Inositol triphosphate (IP<SUB>3</SUB>) receptors, are commonly responsible for release of Ca<SUP>2+</SUP> from intracellular stores. IP<SUB>3</SUB> binding to type 1, but not type 3, IP<SUB>3</SUB> receptors is inhibited by calmodulin in a Ca<SUP>2+</SUP>-independent fashion. Using HSAB-calmodulin to photoaffinity label GST-fusion proteins representing different sequences of the type 1 IP<SUB>3</SUB> receptor, I identified a region that binds calmodulin in both the presence and absence of Ca<SUP>2+</SUP>. This region contains a residue (Ser<SUP>1589</SUP>) that is phosphorylated by protein kinase A, but protein kinase A-catalysed phosphorylation of purified type 1 IP<SUB>3</SUB> receptors was unaffected by calmodulin in either the presence or absence of Ca<SUP>2+</SUP>. I also demonstrated that IP<SUB>3</SUB>-evoked Ca<SUP>2+</SUP> release from cerebellar microsomes and cell types that express predominantly type 1, 2 or 3 IP<SUB>3</SUB> receptors, is similarly susceptible to inhibition by calmodulin. In SH-SY5Y cells, which express largely type 1 IP<SUB>3</SUB> receptors, calmodulin inhibited IP<SUB>3</SUB>-evoked Ca<SUP>2+ </SUP>mobilization only in the presence of Ca<SUP>2+</SUP>. This effect of calmodulin did not result from enhanced metabolism of IP<SUB>3</SUB> since calmodulin also reduced the sensitivity of Ca<SUP>2+</SUP> stores to adenophostin A, a non-metabolizable agonist of IP<SUB>3</SUB> receptors. Thus, in contrast to the inhibition of IP<SUB>3</SUB> binding by calmodulin, inhibition of IP<SUB>3</SUB>-evoked Ca<SUP>2+</SUP>-release occurs only in the presence of Ca<SUP>2+ </SUP>and appears to affect all three subtypes of IP<SUB>3</SUB> receptor. Calmodulin also interacts with type 1 IP<SUB>3</SUB> receptors in the absence of Ca<SUP>2+</SUP>, but the effects of this interaction on IP<SUB>3</SUB> receptor function remain to be determined.

Limbic seizures induced by kainic acid can regulate the level of neurotrophin gene expression in the adult rat hippocampus. I investigate whether non-toxic infusions of glutamatergic or cholinergic agonists that were minimally seizure-behaviourally active can also alter neurotrophin gene expression in the male rat. Stereotaxic intra-ventral hippocampal microinfusions were made of 0.5μ AMPA, carbachol, nicotine and pilocarpine. Animal behaviour was observed for 2 hours post infusion, and was quantified on a scale of limbic seizure activity. After 7 days hippocampal tissue from one group of animals was processed for semi-thin plastic sections, and cell densities calculated using an unbiased disector method. After various survival times, brains from other groups were evaluated by <I>in situ </I>hybridisation using <SUP>35</SUP>sulphur labelled oligonucleotide and ribo-probes for the mRNA of the following genes: NGF; BDNF; NT-3; TrkA; TrkBl; TrkB2; TrkB3 and TrkC. Gene expression levels were measured as optical density values using an NIH image analysis system. AMPA (0.5 nmol), carbachol (3.3 nmol), nicotine (17.3 nmol) and pilocarpine (6 nmol) regulated neurotrophin gene expression at doses that did not cause cellular damage or behavioural apparent seizures. NGF was increased by all agonists, the time course of nicotine differed from the other drugs. The nicotine regulation of NGF was suppressed by the action of glutamatergic antagonists, NK-801 and NBQX. BDNF was only regulated by AMPA and pilocarpine. NT-3 and TrkA were generally depressed by all agonists at earlier time points. The TrkB receptors are all positively regulated by the agonists. These findings suggest sub-toxic activation of glutamate or cholinergic systems can alter hippocampal neurotrophin expression, that there maybe an interaction between the nicotine and glutamatergic systems, and that this regulation may take in the physiology of the septal-hippocampal system.

The research in this thesis investigated the role of different dorsal and ventral striatal regions and their dopaminergic innervations in cocaine-seeking and -taking behaviour under the control of a drug-associated conditioned stimulus (CS). Neurochemical correlates of cocaine-seeking behaviour in rats were first determined by conducting <i>in vivo</i> microdialysis within the nucleus accumbens (NAcc) core and shell subregions during; (1) cocaine self-administration under a second-order schedule of reinforcement whereby cocaine-seeking behaviour was maintained by response-contingent presentations of a drug associated cue and (2) non-contingent presentations of the drug cue. While the results confirmed the involvement of NAcc dopamine (DA) in the pharmacological effects of cocaine, they also revealed a selective DA response in the core, as opposed to the shell, region of the NAcc during non-contingent CS exposure. The same DA system however, did not appear to mediate the ability of the CS to maintain cocaine-seeking behaviour since there was no DA response in either of the NAcc regions to response-contingent CS presentations. The dynamics of DA changes in response to non-contingent CS presentations and cocaine-seeking behaviour in the presence of response-contingent CS presentations were also studied using identical procedures in the dorsolateral striatum, a region implicated in stimulus-response (habit) learning. A robust DA response was observed during the period of cocaine-seeking behaviour under the guidance of the CS, but not during non-contingent presentations of the CS; results that are doubly dissociable from those of the NAcc core region. In the last phase of the studies reported in this thesis, the basis of the differential DA response to non-contingent presentations of the CS in the NAcc core was further investigated by assessing the impact of selectively lesioning the NAcc core and shell subregions on the acquisition of cocaine self-administration, both under a continuous reinforcement schedule (CRf) and a second order schedule of reinforcement.

