Marianne North was a major figure within the history of nineteenth-century botanical illustration. She produced a substantial body of botanical paintings as the result of extensive travels to many different parts of the world and was responsible for the founding of a major purpose-built gallery containing a representative collection of her work which still stands today in the Royal Botanical Gardens at Kew. Despite North's percieved importance as a botanical painter, relatively little of any critical/analytical substance has been written about her life and work from an art-historical or scientific perspective. North's place within nineteenth-century visual culture is arguably a contested one, despite having been a major contributor to nineteenth-century botanical painting. North's work therefore remains problematic to both botanists and art historians because it does not conform wholly to the established nineteenth century conventions of either scientific-botanical illustration or art. This thesis will explore the uncertain positioning of North's painting through a close analysis of its relationship to nineteenth and twentieth-century visual practices. In light of this analysis, it will be argued that North's painting does not successfully combine artistic and scientific perspectives, as some have argued, but instead presents an unidentifiable mode of visual representation that shifts uncertainly between art and science, thereby deconstructing any categorical distinction between the two.

Background. Necator americanus is one of the major causes of human hookworm infection, affecting over 800 million people worldwide. Hookworm infections cause gastro-intestinal bleeding, anaemia and iron deficiency, and are associated with high rates of morbidity, especially in children. Although chemotherapy has proven effective, high rates of reinfection are reported in socioeconomically developing countries, possibly due to the short-term efficacy of anthelmintic drugs in addition to individual predisposition to these infections, raising interests in developing suitable alternatives to chemotherapy which are capable of providing complete, long-term protection against hookworms. Understanding of the molecular mechanisms used by Necator americanus larvae to penetrate the human skin and the vasculature would therefore aid the development of effective vaccines against this important pathogen. Methods. First, Necator americanus larval exsheathing fluid (EF) and excretory/secretory products (ES) were profiled using gel electrophoresis and enzyme assays. Protease inhibitors against the main protease classes were used to determine which proteases are present in larval products. Second, the interaction of larval EF and ES products with human skin and extracellular matrix (ECM) macromolecules including collagens I, III, IV and V, fibronectin and laminin was investigated using western blots and protein separation by gel electrophoresis. Third, the impact of Necator americanus larval EF and ES on the endothelial barrier was examined using human umbilical vein endothelial cells (HUVEC). Permeability, an essential endothelial barrier function, was assessed during treatment with larval products, using transendothelial electrical resistance (TEER), and post-treatment using albumin-tracer flux. Finally, at the cellular level, responses to treatment with larval products were assessed by investigating molecular changes at cell-cell vascular endothelial (VE)-cadherin junctions and actin filaments, and by determining levels of secreted inflammatory cytokines, IL-6 and IL-8, and vascular endothelial growth factor (VEGF) in the culture medium. Results. It would appear that a repertoire of larval proteases, including serine, cysteine, aspartyl and metalloprotinases, caused partial degradation of skin macromolecules, collagens I, III, IV and laminin while fibronectin was fully degraded. Proteolysis of skin- and ECM macromolecules was related to the characteristic presence of proteolytic enzymes in larval products. The presence of transglutaminase activity was confirmed in both EF and ES products. Larval proteases caused a dose related increase in endothelial permeability, characterised by a decrease in monolayer resistance (TEER) with increased permeation of albumin tracer, which was minimal in the presence of a cocktail of protease inhibitors. These barrier changes were associated with disruption of junctional VE-cadherin and F-actin, the formation of intercellular gaps and an increase in endothelial secretion of IL-6 and IL-8. Conclusions. Necator americanus larvae produce a repertoire of proteolytic enzymes which could play an important role in negotiating the skin and breaching the endothelium to gain access to the host’s blood circulation.

