http://hdl.handle.net/10034/31693 Thu, 23 May 2013 15:16:40 GMT 2013-05-23T15:16:40Z http://hdl.handle.net/10034/277691 Title: Interactions between extracellular Hsp72 and blood cells Authors: Williams, Helen Abstract: In recent years, compelling evidence has accumulated suggesting heat shock proteins (HSPs) which are generally believed to be localised and functioning mainly within eukaryotic cells as cyto-protective molecular chaperones, are also localised in the extracellular milieu. Depending on their localisation, on the cell surface (membrance-bound or embedded), or in the peripheral circulation, extracellular HSPs may induce apoptotic cell death, or in contrast protect cells from cell damage and/or cell death when exposed to cellular stress, or may even elicit a stimulatory effect on the innate immune response including cell activiation and cytokine secretion. Hence, the localisation of intracellular and extracellular HSPs appears to be critical in determining their roles in terms of stimulating cell death, cyto-protection, or immune activiation under normal physiological conditions and following exposure to stress stimuli. This thesis describes the intracellular expression, up-regulation, and cell surface localisation of endogenous HSPs: HSP27, Hsp60, Hsp72 and Hsp90 by flow cytometry, florescence microscopy and Western blotting, under control conditions and in response to environmental stress using in vitro and ex vivo models with the intention of determining their physiological roles. The ability of extracellularly administered HSPs (Hsp70 and Hsp72) to protect cultured U937 cells in vitro or peripheral primary human leukogytes or erythrocytes ex vivo from various stress stimuli was demonstrated and was found to be dependent on surface binding and/or internalisation via scavenger receptors (SRs) or phosphatidylserine (PS), which could be blocked by receptor specific ligands. Extracellular HSPs were also shown to be able to stimulate an immune response through the induction of U937 monocyte differentiation into macrophages as evidenced through the up-regulation of the surface receptors: CD36, SR-A1 and CD91 analysed by flow cytometry. These proteins were able to stimulate TNF-x and IL-10 production and secretion by U937 macrophages, shown by ELISA, and chemotatic properties were demonstrated using Boyden chambers. The cyto-protective and immune regulatory effects of extracellular HSPs have potential therapeutic value as treatments in a wide variety of clinical situations. Description: Not to be added to EThOS Wed, 01 Dec 2010 00:00:00 GMT http://hdl.handle.net/10034/277691 2010-12-01T00:00:00Z http://hdl.handle.net/10034/211249 Title: Maternal investment in mountain gorillas (Gorilla Beringei Beringei) Authors: Eckardt, Winnie Abstract: Investigating maternal investment (Ml) and mother-offspring relationships during the period of infant dependency is critically important to furthering the understanding of female reproductive strategies in primates. Infant primates are completely dependent upon their mothers. The way in which a mother allocates her resources therefore is crucial for infant survival, but is balanced Against her need to invest in subsequent offspring. One approach to examining how mothers might invest in their offspring stems from the Trivers & Willard hypothesis (TWH, 1973), which predicts that mothers in good condition should bias their investment towards sons and whereas mothers in poorer condition should bias investment toward daughters. Long-term demographic records on birth sex ratio and inter-birth interval suggest that female mountain gorillas (Gorilla beringei beringei) do not bias investment prenatally, but they may adjust postnatal Ml according to the TWH. This study investigated Ml and mother-offspring relationships in wild mountain gorillas, using behavioural correlates of Ml, including suckling, weaned age, physical contact, "transport, and grooming to redress the lack of understanding about Ml in this species. The appropriateness of TWH was investigated, integrating different indicators of maternal condition. Important determinants of Ml and mother-offspring relationships were considered, such as offspring age, parity, presence of siblings and maternal relatives, group size and lastly, personality, which has been largely neglected in nonhuman primates. The extent, to which the offspring influenced Ml patterns, was examined using the parent-offspring conflict theory (Trivers, 1972) as a theoretical framework. During 2006-2007, 38 mother-offspring dyads were observed in the Virunga massif, resulting in 1210 hours of direct behavioural observation. Additional field data from the previous four decades