Platelets are an essential factor in wound repair and blood clotting, where exposed sub-endothelial extracellular matrix (ECM) proteins induce activation during vascular injury. However, platelets can also be activated by a diverse range of stimuli that share little-to-no resemblance in structure to each other, or to recognized ligands of platelet receptors. These stimuli include diesel exhaust particles (DEP), various peptides including 4N1-1 and Champs, lipoproteins such as PAM3-CSK4, and large polysaccharides for example, fucoidan, and dextran. In this thesis, I demonstrate that this seemingly miscellaneous group of stimuli cause aggregation of human and mouse platelets through Src and Syk tyrosine kinases in association with stimulus-specific tyrosine phosphorylation of the GPVI/FcRγ-chain complex and/or CLEC-2. A critical role for GPVI and/or CLEC-2 in mediating aggregation is shown using platelets from receptor-deficient mouse platelets. Additionally, in double deficient mouse platelets these stimuli activate Src tyrosine kinases independent of GPVI and CLEC-2. DEP, fucoidan and dextran were shown to activate transfected GPVI or CLEC-2 in a cell line model. However, 4N1-1, Champs and PAM3-CSK4 did not activate transfected GPVI or CLEC-2 in a cell line model, nor could they bind to recombinant forms of either receptor. In addition, I demonstrate the unexpected observation that fibrin also activates GPVI revealing a new stage of haemostasis in which the generation of fibrin from fibrinogen reinforces platelet activation.

The effect of the chemoattractant bourgeonal on [Ca\(^{2+}\)]i and chemotaxis in human sperm was investigated. Burgeonal induced a dose-dependent, slowly-developing tonic elevation in [Ca\(^{2+}\)]i, The response was dependent on capacitation. In low-Ca\(^{2+}\) or EGTA-buffered saline the response to bourgeonal was inhibited. Pretreating spermatozoa with bis-phenol (20μM) to release stored Ca\(^{2+}\) did not alter the response. Thus bourgeonal acts primarily by inducing Ca\(^{2+}\) influx. Treatment of sperm with bourgeonal caused an increase in [cAMP]. When cells were pretreted with bourgeonal in low-Ca\(^{2+}\)saline, subsequent introduction of Ca\(^{2+}\) resulted in a single, large [Ca\(^{2+}\)]i transient in >75% of the cells, indicating that sudden influx of Ca\(^{2+}\) caused closure of the bourgeonal-sensitive Ca\(^{2+}\)- channel. This negative feedback was not modulated by IBMX (1mM) or dbcAMP (1mM), indicating that cAMP was not involved and that a direct action Ca\(^{2+}\) was more likely. Both Ni\(^{2+}\) (10μM) and La\(^{3+}\) (100μM) inhibited the action of bourgeonal on [Ca\(^{2+}\)]i, suggesting a possible role of CNG channels. Exposing sperm to a temporal bourgeonal gradient caused a series of transient [Ca\(^{2+}\)]i elevations in >20% of the cells. A gradient of progesterone (another characterised chemoattractant for human sperm) induced similar Ca\(^{2+}\) oscillations (in >20% of the cells), which increased in amplitude and frequency in response to the increasing progesterone concentration. Human spermatozoa responded chemotactically to a 1nM bourgeonal gradient, Chemotaxis was dependent on capacitation. The response was inhibited in low [Ca\(^{2+}\)]o but was unaltered by TMB-8 (an inhibitor of stored Ca\(^{2+}\) store release), thus showing a dependence on Ca\(^{2+}\) influx similar to the [Ca\(^{2+}\)]i signal.

Chronic obstructive pulmonary disease (COPD) is characterized with a poorly reversible airflow limitation. It is a major public health challenge in many developed countries. Cardiovascular complication is a common comorbidity in COPD and is linked to hypoxia and inflammation. Arterial stiffness is also correlated with emphysema severity in COPD patients, with a suggestion that this might be related to systemic inflammation. In this thesis, we have looked to identify the potential interaction of hypoxia and inflammation upon arterial stiffness of alpha-1 antitrypsin deficient subjects. In addition, we investigated the influence of inflammatory mediators and hypoxia on vascular dysfunction in an in vivo and in vitro rat model. Our results have shown that serum TN F-a levels correlated positively and significantly with arterial stiffness as measured by pulse wave velocity (PWV). Regression analysis revealed that TNF-a is not an independent predictor of PWV. However, when combined in a regression model with age, sex, Forced Expiratory Volume in 1 second, systolic pressure and gas transfer factor TLCO, The model was effectively able to predict PWV. There was no significant interaction observed between hypoxia and inflammation to modulate arterial stiffness. In a rat model, we found that TNF-a had significant effect upon pulmonary vascular dilatation in response to carbachol and sodium nitroprusside. Hypoxia applied for 1-2 weeks in vivo had no effect upon of vascular reactivity. However, when combined with hypoxia, the effect ofTNF-a on vasodilation was further impaired. These data suggest that a combination of inflammation and hypoxia can have a combined and detrimental effect upon vascular reactivity and so provide a potential model for studying these exacerbations in vitro.

