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Background: Intrahepatic cholangiocarcinoma (CCA) is a highly treatment-resistant malignancy of biliary epithelium with increasing global mortality. Histologically, CCA is characterised by a pronounced inflammatory stroma of tumour-associated myofibroblasts, macrophages, immune cells and a modified extracellular matrix (ECM). In other solid cancers, the stroma plays a tumour promoting role. The functional role of the stroma in CCA remains unclear. The origin and the proportional contribution to the stroma by haematopoietic and mesenchymal bone marrow (BM) -derived cells is not known in CCA. Intriguingly, reports suggest that mesenchymal stem cells (MSCs) may contribute to the epithelial compartment of malignant tumours. Furthermore, the Notch signalling pathway is known to play oncogenic and tumour suppressive roles in diverse neoplasms but its role in CCA remains unclear. Aims and Methods: The functional role of myofibroblasts and macrophages in the tumour stroma of CCA was investigated together with an analysis of the origin and contribution of BM-derived cells to the stromal and epithelial compartments of CCA. The Notch signalling pathway was studied as a potential signalling mechanism through which the stroma and malignant epithelial compartments of CCA may interact. Results: The thioacetamide rat model of CCA was optimised and found to display excellent histological congruence with human lesions. The tumour cellular microenvironment comprised of myofibroblasts, migratory macrophages and immune cells. During cholangiocarcinogenesis, progressive intrahepatic accumulation of inflammatory cells and proliferation of bipotential progenitor cells preceded the development of invasive CCA. In vitro, CCA lines were identified to contain a side population of stem cells. Adoptive transfer of BM from Enhanced Green Fluorescent Protein (EGFP) transgenic rats to wild type rats to establish chimeras was undertaken. In transplanted rats, persistent EGFP+ chimerism of both haematopoietic and mesenchymal stem cell compartments was established. In tumours, macrophages and neutrophils were overwhelmingly EGFP+ve, whereas myofibroblasts, fibroblasts and benign and malignant bile ducts were EGFP-ve. There was no evidence of cell fusion or EGFP silencing. These findings were confirmed in spontaneous breast, skin and colon tumours in EGFP+ chimeric rats not treated with TAA. In vitro studies to recapitulate the cellular and extracellular elements of the tumour niche identified that ECM components induce characteristic cell proliferation patterns dependent on the matrix component but do not appear to affect chemosensitivity. Bidirectional interaction between CCA cells and hepatic stellate cells (mediated by soluble factors) was identified. Furthermore, in direct co-culture, M2 polarised macrophages appear to enhance CCA cell proliferation compared to M1 macrophages. In considering the Notch pathway, Notch signalling components (particularly Notch3) and target genes were upregulated in human CCA specimens. Immunohistochemical analysis identified apparent distribution of Notch ligand on tumour stroma and Notch receptor subtypes on malignant epithelia. Although direct co-culture of CCA cells with myofibroblasts and M1/M2 polarised macrophages did not clearly demonstrate stromal:epithelial Notch pathway activation, this may have been a function of in vitro experimental limitations. Gamma-secretase inhibition downregulated the Notch pathway, reduced proliferation and appeared to enhance chemosensitivity of CCA cells in vitro. Conclusions: A stereotypical niche forms around CCA in developing and malignant lesions. There was no evidence of a BM-derived stem cell contribution to the epithelial component of CCA, breast, colon or skin malignancies. Haematopoietic but not mesenchymal components of the tumour stroma were of BM origin. Notch signalling is upregulated in CCA and appears to play a tumour promoting role in CCA; pathway inhibition represents a potential therapeutic target.

The published work in this thesis is based on a number of studies that utilise a suite of (related) “best practice” benchmarking tools, providing an evaluation of the North East England’s manufacturing and service sectors and the UK Further Education sector. Within this submission, a supporting commentary in the form of a critical literature review is provided. The complementary review initially provides an introduction and background to the studies that as a whole comprise this PhD by publication. Consideration is then given to the literature specific to benchmarking, particularly in terms of its role in facilitating organisational improvement and learning, as well as its take-up and applications. The studies described above relate to the implementation of related “best practice” frameworks, yielding data from the self-assessing participating organisations. This leads to the third part of the literature review where the association between practice and performance is assessed relating to certain connected themes. The final part of the commentary assesses the contribution to knowledge that is made by this PhD submission in terms of the literature that existed at the time the constituent papers and reports were developed, along with my specific contribution to these outputs and the potential future research that could lead from this contribution. A key contribution of this work to the benchmarking literature rests in the deployment of a framework in two new sectoral contexts, the regional application being underpinned by a novel approach to supported self-assessment. This complemented the case-based literature dominant at the time, the review providing a critical comparison of “best practice” frameworks and the adoption of generic benchmarking metrics. The empirical assessment of practice against performance suggests that the former does impact on the latter, but with greater influence internally. The association between excellence achievement and stakeholder satisfaction is holistically positive, although the findings are perhaps both less than clear-cut and unexpected. The contribution to knowledge provided here relates to the assessment of the broader service sector, including dual consideration with stakeholder perception and examination of additional performance areas, such as corporate social responsibility, thus moving this evaluation into areas under reported at that time.

TGFβ signalling has long been recognised as having a role in heart development. The central TGFβ type II receptor (Tgfbr2) is thought to be essential for epithelialmesenchymal transition (EMT) in the atrioventricular cushions. However, little is known about the roles of Tgfbr2 in heart development in vivo. This is largely because the Tgfbr2 knockout (KO) shows early embryonic lethality during heart valve development. To bypass the early lethality of the TRfbr2 KO mouse and to investigate the role of Tgfbr2 during heart development, a cre/loxP system was used to generate three different conditional knockout mouse models to specifically delete this receptor in (i) the AV myocardium, (ii) the ventricular myocardium and (iii) the endocardium.