transformation” to achieve the aim of a high skill society, suggesting an increased role for government and social partners and the political will for skills upgrading. In this regard, the thesis analyses how adopted policies are used to reduce or reproduce inequalities. The research used quantitative and qualitative methods, and engaged 1,240 people representing VET graduates, students, teachers, counsellors and management of VET institutes, in addition to policymakers on national and regional levels, as well as teachers and principals of general schools, employers, community representatives and government officials. 33 VET institutions were engaged representing the different VET providers being; governmental, non-governmental, semi-governmental and UN bodies. Selected institutes and consulted people presented different identified inequality elements. The Thesis presented empirical evidence on the increased value of VET for the marginalised, and for marginalised communities in adverse conditions. It presented that VET graduates’ achievements in employment, self-employment and different kind of work as well as in their speed in transiting to the World of Work (WOW) in comparison to their peers. Also presented their ability to generate income and other economic resources for poverty reduction and starting new families. In addition, it highlighted the empowerment achievements of the graduates and its link to achieving their aspirations. Confirming the transformation of the graduates through empowering the dis-empowered. Nevertheless, graduates were faced with internal and external structural challenges. Internal in social attitudes and institutional policies and measures, while the external is in the military occupation and its effects on mobility and socio-economic status. The structural challenges have clearly affected graduates functionings, and achievements of empowerment and aspirations. The thesis presented models of institutes and best-practices by others to support VET learners in overcoming some of the internal challenges, and highlighted the deficits in national policies and measures, it also highlighted a major international deficit. In addition, the thesis has presented the VET contribution to marginalised community resilience. The thesis presented methods of measuring empowerment, functionings and achievement of aspirations as voiced out from VET graduates and learners and triangulated with other resources. One of the main contributions of the thesis is in bringing in GAD to CA in HD approach to VET, and in linking them to political economy, providing a holistic framework to examine VET contribution to HD and reducing inequality. The thesis was able to link arguments about VET and inequality in an international context, and provided empirical evidence for the significance of the link between VET and empowerment in the reduction of inequality, thus indicating the importance of VET in the debate on inequality, and the importance of empowerment measures and goals for identifying the role of VET in reducing inequality and advancing human development.