Haemoglobinopathies are inherited disorders, typically detected during neonatal healthcare screening programmes. Haemoglobinopathies are characterised by either a reduction in the synthesis of the globin chains or by point mutations in the globin gene often leading to a single amino acid substitution in the globin chain. Current screening techniques analyse samples from resolubilised dried blood spots (DBS) by HPLC and/or isoelectric focusing. These methods are characterised by lengthy sample preparation and/or ambiguous variant determination. Further analysis is required for unequivocal diagnosis. The work presented here describes a method for the unambiguous diagnosis of haemoglobin variants in neonatal DBS samples using a surface sampling technique called liquid extraction surface analysis (LESA) coupled with high resolution top-down mass spectrometry. LESA allows DBS samples to be sampled directly and the material recovered to be directly electrosprayed into a mass spectrometer. Both the sampling and MS/MS (CID/ETD) parameters were optimised to yield maximum globin chain sequence coverage (up to 81 %) allowing for the unambiguous diagnosis of common Hb variants such as HbS and so-called unknown variants (variants unable to be diagnosed during the standard screening protocol). The LESA sampling technique has also successfully been applied to study Hb non-covalent interactions.

Phosphoinositide-specific phospholipase C enzymes (PLCs) cleave the plasma membrane phospholipid PtdIns(4,5)P\(_2\) to generate two messengers, inositol (1,4,5) trisphosphate [Ins(1,4,5)P\(_3\)] and diacylglycerol (DAG). Ins(1,4,5)P\(_3\) is an important second messenger in animal cells. It releases calcium from intracellular stores by opening Ins(1,4,5)P\(_3\) receptors (InsP\(_3\)Rs) and is formed in response to activation of cell surface hormone and growth factor receptors. It is also important for its immediate phosphorylation to form higher phosphorylated inositol phosphates (InsPs), which are critical for various cell signaling functions. Sub-families of PLC enzymes exist in cells and the regulation of the \(\beta\) and \(\gamma\) type PLCs via G-protein coupled receptors and receptor tyrosine kinases respectively, is well understood. In contrast, the regulation of PLC-\(\delta\)s is still a mystery. This is particularly frustrating because these enzymes are the only isoforms of PLC found in fungi and plants, as well as animals, and thus are likely to perform an ancient and important function. Plc1p is the single PLC of the \(\delta\) family in the budding yeast Sacchromyces cerevisiae. It plays a key role in generating Ins(1,4,5)P\(_3\) in this fungi in response to stress and yet the yeast genome encodes no Ins(1,4,5)P\(_3\) receptor. This suggests that the functions of PLC-\(\delta\)s are mediated in a novel fashion, probably occurs via the higher inositol phosphates that derived upon rapid phosphorylation of Ins(1,4,5)P\(_3\) by a series of kinases (Arg82p, Ipk1p, Kcs1p and Vip1p). This study focuses on the localisation of GFP-Plc1p in order to gain insight into its function. Plc1p is present in both cytoplasm and nucleus. Plc1p is too large to diffuse through the nuclear pore complex, and thus relies on nuclear export signals (NES) and nuclear localisation signals (NLS) to facilitate its passage through the nuclear envelope. By creating mutants of Plc1p that are restricted to one of these compartments: GFP-Plc1p\(^{CAAX}\) for plasma membrane localisation, GFP-Plc1p\(^{NES}\) for nucleus and GFP-Plc1p\(^{PKI}\) for cytoplasm, I investigated which defects persist in yeast expressing these mutants as their sole Plc1p. My data suggest that these mutants do appear to display distinct subsets of phenotypes consistent with the idea of separate pools of inositol phosphates. I showed that both cytoplasmic and nuclear pools of Plc1p are important for function as neither PLC1\(^{NES}\) nor PLC1\(^{PKI}\) rescue the stress sensitivity of plc1\(\Delta\) mutants. Therefore, Plc1p are likely to shuttle in between different compartments to exert its diverse functions. It will be instructive to characterise inositol phosphate metabolism in these mutants to determine in which compartments basal and stimulated rises in inositol phosphates occur in yeast cells. In addition, I report the novel finding that Plc1p interacts with one (or more than one) of its metabolites. Plc1p appears to associate tightly with the plasma membrane in the absence of the inositol phosphate kinase Arg82p, most likely due to the absence of Arg82p-derived inositol polyphosphates, and such association is required for intact PH, X-Y and C2 domain – disruption in any of these domains would result in failure of such association. Hence, higher phosphorylated inositol phosphates seem to influence Plc1p’s localisation, and a physiological feedback cycle of regulation probably exists where Plc1p may be regulated by the products of its own catalytic activity. Future studies will seek to understand the role of this feedback cycle in stress signalling and physiology in yeast cells.

