• Colonisation and development of salt mark in the Dee estuary, NW England: Integrating large-scale pattern and small-scale ecological process

      Marrs, Robert H.; Potter, Jacqueline; Huckle, Jonathan M. (University of Liverpool (Chester College of Higher Education), 2000-07)
      The Dee estuary, one of the most important British estuaries in terms of size and conservation value, has been subject to extensive colonisation and development of intertidal mudflats by salt marsh vegetation. In the last century, acceleration of this process has been attributed to the ability of Spartina anglica C.E. Hubbard to colonise bare sediment. The research in this thesis aims to investigate the ecological patterns and processes involved in the development of salt marsh vegetation. These have been examined using a large-scale approach involving remote sensing techniques and a small-scale approach to examine ecological processes at the level of the individual plant and species. Large-scale temporal patterns in the distribution were investigated by analysing a sequence of monochrome aerial photographs dating from 1955 to 1997. At the marsh apex, initial rapid colonisation was followed by a decreased rate of expansion and a reduction in the pioneer zone. This suggested a steepening of the marsh elevation gradient, which is interpreted as the marsh approaching its natural limit of expansion. The rate of salt marsh expansion was consistent across the time sequence for the second target area, a cross-section of the marsh gradient, but with S. a«g/zca-dominated colonisation of mudflats changing to colonisation by a pioneer community co-dominated by S. anglica and Salicornia europaea. Large-scale spatial distribution patterns were further investigated using multispectral remote sensing data from 1997. Radiometric data were used to define the spectral characteristics of the major types of salt marsh vegetation. Airborne Thematic Mapper data were used to classify the reflectance data from the whole marsh to determine the spatial distribution of plant communities based on their spectral characteristics. Mapping of these communities provided a baseline that will be a useful tool for future management of the salt marsh. An experimental approach was used to examine the role of abiotic and biotic factors on the growth and interactions between S. anglica and Puccinellia maritima (Huds.) Parl. In two series of competition experiments, P. maritima exerted a one¬way effect over S. anglica. The intensity of this interaction was increased in environmental conditions favourable to P. maritima, and was greater in terms of above-ground than below-ground biomass. In both experiments, S. anglica exhibited a disproportionate reduction in below-ground competitive interaction in abiotic conditions less favourable to P. maritima. A corresponding increase in rhizomes suggested that this is a potential mechanism by which S. anglica may evade competitive neighbours at low marsh elevations. An appreciation of the importance of scale has led to a multi-scaled and holistic view of the ecological process of salt marsh colonisation and development. Integration of both large and small-scale approaches has provided valuable information on the ecological patterns and processes, and has important implications for current and future management of salt marsh in the Dee estuary.
    • Heat shock proteins: Interactions with bone and immune cells

      Williams, John H. H.; Davies, Emma L. (University of Liverpool (Chester College of Higher Education), 2004-09)
      Heat shock proteins (Hsps) are increasingly being seen as having roles other than those of intracellular molecular chaperones, particularly with regard to their potential to act as cytokines, and to stimulate the innate immune system. Hsps have also been found to promote bone resorption and osteoclast formation in vitro, although the mechanism has not been previously identified. The overall aims of this thesis were to determine whether Hsps could stimulate bone resorption by affecting the RANKL/OPG pathway, and to address the hypothesis that Hsps can act as a danger signal to the innate immune system. In order for Hsps to affect either the RANKL/OPG system of bone resorption or act as danger signals they would need to be actively released from cells, ideally in a controlled manner following exposure to the source of stress. Hsp60 and Hsp70 were found to be released from a range of immune cells including the cell lines Jurkat and U937, and also PBMCs, T-cells and B-cells. This release was not due to cell damage. The release of Hsp60 and Hsp70 were downregulated by inhibitors of protein secretion, in particular Hsp70 release was reduced by compounds that inhibited lysosomal pathways and Hsp60 release by classical secretion inhibitors. Hsp60, Hsp70, GroEL and LPS all affected the RANKL/OPG system of bone regulation; OPG production and release was down-regulated in the MG63 and GCT osteoblast-like cell lines following treatment with Hsp60, Hsp70 and LPS, and RANKL expression was upregulated following treatment with Hsp60, Hsp70, GroEL and LPS. This effect on the RANKL/OPG system was found to translate into an effect on osteoclast formation when conditioned media from treated osteoblasts was added to osteoclast precursors in the presence of M-CSF. A