Ovarian cancer is a disease which is fatal in the majority of cases. The evolution of surgery and chemotherapy over the past 30 years has resulted in improvements in overall and progression-free survival. However, the rate of relapse in ovarian cancer is very high, suggesting that current treatment strategies are ineffective. Therefore, to overcome the poor prognosis of ovarian cancer, immunotherapeutic strategies have been devised such as the use of anti-CTLA-4 antibody therapy in melanoma. The principle that the immune system can effect either cancer development or clearance has been the subject of debate for over a century. Clinical results of novel immunotherapeutic approaches that aim to exploit and enhance this immunogenicity have had mixed successes such as IL-2 therapy in renal cell carcinoma. It is clear that whilst many tumours possess antigenic component in their make-up, they do not stimulate durable and effective immune responses in vivo. This may reflect the fact that tumours develop a network of escape mechanisms to circumvent tumour-specific immunity. Due to the ineffectual nature of current treatment options and the complexity of the tumour microenvironment a coherent stratagem needs to be composed. This thesis explores, in principle, a contemporary strategy to propagate an anti-tumour immune response within ovarian cancer by using existing drugs in combination to target three different facets of ovarian cancer immunity; Regulatory T cell (Treg) migration, poor release of the tumour associated antigen, MUC1, and reduced cytotoxic T cell (CTL) proliferation. The migration of Regulatory T cell (Treg) to ovarian cancer is principally mediated by the CCR4-CCL22 chemokine receptor-chemokine axis. AZ1, a specific antagonist for the chemokine receptor CCR4, which is highly expressed on Treg, abrogated the migration of these cells to the chemokine. This compound did not alter Treg function suggesting that its activity was specifically against Treg migration. In order to induce an adequate T cell response, sufficient antigen needs to be provided. Camptothecin, a classical topoisomerase inhibitor, demonstrated effective tumour cell death and release of the tumour-associated antigen, MUC1. The increase in tumour antigen release and decrease in tumour load was offset by significant immune toxicity. The incorporation of Camptothecin into a synthetic drug delivery system led to a decrease in immune toxicity while retaining the drug’s anti-tumour activity. Finally, in order to take advantage of tumour antigen release, it would be desirable to stimulate CTL. Imiquimod, the toll-like receptor 7 agonist, widely used in basal-cell carcinoma and melanoma was able to demonstrate a potential enhancement of an anti-tumour response in three ways. Firstly, the drug enhanced the activation and antigen uptake capacity of plasmacytoid dendritic cells. It also had a direct effect on CTL themselves whilst also reducing the suppressive effect of Treg. This thesis illustrates, in principle, the possibility that a poly-pharmaceutical approach can be taken to target ovarian cancer. It indicates that readily available compounds, when used in the correct combination, could be key in developing effective anti-cancer therapy. Future work in this area should focus on using existing chemotherapeutic and immunotherapeutic drugs in combination to illicit enhanced anti-tumour cytotoxicity. Critically, the next step in developing this strategy is to acquire suitable in vivo models. This is key as there is conflicting evidence regarding the efficacy of certain drugs in mice compared to man.

In recent years evidence has emerged that the endocannabinoid system (ECS) may have a significant role in metabolism and energy homeostasis. Several studies have identified upregulation of the peripheral ECS in obesity and type 2 diabetes, but the mechanisms behind this and the consequences of upregulation are unclear. The aim of this thesis was to further elucidate the role of the ECS in mature adipocytes, and its activity in obesity and related metabolic dysfunction. Three adipose tissue depots were dissected from lean, obese and obese diabetic Zucker rats (n=6-8). In human studies, written informed consent was obtained from healthy volunteers within the University of Nottingham and obese surgical patients at the Royal Derby Hospital. Anthropometric measurements and venous blood samples were obtained. In these studies, subcutaneous abdominal adipose tissue was taken from all subjects (n=28 healthy study; n=27 surgical study), and visceral adipose tissue was obtained from some of the