by Wing Shun Cheung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 159-178). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Our ability to learn relies on the potential of neuronal networks to change through experience. The primary visual cortex (V1) has become a popular system for studying how experience shapes cortical neuronal networks. Experience-dependent plasticity in V1 has been extensively studied in young animals, revealing that experiences in early postnatal life substantially shape neuronal activity in the developing cortex. In contrast, less is known about how experiences modify the representation of visual stimuli in the adult brain. In addition, adult experience-dependent plasticity remains largely unexplored in neurodevelopmental disorders. To address this issue, we established a two-photon calcium imaging set-up, suitable for chronic imaging of neuronal activity in awake-behaving mice. We implemented protocols for the reliable expression of genetically encoded calcium indicators (GCaMP6), for the implantation of a chronic cranial window and for the analysis of chronic calcium imaging data. This approach enables us to monitor the activity of hundreds of neurons across days, and up to 4-5 weeks. We used this technique to determine whether the daily exposure to high-contrast gratings would induce experience-dependent changes in V1 neuronal activity. We monitored the activity of putative excitatory neurons and of three non-overlapping populations of inhibitory interneurons in layer 2/3 of adult mice freely running on a cylindrical treadmill. We compared the results obtained from mice that were exposed daily to either a high-contrast grating or to a grey screen and characterized their neuronal response properties. Our results did not reveal significant differences in neuronal properties between these two groups, suggesting a lack of stimulus-specific plasticity in our experimental conditions. However, we did observe and characterize, in both groups, a wide range of activity changes in individual cells over time. We finally applied the same method to investigate impairments in experience-dependent plasticity in a mouse model of intellectual disability (ID), caused by synaptic GTPase-activating protein (SynGAP) haploinsufficiency. SynGAP haploinsufficiency is a common de novo genetic cause of non-syndromic ID and is considered a Type1 risk for autism spectrum disorders. While the impact of Syngap gene mutations has been thoroughly studied at the molecular and cellular levels, neuronal network deficits in vivo remain largely unexplored. In this study, we compared in vivo neuronal activity before and after monocular deprivation in adult mutant mice and littermate controls. These results revealed differences in baseline network activity between both experimental groups. These impairments in cortical neuronal network activity may underlie sensory and cognitive deficits in patients with Syngap gene mutations.

Platelet-derived microparticles, released as a result of platelet activation, promote coagulation through the surface exposure of phosphatidylserine, acting as the catalytic site for the conversion of prothrombin to thrombin by the activated coagulation factors X and V. Although elevated numbers of circulating platelet-derived microparticles can be detected in a number of clinical disorders, the methods for the detection of these microparticles are far from standardised. In addition, recent reports have also speculated that not all microparticles may expose phosphatidylserine, demonstrating that the binding of Annexin V, a phosphatidylserine-specific binding protein, is not detectable on a population of microparticles. The initial stage of this thesis was to establish a flow cytometric method for the detection and enumeration of microparticles based on their capacity to bind Annexin V and to utilise this assay to investigate a number of the issues that have limited assay standardisation. The assay could be performed on either stimulated or unstimulated plasma or whole blood samples. Interestingly, plasma microparticle counts were significantly higher than whole blood microparticle counts. The effects of centrifugation alone could not be attributed as the sole source of this discrepancy. The antigenic characteristics of platelet-derived microparticles were also investigated, with platelet-derived microparticles demonstrated to express the platelet glycoproteins CD31, CD41a, CD42a and CD61. Platelet-derived microparticles also expressed CD42b, and this expression was significantly decreased when compared to their