D-type cyclins in plants are represented by seven conserved subgroups and play a major role in controlling cell division. Relatively little is understood of their role during seed development, although their expression pattern has been characterized and ectopic expression of CYCD3;1 has previously been shown to disrupt normal embryo development. Here the consequences of ectopic expression of CYCD7;1 using the early endosperm-specific promoter FWA in developing Arabidopsis seeds were investigated. Ectopic CYCD7;1 expression in the maternal central cell prior to fertilization, and in the endosperm from fertilization until cellularization resulted in seeds up to 45% larger. Seed enlargement was accompanied seed lethality, shown to be due to a defect of development during early and mid stages of seed development. As expected from the maternal specific expression of the imprinted FWA promoter, seed size and lethality was dependent on maternal origin of the transgene. Larger seed size was correlated to mature embryo and seed coat outgrowth, and was due to cell proliferation rather than cell elongation. In particular, embryo development was accelerated during the early stages, suggesting these may be dependent on cell division rate, whereas later stages progressed at the same rate as WT seeds. Seed-targeted CYCD7;1 expression phenocopies (1) the nucleus proliferation in the endosperm prior to fertilization observed in rbr and fis-class mutants and (2) the seed enlargement observed in paternal genome excess interploidy crosses. These suggest that CYCD7;1 may act through the RBR pathway to promote cell proliferation and modify imprinting in the endosperm, thereby influencing the parental genome balance. Mechanistically, CYCD7;1 did not interact directly with CDKA;1 but the interaction was promoted in presence of the inhibitor of CDK, ICK1/KRP1 or ICK2/KRP2 in a yeast-three-hybrid assay. However, loss of either KRP1 or KRP2 in respective mutant backgrounds did not prevent the seed enlargement phenotype.

Prediction of pathogen dynamics and the design of effective interventions to control and eliminate disease are key goals in epidemiology. While progress has been made towards the elimination of many infectious diseases, only two, smallpox and rinderpest, have been globally eradicated. Mass vaccination can greatly reduce the burden of vaccine-preventable diseases. However, there is relatively little scientific guidance on the optimal duration, frequency and placement of control interventions for achieving elimination. Such insights could greatly inform policy and practice. Rabies is a deadly and terrifying disease that exacts a heavy toll on human lives and national economies, with over 50,000 human deaths each year and many millions more requiring expensive life-saving post-exposure vaccines. Elimination of rabies is feasible through vaccination, and oral rabies vaccination (ORV) campaigns have eliminated fox rabies from Western Europe. However, scientific guidance could improve elimination efforts elsewhere, and is still needed for contingency planning to maintain rabies freedom and for emergency response to incursions. My thesis focuses on two pivotal questions in infectious disease ecology: what are the underlying determinants of disease persistence, and how can vaccination strategies be optimized to eliminate infection? To answer these questions, I analysed a rich and highly resolved spatial dataset of fox rabies cases and ORV efforts over three decades in Germany and neighbouring countries. The long-term, large-scale nature of these data provides a unique opportunity to improve our understanding of wildlife rabies dynamics in response to vaccination using novel spatial modeling techniques. In chapter 2, I create a metapopulation model of regional rabies dynamics that incorporates local transmission (within regions) and spatial coupling (between regions) using a hierarchical Bayesian state-space model. In chapter 3, I extend the model developed in chapter 2 to determine the best vaccination strategy, in terms of scale and duration of ORV efforts for three common epidemiological scenarios: {\bf endemic} circulation of rabies; {\bf high-risk} situations when rabies-free but neighbor endemic areas; and an {\bf endgame} scenario when only a single endemic foci remains. In chapter 4, I develop a space-time model of fox rabies dynamics and explore the effect of scale on estimates of transmission terms by aggregating rabies case data at different spatial resolutions. I then relate these estimates to the scaling of individual interactions to regional dynamics through population mixing. Collectively, the findings from this thesis contribute to our understanding of how infectious diseases persist and can be controlled through vaccination. The methods generated can be used to explore tradeoffs in the scale and duration of ORV efforts, and generate recommendations on the time horizon and investment required to achieve and maintain freedom from disease. The model developed in chapter 4 also presents the first steps to developing a highly resolved spatial model of local rabies dynamics. These findings have immediate application to the design of cordon sanitaires in Europe, and to strategies aiming to rapidly eliminate re-emergence in high-risk countries such as Greece and Turkey. The analytical and statistical framework developed in this thesis is also applicable to answering analogous questions for the elimination of dog-mediated rabies and for other vaccine preventable diseases.

