UQ Theses Collection (RHD) - UQ staff and students only - UQ eSpace

Cellular transformation and expression profiling studies related to nasopharyngeal carcinoma

Whitson, John Murray. — 2011-12-05T00:00:00Z

CEREAL PROCESSING, PARTICLE SIZE DISTRIBUTION AND STARCH DIGESTIBILITY IN ANIMAL FEED

Ghaid Al Rabadi — 2011-02-02T00:00:00Z

Abstract Starch in grains represents the main source of dietary energy for monogastric production animals. However, utilization of this energy is not complete due to factors related to grain source and in vivo gastrointestinal conditions. Incomplete starch digestion is a significant economic issue for the pork industry in Australia and prior evidence suggests that there is room for improvement. The general hypothesis of this thesis is that milled grain particle size fractions obtained by sieving posses a significantly different and wide range of functional and compositional properties in relation to digestion and hydrothermal processing. To identify these properties, sorghum and barley grains were milled separately using a 4 mm hammer-mill and then the ground cereals were segregated by size using a series of analytical sieves. Generated different particle sizes were investigated for their properties before and after cooking using extrusion processing. Sorghum was used as an example of a high starch content grain with low fibre content and barley was used as an example of a grain which contains high levels of fibre. Both grains are widely used as major feed ingredients in Australia, particularly in Queensland. Particle Size Effect on Digestibility The effect of milled uncooked grain particle size on the kinetics of enzymatic starch digestion and starch sources was examined. The in vitro starch digestion for different particle sizes were well fitted by simple first order kinetics for both barley (r2 range 0.90-0.99) and sorghum (r2 range 0.87-0.99). The digestion rate coefficients for different particle sizes decreased with increasing particle size, and were shown to be well fitted by an inverse square relationship for both barley (r2=0.99) and sorghum (r2=0.98). This supports the hypothesis that the rate-determining step in starch digestion is the diffusion of enzyme through the grain fragment. Quantitatively, the estimated (apparent) diffusion coefficient was estimated for both barley and sorghum (1.7 and 0.76 x 10-7 cm2 s-1, respectively). The apparent diffusion coefficient of á- amylase enzyme in grains was lower by an order of magnitude than that obtained in water, thus quantifying the barriers to amylase diffusion caused by grain structure. Particle Size Distribution in Milled Grain The contribution of individual size fractions to the overall hydration, rheological, and enzyme susceptibility properties of milled grain was evaluated. Sieve fractions were obtained from hammer-milled barley and sorghum grains (eight sieves, 0.125 mm to 2.8 mm opening size). Different particle size fractions showed a significant effect on digestion and hydrothermal properties. The extent of starch digestibility varied between different particle size up to 10 fold between the smallest and the largest particle size. When the results obtained for the unfractionated milled grain were compared to the weight average values (calculated from fraction yields and property values for each size fraction), water absorption index (WAI), water solubility index (WSI), and starch digestibility values were not significantly different from values obtained for non-fractionated ground grains of both barley and sorghum. Generally, hydration and digestibility results can be attributed to particle size and composition (starch or soluble fibre) of individual fractions. Response to hydrothermal treatment was different between sorghum and barley. In sorghum, viscosity profiles for the fractions were controlled by starch swelling which became increasingly limited as particle sizes increased. However in barley, viscosity profiles for segregated fractions did not show typical starch-based behaviour and were most likely controlled by the soluble fibre component. The results show the possibility of predicting and manipulating hydration and digestibility properties of ground grains simply by indentifying the particle size distribution of ground grains. Extrusion Processing of Milled Grain Extrusion processing was used in attempts to increase the rate and extent of starch digestibility as well as to characterize processing parameters and extrudate properties. Extrusion processing of three levels of particle size after sieve fractionation (fine, medium and coarse fractions) affected processing parameters (torque, specific mechanical energy [SME], die pressure), extrudate properties (WAI, WSI, expansion index, pasting profiles, and the kinetics and extent of starch digestion). The differences in response to processing parameters and extrudate properties was mainly due to the differences in viscosity during extrusion, while extrusion temperature (100°C versus 140°C maximum barrel temperature) had a smaller effect. The different particle size fractions of sorghum and barley both before and after extrusion showed a good fit to first-order kinetics of starch digestion, with all r2 values above 0.95. Results of the extrusion experiment suggested that the extent of swelling and gelatinisation during the extrusion may have been further improved by incorporating a pre-conditioning stage prior to extrusion. Particle size had a significant effect on the fractional digestion rate for both grains, but extrusion temperature had no effect. Based on fractional digestion rate values, extrusion processing at low temperature (100°C) could be an alternative method to fine milling for achieving a high rate and extent of starch digestion. Feed Conversion in Pigs A novel processing procedure, based on separating the coarse fraction after hammer milling and then regrinding this fraction, was used in an attempt to improve growth performance in pigs offered barley and sorghum based diets. The pig feeding trial examined the effect of (a) grain type (barley; sorghum), (b) particle size (ground grain; ground grain where the coarse fraction was separated and re-ground then added back to the fine fraction), and (c) diet form (mash, pellet), on feed conversion ratio (FCR), rate of growth (ROG) and average daily intake (ADI). Particle size had the largest effect on FCR in pigs. Feeding pigs a barley or sorghum based diet in the mash form after regrinding the coarse fraction improved FCR in sorghum by 10% and in barley by 7.8% compared to diets prepared without regrinding the coarse fraction. The form of the diet showed a smaller effect on FCR. In the pellet form, pigs fed sorghum or barley based diets after regrinding the coarse fraction improved FCR in sorghum by 5.1% and in barley by 4.8% compared to diets prepared without regrinding the coarse fraction. Within grain type, effects of particle size or diet form on ROG and ADI in barley were not statistically significant, although their ratio (FCR) did show significance. In sorghum, pigs offered diets in the ground-mash form had the highest ADI. Pigs offered ground-mash form (ground grain where the coarse fraction was separated and re-ground then added back to the fine fraction) had the highest ROG. The results from this investigation suggest that regrinding the coarse fraction after sieve separation can be used to maximise feed conversion ratio (FCR) for both barley and sorghum based diets. In contrast to single stage grinding through a smaller screen size, the regrinding method used in this work did not result in major increases in fractions with sizes below 0.5 mm and particularly below 0.25 mm. This is important as these very small particles cause dust problems in handling and on ingestion. Desired hydration properties, chemical composition, response to hydrothermal treatment, and extent of starch digestion could be manipulated by modifying the distribution of particular size fractions of ground grains. Extrusion of medium and coarse size fractions can enhance digestibility with efficient utilization of thermal energy compared to extrusion of ground but unfractionated grains. This could be a good processing strategy as this work has found that the rate and extent of starch digestion is relatively high for uncooked small particle size fractions. Alternatively, regrinding the coarse fraction after separation could be an alternative method to conventional steam pelletizing for increasing starch digestibility and energy delivery to growing pigs.

Cerebellar modeling techniques for mobile robot control in a delayed sensory environment

Collins, David Timothy. — 2007-08-24T00:00:00Z

Certain India : An enquiry into a claim to national territory

Grant, William John — 2008-11-21T00:00:00Z

In both intimate and abstract encounters, India is today understood as a land certain, and a land connected naturally with the Indian nation. India is, for many, a land visible in the physical world, a land visible on the world map, and a land the exclusive political domain of a definable group of people. This was not always the case. This thesis enquires into the circumstances and campaign that have made it so; this thesis enquires into the claim to national territory in India. Specifically, I seek in this work to explain how the land between the Himalayas and the Indian Ocean has been produced as the national territory of the Indian nation, and how this national territory is understood by these nationalists. To conduct this enquiry, I take in this work two broad steps: the construction of a framework within which we can theoretically situate the Indian national territory, and the application of this framework to the specifics of the case at hand. To construct the required theoretical framework, I turn first away from the Indian national territory to the more abstract phenomenon of the national territory in general, and to theoretical literature against which this more abstract national territory in general can be situated. In this, I turn to both theoretical works on nations and nationalism, and theoretical works on space. Here I suggest that we can theoretically situate the abstract national territory in general against both our understanding of the essence of the nation and our understanding of the essence of space; that the national territory is both one element of that which is of the nation, and one particular form of space. Located in this way, I argue in this work that the national territory is a form of space emergent in the intersection of the highly abstract, disembodied forms of knowledge of the physical dominant with the onset of modernity, and knowledge of the physical derived from forms of integration more intimate, particular and local. I argue, more precisely, that the national territory is a form of space produced through both the emergence to dominance of highly abstract forms of knowledge of the physical, and the renegotiation of these dominant forms according to interests and understandings more intimate and particular. From this point, I then proceed to locate and describe the specifics of the production and understanding of the Indian national territory. To do this, I draw on the theoretical framework constructed in the first part of the thesis to trace the emergence to dominance in southern Asia of highly abstract, disembodied forms of knowledge of the physical, and, following this, the response to this derived from the more intimate forms of knowledge of the physical marginalised by this emergence. Thus in this second step I chart first the emergence to dominance of highly abstract forms of knowledge of the physical in southern Asia. In this I look in particular at the forms of social integration and knowledge of the physical that emerged to dominance under the British Empire. Following this, I then trace the response to this emergence to dominance offered by two significant political perspectives: one of secular Indian nationalism, the other of Hindu nationalism. With this, I argue that the Indian national territory is a space which has been produced through the emergence to dominance in southern Asia of highly abstract, disembodied forms of knowledge of the physical, and the complicated and ongoing efforts to renegotiate this highly abstract knowledge according to interests and understandings more intimate and particular. The Indian national territory is understood, thanks to this production, as a land natural and concrete, eternal and certain, in both embodied locales and the world at large; a land, that is, for which people have willingly died.

Cervical musculoskeletal impairment in frequent intermittent headache

Amiri, Mohsen — 2012-01-31T00:00:00Z

Challenges for Australian Tribunals; Towards Aretaic Practice Principles

Christou, Alison — 2008-02-28T00:00:00Z

‘…more citizens… receive their justice from agencies than the courts’ (Chitra: 3). The last 30 years of administrative law practice in Australia have been characterised by the proliferation of tribunals and other bodies engaged in quasi-judicial work. These organisations have developed on a largely ad hoc basis, particularly at the state and territory level. Despite this rapid growth – or perhaps as a result of it - a clear articulation of the underlying practice principles pertinent to tribunals has yet to occur in any coherent fashion. This thesis canvasses the current mechanisms used to assist tribunal members in their work, including legislative and ethical parameters. The argument is then mounted that more guidance is needed in relation to the normative excellences of the tribunal member’s role. An analysis of the systemic and cultural challenges faced by the sector is presented, highlighting the lack of sector definition, disparate practices, resource limitations and institutional isolation that characterise Australian tribunal work. Canadian and United Kingdom experiences provide a useful context for Australian impediments and developments. A state-based merits review body is used as a case study throughout the thesis to illustrate these issues. It is posited that greater emphasis upon the philosophical underpinnings of tribunal practice will assist in improving cohesion among sector members, leading to enhanced delivery of administrative justice. A stance from virtue ethics is adopted as a starting point for this endeavour, on the basis that improved decision-making commences with close analysis of the excellences defining the tribunal member’s role. This aretaic approach highlights the importance of ‘practical wisdom’ or phronesis in tribunal work. Methods for implementing and enhancing discussion of relevant tribunal practice principles are then examined, with various innovative legal training tools being nominated as particularly useful in the tribunal context. The tribunal sector is generally seen to have operated adequately to date. A more normative approach to decisionmaking excellence within the tribunal field is required, however, with this thesis forming the staring point of such analysis.

Challenges in management of infections on a burn injury unit.

