The innervation of the salivary gland of the cockroach Nauphoeta cinerea (Olivier) has been investigated with the use of light and scanning electron microscopy (SEM). Light microscopy revealed the presence of a dual innervation arising from the central nerve cord and the stomadeal nervous system; the principal innervation is that from the central nerve cord which passes to the gland via the reservoir ducts. Branches of these nerves form a plexus on the acinar surface, the axons of which exhibit swellings at irregular intervals. The presence of this plexus and the axonal swellings was confirmed by SEM both in normal glands and in those in which the basement membrane had been removed by means of an ICI-collagenase digestion method. Cell bodies associated with the larger axons of the duct nerves were identified in the sub-oesophageal ganglion using an axonal filling method employing cobalt chloride and horseradish peroxidase. No acinar plexus was apparently formed by branches of the stomatogastric nerve associated with the gland. Other branches of this nerve were connected with a network of multipolar neurones on the surfaces of both salivary reservoirs. Intracellular recordings from the gland cells revealed that a hyperpolarizing response evoked by electrical stimulation of the duct nerve was graded according to the number of stimuli. The biogenic amines, adrenaline, dopamine, noradrenaline, 5-hydroxytryptamine and octopamine, produced dose-dependent hyperpolarizing responses. A quantitative study of the inhibition by phentolamine on the responses to nerve stimulation and the bath applied agonists was made. The investigation showed that phentolamine discriminates between two kinds of receptor in this gland, one binding 5-hydroxytryptamine and the other combining with the catecholamines and the neurotransmitter. The inhibition appeared to be competitive and measures of phentolamines affinity constant gave values of 0.015 (,aM)- and 1 (dM)- for each type of receptor respectively. It was concluded that the neurotransmitter in the cockroach salivary gland was probably dopamine.

The principal aim of this thesis was to investigate the role of nuclear factor-kappa B (NF-κB) in the developing nervous system. NF-κB is a ubiquitously expressed transcription factor that plays a key role in regulating the expression of genes involved in a variety of cellular processes, including innate and adaptive immune responses, stress responses, cell survival, proliferation and differentiation. In the nervous system, NF-κB plays a role in regulating neuronal survival and has been implicated in learning and memory. In sensory neurons of the nodose ganglion of newborn mice cultured with BDNF, inhibiting NF-κB activation with super-repressor IκB-α, BAY 11-7082 (IκB-α phosphorylation inhibitor) or N-acetyl-Leu-Leu-norleucinal (proteosomal degradation inhibitor) or inhibiting NF-κB transcriptional activity with κB decoy DNA substantially reduced neurite arbour size and complexity while having no effect on survival. This novel role of NF-κB signalling in regulating neurite growth and morphology was found to be restricted to neurons cultured from mice between the ages of E18 and P1, a phase of development immediately after the phase of naturally occurring neuronal death when the processes and connections of the remaining neurons are extensively modified and refined. Monitoring NF-κB dependent transcriptional activity with a GFP reporter revealed a basal level of activity that was unaffected by BDNF. NF-κB was also found to be involved in promoting neurite growth from nodose neurons grown with CNTF, but not when these neurons were grown with the related cytokine LIF. Investigating the potential role of NF-κB signalling in regulating the growth of sympathetic neurites was complicated by the fact that NF-κB signalling is involved in mediating the survival-promoting effects of NGF, the neutrophin that promotes the survival of these neurons during development. To circumvent this problem, caspase inhibitors were used to prevent the death of superior cervical ganglion neurons in which NF-κB activation was prevented using either the inhibitor peptide SN50 or BAY 11-7082. These treatments resulted in significantly smaller neurite arbors seen in the presence of NGF, suggesting that NF-κB also plays a role in regulating the growth and complexity of developing sympathetic neurons.

This thesis assesses the potential use of PrP^c as a surrogate marker for CJD by an analysis of blood from vCJD patients, sCJD patients, non-CJD neurological controls and healthy adults. PrP^c was measured by DELFIA and cell-associated PrP was measured by flow cytometry. These are differences in free and cell-associated PrP found in blood of CJD patients and control groups, some of which may be useful as surrogate markers of disease DELFIA analysis identified a significant reduction in the concentration of PrP^c in the whole blood of vCJD and non-CJD neurological patients compared with healthy adults. A significant elevation was found in plasma PrP^c in sCJD patients compared with healthy adults and neurological controls. Flow cytometry found no significant differences between groups in the expression of PrP on platelets and lymphocytes. Neurological controls show significantly less PrP on red cells than healthy adults. In addition the development of a DELFIA based test designed for the detection of the disease-associated PrP^Sc in human peripheral blood is the other main focus of research studies detailed within this thesis. Sensitive assays have been developed using existing techniques allowing the detection of PrP^Sc in the central nervous system and peripheral lymophoreticular tissues of patients with vCJD as validatory studies prior to application of these assays to patient blood samples. Atomic dielectric resonance spectroscopy analysis techniques have also been used to investigate potential differences in frequency and atomic resonance, which may allow identification of characteristics distinct to vCJD peripheral blood samples.

