Colorectal cancer (CRC) is the second most common cause of cancer related deaths in the UK. Whilst identification of molecular events that contribute to the initiation and progression of CRC have facilitated the development of predictive biomarker-driven therapeutics, their success in the clinic has been restricted by the lack of anticipated responses. Furthermore, not all genetic signatures of a tumour have been linked to related drug targets, highlighting the need for novel development of compounds and better therapeutic rationales for the treatment of patient subsets. The limited success of targeted therapies, both within the clinic and drug discovery pipeline, has been attributed to a lack of effective preclinical models that are capable of capturing the complexity of deregulated signalling networks. The development of functional readouts that better represent tumour complexity, is therefore imperative to confirm the effects of hypothesis-driven therapeutics. This thesis therefore aimed to investigate whether 3D CRC organoids , which show a degree of greater complexity compared to preceding in vitro models, could be applied as suitable readouts for stratified medicine programmes and novel compounds within the drug discovery setting. To achieve this, a panel of 3D patient-derived CRC organoids cultures were generated. Suitable methodologies were established to facilitate organoids towards quantifiable, robust assay formats. This platform enabled the study of organoids within an in vitro clinical trial setting, based upon treatments administered within the ongoing FOCUS 4 stratified medicine trial, exploring organoids’ capacity to predict responses to targeted therapeutics. Organoids were differentially sensitive to therapies, irrespective of their genotypic background. It will be interesting to see whether prediction-response correlations observed in this study are typical of those seen in patients and whether functional readouts will be required to support stratified medicine approaches. Quantitative image-based analysis was also found to identify signatures of organoid responses against novel Wnt signalling inhibitors, suggesting that organoids may constitute a platform that can be used to study the effects of targeting a prospective cancer stem cell (CSC) population. Taken together, the findings in this thesis highlight the utility of patient derived organoid models as a functional model to evaluate novel therapeutic strategies, potentially generating clinically relevant hypotheses.

An emerging concept in the regulation of signal transduction specificity is the mediation of scaffold proteins embedded in the circuitry of signalling pathways. The multidomainbased architecture of scaffold proteins facilitates the assembly and modulation of protein complexes to regulate cellular signals to bring about an exacting biological output. The work presented in this thesis aimed to investigate the mechanisms of the protein scaffold CNK1 (connector enhancer of Ras 1) in the pro-apoptotic MST2 pathway and the prooncogenic Raf-1 signalling pathways. Here, by using several molecular, biochemical and cell biology techniques, I demonstrated that CNK1 regulates the interaction of the protooncogene Raf-1 and the tumour suppressor MST2 kinase. Perturbations of CNK1 levels exhibit a biphasic signalling response typical of a scaffold protein. Transient expression of CNK1 upon growth factor withdrawal results in a concentration-dependent increase of the Raf-1/MST2 complex, thus preventing apoptosis, but this complex dissociates at higher expression levels, hence promoting an apoptotic response. Moreover, CNK1 is involved in the regulation of Fas-induced apoptosis via the MST2/RASSF1A pathway by influencing the time-scale kinetics of MST2 docking and release from the Raf- 1/CNK1 complex and its eventual activation. SiRNA-silencing of CNK1 destabilizes the Raf-1 and MST2 interaction, and enhanced MST2/LATS1 interaction that promotes apoptosis. Thus, CNK1 is required for Raf-1 inhibitory function, but is also necessary for MST2-mediated apoptosis. Remarkably, CNK1 selects and switches complex formation of opposing anchored proteins depending on the stimulus. In response to Fas ligand stimulation, MST2 is released, whereas Raf-1 is retained in complex with CNK1. Conversely, CNK1 retains MST2 and whilst releasing Raf-1 from the complex following growth factor treatment. Mapping the multidomain binding sites of CNK1 using peptide array demonstrates specific interaction sites of client protein complexes. Specific CNK1 point mutants were generated, and found to alter wild-type regulation of client protein complexes. Thus, the work described in this thesis may reveal a regulatory crosstalk between the MST2 apoptotic pathway and the Raf-1 proliferative pathway through CNK1 by coordinating assembly of appropriate pathway components to possibly drive discrete stimulus-specific responses.

