Programmed cell death is a widespread phenomenon in the developing nervous system. During early development, neurons are initially produced in excess and up to 70% of them are eliminated in later stages of development, during a period of synapse formation with their targets. However, the mechanisms that initiate the death of neurons are not clear. In the avian ciliary ganglion, neurons go through the period of target-dependent cell loss between E8 and E14; however, almost all neurons in the ganglion are prevented from dying by the chronic in ovo treatment with α7-nAChRs specific antagonists, α- bungarotoxin or MLA. Since α7-nAChRs are implicated in the cell death of ciliary ganglion neurons, I tested whether the activation of these receptors directly on the ciliary ganglion neurons facilitates cell death by inducing large increases in intracellular Ca2+. I found that the ciliary ganglion neurons are heterogeneous with respect to their surface α7-nAChR density and, as a result, activation of these receptors by nicotine leads to large increases in [Ca2+]i in some neurons but not in others. Furthermore, immature E8 neurons exhibit slower rates of Ca2+ decay after nicotine stimulation than E13 neurons, suggesting that E8 neurons do not clear [Ca2+]i efficiently and could be more susceptible to Ca2+ overload. Expressing the αbtx that is tethered to the cell membrane via the glycosylphosphatidylinositol anchor (GPIαbtx) in the ciliary ganglion neurons inhibits the increases in [Ca2+]i induced by nicotine through α7-nAChRs specifically. This cellautonomous inhibition of α7-nAChRs prevents cell death of ciliary and choroid neurons. For this to happen, GPIαbtx must be expressed in neurons; the expression of this construct in the surrounding non-neural tissue does not prevent neuronal loss in the ciliary ganglion. Later in development, α7-nAChRs are prevented from inducing cell death by the chicken PSCA molecule that is significantly upregulated in the ciliary ganglion between E8 and E15. The chicken PSCA is neuronal specific molecule that belongs to the Ly-6/neurotoxin superfamily that includes αbtx and lynx1 and compared to other tissues, it is highly expressed in the ciliary ganglion. The expression of the PSCA mRNA in tissues correlates with the expression of α7-nAChR mRNA, suggesting that PSCA modulates the signaling via these receptors. In fact, overexpressing the PSCA in the ciliary ganglion neurons prevents nicotine-induced increases in [Ca2+]i through α7- nAChRs. Misexpressing the PSCA in E8 ciliary ganglion prevents choroid but not ciliary neurons from dying. Therefore afferent inputs can induce cell death by activation of α7- nAChRs in the developing ciliary ganglion by increasing the [Ca2+]i over the threshold for cell death. Upregulation of endogenous prototoxins, such as PSCA, opposes the large increases in [Ca2+]i via α7-nAChRs and prevents these channels from facilitating cell death after the final numbers of neurons have been established. These results indicate that the control of cell death is more complex than originally proposed by the neurotrophic hypothesis and present the mechanism by which cell death in the developing ciliary ganglion is regulated, thus, further highlighting the importance of non-traditional roles of α7-nAChRs during the development of the nervous system.

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Malaria caused by the protozan parasite Plasmodium is one of the most serious infectious diseases in the developing world. It is estimated that malaria causes an annual mortality rate of ~627,000. New drugs are urgently required, as the incidence of resistance is spreading rapidly. Glycosylphosphatidylinositol (GPI) anchored proteins decorate the merozoite surface and several of which, including merozoite surface proteins - 1 and -2 have previously been shown to be essential for erythrocyte invasion and parasite survival. Plasmodium GPI-anchors contain a glycan core consisting of four mannose residues. Therefore, the enzymes involved in the synthesis of activated mannose, guanidine diphosphomannose pyrophosphorylase (GDP-Man PP) and dolichol phosphate mannose synthase (DPMS), are thought to be crucial for GPI-anchor biosynthesis and as such potential drug targets. Double homologous recombination has been exploited to test whether PfGDP-Man PP and PfDPMS are essential during the erythrocytic portion of the parasite's life cycle. Additionally, overexpression parasite lines for both enzymes have been generated and have shown that the regulation of the two enzymes are intricately linked. Focused metabolomics by multi-reaction monitoring of the overexpression lines suggests that the fucosylation pathway may have a novel function within the parasite, possibly as a dynamic store for activated fucose/mannose. In order to determine the cellular concentration of key metabolites within the parasite, the volumes of the intra-erythrocytic stages have been determined and show that the concentration of metabolites in the ring stage parasites is substantially higher than previously thought. Furthermore, the sub-cellular localisation of GDP-Man PP and DPMS has been determined by immunofluorescence assay. The recombinant expression of DPMS in E. coli allowed its active site residues to be probed as well as establishing a platform for inhibitors to be screened against the enzyme. Finally, inhibitors of the T. brucei DPMS enzyme have been screened against the P. falciparum parasites in culture.