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Studies of avian ecological immunology attempt to describe the biotic and abiotic factors which explain natural variation in immune function within and among free-living bird species. Understanding this variation and the trade-offs associated with maintaining appropriate immune defences and individual life history variables has important implications for the conservation of endangered species, many of which are characterised by small population size and reduced genetic diversity. Such species often display increased susceptibility to infectious diseases as a result of inbreeding depression and are prone to the effects of novel parasites and pathogens. This thesis aims to explain the variation in immune function in the endangered, island-endemic Mauritius parakeet (Psittacula echo), a species which has passed through a considerable population bottleneck but now thrives by virtue of ongoing conservation management despite the presence of a highly infectious disease. Identifying the ecological, environmental and genetic elements which define individual immunity offers the potential to predict the survival probability of juvenile individuals in a disease landscape thereby representing an exciting prospect for the field of conservation reintroduction biology. Interactions among indices of immune function are investigated at the individual level for Mauritius parakeets and also at the species level with the sympatrically occurring and non-native Indian ringneck parakeet (Psittacula krameri). Patterns of species-level genetic diversity of the Mauritius parakeet spanning two decades are examined and interspecies variation in immune function and genetic diversity is explored. Productivity and survival of Mauritius parakeets is summarised during and after a disease outbreak and an in depth analysis of the predictors of infection status and immunocompetence in this species is offered. This study highlights the complexity of the immune system and the challenges faced when trying to characterise it among individuals in an ecological context. I reveal a declining trend in species-level genetic diversity among Mauritius parakeets due to low natural dispersal demonstrating the importance of adaptively managing endangered species. I illustrate how, as a result of population bottlenecks or contrasting evolutionary histories that the Mauritius parakeet displays an attenuated immune function when compared to the Indian ringneck parakeet. I find no evidence to suggest that genetic diversity or inbreeding predicts disease infection in Mauritius parakeet nestlings and finally I use long-term monitoring data to cherry pick suitable individuals for reintroduction.

Increasing concentrations of CO2 are being released into the atmosphere from anthropogenic sources which consequently dissolves into the ocean creating carbonic acid. The effect of this is to decrease seawater pH and change the composition of marine carbonate chemistry as a whole. In certain areas of the world’s ocean, there is already a substantial natural variability in carbonate chemistry which, in some cases, can exceed the projected figures for long-term anthropogenic acidification. This is especially true of coastal areas which can be subject to increased human activity but also a larger variation of naturally forced biological activity and hydrographically induced fluctuations of water column properties. Anthropogenic acidification will therefore be layered on top of this natural variability and this could have potentially adverse effects on the marine ecosystem. Studies of coastal areas can aid in ocean acidification research by highlighting how organisms cope under the decreasing levels of alkalinity. Because of this, it is vital that we characterise and quantify the drivers of the natural patterns in the marine carbonate system as well as the anthropogenically forced changes that are now evident. Partial pressure of CO2 (pCO2) is one of the most important parameters to be measured in conjunction with ocean acidification and carbonate chemistry research. High frequency temporal and spatial measurements of pCO2 will provide some understanding of the fluxes and their variability and forcing parameters. To aid the investigation into natural variability of coastal carbonate chemistry, pCO2 sensors are an invaluable tool for ease of in-situ data collection. However, these sensors can require not only specific expertise of utilisation but are also inaccessible to many due to high cost. In lieu of an expensive sensor, the most common way to measure pCO2 in seawater is with discrete sampling of water and subsequent analysis for two of the three parameters of the carbonate system (dissolved inorganic carbon (DIC), Total alkalinity (AT) or pH) which is then used to calculate a final pCO2 value. This method requires a substantial amount of cost, time and labour to not only retrieve seawater from depth, but also employ precise expertise in analyses with each step being potentially fraught with human error. This research addressed these issues by developing a low-cost, easy-to-use sensor which efficiently and accurately measured coastal marine pCO2. This required a research and development stage where the sensor and housing design was tested at The University of Glasgow (Chapter 2 and 3) and also deployed in a temperate (Chapter 4) and tropical (Chapter 5) field environment. Seawater samples were also taken and their carbonate chemistry analysed in conjunction with sensor readings to calibrate and confirm the accuracy of the sensor. Along with the developed sensor and the collection of in-situ pCO2 data, other marine variables were also measured (pH, dissolved oxygen, chlorophyll, salinity, temperature, depth, photosynthetically active radiation, dissolved inorganic carbon and total alkalinity) to obtain a characterisation of the areas and an analysis of the drivers behind these variables. The observed variability in the temperate area of Caol Scotnish, Loch Sween, Scotland was shown to be highly dependent on biological activity and the tidal action which exchanged different water masses into and out of the site. The observed variability in the tropical area of El Quseir, Egypt was shown to be highly dependent on biological activity, temperature and weather events. The sensor coped well in characterising the concentrations of pCO2 in both sites. There is a larger fluctuation of pCO2 in the tropical site than compared with the temperate site which is dictated by the relative hydrography in each area and the particular weather conditions experienced. This research provides industry, scientists and interested parties with a means of monitoring pCO2 levels in the marine environment in an efficient, easy and low-cost manner and contributes to the demand for the development of these sensors to monitor anthropogenically-forced global change which is layered over already in-flux natural carbonate chemistry.

