Soluble guanylate cyclase is a multidimeric enzyme that regulates cardiovascular homeostasis and is the receptor for nitric oxide in the brain. The enzyme is the known target for a new agonist drug used for the treatment of pulmonary hypertension. Whilst drug discovery has been successful for the finding of small molecules that activate the enzyme, the currently available inhibitors lack selectivity as they act through oxidation of a heme prosthetic group in the enzyme, which is conserved amongst other hemeproteins. Nonetheless, it has been suggested that inhibition of soluble guanylate cyclase by small molecules could be useful in the treatment of neurological conditions such as Parkinson’s Disease. In this thesis, new activators of soluble guanylate cyclase have been identified by virtual screening, and a new class of inhibitors has been designed and synthesised. The synthetic routes developed are efficient and take advantage of microwave-assisted organic synthesis. The drug-target interaction was characterised using a biophysical technique based on surface plasmon resonance, which allows the detection of label-free binding between small molecules and biological macromolecules. The biophysical assay has been developed using different constructs of soluble guanylate cyclase and validated through binding of the natural ligands ATP and GTP. The instrument and assay design were validated using the well-defined interaction between natriuretic peptides and the extracellular domain of natriuretic peptide type-C. Additional biochemical characterisation of the ligands allowed discrimination between activators and inhibitors. This combination of biophysical and biochemical techniques allowed the identification of the catalytic domain of soluble guanylate cyclase as the target for binding of the new class of synthesised inhibitors and has given insight into the functional groups necessary for activity and binding to the enzyme.

Introduction: PDE5 inhibitors improve erections by potentiating nitric oxide (NO)- cyclic guanosine monophosphate system. However, long-term diabetic patients have reduced efficacy secondary to dysfunction of NO system. The aim of this thesis was to investigate in-vitro effects of a PDE5 inhibitor (sildenafil), a soluble guanylate cyclase (sGC) activator (BAY41-2272) and an NO-releasing PDE5 inhibitor (NCX-911) on urogenital smooth muscle in conditions of NO deficiency. Method: The effect of these compounds was investigated on tone and electrical field stimulation-induced nitrergic relaxation of cavernosal (human and rabbit) and anococcygeal (rat) smooth muscle and compared to an NO donor (spermine-NONOate) and non-specific sGC activator (YC-1) in the absence/presence of inhibitor of NO synthesis (L-NAME) or inhibitor of sGC (ODQ). In a diabetic rat model, these compounds were assessed in untreated and L-NAME-treated tissues from non-diabetic and diabetic animals. Results: BAY41-2272 was more potent than YC-1 and spermine-NONOate at relaxing rabbit/human cavernosum. ODQ significantly decreased the potency of BAY41-2272 whereas L-NAME did not. BAY41-2272 potentiated nitrergic responses and partially reversed the inhibition of nitrergic responses by L-NAME. NCX-911 and sildenafil were equally potent at relaxing rabbit and human cavernosum. In presence of L-NAME the potency of sildenafil decreased significantly. Both compounds potentiated nitrergic relaxations equally but failed to induce relaxation in the presence of ODQ. Nitrergic relaxation was significantly decreased in the diabetic rats but still potentiated by BAY41-2272 but not by sildenafil or NCX-911. The potencies of NCX-911 and BAY41- 2272 were unaltered but that of sildenafil was significantly reduced in the diabetic animals. Conclusion: The rank of potency in control tissues was BAY41-2272 > NCX-11 = sildenafil; whereas in NO deficiency BAY 41-2272 > NCX-911 > sildenafil. Endogenous NO derived from nitrergic nerves is significantly decreased in diabetes. NO-releasing PDE5 inhibitors and sGC activators may be effective in management of diabetic erectile dysfunction.