Research during the past decade has highlighted the functional role of perivascular adipose tissue (PVAT) in regulating the contractility of the underlying vascular smooth muscle cell layer. However, the mechanisms underlying these observations are poorly understood. As a sexual dimorphism has been identified in the regulation of vascular tone, the aim of this thesis was to determine whether there are sex differences in PVAT-mediated regulation of the porcine coronary arterial (PCA) tone. In the presence of adherent PVAT, which was stored overnight at 4Co, contractions to the thromboxane mimetic U46619 and endothelin-1 were significantly reduced in PCAs from females, but not PCAs from males. In PCAs pre-contracted with U46619, re-addition of PVAT caused relaxation in PCAs from females, but not PCAs from males. This relaxant response in coronary arteries derived from females was inhibited by a combination of both NO synthase inhibitor (L-NAME) and the cyclooxygenase inhibitor indomethacin. Pre-incubation with an anti-adiponectin antibody abolished the relaxant effects of PVAT, indicating that adiponectin is likely to be the mediator released from the fat. There was no difference in either expression or release of adiponectin from PVAT between sexes. On the other hand, the adiponectin receptor agonist adipoRon produced greater relaxation in PCAs from females compared to PCAs derived from males. No adiponectin receptor 1 expression was detected in PCAs whilst adiponectin receptor 2 and adiponectin binding protein (APPL1) were expressed equally in PCAs from both sexes. Pre-incubation with the hydrogen sulphide enzyme inhibitors 4-propargylglycine (PPG) and 2-(aminooxy)-acetic acid (AOAA) did not reduce the anticontractile responses to PVAT. Cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3MPST) are expressed in PVAT from both sexes equally, with a relatively low activity, whilst no cystathionine γ-lyase (CSE) expression has been identified in PVAT from both sexes. These data indicate a clear sex difference in the regulation of coronary artery tone in response to adiponectin receptor stimulation, which may underlie the anticontractile effects of PVAT in females. Although H2S synthesizing enzymes are expressed in PVAT, they have no functional role in PVAT-induced vasorelaxation. In arteries from both male and female pigs, addition of fresh PVAT caused a contraction, which was partially inhibited by the cyclooxygenase inhibitors indomethacin and flurbiprofen. The PGF2α receptor (FP) antagonist AL8810 attenuated the PVAT-induced contractions in arteries from males, whereas the TXA2 receptor (TP) antagonist GR32191B inhibited the PVAT-induced contractions in arteries from females. Although there was no difference in PGF2α levels in PVAT between females and males, PGF2α produced a larger contraction in arteries from males, correlating with a higher FP receptor expression detected by immunoblotting. In contrast, the release of TXB2 from PVAT from females was greater than the release from males PVAT, but there was no difference in the contraction by the TXA2 agonist U46619. Furthermore, there was no difference in the role of extracellular Ca2+ and Rho kinase pathway (intracellular Ca2+) in the action of U46619, or TP receptor expression in arteries from different sexes. Pre-incubation with the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC), a non-selective NADPH oxidase (Nox) inhibitor (DPI), a selective Nox1 inhibitor (ML171), a selective Nox2 inhibitor Phox-I2 and selective Nox1 and Nox4 inhibitor (GKT137831) significantly reduced PVAT-induced vasoconstriction in PCAs from both females and males. Nox1, Nox2 and Nox4 were expressed equally in PVAT from male and females. However, in PCAs, Nox1 expression was greater in females whilst Nox4 was higher in males. Nox2 was expressed equally in PCAs from different sexes. ML171 and phox-I2 reduced superoxide anion (O2-) production in PVAT and PCAs from both sexes. GKT137831 inhibited H2O2 production in PCAs, but not PVAT, from both sexes. Both U46619 and PGF2α had similar effects on ROS production in that they did not increase O2- production in PVAT while they enhanced O2- production in PCA from females only. Functional studies showed that Nox inhibitors could inhibit the PCAs response to both U46619 and PGF2α in females only. The adipose-derived compound chemerin-9 caused a significantly higher vasoconstriction in PCAs from females in comparison with males PCAs. Similarly, chemerin-9 enhanced Nox activity in females PCAs but not in males PCAs. Chemerin mRNA was expressed in the PVAT, and the chemerin receptor ChemR23 was expressed in PCAs, although there were no sex differences. The anti-chemerin antibody can abolish the PVAT-induced contractions in both sexes. The sexual dimorphism in the contraction to chemerin could be explained by differences in the signalling downstream of the chemerin receptor in PCAs rather than the expression of chemerin or its receptor in PVAT and PCA, respectively. In conclusion, this thesis has demonstrated clear sex differences in the regulation of coronary artery tone by PVAT, with a dual effect of PVAT on the contractility of the PCA. PVAT stored overnight enhanced vasorelaxation in PCAs from females only due to the increased adiponectin activity compared to the males PCAs. In contrast, fresh PVAT augments vasoconstriction in PCAs from both sexes. Prostanoids have an important role in PVAT-induced vasoconstriction in which PGF2α and TXA2 are responsible for vasoconstriction in male and female PCAs, respectively. In addition, chemerin may have a role as a PVAT-derived contractile agent in female PCA only, while Nox-derived ROS regulates PVAT-induced contraction in the PCAs from both sexes. This study highlighted the importance of considering potential sex differences in the responses to PVAT and that sex-specific drug treatment may represent a potential strategy in the treatment of cardiovascular diseases.

Inequalities in cancer survival outcomes can partly be explained by prolonged cancer symptom presentation among socioeconomically deprived groups. This PhD aimed to (1) understand the barriers to cancer symptom presentation among low socioeconomic groups and (2) develop a targeted cancer awareness intervention to promote timely symptom presentation. The COM-B (Capability, Opportunity, Motivation-Behaviour) model was selected to guide understanding of the influences on cancer symptom presentation behaviour. Systematic review and qualitative methods (30 in-depth interviews and six focus groups) were employed to identify the factors influencing symptom presentation. Findings from these studies and a scoping review of cancer awareness interventions were used to inform intervention development, guided by the Behaviour Change Wheel. The intervention was tested for acceptability with two groups of potential users. The combination of poor cancer symptom knowledge, fearful and fatalistic beliefs about cancer, and barriers such as problems associated with obtaining and accessing a primary care appointment prolonged cancer symptom presentation among low socioeconomic groups. In addition, the wider social and environmental opportunities available to people from low socioeconomic groups including economic hardship and negative experiences of cancer were identified as key influences on behaviour. An intensive community group based educational session was developed targeted at current or former smokers and family members of smokers, aged 40 years or over from socioeconomically deprived communities. Content was developed to increase cancer symptom knowledge, modify beliefs and enable timely symptom presentation by utilising strong social networks in the community. Findings from user testing confirmed that group education was an acceptable mode of intervention delivery. Understanding the complex interaction between individual psychological characteristics and the wider environment in which people from low socioeconomic groups live in is essential for modifying cancer symptom presentation behaviour. Community education could be used as a strategy to engage low socioeconomic groups in early cancer detection and warrants further feasibility and pilot testing.

