The timely and efficient movement of chromosomes to the spindle equator during mitosis is a prerequisite for accurate chromosome segregation. Recent work has shown that the majority of chromosomes are able to congress and biorientate almost instantaneously after nuclear envelope breakdown due to their position relative to the forming spindle. However, other mechanisms are required to facilitate the congression of chromosomes, which do not congress in this initial wave. Congression of these remaining chromosomes is mediated by multiple mechanisms including: (1) Kinetochore sliding along the microtubule lattice using the Kinesin-7 CENP-E, and (2) kinetochores biorientating near the pole and congressing through microtubule depolymerisation-coupled movement. Here, we show that the constitutive centromere associated network (CCAN) subunit CENP-Q is required for both mechanisms. CENP-Q is required to recruit CENP-E to kinetochores thus explaining the absence of lateral sliding in CENP-Q depleted cells. Because depletion or inhibition of the CENP-E motor does not affect depolymerisation-coupled pulling, we identify a CENP-E recruitment-independent role for CENP-Q in chromosome congression. Following congression we find that biorientated kinetochore movements require both CENP-Q and CENP-E dependent mechanisms. This suggests that as biorientated kinetochores congress they switch into a mode that requires CENP-E motor activity.

The ER is a network of membrane sheets and tubules connected via three-way junctions. Reticulons are integral membrane proteins responsible for shaping the tubular ER. There are four reticulons isoforms in animals, two in yeast and 21 isoforms in Arabidopsis. This study developed an expression profile for isoforms AtRTNLB1-18 by expressing these reticulons driven by their native promoters. The reticulons analysed localised to the ER and coexist in many tissues. Given this result it is possible that the large size of the Arabidopsis gene family may have evolved to afford functional redundancy. It is known that a knock-out of seed specific RTNLB13 does not disrupt the seed ER morphology. Is this due to functional redundancy? In this study knock-outs were made of two other reticulons found non-exclusively in the seed, RTNLB1 and RTNLB2. In accordance with the redundancy theory, there was no apparent disruption in the mutants ER morphology. Knock-out/knock-down mutants of RTNLB1, RTNLB2 and RTNLB13 were also made and verified. The results of this were beyond the time frame of this study. In mammals and yeast reticulons are known to interact with other tubule forming proteins (DP1/Yop1p) and human atlastin. The Arabidopsis homologue of Yop1p is HVA22. In this study the seed-specific isoform HVA22b labels the tobacco ER (and the nuclear envelope), but it does not alter ER morphology or cause constrictions of the tubules (as seen with RTNLB13), suggesting that it is not a structural component. The closest plant homologue of atlastin is RHD3. This study shows that RL2, an RHD3 isoform that is highly expressed in the seed, locates to the ER without perturbing the ER morphology or Golgi body mobility. Over-expression of RL2 bearing mutations within its GTPase domain, however, induces cable-like ER, suggesting that a functional GTPase domain is required for the formation of three-way junctions. Co-expression of RTNLB13 with RL2 resulted in a striking modification of the ER network. This alteration was independent of an active RL2 GTPase domain but required a functional reticulon. RL2 and its GTPase mutants co-immunoprecipitate with RTNLB13. These results indicate that RL2 and RTNLB13 interact and operate synergistically in modulating ER morphology.

Ecotoxicology is currently undergoing a revolution as the result of new technological advances in molecular biology, capable of finely resolving metabolic mechanisms associated with exposure. These high-throughput analyses can detail the evolutionary and ecological implications of exposure in non-model organisms, such as the earthworm, Lumbricus rubellus. This terrestrial sentinel has been observed across former mine sites that are highly contaminated with arsenic and have been found to mitigate toxicity at soil concentrations that cause mortality in unadapted individuals. This is indicative of the adaptive capacity of natural populations recently exposed to persistent and strong selection pressure. However, mechanisms surrounding adaptation to arsenic in L. rubellus have yet to be characterised, and so the effects of exposure are broadly reported with the aim of distinguishing resistance from phenotypic plasticity in natural populations. Unadapted earthworms were initially used to derive basal phenotypic variation associated with arsenic exposure. Variation in life-history parameters was observed among adult and juvenile L. rubellus, establishing relative sensitivity and population-level inferences. A systems biology approach was employed to describe molecular mechanisms associated with arsenic metabolism, encompassing transcriptomic and metabolomic analyses, underpinned by arsenic speciation. Insight into the genetic bases of arsenic resistance, which enable persistence of L. rubellus at highly contaminated sites, was sought. Recombinant inbred lineages derived from adapted populations, were cultivated and their phenotypes relative to arsenic exposure determined. Phylogeographic analyses were used to interrogate genetic variation among populations inhabiting former mine sites as well as proximal control sites. A mitochondrial marker defined cryptic species across the UK, but did not establish soil chemical profiles relative to clade occurrence. RADseq better resolved genetic variation at these sites, determining that soil geochemistry is strongly associated with genetic variation. Furthermore, genomic markers inferred genetic erosion, found to selectively reduce variation at sites relative to a single clade.

