Microglial cells are resident immune cells of the central nervous system (CNS) that participate in the CNS response to systemic inflammation by producing inflammatory mediators, which subsequently contribute to the behavioural and metabolic adaptations to systemic infections collectively termed sickness behaviour. Ageing leads to changes in microglial phenotype and a maladaptive, exaggerated CNS inflammatory and behavioural response to systemic infection has been described in aged rodents, which could have a negative impact on CNS health. However, most studies examining the effects of ageing on microglia have focused on a single region of the brain (the hippocampus) and have used a single model of infection, the bacterial mimetic lipopolysaccharide (LPS). This raises two important questions – are microglia in different parts of the brain equally effected by ageing, and do different models of systemic infections have different effects on sickness behaviour and on microglia? To address these questions we used immunohistochemistry, quantitative PCR and behavioural assays to investigate the effects of region on age related changes in microglial phenotype along a rostral to caudal axis and the CNS inflammatory and behavioural response elicited by LPS was compared to that elicited during a live infection with Salmonella typhimurium. We detected significant differences in the effects of ageing on microglia of different regions of the CNS, with microglia of white matter areas and the cerebellum demonstrating significantly greater changes in expression of activation markers than those of rostral grey matter areas. Co-ordination and balance was impaired in aged mice at baseline and some sickness behaviours were exaggerated in aged mice in response to LPS injection, whereas Salmonella typhimurium infection induced long-lasting reductions in exploratory activity of equal size in young and aged mice and, in aged mice, co-ordination and balance deficits and prolonged weight loss. A low grade, prolonged inflammatory response was detected in the hippocampus which was accompanied by increased expression of microglial activation markers throughout the young and aged CNS, particularly in the spinal cord, where increased axonal stress and changes in the organisation of the paranodal junction were also observed. These changes in cytokine levels and microglial phenotype were mostly of similar magnitude in young and aged mice, contrasting to the effects of LPS. These results highlight regional differences in the sensitivity of microglia to systemic infection and ageing and show extensive differences between the effects of the bacterial mimetic LPS and a live bacterial infection on microglia and on sickness behaviour in young and aged mice. They also have important implications for the study of ageing microglia regarding the selection of the infection models and in deciding which CNS regions to examine.

An investigation was conducted into the factors influencing a soft-sediment intertidal benthic community dominated by the large tube building polychaete Lanice conchilega (Pallas, 1766). The environmental and biological characteristics of a mid-shore sampling site on a relatively sheltered sandflat were monitored over a two year period. A total of 115 taxa were recorded. Temporal variability in the community is discussed in relation to the sporadic occurrence of macroalgae, harsh winter conditions and physical disturbance to the sedimentary environment. Lanice conchilega had a significantly contagious distribution on the shore. The population was analysed using width measurements of both worms and tubes. Recruiting Lanice conchilega juveniles observed during the first year of the study were initially randomly distributed, but as these individuals developed the overall distribution of the population returned to being contagious. The presence of excess tubes towards the end of the sampling period indicated that individual Lanice conchilega were dying, being removed by predation or emigrating. Further sampling was designed to investigate the macrofauna and meiofauna in areas of sediment containing different densities ofLanice conchilega tubes. A consistently significant positive correlation existed between tube density and macrofaunal abundance, which was attributed to sediment-mediated interactions. At higher tube densities, an increase in the depth of sediment above the underlying clay equated to an increase in the volume of available infaunal space. In addition, Lanice conchilega-derived mucus and its associated microbes potentially enhanced levels of food resources available to deposit feeders. Species living at or near the sediment-water interface would also have been positively influenced by any sediment stabilisation in high tube density areas. Possible causes of a positive correlation observed between tube density and Exogone hebes abundance are discussed. No significant relationship existed between tube density and the abundance of macrofaunal juveniles, indicating that any inhibitory or facilitatory Lanice conchilega-juvenile interactions were either insignificant or acting in opposition to to produce an overall neutral effect. Details of the developmental morphology of several polychaete species are described. Meiofaunal abundance (in particular nematode and harpacticoid abundance) was consistently greater in samples taken from areas of high tube density. Potential mechanisms responsible for this relationship include the provision of habitat heterogeneity and areas of refuge by tubes, and the enhancement of sediment stability and food resources by mucus. Particle accumulation in high tube density areas also potentially influenced meiofaunal settlement and resettlement patterns.