The aim of this thesis was to study the response of macrophages to infection with <i>Salmonella enterica</i> serovar Typhimurium and to determine whether TLR signalling contributes to the induction of cell death in infected macrophages. When macrophages were infected with wild-type <i>S</i>. Typhimurium, cell death occurred within two hours of infection. This was dependent on the bacterial protein SipB and required caspase-1 activity. At two-hours post-infection, little or no cell death was observed in macrophages infected with a <i>sipB</i>-deficient mutant of <i>S</i>. Typhimurium C5. Cell death was triggered by this mutant by 24-hours post-infection. The role of TLR4 in <i>S</i>. Typhimurium-induced cell death was investigated using macrophages from TLR4 knockout mice. At 24 hours, induction of cell death by <i>S</i>. Typhimurium C5-<i>sipB</i> in TLR4-deficient cells was reduced compared to wild-type controls. Activated TLR4 recruits the adapter proteins MyD88, Mal. Trif and Tram. The induction of cell death in macrophages deficient for each of these adapters was studied.  Preliminary data suggest that signalling through Trif and Tram contributes to the induction of cell death in infected macrophages. MAPK inhibitors were used to investigate the role of MAPK signalling in <i>S</i>. Typhimurium-induced cell death pathways. Specific inhibition of p38 MAPK by SB203580 increased the level of cytotoxicity in infected macrophages. However, PD98059, which inhibits activation of p38, ERK and JNK, had no effect on cell death suggesting that MAPK proteins are involved in both pro-survival and pro-cell death pathways. Inhibition of p38 signalling alone interferes with survival pathways and tilts the balance towards cell death.

Vascular smooth muscle cell (VSMC) proliferation with neointima formation is the key feature of restenosis after successful percutaneous transluminal coronary angioplasty (PTCA), which severely limits the clinical benefits of a major option in the treatment of coronary heart disease. To date, the prevention and therapy of restenosis in humans remain limited or unsuccessful. Natriuretic peptides (NP) are powerful inhibitors of VSMC proliferation <I>in vitro</I>. However, the mechanisms by which NP inhibit VSMC proliferation are still nuclear, and neither are the <I>in vivo</I> effects of NP on VSMC proliferation known. This dissertation is addressed to the study of the interaction of the NP system with VSMC under pathological conditions of arterial injury. Normal VSMC from different species or different arteries all expressed NP receptors (NPR), although in different subtypes. Normal rat carotid arteries expressed type-A NPR (NPR-A), whereas normal rabbit ear central arteries expressed exclusively type-B NPR (NPR-B). Despite this difference in normal arteries, neointimal VSMC in both rat and rabbit arteries expressed the same subtype of NPR, type-C (NPR-C) implying the involvement of NPR-C in the regulation of arterial response to injury. In the rabbit model of arterial injury, the media of damaged arteries also expressed NPR-C besides the NPR-B that already exists in normal arteries. Time course experiments of NPR expression in this model showed that NPR-C were upregulated between 5 and 7 days after arterial injury. Affinity cross-linking demonstrated that the molecular weights of reduced NPR-A, NPR-B and NPR-C in arteries were 120, 130 and 64 kDa, respectively. Second messenger assays showed that NPR-A and NPR-B in the normal arteries were coupled with cGMP system, whereas NPR-C was not found to be coupled to the cGMP nor to the cAMP system.