G-protein coupled receptors (GPCRs) are integral membrane proteins which represent primary cellular targets for intracellular signalling. Many of these receptors are altered in disease states and hence are the target for over 50% of marketed drugs. Despite their physiological importance, high-throughput, cell-free assays which measure functional or signalling activity are only recently being investigated. The current approach by the pharmaceutical industry to initially screen compounds for functionality is to use heterologous cell-based assay formats. The aim of this work was to reconstitute a cell-free GPCR signalling system on an appropriate platform (surface) as a prototype for future rapid drug screening and other applications. The proof-of-concept approach involved using the �2A-adrenergic receptor (�2A-AR) containing cell membrane preparations as the model GPCR, reconstituted with a set of heterotrimeric G-proteins; G�i1 and �1�2 (the signal transducing complex being termed a �transductosome�). However, other receptors and G-proteins were also investigated. Receptors were initially obtained from natural (tissue) sources, however in the later stages they were expressed in a heterologous system (insect or mammalian expression system). G-proteins were expressed in Spodoptera frugiperida (Sf9) insect cells using the baculovirus expression system. Receptor expression was verified by radioligand binding assays and endogenous G-proteins were removed from membrane preparations using the chaotropic agent urea to allow for reconstitution with purified G-proteins. Signal transduction through the transductosome was measured using the [35S]GTP�S binding assay. Receptor activated [35S]GTP�S binding was used to determine functional reconstitution and to validate that the system was working in the normal physiological manner both on and off a surface (with surface attachment being via histidine attachment on the G�i1 (6xHIS) subunit). Using the captured (surface-attached) transductosomes, the IC50 values for Rauwolscine, Yohimbine (potent �2-AR antagonists), Prazosin (potent �1- AR antagonist) and Propranolol (�-AR antagonist) displayed the appropriate rank order for this class of receptor. This cell-free, surface-attached signalling complex prototype may have use in the future development of drug screening and discovery assay technologies as well as other applications as an alternative to cell-based assays which are not readily amendable to miniaturisation, long term storage and therefore stable robust microarray formats.

Thesis (Ph. D.)--Brigham Young University. Dept. of Chemistry and Biochemistry, 2006. / Includes bibliographical references.

Primarily expressed by trophoblast cells, human leukocyte antigen-G (HLA-G) plays an essential role in maintaining maternal-fetal immune tolerance. Having previously been detected following heart transplantation, we sought to establish the value of HLA-G in identifying freedom from moderate or severe rejection post-heart transplant, and the capability of its expression in vitro. After assessing myocardial HLA-G expression through immunohistochemistry, we demonstrated that it was significantly more prevalent in non-rejecting than rejecting heart transplant recipients. Utilizing vascular endothelial and smooth muscle cell culture models, we also determined that while HLA-G expression remains tightly regulated, its expression in vitro can be induced following progesterone treatment in a dose-dependent manner. Hence, HLA-G may reliably identify patients with a low immunological risk of developing subsequent clinically significant rejection post-heart transplant. Furthermore, HLA-G expression can be induced in cultured endothelial and smooth muscle cells, which might represent a strategy to protect against allograft rejection and vasculopathy.