Calcareous grassland, considered among the most species rich and diverse habitats in Europe, underwent wide scale loss and degradation following post 1950s agricultural intensification. Consequently, they are the focus of conservation efforts and are protected in national and international legislation (e.g. EU Habitats Directive). As elsewhere in Europe, a major cause of upland calcareous grassland loss and degradation in Britain was intensive grazing, typically with sheep. In recent years, conservation organisations have altered grazing practices in an attempt to prevent further loss and degradation by focussing management on conserving characteristic calcareous grassland vegetation. However, the impact of the contrasting grazing regimes used in this internationally important habitat on invertebrates is unknown. This study is the first to investigate the impacts of a range of established grazing regimes (low intensity sheep grazing, low intensity cattle grazing, high intensity sheep grazing and no grazing) on aspects of plant diversity and structural complexity, carabid beetle diversity, and spider diversity in upland calcareous grasslands. It also provides the first evidence based management recommendations for UK upland calcareous grasslands which incorporate both plants and invertebrates. In addition, this study is also the first to assess the biodiversity value of acid grassland and limestone heath habitat patches that occur as 3 part of the calcareous grassland matrix and are not targeted by conservation management, by examining the spider fauna in each habitat in relation to calcareous grassland. Further evidence based recommendations for the management of these non-target habitats are made for the first time.

Basement Membranes (BMs) are specialised extracellular matrix (ECM) structures that underlie all endothelial and epithelial cells, and provide structural support to tissues as well as influence cell behaviour and signalling. Mutations in the BMs major component collagen IV cause eye, kidney and cerebrovascular disease including intracerebral haemorrhaging (ICH). Haemorrhagic stroke accounts for 15% adult stroke and 50% paediatric stroke, and carries the worst prognosis and there are no therapeutic strategies. Mutations in the genes COL4A1/COL4A2 (collagen IV alpha chain 1 and 2) cause BM defects due to mutant protein incorporation in the BM or its absence by ER retention, and ER-stress due to intracellular accumulation of collagen IV. Despite this, the mechanism(s) of collagen IV mutations disease remain poorly characterised. To provide novel insights into mechanisms of collagen diseases, this study investigates the effect of defined engineered biointerfaces on cell behaviour/signalling, collagen secretion in COL4A2 mutant and wild-type cells. Atomic force microscopy and spectroscopy were employed together with confocal and biochemical analyses of cells cultured on engineered synthetic polymers, poly(ethyl acrylate) and poly(methyl acrylate), coated with ECM proteins, namely laminin, collagen IV and fibronectin. This enabled us to address the hypothesis that biomaterials may alter the behaviour of COL4A2+/G702D mutant cells by overcoming some of the defects caused by the mutation and rescuing the downstream effect of the ER stress. Of the ECM proteins that were used, only fibronectin was observed to undergo a drastic structural change depending on the substrate chemistry. On poly(ethyl acrylate), fibronectin was assembled into fibrillary networks upon adsorption, and these nanonetworks induced increased secretion of Col4a2 in COL4A2+/G702D cells than on poly(methyl acrylate) or control glass. The behaviour of the mutant cells appeared to be influenced by the underlying biointerface, increased levels of molecular chaperones and reduced ER area suggested an increased collagen IV folding capacity when the cells were cultured on the FN nanonetworks compared to the other surfaces. COL4A2+/G702D cells interacted with the adsorbed proteins and were able to mechanically translocate them. Enhanced formation of focal adhesions was also seen on FN-coated polymers, where ligand density and actin-myosin contractility accounted for the observed increase in cell adhesion strength. The stiffness of the mutant fibroblasts and of their ECMs was found to be 10 times lower than that of the wild-type cells; interestingly, mutant cells cultured on FN nanonetworks on poly(ethyl acrylate) were able to deposit a protein matrix with significantly higher Young modulus than on glass or poly(methyl acrylate). These findings suggest that biomaterials are able to influence the behaviour of these mutant cells through changes in the interfacial layer of adsorbed proteins presented to them. Collectively, these data provide an understanding of the effect of mutations on cell characteristic and a basis of concept that material may be employed to modulate effects of mutations of collagen/ECM molecules. Understanding the mechanisms through which these surfaces trigger a change in cell response will prove valuable for the development of new therapeutic approaches to address pathologies due to collagen IV mutations. In this respect, further investigation is needed to dissect the signalling pathways involved.