Bhavik Patel — 2012-06-29T00:00:00Z

Burn injury patients are a special subset of patients with an increased likelihood of developing infections as well as reduced capacity to fight them. This Thesis focuses on two aspects of infections in burn injury patients, the issues surrounding blood stream infections (BSI) which were defined according to The American Burn Association Consensus Conference on sepsis and infections in this subset of patients and a possible method to optimise drug dosing in burns patients using therapeutic drug monitoring (TDM). BSI is a known negative predictor in the outcome of burn injury patients. This is the first description from a tertiary referral burn centre in Australia of the organisms responsible, the antibiotics prescribed and the various other factors that are associated with clinical outcome in burn injury patients that develop BSI. Data were collected from various databases on admitted patients with burn injuries from January 1998 to December 2008. Selected information from databases was analysed using SPSS version 17 (SPSS Inc. Chicago). Descriptive, univariate and multivariate analysis was undertaken to determine factors predictive of clinical outcome. The factors analysed by univariate analysis were selected on clinical plausibility. Univariate analysis used a crosstabs procedure initially to estimate maximum likelihood. Factors that were associated with a P value <0.15 were entered into a binary logistic regression to detect which covariates were independent predictors of mortality in BSI and outcome according to specific organisms. Ninety-nine out of 2364 (4%) patients developed 240 documented BSI. The median time from burn injury to BSI was 7 (interquartile range 3-16) days. There were 13 deaths during the hospital stay for this cohort; Pseudomonas aeruginosa BSI was associated with six deaths. Factors associated with P.aeruginosa mortality by univariate analysis included inhalational injury, percentage total body surface area burn (%TBSA), days of antibiotic prescribed and Acute Physiological and Chronic Health Evaluation (APACHE) II scores. Univariate analysis of the 2364 patients admitted to our center over the 11-year period found BSI to be predictive of mortality (p < 0.001). Multivariate analysis identified inhalational injury to be the only factor associated with BSI-related mortality. The second part of this Thesis was a prospective study where TDM was performed on all patients admitted to a tertiary referral burns unit that were prescribed a beta−lactam antibiotic. Steady state blood samples and Creatinine levels were collected immediately prior to a scheduled dose. Therapeutic concentrations were based on pharmacokinetic (PK) / pharmacodynamic (PD) data from previous studies. For the purpose of this study, treatment outcome was considered positive if there was completion of treatment course without change or addition of antibiotic therapy or commencement of additional antibiotics within 48 hours of discontinuation of antibiotic therapy. The PK/PD targets assessed were: (a) unbound trough antibiotic concentrations exceeding the minimum inhibitory concentration (MIC; f T >MIC) and (b) unbound concentrations greater than or equal to 4 x MIC of the known or suspected pathogen (f T>4xMIC). Duration of therapy was independently determined by the treating clinician independent of dose adjustment. A total of 50 patients were included for TDM over a 12 month period. The mean (+ standard deviation) age was 49 + 16 years. The mean % TBSA burn was 17+13%. The mean serum creatinine concentration was 86 + 20 umol/L. Sixty percent of patients did not achieve f T >MIC and only 18% achieved the higher target of f T>4xMIC. Although all patients achieved a positive clinical outcome, the duration of antibiotic treatment was shorter in patients that achieved f T >MIC compared with those that did not (4.2 + 1.1 v/s 5.3 + 2.3 days; P= 0.03). This study demonstrates that empiric beta-lactam dosing does not consistently achieve therapeutic targets and there is significant antibiotic trough concentration variability in burn injury patients. The clinical ramifications of this clinically remain unclear, although patients with therapeutic beta-lactam concentrations did have a shorter duration of antibiotic therapy. This data does suggest that a TDM programme is a useful intervention to optimise beta-lactam dosing in stable ward based burn injury patients.

CHALLENGING GENDER EQUITY IN HIGHER EDUCATION: A PLAN FOR PAPUA NEW GUINEA

Tuaru, Velepat Gutuma — 2008-07-21T00:00:00Z

Change in lumbar dysfunction in surgical patients and the effect of two exercise programs

Kelley, Susannah M — 2013-02-01T11:14:21Z

Change in the Cultural Identity of German Settlers of the Logan and Maroochy Rivers, Queensland, 1860-1914

Jasmine Sommer — 2010-02-01T00:00:00Z

This thesis concentrates on the 1860s migration and settlement experience of the first German settlers of Gramzow on Queensland’s Logan River. It also describes the internal migration of some among them to the North Arm of the Maroochy River in the early 1880s. The latter journey was undertaken in the company of other Germans from the Logan River district and formed part of a pattern of cluster and chain migration to the North Coast. The first chapter in this thesis discusses the early German settlers’ decision to migrate from their homelands, and their economic and societal reasons for migration. The role played by Johann Christian Heussler in the Germans’ choice of Queensland as a destination, and his contributions to the economic development of Queensland through his position as Emigration Agent to the German States, are reviewed. This thesis also attempts to bring balance to the reputation of Godeffroy and Son, the Hamburg shipping line engaged by Heussler, which brought most of the German settlers to Queensland in the 1860s. The company’s visible commercial strengths such as their size and experience in the Pacific, and their private, internal weaknesses such as failure to adopt new technologies, are examined. Conditions on the Godeffroy vessels are compared with the conditions on ships sailing from Hamburg to America. This approach avoids the usual comparison of German with British sailing ships coming into Moreton Bay. Britain’s exemplary standards for passenger health were beyond the reach of emigrant fleets who operated under Hamburg’s older regulations. The research concludes that, in the early 1860s, conditions on the Godeffroy ships for Queensland were superior to Hamburg ships for New York. Furthermore, this thesis describes the 1868 German settlement of Gramzow on the Logan River and compares it to Bethania. The significance of Queensland’s 1868 lands legislation to the German settlers is explored. It is suggested that the 1868 Crown Lands Alienation Act is connected to the U.S. Homestead Act, 1862, and a comparison is drawn between the Australian, American and Canadian lands settlement legislation. This comparison enables the further suggestion that homestead selectors of the Logan were part of an international group of homesteaders whose occupational identity was tied to opening the land to agricultural smallholding at little cost through many similar or identical legislative rules that predominantly impacted their economic standing positively. How land orders enabled Logan settlers to increase their land holdings is discussed, as are the negative aspects of the lands legislation such as the upper 160 acre limit on land holding. The migration of early German settlers of the Logan district north to Maroochy occurred under Queensland’s 1876 lands legislation. This thesis examines the settlement of Germans on the Canando Run along the North Arm of the Maroochy River in the early 1880s, and describes their settlement conditions. Their motives for moving are examined, how the discovery of gold at Gympie affected them is explored, and the establishment of three German businesses at Maroochy is described. A chart comparing the settlers’ land holdings on the Logan with those at Maroochy illustrates that by moving north, some settlers were able to increase their land holdings threefold. The disappearance of Deutschtum (‘German culture’) after the turn of the century is examined in the final chapter. This thesis asks whether it is appropriate to continue to use the term ‘assimilation’ when speaking of Queensland’s German settler community before and during the First World War. The term appears to draw a veil over the political and economic subjugation of the community during this period. The thesis proposes that it was easier to survive the difficulties of war in rural rather than in urban communities. Although the historiography of the German settlers of Queensland supports an academic conversation on topics such as German emigration, land acquisition and settlement, this thesis focuses on issues outside the boundaries of the current academic thrust. These issues include the settlement of Gramzow in 1868, the homestead provisions in the 1868 Crown Lands Alienation Act and their origins in lands legislation in America, the services provided through Johann Christian Heussler by the German emigrant shipping line ‘Godeffroy and Son,’ the settlement of the North Arm of the Maroochy River by Logan Germans in the early 1880s, and a rejection of the term ‘assimilation’ to describe the eradication of German culture in Queensland after 1914. The leitmotif of this thesis is cultural identity and it explores change in German settlers through various aspects of their identity such as their psychological identity, diasporic experiences, language, and legal and political identity after taking citizenship.

Changes in Bacterial Communities, Carbon and Nitrogen Dynamics on Coral Surfaces Following Mortality: Potential Implications for Reef Systems

Davey, Andrew Mark — 2008-11-21T00:00:00Z

Prokaryotic energy and nutrient cycling is critical to coral reef ecosystems. Living coral surfaces can harbour diverse but markedly different bacterial communities than other marine surfaces and, these communities may alter considerably following a disturbance. The consequences of these changes may include; alteration of fluxes and routes of organic and inorganic matter cycling, accelerated eutrophication and proliferation of algal blooms, all of which may ultimately lead to changes in trophic interactions. Coral reefs worldwide are experiencing a decline in health of a magnitude not seen in recorded history. In view of current trends and events over the last 30 years, it appears inevitable that we will lose a large percentage of living coral in the coming decades. It is therefore necessary to gain a greater understanding of the consequences associated with a substantial increase in the area of dead coral substratum. In particular the changes in the bacterial associates of coral surfaces following mortality may lead to dramatic changes in carbon and nutrient cycling, which may have implications throughout the ecosystem. This study investigated the primary colonization and early succession of bacteria on dead coral surfaces following mortality. This included an examination of the associated changes in production, carbon and nitrogen dynamics of the developing biofilms, and the consequent contributions to reef carbon and nutrient budgets. Inferences were made to the ecosystem changes that may be associated with a decrease in living coral and concomitant increase in bacterial mediated carbon and nutrient processes. Coral mortality via thermal stress resulted in the development of markedly different bacterial communities to those of live corals. Analysis of bacterial primary recruitment and early succession using Fluorescence in situ hybridisation (FISH) allowed direct visualization of bacteria and their spatial heterogeneity within and across samples. Members of the Cytophaga-Flavobacterium-Bacteriodes (CFB) and the gamma Proteobacteria dominated samples from dead corals, while members of the alpha Proteobacteria were apparent over time after coral mortality. Profiling of bacterial populations using Denaturing Gradient Gel Electrophoresis (DGGE) and Terminal- Restriction Fragment Length Polymorphism (T-RFLP) showed significant differences in surface bacterial populations between live and dead coral and that the bacteria on dead corals were dynamic over time post-mortem. In addition, T-RFLP allowed the identification of bacterial species that have previously been linked to nitrogen processes. The results of molecular analysis suggest that significant changes in surface biogeochemistry may occur concurrently with the changes in coral-surface bacteria following coral mortality. Surface associated oxygen metabolism was investigated using a combination of oxygen microelectrode profiles and Pulse Amplitude Modulated (PAM) Fluorometry. Results demonstrated a significant increase in oxygenic photosynthesis from dead coral biofilms. The associated P/R ratios implied a net increase in autotrophic carbon fixation, which was also demonstrated by increases in photosynthetic pigments and algal biomass. The developing biofilms also had greater photosynthetic efficiency, particularly at lower light levels, suggesting a greater capacity for carbon fixation. Changes in nitrogen processes were investigated by nitrogen fixation assays as well as fluxes of NH3/NH4[superscript]+ and NO[subscript]x. A significant increase in nitrogen fixation was observed from dead coral surfaces. These nitrogen fixation rates represented a significant input of new nitrogen to coral reefs, particularly in the initial two weeks after coral mortality. Analyses of nitrification and denitrification processes indicated these processes were absent in the initial development period of the dead coral algal-bacterial community. Assessment of the coral surface algal-bacterial biofilm showed an increase in carbon and nitrogen within the biofilms, which suggested that biochemical activity within the biofilm was primarily responsible for the increase in algal biomass observed. Overall, the results demonstrate a change in coral surface biogeochemical processes, which in the context of coral bleaching, may have ecosystem wide consequences. Coral bleaching has already been responsible for the death of large areas of coral. Death of coral over large spatial scales and the consequent increase in bacterial-mediated nutrient cycling may provide a significant input of new nitrogen to the system. This thesis explores the potential significance of these changes for the long term alteration of coral reefs subject to thermal bleaching.

CHANGES IN GABAA RECEPTOR EXPRESSION AND BRAIN CYTOSKELETON IN NORMALLY GROWN AND INTRAUTERINE GROWTH RESTRICTED PIGLETS ACROSS THE PERINATAL PERIOD