Colorectal cancer (CRC) patients survive and stay free of disease for longer after surgery if their primary tumours were infiltrated with an increased density of T cells. Studies of breast tumours and melanoma have also shown that the presence of specialised blood vessels named high endothelial venules (HEVs), within tumours are associated with a high density of infiltrating T cells and a positive prognosis. It is therefore possible, that presence of HEVs within CRC is associated with a high density of infiltrating T cells and a good patient outcome. To test this hypothesis, primary tumours, resected from sixty-two CRC patients were analysed for the presence of HEVs. These were studied with respect to the numbers and distribution of intra-tumoural T cells as well as tumour stage and patient survival. The results showed that HEV developed in response to CRC but were found within the extra-tumoural area and not the tumour mass. HEVs were also always present within a concentration of T and B cells, namely lymphoid aggregates which resemble ectopic lymphoid structures (ELS). These ELS were associated with more advanced disease and hence did not necessarily identify patients with a better prognosis. Recent studies have suggested that the type of T cells infiltrating the tumours is a determinant for patient outcome indicating that not all T cells confer benefit. IL-17A producing T cells are thought to drive CRC development. Moreover, our laboratory has previously shown that detection of a CEA (Carcinoembryonic antigen)-specific T cell response by in vitro secretion of IFN-γ is associated with tumour recurrence whereas the opposite is true for the 5T4 tumour antigen. This study therefore set out to determine whether IL-17A producing T cells are present at higher frequencies in CRC compared to normal bowel and whether IL-17Aproducing T cells are CEA-specific. The experiments revealed that IL-17A-producing T cells are present at a higher frequency within CRCs, but the prevalence of Th17 responses specific for 5T4 was slightly higher than for CEA, implying that IL-17A secretion by CEA-specific T cells was not responsible for the tumour recurrence. Tumours from CEA-responsive patients were less immunogenic than those from CEA non-responsive patients reflecting the aggressiveness of the tumour.

The work contained within this thesis explores the relationship between clinical and technological parameters of radiotherapy treatment planning and patient outcome in patients who were treated for cancer of the oesophagus as part of the SCOPE 1 clinical trial. However the methods and concepts of the work could also have applications at other tumour sites. By developing ideas from previous studies, a novel method of applying a conformity index found a significant relationship between the quality of a radiotherapy plan and patient outcome in terms of overall survival. Furthermore it was found that the plan quality could be improved by utilising a relatively new method of dose delivery. This dose delivery method also allowed the improving of tumour control via dose escalation to be explored via radiobiological modelling. The results of this work showed that although the probability of controlling the tumour is increased, there is also a significantly higher risk of increased gastric toxicity for patients with lower oesophageal tumours. Interfraction gastric movement was also investigated with the end result being a recommendation for stomach movement and toxicity to be minimised by using a pre-treatment protocol. This is being taken forward in a nationwide multicentre clinical trial. Finally a texture analysis software package was used to investigate whether there was relationship between the image heterogeneity parameters of computed tomography images and patient outcome. This work could potentially aid the decision making process of radiotherapy treatment, allowing a more informed judgement to be made on the most beneficial treatment for the patient.

Colorectal cancer (CRC) continues to cause significant global health burden, despite advances in our understanding of tumour biology, the development of screening programs and increasing public awareness about the disease. Previous studies investigating CRC pathogenesis have been criticised for focussing on the tumour tissue itself. Investigators have proposed that if early biomarkers of disease are to be identified, efforts need to be undertaken in examining pre-neoplastic tissue prior to malignant transformation. Based on the field cancerisation concept, the research hypothesis was that the macroscopically normal mucosa (MNM) around colorectal cancer and polyps is biologically altered. The aims of the study were to determine if the presence of colorectal adenomas at time of cancer diagnosis was predictive of future risk of colonic neoplasia and to characterise the global gene expression profile of MNM adjacent to CRC and adenomas. A retrospective cohort study of CRC patients demonstrated that synchronous adenomas were associated with a higher risk of future adenomas at short term follow up but were not predictive of local recurrence. Thus, other more reliable biological markers of field effect need to be identified. Global gene expression profiles of MNM around cancer, polyps and in control subjects were significantly different when evaluated with micro-array. The differentially expressed genes were involved in immunity, metabolism, epithelial-mesenchymal transition and RNA transcription. CXCL2 and FGF7 were identified as being upregulated in MNM adjacent to CRC suggesting that they could be utilised as markers of field cancerisation in the colon. Further investigation demonstrated that the FGF7-FGFR2 axis was disrupted only at the tumour site with downregulation of some of its downstream targets emphasising the potential role of this signalling axis in CRC formation. Collectively, these findings support the field cancerisation concept in CRC and highlight the importance of signals released by stromal cells in facilitating epithelial growth. These genes may be utilised to develop early biomarkers of disease or could be targeted with pharmacotherapy to modulate future CRC risk.