This study examined the ecological role of herbivory on coral reefs of the Saudi Arabian Gulf coast. Herbivory is a ubiquitous process and important in regulating benthic marine communities. Three reef sites were studied over a 12-month period; one on an inshore fringing reef (1.5 m depth), and two on the fringing reef surrounding an offshore island (3 m and 11 m depth). The inshore site experienced greatest extremes in temperature, salinity and sedimentation. The ecological role of herbivory was determined from algal settlement plates and their selective exclusion from herbivores. The inshore site was naturally dominated by filamentous algae, while both offshore sites supported a higher proportion of crustose forms. Location (i. e. distance from shore) appeared to be more important than seasonality in determining the structure and composition of the epilithic algal community. At the inshore site herbivorous fish (dominated by Siganus spp. ) imposed a uniform, wide-ranging grazing pressure of intermediate intensity. Herbivorous echinoids (Echinometra mathaei) imposed an intensive but localised grazing pressure. At the shallow offshore site, only herbivorous fish (dominated by Scarus sp. ) appeared responsible for grazing impacts, which were also intensive. At the deep offshore site both herbivorous fish (dominated by Pomacentrus spp. ) and echinoids (Diademo setosum) were responsible for limiting algal growth, although other factors (e. g. light penetration) may also inhibit algal productivity at this site. An experiment assessing the effects of extreme perturbations (i. e. removal of the algal community), showed that seasonal life-history strongly affected generic succession and rate of re-colonisation. Perturbation effects were temporary and did not precipitate permanent alternative stable communities. An important secondary effect of herbivory is bioerosion. The mean erosion rate by E. mathaei was comparable to rates recorded elsewhere. Behavioural studies revealed that burrow defence and fidelity were positively correlated with burrow complexity, and that the frequency of agonistic behaviour was low. Foraging range was negatively correlated with burrow complexity. In addition, the risk of mortality by finfish predators at the inshore site was estimated to be very low. The study has shown that Saudi Arabian Gulf reef communities may be particularly vulnerable to the depletion of herbivores, for example by overfishing. Management plans should therefore safeguard the herbivorous community, in order to maintain natural bioerosion rates and other reef processes.

This study was undertaken to investigate the effects of different environmental variables on the association between the temperate anemone, Anemonia viridis and its symbionts. The effects of exposure to ammonium enrichment, changes in light intensity, feeding and starvation were studied. Many studies involving tropical associations have addressed this question by monitoring changes in the symbiont population density. However, the symbiont population density can change as a result of changes in the zooxanthella population or changes in the host biomass or host surface area. In addition, the zooxanthella population is determined by the rate of division and the rate at which cells are lost from the population. Anemones maintained at light intensities of 20 and 300E.m-2s-1 had similar population densities measured as cells.g host protein-1 in tentacles, however the zooxanthella division rate, measured as the mitotic index, increased from 2 to 4% with increasing light intensity within the range 20 to 300E.m-2s-1 after 3 weeks exposure. The specific expulsion rate also increased with light intensity over the range 50 to 300E.m-2s-1 from 0.0003 to 0.002 cells.cell-1.d-1. Although the mitotic index of zooxanthellae increased with increasing light intensity, the increase was much larger in animals receiving ammonium enrichment for 4 weeks. Under ammonium enrichment, the symbiont mitotic index increased from 2% at 20E.m-2.s-1 to 9% at 300E.m-2.s-1. The rate of ammonia uptake in A.viridis has previously been shown to be determined by the level of illumination and therefore the above observations indicate that the rate of cell division may be limited by low light intensity through limitation of ammonium uptake even under ammonium enrichment.