surgical patients (n=14). In all studies, collagenase was used to isolate mature adipocytes from the adipose tissue, and FAAH and MGL activities in the adipocytes were assayed using tritium labelled substrates. Human subcutaneous preadipocytes (Promocell, Germany) were cultured and differentiated. Adipocytes were cultured with high concentrations of glucose (15 mM) and/or insulin (1 μM) for 24 hours, in combination with anandamide or 2-AG for 2 or 24 hours. Adiponectin, leptin and resistin in the cell culture media were then measured using sandwich ELISAs. In another study, anandamide and 2-AG uptake were measured in differentiated adipocytes after 2 or 24 hours’ stimulation with glucose and/or insulin. FAAH and MGL activities in the cultured adipocytes were also measured in this study. In rats, FAAH and MGL activities correlated with body mass. In healthy humans, FAAH activity in subcutaneous adipocytes correlated with BMI and waist circumference, but not with other anthropometric measurements, serum glycaemic markers or adipokines. In obese patients, the enzyme activities had no relationships with any of the anthropometric or metabolic markers investigated. Furthermore, there were no differences in activity between patients with metabolic syndrome or diabetes and those without. In both rats and humans, there were no significant differences in FAAH and MGL activities between subcutaneous and visceral adipocytes. In the cell culture studies, anandamide and 2-AG did not alter adipokine secretion under normal, high glucose or high insulin conditions. Chronic insulin exposure increased anandamide uptake, but none of the other acute or chronic treatments with glucose and/or insulin affected anandamide or 2-AG uptake. Glucose and insulin were found to reduce MGL activity. These studies suggest that the rate of anandamide hydrolysis in mature adipocytes is increased in obesity. This relationship was not apparent in a morbidly obese sample. MGL activity in humans does not have relationships with adiposity or metabolic markers, and this may reflect its role as a major component of lipid metabolism, particularly lipolysis. Anandamide and 2-AG are unlikely to be direct mediators of adipokine secretion, at least in cell culture. Insulin may affect endocannabinoid signalling in adipocytes by increasing anandamide uptake and suppressing MGL activity. Overall, these results support the notion that the ECS in adipocytes is dysregulated in obesity, but this is not driven by specific factors associated with obesity.

This thesis investigates the influence of regional culture on young people's decision making when considering post-sixteen educational choices and directions from school. The data is provided by life story interviews with young people - aged eighteen to twenty years, 'born and bred' in Lincolnshire - who have followed four pathways from compulsory education. Within the context of Lincolnshire the influence of rurality is a major element of regional culture and figures in much of the discussion and analysis. The work of Pierre Bourdieu in defining culture through field and habitus is used as a theoretical perspective in the data analysis and conclusions. The research highlights the continued importance of family and community habitus in the decision-making processes of young people. The interviews are used to consider the relative field positions important in defining individuals' post-sixteen pathways. The nature of rurality as a social construct rather than simply a reflection of physical geography is discussed and conclusions offered as to its possible effect on preferred post-sixteen pathways. The relative importance given to physical and social characteristics of rurality is used to construct a series of cultural indicators for rural communities. The data would support the conclusion that new initiatives designed to increase participation rates in post-sixteen education are having some effect, but only among those young people predisposed through family habitus to continuing education. Those young people whose family habitus most closely coincides with pedagogic authority are most likely to operate comfortably within the educational habitus and hence continue with formal education beyond sixteen. The thesis suggests the real differences in habitus between urban and rural communities requires a shift in the policy debate if rural people are to participate fully in the notion of lifelong learning. NB. This ethesis has been created by scanning the typescript original and contains some inaccuracies. In case of difficulty, please refer to the original text.