The aim of this study was to. further elucidate the mechanisms whereby growth .. . hormone (GH) exerts its ~hronic effect on adipose tissue metabolism, and in particular the effects of GH on lipolysis. Previous studies had shown that turnout necrosis factor alpha (TNFa.) chronically increases basal lipolysis in rat epididymal cidipocytes: an effect that is similar to that of GH. Other research had implicated a protein with a halflife of less than 3 hours in the regulation of lipolysis and lipogenesis by GR. This led to the investigation if TNFa. might be this putative protein involved in mediating the chronic metabolic effects of GH. Initial studies used ovine adipose tissue explants. TNFa. caused a small increase in basal lipolysis and attenuated insulin effects on lipogenesis. However, the effects ofTNFa. were smaller than those ofGH and TNFa. did not appear to mimic the effects of GH on isoproterenol-stimulated lipolysis in this system. Therefore TNFa. was not the protein involved in GH regulation of lipolysis and lipogenesis. Previous studies in the laboratory on the mechanism of GH action had used inhibition of signal transduction components in an ex.plant system. More specific effects could be observed by using an antisense approach, but this required the use of a cell culture system rather than adipose tissue explants. The suitability of an ovine cell culture system was established for investigating the molecular basis of the lipolytic effects of GH; in particular the inhibitory effects of GH on adenosine inhibition of lipolysis. The lipolytic system partially developed in primary ovine adipocytes, but the antilipolytic system did not appear. to develop. However, by. manipulating the differentiation conditions, I significantly improved both cell differentiation and the lipolytic response and sensitivity to isoproterenol, but there· was no improvement in response to adenosine. As an alternative, the suitability of the murine cell line, 3T3-F442A, was .. . investigat.ed ·for determining the molecular basis of the lipolytic effects of GH. However, although the lipolytic system did· develop in differentiated 3T3-F442A adipocytes and response to isoproterenol was observed, the antilipolytic system did not appear io develop either. This line of investigation was not pursued further. Therefore, I decided to investigate the effects of GH and insulin on the lipogenic system in 3T3-F442A adipocytes i.nstead, with a view to extending previous observations by others (Millar, 1998) in the laboratory on the roles of specific isoforms of protein kinase C (PKC) on the modulation of lipogenesis by insulin and GH. The main objective was to determine the role of PKC isoforms in the modulation of the effect of insulin and GH on activation' and expression (mRNA) of the lipogenic enzyme . acetyl CoA carboxylase (ACC). However, the effect of the hormones on lipogenesis, and especially ACC, was considered to be too small to investigate the roles of specific PKC isoforms, despite trying many different ways of improving the hormone effects. A possible explanation for the poor response to insulin was that the lipogenic system was not "switching off' in the absence of insulin, so isoproterenol was added to the 3T3-F442A adipocytes to decrease lipogenesis. Isoproterenol did reduce the rate of lipogenesis, but the effect of insulin was still small. Therefore, modulation of the effect of specific phosphodiesterase (POE) isoforms on lipogenesis was explored as an alternative. The use of specific POE inhibitors showed that both POE3 and PDE4 enzymes were involved in the modulation of lipogenesis in 3T3-F442A adipocytes.

