Clathrin mediated endocytosis (CME) is an integral process in eukaryotic cells and governs a wide range of processes in higher organisms including neurotransmitter release and cell signalling, to development and cell polarity. Due to its wide ranging roles this mechanism has been implicated in various disease states such as Huntington’s disease and various cancers as well as being a method of entry into cells for many viruses and bacteria. The process of CME involves the formation of a clathrin coat, primarily consisting of the protein clathrin, that drives uptake of cargo at the plasma membrane. These interactions are facilitated through a large family of proteins known as adaptor proteins, which drive the process of CME through specific interactions with clathrin, cargo, the plasma membrane and the cytoskeleton. Once the cargo has been endocytosed the process must be reversed through the actions of the disassembly proteins auxilin/GAK and Hsc70. A number of questions remain as to how adaptors promote assembly and how auxilin and Hsc70 drive disassembly through interaction with clathrin and potentially through interactions with the adaptor proteins. By purifying adaptor proteins and clathrin I have used various biochemical and biophysical techniques to investigate these interactions in vitro. Using a novel assembly assay based on dynamic light scattering I have shown that it is possible to measure the effect of adaptors on clathrin cage size distribution during assembly. In disassembly I have shown how mutations in the disassembly protein auxilin affect its ability to catalyse the disassembly of clathrin cages and how the presence of various adaptor proteins alters the ability of auxilin and Hsc70 to disassemble these structures. Finally, I demonstrate an inhibitory effect on disassembly by the adaptor protein epsin and propose a mechanism of interaction with clathrin that can be disrupted through mutations to epsin clathrin-binding motifs and discuss the implications of this effect for the role of adaptors in vivo.

The Negativicutes are a class of bacteria within the predominantly Grampositive phylum Firmicutes that possess a diderm Gram-negative cell envelope along with lipopolysaccharides. Remarkably, some members of this class of Gram-negative bacteria can form endospores. The evolutionary origins of this enigmatic class were investigated through bioinformatics analyses, which defined and characterised their Gram-negative and Gram-positive toolkits, while analyses of Gram-negative signature proteins provided insights into the origins of the Gram-negative toolkit. The genome of Sporomusa sphaeroides, a sporulating species from the Negativicutes, was sequenced and annotated for the first time. In addition, the transcriptional landscapes of Veillonella parvula and Sporomusa sphaeroides during logarithmic and stationary phase growth were investigated using RNA-Seq, expanding the repertoire of known genome features, including non-coding RNAs within these species.

Food supplementation of birds in gardens is widespread and UK householders have recently been advised to supplement birds throughout the spring and summer. This coincides with reproduction of many avian species and supplementation with specific foods (e.g. live invertebrates) is encouraged to support breeding attempts in gardens. To investigate this further I mimicked food supplementation in gardens by providing two commercial bird foods (peanut cake and mealworms \(Tenebrio\) \( molitor\) to blue tits \(Cyanistes\) \(caeruleus\) and great tits \(Parus\) \(major\) breeding in woodland in central England from 2006 to 2008. Supplementation advanced laying and reduced the number of young fledged significantly in both species, but provisioning with mealworms during the nestling phase increased apparent survival of fledglings. Intriguingly, however, stable isotope analysis revealed that supplement use was insubstantial and similar between birds on supplemented and non-supplemented territories. Analyses of data from the British Trust for Ornithology’s Nest Record Scheme demonstrated strong parallels between findings of my field study and patterns of reproduction of blue and great tits in urban habitats across the UK. I discuss the implications of my findings, including the use of food supplementation in avian conservation and in pure scientific research, and I outline exciting future directions.

Typhlodromips montdorensis is a non-native predatory mite used for control of red spider mite and thrips, but is not yet licensed for use in the UK. Current legislation requires that non-native glasshouse biological control agents may not be introduced into the UK without a risk assessment of establishment potential outside of the glasshouse environment. This work focuses on the application of a recently developed protocol to assess the establishment potential of T. montdorensis in the UK. Further, the use of alternative prey outside the glasshouse by, Macrolophus caliginosus is examined, and interactions between Neoseiulus californicus, Typhlodromus pyri and T. montdorensis are investigated. Laboratory results demonstrate that T. montdorensis has a developmental threshold of 10.7°C, lacks cold tolerance and is unable to enter diapause when tested under two different regimes. Field studies indicate that no reproduction occurs within the field in winter (November to March) and 100% mortality of eggs, larvae and adults occurs within two weeks of release during this period. It is suggested that T. montdorensis would be a ‘safe candidate’ for introduction as a glasshouse biological control agent in the UK as outdoor establishment is unlikely to occur. Macrolophus caliginosus is able to feed and reproduce on Trialeurodes vaporariorum, Myzus persicae and Aleyrodes proletella although performance (lower fecundity and longevity) is reduced on the latter prey source. Both N. californicus and T. pyri were able to feed on larval stages of each other but when given a mixed diet, showed a preference for Tetranychus urticae over their phytoseiid prey. Neoseiulus californicus showed a preference for T. urticae over T. montdorensis, whereas the latter species showed no preference between T. urticae and N. californicus. This work has identified a robust experimental protocol for predicting the establishment potential of non-native biological control agents outside the glasshouse and has begun to develop a further experimental system for assessing the possibility of non-native biological control agents feeding on alternate hosts outside the glasshouse.

