The purpose of this study was to investigate ROCK2 activation in squamous cell carcinogenesis and assess its co-operation with rasHa and fos oncogene activation together with loss of PTEN mediated AKT regulation. The analysis of ROCK deregulation with these genes in the MAP Kinase and PI3K pathways, two of the most commonly deregulated signalling systems, employed a well-characterised, transgenic mouse skin model of multi-stage carcinogenesis. A major goal was to study co-operation between these genes in the conversion of benign tumours to malignancy and investigate subsequent progression to aggressive carcinomas, given these are the most significant clinical stages of carcinogenesis from a patient’s viewpoint; and also investigated effects of ROCK2 deregulation on the processes of normal epidermal differentiation. ROCK2 is an effector protein of RhoA, which is a member of the ras superfamily and ROCK2 activation has been associated with tumour progression via an increase in tissue stiffness mediated by changes in actomyosin cytoskeleton leading to increased cellular motility. Thus, ROCK2 expression is commonly associated with the later events in cancer. Furthermore, there are many studies investigating ROCK2 in cancer given that it may be a useful therapeutic target in being downstream of oncogenic ras signalling. Yet, relatively few studies have explored the possibilities of a definite link that confirms the co-operation status of ROCK2 activation with ras/MAPK/fos and /or PTEN/PI3K/AKT pathways in SCC aetiology. Thus, questions exist as to exactly when does ROCK2 activation become causal; and what are the collaborating genes involved in the mechanism that drive the early or late stage events in carcinogenesis. To begin to answer these questions, inducible ROCK2 activation has been introduced into a well-characterised transgenic mouse skin carcinogenesis model that expressed a combination of ras and fos activation, driven by a modified human keratin 1 vector (HK1). Thus, exclusive epidermal expression of activated rasHa and fos oncogenes, in proliferative basal layers, gave hyperplasia and papillomatogenesis; but with no evidence of spontaneous malignant conversion. This stability of phenotype is thus ideal to assess roles for multiple transgene co-operations in the development of benign tumours and their conversion to malignancy. Hence, ROCK transgenic mice that expressed a conditionally active, 4-hydroxytamoxifen (4-HT)-regulated of human ROCK2 transgene were crossed with mice expressing activated rasHa and /or fos exclusively in epidermal transit amplifying keratinocytes (HK1.ras, HK1.fos). Inducible PTEN tumour suppressor gene mutation via exon 5 ablation (K14.Cre/D5PTENflx) and thus loss of AKT regulation was also incorporated into this model. This was achieved via deletion of exon 5 employing the RU486-mediated cre/loxP system, driven by keratin K14 promoter expression in basal layer keratinocytes. Therefore, to facilitate this investigation, a new and unpublished inducible ROCK2 system was employed in order to target the identical keratinocytes as PTEN loss. This new transgenic line of lsl.ROCKer transgenic mice employed the same 4-HT inducible ROCKer transgene but now driven by a generic CAG promoter following cre mediated ablation of the stop cassette once treated with RU486. In bi-genic K14.ROCKer/HK1.ras1205 mice, synergism between ROCK2 activation and (wound-promotion) sensitive HK1.ras1205 line showed direct co-operation and achieved malignant conversion of benign papillomas to well-differentiated squamous cell carcinoma (wdSCCs) histotypes (12 weeks of 4-HT treatment). This placed ROCK2 activity as the causal event driving malignant conversion, but in the absence of a wound promotion stimulus (loss of ear tag), papillomas did not convert. The correct papilloma context was required for ROCK to become causal proved to be the case on employing the wound insensitive HK1.ras1276 line. Here, K14.ROCKer/HK1.ras1276 mice failed to exhibit any papillomas and required the constitutive promotion stimulus from additional fos activation. Interestingly, following cessation of 4-HT, two intriguing observations were recorded. Firstly, that once bi-genic ROCK/ras1205 achieved malignancy, exogenous ROCKer expression appeared to show no involvement once squamous cell carcinomas (SCCs) progressed to poorly differentiated squamous cell carcinomas (pdSCCs), given the elevated expression of endogenous ROCK upon malignant progression. Secondly, the rapid growth of papilloma appeared upon cessation of 4-HT with highly intense p21 expression indicated the requirement of exogenous ROCK2 for malignant conversion and the possibility of a papilloma inhibition by 4-HT anti-cancer activity. Another major novel finding demonstrated ROCK2 activation could act as an initiator in co-operation with fos activation. Direct co-operation between ROCK2 and fos activation produced benign squamous papillomas yet, whereas ROCK2 activation alone induced hyperplasia as did fos activation alone at this time point, given papilloma formation in HK1.fos mice occur over 4-5 months. However, unlike deregulation of MAP Kinase signalling in bi-genic ROCK/ras1205 mice, in bi-genic ROCK/fos mice, no malignant conversion was observed due to high levels of compensatory p53/p21 expression. Thus, this bi-genic K14.cre/lsl.ROCKer/HK1.fos model suggests the requirement of additional mutation event for malignant conversion. An unexpected result appeared in bi-genic ROCK/Δ5PTENflx co-operation experiments where K14.cre/lsl.ROCKer/Δ5PTENflx cohorts exhibited epidermal hyperplasia with folded papillomatous appearance to the epidermis, but without papillomatogenesis even after seven month of period. This either indicates a weak co-operation between ROCK2 and Δ5PTENflx which may be due to the unexpected low levels of p-AKT from a compensatory increased p21 expression; and additional events needed to fill in the oncogenic gap in this bi-genic ROCK/Δ5PTENflx model, or may possibly highlight redundancy in the oncogenic hits provided by ROCK and PTEN. This latter suggest similar links exist between their normal roles in the epidermis which may be accountable for the alterations observed in keratinocyte differentiation. In both in vitro and in vivo experiments, K14.cre/lsl.ROCKer/Δ5PTENflx cohorts showed alterations in epidermal differentiation via anomalous K1 and low levels of K6 expression. Interestingly, activated ROCK2 appeared to induce or accelerate differentiation activity in K14.cre/lsl.ROCKer keratinocytes via increased K1 (early differentiation marker) and reduced K6 (proliferation marker) expression profiles. These results were consistent with in vivo data where K6 was expressed in low levels in K14.cre/lsl.ROCKer hyperplasia histotypes. In contrast, in K14.cre/lsl.ROCKer/Δ5PTENflx keratinocytes, inactivation of PTEN-mediated AKT activity may be accountable for restored keratin K6 and anomalous keratin K1 expression; as keratin K1 was expressed in a similar fashion of normal keratinocytes in K14.cre/lsl.ROCKer/fos keratinocytes. Interestingly, all tri-genic cohorts: K14.cre/lsl.ROCKer/ras1276/fos, K14.cre/lsl.ROCKer/fos/Δ5PTENflx and K14.cre/lsl.ROCKer/ras1276/Δ5PTENflx synergisms exhibited malignant conversion and /or malignant progression in all animals highlighting a novel role of ROCK2 activation. Further, the stage-specific consequences in each model in this study were shown to be influenced by p53/p21 status, where typically p53 expression disappeared in late stage papillomas yet, p21 expression persisted. This demonstrated the importance of compensatory p53/p21 expression in modulating tumour pathogenesis in all these models. Given that this study incorporated PTEN mutation, the influence of AKT activity was investigated in the SCC progression of tri-genic ROCK/ras1276/PTENflx and ROCK/fos/PTENflx cohorts; revealing a crucial antagonism between p21 and AKT. However, this study revealed that the malignancy in tri-genic ROCK/ras1276/fos cohorts was not influenced by p-AKT expression, and as this tri-genic model achieved wdSCCs only. This suggests that as the roles of ROCK in altering cytoskeletal organisation leading to increase in tissue stiffness are overlaid onto both MAP Kinase and AKT deregulation, the outcome is a very aggressive pdSCC. Thus, suggesting ROCK signalling to be a potential therapeutic target for ras/MAPK/fos pathway in carcinogenesis. Overall, this study showed the involvement of ROCK2 activation in the initiation stage for papillomatogenesis with fos oncogene, and demonstrated ROCK2 as a converter again and also in malignant progression with ras/fos/Δ5PTENflx mutations. This indicates the link of ROCK2 signalling with both MAPK and PI3K pathways, thus targeting ROCK2 would aid in development of cancer therapy.