Germ cell tumours (GeTs)affect both paediatric and adult populations, and can occur either in gonadal or extragonadal regions along the body's ventral midline. These tumours can be broadly categorized into two subgroups, seminomatous (SEM) or nonseminomatous (N-SEM). The latter can be further subcategorized into embryonal carcinoma (EC), teratoma, yolk sac tumour (YST) and choriocarcinoma (eC) according to their differentiation. As in many other tumours, DNA methylation has been proposed to be involved in GCTdevelopment. However to date, most studies were performed using adult testicular GCTs. Furthermore, these studies only include a handful of genes in their analysis. Thus, the roles of DNA methylation in paediatric and extragonadal GeTs have not been explored. Therefore, this project attempted to fill this gap in knowledge by performing methylation analysis in a cohort of paediatric GCT samples and GCTcell lines. Although paediatric GCTsmostly consist of teratomas, seminomas or YSTs, only the latter two were included in the methylation analysis as they were the only samples in the available tumour bank. Using the methylation level of L1NE-l repeat elements as a measurement of global genome methylation, we found that both paediatric seminoma and YST samples displayed global hypomethylation as compared to somatic controls. However, when methylation at gene promoter regions was investigated using Illumina Golden Gate methylation arrays, seminoma and YST exhibited very different methylation features. YSTs were found to be highly methylated at many of the sites investigated. Surprlslnglv, we found that the methylation features in seminoma were similar to the somatic controls. From this analysis, we identified 85 genes that were differentially methylated in the VSTs. However, by correlating our methylation data with the expression array data performed by our collaborators on the same samples, only eight of these genes (PYCARO, CASPB, C02, HOAC9, TFAP2C, ETV1, EV/2A, HLA-F) were differentially expressed. As in previous GCTstudies, our analysis was focused on the methylation at epG islands. During the course of this project technological advancement led to the creation of new methylation arrays that offer wider genome coverage. One example is the Infinium Methylation 450K array that covers more than 450,000 CpG sites and includes regions flanking the CpG islands such as the CpG shores and CpG shelves. Since no previous GCT studies have attempted to investigate methylation in those regions, we utilized this methylation array on four GCTcell lines; TCAM2 (seminoma), NT2Dl (teratocarcinoma), GCT27 (embryonal carcinoma) and GCT44 (yolk sactumour). Similar to previous GCT studies, we found that nonseminomatous GCT cell lines displayed higher methylation at the CpG islands as compared to the seminoma cell lines. Strikingly, expanding our analysis to other regions (CpG shores and shelves etc.) revealed that each GCT subtype exhibited distinct methylation features. Both ECand teratoma cell lines displayed higher methylation than the seminoma and YST cell lines at all regions. Interestingly, the YST cell line only showed higher methylation than the seminoma cell line at the CpG islands and to a lesser extent at the CpG shores while the seminoma cell line exhibited higher methylation at the CpG shelves as compared to the YST cell line. This is the first time such features have been reported for GCTs. From this Infinium methylation data, we have also identified a high number of hypermethylated genes including those that are uniquely methylated for each cell line. By correlating this methylation data with Affymetrix gene expression data, 98 genes that were differentially methylated and differentially expressed in the YST cell line have been identified. However, further analysis needs to be performed to understand the role of these genes in YST development. As in other types of tumour, the hypermethylation observed in the YST cell line might be caused by many epigenetic modifiers. Using real-time RT-PCR on three epigenetic modifiers (DNMT38, EZH2, SUZ12), we found that DNMT38 was highly expressed in the YST samples and cell line as compared to the seminoma samples and cell line. This suggests that DNMT38 might contribute to YST hypermethylation and resulting differences in their biology. However, knockdown of DNA methyltransferases (DNMTs) and DNMT38 using 5-azadeoxycytidine and microRNA-29b respectively, did not seem to have any effect on the response of all four GCT cell lines towards cisplatin. On the other hand, both knockdowns only caused little effect on cell migration; affecting only the seminoma and YST cell lines. Nonetheless, further analysis is still needed to fully assess the role of DNA methylation in regulating cell behaviour. In summary, paediatric YSTs displayed hypermethylation at many promoter regions as compared to seminomas. Meanwhile, methylation analysis at regions outside of CpG islands in GCTcell lines revealed unique methylation features for each GCT subtype which might indicate different underlying mechanisms in their development. Further analysis on genes found to be differentially methylated and differentially expressed in both paediatric and GCTcell lines are now needed to fully establish their role in GCT development.