No description available.

Protein G (SpG) is a Type III bacterial Fc receptor that binds selectively and non-antigenically to both the Fc and Fab regions of IgG. Here studies are described aimed at understanding the individual contributions to complex stability made by amino acid residues known to make contacts with either the Fab or Fc. Specific amino-acid residues that contribute to these binding interactions via a network of hydrogen bonds have been identified and studied by a programme of site directed mutagenesis. Unique tryptophan residues (W42 or W14) have been used to facilitate equilibrium and pre-equilibrium fluorescence studies to observe binding to Fc or Fab and to determine the Kd for the various equilibria. The effect of secondary substitutions of residues implicated in bond formation to either the Fc or Fab IgG fragment have been determined in order to generate SpG domains with a range of binding affinities for biotechnological uses. SpG species that only bind to Fc or Fab have also been developed. The mutant W42F has been shown by ITC studies to have lost the ability to bind to Fc, and the double mutant T15A-T16A shows no detectable binding to Fab. ITC and kinetic studies at various temperatures have been employed to characterise the thermodynamic parameters underlying binding of wild type or mutated SpG to Fc or Fab. Some restoration of Fc binding activity has been brought about by the placement of a tryptophan at residue 14 (E14W), which may be a useful protein for affinity chromatographic applications as a Kd in the micromolar range is ideal for affinity ligands where easy elution of the bound target protein is required. A dramatic reduction in the affinity of SpG for Fc has been obtained for the E26A mutation, and the N34 residue has been found to be significant in Fc binding, with a 50-fold decrease in affinity for the N34A mutation. The electrostatic nature of the interactions and their sensitivity to changes in pH has also been studied and results showed that Q31E has increased Kd by approximately 6-fold, demonstrating that a negative charge at this position is detrimental to binding. The lower affinity and the influence from the ionisable group would offer advantages for the elution of the Ig from the column.

The preimplantation mammalian embryo is sensitive to its immediate surroundings: alterations to its in vitro or in vivo environment can affect not only the immediate events of blastocyst formation, but can also give rise to long-term phenotypic consequences during fetal and/or postnatal life. For example, rodent studies have shown that maternal under-nutrition during preimplantation gestation can lead to increased risk of cardiovascular, metabolic and behavioural abnormalities in the adult offspring. Do other types of maternal challenge similarly impact on the developmental programme with long-lasting consequences? Infection and injury are common in everyday life and normally result in altered homeostasis and generation of an inflammatory response. The aim of my thesis was to study the effects of an inflammatory environment during preimplantation development on the phenotype of the blastocyst, fetus and offspring postnatally. In the first part of the study mouse embryos were cultured in vitro in the presence of medium only (control) or increasing concentrations (1-1000 pg/ml) of the inflammatory cytokine, interferon γ (IFN-γ). The second part of the study focused on an in vivo model of maternal systemic inflammation where saline (control), 10, 50 or 150 μg/kg lipopolysaccharide (LPS) was administered intraperitoneally (i.p.) to female mice on gestational day 0.5 (GD 0.5). In vitro culture of mouse embryos with select higher concentrations of IFN-γ resulted in a greater proportion of cavitated embryos (1000 pg/ml) and reduced inner cell mass (ICM) cell number (10 and 1000 pg/ml) without affecting trophectoderm (TE) cell number. In vivo, generation of a maternal systemic inflammatory response to LPS administration was confirmed initially. LPS treatment induced sickness behaviour, weight loss and increased the serum concentration of several cytokines, e.g. interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α). Prenatally, the maternal inflammatory challenge resulted in reduced ICM cell number and reduced ICM:TE cell ratio in GD 3.5 blastocysts, but did not affect the number of embryos generated or GD 17 litter size. Furthermore, the GD 17 conceptus was normal in terms of weight of the extra-embryonic tissues and fetal organs. Postnatally, the systemic maternal inflammatory challenge did not alter litter size, birth weight or growth, but did result in altered behaviour, organ/body weight ratios and immune status of adult offspring. In particular, male offspring from 150 μg/kg LPS treated mothers displayed reduced levels of locomotor and exploratory related activity, increased mass of specific fat-pads and increased body mass index (BMI). Furthermore, male offspring from 150 μg/kg LPS treated mothers displayed altered splenic T and B lymphocyte populations with the percentage of B lymphocytes reduced and the percentage of T lymphocytes increased. Both male and female offspring from LPS treated mothers had lower concentrations of 3 a number of serum cytokines and chemokines, either basally or after directly receiving their own LPS challenge. My work using a mouse model has shown that maternal inflammation during preimplantation gestation can permanently change the developmental programme, leading to altered adult phenotype, affecting diverse physiological systems. This study implicates maternal immune status during very early gestation as critical in the health of the next generation.