progenitor platelets. The expression of the platelet activation markers CD62p, CD63, CD40L and PAC-1 was dependent upon the sample milieu, suggesting that the centrifugation conditions required to generate platelet-poor plasma may lead to artefactual increases in the expression of platelet activation markers. An investigation of the role of the GpIIb/IIIa complex on the formation of platelet-derived microparticles was also performed. A monoclonal antibody to the GpIIb/IIIa complex (Abciximab) significantly inhibited in vitro collagen-stimulated platelet-derived microparticle formation. Interestingly, platelets obtained from two subjects with impaired GpIIb/IIIa activation, demonstrated normal microparticle formation following collagen stimulation, suggesting that the presence of GpIIb/IIIa complex, but not its activation, is required for collagen-induced microparticle formation. A novel mechanism for microparticle formation was also investigated, with platelet-derived microparticles demonstrated to form in response to the sclerosing agents sodium-tetradecyl sulphate and polidocanol. Interestingly, the removal of plasma proteins by the washing of platelets left platelets more susceptible to sclerosant-induced microparticle formation, suggesting that plasma proteins may protect platelets from microparticle formation. The procoagulant activity of platelet-derived microparticles was also investigated using a novel coagulation assay (XACT) specific for the procoagulant phospholipid. An evaluation of this assay demonstrated a significant correlation between Annexin V binding microparticle counts and procoagulant activity in both whole blood and plasma samples. There was more procoagulant activity in whole blood samples than in plasma samples, suggesting that the procoagulant phospholipid activity was also associated with erythrocytes or leukocytes. To further investigate this phenomenon, a whole blood flow cytometric assay was developed to assess Annexin V binding to erythrocytes, leukocytes, platelets and microparticles. This assay demonstrated that a large proportion of Annexin V binding (51.0%) was associated with erythrocytes. Interestingly, a proportion of the Annexin V binding erythrocytes (24.5%) and leukocytes (78.8%) were also associated with platelet CD61 antigen, suggesting that they also bound a platelet or platelet-derived microparticle. The effect of sample anticoagulant on microparticle procoagulant activity was investigated. Microparticle counts were most stable in EDTA anticoagulated samples, but were stable in sodium citrate for up to 15 minutes following sample collection. The procoagulant activity of microparticles was significantly inhibited by EDTA in collagen-stimulated platelet-rich plasma samples, when compared to sodium citrate anticoagulated samples. Although the initial method used to investigate microparticles was based upon their ability to bind Annexin V, it was consistently observed that a large proportion of events in the size region of a microparticle were Annexin V negative. An investigation was therefore commenced into the procoagulant activity of microparticles based on their capacity to bind Annexin V. The presence of Annexin V negative microparticles was confirmed by flow cytometry and the proportion of microparticles that bound Annexin V was dependent upon type of agonist used to stimulate microparticle formation. Varying the assay constituents (calcium concentration / Annexin V concentration / buffer type) did not alter the proportion of Annexin V binding microparticles. When compared to Annexin V positive microparticles, Annexin V negative microparticles expressed significantly higher levels of CD42b on their surface, but possessed significantly decreased expressions of CD62p, and CD63. A significant correlation between the percentage of Annexin V binding and XACT procoagulant activity was found (p=0.03). Furthermore, Annexin V binding inhibited greater than 98% of procoagulant phospholipid activity, suggesting that Annexin V binding was a true reflection of procoagulant activity. Microparticles could be sorted using either a flow cytometric or magnetic sorting strategy. By electron microscopy, Annexin V negative events isolated following magnetic sorting were vesicular structures and not small platelets or the remnants of activated platelets. In summary, this thesis has demonstrated the ability of the flow cytometer and XACT assays to detect microparticles and their procoagulant activity. It has also shown that the use of Annexin V to detect microparticles may warrant further investigation.