The methylated sulfur compound dimethylsulfide (DMS) plays a major role in the biogeochemical sulfur cycle and atmospheric chemistry. Bacteria are a main sink for DMS in the global sulfur cycle and can utilise DMS as a sole carbon and energy source. This study investigated the diversity and activity of bacteria capable of DMS degradation and associated with the salt marsh plant Spartina anglica known to be a producer of the DMS precursor dimethylsulfoniopropionate (DMSP). Initially, it was shown that S. anglica is rich in DMSP throughout the entire seasonal cycle in the Stiffkey salt marsh providing a likely hotspot for DMSP- and DMS-degrading bacteria. DMS uptake experiments demonstrated that DMS degradation takes place in the phyllosphere and rhizosphere of S. anglica and denaturing gradient gel electrophoresis (DGGE) and high-throughput amplicon sequencing of the 16S rRNA revealed the dominance of bacteria related to α - and γ- Proteobacteria, as well as Flavobacteria in the phyllosphere of S. anglica, whereas the rhizosphere was mainly colonised by members of the classes γ-, δ-, α-, and ε-Proteobacteria and Bacteroidia. The diversity of DMS-degrading bacteria associated with S. anglica was first assessed by enrichment culture. DGGE analysis and high-throughput sequencing diversity of DMS enriched samples using the 16S rRNA gene as a marker suggested the dominance of Piscirickettsiaceae, Methylophaga and Methylophaga-like bacteria in DMS-enrichments of phyllosphere and rhizosphere samples of S. anglica. A functional gene marker analyses was carried out using the gene encoding methanethiol oxidase (mtoX), a key enzyme in DMS degradation and the gene encoding a DMSP lyase (dddP) to determine the diversity of bacteria degrading DMS and DMSP, respectively, in the phyllosphere and rhizosphere of S. anglica. The analysis for mtoX showed a great diversity of this gene in phyllosphere and rhizosphere of S. anglica and that major clades of mtoX clustered together with Sedimenticola, Methylohalobius, Methylophaga and other mtoX clones previously detected in surface sediments of the same salt marsh. The results for the functional marker gene analysis for the dddP gene suggested the dominance of Ruegeria-like species and Roseobacter-like bacteria but also of unidentified Ddd+ bacteria in the phyllosphere and rhizosphere of S. anglica. In order to identify the active DMS degraders in the phyllosphere and rhizosphere of S. anglica stable-isotope probing (SIP) combined with DGGE and highthroughput sequencing of the 16S rRNA gene was carried out. The SIP experiments revealed the dominance of Piscirickettsiaceae, Methylophaga and Methylophaga-like microorganisms in the rhizosphere of S. anglica. However, the DMS-degrading microbial community in the phyllosphere seemed more diverse than in the rhizosphere and microorganisms like Halothiobacillus, Xanthomonadaceae, Rhodanobacter but also Piscirickettsiaceae seemed to be involved. A comparative proteomic and transcriptomic experiment of Methylophaga thiooxydans, a microorganism found in phyllosphere and rhizosphere of S. anglica, revealed the general pathways involved in methanol but especially DMS degradation. During DMS cycling the protein and the protein-encoding gene for the methanethiol oxidase (MtoX/mtoX) was highly expressed. A metaproteogenomic experiment provided an insight into the taxonomy and functional diversity of the microbial community associated with the Spartina anglica phyllosphere. Analysis of the metagenome provided evidence that the microbial community associated with S. anglica is dominated by γ-Proteobacteria such as Halomonadales, Alteromonadales, Oceanospirillales, and Thiotrichales and the alphaproteobacterial order Rhodobacterales and showed therefore a major difference to the bacterial community composition in the phyllosphere of for instance A. thaliana, clover, soybean and rice. The detection of DMSP lyase encoding genes and genes encoding proteins for DMS degradation confirmed the genetic potential for the observed DMSP and DMS degradation activity previously measured in the phyllosphere of S. anglica. The metaproteomic experiment allowed a first insight into the proteins expressed in the phyllosphere of S. anglica which also suggested that mainly γ-Proteobacteria and α-Proteobacteria are dominant populations occurring in this habitat. New insights were gained into the activity and diversity of DMS-degrading microbial communities associated with a salt marsh plant that represents a significant component of salt marsh plant communities world wide. Not only was the taxonomic and functional diversity of DMS-degrading microorganisms associated with S. anglica greater then previously realised, the observation of considerable potential of above-ground plant-associated DMS degradation in the phyllosphere demonstrates a previously unrealised sink in the DMS cycle in coastal ecosystems, which is clearly more complex than previously appreciated.