range of different factors that affected Hsp release were identified; PHA activation of PBMCs was found to upregulate Hsp60 release from PBMCs. GroEL and LPS caused an upregulation in Hsp70 release from PBMCs and GCT osteoblast like cells, and Hsp70 was found to stimulate Hsp60 release from PBMCs and GCT cells. These responses of Hsp release were used to form a theory of a cascade-like danger signal that may occur when cells are exposed to bacterial infection and which would result in activation of antigen presenting cells via previously identified receptors for Hsps such as CD14/TLR4 or by unidentified pathways. The elevated release of Hsps in response to GroEL and LPS was also identified as a mechanism that could stimulate bone loss during infection or autoimmuniry by affecting the RANKL/OPG system. hi conclusion, Hsp60 and Hsp70 can be released from immune cells under normal conditions, and from both immune and osteoblast-like cells following stimulation with LPS and other Hsps. The observed release responses provide a mechanism through which Hsps can act as danger signals to the innate immune system, and also as promoters of bone resorption via the RANKL/OPG system.
    • Hsp72 translocation and secretion in in vivo and in vitro models

      Williams, John H. H.; Andrew, Sarah M.; Leoni, Francesca (University of Liverpool (Chester College of Higher Education), 2009-03)
      Evidence suggesting that Hsp72 is actively participating in cellular signalling as well interacting with immune system dynamics has been increasing. This is true in healthy, stressed and diseased cells but to different degrees. Modulation of the plasma membrane association and secretion in the extracellular environment by different types of stressors is the key event that leads to different degrees of immune system activation. Hence a better understanding of the mechanisms of Hsp72 secretion and association with plasma membrane is crucial. This thesis investigated the tissue source and mechanism of Hsp72 surface presentation to plasma membrane structures and release in relation with different cellular and physiological stressors. In vivo models confirmed that different tissue types determine specific Hsp72 responses following the same stress and increase serum Hsp72 dependant on intensity and duration of the stress. Diseases models confirm that Hsp72 responses in specific cell populations is related to disease progression, while in vitro models clearly showed that there are multiple mechanisms of secretion and surface presentation, dependent on the nature of the stressor as well as the intensity and duration. This observations clearly change the view of extracellular Hsp72 as a danger signal and lead to a revision of the original danger model. It also suggests that manipulation of Hsp72 translocation through the different pathways involved may prove effective therapeutically.
    • An investigation of the test-retest reliability of an ultrasound densitometer

      Lamb, Kevin L.; Owen, David G. (University of Liverpool (Chester College of Higher Education), 18/10/1998)
      Army recruits undertake a rapidly increasing amount of exercise in their initial basic training period. Injuries due physical training forces many recruits out of the Army and costs the Ministry of Defence millions of pounds. Stress fractures are one of the most commonly diagnosed injuries amongst Army recruits. Low bone mineral density has been identified as a risk factor for stress fractures. A technique which can measure bone mineral density is Ultrasound Densitometry (US). This study will address a gap in the research by assessing the inter-observer and intra-observer reliability of the two US measurements, broadband ultrasound attenuation (BUA) and the velocity of sound (VOS). Ninety eight white male recruits, median aged 18 (I.Q. range 1yr) were measured at the calcanea of the non-dominant foot. A repeated measures design was used, BUA and VOS were measured in 55 subjects by both researcher 'A1 and 'B' for inter-observer (inter-BUA & inter-VOS), and 43 subjects were measured for BUA and VOS twice by researcher 'A' for the intra-observer analysis (intra-BUA & intra-VOS). The results from this study found that a coefficient of variation (CV) analysis was not appropriate for assessing measurement error, this was due to the homoscedasity of the data. An alternative method the '95% limits of agreement' found that only VOS was reliable. The '95% limits of agreement1 results (bias ±1.96 x s) were 0.74 ±22.77 m/s for intra-VOS and 4.85 ±23.44 m/s for inter-VOS, the variance in scores were judged to be acceptable, f-test confirmed this with a non-significant difference between measurements (t=0.83, p=0.477; t=0.42, p=0.677, respectively). The '95% limits of agreement1 results for BUA were -0.22±11.56 dB/MHz (inter-BUA) and -1.39 ±11.11 dB/MHz (intra-BUA). These results represent an unacceptable variability in the range of scores obtained. This is highlighted when expressed as a proportion of the mean measurement: inter-BUA ±11.41% and intra-BUA ± 11.91%. However, the West's for inter- and intra-BUA indicate no significant difference (t = -0.07, p = 0.091; t = 1.60, p = 0.116). This insignificance may be the result of the inappropriateness of a statistical method that reliance on a comparison of means. The CV results for BUA indicate that both inter- and intra-BUA are reliable (4.08% & 4.38%, respectively), even though as already stated that the BUA measurements are not deemed reliable when analysed by the '95% limits of agreement'. The results of this study suggest that VOS measurements are reliable and that BUA measurements are non-reliable. As both BUA and VOS would have been used to assess those at risk of suffering stress fractures it was essential that both were found to be reliable. Thus US's appropriateness in individual diagnosis is questioned. This study has also highlighted how the use of an inappropriate statistical method, in this case the CV, can effect the interpretation of data and cause false claims over e.g. reliability.