Here the use of the in vitro spheroid conformation model, is carried out in such a way as to make use of confocal laser scanning microscopy on vital tissues. Its use in the observation of the invasion of stromal tissues by cancer tissues and the progression of this invasion dynamically was investigated. The employment of simple routine laboratory techniques and the adaptation of this method for the dynamic investigation of human tissues and cancers during the invasion process was paramount. The spheroids for the confrontations were produced from re-aggregates of cells grown in monolayer culture stained with vital fluorescent dyes CMFDA and CMTMR.. The confrontation method seemed appropriate for the examination of invasiveness as it enables the observation of the invasive process on a three dimensional structure which emulates the situation in vivo. The use of the clinically significant parameter INVASLOG is described here and its limits have been elucidated. It has been demonstrated that the use of the INVASLOG enables an objective estimation of the invasive process in vital cultures. Autologous clones of mouse melanoma K-1735, of varying invasive behaviour in vivo and human melanomas A375 and A2058 can be distinguished between in their behaviour. The investigation of the effect of retinoic acid has also demonstrated and the usefulness of this method and the invaslog parameter in investigating invasion. Its use in the investigation of and its application to, diagnosis and prognosis of cancer should be investigated particularly where specific therapies for specific cancers can be tested and the treatment regime adapted for each patient individually.

In the last three decades vitamin D, or calcitriol, has been found to have important anticancer role in different cancer types. Unfortunately, a therapy using calcitriol remains a challenge due to increased drug resistance as a consequence of the up-regulation of CYP24A1, which metabolises and inactivates calcitriol. Moreover, the hypercalcaemia associated with an elevated dose of calcitriol does not allow the use of vitamin D at a high concentration. Analogues of calcitriol have enhanced anti-tumour activity, reducing the calcaemic undesired effect. The use of CYP24A1 selective inhibitors could be the appropriate strategy to increase the lifetime and thereby! the anti-cancer functions of calcitriol and its derivatives. Consequently, the aim of this project is to develop new, potent and selective inhibitors of CYP24A1 that could be used in the treatment of different types of cancer in order to enhance endogenous vitamin D levels and favour its anti-tumour activity. Through molecular modelling studies, a new CYP24A1 homology model has been prepared and the active site has been characterised examining the disposition of (R)-VID400, a CYP24 inhibitor, (E)-N-(2-(1H-imidazol-1-yl)2-phenylethyl)-4-styrylbenzamide (MCC165), a compound previously synthesised in our laboratory that showed a potent CYP24A1 inhibitory activity (IC50= 0.3μM), and the natural substrate calcitriol. Different series of potential CYP24A1 inhibitors were designed in order to mimic completely the calcitriol disposition in the binding pocket and to interact with the haem iron of the enzyme catalytic site. For each series a synthetic pathway was developed. The synthesis was followed by a CYP24A1/CYP27B1 inhibition assay. All the compounds occupy the same hydrophobic tunnel as calcitriol and access the active site through the same channel. Moreover the substituents in the lateral chain bind directly to the haem iron via a lone pair of electrons. The different syntheses were obtained after several optimisations of reactions and routes. The CYP24A1/CYP27B1 inhibitory activity (IC50) using a cell-free assay and the value of the Ki (dissociation constant) of the different series of compounds, compared with ketoconazole (Ki= 0.030 μM, IC50= 0.47 μM) as the standard, were evaluated. Selectivity of CYP24A1 over CYP27B1 was also calculated. New potent CYP24A1 inhibitors were found.!Selectivity gave a range from poor to moderate results with selectivity improved in some case compared with ketoconazole (selectivity: 1.6).