To understand the information flow in neural circuits, it is essential to determine how neurons translate synaptic input into spike output. In the cerebellum, Golgi cells are the only interneurons that inhibit granule cells in the input layer, where mossy fibre (MF) signals converge onto both cell types. Golgi cells also receive inputs from parallel fibres (PFs, granule cell axons) that form synaptic contacts with the molecular layer interneurons and Purkinje cells. This synaptic organisation suggests the Golgi cells may be an important regulatory element in the cerebellar circuit. In this thesis, I used targeted patch-clamp recordings guided by 2-photon microscopy to examine the synaptic input and spike output patterns of Golgi cells in anaesthetised transgenic mice. I found that Golgi cells received bursty and occasionally rhythmic excitatory inputs and sparse inhibitory inputs in vivo. My results also revealed that Golgi cells exhibit low spontaneous firing rates, and their spiking activity can display 1 Hz rhythmicity and synchrony with millisecond precision. Remarkably, Golgi cells usually generated only a single spike time-locked to the stimulus by integrating multiple synaptic inputs during sensory stimulation. These results suggest that Golgi cells encode temporal information in their spikes, and will therefore transfer this message throughout the cerebellar network by controlling granule cell (PF) activity. Purkinje cells, the sole output neurons of the cerebellar cortex, often display large, single "all-or-none" synaptic responses from the strong excitation by climbing-fibre (CF) inputs in vitro. However, the transmission at the CF-Purkinje cell synapse in the intact brain remains poorly understood. Using whole-cell voltage clamp recordings from Purkinje cells in anaesthetised rats, I revealed that the spontaneous CF excitatory postsynaptic currents (CF-EPSCs) can be bursty, indicating that high-frequency olivary axonal signals are transmitted to the cerebellum in vivo.

Septic shock is characterized by systemic vasodilatation and an attenuated pressor response to catecholamines. As the hyperactivity (i.e. more channels remain in the open state) of the vascular adenosine triphosphate (ATP)- sensitive type of potassium (KATP) channels results in vasodilatation, this channel has been implicated in the pathogenesis of septic shock. The studies present in this thesis sought to demonstrate that sepsis activates vascular KATP channels, that channel inhibition reverses hypotension, and that channel hyperactivity is related to an increased gene expression. Rat aortic smooth muscle cells exposed to bacterial endotoxin and the inflammatory mediator interleukin-1β were found to have an increased gene expression of KIR6.1, the channel’s pore-forming subunit, at 48h. This gene induction could be reversed by the nitric oxide synthase inhibitor, 1400W. KIR6.1 mRNA was also increased in mesenteric arteries from rats subjected to faecal peritonitis for 24h. 86Rubidium efflux experiments revealed an increased channel activity in these vessels when stimulated by the KATP-channel opener, levcromakalim; the basal activity, however, remained unchanged. Conscious rats subjected to faecal peritonitis developed an attenuated pressor response to norepinephrine that, unexpectedly, could not be reversed by the channel inhibitor, PNU-37883A; this agent could not either elevate the baseline blood pressure of septic rats. Surprisingly, this attenuated pressor response to PNU-37883A could be partially reversed by the autonomic ganglion blocker, pentolinium, suggesting that during sepsis the vascular KATP channel may be inhibited in vivo by the high sympathetic tone. Therefore, the inhibition of vascular KATP channels may assume a limited role in the treatment of septic shock.

The inferior olivary nucleus is the source of the climbing fibres, one of the two major afferent pathways into the cerebellum. This thesis is concerned with aspects of the cellular anatomy and physiology of neurons in the inferior olive. In the first chapter, I report on the first direct patch-clamp recordings from olivary axons, and show that they fire in short bursts that can relay information about the state of olivary network and modulate plasticity in the cerebellar cortex. A remarkable feature of the olive is the widespread electrotonic coupling between neurons underlying their synchronous firing. In the second chapter I combine electrophysiological and immunohistological methods to characterize the coupling. I reveal the first morphological reconstructions of coupled pairs of olivary neurons, and show that the dendritic spines responsible for coupling neurons have very heterogeneous morphologies. Furthermore, I show that olivary dendrites may contact olivary somata and oligodendrocytes. In the third chapter, I use pharmacology and modelling to study the effect of inhibitory synapses on the coupling between olivary neurons. Confirming a popular theory, I show that GABA-A receptor activation reduces coupling between neurons, and use models to study the effect of location, timing and stochastic properties of the inhibitory input on electrical coupling. The common theme for all our findings is that the remarkable interplay between the anatomy and electrophysiological characteristics of the inferior olive underlies a unique computational unit in the central nervous system.