No description available.

Breast cancer is the third most common cause of cancer death in the UK, accountable for more than 11000 deaths in 2010 alone (www.cancerresearchuk. org). Developmental pathways commonly required for normal development are often hijacked during tumour progression, so a better understanding of mammary gland development is necessary to fully understand the roots of breast cancer. The Runx gene family are known to be important regulators of development in different lineages. In particular RUNX1 and RUNX2 have been widely studied in the context of haematopoiesis and osteogenesis respectively, but their role in epithelial tissue is much less well understood. In this thesis a role for RUNX1 and RUNX2 in mammary development and breast cancer has been identified. The first part of this study is focused on characterizing the expression and function of the Runx genes in the mammary epithelium. RUNX1 and RUNX2 protein levels fluctuate during embryonic and adult mammary development, and an in vivo conditional knockout strategy shows that both genes are important for maintenance of mammary epithelium homeostasis. Moreover, combined loss of RUNX1 and RUNX2 significantly perturbs the normal mammary architecture with an expansion of the basal population in vivo and the appearance of preneoplastic lesions in aged mammary glands. An exciting new role for RUNX2 in mammary stem cells has also been revealed showing that RUNX2 is important for the regenerative potential of mammary epithelial cells in vitro. Evidence is also presented to indicate that RUNX2 could be linked to regulation of quiescence and Wnt signalling in the stem cell compartment and during transformation. Finally, the role of these genes in breast cancer is discussed demonstrating involvement of RUNX1 and RUNX2 specifically in the triple negative (ER-PR-HER2-) subtype. In particular, for the first time, RUNX1 is revealed as an independent prognostic indicator correlating with poor prognosis in triple negative tumours. Meanwhile, evidence from various mouse models demonstrates that RUNX2 may be specifically involved in the squamous metaplastic form of this disease.

How are processes working at the individual level, the species level and the macro-ecological level connected? This thesis explores the theoretical and structural constraints on biological evolution. It does this by developing an evolutionary program to model biological form. This development was necessary as the existing models of evolution are poorly suited to modelling morphological constraint. The model of biological form developed in this thesis uses graphs to abstractly represent organisms and the relationships of their internal structure. We show that by increasing the number of degrees of freedom, or by increasing the ruggedness of the fitness landscape, higher levels of diversity are supported - particularly when there is strong directional selection. We explore whether meta-regulation is bounded in the model by using an analytical framework. We show that there is no analytical steady state, but that one can be induced in the model by selection effects. We find that a mixed strategy between increasing object complexity and increasing hierarchical complexity maximises the average degree of a vertex. This agrees with the evolutionary history of meta-regulation. We claim that the macro-ecological response to environmental perturbation is determined by both the characteristic time scale of mutation and the time scale of the environmental change. We show that for high amplitude changes the system can adapt provide the mutation time scale is smaller than the environmental change. We also show that low amplitude environmental changes cause rapid turnovers in species' diversity. Finally, we show that mass extinctions can be the result of species' interactions and background rates of extinction, and do not need large external perturbations to occur. This, combined with the results above, suggests that many of the trends seen over geologically long time periods can be explained as a result of the interacting processes at the individual and species level.