The Buffer Zone (BZ) concept has been introduced in Nepal as a key component of the national biodiversity conservation strategy to mitigate the impacts of protected areas on local communities, and thereby reduce adverse impacts of local people on protected areas. Unlike traditional Buffer Zone programmes which are mostly limited to creating a protective layer and/or distributing economic benefits to local people, the Buffer Zone management approach in Nepal integrates livelihoods and conservation issues and their linkages in a more holistic and balanced manner. The programme has been successful in establishing a network of community institutions and in mobilising large numbers of local communities in conservation and community development. The research findings clearly indicate that the current Buffer Zone management approach based on park revenue sharing for community development has been successful in developing positive attitudes among local people towards protected areas. There is also evidence of improvement in the condition of forests and biodiversity in the Buffer Zone and a decrease in pressure inside the protected areas for basic forestry resources. The BZ communities also feel empowered by the Buffer Zone management programme. These outputs suggest that if properly designed, the Buffer Zone management programme can achieve both conservation and development objectives ensuring the long-term integrity of the protected areas. At the same time, however, the research has also revealed that the existing incentives and institutional arrangements adopted in the Buffer Zone management programme were necessary but not sufficient to address present and potential challenges in Chitwan National Park. There is a need to use additional instruments to demonstrate Buffer Zone management as a viable conservation governance strategy to expand conservation into the areas beyond park boundaries ensuring greater stability of the Park. Any park management strategy seeking to make tangible impacts on conservation, livelihood and governance should have five elements, namely; incentive, empowerment, education, enforcement and integration (IEEEI); and appropriate policy and institutional frameworks to implement them in an integrated way. If issues such as inclusion, equity, empowerment and integration are properly incorporated into the policies and programmes of the Buffer Zone management, the Buffer Zone management strategy adopted in Chitwan could be promoted as a viable model for the sustainable management of protected areas situated in a human dominated landscape.

Stem cells have potential use in tissue engineering and regenerative medicine, and as they underlie the development and maintenance of tissues throughout life, how they function is also of interest. The extracellular matrix presents a variety of physical and chemical signals to stem cells to regulate their behaviour in vivo. Recapitulation of these signals in vitro could enable the control of explanted stem cells to facilitate their study. Biomaterials that display extracellular-matrix inspired cues are one way to do this. By combining surface chemistry and fibronectin, an extracellular matrix protein with cell binding and growth factor binding domains, the conformation of fibronectin was controlled to create artificial extracellular matrices. Adsorbed on a film of poly(ethyl acrylate), fibronectin adopted a network-like conformation which ostensibly increased the exposure of its functional domains, whereas on poly(methyl acrylate) it had an unconnected organisation with more concealed domains. The growth factors bone morphogenetic protein 2 and vascular endothelial growth factor, known to bind to fibronectin, were adsorbed to the network conformation. Prior studies have reported that these artificial extracellular matrices differentially affected cell behaviour. In this work, the growth and differentiation of human bone marrow stromal cell surface marker-1 positive mesenchymal stem cells was characterised on these substrates. It was shown that all combinations of fibronectin conformation and growth factors supported cell adhesion and growth. A high-content image processing and analysis pipeline was developed to take advantage of automated fluorescence microscopy to show that cytoskeletal, nuclei, and differentiation-associated protein features distinguished cells cultured on the artificial extracellular matrices. Those on the isolated conformation and the network conformation with vascular endothelial growth factor were particularly distinct. Further, metabolomics revealed several metabolic pathways that differed in activity between the fibronectin conformations. To analyse the metabolomics data a Quick Results web application was built, which extended the existing Polyomics integrated Metabolomics Pipeline. The application improves the visualisation and interpretation of untargeted liquid chromatography—mass spectrometry metabolomics data. This work gives insights into how these artificial extracellular matrices can control stem cell behaviour, and developed and demonstrated several tools to improve the understanding of these biomaterials and the use of metabolomics data.