Viskasari-Pintoko Kalanjati — 2011-02-10T00:00:00Z

Intrauterine growth restriction (IUGR) is one of the commonest causes of perinatal mortality and neuromorbidity, including a higher propensity to develop seizures and epilepsy. Impairment of GABA (g-aminobutyric acid) and glutamate systems and abnormalities of the brain cytoskeleton have been reported in humans, particularly in association with seizures and epilepsy. Whether impairments to the GABA and glutamatergic systems and brain cytoskeleton take place in spontaneously occurring IUGR has yet to be elucidated. This thesis is directed at understanding the pathophysiology of the neuromobidities which occur in IUGR infants. GABAA (g-aminobutyric acid type A) receptor a1, a3 and b2 subunit protein expression levels and distribution in parietal cortex and hippocampus of normally grown (NG) piglets (n=34) were analysed and compared to those of spontaneously occurring IUGR piglets (n=31) at 14 days preterm (P-14), 10 days preterm (P-10), birth (P0) and post-natal 7 days (P7) by western blotting and immunohistochemistry. The four time-points were chosen because GABAA receptor subunit expression is known to be developmentally regulated around birth. Immunohistochemistry of GAD67 (glutamic acid decarboxylase 67) was used to compare GABAergic-positive cell expression in NG and IUGR piglet brains in the four age groups; with GLAST1b (glutamate-aspartate transporter 1b) performed to label dysfunctional neurons. Gray and white matter brain cytoskeleton was studied by MAP2 (microtubule-associated protein 2) and MBP (myelin basic protein) immunolabelling to allow visualisation of any impairments to neuronal somatodendrites and myelinated axonal fibres respectively. In NG piglets, significantly higher a1 expression in P7 cortex was observed compared to all other age groups (p<0.05); whilst in hippocampus it was significantly higher in P7 compared to the P-14 and P-10 groups and in P0 compared to the P-14 group (p<0.05). In IUGR piglets, a1 expression in cortex increased in preterm groups to P7; a similar trend was seen in hippocampus with significant differences found only in P7 compared to the P-10 group (p<0.05). In NG piglets, a3 subunit expression in cortex peaked at P0, when it was significantly higher than at P7 (p<0.05); a similar trend was seen in hippocampus. In IUGR cortex and hippocampus however, a3 expression was highest at P-14 compared to the other age groups but this was not statistically significant. In P7 IUGR cortex, the a3 expression was significantly higher than in P7 NG cortex (p<0.05) whilst the a1 subunit expression tended to be lower (p=0.1). There was a significantly lower a1/a3 in P7 IUGR cortex than P7 NG cortex (p<0.05). In NG and IUGR piglets, b2 expression in cortex and hippocampus did not differ from the P-14 to the P7 group. vi There were no obvious differences on the laminar and cellular distributions of GABAA receptor a1, a3 and b2 subunits in NG and IUGR piglet parietal cortex and hippocampus across the age groups. Immunolabelling of the a1, a3 and b2 subunits were shown throughout all cortical layers with an intense b2 labelling observed in layer IV, whilst the a3 subunit was predominantly observed in layer V-VI. The a1 and b2 subunits were widely distributed in all strata in the hippocampus of NG and IUGR piglets, whereas the a3 subunit was limitedly expressed. At the cellular level, these subunits were observed in the neuropiles, cell bodies and processes of pyramidal and non-pyramidal neurons in both NG and IUGR cortex and hippocampus. No obvious differences in GLAST1b expression were found in NG and IUGR piglets. However neuronal somatodendrite labelled by MAP2 was impaired in P7 IUGR parietal cortex and CA1 hippocampus compared to the P7 NG. An obvious reduction in myelinated axonal fibres labelled by MBP was observed in subcortical parietal white matter of P-14, P-10 and P0 IUGR piglets when compared to NG piglets at each age group. However, there was no clear difference in the P7 group or in hippocampal MBP labelling between the IUGR and NG piglets in each age group. In conclusion, GABAA receptor a1, a3 and b2 subunit protein expression is developmentally regulated in a specific manner in NG piglet cortex and hippocampus; with significant impairments observed in P7 IUGR cortex when compared to P7 NG cortex. Brain cytoskeleton abnormalities detected using MAP2 and MBP in IUGR piglet cortex and hippocampus, were observed during the perinatal time period. These findings collectively contribute to understanding the underlying mechanisms involved in IUGR neuromorbidity and may partly explain the propensity to seizures and epilepsy found in the clinical condition of IUGR.

Changes in translational norms in postwar Japan

Furuno, Yuri — 2012-01-31T00:00:00Z

Changing agricultural productivity in Bangladesh : its nature and implications for poverty, women, off-farm employment and the environment

Jahan, Nilufar — 2010-12-07T00:00:00Z

Changing Complex Documents

Carter, Simon Matthew James — 2008-11-21T00:00:00Z

Change management is a discipline fundamental to the task of building ever more complex computing systems. Properly managed change provides a means whereby alterations to existing components of a complex artefact and their relationships can be evaluated, managed and evolved. This thesis takes as its example Official RAAF Publications, some of which need to be revised as a result of changes to the system they describe. The thesis develops a model of change propagation providing a set of operations to examine and record the changes to a set of publications. Additional operations enable coping with reversing decisions and handling the unexpected arrival of externally generated amendments. The model is extended to cover a finer granularity of entities (at the page level) to determine whether this greater level of detail would ease some tasks. A further extension provides the notion of relationships between the publications of concern, focusing on a dependency relationship between two publications. This enables exploration of the possibility of improving the process by reducing the risk of missing publications needing revision and providing a means by which some tasks can be partly automated thus speeding up the process. The models presented were developed in Sum, a variant of the Z specification language, to gain greater insight into the essential details of the operations and data structures involved. By ignoring implementation details the essential logical steps of each model can be emphasised and their differences and similarities contrasted. This thesis demonstrates that fine-grained change management is feasible. The thesis develops processes that automatically track the status of changes as they are propagated through a set of documents. The greater knowledge of work done on individual pages allows only the page(s) of concern to be affected. The work also enables recommendations to be made as to the applicability of each model and, by comparing the models, provides insight into the amount of work and resources required for tackling change at different levels of granularity.

Changing Musical Emotion through Score and Performance with a Computational Rule System

Steven R. Livingstone — 2008-09-05T00:00:00Z

Chaplaincy in the Royal Brisbane Hospitals. The genesis and evolution of hospital chaplaincy in a Queensland hospital complex.

John Pearn — 2010-11-23T00:00:00Z

CHAPLAINCY IN THE ROYAL BRISBANE HOSPITALS The genesis and evolution of hospital chaplaincy in a Queensland hospital complex John Hemsley Pearn A Thesis submitted as fulfilment for the Degree of Master of Philosophy School of History, Philosophy, Religion and Classics The University of Queensland June 2010 ABSTRACT This thesis is an account of the genesis and evolution of the developed discipline of hospital chaplaincy in the Herston Hospitals complex, Brisbane. This multihospital campus comprises the hospitals of the Royal Brisbane and Women’s Hospital and the Royal Children’s Hospital and their predecessors. The Brisbane Hospital was founded in 1867 as a charity hospital. Since that time it has formed the epicentre of an extending healthcare campus where more than 300 chaplains have provided solace and sacraments, conducted rites and offered counsel to hundreds of thousands of inpatients. Their history and the history of hospital chaplaincy more generally has hitherto been unrecorded. This thesis documents the operational role and analyses the influences which have impinged on the development of hospital chaplaincy as a specialty discipline within a secular healthcare system. The central question addressed in this thesis is “What has been the shifting role and institutional position of hospital chaplaincy in a state-funded, avowedly secular, inpatient healthcare service in the twentieth century?” The unifying hypothesis tested in this thesis is that in spite of the tenet of secularity central to healthcare provision in a typical Australian state hospital, religious support in the form of hospital chaplaincy has been an important and evolving component of inpatient care. The research embodied in the thesis has indicated that hospital chaplaincy in the Herston Hospitals has passed through three distinct phases. Part I of this thesis describes the influences which operated from 1867 to 1924 –– the era between the foundation of the Brisbane Hospital at Herston and the imposition of government funding and control as a consequence of the Queensland Hospitals Act of 1923. Pastoral care, exclusively Christian in that era, was provided by visiting clergy, by visiting nuns (from both Church of England and Catholic orders) and by devout lay visitors, all drawn from the principal denominations which comprised the more general Queensland population. The second distinct era of hospital chaplaincy in the Herston Hospitals encompassed the period from 1924 to 1981. It began with the nationalisation of healthcare, imposed by consequential regulations which followed the Hospitals Act of 1923 and implemented by the formation of the Brisbane and South Coast Hospitals Board in 1924. It ended in 1981, the year in which the hospital chaplains themselves, hitherto independent and separate agents, established the Chaplaincy Department of the Royal Brisbane Hospitals complex. That era saw the emergence of a dedicated, onsite chaplaincy service. The first chaplain formally appointed to the Herston Hospitals was a Church of England chaplain who began his full-time hospital ministry in 1931. Other churches followed with the establishment of both full-time and part-time hospital chaplaincy appointments. This development was the genesis, not only within the ecclesiastical world, but also within the hospital world of specialist services, of the discipline of hospital chaplaincy as this specialty became known. Research on hospital chaplaincy in this period has indicated that chaplaincy endured, but did not develop beyond that of a visiting, sacramental or occasional pastoral role. By the end of that era, the Herston Hospitals had grown to comprise an extensive (almost 2,000 inpatients), avowedly secular, state hospital complex. Nominal rolls of the chaplains who served the Herston Hospitals have been compiled from primary sources and documented in the Appendices to this thesis. The third era of hospital chaplaincy, that encompassing the period from 1981 to the first decade of the twenty-first century, was a period of dramatic change. It saw both (a) the first identity and the rapid evolution of the specialised professional discipline of modern hospital chaplaincy; and (b) the modifying effects, indeed powerful imposts, of societal influences on the delivery of pastoral care in the Herston Hospitals. In Part II, the thesis describes influences which led to the genesis of the Hospital Chaplaincy Department (7 August 1981). This institution both reflected, and was itself to become influential in, the development of hospital chaplaincy as a recognised sub-specialty. From that base, chaplains at the Herston Hospitals were the catalysts for the establishment of the Ecumenical Hospital Chaplaincy Committee (from 1982) and the Multifaith Academy for Chaplaincy and Community Ministries (1993). In that era, the hospital chaplains themselves instituted the first specialty training which was to characterise the emergent discipline of modern hospital chaplaincy. This thesis argues that two features set the specialist profession of hospital chaplaincy apart from the general pastoral and sacramental work of ordained clergy in the general community. One of these was the particularly vulnerable and supplicant relationship of sick and injured inpatients, sometimes for the first time in their lives, with a chaplain. The second feature which came to define the specialty of hospital chaplaincy was the necessary acquisition of specialised knowledge of disease processes and healthcare which predicated a patient’s admission to hospital. Occasionally, chaplains became counsellors not only to patients but also to medical and nursing staff, in circumstances where a patient’s religious beliefs conflicted with medical treatment. In Part III, this thesis argues that (from 1981) three external influences imposed significant changes on both the organisational structure of hospital chaplaincy and on the professional role of chaplains themselves. These three influences comprised (a) the secular anti-discrimination movements of the last decades of the twentieth century, influences which followed some Faith-based trends towards a modified ecumenism; (b) the more general societal privacy movement of the last three decades of the twentieth century and its pragmatic consequences upon the preservation of inpatient confidentiality; and (c) the emergent discipline of bioethics and the widespread involvement of hospitalised patients in medical research. This thesis also documents and analyses a ‘sea-change’ of government attitude to the regulation of inpatient access to Faith-based pastoral care, introduced first in the Herston Hospitals in 1991 by Queensland Government regulations. Prior to 1991, Queensland Health (the State Government Department of Health) had taken no part and provided essentially no services for inpatient pastoral care. As formal state policy, it left this service exclusively to the discretion and will of external religious bodies and to devout members of the lay public. The first Hospital Chapel at the Royal Brisbane Hospital had not been constructed until 1976, more than a century after the Hospital had opened in 1867. Even at that late stage, the first chapel was made possible only by fundraising undertaken exclusively by volunteer members of the hospital staff and by the lay public. By contrast, in a major change of policy in 1991, Queensland Health responded to and embraced the secular movement of anti-discrimination and trends towards religious ecumenism. For the first time, the state acknowledged that the provision of chaplaincy services was integral to holistic inpatient healthcare. From that era, government regulation guaranteed the provision of services for and access to a pastoral carer of an inpatient’s choice. For the first time the state provided physical resources, specifically multi-Faith resources, to ensure this. This thesis argues that the historical evidence –– archival, documentary and oral –– indicates that a specialised hospital chaplaincy has evolved to be an ubiquitous component of healthcare, in secular as well as Faith-based hospitals. As such, hospital chaplaincy has developed to be a true professional sub-specialty which complements the other traditional specialties of the entire healthcare domain. ___

Characterisation and Environmental Risk Assessment of Metals and Metalloids Related to Mining Activities at Wainivesi,Fiji

Raijieli Taga — 2010-03-26T00:00:00Z

Characterisation and expression of zebrafish frizzled-3a (zfzd3a) during embryonic development

Wolfgang Hofmeister — 2008-07-22T00:00:00Z

Characterisation and Functional Analysis of Osteal Macrophages: Resident Tissue Macrophages are Intercalated throughout Mouse Bone Lining Tissues and Regulate Osteoblast Function In Vitro

