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This thesis concerns synthetic melanin formation using 3,4-dihydroxyphenylalanine (DOPA), with respect to its possible fibril formation. This study aims to bridge the gap between the understanding of L-DOPA as a precursor molecule and its formation into melanin. Thus, an overview of L-DOPA is presented in three distinct stages: its fluorescence properties as a precursor, the formation of melanin from L-DOPA, and the dependence of melanin’s eventual structure based on environmental properties. Throughout this study, the aim is to directly correlate fluorescence properties with the morphology of melanin and its precursors. In particular, there is an examination of the absorption, fluorescence and lifetime properties of L-DOPA correlated with well-studied naturally fluorescent amino acids – namely tryptophan, tyrosine and phenylalanine – thus allowing examination of the effect of its molecular structural characteristics on fluorescent properties. Next, the role of environmental conditions on the formation of melanin from L-DOPA is examined, and in particular study two systems: one at pH 10 with the addition of ammonia; the other during heating to 37oC . Lastly, research is reported on novel fibril structures observed during synthetic eumelanin formation. To allow direct comparisons of structure and photophysical properties, formations were measured using multiphoton fluorescence lifetime imaging, atomic force microscopy and electron microscopy, thus finally proposing an explanation for melanin fibril synthesis. These studies reveal the continuing importance of the study of L-DOPA and melanin’s chemical and structural properties in the battle against both Parkinson’s disease and malignant melanomas.

Background. Experiments in both humans and animals indicate a prominent role for serotonin in social behaviour. However, little is known about its role in human group interactions, or cognitions about important social relationships including those with close intimate partners. Methods. I developed resource dilemma games in which participants harvested valuable but depletable resources, independently and as part of a social group. I used these games to explore the neural correlates of group resource management in healthy adults; I also investigated the role of serotonin in resource management using Acute Tryptophan Depletion (A TD) and sub-chronic (8 day) treatment with citalopram. Finally, my experiments investigated how serotonin activity influenced cognitive judgments about other peoples' close intimate relationships and their own relationships. Results. The value of a shared resource was represented in distinct neural structures depending upon the use to which this information was put: within reinforcement-related regions including the ventral striatum while harvesting, but within medial prefrontal regions while considering the harvesting behaviour of social partners. Tryptophan depletion was associated with increased frequency of exhausting a shared resource, lower personal gains, and increased sensitivity to others' past harvesting behaviour relative to personal, past harvesting choice. A TD and citalopram had opposite effects on independent resource management in females, enhancing and diminishing, respectively, sensitivity to recent changes in the resource size when selecting harvests. A TD decreased ratings of intimacy and romance in others' relationships, whilst treatment with citalopram reduced perceptions of others' physical relationship quality, the importance of a good physical relationship, and the importance of intimacy with current partners. Both A TD and citalopram treatment modulated ratings of partners' relative dominance, but differently for men and women. In men, citalopram was also associated with reduced perceived discord in others' relationships. Conclusions. Serotonin plays a significant role in the representation and management of valued resources both individual and group settings. Serotonin activity also supports appraisals of the quality of, and power within, close relationships, and modulates the perceived importance of trusting and intimate aspects of interaction between sexual partners. These findings may be relevant to the pathophysiological bases and pharmacological treatment of depression.

Enteric neurons are derived from vagal and sacral neural crest cell populations. Using human embryonic tissue aged 4-14 weeks of gestation the timing and pattern of gastrointestinal colonisation by vagal neural crest cells, and the development of interstitial cells of Cajal and the smooth musculature of the gut, were investigated using immunohistochemistry. Formation of both the enteric nervous system and smooth muscle involves rostrocaudal migration and maturation of these tissues. The gut is fully colonised by neural crest cells at week 7 and these cells first form the myenteric plexus external to the future circular muscle layer. Secondary centripetal migration to the submucosal plexus occurs at week 8 in the oesophagus and week 11 in the midgut. Smooth muscle appears at week 8, first as the circular muscle layer, with secondary formation of the longitudinal layer. ICCs are present from week 8 and develop in close association with the myenteric plexus in the midgut and hindgut. At week 14 all components required for a mature gut are present. Rearranged during transfection (RET), a tyrosine kinase receptor and the most commonly linked gene to the enteric nervous system developmental disorder, Hirschsprung's disease, has been described in vitro as a dependence receptor, i.e.: expression of the receptor causes a cellular dependency on its ligand. In order to test whether RET functions as a dependence receptor in vivo constructs designed to perturb the caspase dependent cleavage of RET were introduced by electroporation of neural crest in E1.5 chicken embryos. Inhibition of Caspase-9 dependent apoptosis reveals a role for cell death in early control of neural crest cell numbers. No effect is observed when intracellular wildtype RET is introduced. However, introduction of a mutated form of RET induces a delay in the migration and differentiation of enteric precursors. These observations, combined with Tunel and ret in situ data, suggest a role for RET as a dependence receptor in vivo via a caspase-9 dependent mechanism.