Multi-host pathogens of domestic and wild mammals have significant socio-economic, animal health and conservation consequences. However, despite the interest in diseases at this interface, most research has focused on only a few key pathogens. Studies of focal systems provide limited information on the broad scale ecological patterns of pathogen occurrence and the factors that might drive these distributions, yet large scale studies may have important consequences for disease control and pathogen surveillance. This thesis aims to quantify the abundance and distribution of pathogen sharing among domestic and wild mammals, and develop and understanding of the processes that determine this distribution. Using comparative methods and comprehensive databases this thesis provides the first systematic assessment, on a global scale, of which domestic mammal pathogens have been reported in wild mammals in natural settings. Assessing the extent of pathogen sharing and the characteristics of the pathogens involved showed that the occurrence of domestic mammal pathogens in wild mammals was greater than previously recorded, with an additional 28.5% of domestic pathogens reported to infect wild hosts. Pathogens with the broadest host range had the greatest sharing probability and, in general, pathogen transmission strategies did not limit the degree of sharing. Importantly, from analysing reporting trends, the majority of shared pathogens are already likely to be known, but these are still being reported from novel host-parasite combinations suggesting that the opportunities for pathogen transmission continue to occur, especially since the majority of pathogens have been reported in wildlife multiple times. Most wild ungulates (artiodactyla and perissodactyla) have evidence of infection with domestic livestock parasites, and these hosts are also dominated by those more closely related to livestock. However, phylogenetic relatedness did not appear to be a barrier of infection. The diversity of sympatric wild species was associated with a greater proportion of shared viruses and bacteria, but a lower proportion of shared helminths. These differences among parasite groups are potentially due to variation in parasite transmission strategies. Hosts of conservation concern were not more likely to be infected with domestic mammal pathogens than un-threatened species, suggesting that domestic hosts do not directly contribute to parasite driven declines of wild mammals. Assessing the spatial reporting of wild mammal parasites and what global environmental drivers determines the occurrence of shared parasites may have important implications for disease control and surveillance. Although there are bias in reporting, the majority of wild mammal sampling locations reported pathogens also found in domestic mammals. Livestock densities did not predict the occurrence of pathogens in wild ungulates, but human densities (a proxy for domestic carnivores) did predict the occurrence of pathogens in wild carnivores. For both host groups economic variables were also informative. The probability of parasite sharing was lower in protected area systems, suggesting that these areas may have an valuable role in wildlife disease management. Pathogen sharing among domestic and wild mammals is ubiquitous. Therefore, systematic surveillance for shared pathogens or those shared pathogens that are likely to cross the species barrier in the future is arguably not beneficial. Instead, the informative drivers determined from this macro-ecological analysis, and the identification of areas and hosts that have an increase risk of pathogen sharing may help inform disease management and surveillance strategies.