Cultures of two Dipteran flies (Calliphora vicina (R-D) and C. vomitoria (L.)) were established to answer questions in regards to responses to thermal and desiccation stress, effects of diapause and the mechanisms which underpin diapause. The findings are divided in to two sections. Unequivocal new findings – Calliphora vomitoria were seen to depend on water being present in culture medium for increased survival. Furthermore, C. vomitoria were found to have lower desiccation resistance than C. vicina. Larvae of C. vicina and C. vomitoria showed different cold tolerance strategies, with C. vicina being freeze-avoiding and C. vomitoria ‘partially’ freeze-tolerant. Metabolomics, using \(^1\)H-NMR, revealed that diapause and non-diapause had distinct metabolic profiles. Diapause larvae were seen to reduce energy synthesis from the Krebs cycle and increase glycolysis. Calliphora vicina and C. vomitoria also exhibited different diapause phenotypes; C. vicina entered a maternally regulated facultative diapause as an L3 larvae, Calliphora vomitoria had a less distinct diapause, with maternal conditions having little effect. Speculative new findings - Despite the above differences C. vicina and C. vomitoria were able to produce a viable cross, though field fresh C. vomitoria were not used, as such it cannot be confirmed if this could occur in the wild. Increased temperatures due to climate change may affect both phenology and survival of insects; C. vicina was seen to have a delayed induction to diapause and a reduction in the proportion entering diapause. Diapause conferred increased cold tolerance; therefore those insects that overwinter not in diapause may suffer increased mortality.

This work is focused on the recognition of natural lipopolysaccharide (LPS) by the innate immune protein human lung surfactant protein D (hSP-D) in the form of a biologically active recombinant fragment (rfhSP-D), containing the α-helical coiled-coil and three carbohydrate recognition domains (CRD). Intact LPS from two bacterial strains, S. minnesota (R5 mutant) and H. influenzae type b Eagan (CA7 mutant), were delipidated by means of mild acid hydrolysis, leaving the purified polysaccharide (PS) to be used in X-ray diffraction studies by means of co-crystallisation with rfhSP-D. S. minnesota R7 full LPS was also investigated following development of a suitable solubilisation method which also utilised the LPS from E. coli O111:B4. The structural studies of rfhSP-D bound to H. influenzae Eagan CA7 PS (solved and refined at 2.98 Å) and to S. minnesota rough mutant LPS/PS (solved and refined at 3.3 Å) reveal that rfhSP-D binds to LPS preferentially through the non-terminal inner core heptose HepI via the O6’ and O7’ hydroxyls. rfhSP-D recognition of S. minnesota HepI shows a similar bound heptose orientation to that previously reported for heptose binding by rfhSP-D in the literature with an indication of normal Kdo in the inner core Kdo-Hep-Hep trisaccharide. rfhSP-D recognition of the HepI of H. influenzae Eagan CA7 reveals a novel bound heptose orientation, with the heptose rotated by 180° about C5-C6, resulting in the O6’ and O7’ hydroxyls being interchanged with respect to coordination to Ca1 and protein. The novel orientation of HepI is accompanied by a salt bridge being formed between the flanking residue Arg343 and Glu347, both of which adopt a previously unseen conformation. The novel binding mechanism of rfhSP-D for Eagan CA7 suggests flexibility in recognition and offers evidence to explain why this mutant binds more weakly than the Eagan 4A mutant to both rfhSP-D and hSP-D.

The 'Vegetable Substances' collection was formed by the physician and collector Sir Hans Sloane (1660-1753) between the 1680s and the 1750s. All sorts of people ranging from ship's captains in the Americas to surgeons in the East Indies sent natural material from around the world to London. Sometimes this involved a variety of means and intermediaries, and in other instances individuals, including aristocratic women in London and Royal Society Fellows across England, gave items directly to Sloane. When Sloane received these samples of botanical items, he had them sealed into small glass and wood boxes. He then numbered these items and described the sample in a three-volume manuscript catalogue. 12,523 items are listed in Sloane's hand in this catalogue with varying degrees of information relating to their identification, contributor, provenance and use. Today, the Natural History Museum in London holds Sloane's surviving catalogue and over 8000 of these 'Vegetable Substances' objects. Considering the collection as a whole, this thesis explores the role of the 'Vegetable Substances' in early eighteenth-century natural knowledge. Using data provided by the catalogue and Sloane's surviving correspondence at the British Library, this thesis explores what is in the 'Vegetable Substances' and identifies how Sloane formed the collection by surveying the connections he developed with people across the world and how he managed these different relationships. Drawing on these exchanges, this thesis also focuses on the uses of the 'Vegetable Substances' by examining its contents in particular eighteenth-century contexts including gardening spaces and medicine.