Approximately 50 % of current pharmaceutical drugs target the G protein-coupled receptor (GPCR) family, rendering an understanding of the mechanisms behind their actions critical. Dopamine is a key neurotransmitter involved in motor function and behaviour and has been associated with disorders such as Parkinson's disease and schizophrenia. Within the present study, a Fluorescence Resonance Energy Transfer (FRET)-based conformational change sensor was created to study dopamine D2S receptor responses to ligands to gain an understanding of the mechanisms underlying ligand-induced activation. Chimaeric receptors were created with a tetracysteine sequence FlAsH binding motif at varying positions within intracellular loop 3 and CFP fused to the C-terrninal where FlAsH and CFP formed a convenient FRET pair. The constructs were transiently transfected into HEK293 cells and characterised using confocal and FRET microscopy. The 353-CFP-D2s receptor was well expressed at the cell surface and produced agonist-induced FRET changes and therefore was used to create a stably expressing HEK293 cell line. Radioligand binding and eSS]GTPyS binding assays showed that the 353-CFP-D2s receptor had a pharmacological profile similar to the native D2S receptor. On application of agonist (NP A, dopamine, m-tyramine, p- tyramine and (- )-3-PPP) to the 353-CFP-D2s receptor, a concentration dependent decrease in FRET was detected which was completely reversed when agonist was removed. The extent of FRET response was found to broadly correlate with the extent of G protein activation. The forward rate constant of the FRET change was also found to alter in an agonist-concentration dependent manner while the reverse rate was concentration independent. Throughout the project, the control and optimisation of experimental variables was found to be crucial for successful use of this FRET -based system. In conclusion, this sensor and the use of this technique could be very useful to aid understanding of activation of this important GPCR. 111.

The post-weaning social isolation reanng rat model utilises early-life, environmental adversity to mimic the neurodevelopmental aetiology of schizophrenia. The overall aim of this thesis was to investigate behavioural and neurochemical changes associated with this model in to deduce whether manipulation of central glutamatergic pathways could account for some of the 'schizophrenia-like' alterations seen. Results confirmed that postweaning-social isolation rearing during adolescence induced a range of 'schizophrenia-like' behavioural changes in the adult rat. Isolation- rearing from weaning induced locomotor hyperactivity in a novel arena which was reversed by the mGluR2/3 agonist, LY379268, impaired performance in a novel object discrimination task which was selectively reversed by LY379268 and the NMDAR antagonist, memantine, reduced prepulse inhibition of the acoustic startle response and reduced conditioned emotional learning in a contextual fear conditioning paradigm. Neither drug had an effect on PPI or emotional processing deficits. Acute systemic administration of PCP to adult rats, with the exception of worsening PPI deficits, had no effect on isolation-rearing induced behavioural changes. Discrete administration of memantine into the mPFC of isolation-reared rats by reverse in vivo microdialysis reduced impairments in novel object discrimination and was accompanied by an apparent increase in extracellular dopamine compared with that in vehicle-treated rats. No task- related changes in extracellular amino acids were seen in either group, thus implying a dopamine-dependent mechanism of action of the drug that warrants further investigation. From these results, it could be hypothesised that dampening ex citatory activity in key brain regions either by voltage-dependent blockade of postsynaptic NMDA receptors or via the activation of presynaptic mGluR2/3 receptors, may have beneficial effects on cognitive performance in this animal model.