    • Physiological and behavioural measures of stress in domestic horses

      Young, Tamsin (University of Liverpool (Chester College of Higher Education), 2011-09)
      The welfare of domestic horses has been scrutinised by the scientific community in recent years. Traditional riding and stable management practices have been recognised to be at odds with the physical and behavioural adaptations of the horse. There is, therefore, a growing need to understand how modern horse management can impact on horse welfare. The first study in this thesis assessed the impact of common management practices on physiological stress in the horse. Faecal cortisol was higher in horses that were stabled and exercised, than turned out to grass with no exercise. The effect of exercise alone was also seen to increase levels of salivary cortisol. No change was seen in cortisol following short-term routine husbandry procedures such as exposure to the sound of electric coat clippers, but it was suggested that this required further investigation. The study confirmed exercise increased stress as reflected by cortisol concentration, and indicated that individual stabling may also contribute to elevated stress. The study recommended that horses may benefit from periods of rest and turn out to grass, to reduce stress levels and improve welfare. The measurement of stress for the purpose of welfare assessment is, however, best carried out using an integration of both physiological and behavioural measures. Behaviour scores offer non-invasive, quick and easy methods of assessing stress in domestic animals, but have typically been developed using only behavioural assessment of the stress response. In the second study a scale of behavioural indicators of stress was developed using behavioural and physiological measures for the purpose of assessing stress in stabled domestic horses. Principal component analysis of behavioural reactions and changes in salivary cortisol concentration in response to routine husbandry procedures, revealed three meaningful components that were used as the basis to the stress scale. Behavioural reactions to the husbandry procedures were further analysed by a panel of equestrian professionals using free choice profiling, and results were added to the appropriate components. The final scale comprised of four levels of stress (no stress, low, iii medium and high stress), and each category was further sub-divided into behaviour scores (BS). The scores represented accumulating levels of behavioural indicators of stress within each stress level, and provided indices of physiological stress. The scale offers an easy to use method of welfare assessment in horses, and reduces the need for additional physiological measures to be taken. The scale represented a novel approach to measuring stress, and was used in the final study to measure stress in horses stabled individually, group housed, and in horses moved from stabling to group housing. The effectiveness of the scale at measuring stress, was compared to the effectiveness of measures of heart rate variability (HRV) and faecal cortisol at measuring stress in the same horses. Lower levels of stress were recorded in group housed horses as measured by the BS, but measures of HRV and faecal cortisol showed no difference between those stabled or group housed. Stress levels were unaffected by the move to group housing, but BS declined significantly over the three weeks that the horses remained group housed. The physiological measures did not, however, reflect such a decrease in stress. Stress levels were also compared between horses housed in both environments whilst waiting to be fed. Group housed horses had lower stress levels as measured by the BS. Results provided by the BS were supported by relevant literature, and the scale appeared to be more sensitive than the physiological measures which did not yield significant results with the small sample sizes used in the study. The research confirmed short-term management practices horses are typically exposed to daily, can elevate their stress levels. Further research into which practices put horse welfare at a particular risk, and thus require modification or need to be avoided where possible, is necessary. The findings also suggest horse-owners may need to pay more attention to their horse’s stress levels, to avoid repeated or on-going stress that can jeopardise health and welfare. The scale of behavioural indicators of stress would provide a suitable method by which stress could be monitored and thus become a part of horse management.