Ming-Kang Chang — 2009-03-03T00:00:00Z

Resident tissue macrophages are an integral component of many tissues and are important in development, homeostasis and repair. Macrophages are present at sites of both pathologic bone deposition and loss, and can produce osteo-active factors. These observations link macrophages to bone disease, however their contribution to bone dynamics is poorly understood. The molecular and cellular mechanisms driving osteoblast differentiation, matrix deposition and mineralization in vivo are incompletely understood and this deficiency is translated to limited ability to clinically manipulate bone formation. The emerging understanding of the bi-directional interactions between the osseus and immune systems (osteoimmunology) provides a novel avenue to identify mechanisms involved in the regulation of bone formation. In this study, the presence and distribution of macrophages on bone surfaces was systematically analysed and their functional contribution to the bone microenvironment was investigated. Using immunohistochemistry a discrete population of mature resident tissue macrophages was demonstrated throughout resting murine osteal tissues, termed OsteoMacs. Utilising MacGreen mice (csf1r promoter drives eGFP transgene expression in macrophages and other myeloid cells), it was demonstrated that OsteoMacs were intercalated amongst other bone lining cells in both the endosteum and periosteum. OsteoMacs were TRAPneg in situ and had limited osteoclastogenic ability in vitro therefore they are unlikely to serve as the immediate physiologic osteoclast precursors in vivo. Microarray gene expression profiling demonstrated that macrophage gene expression was regulated in response to a characteristic feature of the bone microenvironment, elevated extracellular calcium. Quantitative PCR validated upregulation of sphingosine kinase 1, interleukin 1 receptor antagonise, progressive ankylosis, vascular endothelial growth factor c and dipepetidase 2 mRNA in response to elevated extracellular calcium, suggesting the potential roles of these genes in this unique niche. GNF Symatlas microarray and quantitative PCR demonstrated the expression of macrophage-restricted genes throughout a 21-day primary osteoblast differentiation time course, suggesting co-isolation of OsteoMacs with primary osteoblasts. Flow cytometry analysis confirmed that over all 15.9% of the digested primary calvarial cell preparations were OsteoMacs. Immunocytochemistry demonstrated that OsteoMacs persisted and expanded in standard 21-day osteoblast differentiation assays. Contrary to previous studies, we demonstrated it was the OsteoMacs, and not osteoblasts, within calvarial preparations that selectively detected patho-physiological concentrations of the bacterial product lipopolysaccharide (LPS). A protocol was developed to deplete OsteoMacs from calvarial digests to determine if their presence within these cultures facilitates osteoblast differentiation or function. OsteoMac removal did not affect expression of the early osteoblast differentiation marker genes collagen type I or alkaline phosphatase. However, OsteoMac removal significantly decreased gene expression of the osteoblast mineralisation marker osteocalcin and mineralisation function, assessed by von Kossa staining. Microarray gene expression profiling demonstrated that osteoblast enrichment had a broad impact on transcription within the culture, identifying both candidate OsteoMac marker genes as well as osteoblast expressed genes that are regulated by OsteoMacs. Potential OsteoMac-enriched candidate genes insulin-like growth factor a, dipepetidase 2, glycoprotein NMB, and macrophage expressed gene 1 as well as osteoblast-specific genes bone sialoprotein and thrombospondin 1 were selected based on their potential involvement in osteoblast function. In a transwell co-culture system of enriched osteoblasts and macrophages, it was demonstrated that macrophages were required for osteoblast mineralisation in response to the physiologic remodelling stimulus, elevated extracellular calcium. A blocking soluble receptor strategy provided evidence that this is mediated in a BMP-2 and -4 independent manner. To investigate the relevance of OsteoMacs to bone formation in vivo, immunohistochemistry staining for the mature tissue macrophage marker F4/80 was performed in long bone sections from rapidly growing mice. OsteoMacs were closely associated with areas of bone formation in situ, forming a distinctive canopy structure over mature cuboidal osteoblasts (collagen type I+, osteocalcin+) on endosteal cortical surfaces. Using adapted histomorphometic analysis, we determined that 77 ± 2.1% (n = 7) of the endosteal mature osteoblast surface was covered by the F4/80+ OsteoMac canopy. This observation suggested that OsteoMacs are optimally located to regulate osteoblast function in vivo. In summary, we have demonstrated that OsteoMacs are an integral component of bone lining tissues and play a novel role in bone dynamics through regulating osteoblast function. These observations implicate OsteoMacs, in addition to osteoclasts and osteoblasts, as principal participants in bone dynamics. Further delineation of OsteoMac functions is likely to provide new avenues for treating bone disease and assisting bone repair.

Characterisation and recombinant expression of antigens for the rapid diagnosis of West Nile virus infection

Jody Hobson-Peters — 2010-04-21T00:00:00Z

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.

Characterisation of a haemagglutinin from Haemophilus paragallinarum

Hobb, Rhonda I. — 2007-08-24T00:00:00Z

Characterisation of a novel p75 neurotrophin receptor death signalling pathway

Linda May — 2012-04-30T00:00:00Z

The Alzheimer’s disease (AD) peptide amyloid beta1-42 (Aβ42) has been shown to initiate death signalling through the p75 neurotrophin receptor (p75NTR), however little is known about the Aβ42-p75NTR mechanism of cell death or how such a mechanism would fit with what is known about amyloid pathology. Using a combination of in vitro and in vivo techniques, we demonstrate here that Aβ42 has excitatory properties which promote an increase in intracellular levels of calcium in neurons, which in turn causes an increase in G-protein activated inwardly rectifying potassium (GIRK) channels at the plasma membrane. p75NTR activates these GIRK channels to cause a sustained potassium efflux which leads to caspase activation and cell death. We found that the NMDA receptor is involved in the Aβ42-initiated calcium overload in neurons, which drives the GIRK channel response. Further, chronic activation of the inhibitory GABAb receptor removes GIRK channels from the surface of the neuron and overrides neuronal death in vitro. We also show that GIRK channels are upregulated in response to excitotoxic insults in scenarios where p75NTR death signalling occurs. GIRK channels are increased in vivo in the dentate region of the hippocampus after pilocarpine-induced seizures in mice, and in vitro in hippocampal neurons where excessive glutamate challenge potentiates p75NTR-dependent death signalling. We found that p75NTR expression in the mouse hippocampus is increased in vivo due to normal ageing and further increased in a transgenic mouse model of AD and in human AD post-mortem tissue. Finally, we report that in human post-mortem tissue, the levels of SDS-soluble Aβ42, p75NTR and GIRK proteins together accurately predicted clinically diagnosed cases of AD. Conclusion: Our results demonstrate that Aβ42 activates p75NTR and upregulates GIRK channels which activated p75NTR uses to cause potassium efflux and cell death. By precipitating cellular dysregulation of calcium and subsequent inhibitory responses, Aβ42 provides both the impetus and the means for p75NTR death signalling. The p75NTR-GIRK channel death mechanism may provide a paradigm for understanding other forms of excitotoxic neurodegeneration.

Characterisation of a putative G-protein coupled receptor and its protein interacting partner in Arabidopsis

Humphrey, Tania Vivienne. — 2010-12-07T13:17:59Z

Characterisation of a type IV fimbrial gene, fimv, and a re-examination of twitching motility in Pseudomonas aeruginos

Semmler, Annalese Barbara Trudy. — 2007-08-24T00:00:00Z

Characterisation of Australian bat lyssavirus and an Evaluation of the Rabies Vaccines.

Peter Moore — 2012-05-21T00:00:00Z

Australian bat lyssavirus (ABLV) is an endemic Lyssavirus first identified in Australia in 1996 and is most closely related to Rabies virus (RABV). There are two genetically distinct strains of ABLV, the first is transmitted by the four flying foxes (Pteropus sp.) found on the Australian mainland and the second is transmitted by the insectivorous bat Saccolaimus flaviventris. Australian bat lyssavirus has been responsible for the deaths of two Queensland women, in 1996 and 1998 and as such is of public health significance. The main aim of this thesis was to evaluate the efficacy of the rabies vaccines currently used in Australia to vaccinate humans and animals against ABLV infection. The neuroinvasiveness, phenotypic and genetic characteristics of a number of ABLV isolates was also evaluated and compared with specific RABV strains. The full genome of ABLV isolated from a Yellow bellied sheath tailed bat (Saccolaimus flaventris) was sequenced for the first time (chapter 2). Additionally the full genomes of ABLV isolates from each of the four mainland hosts (Pteropus alecto, P. scapulatus, P. conspicillatus and P. poliocephalus) were sequenced for the first time. Phylogenetic trees of each of the five genes were constructed along with the pseudo gene (thought to be a remnant gene located between the G and L genes on the ABLV genome) and a whole genome comparison undertaken. Previous studies of Lyssaviruses had grouped viruses based on host species or geographic location after phylogenetic analysis of whole genome sequences and pseudo gene sequences. However, there was not enough variation between ABLV isolates of the same strain to identify isolates from different bat species or location by sequence data, which correlates with previous studies of individual genes. Chapter 3 identified a pteropid ABLV (Pt-ABLV) isolate with a high neuroinvasiveness (ability to cause disease when the virus is inoculated peripherally) for the vaccine trial. This Pt-ABLV isolate along with a Yellow bellied ABLV (Yb-ABLV) isolate and three Rabies isolates, challenge vaccine standard 24 (CVS-24), a bat Rabies isolate (BRV) and a coyote Rabies isolate (COSRV) were titrated in mice by intra-cerebral (i.c.) and peripheral (footpad f.p.) inoculation. These titrations allowed the neuroinvasiveness of each virus to be calculated for comparison with previous studies. A Pt-ABLV isolate from the salivary gland of a P. alecto was most neuroinvasive followed by the brain isolate of the same Pt-ABLV virus, BRV and CVS-24. The neuroinvasiveness of COSRV and Yb-ABLV could not be calculated as these viruses did not cause sufficient disease by f.p. inoculation. This chapter also examined the difference between incubation periods of Pt-ABLV isolates isolated from the brain and salivary gland of the same flying fox. Additionally, despite Yb-ABLV displaying a low titre by f.p inoculation in mice it had a shorter incubation period on average when compared with Pt-ABLV. Chapter 4 is based on studies of a RABV isolated from a Silver haired bat (Lasionycteris noctivagans) that was responsible for a number of human deaths in the United States of America that had no history of exposure to rabies vectors. It was found that this virus had a higher affinity for epithelial and fibroblast cells when compared with a terrestrial RABV strain and it was concluded that this virus could cause disease from less severe bite wounds. These cell infection assays were repeated with both ABLV strains along with the three RABV strains CVS-24, COSRV and BRV. It was concluded that both ABLV strains possessed similar phenotypic characteristics to SHBRV with Yb-ABLV displaying a high affinity for epithelial cells. In chapter 5 the efficacy of the Rabipur® (human) and Nobivac® (veterinary) rabies vaccines was evaluated, to ascertain protection against Pt-ABLV, the stain of ABLV to which humans are most likely to be exposed. Three vaccination methods, pre exposure, post exposure and post exposure with anti-rabies immune globulin (RIG) were also compared. The study showed that the Rabipur® vaccine protected 100% of mice against Pt-ABLV challenge by both pre exposure and post exposure vaccination with RIG. One hundred percent of mice challenged with CVS-24 were protected by pre exposure vaccination but only 75% were protected by post exposure vaccination with RIG. Additionally when post exposure vaccination without RIG was used only 5% of mice were protected against CVS-24 and 95% against Pt-ABLV. The Nobivac® vaccine protected 100% and 95% of mice vaccinated by pre and post exposure vaccination and challenged with Pt-ABLV, with 90% and 0% protected against challenge with CVS-24. It was also observed that although all mice were challenged with 25MFPLD50 of each virus, the incubation period of Pt-ABLV was four to five times longer than that of CVS-24, aiding successful vaccination.

Characterisation of blood myeloid dendritic cells in mannose binding lectin-sufficient and mannose binding lectin-deficient individuals

Melinda Dean — 2009-11-04T00:00:00Z

Mannose binding lectin (MBL) belongs to the collectin family of soluble pattern recognition molecules that elicit diverse biologic activities. Via multiple carbohydrate-recognition domains (CRD), MBL binds to mannose and N-acetyl-glucosamine oligosaccharides present on the surface of bacteria, fungi and yeast. Following pathogen recognition, MBL activates the complement system via MBL associated serine proteases in a manner independent of antibody and C1 complex. Deficiency in function and level of MBL is found in 25% of otherwise apparently healthy individuals, representing the most prevalent innate immune deficiency. MBL deficiency is a risk factor for the development of infections in humans and mice. The role of MBL as a modulator of infection is complex. MBL deficiency may influence proinflammatory cytokine production, expression of leukocyte adhesion molecules, or vascular damage, during the course of infection. Given that dendritic cells (DC) are antigen presenting cells (APC) with potent capacity to respond to microbial stimulation, I hypothesized that MBL deficiency may be reflected in DC functions associated with microbial stimulation. Initially, I investigated the association of MBL with human immune cells and demonstrated that in both MBL-Sufficient (MBL-S) and MBL-Deficient (MBL-D) individuals, MBL was particularly associated with monocytes. RT-PCR analysis demonstrated MBL was not transcribed by monocytes or other immune cells investigated (T, B, and NK cells, CD11c+DC, immature monocyte derived DC [MoDC], LPS matured MoDC, and granulocytes), suggesting MBL association with the cell surface may be via an adapter or co-receptor. Magnetically separated monocytes but not MoDC bound exogenous purified human plasma MBL (hpMBL). Addition of hpMBL (5 -15 µg/mL) did not induce MoDC activation, and MBL added together with lipopolysaccharide (LPS) did not induce MoDC activation above the level induced by LPS only. In the second part of this study, I used the particulate MBL ligand zymosan (Zy) as a pathogenic stimulus in a whole blood model to gain a greater understanding of the consequences of MBL deficiency. I compared surface phenotype, inflammatory cytokine production and antigen presenting capacity of blood myeloid (M)DC of MBL-D and MBL-S individuals following stimulation with Zy and MBL opsonised Zy (MBL-Zy). Blood MDC in MBL-D individuals, unlike their counterpart in MBL-S individuals, displayed unique functional characteristics, including higher production of proinflammatory cytokines IL-6 and TNF-, but poor capacity for allo-T cell effector cell induction. It appeared that stimulation with MBL-Zy reduced elevated production of IL-6 but not TNF- by blood MDC in MBL-D individuals. In the third part, expression microarray analysis was utilised to provide broad information on the genes and potential signalling pathways involved in the MDC responses in MBL-D and MBL-S individuals following stimulation with Zy and MBL-Zy. MBL-S individuals demonstrated greater capacity to induce T cell and NK cell signalling pathways than MBL-D individuals. Further, MBL acted as a regulator of important inflammatory molecules, namely T-cell receptor zeta (CD247), IFN-γ and perforin 1. The data presented in this study provides novel information on blood MDC function in MBL-S and MBL-D individuals in response to pathogen stimulation, and provided insight into mechanisms involved in the increased frequency of infection observed in MBL-D individuals.