No description available.

Colorectal cancer is the second most common cause of cancer death in the Western World. Although staging and prognosis is presently based on pathological assessment of primary tumour invasion and the presence of lymph node and distant metastases, it is increasingly recognised that other factors pertaining to both the tumour and host may similarly affect outcome. The local and systemic environment, encompassing host inflammatory responses and the tumour microenvironment, are examples of such. However, how such measures may compliment present TNM-based staging are not clear. Furthermore, tumour and host factors, both modifiable and non-modifiable, which may determine the local and systemic environment, remain to be fully determined. The present thesis examined the clinical and prognostic utility of assessment of the local and systemic environment, and potential tumour and host factors which may determine these responses. The following conclusions were drawn: Examining patients from the United Kingdom and Japan, Chapter 2 and 3 concluded that assessment of the systemic inflammatory response, utilising the modified Glasgow Prognostic Score, provides further prognostic stratification in addition to TNM stage. Although the proportion of patients exhibiting an elevated systemic inflammatory response differed between populations, the prognostic value was comparable. Chapter 4 validated assessment of the tumour stroma percentage as a prognostic factor independent of TNM stage and the local inflammatory cell infiltrate (cancer-specific survival HR 1.84, 95% CI 1.17-2.92, P=0.009). Chapter 7 further confirmed the prognostic value of a combined tumour microenvironment score, based on assessment of the generalised inflammatory cell infiltrate and tumour stroma percentage, in patients with primary operable colorectal cancer. This score, termed the Glasgow Microenvironment Score, was able to stratify patients into a good prognostic group, with five-year survival of 89%, an intermediate group with a two-fold increased risk of death and five-year survival of 75%, and a poor prognostic group, with a four-fold increased risk of death and five-year survival of 51%. Chapters 5 and 6 identified the presence of mismatch repair deficiency and activation of the JAK/STAT3 as two potential mechanisms which may determine host local and systemic inflammatory responses. However, the prognostic value of such candidate mechanisms was weak, suggesting that other pathways and tumour characteristics are implicated, and that molecular heterogeneity is likely to play an important role in determining not only the local and systemic environment, but also outcome. Chapter 9 concluded that the Immunoscore, an immunohistochemistry-based assessment of T-lymphocyte density within the tumour microenvironment, held greater prognostic value than assessment of the generalised inflammatory cell infiltrate using the Klintrup-Mäkinen grade. However, assessment of tumour stroma percentage provided additional prognostic value irrespective of the methodology employed to examine the local inflammatory cell infiltrate. Furthermore, the results of Chapters 7, 8 and 9 together suggested that loss of the local, anti-tumour immune infiltrate was the primary event which allows continued tumour growth, development of a tumour-supportive microenvironment and propagation of a systemic inflammatory response. Chapter 10 concluded that pre-diagnosis use of aspirin but not statins was associated with a lower modified Glasgow Prognostic Score, despite strong associations with comorbidity and BMI. This did not translate into an improvement in survival, potentially reflecting the underlying indication for use of these drugs primarily as cardiovascular secondary prevention medications. Finally, Chapter 11 examined the clinical utility of assessment of the tumour microenvironment using colonoscopic biopsy specimens, concluding that the use of biopsy-derived specimens was feasible. Furthermore, in addition to identifying patients who may benefit from therapies targeting the tumour microenvironment, assessment of a biopsy-derived Glasgow Microenvironment Score had comparable prognostic value to full section assessment of the tumour microenvironment.

Subclinical thyroid dysfunction (SCTD), characterised by abnormal serum thyrotrophin concentrations (TSH) with normal free thyroxine (FT\(_4\)), is regularly encountered in primary care. The clinical manifestations of SCTD are not well established, particularly in older individuals in whom SCTD, co-morbid conditions and symptoms frequently occur. This thesis clarifies the clinical expression and pathophysiological consequences of SCTD in the elderly with reference to existing evidence and a cross-sectional study evaluating thyroid function (TF), health status and specific symptoms in community dwelling individuals aged 65 years and above. TF for 2870 participants was categorised, 2703 (94%) euthyroid, 138 (4.8%) subclinical hypothyroidism and 29 (1%) subclinical hyperthyroidism. No significant differences in the prevalence of individual symptoms, pairs of symptoms or multiple symptoms were observed between TF groups. In the presence of individual or multiple symptoms, health status scores were significantly lower. In conclusion, symptoms and impaired health status were not associated with SCTD in this study. Results suggest that assessment of symptoms and health status does not aid clinical decision making with respect to management of SCTD in the elderly. Coupled with weak evidence demonstrating pathophysiological consequences in SCTD, overall findings suggest this population is unlikely to benefit from treatment for SCTD.