The Convention on Biodiversity (CBD) signed at the 1992 Earth summit in Rio formally recognized biodiversity at the habitat, species, and genetic levels. For species and habitat biodiversity there is a well-established set of frameworks under which conservation programmes are constructed and delivered. From a genetic biodiversity perspective, however, there is no clear consensus on how best it should be measured, or how conservation programmes should be implemented. The major reasons for conserving intra-specific genetic biodiversity can be summed up under two inter-related themes, (1) Protecting a broad spectrum of genetic biodiversity, and (2) Maintaining evolutionary fitness and adaptive variation. This thesis takes a case-study approach and explores the issues surrounding these themes for conservation strategies in two angiosperm species: Saxifraga hirculus and Lathyrus japonicus. 1) Protecting a broad spectrum of genetic biodiversity: This section of the thesis considered the evidence for major intra-specific genetic races in Saxifraga hirculus and the spatial distribution of its genetic biodiversity. Variation in Saxifraga hirculus chloroplast DNA was assessed in order to gain information on the biogeography of the British populations in the context of the wider European gene pool, and also to compare this with populations from Alaska and Colorado. In a European context, British popUlations have a high level of chloroplast diversity (three haplotypes) and contain a highly divergent lineage that was previously unsuspected. Seven haplotypes were found in total from 17 popUlations in Europe with marked inter-population differentiation (FST = 0.92). Higher diversity and lower popUlation differentiation was detected in Alaska (33 haplotypes /12 populations; FST = 0.46). Since most popUlations in Europe had unique haplotypes it is not possible to track migration routes or pinpoint refugia for the European popUlations, but the much higher diversity in Alaska compared to Europe indicates that the Beringia region may have acted as a refugium for this species throughout the Pleistocene. This highlights the importance of Alaska for the conservation of intra-specific genetic biodiversity in this species. (2) Evolutionary fitness and adaptive variation: To assess the relationship between population size, genetic variation, morphological variation and fitness, genetic studies were undertaken on populations of Lathyrusjaponicus. Eleven populations of L. japonicus were examined for variation using nine microsatellite loci. The populations show genetic isolation by distance across the distribution of the species in Britain, although isolation by distance breaks down when only the range centre populations are considered. There was no relationship between population size or isolation and genetic variation, with some small and/or isolated populations having high diversity, and large and/or range centre populations having low diversity. There was, however, a significant difference in the inbreeding coefficient of adult versus seedling plants. The heterozygosity of adult plants sampled in the field was significantly higher than seedlings grown in cultivation, indicating a survival advantage for heterozygotes. Significant differences were found between populations for seed weight, number of seeds per pod, number of pods per cluster, and leaf shape of L. japonicus individuals in the field. For seedlings grown in common conditions significant differences were found in leaf shape, pigmentation, and dry weight after two season's growth. Morphological and genetic differentiation were well matched in this species, and gave similar signals. Seedlings from Carnoustie (Scotland) grew much more vigorously in cultivation in Edinburgh than seedlings sourced from English populations, indicating local adaptation. However no significant relationship was found between any fitness associated traits or morphological variation with genetic variation, in spite of the heterozygote advantage revealed by the genetic data. The results from both research themes are discussed highlighting the difficulties in equating patterns of genetic marker variation to traits likely to be of evolutionary and ecological relevance.

In this thesis, I describe work to resolve issues of bone identifications that have been outstanding since Smith Woodward's initial description in 1889, to assess the taxonomic validity of material assigned to the hypodigm of Leedsichthys and the interrelationships of the members of Family Pachycormidae. In addition I look at the palaeoecology of this animal on the basis of its size and growth and its locomotion capabilities and its likely feeding abilities and behaviour. Chapter 2 includes a review of the history of work on Leedsichthys, with particular reference to the discoveries made in the Peterborough district. In chapter 3, archival photographs and papers are used to establish the distinction between the type material, the tail specimen, and the gill basket specimen. In chapter 4, occurrences of Leedsichthys outwith the Peterborough district are considered, including the announcement of a new locality extending the range of the taxon into the Kimmeridgian. Some identifications of previously misidentified bones are made, specifically the hypobranchial and dorsal fin-rays. Feeding trace fossils are interpreted in the context of Leedsichthys. In chapter 5, a new Callovian pachycormid is described from the Oxford Clay of Peterborough district, and used in a reworking of Lambers' 1992 phylogenetic analysis of the interrelationships of the Pachycormidae. The Pachycormiformes are redefined on the basis of derived characters. In chapter 6, the value of gill rakers as a source of taxonomic characters is considered, with specific reference to their use in Lambers' 1992 character set, and the validity of Leedsichthys notocetes as a distinct species. In chapter 7, specimens are analysed using growth marks and scaling, in order to establish estimates of length-at-age for Leedsichthys. In chapter 8, the bone identifications of Smith Woodward (1889b) are revised, and further bone morphologies identified from within the hypodigm of the genus. In chapter 9, the size estimates derived in chapter 7 are used to inform interpretation of Leedsichthys palaeoecology, focussing primarily on locomotion and feeding. In the conclusions, an up-to-date reconstruction is presented.