The pathogenic fungi Cryptococcus neoformans and Cryptococcus gattii are two of the main causes of life-threatening meningoencephalitis in immunocompromised and immunocompetent individuals respectively. Following inhalation, cryptococci are engulfed by phagocytic cells in the lung and previous studies by our group and others have demonstrated that they are then able to survive inside these cells (especially macrophages), thus acting as intracellular parasites. This intracellular phase is thought to underlie the ability of the pathogens to remain latent for long periods of time within infected individuals. Here, we demonstrate that cryptococci can also manipulate host macrophages in order to mediate an exquisitely controlled ‘escape’ process. This expulsive process, which we have termed ‘vomocytosis’, can occur either into the extracellular milieu or, remarkably, into neighbouring host cells, thus resulting in direct cell-to-cell transmission (‘lateral transfer’). After vomocytosis, both the host macrophages and the expelled cryptococci appear morphologically normal and continue to proliferate. Vomocytosis therefore represents an important mechanism by which pathogens are able to escape from phagocytic cells without triggering host cell death and thus inflammation. Moreover, direct cell-to-cell spread of cryptococci allows the pathogen to remain concealed from the immune system and protected from antifungal agents, thus achieving long-term latency. This project has also provided a possible explanation for the molecular cause of a recent C. gattii outbreak on Vancouver Island, Canada. We found that isolates from the outbreak have dramatically increased their ability to replicate within macrophages in comparison with other C. gattii strains, despite the fact that they are genetically very similar to each other. We further demonstrate that such enhanced intracellular parasitism is directly linked to virulence in a murine model of cryptococcosis, suggesting that this ability might be the cause of the outbreak. Finally, application of high-density whole genome tiled arrays, confocal microscopy and mating assays reveal regulation of mitochondrial activity to be a major driver of virulence in this pathogen group. Taken together, these data indicate that a recent change in mitochondrial regulation within the C. gattii lineage has led to an increased intracellular proliferative capacity, resulting in the hypervirulent phenotype that underlies the outbreak. Such shifts in intracellular replication capacity may be a widespread phenomenon in other human pathogens and could potentially underpin disease epidemics caused by otherwise unrelated pathogens.

Thyme is one of the best known genera because of its diverse medicinal and culinary uses. To understand plant response to drought, a range of genotypes of thyme was examined including Thymus vulgaris, T. serpyllum, T. daenensis, T. kotchyanous, T. capitata and T. zygis. Drought stress was imposed on 30 day old plants and some morpho-physiological traits were measured. Together these traits indicated that T. serpyllum was the most tolerant and T. vulgaris the most susceptible populations. Metabolite profiling using direct-infusion FT-ICR mass spectrometry identified differences in both polar and non-polar fractions. These results suggested that mechanisms adapting thyme to drought may include osmotic adjustment, ROS scavenging, cellular components protection, membrane lipid changes and hormone activity in which the key metabolites were proline, betaine, mannitol, sorbitol, ascorbate, JA, SA, ABA precursor, unsaturated fatty acids and tocopherol. Profiling of volatiles using GC/MS, showed an increasing-decreasing trend at major terpenes apart from thymol, alpha-cubebene and germacrene in sensitive plants. These results suggests that tolerant and susceptible populations of thyme employing different strategies in response to drought. In conclusion, the combination of metabolite profiling and physiological parameters contributed to a greater understanding of the mechanisms of thyme plant response at metabolomics level.