Known for over a hundred years, the human fungal pathogen, Cryptococcus neoformans causes cryptococcosis, a life threatening disease. Infection is acquired through inhalation of spores or dried yeast cells into the lungs from which the fungus can potentially transmit to all body parts of which the brain is the most affected organ. Once in the brain, the yeast C. neoformans causes meningoencephalitis, a fatal condition even with optimal treatment. The mechanism by which C. neoformans penetrates the normally impermeable blood brain barrier (BBB) to cause brain infection is not understood. This thesis presents two aspects of investigation: 1) the extent to which binding and uptake of cryptococci by brain microvascular endothelial cells (BMEC) explains transcytosis as a mechanism for cryptococcal traversal of the BBB and 2) the relationship between Cryptococcus – macrophage interaction and Cryptococcal meningoencaphalitis (CM) disease. We show that adherence and internalization of cryptococci by brain microvascular endothelial cells is a rare event characterized by a small number of cryptococci, an indication that C. neoformans most likely uses multiple routes to traverse the BBB. Secondly, by studying clinical isolates from cerebral spinal fluid (CSF) of HIV- associated CM patients, we demonstrate that high rate of cryptococci uptake by macrophages is associated with patient fungal burden whilst the intracellular proliferation rate is inversely associated with TNF- \(\alpha\) levels in the patient CSF. Interestingly, the high uptake – high fungal burden isolates were less encapsulated but more rapid melanin formers, traits known to modulate phagocytosis and protection from host-induced oxidative stress respectively. We therefore hypothesize that highly phagocytosed C. neoformans strains use phagocytes to disseminate faster to the brain resulting in high fungal burden.

Salmonella typhimurium is one of the most common causes of bacteraemia in children in sub-Saharan Africa and is prevalent in HIV-infected individuals. However, symptoms of this systemic infection are unclear, and while fatalities are frequent, how infection kills is unknown. Here we use a mouse model of systemic (but resolving) infection to investigate physiological and immunological aspects of the host response to infection. The liver is colonised during systemic infection, and in the model used, bacterial numbers peak at day 7 and are largely resolved within a month. Inflammatory lesions, consisting of multiple leukocyte populations, develop within the liver. These persist and are more severe once bacterial clearance is established. Whilst lesions can develop in the absence of T and B cells, these cells contribute to the regulation of inflammatory foci. In the absence of interferon-γ, lesions do not develop and inflammation in the liver is largely absent. In parallel, extensive platelet thrombosis occurs in the liver venous system and the shared kinetics with lesion formation suggest these phenotypes may be co-regulated. Here we describe how parenchymal and vascular inflammation are anchored by inflammatory up-regulation of podoplanin expression in the liver. Thrombosis is substantially abrogated in the absence of C-like lectin-type receptor-2 (CLEC-2) expression on platelets and we show that podoplanin (the physiological ligand for CLEC-2) expression on clodronate-sensitive myeloid populations is necessary for thrombus development. Therefore, the parallel association between inflammation and platelet activation could be the basis for developing novel treatments for systemic bacterial infections in humans.

Staphylococcus aureus is a Gram-positive human pathogen that has developed resistance to all traditionally used antibiotics. Sortase A (SrtA) is a 'house-keeping' enzyme present in a number of Gram-positive organisms including S. aureus that is responsible for the covalent anchoring of proteins to the cell wall through the recognition of a highly conserved LPXTG motif. Many of the proteins it anchors are involved in virulence and immune evasion suggesting that it is an attractive target for potential anti-infective therapies. Moreover, as SrtA is not vital for bacterial growth or survival, inhibition may not select for the development of drug-resistance, unlike conventional antibiotics. This study evaluated different assays to assay SrtA activity and includes an in vitro Fluorescence Resonance Energy Transfer (FRET) assay using purified recombinant SrtA protein and an in vivo whole cell-based assay that measured SrtA-mediated anchoring of Gaussia luciferase (GLuc) in S. aureus. A further in vivo assay was evaluated, which measured SrtA activity using fluorescence as a reporter and analysis by flow cytometry and structured illumination microscopy. In this study three novel small molecules were identified as potential inhibitors of SrtA using in silico computational docking and SAR analysis; NCC-00014270, NCC-00076932 and NCC-00032784. These compounds were shown to inhibit SrtA in vitro in a dose-dependent manner with IC50s of 140 ± 24.6 µM, 172 ± 28.1 µM and 628 ± 122 µM respectively and were shown to act as competitive inhibitors of the SrtA. With the use of an in vivo reporter, it was shown that all three compounds negatively affected SrtA-mediated anchoring in S. aureus whole cells. Moreover, the cytotoxicity of these lead compounds against eukaryotic cells was assessed. Overall, these data suggest that two of the three lead compounds were potential 'hit' molecules for further structural modification for increased inhibitory activity against SrtA.