Characterisation of brain injury and the role of neuroinflammation in a rodent model of preterm hypoxia-ischemia

Carty, Michelle Linda — 2012-06-21T00:00:00Z

Characterisation of Cells in the Foreign Body Response

Ms Sarah Le — 2008-11-13T00:00:00Z

Characterisation Of Intracellular Replication And Pathogenesis of Murine Norovirus 1

Jennifer Hyde — 2011-06-21T00:00:00Z

Human Noroviruses (HuNoVs; family Caliciviridae) are the leading cause acute of non-bacterial gastroenteritis worldwide. Despite the prevalence of these viruses within the community there are no specific antiviral therapies or vaccines available to treat or prevent HuNoV infection. As there are no reliable HuNoV culture systems in existence, study of NoV replication, pathogenesis, and immunity has been largely hindered, and relatively little is known about the intracellular events associated with NoV replication. In 2003, isolation of a novel mouse NoV (MNV-1) from immunodeficient laboratory mice provided the first opportunity to study NoV pathogenesis and immunity in a small animal model. This study highlighted the importance of the signal transducer and activator of transcription 1 (STAT1)-mediated innate immune response in protection against lethal MNV-1 infection in immunocompromised mice. Subsequent to this discovery, the identification of the tropism of MNV-1 for macrophage and dendritic cells provided the first reliable tissue culture system for the study of NoV replication, pathogenesis, and immunity. Using MNV-1 as a model for the study of NoV replication, key events associated with MNV-1 replication and pathogenesis were identified and characterised. Specifically, the subcellular localisation of the viral replication complex (RC) as well as the individual open reading frame 1 (ORF1) proteins was identified. It was observed that MNV-1 RNA replication is closely associated with virus-induced vesicular clusters that accumulate in the cytoplasm of infected cells. It was subsequently demonstrated that these virus-induced membranes structures were derived from components of the host secretory pathway, namely the endoplasmic reticulum, the Golgi apparatus, endosomes, and lipids. Individually expressed ORF1 proteins were also shown to exhibit unique subcellular localisation profiles, with NS1-2 localising with markers for the endoplasmic reticulum, NS3 with the Golgi apparatus and intracellular lipids, NS4 with endosomes and the Golgi apparatus, and NS6 with mitochondria. Based on the subcellular localisation patterns observed, potential roles for the individual viral ORF1 proteins in membrane association and proliferation, host cell trafficking, and immune signalling and apoptosis have been proposed. These studies were subsequently extended to examine the role of other cellular components in MNV-1 replication, particularly the role of the cytoskeleton, as it was observed that the MNV-1 RC was closely aligned with the centrosome. It was demonstrated that the microtubule and actin networks play an important role in efficient MNV-1 replication and that the virus also interacts with and recruits acetylated tubulin to the RC. It was subsequently demonstrated that NS3, NS4, and the major structural protein VP1 interact with acetylated tubulin and therefore likely mediate this interaction between MNV-1 and acetylated microtubules. In addition to exploring the role of host components during MNV-1 replication potential strategies used by MNV-1 to evade the host innate immune response was also examined. MNV-1 replication was shown to prevent the phosphorylation of STAT1 in the presence of interferon (IFN) thereby inhibiting the IFN response. It was subsequently demonstrated that the individual N-terminal ORF1 proteins (NS1-2, NS3, and NS4) were able to prevent the translocation of STAT1 and phosphorylated STAT1 to the cell nucleus in the presence of IFN. Additionally, the ability of MNV-1 to modulate other arms of the innate immune response was examined, principally the production of key innate immune cytokines. Here it was shown that during infection MNV-1 was able to inhibit the transcriptional upregulation and production of tumour necrosis factor alpha (TNF-α), interleukin-6 (IL-6), Regulated upon activation, Normal T-cell Expressed, and Secreted (RANTES), and IFN-β, cytokines which play key roles in activating and regulating the innate and adaptive immune responses. Overall, this study illuminates some of the key intracellular events associated with MNV-1 replication and pathogenesis which were previously unknown, including the intracellular localisation of the MNV-1 RC, the potential role of the ORF1 proteins in replication, the exploitation of specific cellular components during replication, and the perturbation of the innate immune response. In the absence of a viable HuNoV culture system, MNV-1 provides an ideal model to study many aspects of NoV biology and replication, and the findings outlined in this study provide important preliminary insights into key events associated with NoV replication and pathogenesis. By better understanding the replication events associated with NoV infection, effective treatments, vaccines, and containment strategies can be employed to control the incidence and spread of this group of medically important viruses.

Characterisation of kunjin replicon vaccines

Ms Paweena Rattanasena — 2008-12-03T00:00:00Z

Characterisation of Lipid Droplet Metabolism

Samantha Murphy — 2011-09-16T00:00:00Z

As obesity reaches epidemic proportions globally, the need for a more thorough understanding of cellular, tissue and whole body lipid regulation is becoming apparent. Within the cell, lipid droplets (LDs) are the primary sites for the storage of excess fatty acids. LDs have only recently been ascribed many of the functional characteristics of bona fide organelles and despite their importance in maintaining lipid homeostasis; many fundamental aspects of LDs remain largely unknown. One of the characteristic features of LDs is their size and distribution, which can vary depending on cell type and metabolic state. Recent studies have suggested that LDs can undergo constitutive homotypic fusion under basal conditions. However, in lipid-loaded cells and in model cells lines such as 3T3-L1 adipocytes, LDs frequently form closely packed clusters, implying that LD fusion does not readily occur. In Chapter Three of this thesis, we developed refined light microscopic methods and strict quantitative criteria to investigate LD fusion. We have now shown that under normal growth conditions, LDs in diverse cell types exhibit very low fusogenic activity. However, by screening a number of pharmacological agents we found that homotypic LD fusion could be triggered in a variety of cell types. This provided a novel cell system in which we could study the regulated fusion of phospholipid monolayers. Using this system we have shown that LD fusion involves a two-step process to produce a single spherical LD. Analysis of LD fusion events revealed that fused LDs maintained their initial volume, with a corresponding decrease in surface area, which suggests rapid removal of membrane from the fused LD. These results highlighted the lack of LD fusion observed under control conditions as well as the extent of LD restructuring that occurred when homotypic LD fusion was triggered. After demonstrating that LDs could undergo regulated homotypic fusion, and in view of the number of studies suggesting a link between LD size and lipid metabolism, we went on to investigate the regulation of LD size and fusion during fatty acid release and lipid storage in adipocytes. Despite being fundamental to adipocyte biology, very little is known about the morphological changes that occur during these processes. The remodelling of lipid droplets to form microlipid droplets (mLDs) is a striking feature of lipolysis in adipocytes. However, once lipolysis ceases, the cell must regain basal morphology. In Chapter Four of this thesis, we have characterised the mLDs that appear throughout the cell during lipolysis. We have shown for the first time that mLDs are bone fide LDs that form in both cultured and primary adipocytes. Using high-resolution electron microscopy and tomography techniques we have demonstrated that mLDs have essentially the same ultrastructure as large LDs and identified novel tethering structures between mLDs and other organelles. We also show that contrary to popular assumption, mLD biogenesis does not involve fission from large LDs or fatty acid esterification, suggesting a role for triglyceride transfer to peripheral sites of mLD formation. Analysis of mLD formation in live cells revealed that an average of 6 mLDs were formed per minute, increasing the total LD surface area by 36.5% after 30 min. We propose that mLDs are formed specifically in response to activation of the lipolysis pathway to increase the surface area available to lipases to augment lipid hydrolysis. We then went on to examine the changes in LD morphology that occurred when lipolysis ceased and lipid storage was promoted. In Chapter Five we have shown that insulin inhibited the lipolytic signalling pathway, rapidly reversing the phosphorylation of perilipin and returning the cell to basal morphology. Further analysis showed that the return to basal morphology was achieved through the formation of macroLDs. For the first time we have demonstrated insulin- and microtubule-dependent restructuring of LDs following lipolysis and shown that this occurred via two mechanisms; mLD growth and homotypic mLD fusion. mLD growth was defined as the gradual increase in mLD size without any apparent interaction with other LDs, suggesting the addition of lipid directly into the mLDs. Two types of mLD fusion were also observed, complete fusion analogous to that triggered by the addition of chemical fusogens where two LDs rapidly fuse to create one spherical LD, and controlled fusion where lipid appeared to be transferred from a donor LD to an acceptor LD. These results demonstrate that in adipocytes LD morphology is altered in accordance with changes to the metabolic state of the cell. Altogether this study highlights the inherent stability of LD structure, providing evidence against LD fission and fusion in cells under normal growth conditions, whilst demonstrating the dynamic restructuring that occurs during the cycle of lipolysis and lipid storage. This work provides novel insights into fundamental characteristics of LD biology.

Characterisation of mammalian cells during protein production - a systems biology approach

Dietmair, Stefanie — 2012-08-21T00:00:00Z

Characterisation of microbial community structure within anaerobic biofilms on municipal sold waste

Song, Hyohak H. — 2007-08-24T00:00:00Z

Characterisation of Myeloid Cells in the Peritoneal Foreign Body Response: Evidence for Phenotypic Plasticity