Background: Successful recruitment, adherence and retention are essential for randomised controlled trials (RCTs) to produce robust and meaningful findings. Studies exploring trial participation predominantly focus on the characteristics and views of participants and staff and often reveal contradictory findings. To date little is known about adherence and retention to RCTs. Aim: To further understanding of recruitment, adherence and retention to non-therapeutic paediatric RCTs, with particular emphasis on the role of social context. Methods: An ethnographic approach was taken using two RCTs as case studies. Participant observation (130 hours) was conducted on a clinical trials unit. Twenty-six trial staff and 56 parents who considered or had participated in the RCTs were interviewed and relevant documents collected. Data were analysed using the principles of thematic analysis. Results: Drawing on Bourdieu’s (1977; 1990) ’Theory of Practice’ and Titmuss’ (1970) ’The Gift Relationship’ it was evident that recruitment, adherence and retention were influenced by the values and beliefs of parents and staff and by the wider context in which the RCTs were conducted. Recruitment and adherence were influenced by the degree of concordance between the philosophies of the trials’ fields and those of the wider fields of parenting, infant feeding, medical research and allergy healthcare. Perceptions of personal and societal benefit were relevant to participation but, reflecting the philosophy of the parenting field, families often prioritised personal benefit.<br/>Conclusion: Open and regular personal communication between parents and staff was particularly important for retention. Trials that maximise personal contact may have more success retaining participants. Comparing recruitment, adherence and retention between the two RCTs illuminated the relevance of the wider context for participation, particularly recruitment and adherence. Conducting a thorough assessment of the context in which an RCT will take place will allow potential barriers to participation to be identified before trial commencement.

Delivery of proteins and nucleic acids into cells is a major challenge to the development of biological therapeutics. Cell penetrating peptides (CPPs) and cationic liposomes have been shown to internalise short interfering RNA (siRNA) to achieve gene silencing, but no standard reagent exists which can safely deliver macromolecules both in vitro and in vivo. Small Molecule Carriers (SMoCs) are amphipathic α-helix mimetics displaying guanidine groups in order to mimic the structure of the CPP penetratin. Previously, SMoCs were shown to effectively deliver active proteins into cells. It is hypothesized that SMoCs may also be applied to the delivery of siRNA into cells in order to knockdown target genes. In addition, since cell surface binding is thought to be a crucial step in CPP internalisation, a new SMoC which optimises binding to proteoglycans may be more efficiently taken up. The aims of this thesis are to optimise the synthesis of the SMoCs in order to increase the quantity of product; to demonstrate that SMoCs may be used as siRNA delivery agents; and to design and synthesize a new SMoC which maximises siRNA uptake. The synthesis of SMoCs has been significantly enhanced, with the development of new reagents to improve the yields and cost of production. The electrostatic interactions of SMoCs with siRNA have been characterised, using NMR to examine a π-cation interaction which may contribute to anion binding, as well as determination of binding affinities using ITC and gel shift assays. Initial experiments using SMoC-siRNA complexes show significant mRNA knowdown which demonstrates the potential of SMoCs as siRNA delivery vectors. Finally, a new SMoC has been designed and synthesized which represents the first in a new class of dendritic SMoCs which are designed to maximise binding to the cell surface. This SMoC is also capable of delivering siRNA into cells, and may also be expanded by the addition of targeting peptides.