were integrated into the dataset for the analysis of suckling and weaned age. Gorilla personality was assessed through the Hominoid Personality Questionnaire. Findings relating to suckling frequency, weaned age, and maternal feeding activities were consistent with the TWH: sons suckled more often than daughters when they had mothers in good condition, whereas the reverse sex-pattern occurred in offspring with mothers in poorer condition. In addition, daughters were weaned at an earlier age than sons when mothers were in better condition, although this sex-difference reduced in older mothers that were categorised as being in good condition. Maternal feeding time and feeding efficiency revealed that mothers in poorer condition spent more time ingesting food when they had daughters, whereas mothers in better .condition spent more time ingesting food when they had sons. Furthermore, group size affected lactation duration with offspring in small groups being weaned earlier than offspring in large groups. Behavioural conflicts over Ml showed that the mother and offspring influenced Ml patterns during the period of dependency. Finally, six personality dimensions were identified, of which five revealed effects maternal behaviour, such as maternal retrieval, responsiveness and rejection, although their relative importance varied between those behaviours. In general, mother and offspring personality effects were complex due to their interactions with the developmental stage of offspring. In conclusion, my thesis research has made several novel contributions to furthering the understanding of female reproductive strategies in the highly endangered mountain gorilla. I presented the first evidence using behavioural data that females bias their postnatal investment towards the sex with the greatest fitness return as predicted by the TWH. My findings are discussed in the light of alternative Ml strategies, such as the local resource competition and enhancement model. My research has highlighted the importance of integrating anthropometric and physiological measures and demographic long-term data into future Ml studies to assess direct costs and benefits of Ml. The examination of mother-offspring behavioural conflicts showed that offspring have a strong impact on the level of Ml they receive. I have also examined the personality of a wild mountain gorilla population for the first time. My findings demonstrate that personality-parenting links are evident in several respects and I have demonstrated the great potential of personality as a determinant of maternal behaviour and mother-offspring relationships. Mon, 01 Feb 2010 00:00:00 GMT http://hdl.handle.net/10034/211249 2010-02-01T00:00:00Z http://hdl.handle.net/10034/200750 Title: The classification and management of limestone pavements - an endangered habitat Authors: Willis, Sue D M Abstract: This thesis describes an in-depth study of limestone pavements across North West England and North Wales. The aim was to combine elements of geodiversity and biodiversity in order to create a holistic limestone pavement classification to inform future management. A field-based research protocol was used to assess a stratified random sample (46 pavements), accounting for approximately 10% of the limestone pavements in the geographical area. Detailed analyses of key elements are presented, along with important issues that continue to pose threats to this Annex One Priority Habitat. This research resulted in a comprehensive classification, using TWINSPAN analysis and Nonmetric Multidimensional Scaling, identifying six distinct holistic functional groups. The prime factors driving limestone pavement morphology, and hence the classification, were established to be lithology, proximity to structural fault, altitude and human intervention, particularly in terms of grazing intensity. Three upland, open limestone pavement classes were formed. Of these, the richest in terms of geodiversity and biodiversity was the group with the thickest bedding planes and hence the deepest grikes, typically greater than 1m. The class that was most species-poor was "at the highest altitude (above 450m), formed on the thin limestones of the Yoredales. These were characterised by shallow, wide grikes. The third upland limestone pavement group had mid-range grikes, generally 0.5-1m in depth, and small clints. Two wooded classes were identified. One was a lowland 'classic' wooded limestone pavement group with deep, narrow grikes and shallow soils. Indicator species included Juniperus communis and Taxus baccata. The second wooded group was situated proximal to a major structural fault. In this group the pavement dip ranged between 10°-40° with