This study assesses the adaptation of Populus trees to flooding stress. Due to current and predicted changes in the climate, there is an increased need for research into tolerant species to be grown for both flood defence and as crops. Two species of Populus that naturally hybridise were used to assess the adaptation to flooding stress. Due to differences in their native habitat, Populus alba and Populus tremula, are predicted to be relatively flood tolerant and flood sensitive species respectively. Here, they were used to highlight key physiological mechanisms related to flood tolerance and to elucidate genes underlying these processes. This will aid the development of flood tolerant crops and improve understanding of adaptation to flooding in sensitive and tolerant tree species. This research employed physiological and morphological monitoring techniques, along with global gene expression microarrays to identify the key responses related to flooding stress acclimation and adaptation. Many of the traits measured including stomatal conductance, transpiration, leaf size, leaf number, height and chlorophyll content index (CCI) revealed family differences in flooding response though these were not consistent within species therefore failing to reveal why the species differ in natural environments. Furthermore, these responses varied between flooding events suggesting epigenetic changes were in play. Adventitious roots (AR), able to grow and function in oxygen-deficient flooded soil, probably by virtue of their aerenchyma, were produced in response to both summer and winter flooding. During winter flooding, only two P. tremula individuals produced them whereas in P. alba they were produced by 16 individuals. The final focus was on the transcriptomics of root flooding. A set of gene expression changes that occur in roots in response to flooding were identified. This was in addition to a set of gene expression differences between flooded root types. The flooding response revealed 3,687 genes were differentially expressed, of which 1,961 were up-regulated in flooded roots. Similarly the comparison of flooded root types revealed 7,589 genes were differentially expressed, of which 3,866 genes were up-regulated in adventitious roots (AR). The results of this project may be due to epigenetic regulation and therefore future analysis of the DNA methylation may elucidate the effects of repeated flooding. I can conclude that the flooding stress response does differ within the population but there are no significant differences between the two Populus species. Therefore the two species are similarly flood tolerant but where present the flooding stress responses expressed were adventitious root formation (ARF), stomatal closure, and shoot and leaf growth reduction.