MicroRNAs (miRs) are a group of short non-coding RNAs, on average 22 nucleotides in length, that form an important axis of post-transcriptional regulation of gene expression. They have been identified as major modulators of all biological processes including development, cell differentiation, growth and apoptosis as well as diseases such as cancer, diabetes and cardiovascular disease (CVD). In the developed world CVD remains the leading cause of morbidity and mortality, and a substantial burden on healthcare. Left ventricular hypertrophy (LVH) is defined as an increase in thickness of the myocardium and is an important risk factor in CVD. The stroke-prone spontaneously hypertensive rat (SHRSP) is an animal model of essential hypertension used in research of CVD together with a normotensive reference strain Wistar-Kyoto (WKY). The SHRSP animals exhibit an increase in the size of myocardium prior to the onset of hypertension and have established LVH at 16 weeks of age thus are a good model for investigating the genetics of this condition. The aim of this project was to identify signature expression patterns of novel and previously implicated microRNAs and to investigate their role in the development of LVH in the SHRSP. Furthermore, potential gene targets of candidate selected microRNAs were identified to investigate biological pathways involved in the disease process. MicroRNA microarray profiling was performed by Dr. McBride in the hearts of 5 week old SHRSP and WKY male rats using the LC Sciences (LCS) multispecies chip based on Sanger miRBase 11.0. The data were analysed (Drs. McBride and McClure) using Rank Product (RP) analysis method and evaluated in combination with the statistical analysis provided by LC Sciences (LCS). LCS data indicated 103 microRNAs differentially expressed at 5 weeks of age, 64 at 16 weeks of age, with 9 in common. The RP analysis identified 72 microRNAs differentially expressed between WKY and SHRSP at 5 weeks of age and 51 at 16 weeks of age, and 21 microRNAs were differentially regulated at both time points. Both methods identified a subset of 35 microRNAs in 5 week old hearts and 8 in 16 week old samples. TaqMan® microRNA assays were used to confirm these expression patterns. Based on these data and published literature candidate microRNAs – miR-195, miR-329 and miR-451 were selected for further experimental investigation. Expression of candidate microRNAs (miR-195, miR-329 and miR-451) in neonatal hearts of SHRSP and WKY rats was also investigated. It was found that all three candidate microRNAs were differentially expressed at this time point and there were significantly increased levels in the SHRSP compared to WKY. Cardiac cell line H9c2 AngII model of hypertrophy was used to investigate the effect of AngII on our candidate miRNA expression levels. A 96 hour stimulation of H9c2 cell with AngII resulted in a significant increase in cell size. Levels of miR-195 and miR-329 were not affected by addition of AngII; expression of miR-451 was significantly down-regulated immediately post stimulation, however levels were increased at the final assessment at 96 hours. Adenoviral vectors over-expressing miR-195, miR-329 and miR-451 were designed and generated. These vectors were used to investigate if overexpression of each individual miR could affect cell size in the selected in vitro model of cardiomyocyte hypertrophy. It was found that all candidate microRNAs reduced AngII mediated hypertrophic cell growth at higher doses. Identifying pathways and specific gene targets affected by changes in microRNA levels is of paramount importance. Availability of such data not only provides information about regulation of cardiac homeostasis, but also possible therapeutic approaches for treatment and prevention. Target prediction algorithms (DIANAmT, miRanda, miRDB, miRWalk, PICTAR5, PITA, RNA22, RNAhybrid and Targetscan) were used to identify potential gene targets for candidate microRNAs. To refine these lists to genes relevant to the experimental design Ingenuity Pathway analysis (IPA 9.0) software was used to overlay microRNA microarray data with results of heart mRNA gene expression data (M. McBride, personal communications) from the