Telomeres cap the end of eukaryotic chromosomes and prevent the natural end of a chromosome from being recognised as a double-stranded DNA break. Dysfunctional telomeres may trigger replicative senescence, or fuse with other telomeres or with non-telomeric DNA breaks. The length of a telomere plays a key role in telomere function. Relatively little is known about how telomeric chromatin influences telomere length and function. A number of studies in mammalian cells have identified a handful of chromatin remodelling proteins and the chromatin marks they deposit in telomere length regulation. However such examples at human telomeres are scarce. The primary aim of this thesis was to investigate whether the chromatin structure of a telomere is a determinant of its length in human cells. Two approaches were taken to address this issue: Firstly, the chromatin structure of telomeres of differing lengths were directly analysed by measuring enrichment of histone modifications known to be prominent at telomeres in other model organisms. Secondly, selected chromatin remodelling proteins were studied to determine whether they play a role in telomeric chromatin structure and telomere length. Single Telomere Length Analysis (STELA) provides a high resolution method to measure telomere length distributions at individual chromosome ends. STELA assays were previously designed for the 2p, 9p, 11q, 12q, 16p, 17p and 18q telomeres. An allele‐specific STELA assay has also been designed for the XpYp chromosome end. In this study novel telomere and telomeric allele‐specific qPCR assays were developed for the same chromosome ends. These qPCR assays, when used in conjunction with ChIP provide a tool for analysing telomeric chromatin structure at individual chromosome ends. Applying this ChIP‐qPCR approach alongside STELA allows any correlations between telomeric chromatin structure and telomere length to be identified. This approach suggested differences in telomeric chromatin structure between telomeres of different lengths in telomerase‐positive HT1080 fibrosarcoma cells. Shorter HT1080 telomeres were less abundant in H3 and TRF1 and also had lower levels of H4K20me3 and, to a lesser extent, H3K4me3 compared to longer telomeres. Differences in chromatin structure were not observed between telomeres of different lengths in telomerase negative MRC5 fibroblasts. Changes in chromatin structure were observed at individual telomeres/telomere alleles were observed between actively proliferating cells and in cells undergoing senescence. Telomeric enrichment of H3 and TRF1 as well as the histone methylation marks H3K4me3, H3K9me3 and H4K20me3 were reduced in senescent cells. The degree of chromatin structural change as the cells entered senescence differed between chromosome ends. This highlights the benefits of using the telomere-specific ChIP-qPCR approach over the more traditional ChIP-dot blot assays which would not be able to differentiate between the chromatin structure of different chromosome ends. To identify roles for chromatin remodelling proteins in telomere length maintenance siRNA mediated knockdown of selected chromatin remodelers was performed in a clonal population of HT1080 cells followed by STELA analysis. RNAi-depletion of the histone methyltransferase (HMTase) III EHMT2 resulted in an increase in very short 17p telomeres whereas loss of another HMTase, DOT1L caused a divergence in the 17p telomere length distribution suggesting the presence of two subpopulations of cells each with differing telomere length distributions. Subtle changes in mean telomere length was observed after siRNA mediated knockdown of the HMTases MLL and EZH2, the histone deacetylases (HDACs) HDAC1 and SIRT6, the ATP dependent chromatin remodelling complex subunit BAF155 and the H3.3 histone chaperone DAXX. However due to certain limitations of the RNAi screen the validity of these observations is questionable and more work would have to be performed to confirm whether these chromatin remodelers have an effect on telomere length. Finally, dramatic telomere shortening was observed in a keratinocyte holoclone population after siRNA mediated knockdown of DAXX at number of chromosome ends. Prolonged depletion of DAXX also caused an increase in telomere‐to‐telomere fusions. A similarly dramatic loss in telomere length was seen in these cells after knockdown of EHMT2.

Tamoxifen has made a significant contribution in decreasing breast cancer related deaths for over 30 years and until recently was the gold standard for treatment of ER positive breast cancer (Fisher et al, 1998). Resistance to tamoxifen is however a considerable issue with cells utilising a number of molecular mechanisms to bypass the growth inhibition caused by blocking ER activity. This move towards an anti-hormone resistant state from an antihormone responsive state is associated with the transition to a much more aggressive phenotype including increased proliferation and also invasiveness. Thus unfortunately, acquisition of tamoxifen resistance is not only associated with a recurrence of breast cancer, but this cancer is also much more aggressive in nature with fewer treatment options available than the initial cancer. This study has identified Lyn kinase as increased in tamoxifen resistant breast cancer cells compared to oestrogen-responsive breast cancer cells. Subsequent removal of Lyn kinase from tamoxifen resistant breast cancer cell lines using RNAi technology led to a significant decrease in cell proliferation, increased apoptosis and also a decrease in migration and invasion. A mechanism has been suggested whereby Lyn kinase is involved in a calcium dependent zinc wave which ultimately leads to the activation of tyrosine kinases. Metastasis to other sites in the body is ultimately responsible for fatalities due to breast cancer and so being able to block its action is key to treating breast cancer in the clinic. Therefore identifying Lyn kinase as a gene target that leads to the advancement of breast cancer to a more aggressive state provides a powerful tool for treating breast cancer in the clinic.