Nitric oxide (NO) is a freely diffusible transmitter acting throughout the mammalian nervous system via guanylyl cyclase activation and cGMP production. Since neuronal NO synthesis is linked to NMDA receptor activation, much research has focused on the role of NO in NMDA receptor-dependent long-term potentiation (LTP). The proposed role predicts that exogenous NO, paired with a standard LTP induction protocol, should restore the NO-dependent component of LTP when NMDA receptors are blocked. Surprisingly, however, tests of this prediction have not been reported. Here, it was found that exogenous NO, paired with a 1-s, 100-Hz tetanus during NMDA receptor blockade yielded a slowly-rising, long-lasting potentiation of CA1 field EPSPs in hippocampal slices. Like NO-dependent LTP, this potentiation required the tetanus and was guanylyl cyclase-dependent. Contrary to predictions, however, the NO-induced potentiation was additive with subsequent LTP. At CA1 and other synapses, NO is viewed as a putative retrograde transmitter, generated postsynaptically and acting presynaptically. Discordant with this role, the NO-induced potentiation was not associated with a persistent change in paired-pulse facilitation, an index of presynaptic function. However, endogenous NO did appear to facilitate neurotransmitter release under conditions of basal stimulation. In this case, NO generated by endothelial cells was responsible, perhaps explaining the requirement for endothelium-derived NO in LTP. An NMDA receptor-independent form of LTP involving L-type voltage-gated Ca2+ channels has previously been described at CA1 synapses. Unexpectedly, we found that this type of LTP also required NO, apparently derived solely from neurons. Unfortunately, supposed inhibitors of neuronal NO synthesis, though widely used, were found to be inadequately selective to be of use diagnostically. Finally, presynaptic effects of NO, such as those described above, have been reported to require the guanylyl cyclase α1 subunit. Accordingly, immunohistochemistry was used to investigate the location of this subunit in the hippocampus.

A central goal of systems neuroscience is to understand how brain function can be explained by the activity of intricate neuronal circuits. A major hurdle towards addressing this question has been the lack of appropriate techniques to specifically manipulate individual neurons within functioning networks and to measure their connectivity at single cell resolution. Here I present a new technique, two-photon guided single-cell electroporation, to perform genetic manipulations of individual neurons in the intact mammalian brain in vivo. I demonstrate how stable transgene expression can reliably be induced with high success rates both in single neurons as well as in spatially defined groups of neurons in the cerebral cortex of mice. Furthermore, I demonstrate how single-cell electroporation can be used in combination with retrograde viral tracing techniques to label the microcircuit impinging on a single postsynaptic target neuron. Using this approach, I present the first data of monosynaptic neuronal tracing at single-cell resolution in intact networks in vivo. These complementary techniques will provide researchers with a new approach to manipulate the function of single neurons within intact networks and to link the function of a neuronal network to the underlying circuitry.

Electrical signals are the basis of information processing in the nervous system. Action potentials (APs), the output signals of neurons, are generated in and propagate along the axon to transmit information to downstream neurons. Identifying where in the axon APs are initiated and how they propagate throughout the axonal arbour is critical to understanding neuronal function. This thesis addresses these questions for the Purkinje cell (PC) axon, the sole output route of the cerebellar cortex. Using combined whole-cell patch clamp and multisite recordings of extracellular APs (eAPs) from PCs in mouse cerebellar slices, I present evidence that PC APs are initiated close to the soma, at the axon initial segment. With the help of theoretical modelling I show that eAP recordings are a valid method for identifying the axonal AP initiation site. Besides long-range projections to the deep cerebellar nuclei (DCN), PCs also innervate neurons of the cerebellar cortex via recurrent axon collaterals. I present data showing that simple spikes propagate reliably even at high frequencies (250 Hz) into recurrent axon collaterals of PCs of mature and young animals, whereas complex spikes fail to propagate fully. This is comparable to previous reports of propagation along the main axon and suggests that PC axons relay information similarly between the DCN and the local network. PCs express axonal voltage-gated Ca2+ channels (VGCC) early in postnatal development, but their role post-myelination is unknown. I demonstrate spatially-restricted activity-dependent Ca2+ influx at branch points of PC axons, which are typically nodes of Ranvier. I show that baseline nodal [Ca2+] depends on spontaneous firing frequency, suggesting that nodal [Ca2+] reports neuronal activity. Finally, I present data demonstrating that blockade of nodal VGCCs reduces axonal spike propagation velocity and can lead to propagation failure, showing that nodal VGCCs are crucial to facilitate and safeguard axonal spike propagation.