Platelets are small anucleate cell fragments found in blood whose physiological role is important in maintaining haemostasis. In vivo, platelet surface glycoproteins mediate the mechanistic roles of platelets, and polymorphic changes to these glycoproteins have been observed to have significant effects on the platelet cellular function and such changes may include over-expression, under-expression and antigenicity of the protein. Human platelet antigens (HPA) are a result of polymorphic differences in platelet surface glycoproteins which have been found to be variably expressed in the population. Foetal maternal alloimmune thrombocytopaenia (FMAIT) is a condition that is observed in the unborn foetus and neonates due to HPA incompatibility between the mother and the foetus. HPA incompatibility accounts for a majority of severe thrombocytopaenic cases in neonates, and delayed diagnosis and treatment of such a condition often lead to intracranial haemorrhage. The risk in neonates diagnosed with FMAIT becomes increasingly significant in cases where intra-uterine (during pregnancy) platelet transfusion is the only effective therapeutic option. There are currently no antenatal screening programs for this condition, and laboratory diagnosis of FMAIT relies on the detection of maternal alloantibodies and parental HPA typing. For these reasons a significant amount of research is currently being invested into the isolation of recombinant antibodies with specific reactivity against FMAIT-related platelet antigens. Stable and specific recombinant platelet antibodies have great potential as a diagnostic agent in antenatal screening and broad-scale HPA typing of blood donors for platelet transfusion. Further characterisation of the isolated antibody may lead to a possible therapeutic agent. Studies by previous researchers have shown that the traditional methods (ie. Mouse monoclonal and EBV transformation) of obtaining monoclonal antibodies against FMAIT-related antigens have proven unsuccessful. The continuing progress in the discipline of phage display has produced several novel antibodies against self and non-self antigens. A further advantage in the application of phage display technology for the isolation of novel antibodies is the easy transition from bacterial to mammalian expression for the characterisation of glycosylated antibodies. The main focus of this project was to create and isolate a recombinant human anti-HPA-3a antibody using phage display for its possible application as a therapeutic or diagnostic agent.

It is increasingly being recognised that global natural resource consumption levels exceed planetary limits and that the present trajectory of industrial development is not sustainable. To achieve a more viable existence necessitates a fundamental shift in priorities from the prevailing economic growth-centred, consumer driven philosophy to one that marries aspirations for economic growth with long-term environmental and social considerations. This shift in priorities requires significant contributions and action at the global, national and local levels by the primary 'wheels' of sustainability: government, corporations and civil society. Over the past 100 years, corporations have become the most powerful institution on the planet with both the financial resources and institutional capacity to take the lead role in shaping a sustainable future for humankind. Yet, within and between industry sectors and across geographic locations there has been great diversity in the extent and level of corporate commitment and engagement in societal efforts relating to planetary sustainability. Hence, greater understanding of what drives corporate interest and involvement in ecological sustainability will become increasingly critical to promoting corporate engagement in processes and practices to secure a long-term future for humanity. However, there has been limited explanatory research oriented upon developing an understanding of the processes and factors associated with corporate 'eco-change'. In recognition of this shortcoming in the literature, the present study utilised the case of the non-renewable resource extractive sector of Australia to examine corporate engagement with processes and practices for planetary sustainability. Specifically, it sought to construct and evidence an explanation of the external and internal factors that have promoted and/or retarded corporate engagement with planetary sustainability in the non-renewable resource extractive sector (NRRES) of Australia. Guided by grounded theory methodology, an instrumental case study of the NRRES in Australia was undertaken. The NRRES was chosen as this sector's profile, visibility and activities over the past twenty years have meant it has come under mounting pressure to incorporate the concept and principles of planetary sustainability into its ethos and operations. As such, the sector represents the opportunity to study this phenomenon within a dynamic context of sectoral and corporate responses to evolving societal