Effects of external vibrations (called g-jitter) on Marangoni convection in a liquid bridge were investigated on the International Space Station (ISS) and in ground-based experiments. In ISS, most dominant g-jitter frequency was noted to be ~110 Hz. ISS experiments suggested that the surface vibrations were mainly affected by the aspect ratio (length/diameter ratio), but not the imposed temperature gradient. Liquid bridge surface vibrations agreed well with Ichikawa et al.’s model. Ground-based experiments confirmed that increasing the volume ratio would cause the resonance frequency to increase. When a temperature difference was imposed between the upper and lower disks, for constant aspect and volume ratios, the resonance frequency tended to increase with the decreasing temperature difference. Furthermore, the shift in the resonance frequency due to a temperature difference, was found to be due to Marangoni convection and not due to reduced viscosity or surface tension of the fluid.

Effects of external vibrations (called g-jitter) on Marangoni convection in a liquid bridge were investigated on the International Space Station (ISS) and in ground-based experiments. In ISS, most dominant g-jitter frequency was noted to be ~110 Hz. ISS experiments suggested that the surface vibrations were mainly affected by the aspect ratio (length/diameter ratio), but not the imposed temperature gradient. Liquid bridge surface vibrations agreed well with Ichikawa et al.’s model. Ground-based experiments confirmed that increasing the volume ratio would cause the resonance frequency to increase. When a temperature difference was imposed between the upper and lower disks, for constant aspect and volume ratios, the resonance frequency tended to increase with the decreasing temperature difference. Furthermore, the shift in the resonance frequency due to a temperature difference, was found to be due to Marangoni convection and not due to reduced viscosity or surface tension of the fluid.

Primarily expressed by trophoblast cells, human leukocyte antigen-G (HLA-G) plays an essential role in maintaining maternal-fetal immune tolerance. Having previously been detected following heart transplantation, we sought to establish the value of HLA-G in identifying freedom from moderate or severe rejection post-heart transplant, and the capability of its expression in vitro. After assessing myocardial HLA-G expression through immunohistochemistry, we demonstrated that it was significantly more prevalent in non-rejecting than rejecting heart transplant recipients. Utilizing vascular endothelial and smooth muscle cell culture models, we also determined that while HLA-G expression remains tightly regulated, its expression in vitro can be induced following progesterone treatment in a dose-dependent manner. Hence, HLA-G may reliably identify patients with a low immunological risk of developing subsequent clinically significant rejection post-heart transplant. Furthermore, HLA-G expression can be induced in cultured endothelial and smooth muscle cells, which might represent a strategy to protect against allograft rejection and vasculopathy.

Neuroinflammation and excitotoxicity are frequently regarded as the classical hallmarks of all major central nervous system (CNS) diseases such as stroke and neurodegenerative disorders. However, the limited number of current clinical options for the treatment of these diseases and the side effects associated with these treatment options indicate that there is an urgent and important need to develop drugs that delay neurological diseases. Although the molecular mechanisms underlying these neurological diseases remain poorly understood, it is widely accepted that alterations in microglia function is the key causative factor. It was recently reported that granulocyte colony-stimulating factor (G-CSF) and a natural marine compound, GB9, show great potential as anti-inflammatory agents. In the present study, we used a model of neuroinflammation to investigate the neuroprotective effects of G-CSF and GB9, and whether they exert an anti-neuroinflammatory effect on IFN-£^-stimulated microglia (BV2). Our results revealed that both G-CSF and GB9 attenuate the upregulation of proinflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in IFN-£^-stimulated microglia. Furthermore, Western blot and immunohistochemical analyses revealed that G-CSF or GB9 prevent downregulation of the glutamate transporter (Glu-Asp transporter, GLAST) and activation of the glutamate receptor in the IFN-£^-stimulated microglia. Additionally, our in vivo analyses revealed that centrally administered G-CSF could reverse the increase of OX-42 immunoactivity, which is the marker of IFN-£^-stimulated microglia. In summary, our findings support the hypotheses that G-CSF and the marine compound, GB9, possess anti-neuroinflammatory properties and could be pursued as potential therapeutic agents for CNS diseases.