same cardiac tissue and to relate these to appropriate pathways and cellular functions. A list of 12 genes was generated: similar to CG4768-PA (RGD1309748), KN motif and ankyrin repeat domains 1 (Kank1), sterile alpha motif domain containing 4B (Samd4b), dual specificity phosphatase 10 (Dusp10), follistatin-like 3 (secreted glycoprotein) (Fstl3), jun D proto-oncogene (JunD), forkhead box M1 (Foxm1), SIN3 homolog A transcription regulator (yeast) (Sin3a), cyclin-dependent kinase 1 (Cdk1), kinesin family member 23 (Kif23), bone morphogenetic protein receptor type IA (Bmpr1a) and sestrin 1 (Sesn1). Expression of these candidate targets was assessed in heart tissues from neonates, 5 and 16 week old rats. Six out of ten of these targets were differentially expressed at one or more time points. To further investigate the proposed targeting of these genes by candidate microRNAs, expression levels were measured in each of the predicted targets in H9c2 cell transduced with miR over-expressing viruses. The expression patterns of Cdk1, Kif23, Kank1 and Sin3a were consistent with overexpression of the targeting microRNA, i.e. expression of each gene was down-regulated. In summary, data presented in this thesis elucidate the role of miR-195, miR-329 and miR-451 in the development of LVH in the SHRSP. Understanding the underlying cause for differential expression of these candidate microRNAs, confirming gene targets and identifying relevant pathways will improve the understanding of LVH at the molecular level. It will also help explain the pathophysiology of cardiovascular disease development in this rat model of human hypertension providing a basis for the development of novel therapeutic approaches to treat or prevent LVH.

Saprotrophic fungi are the main agents of primary decomposition and nutrient cycling in woodland ecosystems. Powerful enzymatic capabilities enable then to break down the most recalcitrant components of wood and leaf litter, such as lignin and cellulose. Nutrients are retained by dynamic networks of mycelium, which are vulnerable to grazing by soil invertebrates. The studies reported in this thesis employed laboratory microcosm, mesocosm and field manipulations to further mechanistic understanding of climate change effects on basidiomycete fungal-dominated woodland decomposer community dynamics and ecosystem processes. Increased mycelial growth at elevated temperature can be prevented by collembola grazing in soil microcosms. The strength of this top-down effect varied with fungal palatability, which had a bottom-up effect on collembola populations and their responses to warming. A mesocosm multispecies collembola population was more strongly regulated by the bottom-up effect of inoculation with cord-forming fungi than climate change (warming, in combination with soil wetting or drying). Collembola can graze fungal cords, but thickness and chemical defences make them less palatable than soil microfungi, which are outcompeted by basidiomycete mycelia. In the absence of fungal biomass limitation by collembola, abiotic conditions regulated microbial community functioning. Warming stimulated fungal-mediated wood decomposition, particularly in drier soils. Moisture was the most important determinant of enzyme activity and displayed an interaction with temperature analogous to that for wood decay. Macro-invertebrates, such as woodlice, are better able to exploit nutritious, but thick and defensive, fungal cords. The consequences of macro-invertebrate grazing for fungal-dominated microbial community function were tested in a field manipulation of woodlouse (Oniscus asellus, Isopoda) population densities, predicted to increase due to climate warming. This provides the first evidence for bottom-up effects of fungal palatability on woodlouse populations. Body lipid analysis revealed fungi as a major component of the generalist woodlouse diet. Despite low population densities at the site, altered O. asellus abundance influenced aspects of microbial community functioning. The importance of biotic effects on decomposition may be more heterogeneous than abiotic influences, depending on microbial community dominance and the abundance of key macro-invertebrate taxa.