expectations. The research was undertaken in three phases and the principal research method was in-depth key informant interviews with purposively sampled members of the sector's stakeholder groups. Each NRRES corporation is situated at the centre of a web of interconnected interests or 'stakes' necessitating efforts to balance the various stakeholder interests to maintain the institution's license-to-operate and secure a long-term existence. The thesis constructs an explanation of the societal drivers of NRRES corporate engagement with planetary sustainability, organised as the three categories of government, civil society and the corporate sector. These three groupings of stakeholders have been clustered into the broad category or theme of Activating Engagement, which recognises their collective role as the stimuli for NRRES corporation engagement in processes and practices for planetary sustainability. While the theme of Activating Engagement emphasises the importance and interrelatedness of the roles and actions within and between the three primary wheels of sustainability, of particular note is the evident rise of civil society as a more active societal stakeholder and more salient driver of corporate uptake of social and environmental issues. As the identified external drivers play a critical role in motivating NRRES corporation engagement, it is a corporation's particular characteristics that ultimately determine the extent and level of uptake of strategies to demonstrate corporate social responsibility. The thesis develops an explanation of the internal factors mediating NRRES corporate engagement comprising the factors of leadership, resources, structures, culture and understanding. These factors are conceptualised as the theme of Capacity for Engagement, which identifies their collective importance in a NRRES corporation's preparedness, impetus and capability relating to interest and participation in planetary sustainability. While all of the five factors are seen as essential to meaningful NRRES corporate engagement, the thesis identifies leadership as the most critical factor in Capacity for Engagement. Based on the findings of the research, several explanatory frameworks are developed. These frameworks aid in deepening our understanding of the NRRES corporate engagement process, in particular, the interconnections between the factors impeding and facilitating corporate interest and engagement with processes and practices for planetary sustainability. As such, these frameworks will make a substantial contribution to building our understanding of how the various factors and their components or 'pieces of the puzzle' interact and interrelate with each other to generate corporate engagement. The frameworks are the culmination of the research and, coupled with the more detailed explanations of their constituent factors, enhance our knowledge and understanding of the dynamics of NRRES corporation engagement with planetary sustainability. This enhanced understanding is significant and could be of considerable value in informing and targeting efforts to advance the depth and breadth of NRRES corporation engagement with processes and practices for planetary sustainability. To advance the standing of the study's findings, a series of case studies could be undertaken targeting the investigation of NRRES corporate engagement in other geographic locations and within different industry sectors.

We have recently established that aberrant alkylacylglycerophosphocholine metabolism results in the increased tissue concentration of platelet activating factors (PAFs) in the temporal cortex of Alzheimer Disease (AD) patients and in TgCRND8 mice over-expressing mutant human amyloid precursor protein. PAF lipids activate a G-protein coupled receptor (PAFR) reported to be expressed by microglia and subsets of neurons in rat. It is not known whether this same expression pattern is recapitulated in mice however, as the expression has only been inferred by use of pharmacological PAFR antagonists, many of which impact on both PAFR-dependent and PAFR-independent signalling pathways. PAFR plays a role in long term potentiation (LTP) induction in rats. PAFR has also been implicated in behavioural indices of spatial learning and memory in rats. Contradictory reports using mice provide ambiguity regarding the role of PAFR in LTP induction in mice. To assess whether PAFR is expressed in murine neurons, I localized PAFR mRNA in wild-type C57BL/6 mice using PAFR KO mice as a negative control. I further showed that the loss of PAFR did not impair learning and memory although this assessment must be considered preliminary as the behavioural test employed was not optimized to detect changes in learning and memory of C57BL/6 mice over time adequately.Finally, I showed that the loss of PAFR in TgCRND8 mouse model of AD had no impact upon Aβ plaque number. My observations suggest that PAFR is restricted to microglial-like cells in mouse hippocampus and as such, it may not play a role in learning and memory.