Previously, disruptions of the cyclic adenosine monophosphate (cAMP) signalling pathway have been found to affect olfactory learning in Drosophila. On a neuroanatomical level, the mushroom bodies and central complex have also been implicated in this process. In this study, four P-GAL4 enhancer trap lines were identified, which when used to drive expression of a constitutively activated stimulatory heterotrimeric GTP-binding protein alpha subunit (Gs), were disrupted in their ability to perform an associative olfactory learning task. In three of these lines, expression of activated Gs eliminated learning. In the fourth line, expression of activated Gs reduced learning by about half compared with controls. By contrast, expression of wild type Gs in these P-GAL4 lines had no effect on associative learning. While no P-GAL4 insertion was exclusively expressed in the mushroom bodies, all four showed prominent preferential expression in these structures. In addition, the P-GAL4 insertion producing a partial learning defect was expressed in a restricted subset of mushroom body neurons. Gross mushroom body morphology was not obviously disrupted in these lines, although more subtle defects in mushroom body morphology or development could not be excluded by this analysis. Expression of activated Gs in components of the central complex had no effect on associative learning, suggesting that such disruption to Gs signalling were insufficient to interfere with olfactory learning. Taken together, these data represent functional evidence that regulated Gs signalling within mushroom body neurons of the Drosophila brain is required for associative olfactory learning.

We have developed upon existing immobilised metal-affinity chromatography (IMAC) techniques for capturing phosphopeptides, to selectively purify phosphoproteins from complex mixtures. Using this novel approach, combining both protein and peptide IMAC and MS data acquisition strategies, a comprehensive map of the mouse forebrain cytosolic phosphoproteome was achieved. This new approach was applied to purified mouse forebrain synaptosomes and resulted in the first large-scale map of the mouse synapse phosphoproteome. We have detected over 650 phosphorylation events, corresponding to 331 unique phosphorylation sites on synaptic proteins. 92% of these phosphorylation sites are novel, indicating a previously underestimated complexity in synaptic signalling. Bioinformatic and in vitro phosphorylation assays of peptide arrays suggest a small number of kinases phosphorylate many proteins and each substrate is phosphorylated by many kinases. In recent years, mass spectrometry (MS) based analysis of the postsynaptic density (PSD) and receptor complexes, have established for the first time a detailed list of its molecular components. In order to provide a coherent map of the molecular components of the synapse proteome, a bioinformatic approach was used to combine six PSD and three postsynaptic receptor complex datasets into a single database of synaptic proteins. This process of data integration allowed comparisons of analytical approaches used and revealed the most effective biochemical and MS-based methodologies. This data was used as a framework on which multiple data sources were integrated and allowed the derivation of proteome-wide molecular network maps at the level of gene regulation, protein interaction and protein phosphorylation. These maps were merged with functional or phenotype data from individual molecule studies to derive new models of synapse function.