Jane Mooney — 2012-04-30T00:00:00Z

The implantation of foreign material in the peritoneal cavity initiates a sequence of events including inflammatory cell recruitment and wound healing-like processes, collectively known as the foreign body response (FBR). Previous research has demonstrated that bone marrow-derived cells, many with monocyte/macrophage morphology, are recruited to the implant. This continues until the material is either phagocytosed or encapsulated in tissue comprised predominantly of myofibroblasts. Given that plasticity is a hallmark of cells of the myelomonocytic lineage, and cells intermediate in morphology between macrophage and fibroblast have been detected within the tissue capsule, monocyte/macrophages have been proposed as a possible source of tissue capsule myofibroblasts. However, to date, definitive evidence to support this has not been provided. Thus, this thesis investigates the hypothesis that monocyte/macrophages transdifferentiate to tissue capsule myofibroblasts in the peritoneal FBR. The transgenic MacGreen mouse, in which EGFP expression is controlled by the Cfms (Csf1r) promoter, was used to investigate the time-dependent accumulation of myeloid cells in the peritoneal FBR. An influx of monocytes and neutrophils into the peritoneal cavity within 48 hours of implantation was identified by surface marker expression and FACS analysis of the inflammatory infiltrate. These cells could be distinguished by variable expression levels of EGFP and Ly6C; neutrophils were EGFPlo Ly6Cmed-hi whilst monocytes were EGFPhi and expressed variable levels of Ly6C. Over time (days 7-14), Ly6C expression was down-regulated, concomitant with up-regulation of F4/80, indicative of monocyte-to-macrophage maturation. At the same time, up-regulation of F4/80 on the EGFPlo subset suggested neutrophil-to-macrophage transdifferentiation. The infiltrating EGFP+ Ly6C+ subsets rapidly adhered to the surface of the implanted material, and a similar process of differentiation and maturation was observed for cells in the encapsulating tissue. Over time, EGFP+ cells within the tissue capsule changed morphology from a rounded macrophage-like appearance to an elongated phenotype characteristic of myofibroblasts. Co-expression of EGFP and the myofibroblast marker alpha-smooth muscle (α-SM) actin was also demonstrated with the proportion of EGFP+ α-SM actin+ cells increasing from 11.13 ± 0.67% at day 14 to 50.77 ± 12.85% of total cells at day 28. The critical role of monocyte/macrophages in tissue capsule development was confirmed by clodronate-liposome depletion experiments which resulted in almost complete abrogation of capsule development. Microarray technologies were employed to determine the time-dependent gene expression profile of monocyte/macrophages within the peritoneal FBR. BioLayout Express3D network analysis identified clusters of co-expressed genes associated with EGFPhi cells from the inflammatory infiltrate and tissue capsules over a 28 day time-course. The expression of Acta2 and other genes associated with the actin cytoskeleton (Cnn2, Tnni1 and Tnni2) was demonstrated in early infiltrating cells, suggesting that this transcriptional programme is switched on very early in the inflammatory response, before encapsulation and well before the translated proteins can be detected. Whilst Acta2 was not expressed by tissue capsule cells, a number of other mesenchymal-related genes were expressed including Actc1, Tnni3 and Twist1. Also identified as significant during the process of tissue encapsulation were genes related to angiogenesis and wound repair. Encapsulating cells co-expressed genes characteristic of M1 and M2 macrophages, shifting from an M1-dominated expression profile during the early inflammatory stage of foreign object recognition and encapsulation, to a more reparative M2 phenotype during the later stages of fibrotic tissue capsule formation. Macrophages associated with early encapsulation (day 7) expressed the classical (M1) activation marker Nos2, as well as low levels of the alternative (M2) activation markers Chi3l3 and Chi3l4 (Ym1/2), Ccl17 and Il10. Nos2 expression declined after 7, whilst Chi3l3, Chi3l4, Ccl17 and Il10 expression increased over time, reaching maximal levels after day 21; expression of the classical activation marker Il23a also increased. Taken together, the results show that tissue capsule macrophages possess a mixed M1-M2 activation phenotype that is dynamic and exhibits phenotypic duality. In conclusion, macrophages participating in encapsulation of sterile foreign material implanted in the peritoneal cavity possess significant phenotypic plasticity and display a complex progression of phenotypes, with overlap in M1/M2 gene expression. This M1-M2 switch is coincident with morphological progression from rounded macrophage-like appearance, to a spindle-shaped morphology and acquisition of α-SM actin expression. Whilst the research findings do not support macrophage-to-myofibroblast transdifferentiation, a dual role for macrophages within the peritoneal FBR is clearly established, as evidenced by the progression from a pro-inflammatory to wound healing, or ‘fibroblastoid’, phenotype. Taken together, the results presented in this thesis contribute to the growing body of literature describing distinctive macrophage phenotypes that are dependent on the environmental milieu and coupled to changes in transcriptional output.

Characterisation of Neogenin signalling pathways in polarised epithelial cells

Hayley Cox — 2012-04-21T00:00:00Z

Neural tube formation in the early vertebrate embryo requires highly-tuned regulation of neuroepithelial cell-cell adhesion. The classical cadherins are key mediators of cell-cell adhesion in the neural tube, therefore, a mechanistic understanding of their regulation and dysregulation is paramount for gaining insight into their role in this developmental process. Compelling evidence in the literature identified Neogenin as a potential mediator of cadherin-based cell-cell adhesion, however, the nature of its interaction with the cadherins and its precise role in the regulation of cadherin signalling was largely unknown. The current investigation has used an in vitro epithelial cell model to reveal a number of potential proteins that may mediate co-operation between Neogenin and the cadherins in the early vertebrate brain. Immunofluorescence analysis revealed that Neogenin localised to the epithelial cell-cell junction of CaCo2 cells, where it colocalised with E-cadherin. This placed Neogenin in an ideal subcellular localisation to be able to interact with E-cadherin. The potential for a mutual co-operation between Neogenin and E-cadherin was identified, whereby E-cadherin depletion using siRNA resulted in a loss of Neogenin from the cell-cell junction, and conversely, Neogenin depletion resulted in a severe disruption to the integrity of the cell-cell junction basal to the zonula adherens. The current investigation also revealed that depletion of Neogenin disrupted the integrity of the microtubule cytoskeleton. More specifically, the disruption appeared to be restricted to the dynamic pool of microtubules within the cell, while the stable pool of acetylated microtubules appeared unaffected. Further, a marked increase in the number and length of dynamic microtubule plus-ends identified by the +TIP EB1/3 antibody reiterated that Neogenin may be required for the regulation of the dynamic pool of microtubules specifically. This mechanistic insight has provided a foundation for further investigation into the activity and regulation of Neogenin to determine how it may translate into more complex developmental processes, such as neurulation.

Characterisation of Newly Discovered Human Polyomaviruses WU and KI

Seweryn Bialasiewicz — 2010-11-22T00:00:00Z

Background: Until recently, only two polyomaviruses, JC virus (JCV) and BK virus (BKV), were known to commonly infect humans. In 2007, two new human polyomaviruses, KI polyomavirus (KIV), and WU polyomavirus (WUV) were discovered in respiratory samples of children suffering from acute respiratory tract disease. Due to their relative novelty, many details about KIV and WUV’s viral infectious cycles, pathobiology, and epidemiology could only be assumed by parallel comparison with other polyomaviruses. Thus, further studies were needed to elucidate the epidemiology, and pathogenesis of KIV and WUV. Methods: Epidemiology: Nearly 3000 respiratory samples collected over an entire year were screened for WUV and KIV by PCR, along with 8 other common respiratory viruses. An encapsidation assay was used to determine if whole viral particles were present in the respiratory tract of patients. Detection methods: Two KIV and six WUV real-time PCR (rtPCR) assays were designed and evaluated against a panel of other respiratory and blood pathogens, and clinical samples to establish sensitivity and specificity. Plasmid controls were created to establish analytical sensitivity. Tissue tropism: Collections of blood, urine, upper and lower respiratory tract secretions, faeces, and cerebrospinal fluid from both immunocompetent and immunocompromised adults and children were screened for WUV, KIV, JCV and BKV, and detection rates were compared. Representative WUV and KIV isolates had their VP1 and non-coding control regions (NCCR) sequenced, and analysed for changes respective to their site of detection. Transcription factor binding sites (TFBS) were predicted and compared to those of JCV and BKV. Global WUV diversity and genotyping: Forty nine WUV isolates collected across four continents had their genomes fully sequenced and analysed for levels of diversity, existence of genotypes, impact on predicted protein functional sites and association with disease or patient demographics. A genotyping scheme was also evaluated for fidelity against the whole-genome phylogenetic tree. Results: KIV and WUV showed a 2.6% and 4.5% average annual prevalence, respectively, in the study population. Predominance of WUV (93%) and KIV (78.6%) detections were in patients under the age of five. There was no significant seasonal variation of KIV, or WUV. The level of co-infection of KIV or WUV with other respiratory viruses was 74.7% and 79.7%, respectively. Respiratory symptoms were indistinguishable from those of other common respiratory infections. The encapsidation assay demonstrated that WUV DNA was fully protected from digestion in a respiratory sample with high WUV load. Seven rtPCR assays did not cross react with unrelated organisms, and all could reliably detect 10 copies of genomic DNA equivalents per reaction, which was more 10x more sensitive than conventional methods. Compared to the conventional PCR assays, clinical sensitivity was 100% in one of the KI and two of the WUV rtPCR assays. Clinical specificity was higher than 95% in four of the assays; the calculations suffered from poor sensitivity in the gold standard however. WUV and KIV were predominantly detected in upper and lower respiratory tract specimens, and faeces from paediatric patients. JCV and BKV were primarily detected in blood, urine and faeces from adult patients. WUV and KIV NCCR/VP1 sequence similarity ranged from 99.5-100% and 97.5-100%, respectively. No substantial difference was noted between the TFBS of JCV, BKV and WUV and KIV. Three main WUV genotypes and five subtypes were identified, provisionally termed Ia, Ib, Ic, II, IIIa and IIIb. Overall nucleotide variation was low (0-1.2%), however individual protein sequence variation between subtypes was as high as 3.0%, with VP1 being the most variable gene, indicating a possibility of multiple serotypes. A general geographical association was also noted. The discriminatory power of the previous VP2 fragment typing method was found to be limited and a new larger genotyping region within the VP1 was proposed. Conclusions: Both of the newly-discovered human polyomaviruses are frequently present in the respiratory samples of children suffering from upper and lower respiratory tract disease. The detection peaks for both WUV and KIV indicates an early age of primary exposure followed by reactivation in older age and under immunosuppression, similar to that seen in JCV and BKV. Clinical symptoms were indistinguishable from other respiratory infections, and the high rate of co-infections confounded any direct association with disease, however evidence of active WUV replication was observed. Multiple real-time PCR assays were developed which were sensitive and specific for use in the detection of WUV and KIV. Both novel viruses have a more restricted tissue tropism range in comparison to JCV and BKV, but VP1/NCCR sequences alone do not appear to offer suggestions as to why. The first extensive genomic survey of global WUV diversity revealed multiple genotypes which were more associated with geo-ethnicity rather than clinical disease. Overall, this series of studies has contributed substantial knowledge to the epidemiology and pathobiology of WUV and KIV, and has created validated tools to facilitate further studies into both of these viruses.

Characterisation of novel Australian rhabdoviruses isolated from vertebrates and insects

Aneta Gubala — 2010-02-03T00:00:00Z

As an outcome of very active arbovirus monitoring programs that began in Australia in the 1950s, some of the most diverse and unusual rhabdoviruses in the world have been isolated from this continent. These novel rhabdoviruses represent an important and valuable pool of highly diverse viruses; however, most of them have remained poorly characterised. In light of the significant disease potential of numerous rhabdoviruses, the characterisation of novel rhabdoviruses is indispensable for threat assessment to livestock, wildlife and humans and preparedness for outbreaks. The genetic characterisation of novel viruses is also an essential step for the development of molecular detection assays for improved monitoring and investigations into unidentified disease cases. In this study, the complete genomes of four novel rhabdoviruses have been sequenced and a fifth is close to completion. The substantial new data generated has significantly extended the understanding of the biology and evolution of the Rhabdoviridae. Wongabel virus (WONV), isolated from the biting midge Culicoides austropalpalis, was found to contain a unique genome structure encoding ten genes, including five novel genes (Chapter 2). Analysis by western blotting suggested that four out of the five novel genes were expressed in infected cell cultures. Ngaingan virus (NGAV), isolated from Culicoides brevitarsis, was found to have the largest genome of any rhabdovirus sequenced to date, and with thirteen genes has the largest number of genes of any (-) ssRNA virus sequenced to date (Chapter 3). Seven of the thirteen genes are novel. Similar to viruses in the genus Ephemerovirus (bovine ephemeral fever virus and Adelaide River virus), NGAV contains a second glycoprotein with an unknown function. Phylogenetic analysis places this virus alongside WONV and the north-American bird and mosquito-associated Flanders virus within the Hart Park group that remains to be classified by the ICTV. Screening of various wildlife and livestock sera collected in northern Australia indicated a strong association of NGAV with macropods. Tibrogargan virus (TIBV) and Coastal Plains virus (CPV) were isolated from cattle and Culicoides brevitarsis (TIBV). Past serological surveys reported both viruses to be highly prevalent in cattle in northern Australia and demonstrated that the two viruses share a relatively close relationship at the antigenic level. The genomic analyses revealed that these two viruses have a unique genome organization, with three additional genes (Chapter 4). These additional genes are highly diverged at the sequence level but the encoded putative proteins share a significant conservation of secondary structure elements. The sequencing of these two related viruses has provided a unique opportunity to gain insights into the characteristics and evolution of novel proteins in two different rhabdoviruses. Phylogenetic analyses showed that TIBV and CPV form an independent cluster which does not appear to belong to any of the current genera, but which is most closely related to the genus Ephemerovirus based on N protein analysis. Although neither virus has been associated with disease, a serological survey of various animal sera collected in northern Australia showed that these viruses are currently highly prevalent in sentinel cattle and buffalo. Oak Vale virus (OVRV) was isolated from mosquitoes, Culex edwardsi and Ochlerotatus vigilax, from two geographically diverse regions of Australia located approximately 3000 km apart. The genome of OVRV was found to contain only one novel gene (Chapter 5). Comparatively, the genome of this virus is much less complex than the others in this study, but this virus displays considerable divergence from all other rhabdoviruses. A high seroprevalence for this virus was found in the feral pig population in northern Australia. The data generated from this study represents a considerable increase in the quantity of genetic data available for this viral family, and has revealed the existence of a large number of previously unidentified genes, highlighting that that the potential for complexity within the prototype genomic model of a rhabdovirus is much greater than previously thought. The novel nature of the additional genes provides grounds for further research into rhabdovirus evolution. Analysis of this new data suggests that these viruses cannot be classified into existing genera under the current criteria and it is clear that the taxonomy of the Rhabdoviridae requires revision. The observation that these viruses are currently circulating in livestock and wildlife in northern Australia accentuates the need for closer monitoring of animals and the need for further study of this diverse and fascinating group of viruses.