Detergent-free solubilisation using a polymer of styrene maleic acid (SMA) has proven useful in the study of membrane proteins. SMA was employed to solubilise G-protein-coupled receptors (GPCRs) from mammalian cells, into SMA lipid particles (SMALPs). Optimal SMALP solubilisation conditions were determined to be 2 % (w/v) SMA, at 37 °C for 1 h retaining wildtype (WT)-like pharmacological profiles and conferring improved stability on GPCRs over detergent micelles. Endoplasmic reticulum (ER)-retention motifs –KDEL and –KHILFRRRRRGFRQ were found not to be applicable to all proteins. Study of SMALP-solubilisation of intracellular membranes was prevented by the inability to retain the adenosine 2a receptor (A2aR) in the ER. A cysteine-nul A2aR construct was produced containing two reporter groups and behaved as WT-A2aR. This construct is ready for use in fluorescence studies to further understanding of A2aR and the use of SMALPs in biophysical techniques. A range of GPCRs, from different GPCR subfamilies, were SMALP-solubilised with retention of ligand binding capability. Methods were successfully developed to reduce non-specific binding arising from ionic interactions of ligand and SMA. Finally, in a world first, the SMA analogue styrene maleimide, was shown to solubilise GPCRs with retention of ligand binding capability.

The canonical vertebrate neurotrophin receptors — the Trk and p75\(^N\)\(^T\)\(^R\) protein families — have not been found in Drosophila, thus it is unclear how neurotrophic signalling is implemented in fruit flies. The Drosophila genome encodes 9 proteins with extracellular domain compositions resembling vertebrate Trks, but lacking an intracellular tyrosine kinase. Here, I investigated whether these 9 proteins, plus 3 further candidates, can function as receptors of the Drosophila neurotrophins (DNTs). Initial characterisation of the candidate proteins highlighted the Kekkon (Kek) family as potential receptors. After tool and mutant generation, I showed that: (1) kek2, kek3 and kek6 overexpression rescues the semi-lethality of DNT1DNT2 double mutants; (2) Kek3, Kek4 and Kek6 interact with DNT2 ligand, eliciting a luciferase readout in S2 cells; and (3) kek3, kek4 and kek6 genetically interact with DNT1 and DNT2. Further analysis revealed Kek6 is expressed in, and required for targeting of, motor neurons, and has a role in locomotion. Surprisingly, Kek4 was enriched in the larval ring gland and affected developmental timing by inhibiting juvenile hormone. Together, data revealed the Keks function in neurodevelopment and growth regulation, and interact with the DNTs. The work uncovered functional conservation of protein domains in neurodevelopment despite domain shuffling throughout the animal kingdom.

NMR spectroscopy is a primary analytical approach of metabolomics. Although 1D 1H NMR spectroscopy is versatile, highly reproducible and widely used, analysis of complex biological samples yields congested spectra with many overlapping signals. This makes metabolite identification and quantification challenging. 1H J-resolved (JRES) experiments spreads this high signal density into a second dimension, simplifying the spectral analysis. This thesis analyses the approaches and suitability of JRES spectroscopy to analyse metabolomics data. Firstly, the robustness of the JRES experiment is investigated. Using spectral relative standard deviation, benchmarks of spectral robustness can be compared between disparate processing techniques, sample types and analytical platforms. JRES spectra were found to be suitable for metabolomics experiments. Secondly, the application of standard metabolomic analysis methods to JRES spectra was examined. Using principal component analysis, the classification accuracy of 1D 1H and JRES spectra were investigated using several data sets. Alongside, three scaling methods were also evaluated. It was found that 2D JRES spectra and the glog transformation could produce 100% classification accuracy. Finally, spectral deconvolution of 2D JRES spectra from line-shape fitting was investigated Here, the mathematical functions describing the JRES line-shape, under several different processing conditions, are derived and used to create a semi-automated metabolite identification and quantification algorithm. Furthermore, possible quantitation errors arising from using JRES spectra are investigated, evaluating effects such as the overlapping of dispersive tails of nearby signals. In conclusion, the JRES experiment is a suitable for use in the field of metabolomics.