We have recently established that aberrant alkylacylglycerophosphocholine metabolism results in the increased tissue concentration of platelet activating factors (PAFs) in the temporal cortex of Alzheimer Disease (AD) patients and in TgCRND8 mice over-expressing mutant human amyloid precursor protein. PAF lipids activate a G-protein coupled receptor (PAFR) reported to be expressed by microglia and subsets of neurons in rat. It is not known whether this same expression pattern is recapitulated in mice however, as the expression has only been inferred by use of pharmacological PAFR antagonists, many of which impact on both PAFR-dependent and PAFR-independent signalling pathways. PAFR plays a role in long term potentiation (LTP) induction in rats. PAFR has also been implicated in behavioural indices of spatial learning and memory in rats. Contradictory reports using mice provide ambiguity regarding the role of PAFR in LTP induction in mice. To assess whether PAFR is expressed in murine neurons, I localized PAFR mRNA in wild-type C57BL/6 mice using PAFR KO mice as a negative control. I further showed that the loss of PAFR did not impair learning and memory although this assessment must be considered preliminary as the behavioural test employed was not optimized to detect changes in learning and memory of C57BL/6 mice over time adequately.Finally, I showed that the loss of PAFR in TgCRND8 mouse model of AD had no impact upon Aβ plaque number. My observations suggest that PAFR is restricted to microglial-like cells in mouse hippocampus and as such, it may not play a role in learning and memory.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The liver is a vital organ, playing numerous critical roles in the body. The liver's ability to perform essential functions is disturbed by injuries that are often associated with many complications such as calcification. Although many reports in the literature document observations of liver calcification, the mechanisms regulating this phenomenon remain unclear. Herein, we aim to investigate the cellular and molecular events that occur during pathological calcification of the liver. / To study the mechanisms of calcification, assessments included histological-staining, immunolabeling, and biochemical and electron microscopy analyses. The findings suggest that calcification may result from hydroxyapatite precipitation in necrotic or apoptotic hepatocytes. Similarly, calcification may be associated with differentiated myofibroblasts expressing bone matrix proteins downstream of TGFbeta signalling. / To identify specific protein regulators linked to the various stages in calcification, and to assess the protein composition of the tissue, a proteomic analysis was used. This analysis identified IQGAP1, an effector of the Rho-GTPases and a master regulator of cell adhesion and migration. IQGAP1 is strongly expressed in myofibroblasts, suggesting that IQGAP1 may be implicated in myofibroblasts migrating towards calcification. Studies on IQGAP1 interactions with its binding partners reveal that it is part of a protein complex that includes beta-catenin, an adhesion protein, and Rac1, a cytoskeletal regulator. These results suggest that IQGAP1 may play an important role in myofibroblast migration upon liver injury. / Having identified that activin and TGFbeta signalling are activated in myofibroblasts, we hypothesised that they may stimulate myofibroblast differentiation and proliferation. Studies using a C3H/10T1/2 cell model reveal that both activin and TGFbeta stimulate differentiation, but only activin induces cell proliferation in a Smad-independent fashion, which requires activation of the ERK/MAPK pathway. / In summary, this work provides new mechanistic insights on the global regulation of liver calcification. The various phases of this work collectively cover the central role of myofibroblasts in liver injury: association with calcification, rapid proliferation, differentiation to an activated form, and migration toward the injured area. The findings allow us to better understand the mechanisms by which liver myofibroblasts are regulated in a specific pathological context.

We have recently established that aberrant alkylacylglycerophosphocholine metabolism results in the increased tissue concentration of platelet activating factors (PAFs) in the temporal cortex of Alzheimer Disease (AD) patients and in TgCRND8 mice over-expressing mutant human amyloid precursor protein. PAF lipids activate a G-protein coupled receptor (PAFR) reported to be expressed by microglia and subsets of neurons in rat. It is not known whether this same expression pattern is recapitulated in mice however, as the expression has only been inferred by use of pharmacological PAFR antagonists, many of which impact on both PAFR-dependent and PAFR-independent signalling pathways. PAFR plays a role in long term potentiation (LTP) induction in rats. PAFR has also been implicated in behavioural indices of spatial learning and memory in rats. Contradictory reports using mice provide ambiguity regarding the role of PAFR in LTP induction in mice. To assess whether PAFR is expressed in murine neurons, I localized PAFR mRNA in wild-type C57BL/6 mice using PAFR KO mice as a negative control. I further showed that the loss of PAFR did not impair learning and memory although this assessment must be considered preliminary as the behavioural test employed was not optimized to detect changes in learning and memory of C57BL/6 mice over time adequately.Finally, I showed that the loss of PAFR in TgCRND8 mouse model of AD had no impact upon Aβ plaque number. My observations suggest that PAFR is restricted to microglial-like cells in mouse hippocampus and as such, it may not play a role in learning and memory.