The work in this thesis is directed towards understanding more about the interplay between glucocorticoids and serotonin in regulating the proliferation of neural progenitor cells in the adult hippocampus. Depleting serotonin (5-HT) by ICV 5,7-DHT (a 5-HT neurotoxin) did not change the number of proliferating cells in the dentate gyrus. Treatment with PCPA (a tryptophan hydroxylase inhibitor) resulted in reduced proliferation as measured by Ki-67 after 3 days treatment, but not by BrdU uptake, and not after 14 days treatment by either method. These results argue against ‘basal’ levels of serotonin playing a role in neurogenesis. Injecting corticosterone for 8 days significantly reduced proliferation in the dentate gyrus. However, serotonin alters the sensitivity of the dentate gyrus to corticosterone. Adrenalectomized (ADX) rats given low doses of replacement corticosterone for 7 days showed reduced proliferation, but this was prevented by depleting 5-HT (ICV 5, 7-DHT). A dose-response study showed that progressive doses of corticosterone (0-40 mg/kg/day) in ADX rats resulted in corresponding reductions in proliferation in 5-HT intact but a reduced effect (shift to the right) in 5-HT depleted rats. These results strongly suggest that serotonin regulates the sensitivity of proliferating cells in the dentate gyrus to corticosterone. Next, we explored the role of 5-HT_1A receptors in the regulation of cell proliferation in the dentate gyrus of the intact and ADX adult rat. Depleting 5-HT with PCPA or stimulating 5-HT­_1A receptors with 8-OH-DPAT by sc injection for 14 days had no effect no cell proliferation in the dentate gyrus. However, combined treatment with PCPA followed by 8-OH-DPAT significantly increased cell proliferation compared to PCPA alone. This suggests that reducing serotonin increases the sensitivity of progenitor cells to 5-HT_1A agonists. Micro-injection of 5,7-DHT in to the fimbria-fornix and the cingulate bundle depleted hippocampal 5-HT (but not other areas of the brain) but did not change cell proliferation 3 weeks after the surgery, thus reinforcing conclusions about ‘basal’ 5-HT (see above).  However, 8-OH-DPAT stimulated cell proliferation in the dentate gyrus of hippocampal 5-HT depleted rats compared with controls. These results suggest that 5-HT_1A modulates cell proliferation in the hippocampus by a direct post-synaptic effect, 8-OH-DPAT delivered by subcutaneous osmotic pumps (rather than sc injection) increased proliferation in intact rats. The 5-HT_1A antagonist WAY-100635 by itself did not alter cell proliferation, but blocked the effect of 8-OH-DPAT. However, WAY-100635 could not block the stimulating action of ADX cell proliferation. 5-HT_1A mRNA expression was not altered in the hippocampus by ADX.

Quiescent, or G0, cells are present as subpopulations within some solid human tumours and exhibit innate resistance to a range of anticancer agents including those which target the enzyme DNA topoisomerase II. They are thought to be chemoresistant due to a) the decreased levels of the target enzyme, b) a requirement for active cell cycle progression for the fixation and recognition of cellular damage and c) the pharmacodynamics of the agent. Drugs which can persistently target DNA cause cellular effects over a time period which extends beyond the initial exposure to drug. Persistence is an important factor for topoisomerase-interactive agents since growth factor stimulation of quiescent breast tumour cells, for example, is associated with an elevation of target enzyme levels. This thesis addresses the effects of topoisomerase inhibition in both asynchronous and quiescent cultures using anthraquinones which persistently trap topoisomerase IIα and induce DNA damage, and uses the techniques of flow cytometry and confocal imaging to examine the subcellular distribution and persistence of these agents. N-oxide derivatives of these agents have been evaluated in an attempt to develop drugs which can be bioreductively-activated under hypoxic conditions to produce persistent topoisomerase II poisons which selectively target G0 cells. The dioxopiperazine, ICRF-193 prevents enzyme activity without generating DNA damage, and has enabled us to investigate the role of DNA topoisomerase IIα in the formation of drug-induced damage. The studies were carried out in a human breast tumour cell system which presented differences in checkpoint integrity, by virtue of p53 mutation, and resistance associated with modified topoisomerase II expression.

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Chapter 1 introduces the concept of response inhibition, while Chapter 2 describes the rodent version of the SST. Chapter 3 clarifies the interpretation of SST variables and their relevance as measures of impulsivity and attention. In Chapter 4, testing various classes of pharmacological agents suggested that noradrenaline (NA) is primarily implicated in ‘stopping’, whereas dopamine (DA) is more involved in optimizing ‘go’ performance. In Chapter 5, inactivation of the dorso-medial (dmPFC) and orbitofrontal cortex impaired stop and go processes, respectively, while enhancing NA transmission in either area speeded SSRT. Within dmPFC, inactivation of the anterior cingulated cortex disrupted action cancellation, whereas silencing the prelimbic cortex impaired action restraint. Infusion of specific pharmacological agents in dmPFC revealed that NA improves stopping performance by modulating sustained attention, the monitoring of performance and stimulus detection, effects that were mediated by α1, α2 and β-adrenoceptors, respectively. Moreover, forebrain NA depletion impaired SSRT only when the attentional demand of the task was increased, whereas dmPFC-lesioned animals were impaired under all load conditions. In Chapter 6, I show that the atypical stimulant modafinil improves inhibitory processes at least in part via α1 noradrenergic receptors located in dmPFC. It is concluded that NA neurotransmission in dmPFC aids response inhibition by increased sustained attention and by facilitating attentional and behavioural shifts. Overall, this Thesis has helped to define the neural and neurochemical basis of inhibitory response control, through the study of pharmacological effects of relevance to human clinical conditions such as attention deficit/hyperactivity disorder.