As the principal vector for the transmission of the Plasmodium falciparum parasite, and hence the spread of malaria in Sub-Saharan Africa, Anopheles gambiae is a globally significant species of mosquito. Over recent years, the efficacy of established insecticides has waned and there is a constant need for novel effective compounds. Cytochrome P450 reductase (CPR) is a diflavoprotein known to have a central role in phase I metabolism of xenobiotic compounds and, in mosquitoes, this involves the detoxification of insecticides. Due to an inherent lack of understanding regarding the mechanisms of action of A. gambiae CPR, the selective inhibition of this enzyme is a previously untried approach. This project aims to biochemically characterise A. gambiae CPR in direct comparison to the human enzyme. It was found that A. gambiae CPR was deficient in bound FMN, and to a lesser extent FAD, relative to human CPR with 20 % less FMN bound to the purified mosquito protein. Following the dissection of A. gambiae CPR into its constituent FMN- and FAD-binding domains, and using Isothermal Titration Calorimetry (ITC), a 4-fold decreased was observed for the binding affinity for FMN in the A. gambiae FMN-binding domain as compared to the equivalent human protein. The redox potential of the oxidised/semiquinone transition of the A. gambiae FMN-binding domain was -92 mV, much more negative than the published value for human FMN-binding domain. These data suggest a clear difference between these enzymes in the binding strength of FMN and its propensity to accept electrons. The binding characteristics of NADPH nucleotides were probed in some detail. Comparison of the binding of NAD+ and NADP+ revealed a strong bias for the phosphate containing NADP+. In addition, the position of the phosphate was important as 3’-AMP bound very poorly whilst 2’-AMP bound more strongly. 2’, 5’-ADP binding highlighted the importance of additional stabilising interactions involving the 5’-phosphate. Comparison of 2’, 5’-ADP and NADP+ binding confirmed that the 2’-phosphate interaction was the principal site for NADPH recognition and provided the majority of the binding energy for this interaction. A. gambiae CPR was shown to bind NADPH nucleotide analogues 2’-AMP, 2’, 5’-ADP and NADP+ much less strongly than the human enzyme highlighting a potentially significant difference in coenzyme binding. Binding affinities for the nucleotide ligand to intact CPR and the isolated FAD domain showed that the FAD-binding site is fully contained within the FAD-binding domain However, differences in the thermodynamic parameters between the intact enzyme and the isolated FAD-binding domain suggest that, although not directly involved in NADPH binding, the presence of the FMN binding domain had an effect on the overall binding energetics. Despite an apparent difference between A. gambiae and human CPR in flavin incorporation and NADPH binding affinity, it was interesting that the activity of cytochrome c reduction of both enzymes was similar. The measured Km with respect to NADPH corroborated the ITC data by suggesting a stronger interaction of the coenzyme with human CPR compared to A. gambiae CPR. There was an approximate 2-fold increase in potassium ferricyanide reduction with the isolated A. gambiae FAD-binding domain compared to the intact enzyme with the presence of the FMN-binding domain again seemingly imparting an effect of events involving the FAD-binding domain. In order to fully understand and rationalise all of the data, a comprehensive structural determination of A. gambiae CPR is required. With this in mind, isotopic labelling and subsequent biophysical analysis was carried out on the intact CPR and its FMN- and FAD-binding domains. Successful labelling was achieved for all samples, including the deuteration of the intact CPR and FAD-binding domain However, the greatest success involved the FMN-binding domain with sufficient triple resonance spectra collected for backbone assignments. Although this success could not be matched for the intact CPR and FAD-binding domain, the work has provided a solid base for more a comprehensive study in the future.

There is increasing interest regarding the impacts of human activities on the functioning of marine systems. A primary driver of change to marine systems is through the impacts of fishing. Biomass-dominant target species have the potential to mediate a number of ecosystem functions, either directly or indirectly, through the influences that taxa have on ecological processes and/or other biotic or abiotic components of the system. This thesis investigates the roles of the cockle, Cerastoderma edule on ecosystem functioning within intertidal sedimentary systems. A series of investigations revealed that cockles have the potential to mediate benthic primary productivity through their roles in the recycling of nutrients and effects of sediment structure and have impacts upon assemblage biomass and functional diversity. However, the roles of cockles on other aspects of ecological functioning were less apparent. An investigation also assessed the suitability of the use of assigning taxa to functional groups when assessing functional diversity. Taxa were shown to have the potential to change their feeding activity following disturbance, with evidence suggesting a change to benthic-pelagic coupling. This change however, would not have been observed with investigations of functional traits alone and thus supports the use of direct measures of functions to support functional trait diversity measures. It is imperative for ecological investigations to consider long-term changes to population dynamics. However, particularly in marine systems, such data are generally lacking. This thesis presents a novel approach, using information from sea fisheries reports to gain a semi-quantitative 30-year data set on cockle landings within Morecambe Bay, north-west England. This technique revealed evidence of temporal changes to the functional processes at a large scale. This thesis provides evidence for cockles acting as key contributors to ecological functioning. The functional role of cockles is tempered by the high degree of redundancy within these assemblages, highlighting a number of issues relating to the use of field studies and encouraging a move towards increased use of traditional ecology in future studies.