The expression of Tumor protein D52 (TPD52) family members is deregulated in many types of cancer. When overexpressed, it is suggested that they increase cell proliferation and migration/invasion as well as avoid apoptosis. Deregulation in the expression of the TPDs is therefore linked to poor prognosis. Little characterisation has been carried out to date, but it is known that the TPDs are found in association with components of the membrane trafficking pathway. The aim of this work is to uncover how the least studied member of the family, TPD54, affects cellular processes involved in carcinogenesis, such as cell migration and invasion. By using the knocksideways method, we have been able to map the cellular localisation of TPD54 and have identified association partners. These associations have been confirmed by immunoprecipitation and mass spectrometry analysis. Amongst these was the small GTPase Rab14. We have also found that TPD54 is involved in the trafficking of receptors containing a dileucine motif in their cytosolic tail, but not a tyrosine-based or NPXY motif. With the mapping of the localisation of TPD54, we hypothesise that TPD54 is on the recycling route following the Golgi apparatus, and in association with Rab14, regulates the trafficking of receptors containing a dileucine motif. Integrins are receptors controlling cell migration. They can be trafficked through the Golgi apparatus before being recycled back to the plasma membrane. This recycling route is not well characterised. We therefore hypothesise that TPD54 regulates this route with Rab14, and that this is the reason why TPD54 is important for cell migration, and that a defect in its function can cause cancer.

Grasslands are considered to be the most endangered terrestrial ecosystem in the world. In the United Kingdom, substantial losses in unimproved grasslands and the abandonment of traditional grazing has resulted in the decline of ecosystem services, such as pollination. A plant trait-based approach was conducted to study the community ecology and restoration of temperate grasslands, with a focus on the convergence/divergence patterns in response to environmental and management factors, and how these scale to the provision of ecosystem processes and services –biomass production and livestock. The role of seven plant traits, obtained from the TRY-database, was investigated using the botanical data of the National Vegetation Classification, the Park Grass Experiment and the North Wyke Farm Platform. Trait-based analyses were conducted on the latter two to investigate the provision of biomass production and livestock production. A microcosm experiment was conducted to test the effects of agricultural soil legacies and restoration seed mixture on the reassembly of grassland communities, and their associated functional structure and composition. It was found that the seven traits investigated were independent and countered the conceptualisation of ecological axes of specialisation and ecological strategies. The type of fertiliser used to improve grasslands was found to be a significant factor driving the convergence/divergence patterns of temperate grassland communities, together with grazing. Biomass production was found to be best explained by statistical models incorporating climate and environmental factors, community-weighted means and different facets of functional diversity. In essence, environmental and management pressures resembling intensely managed, especially with nitrate-based fertilisers, temperate grasslands and an exploitative community best supported greater amounts of high quality biomass. Livestock production was found to be best explained by the Functional Diversity Hypothesis: higher yields from cattle and sheep were found from diverged grazing pastures. A trade-off between cattle quantity and quality was also highlighted. Agricultural soil legacies were found to greatly hinder the progression towards vegetation and functional restoration targets, producing ruderal communities dominated by weak competitors and opportunistic weedy plant species. The work has important implication for the management and restoration of grassland communities.

Infochemical communication is ubiquitous amongst all living organisms, and particularly important in insects. Because smell being the most common basic means of chemical communication, infochemical blends must be constantly decoded in order to proclaim their readiness to mate, to mark out territorial boundaries, to warn off intruders and predators or, in some cases, to locate food or predators with millisecond precision. The central challenge of the thesis was to mimic nature in both cellular and molecular levels on to a technological platform that aids in the development of a new class of technology employing chemicals alone to communicate over space and time. This thesis describes a body of work conducted in the development of a miniaturised, smart and label-free cell-based chemoreceiver for artificial insect olfaction, as part of the development of a novel biomimetic infochemical communication system. A surface acoustic wave based microsensor has been utilized to engineer and develop a chemoreceiver system that mimics the cellular and molecular mechanisms occurring during infochemical detection and decoding in insects. Successful recovery of ratiometric information with the aid of polymer-based gas-phase measurements, established the concept of chemical communication. Thus, small scale, high-throughput infochemical communication has been realized by a combination of precise spatiotemporal signal generation using fruit volatiles and insect sex pheromones with highly sensitive detection and signal processing. This was followed by the investigation of the feasibility of using the prototype cell-based biosensor system in a static mode for artificial insect olfaction applications, mimicking the cellular detection in the receptor/antenna apparatus of insects. Finally, as part of the development of a compact and low-power portable chemoreceiver system, the discrete sensor drive and interface circuitry was deployed in an analogue VLSI chip, thereby overcoming the associated measurement complexity and equipment cost, in addition to extending the reach and functionality of point of use technologies.