Chapter I contains a summary of solidification theory, including theories of the interface processes in pure materials and alloys and applications of analytical diffusion treatments to complex solidification problems. Chapter II contains more detailed literature surveys related to the present work. Section (a) gives a summary of work on dendritic growth, spacing and segregation, including a collation of spacing data, to complement an earlier, more discursive review.⁽¹⁾ Section (b) gives an extensive discussion of theoretical and experimental studies of temperature gradient zone melting and similar migration phenomena. Section (c) is a summary of experimental studies using transparent organic analogues for metallic solidification as in the present work, while section (d) covers previous work⁽¹⁾ by the author. Literature surveys also appear in chapter IV(a) (dendrite tip shapes, effects of convection and the container surface), Chapter V(a) (interdendritic solidification models), chapter VIII(c) (theory of fibre coarsening) and chapter VIII(d) (temperature gradient zone melting during crystal growth.) Chapter III describes the temperature gradient stages for the optical microscope used in the experimental work of chapters IV and VII. A new type of stage was designed for fixed-area studies during solidification, in which the hot and cold plates were together driven across the stage while the specimen cell was held stationary in the microscope viewfield. Horizontal and vertical thermal gradients in specimen cells, with and without the plates moving, were measured with thermocouples and by observing temperature gradient zone melting. As a result, the apparatus was modified to reduce local convective heat losses, to control the heating plate temperatures and reduce heat transfer problems in order to make the thermal field reproducible, and to minimize vertical thermal gradients in cells. Some observations on optical perfection and image contrast are also presented. Chapter IV describes experimental studies on dendritic growth of succinonitrile and CBr₄ alloys in thin specimen cells using the above apparatus. The effects of constraint by the cell walls on growth direction were studied, partly to test the effectiveness of the technique in reproducing bulk dendritic growth features but also to investigate the crystallography of preferred dendritic growth direction. For a given material, dendritic growth form depended largely on grain orientation, and some orientations produced growth forms reasonably similar to bulk growth. There were, however, distinct differences between different alloys although all were cubic, non-faceting, with preferred growth direction [100]. Dendrite spacings and tip temperatures were also measured. There was clear evidence of the influence of convection on primary spacing. Growth temperature measurements were in reasonable agreement with theory. ⁽⁵¹⁾ Chapter V covers theoretical developments of established models of interdendritic solidification. Liquid-state diffusion is discussed in order to correct errors in earlier work and integrate the diffusion analysis with the more intuitive "complete mixing"⁽⁴⁹⁾ treatment of dendritic solidification. An analytical treatment⁽⁴⁹⁾ of solid-state diffusion is marginally improved and included in the analysis but found to remain quite inaccurate. Previous calculations on microsegregation in aluminium-copper alloys⁽¹⁰³⁾ are re-assessed, using self-consistency of the results to obtain a reliable estimate of the solid-state diffusion coefficient, and it is concluded that only one-quarter of the observed⁽¹⁰⁴⁾ homogenization during solidification can be explained by the solid-state diffusion model. Chapter VI describes theoretical work on temperature gradient zone melting. In previous work, the concentration gradient across a migrating region has generally been assumed, e.g. as linear. Such an assumption is not adequate to discuss TGZM during solidification. In this chapter, exact TGZM diffusion solutions are presented for special cases. These are then used to derive approximate solutions of wider validity. Besides describing TGZM during solidification, these solutions show serious faults in previous treatments of fibre coarsening by differential migration⁽¹²⁴⁾ (see also chap. VTII(c)), droplet shape distorsions,⁽¹⁴⁴⁾ and interface stability during TGZM.⁽¹²⁹⁾ Solid-state diffusion outside migrating zones and droplets is also analyzed: "forces" on migrating droplets and droplet shape distorsions and retardations during TGZM are discussed. An expression is given for the distance migrated during solidification by an interdendritic liquid pool. Chapter VII covers experimental studies of TGZM in organics. Migration of interdendritic liquid pools during solidification was shown experimentally to be similar to droplet migration in a static thermal gradient. Detailed observations of migration velocities as a function of droplet size in succinonitrile showed considerable variations at low temperatures. Possible explanations in terms of interface kinetics affected by adsorption, or dislocation dragging by migrating drops, are presented and discussed. Much smaller size dependences of migration velocity were obtained at higher temperature and these were satisfactorily explained in terms of the curvature effect alone. Grain boundaries in succinonitrile were sometimes observed to migrate up the temperature gradient like liquid drops, suggesting that they (and perhaps also dislocations) can behave as microscopic liquid zones. Some observations on gas bubbles and diffusion coefficient measurements in TGZM are also presented. Chapter VIII includes various applications of TGZM theory. The simplicity of the theory is used to advantage in calculating the purity required of a "pure" material in which phase transformations can be said to be "controlled" by heat flow rather than solute diffusion: a purity ≳ 99-99% is obtained. It is shown that despite their very different thermal properties, organic materials remain good analogues for metals in this respect. The theory of chapter VI is shown not to invalidate theories⁽¹⁸⁾ of interface kinetics in TGZM, but some special features of the melting kinetics are discussed. The theory⁽¹²⁴⁾ of fibre coarsening by differential migration is extensively amended but shown not to account for experimental observations satisfactorily. Using insights gained from the introduction of a dimensionless parameter unifying the theories of various solidification phenomena in a thermal gradient (chap. IV(e)), it is shown that droplets inside a crystal growing with a planar interface often migrate as fast as the crystal grows. Implications for high-quality crystal growth are discussed. Finally, some amendments and extensions to earlier work⁽¹⁾ on homogenization and coarsening by TGZM during dendritic solidification are presented.