Microphytobenthos are important primary producers on tidal flats - ecosystems which are characterised by steep physical and chemical gradients. In this study some of the environmental factors that control the distribution and primary production of benthic microalgae were investigated in a series of field surveys conducted on Hythe intertidal mudflat, Southampton Water. The ecological and photo-physiological responses of microphytobenthos to changes in physical factors were studied in the field and in controlled laboratory experiments. Results from a weekly sample program at four stations along an intertidal transect were used to address the hypothesis that changes in incident irradiance defined by a combination of meteorological factors and the tidal cycle cause significant changes in the biomass of microphytobenthos. The light regime at each station was characterised by a model which combined the changes in daylight irradiance with the timing and duration of low tide. At the low shore stations total light availability during low tide was limiting in spring and autumn/winter, when daily photo-periods ranged between 0 and 6 hours. Microalgal biomass exhibited a strong seasonality with a distinct spring bloom in April which coincided with high irradiances at spring low tide. At the high shore station which was exposed to three times longer photo-periods light availability did not correlate with microalgal biomass at any time of the year. Biomass was three times higher at this station than at the three low shore stations and showed little seasonal variation. Taxonomic marker pigments were used to describe the changes in microphytobenthos composition. High correlation between chlorophyll a and fucoxanthin on a temporal and spatial (horizontal and vertical) scale combined with taxonomic analysis gave evidence that diatoms were the main primary producers on Hythe intertidal mudflat. Chlorophyll a and Ik (defined as PBmax/ aB) correlated strongly with incident irradiance at low tide, indicating that photoadaptation of benthic microalgae was occurring. A spatial comparison of photosynthetic parameters showed that benthic microalgae inhabiting the low shore compensated for reduced photoperiods by increasing their photosynthetic capacity (PBmax) and efficiency (aB) and decreasing their Ik. Photosynthetic parameters of microphytobenthos significantly changed during the low tide period under controlled laboratory conditions, suggesting that an endogenous photosynthetic rhythm was present, which caused an increase in PBmax and ocB towards the middle of the photoperiod. Irradiance levels at low tide enhanced the short-term variation in photosynthetic parameters due to photo-adaptation. The change in rates of photosynthesis was accompanied by a significant alteration of the cellular pigment composition, as the photoprotectant xanthophyll diatoxanthin increased and diadinoxanthin decreased in proportion. Short-term changes in cellular pigment composition induced in the laboratory were greater than seasonal pigment adaptation of microphytobenthos isolated from the field. Variation of PBmaxand ocB during one photoperiod occurred on a similar scale to the seasonal and spatial changes, suggesting that short-term photo-physiological adaptations are an important feature of microphytobenthos, aiding their successful survival in the intertidal environment, where strong gradients of incident irradiance occur on small temporal and spatial scales.

The feeding regime of the franciscana dolphin was investigated from stomach contents of incidentally caught animals in gillnets along the southern Brazilian coast, from 1994 to 2000. The analyses were based mainly on Linear and Generalised Linear Models (LM and GLM). There were some diet differences within the population, as juveniles had eaten more small teleosts and shrimps, and adults consumed larger fish prey and more cephalopods. Adult females had similar diet to juveniles, possibly related to parental guidance. Feeding regimes from northern and southern areas also revealed significant differences, where the most important fish prey from the northern area only occurred once in the diet of the southern animals. More detailed analysis dividing the region into 7 smaller latitudinal zones confirmed many differences, with higher occurrence of warm water species in the diet of northern zones, and cold water species in the southern zones. Additionally, the prey frequency and size do not change gradually along the coast, and different zones revealed a diverse diet composition between northern and southern. This raised the question whether variations in environmental conditions along the coast may affect the distribution of franciscana prey, and consequently its feeding regime. To investigate the spatial and temporal patterns of environmental conditions off the study area, Sea Surface Temperature (SST) images from Advanced Very High Resolution Radiometer , and chlorophyll-a (chl a) images from Sea-viewing Wide Field-of-view were extracted. The analysis (MANOVA, PCA, and time series) revealed a marked temporal influence of warm temperatures from the Brazil Current (BC) in the northern zones, and cold temperatures related to Malvinas/Falkland Current (MC) in the southern zones. The central zones showed a dynamic-edge region in terms of temperature behaviour probably caused by the mixing of BC and MC. Northern zones showed low values of chl a concentration and southern zones high values, whereas central zones revealed intermediate values and less clear seasonal variation. The characteristics of the franciscana habitat had shown potential factors affecting the distribution and abundance of its prey, thus the prey species and size were treated as a function of the variables SST, chl a, latitude, depth, and season, using GLM and LM. Models suggested that habitat parameters influence the franciscana prey species and size. As expected, warm water species are more likely to occur in the diet for longer periods at lower latitudes, and cold water prey at higher latitudes. The SST effect on prey specimens size shows that bigger cephalopods and fish were most frequent in warmer temperatures. The chl a only influenced Engraulidae species. The latitude was the most important spatial predictor for many species, and bigger cephalopods were consumed in large quantities in central latitudes. The depth had great influence in the abundance of some prey species, with smaller fish and cephalopods being in higher numbers in shallower. The influence of the predictors on the franciscana prey is very similar to that found in the habitat by research cruises. Because some structures analysed may remain longer in the stomachs, the findings not only suggest a fairly opportunistic behaviour but it is likely that franciscana may occupy, possibly for long periods, small spatial ranges. This behaviour might be a strategy of minimizing energetic costs, ensuring low maintenance and thermoregulatory costs, and these include limiting excursions to distant areas.