SHOOT MERISTEMLESS (STM) encodes a transcription factor in Arabidopsis essential for ensuring correct stem cell fate. STM is known to impinge on a number of key regulatory processes such as cytokinin synthesis and the cell cycle, and interacts with other core regulatory genes such as CUP-SHAPED COTYLEDON1 (CUC1). In this study inducible STM over-expression and RNAi-mediated downregulation over a time course experiment have been used to identify the genes which form STM's gene regulatory network (GRN). These results reveal for the first time how STM over-expression and knockout phenotypes are mediated and identified the temporal order of transcriptomic changes following STM over-expression. A Bayesian network approach further refined the GRN - identifying conditional dependencies among regulated TFs and core signalling components from an independent dataset (>2,000 experiments). Predictions of direct targets from the network have been tested, demonstrating a high degree of accuracy. Interplay between STM and CUC1 is a biologically interesting sub-module of the STM GRN, with unusual dynamics. Via gene expression and microscopy experiments it has been shown that STM positively regulates the CUC1-targetting microRNA miR164c. Mathematical modelling approaches show that this is consistent with a model in which the boundary is the site of highest STM mRNA production via CUC1, and STM movement with miR164c upregulation produces the observed spatial distributions of both proteins. These relationships recast the boundary zone as a particularly dynamic region of the shoot apical meristem (SAM) and significantly develop our understanding of STM developmental context.

In recent years, the microbiology of white mineral dispersions and the application of microbiocides for their preservation have taken a central role for the producer and user with the aim of maintaining high quality requirements such as brightness, rheological parameters, and odour neutrality. Additionally, new applications of mineral dispersions set to open up markets in food, cosmetics and pharmaceutical applications have aroused the interest in the microbiology of white mineral dispersions. Due to the occurrence of biocide resistant bacteria, technical limitations in the usage of biocides, as well as the more rigorous regulatory situation created by the BPD, the demand for new biocide research to ensure continuing effective WMD preservation is increasing. Despite efforts to optimise the application of microbiocides for the storage and protection of mineral dispersions, costs for preservation and disinfection are escalating. These are reasons why the current preservation strategies have been revisited and new preservation strategies have been designed. The work described in this thesis demonstrates that the microbial diversity of white mineral dispersions is greater than previously assumed and gives detailed insight about the microbial diversity of mineral dispersions. The occurrence of microbial contamination in mineral dispersions is of a seasonal nature rather than manufacture site or product type specific. Furthermore, the incidence of biocideresistant bacteria in mineral slurries is increasing and the microbial degradation products of biocidal compounds are disadvantageous for dispersion stability (pH and viscosity). New strategies for the preservation of mineral dispersions have been developed and biocide performance against biocide-resistance bacteria has been enhanced by combining in-use biocides with a range of non-biocidal additives. The industrial application of these new findings contributes to a more efficient preservation of white mineral dispersions with respect to both environmental as well as financial resources and opens up a basis for alternative preservation strategies of white mineral dispersions.

Cell-to-cell adhesion is critically important for the improved secretory function of endocrine pancreatic beta (β)-cells and for the progression of fibrosis in the renal proximal tubule in Diabetic Nephropathy. In this research project the effects of specific biochemical treatment on functional cell-to-cell adhesion and single cell mechanics were systematically investigated. Atomic Force Microscopy (AFM) Single Cell Force Spectroscopy was applied to quantitatively characterise E-cadherin mediated surface ligation and cytoskeletal reorganisation in the pancreatic mouse insulinoma MIN6 and human kidney proximal tubule HK2 cell model. AFM tipless cantilevers were functionalised with a single cell or a spherical microbead for performing cell-to-cell adhesion and single cell indentation experiments respectively. The impact of elastic deformation of single cells into cell-to-cell adhesion was examined by per-forming adhesion experiments at various retraction speeds. The results illustrate that both adhesive and mechanical properties of single cells constitute important underlying factors of the physiological and pathological conditions under investigation since they were significantly affected by biochemical changes. More specifically, it is suggested that the enhanced secretory function of MIN6 cells upon calcium-sensing re-ceptor activation is owned to a combination of increased E-cadherin mediated cell-to-cell adhesion and decreased elastic (E)-modulus of single cells. In addition, it was shown that treatment of HK2 with the cytokine TGF-β1 decreased E-cadherin mediated cell-to-cell adhesion and increased E modulus of single cells, suggesting a mechanism that initiates early fibrotic changes in the tubular epithelia. Overall, both studies demonstrate that alterations of biological states evoke complex interactions between E-cadherin and actin cytoskeleton as manifested by the interplay between the mechanistic behaviour and surface binding of the cells. Therefore single cell mechanics have profound effects on cell-to-cell adhesion characterisation, particularly when physiological versus pathological states are to be investigated.