The sand lizard (Lacerta agilis) is a rare, elusive and cryptic reptile species of conservation importance in the UK. Knowledge of its ecology and behaviour has limited the development of a reliable and effective methodology for population monitoring; this threatens to compromise conservation effort. The behaviour of sand lizards varies seasonally, with sex and with environmental conditions, none of which are fully understood. This aim of this thesis is to further our ecological knowledge of the sand lizard, specifically by investigating factors which influence the detection probability of this species and through exploration of population monitoring and estimation methods. The detection rate of both male and female lizards in a captive population was found to be heavily dependent on their sex and reproductive stage. The detection probability of males was higher (39%) before mating than after mating (33%): with pre-mated males this was strongly associated with temperature and time of day; and with post-mated males it was linked to changeable conditions with solar radiation values between 200-700Wm-2. The detection probabilities of females were higher after mating (40%) than before (25%) with additional variations in detection rate during and after egg laying. Pre-mated females were most commonly observed basking in a preferred range of ultra-violet light and post-mated they favoured conditions where the ground surface temperature ranged between 17.5oC and 27.5oC. An intensive capture-mark-recapture study was performed on independent sand lizard populations and pattern matching software was used to assist with the identification of individual sand lizards from their dorsal patterning. The low recapture rate of sand lizards proved problematic and population estimates were generated using Program MARK following amalgamation of the data from each field season: but this nonetheless generated the first estimates of sand lizard populations in the UK, with a maximum mean density of 222Ha-2. The survivorship of males was consistently higher than females (0.67 vs. 0.26) and the detection rates and populations estimates differed between years. A high number of individuals were encountered only once, resulting in wide confidence limits of abundance estimates for this species. The range of microclimates available within a structurally diverse array of heathland vegetation were quantified and found to be more extensive than previously thought. The range of microhabitats within the preferred temperature range for sand lizards increased from Spring to Summer, so the need for a lizard to be in the open, and thus available for detection decreased. The obstacles encountered when attempting to monitor sand lizards populations arise from the unpredictable nature of the species. Future sand lizard surveys should be conducted at times when the detection probability maximised, as described in this thesis. A national monitoring scheme should steer away from absolute abundance estimates of populations and consider the use of occupancy estimation to monitor our remaining populations.

This thesis presents advances made to the scanning ion conductance microscope (SICM), a tool predominantly used to date for topographical imaging of biological samples. This technique is demonstrated to be a powerful tool for non-invasive surface charge mapping as well, through probing of the diffuse double layer formed at charged interfaces. Surface charge mapping with SICM is demonstrated for a range of samples, including biological systems, and it is shown that through the use of a novel feedback technique, also introduced herein, and newly implemented scanning regimes, that the surface charge information can be elucidated unambiguously, together with topography. Through adopting a characterisation protocol presented in this work, which helps provide a fuller understanding of the used nanopipette probe, the SICM response to charged interfaces and also in bulk solution can become quantitative, allowing for surface charge values for cell membranes and other substrates to be determined. This combination of: SICM experiments, complete probe characterisation and FEM simulations serves as a robust platform for investigating biological and other charged interfaces. The surface charge mapping protocols used allow for unseen surface charge heterogeneities, presented on cell membranes, to be identified and are amenable to future studies, performed in combination with other microscopy techniques, that could help correlate charged domains with physiological function. Finally, the nanopipette probe is also used as a reaction centre for driving the crystallisation of calcium carbonate, as an exemplar system. Through partitioning the constituent ions of calcium carbonate, with calcium present in a bath solution, and carbonate ions in a nanopipette, a bias can subsequently be applied to drive the ions together, leading to the formation of a crystalline entity, which blocks the nanopipette. Changes in the nanopipette conductance can then provide information about the growth process or subsequently the dissolution as the applied bias is reversed. FEM simulations can allow for an understanding of the underlying mixing problem and the technique is shown to be powerful for the screening of growth additives.