Transmissible spongiform encephalopathies (TSEs) are a group of fatal infectious neurodegenerative diseases affecting both humans and agricultural animals. TSE transmission via blood transfusion has been demonstrated experimentally in rodent, primate and sheep models. Additionally, in humans, four variant Creutzfeld-Jakob disease (vCJD) cases have been reported which probably resulted from infected blood transfusions. Although TSEs can be transmitted via blood transfusion, little is known about which blood cells are involved in the replication of the TSE agent and how infectivity is spread throughout the body prior to neuroinvasion. There are no currently validated diagnostic tests for TSE infection in blood. Detection of PK-resistant PrPSc has been extensively used as a biochemical marker for TSE infectivity. However, when this project was started it was not known if PKresistant PrPSc was present in TSE-infected sheep blood in sufficient quantities to explain the infectivity levels shown by bioassay. Following the development of an optimised Western blot method, this project has demonstrated that the pattern of protein detected with novel anti-PrP monoclonal antibodies is very different from the conventional triple banded pattern of PK-resistant PrPSc. High molecular weight bands were apparent in phosphotungstic acid (NaPTA) concentrated scrapie-infected and uninfected blood and may represent a novel form of blood-specific PrP. PK-resistant PrPSc is not therefore a suitable marker for TSE infection in blood. Other proteins in TSE infected blood were also investigated. Using a proteomics approach three protein markers, lactate dehydrogenase, elongation factor 1 and annexin 1 had altered expression patterns in scrapie infected blood. These proteins, in addition to the novel forms of PrP found in blood, may provide new information on the mechanisms of pathogenesis in scrapie-infected sheep and might prove to be useful molecular indicators of diagnostic value.

Patellid limpets are harvested around the world and many stocks are currently overexploited. This study investigated the influence of environment and harvesting upon the biology of target species (Patella spp.) and upon the macro-community structure of non-target species. A broad-scale survey of Patella spp. across the British Isles and Portugal revealed that non-exploited Patella vulgata populations from England had traits indirectly associated with protandry. Females predominated in larger size classes; cumulative frequency distributions of males and females were different; sex ratios were biased towards the first sex and smallest sizes of males were smaller than the smallest sizes of females. In Portugal, P. vulgata populations did not show these patterns, suggesting that protandry was not occurring in those P. vulgata populations, although alternative explanations include the influence of low recruitment leading to fewer small males. In the England Patella depressa appeared to be gonochorist with a sex ratio of 1:1. In Portugal, however, P. depressa also showed some patterns indicating the possibility of slight protandry. In a manipulative experiment to simulate size-selective harvesting of limpets by humans, changes in sex ratio of P. vulgata in the south-west of England were monitored over an 18-month period of repeated removal of bigger limpets. There was strong evidence that the size at sex change decreased in response to the exploitation treatment, emphasizing its plasticity. Sex change occurred at a bigger size than expected from the overlap in male and female size classes. The limpet exploitation led to establishment of Fucus spp. At the end of the experiment, control plots had lower percentage cover of Fucus spp. than both low- and high-exploitation plots. Univariate and multivariate analyses confirmed that the communities on the two shores responded differently to the same source of disturbance. A comparative survey of populations across a gradient of exploitation in the Canary Islands indicated that the abundance of Patella aspera decreased from 1992 to 1999, while for Patella candei crenata no differences were detected, suggesting that P. aspera was under a higher harvest pressure during that period. The observed differences in catches between islands in 1999 indicated that overexploitation of limpet stocks at that time was not yet evident. The results of the field surveys and the manipulative experiment are discussed with respect to the role of ecological experimentation and aquaculture in resource management, and suggestions are made for key issues in future research and conservation.