Characterisation of Novel Starch Materials: Structure-Functionality Relationship

Tan, Ihwa — 2008-11-21T00:00:00Z

Starch is an attractive raw material for biodegradable plastic applications due to its low cost, its availability in large quantities and its excellent thermal process-ability using conventional plastic processing equipments. Despite its attractive potential as a biopolymer material, the use of starch in biodegradable plastic applications is yet limited by its structural and functional properties, which are dictated by its genetic make up. This dissertation involves in-depth characterisations of a range of biotechnologically derived novel starches from different cereal sources to elucidate the relationship between starch structure and functionality. The importance of understanding starch structure-functionality relationship to further the development of starch biodegradable plastics are discussed to identify the research questions, which underlie the motivation of this dissertation and to contextualize the objectives of this dissertation. Diversities in starch macromolecular properties namely the amylose content and amylopectin chain length distribution are evident in these novel starches. The variation in amylopectin structure in these novel starches is explicable by considering the particular inhibition of starch biosynthesis gene expression in the generation of these starch mutants. Amylose content and amylopectin chain length distribution are two separate structural parameters in starch, which influence the granular and functional properties of starch. An improved method to analyse the 13C solid state NMR spectra for native starches was developed in this dissertation and provides the first elucidation on the occurrence of V-type polymorph, which is significant in high amylose starches. An increase in starch amylose content (or decrease in amylopectin content) leads to a decrease in the double helix content and crystallinity. A transition in the double helical packing arrangement of amylopectin side chains from A-type to B-type polymorph is noted for high amylose starches. This can be attributed to the changes in their amylopectin chain length distribution, which leads to the tendency of the glucan chains to form the B-type polymorph during crystallisation from thermodynamic considerations. The application of MTDSC provides the first elucidation on the step transition or heat capacity change, which is noted to occur within the gelatinisation endotherm for all starches. The use of Rheoscope, which allows for simultaneous monitoring of the changes in starch granular and rheological properties during gelatinisation, reveals that the manifested changes in viscosity can be attributed to the increase in the granules size as a result of swelling, the change in granules properties from rigid to more deformable granules due to water penetration and the increase in the viscosity of the continuous phase due to leaching of amylose. The variation in starch gelatinisation thermal properties namely the onset temperature, enthalpy and heat capacity change can be attributed to the variation in amylopectin chain length distribution, amylose content and the amount of starch structural order. A reduction in swelling power with increasing amylose content is consistently noted for all starch types. The variation in starch rheological responses during gelatinisation can be mainly attributed to the swelling ability of starch granules and their granule size distribution (to a lesser extent). Further MTDSC investigations on starch gelatinisation in the presence of water and glycerol with different concentrations indicate that plasticisation of starch granules prior to gelatinisation does not occur. The observed mid-temperature of the step transition (heat capacity change) is more likely due to a change in state of the starch macromolecules from being highly restrained within the granular packing to entangled macromolecules (as the order to disorder transition occurs) rather than due to glass transition. The addition of glycerol promotes starch gelatinisation in a similar way as the addition of water, which suggests that the same structural changes occur during gelatinisation regardless of the solvents used. In summary, the following starch structure-functionality relationships are deduced. The variation in starch macromolecular properties can be attributed to their corresponding mutation of starch biosynthetic genes expression. The variation in starch amylose content affects the extent of structural order inside the granules while the double helix packing arrangement is influenced by the amylopectin chain length distribution. Starch gelatinisation thermal properties are mainly influenced by the amylopectin chain length distribution while the swelling power and rheological properties are mainly affected by the amylose content.

Characterisation of photo-oxo-degradable polyethylene films

Yu-Chieh Hsu — 2012-03-21T00:00:00Z

crop propagation films - a tool to enhance crop growing conditions by minimising the effects of climate fluctuation. It is an economical method to reduce the use of irrigation, pesticide, and fertiliser and to increase crop yield. However, the removal of non-degradable PE films is laborious and the remaining films become a waste problem. A potential solution, such as photo-oxo-degradable PE films, is available but their rate of degradation is not well controlled and would not be suitable for varying crop cycle lengths and farm sites. PE degradation is affected by many factors - the primary structure of PE, type and concentration of pro-degradants used, film processing method and processing conditions, cold drawing, and ageing method (accelerated or natural ageing). One of the key tasks of developing a time-controlled PE degradable film is to understand how various factors play a role in the rate of PE degradation and to understand the change in film properties as the film becomes brittle - termed the embrittlement point. This is the critical feature for crop propagation films where PE transitions from being ductile to brittle, allowing the crop to penetrate and grow through the film. This project identified the dominant factors that influence the degradability of PE - by studying a commercial linear low density PE (LLDPE) grade, Dowlex 2045G catalysed by a Ziegler-Natta catalyst (1-octene/ethylene co-polymer) and a commercial pro-degradant, Ampacet 30091, an iron-stearate based pro-degradant. The Dowlex-Ampacet system was compared with an Elite-Ampacet system to understand the effect of short-chain branching distribution (SCBD), where Elite 5400, is also a 1-octene/ethylene LLDPE catalysed by a metallocene catalyst but with a narrower SCBD than Dowlex. The effects of processing method and cold drawing were also studied. Films were cast extruded and aged in an accelerated ultraviolet light device, QUV, until embrittled. Films were characterised at various stages of degradation by evaluating their mechanical properties, molecular mass, oxidative products, crystal morphology and surface properties. From these results, a conceptual model was developed to understand how PE degraded and became brittle. In a semi-crystalline system, it is well established that the amorphous phase and tie chain molecules are the most susceptible to photo-oxo-degradation, compared to the crystalline phase that is too dense for oxygen diffusion. When PE with added pro-degradant was exposed to UV, free radicals were generated from the decomposition of hydroperoxides initiating chain scission reactions, along with some crosslinking and oxidation. Over time, small chain fragments formed from chain scission diffused together, where this process is known as recrystallisation. Results demonstrated that film embrittlement had little correlation with its oxidative products (as indicated by the carbonyl index calculated from infrared spectroscopy spectra) but correlated well with its crystal morphological properties (from differential scanning calorimetry). Further studies via small angle and wide angle X-ray diffraction showed the inter-lamellar distance decreased significantly as the system became denser, with the critical inter-lamellar spacing reaching about 5 to 6 nm when the film became brittle, regardless of Ampacet pro-degradant concentration. This result correlated well with the changes in double yield points seen in the tensile data, where the absence of the second yield point signified that the tie molecules at the lamellar interface underwent chain scission and could no longer transfer the tensile stress to reach c-axis slip of the lamellar crystals. The key factor that influenced the rate of PE degradation was Ampacet, followed by the initial degree of crystallinity, crystal morphology, chain mobility and regularity of the small chain fragments (formed from chain scission). The main factors affecting the crystal morphology were co-monomer concentration, SCBD, processing method, and cold drawing. This fundamental study on a commercial grade of PE with Ampacet pro-degradant has allowed to gain a better understanding of PE photo-oxo-degradation. This knowledge could assist to design a time-controlled crop propagation PE film suitable for varying crop cycle lengths and for different crop sites across Australia. Not only that, the knowledge gained from this project could be transferred to develop other degradable PE film products such as plastic bags, magazine wraps, and other packaging products.

Characterisation of polarised macrophages and their influence on hepatocyte phenotype and function

Walker, Gene — 2012-07-27T00:00:00Z

Characterisation of samples of ore particles using x-ray micro-tomography

Cakici, Murat — 2010-04-14T00:00:00Z

The degree of mineral liberation is important for the efficiency of subsequent physical separation processes such as froth flotation. Mineral liberation studies involve determining the volumetric abundance or volumetric grade distribution of a specific mineralogical phase in a particular mineral. Currently, methodologies for assessing mineral liberation are laborious regarding sample preparation, analysis time (from weeks to months), and the need for stereological correction. These constraints can be eliminated by using X-ray CT which gives the cross-sections directly from three-dimensional data in shorter time (from ten minutes to hours) with minimal sample preparation. X-ray computed tomography (CT) is a non-destructive technique which allows three-dimensional visualisation of inner structures of an object based on the variations in density and atomic composition. Initially, it was developed as a medical tool for imaging soft tissue and bone. During the last decade, the number of X-ray CT applications in engineering and geology has steadily increased, with the improvements in performance and imaging capabilities. The aim of the present work is to apply X-ray CT technique for finely divided ore samples and to study the relationship between mineral liberation and CT results. Four different ore types were used in this study: Northparkes ore (Australia), Ernest Henry ore (Australia), Keetac ore (USA) and Cannington ore (Australia). Different settings of the desktop X-ray CT technique were applied for each particular ore sample for several ore liberation (particle size distribution) properties. Two dimensional CT images were reconstructed from the three-dimensional X-ray CT data. It was found that the settings for CT technique were a function of the ore type. Particularly in the case of Cannington (high density ore) the best setting conditions split from the rest of the ores tested. The appearance of different artifacts occurring during the analysis were studied and kept to the minimum. A functionality between mineral liberation and CT results was found. The variables affecting the most the results were the Voltage and Minimum Intensity Percentage. Contrary to the expected trends, variables having a negligible effect on the results were found to be exposure time / equivalent Al filter thickness.

Characterisation of shale ash packed-column biofilm reactor for shale wastewater treatment

Ho, Ngai Man — 2013-02-05T09:43:18Z

Characterisation of the epidemiology and molecular biology of Koala retrovirus (KoRV)

Tarlinton, R. E. — 2008-11-21T00:00:00Z

Gammaretroviruses are known to cause leukaemia, lymphoma and immunosuppression in many species and the original isolation of KoRV (koala retrovirus) (Hanger et al 2000) was made as part of a study into the cause of the high rate of leukaemia and lymphoma in koalas. The virus however displayed some unusual features. While classified as an endogenous (inherited) gammaretrovirus it was unusually active. While endogenous viruses are very common with up to 8% of the human genome consisting of retroviral material they are usually mutated and inactive (Gifford and Tristem 2002). KoRV possessed a full length replication competent genome, was actively transcribed and produced viral particles. KoRV was also very closely related to the exogenous (horizontally transmitted) retrovirus of Gibbons (Gibbon ape leukaemia virus or GaLV). Normally closely related viruses are found in closely related species. This unusual similarity between KoRV and GaLV raised the possibility of a recent host species jump (Hanger et al 2000). This thesis makes a case for the recent introduction of KoRV into the koala genome and ongoing endogenisation into the koala genome. KoRV is demonstrated definitively as an endogenous virus but present at a mixed prevalence within the Australian koala population. The thesis also further characterizes KoRVs activity and establishes an association between KoRV and disease in koalas. It is difficult to prove that a virus present in all animals in a population (as is the case for most endogenous viruses) is the cause of a particular disease syndrome. Here real time PCR was used to quantify the level of viraemia and proviral DNA load in the blood of individuals. A significant association between high viral load and leukaemia and lymphoma was demonstrated. This was further confirmed in a follow up study of a group of animals where those with a high viral RNA level were at a greater risk of dying from neoplasia. The real time PCR study also indicated that animals suffering from clinical chlamydiosis had higher viral RNA levels than their healthy counterparts though this was not significant. This disease association was further strengthened when KoRV free populations were identified and were shown to have lower incidences of these diseases. The real time PCR studies demonstrated a considerable variation in the proviral copy number in individual animals. With most endogenous retroviruses the proviral copy number is fixed within the species or population (Boeke and Stoye 1997). Southern blotting was used to confirm this variation in copy number and also demonstrated variation in the pattern of these proviral inserts between unrelated animals. The random insertion pattern of KoRV provirus loci was further confirmed using cytogenetics. Sequencing of the KoRV envelope gene revealed marked variation in sequence within individual animals. These sequences despite considerable mutation in some cases were all potentially functional and indicate positive selection pressure for active virus within the individual animals. Inverse PCR was used to identify the koala genomic sequence interrupted by the proviral loci. While koala genomic sequence was successfully amplified no potential oncogenes were found. This study did however demonstrate the presence of truncated KoRV sequences missing a large part of the gagpro- pol gene. Electron microscopy also demonstrated KoRV viral particles in the bone marrow of a leukaemic animal. KoRV had originally been classified as an endogenous virus based on the fact that it was present in all animals tested and in all tissues of individual animals. However the accepted definition of an endogenous virus is one that is present in the germ line DNA and is inherited over several generations (Boeke and Stoye 1997). Single cell PCR of koala sperm was used to demonstrate that KoRV is present in germ line DNA. This was confirmed using fluorescent in situ hybridization (FISH) techniques. Southern blotting of blood obtained from a group of related captive animals showed the inheritance of proviral loci over several generations. To determine whether all koalas carried KoRV loci, samples from geographically diverse populations were obtained. As expected all animals in Queensland were positive for KoRV using PCR techniques. Unexpectedly animals from Kangaroo Island in South Australia were free of KoRV. Samples obtained from Victorian animals demonstrated a mixed KoRV status. The animals on Kangaroo Island have been isolated from other populations since the 1920s and this raises the possibility that KoRV has integrated into the mainland koala population during the last 100 years. This is unprecedented for an endogenous virus with the most recent estimate for integration in other species being the type C viruses of pigs 5000 years ago (Mang et al 2001). The mixed prevalence of KoRV and its high activity indicate that this virus is still undergoing the process of endogenisation and provides a unique opportunity to study this process in a wild animal population.