well-runnelled clints that were heavily moss-covered. The sixth group was low altitude, proximal to the coast, characterised by low moss growth, un-vegetated clints and the presence of Ulex europaeus. Conservation management was identified as key to the quality of the limestone pavement habitat and this thesis identifies best management practises and links these to the holistic limestone pavement classification. Finally, as a sample case study, this thesis presents mollusc species and diversity from eleven of the Yorkshire limestone pavements. Analysis establishes significant links between geodiversity and mollusc populations, with key drivers for mollusc communities echoing those of plant species on limestone pavement. Sat, 01 Jan 2011 00:00:00 GMT http://hdl.handle.net/10034/200750 2011-01-01T00:00:00Z http://hdl.handle.net/10034/200730 Title: The regulation of osteoprotegerin and dickkopf-1 production in osteoblastic cells Authors: McCarthy, Helen Samantha Abstract: Bone is a highly specialised living tissue and has both mechanical and metabolical functions. Remodelling of the bone ensures a healthy bone mass and is regulated by a trio of secreted proteins, namely receptor-activator of NFKB (RANK), receptor-activator of NFKB ligand (RANKL) and osteoprotegerin (OPG). OPG, a major regulator of osteoclastogenesis, bone resorption and vascular calcification, is produced by various cell types including mesenchymally derived cells, particularly osteoblastic cells. Wnt signalling also plays a role in maintaining healthy bone mass. Dickkopf- 1 (DKK-1) is a soluble inhibitor of Wnt signalling and its excessive expression contributes to bone loss in rheumatoid arthritis and multiple myeloma. Recently, NDKK-1 has been demonstrated to be over-produced in osteoblasts of patients with Paget's disease of bone (PDB). The osteoblastic cell lines MG63 and Saos-2 were subjected to a series of different growth factors, hormones and cytokines to investigate the production of OPG, DKK-1 and the expression of various Wnt proteins. These results demonstrate that during standard culture conditions, both OPG and DKK-1 production in osteoblastic cells depend on a factor present in serum. Serum deprivation resulted in the up-regulation of Wnt4 and Wnt11, while down-regulating the expression of Wnt7b. Serum-induced OPG and DKK-1 production and Wnt expression was found to be regulated via a number of different signalling pathways. OPG production and expression was stimulated by platelet-derived growth factor-AB (PDGF-AB) not only in MG63 and Saos-2 osteosarcoma cells, but also a mouse pre-osteoblastic cell line (MC3T3-E1) and human bone marrow stromal cells (BMSC). PDGF-AB was shown to act through the PDGF receptor, PKC, PI3K, ERK and P38 and not via NFKB or JNK. PDGF isoforms AA, BB and AB demonstrated a similar stimulation of OPG production. The importance of PDGF in fracture healing suggests a role for OPG production in countering bone resorption during the early phase of this process. BIO, an inhibitor of canonical Wnt signalling resulted in the down-regulation of DKK-1 and the up-regulation of WntSa. Phorbol ester (PE), a known stimulator of PKC resulted in the up-regualtion of DKK-1, Wnt4, WntTa and Wnt16. The effects of PE were inhibited by bisindolymaleamide but not staurosporine. DKK-1 production, but not expression, was observed to be stimulated by calcium along with an up-regulation of WntTb and a down-regulation of WntWa and Wnt11. Incubation of pre-stimulated cells with Triton-X demonstrated the ability of calcium to increase DKK-1 secretion. DKK-1 was shown to be significantly elevated in the serum of PDB patients compared to healthy controls and did not correlate with ALP levels. Immunohistochemistry demonstrated that DKK-1 production is increased in both osteoblasts and fibrotic cells within the marrow cavity in PDB patients compared to fracture callus. B-catenin was found to be localised to intercellular membranes of plump osteoblasts, demonstrating its alternate role as a cell adhesion protein. DKK-1 therefore may be a useful biomarker of PDB and that Dkk-1 may play a central role in the aetiology of PDB. In summary, the results presented in this thesis have investigated the ways in which OPG and DKK-1 production in osteoblastic cells can be modulated with various effectors and the effect of Wnt signalling. These results may therefore be beneficial to increase the understanding of bone biology, improve fracture repair and generate further research into the role DKK-1 and the osteoblast in the aetiology of PDB to enable improved treatments to be developed. Wed, 01 Jun 2011 00:00:00 GMT http://hdl.handle.net/10034/200730 2011-06-01T00:00:00Z