    • Studies in cancellous bone in osteoarthritis

      Williams, John H. H.; Brown, Sharon J. (University of Liverpool (Chester College of Higher Education)Charles Salt Research Centre, Robert Jones & Agnes Hunt Hospital NHS Trust in Oswestry, 2002-06)
      Osteoarthritis (OA) is a common musculoskeletal disability and represents a major health burden to society. Pathological changes are found in all tissues of the joint; however studies on bone are few. The aims of this thesis were to characterize some of the densitometric, mechanical and compositional properties of cancellous bone from the Superior (Sup) and Inferior (Inf) regions of human femoral heads, and to compare age-selected healthy bones with those from patients with end-stage OA. From coronal sections of the femoral head, bone cores were drilled out along the anterior-posterior axis and non-adherent fatty tissues removed. Measurements of apparent (PA), true (pj) and volumetric bone mineral density (BMDv) were made prior to determining the ultrasound-derived elastic modulus (Eu) and the compression-derived parameters, compressive modulus (Ec), yield strength (CTY) and yield energy (Wy). From aliquots of powdered bone, calcium and hydroxyproline were determined and a minerahcollagen ratio calculated, and osteocalcin, IGF-I, IGF-II and IGFBP-5 measured by RIA. Another aliquot was processed to remove soft tissue, demineralized in EDTA and the resulting collagen matrix digested with trypsin. From these crude tryptic digests (CTDs), divalently and trivalently (ICTP, IIINTP) crosslinked collagen telopeptides and procollagen propeptides of collagens type I and III were analysed by RIA. PA, BMDv, EC, ay, Wy and EU were systematically increased in cores from the Sup region of both Normal and OA groups (all/KO.05) and reflects differences in loading experienced by this region. In OA bone, PA, BMDv and EU were increased in the Inf region (all/KO.05) possibly as a result of adaptation of the bone to altered loading patterns at the hip. Furthermore, the relationship between EC and PA was different to those in Normals (p=0.019) indicating that other factors, such as architecture, may influence the stiffness of cancellous bone. Of the compositional measures, IGFBP-5 was systematically increased in both regions of OA bone (all /?<0.005) and may be the factor responsible for maintenance of bone mass in OA. The minerahcollagen ratio was decreased in the OA Sup region (p=0.008) indicating undermineralization of bone. In the mineralized matrix of OA bone, concentrations of ICTP:collagencTD and type III collagen antigens were increased in both Sup and Inf regions (all/><0.05) suggesting an increase in type I collagen trivalent crosslinking at the Qtelopeptide and an overall increase in type III collagen respectively. Of the relationships between the various properties, inverse correlations were found between mineral.'collagen and ICTPrcollagenciD ratios (rs= -0.36,/?<0.05) in the OA group indicating increased trivalent collagen crosslinking when bone is under mineralized. In the Normal group PA correlated inversely with IGFBP-5 (rs=-0.64 and -0.72, bothp<0.05) and OC (rs=-0.59,/7=0.056 and -0.71, p<0.05) at both Sup and Inf regions respectively, but were lost in the OA group suggesting loss of regulation at this level in OA. hi conclusion, the cancellous bone in OA femoral heads is denser, but not stiffer than that of Normal bone, and has an altered composition with respect to both structural (ICTP) and regulatory (IGFBP-5) factors which may affect the quality of the bone matrix.