Gene delivery vectors that are safe, efficient and affordable could significantly enhance the prospects for genetic-based therapies. Here we describe an approach to such vectors, using new variants of polyphosphazene materials and describe the synthesis of a series of degradable polyphosphazenes with both cationic and anionic side-chains, and report their use as mixed polyelectrolyte complexes for DNA and RNA delivery to glioblastoma cells in vitro and in vivo. Precursor poly(allylamino-phosphazene)s were converted to cationic and anionic derivatives via a,w-thiolated alkylamines and alkylcarboxylates, respectively. Simultaneous co-incubation of alkylamine- and alkylcarboxylate-poly(phosphazenes) with nucleic acids generated polyelectrolyte complexes which were more compact than poly(alkylamino-phosphazene):DNA analogues but with similar positive surface charges. Screening of a series of these complexes for transfection of U87MG glioblastoma cells, showed that 6-mercaptohexanoic acid substituted poly(phosphazene)s mixed in the polycation/DNA complexes resulted in the highest luciferase expression in the cells. These data were consistent with an increased buffering capability of the 6-mercaptohexanoic acid substituted polymer across the early endosomal pH range in comparison with other anionic side-chain substituted polymers. Transfection assays in 3D spheroid models and in subcutaneous xenograft U87MG tumours confirmed higher transgene expression for these mixed cationic and anionic poly(phosphazene)s compared to the related poly(alkylamino-phosphazene)-DNA complexes, and also to PEI-DNA complexes. Extension of the approach to siRNA delivery showed that the mixed cationic and anionic poly(phosphazene)s were able to silence a gene encoding for a kinase implicated in tumour progression (DYRK1A), resulting in a reduced renewal ability of U87MG cells in vitro and in delay of tumour growth in a xenograft model.

Improving the survival of patients with pancreatic ductal adenocarcinoma (PDAC) remains an oncological and surgical challenge. PDAC pathogenesis is underlined by numerous molecular aberrations occurring at a genetic and epigenetic level, however their spectrum of occurrence and clinical impact has not yet been fully elucidated. The majority of patients present with locally advanced or metastatic disease and even the 15-20% of patients who undergo resection for cure have a median survival limited to 18-24 months. Surgical treatment carries a high morbidity and identification of patients expected to have a poor prognosis could assist in the decision making process. The present thesis examines the prognostic importance of pathological and molecular factors in PDAC, specifically: 1. An examination of the frequency, the prognostic impact of resection margin involvement, and furthermore the prognostic influence of tumour involvement at individual margins. 2. Determination of the prognostic impact of peripancreatic fat invasion following resection of PDAC. 3. The investigation of the relationship of candidate protein biomarker expression with overall survival in a large PDAC tissue microarray cohort using immunohistochemistry. 4. Determining gene expression profiles associated with pancreatic cancer compared to normal tissue using gene expression microarray analysis with subsequent development and validation of a prognostic gene signature. 5. microRNAs were identified that associated with pancreatic cancer clinicopathological factors including survival. 6. Copy number aberrations were identified using array comparative genomic hybridisation that correlated with clinicopathological factors following resection for PDAC. 7. Finally the identification of potentially important regulator genes contributing to pancreatic tumourigenesis, was made by integrating the gene expression, microRNA expression and copy number data from previous sections using a bioinformatic approach. In this work a combination of enhanced pathological staging criteria along with the correlation of molecular marker expression and genomic profiling signatures with clinical outcome data has yielded interesting results in patients undergoing resection for pancreatic cancer that allowed detailed disease characterisation and subsequent clinically relevant outcome stratification.

Duchenne muscular dystrophy (DMD) is a muscle-wasting disease caused by the loss of sarcolemmal protein dystrophin. In DMD and the mouse model of the disease mdx, there is an increase in an associated protein, caveolin-3. In this study, mdx mice with deficiencies in caveolin-3 were generated to allow a distinction to be made between the pathology caused by the loss of dystrophin and that caused by an excess of caveolin-3. It was found that in late gestation embryos, there were perturbations in skeletal muscle stem cell populations and depletion of respiratory muscles in mdx and mdx/cav3\(^{+/-}\), both of which were more severe in mdx/cav3\(^{+/-}\) embryos. In post natal skeletal muscles, there was a trend in that the level of regeneration, believed to be indicative of previous degeneration, was consistently greater in mdx than mdx/cav3\(^{+/-}\). Taken together it would appear whereas increased caveolin-3 may compensate for the lack of dystrophin in embryonic mdx muscle; post natally, it may contribute to the muscle regeneration observed in mdx. The data presented in this thesis should help towards clarifying the contribution of caveolin-3 in the pathogenesis of DMD and in doing so expand on the understanding of the molecular aetiology of the disease.