A study has been made of the secretory response and the electrical reponse (a hyperpolarization followed by a depolarization) mediated by dopamine receptors of the cockroach (<i>Nauphoeta cinerea</i> Olivier) salivary gland <i>in-vitro</i>. Although domperidone did not inhibit the electrical response to dopamine, three other actions were observed: one, post-synaptic, led to the potentiation of the hyperpolarization; this action was shared by (±)sulpiride. A separate post-synaptic action resulted in the inhibition of the depolarizing phase of the response. Finally a pre-synaptic action led to the abolition of the response to nerve stimulation.<i>Effects of the calmodulin antagonists W7 and calmidazolium</i>. In an attempt to investigate the role of calmodulin in stimulus-secretion coupling with the salivary gland, the actions of two calmodulin antagonists, W7 and calmidazolium, were studied. In high concentrations, but within the range in which they are known to inhibit calmodulin, W7 and calmidazolium were found to inhibit dopamine-induced secretion and hyperpolarize the acinar cells. The hyperpolarization was not inhibited by SGH23390, and resulted from an increase in cytosolic free calcium, released from the same source as that accessed by dopamine. Lower concentrations of these two antagonists caused submaximal secretion and potentiated dopamine-induced hyperpolarizations. An interpretation of these results is that calmodulin promotes secretion, and exerts a negative control on cytosolic free calcium by an independent process which can be selectively inhibited.

It has previously been shown that 5-hydroxytryptamine (5-HT; serotonin), is able to inhibit high threshold voltage dependent (HVA) calcium currents in 5-HT containing dorsal raphé(DR) neurones via 5-HT<SUB>1A</SUB> receptors (Penington & Kelly, 1990, Neuron, 4,751). This inhibition involves a retardation of current activation, and a partial reduction in amplitude. These calcium currents, and the action of 5-HT, have been further characterised using the whole cell patch clamp method in acutely isolated adult rate DR cells. The effect of temperature on HVA currents has been studied in considerable detail. Results of the temperature dependency are in keeping with the presumed heterogeneous nature of HVA current in neuronal cells. In addition, it was found that a fast inactivating component is uncovered by increases in temperature to 25<SUP>o</SUP>C or above, and this component is voltage sensitive, decreasing in prominence with increasing sizes of test pulses. This is in keeping with at least three HVA components. While the temperature coefficient (Q<SUB>10</SUB>) of current amplitude and inactivation rate was found to be in the expected 2-3 range, the activation rate was discovered to be much more sensitive, being between 10 and 12. Stimulation of 5-HT<SUB>1A</SUB> receptors, or direct G-protein activation had no effect on HVA calcium current amplitude Q<SUB>10</SUB>'s, but led to a dramatic reduction in the activation rate Q<SUB>10</SUB> down to 2-3. This could be reversed by the prior application of large depolarising prepulses prior to the test pulse. The possible involvement of phosphorylation in the maintenance of HVA calcium currents and the action of 5-HT was investigated using phosphatase inhibitors. It was found that phosphatase inhibition tended to lead to a gradual increase in current amplitude over a period of some minutes, but then had no clear effect on the subsequent rate of time dependent decrease in current amplitude ('rundown'). With the single dose of inhibitor used, it was found that 5-HT<SUB>1A</SUB> receptor stimulation, while still causing some reduction in current amplitude, was unable to lead to a reduction in activation rate Q<SUB>10</SUB>.

The guanylyl cyclase-coupled nitric oxide receptor (GC) acts like a classical neurotransmitter receptor, binding the ligand NO and forming the second messenger cGMP. This thesis investigated the regulation of the NO receptor by endogenous regulators and two groups of pharmacological activators. Haem-mimetic compounds activate the NO-insensitive, haem-free form of the receptor. BAY58-2667 activated the haem-free receptor with half the efficacy of NO activating the haem-reduced receptor. These findings prompt reassessment of physiological and pathophysiological roles of the haem-free receptor, and contradict reports that haem mimetics also activate the haem-oxidised receptor. The second group of compounds activates GC by inhibiting receptor deactivation. BAY41-2272 activated purified GC with EC50= 43 23 nM in the presence of maximally stimulating NO concentrations, and this activation was prevented by NO scavengers. BAY41-2272 renders GC the most potent known NO detector (EC50 = 47 3 pM), confirming earlier theoretical predictions (Garthwaite, 2005). Recently a dual-site model for NO-stimulation of GC was proposed (Cary et al., 2005). Predictions of the Cary model were tested on rat cerebellar cells and platelets, using a new technique for delivering repetitive pulses of NO. The findings suggest that the proposed model is of doubtful relevance, and support the existing one site, two states model. The simple model is further refined by incorporation of regulation by nucleotides and Ca2+. In this new model both ATP and substrate GTP act as allosteric regulators. Inhibition by Ca2+ proved rather complex, involving two inhibitory sites that predominately affected NO-stimulated GC activity and also inhibited receptor deactivation. The new model also partially reconciles the different behaviour of GC when purified from its cellular environment. While this new model appears kinetically robust, further investigation is required to properly incorporate Ca2+ into the scheme and also to link these regulatory changes to the underlying structural modifications.

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