Characterisation of the epidermal differentiation complex (EDC) on mouse chromosome 3 and human chromosome 1q21 : characterisation of Flg-2, a novel epidermal protein that is abundantly expressed in mammalian epidermis and functionally related to filaggr

Listwan, Pawel — 2007-08-24T00:00:00Z

Characterisation of the G3BP family of proteins

French, Juliet Danielle. — 2007-08-24T00:00:00Z

Characterisation of the Host Immune Response in Cystic Fibrosis Mice

Palmer, James — 2008-11-21T00:00:00Z

Lung disease is the major cause of mortality in cystic fibrosis patients. Lack of functional cystic fibrosis conductance transmembrane conductance regulator (CFTR) in airway epithelia alters the environment of the lung, predisposing patients to chronic infections predominated by the opportunistic pathogen Pseudomonas aeruginosa. Such infections cause an exaggerated host inflammatory response characterised by a sustained neutrophilic influx which ultimately destroys the lung tissue resulting in respiratory failure. In addition to the inability for CF patients to effectively clear airway infections evidence exists suggesting an innate defect in inflammatory signalling pathways due to a lack of CFTR, priming the CF lung for a heightened state of inflammation even in the absence of detectable infection. In order to develop effective strategies for dealing with CF lung disease a greater understanding is required of the dysregulation in the molecular signalling pathways that leads to the uncontrolled state of inflammation in the CF lung. The G551D CF mouse previously generated in our laboratory exhibits characteristic CF phenotypes including altered chloride conduction in the airways, reduced ability for bacterial clearance and a dysregulated lung inflammatory response upon bacterial challenge. The work presented in this study further characterises the response in these animals upon stimulation with an intratracheal dose of bacterial lipopolysaccharide (LPS). This treatment regime was sufficient to initiate an acute inflammatory response in the lungs of both WT and CF mice, from which animals of both genotypes routinely recovered. G551D animals consistently lost a greater amount of weight than their WT counterparts and exhibited enhanced recruitment of inflammatory cells, predominantly neutrophils, to the lungs. To profile transcriptional responses occurring in the lung after exposure to inflammatory stimulus both CF and WT animals were treated with LPS and sacrificed six hours post-treatment. Six hours marks a point where no discernable difference existed between the two genotypes, as measured by cellular infiltrate and cytokine levels in the airways. cDNA and oligonucleotide microarrays were utilised to determine relative differences in transcriptional activity between CF and WT mice. A series of comparisons was performed to incorporate potential differences in gene expression levels between the genotypes in the absence of stimulus. A robust transcriptional response was detected in both WT and CF animals in response to LPS. The majority of genes were similarly regulated in both genotypes, leaving overall levels of transcript comparable after stimulus. Similarly, few differences were detected between CF and WT mice not exposed to LPS. Analysis of microarrays directly comparing transcript levels in the lungs of WT and CF mice exposed to LPS revealed a number of genes expressed at lower levels in the CF samples which shared the common feature of being known targets of interferon signalling. Independent validation of a series of molecules integral to interferon beta signalling confirmed genes downstream of interferon beta, namely Stat1, Irf7 and Tap2, were consistently expressed at lower levels in the CF lung compared to WT after exposure to LPS. While all three of these genes were transcriptionally upregulated by LPS in both genotypes the response was diminished in the CF lung. Whether or not this perturbation in signalling stems from a deficiency in production of interferon beta itself or is a result of impaired downstream events remains to be elucidated. Nevertheless, with increasing evidence promoting interferon beta as an important molecule in inflammatory regulation the data presented in this thesis suggests this as a worthy point of intervention in managing the persistent deleterious effects associated with CF lung disease.

Characterisation of the immunopathology associated with cerebral malaria

Louise Randall — 2009-03-02T00:00:00Z

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection, predominantly experienced by children in sub-Saharan Africa. Patients with CM are comatose and often convulse, develop retinal haemorrhages and motor abnormalities. Recent histological studies on brain tissue obtained from patients who have died from CM have identified heterogeneity in brain pathology. As a result, CM is considered to be a complex disease that may be comprised of a number of syndromes. Patients admitted to hospital with CM are treated with anti-malaria drugs; however, even in the best equipped hospitals, a large number of CM patients die within the first 24-hours following hospital admission before the anti-malarial treatment can have an effect. For this reason, it is critical that the mechanisms leading to CM are elucidated in order to develop effective adjunct therapies. Experimental cerebral malaria (ECM) caused by P. berghei ANKA (PbA) infection of susceptible mice displays many features of human CM. A key feature of this model is the pivotal role of the host immune response in pathogenesis, particularly the involvement of T cells. Evidence, predominantly from ECM studies, suggests that tumour necrosis factor (TNF) superfamily (TNFSF) members play critical roles in the immunopathology associated with CM. The first hypothesis investigated in this thesis was that key immune response pathways contribute to the development of CM and, despite the heterogeneity observed between CM patients, common pathways exist that may be targeted to prevent CM. The second hypothesis tested was that members of the TNF superfamily modulate the immune response to infection and are involved in the development of pathology observed in severe malaria (SM). In order to investigate the above hypotheses, three projects were carried out. First, we examined the great heterogeneity in brain expression profiles between ECM-susceptible CBA/CaH (CBA) and C57BL/6 (B6) mice at the peak of disease, as well as the significant differences in circulating cytokine expression and expansion of microglia in brain tissue. We found that, despite these differences, common therapeutic and preventative strategies existed to disrupt the development of ECM in the two ECM-susceptible mouse strains. Second, studies in ECM mice have identified T cells and TNFSF members, TNF and lymphotoxin (LT)-a, as critical mediators of ECM pathology. We extend these studies to examine the role of the TNFSF member LIGHT in ECM. Specific blockade of LIGHT signalling through its receptor, LTβR, in PbA-infected B6 mice abrogated the hallmark features of ECM brain pathology and improved the control of parasite growth. Importantly, specific blockade of LIGHT-LTβR signalling caused the expansion of splenic monocytes and an overall enhanced capacity to remove and process antigen during infection. Together, this study discovered a novel pathogenic role for LIGHT and LTβR in ECM and identified this TNF family receptor-ligand interaction as a potential target for therapeutic intervention in SM. Finally, we investigated the role of LTa in human SM and, more specifically, CM. We tested whether the polymorphisms within the gene encoding LTa (LTA) were associated with susceptibility to SM in Papuan Highland children and adults who had migrated from an area without malaria pressure to a region where malaria is endemic. Despite a lack of association between single nucleotide polymorphisms (SNPs) in the LTA/TNF locus and susceptibility to SM in Papuan Highland children and adults, we found a significant association between a SNP in the LTa-related gene encoding galactin-2 (LGALS2) and susceptibility to CM in children, but not adults in this study population. Interestingly, no association was found between this SNP and susceptibility to CM in Tanzanian children originating from and living in a malaria endemic region. These results suggest that there may be differences in the mechanisms leading to CM in adults and children, as well as between individuals from malaria endemic and non-endemic areas. Together, the findings outlined in this thesis are important to both the understanding of the underlying mechanisms leading to CM and to the development of improved interventions and adjunct therapies.

Characterisation of the mechanical properties of thin solid films

Liu, Sheng — 2013-01-15T15:19:52Z

Characterisation of the mouse hnRNP A2/B1 gene and its protein isoforms

Hatfield, Jodie — 2008-11-21T00:00:00Z

The heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 gene participates in at least six cellular processes: telomere biogenesis and maintenance, packaging of nascent premRNAs, alternative splicing, mRNA stability, mRNA cytoplasmic trafficking and translation initiation. At the start of this project the mouse gene had not yet been described, however, the human gene was known to produce two protein isoforms, A2 and B1, but the functional boundaries between these two proteins were ill defined. The characterisation of the mouse hnRNP A2/B1 gene and its protein isoforms was, therefore, undertaken to facilitate research in mouse which would help to define functional differences between the protein isoforms. The mouse gene was found to be a 14 kb, single-copy gene located on chromosome 6 B3, comprised of 12 exons and 11 introns. This gene was regulated by a housekeeping-style promoter which was predicted to be affected by processes such as cell differentiation and maturation, cell cycle progression and stress responses. Transcription from this gene, and the human orthologue, gave rise to a number of RNAs that encoded four protein isoforms through alternative splicing of exons 2 and 9: B1, A2, B1b and A2b. In mouse, the expression and alternative splicing of the hnRNP A2/B1 transcripts were influenced by tissue-type and age. A subset of these transcripts also exhibited alterations to their 3 UTRs such as retention of the 11th intron, alternative polyadenylation and 3 UTR extension. The extended 3 UTR region, intron 11 and sequence flanking the alternatively spliced exons were highly conserved between mouse and human (over 90%) and were predicted to contain regulatory motifs that governed post-transcriptional modifications to the RNA messages. The mouse genome was also shown to contain at least six processed pseudogenes derived from the hnRNP A2/B1 gene, five of which were unlikely to be functional due to frequent variations from the real gene sequence, including insertions, deletions and rearrangements. The remaining pseudogene, pseudogene 1, could give rise to an independent source of A2 proteins and its status is yet to be determined. To date, it is unclear what role each of the four hnRNP A2/B1 protein isoforms play in the cell. It was proposed that protein isoforms with a unique functional role would exhibit differences in subcellular localisation and expression patterns. In order to test this hypothesis, a number of biological tools were developed to distinguish between the highly similar proteins. In addition to two rabbit polyclonal antibodies previously raised in our lab that recognised all four protein isoforms (HA2) and isoforms B1 and B1b (HB1), another four polyclonal antibodies were raised in rabbits and chickens. These antibodies were raised to recognise the following combinations: all four proteins (chicken HA2), A2 and A2b (chicken Hx1/3), B1 and A2 (rabbit Hx9) and A2b and B1b (rabbit Hx8/10). These antibodies were demonstrated to be suitable for Western analyses and immunostaining studies, although antibodies Hx1/3, Hx8/10 and chicken HA2 will require further purification to reduce nonspecific signals seen in immunostaining experiments. A number of fluorescently tagged fusion proteins were also engineered for each of the A2/B1 protein isoforms. Western analyses showed that in mouse tissues and a number of immortalised cell lines, A2 and B1 were the major hnRNP A-type proteins present. Their abundance varied between tissue- and cell-type, but both were constitutively expressed. In contrast, A2b was only present in younger mouse tissues and was absent from most immortalised cell lines, while B1b was not detected in the samples examined. Localisation studies showed that in the immortalised cell lines, HaCaT and PC12, the expressed protein isoforms and related fusion proteins localised to the cell nucleus but were excluded from the nucleoli and exhibited distinct perinucleolar staining. Little difference was observed between the localisation patterns of the protein isoforms A2 and B1 in these cell types. Interestingly in oligodendrocytes, a cell type known to carry out A2-dependent mRNA trafficking, B1 was localised to the nucleus, A2 was predominantly nuclear but also exhibited some cytoplasmic localisation and A2b was distributed almost evenly between the nucleus and cytoplasm (M. Maggipinto and R. Smith, personal communication). Hence, differences were observed in the localisation of these proteins but were cell-type dependent. In conclusion, the hnRNP A2/B1 splice variants and their protein products were differentially expressed temporally and spatially, suggesting that these highly similar protein isoforms were likely to possess unique functional roles. Future research using the biological tools developed and described in this thesis, will be directed towards further defining the different roles each of the protein isoforms may have in the cellular processes associated with the hnRNP A2/B1 gene. Lastly, the hnRNP A2/B1 gene belongs to a sub-family of hnRNP genes that have arisen through chromosomal duplication events and include hnRNPs A1 and A3. Comparisons of their gene structures and subsequent RT-PCR assays have revealed that, in addition to the two previously described splice variants of the hnRNP A3 gene, A3a and A3b, the eighth exon of the A3 transcript was also alternatively spliced in humans and gave rise to two new variants termed A3c and A3d.