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No description available.

The requirement for complex, therapeutic proteins that possess the correct post-translational modifications has resulted in mammalian cells, in particular Chinese hamster ovary (CHO) cells, being widely used in die biopharmaceutical industry, In the creation of mammalian cell lines plasmid DNA carrying the gene of interest integrates randomly into die host cell genome. Integration into different chromatin domains results in variable levels of gene expression between cell lines due to gene silencing mechanisms. In addition, cell lines often show unstable protein production during long-term culture. Variable and unstable recombinant protein expression means that a large number of clones need to be screened in order Therefore there is a necessity to overcome these gene silencing mechanisms, with the intention to accelerate process development of recombinant protein production in mammalian systems.

In drug development, ADME issues are still large contributors to drug attrition. The use of in vitro systems, such as microsomes and hepatocytes, for the assessment of metabolic stability and prediction of in vivo clearance, are well established for substrates of the cytochrome P450 enzymes (CYPs). However, these systems have been less explored for conjugation reactions, such as glucuronidation by uridine diphosphate glucuronosyltransferases (UGTs). The aim of this study was to increase the understanding of glucuronidation kinetics in vitro and the factors influencing the prediction of metabolic clearance by UGTs.

Atypical antipsychotic treatment in both children and adults carries significant risk for excessive weight gain that varies widely across individuals. This variation suggests genetic and/or environmental factors are involved in antipsychotic-induced weight gain. This thesis aimed to: 1) provide a systematic review and meta-analysis of the effects of atypical antipsychotics in children and adolescents on weight gain; 2) genotype three specific genes (HTR2C receptors, LEP and LEPR) which are thought to be likely candidates for association between risperidone and weight gain in young people; 3) assess the association between the genotypes of each gene and weight gain. A genetic study was conducted in outpatient mental health clinics/hospitals in the UK and the Kingdom of Saudi Arabia. In this study we investigated the association of 5 single nucleotide polymorphisms (SNPs) with weight gain in a cohort composed of 200 children and adolescents undergoing first treatment with the antipsychotic risperidone over 36 weeks. Of the 200 patients, DNA samples were successfully collected from 197 patients. For all genes we found no significant association with risperidone–induced weight gain after controlling for baseline weight, age, gender, diagnosis and ethnicity. A significant association was found between baseline body mass index (BMI)-standardised z scores (BMIz) and age at the onset of risperidone treatment and weight gain, patients with a lower baseline BMI gained more weight, (p<0.001), and younger patients tended to gain more weight (p<0.001) for all 5 SNPs tested. There was a significant association between weight gain and ethnicity, with individuals of Arab origin being more likely to gain weight than young British for all SNPs. No association between weight gain and gender was found. In this sample no significant association between risperidone-induced weight gain and any of the genotypes tested was found. The novel aspects of this study are the ethnicity of the sample and the age group. Future directions will include genotyping of other relevant variants, such as in MC4R and FTO. The goal of such work includes identifying segments of patient groups for appropriate targeted clinical interventions.

The nucleus tractus solitarus (NTS) integrates afferent information to maintain cardiovascular homeostasis. Pharmacological experiments have indicated that 5-HT is released in this process. In vitro and in vivo experiments were carried out to measure this release. 5-HT was detected in the NTS indirectly, by measuring glutamatergic inward currents in the slice and directly by voltammetry in the anaesthetised rat. In the slice, the presence of tonic release of 5-HT was confirmed which, in part, is responsible for basal release of glutamate via activation of 5-HT3 receptors. Blockade of the high-affinity, low-capacity 5-HT transporter (5-HTT; SERT), with citalopram and the low-affinity, high-capacity transporters (organic cation transporter 3; OCT3 and the plasma membrane monoamine transporter; PMAT), with decynium-22 caused a 5-HT1A mediated decrease in glutamate release. Evoked glutamate release was only augmented by decynium-22. In experiments with anaesthetisia, a voltammetric scan optimised for 5-HT was used. Stimulation of cardiopulmonary, chemoreceptor and baroreceptor afferents as well as electrical stimulation of vagal afferents increased the electrochemical signal, which was calcium and frequency dependent. Synthesis inhibitors for 5-HT, but not for noradrenaline, decreased the signal confirming it was 5-HT. Decynium-22 increased the evoked signal but neither citalopram nor desipramine, at doses shown to selectively decrease the removal of 5-HT or noradrenaline, had any effect. Blockade of glutamate receptors with kynurenate reduced the evoked 5-HT by ~50% and this remaining 5-HT was potentiated by decynium-22. Preliminary data from rats with heart failure induced by coronary artery ligation found that glutamate and 5-HT transmission to be augmented within the NTS. These data show that 5-HT release is increased by activation of afferent input and this release is regulated by OCT3/PMAT not 5-HTT. This suggests that 5-HT may act, in part, as a volume transmitter in the NTS. This regulation may be affected by disease such as heart failure.

Neuronal nicotinic acetylcholine receptors (nAChRs) are excitatory ligand‐gated ion channels that perform important roles throughout the nervous systems of both vertebrate and invertebrate organisms. Impairments to human nAChRs and cholinergic transmission are thought to underlie the pathophysiologies of several neurological and psychological diseases including schizophrenia, Alzheimer’s disease, Parkinson’s disease and certain forms of epilepsy. They are also the receptors that mediate the effects of tobacco smoking and contribute to the physiological and psychological changes associated with nicotine addiction. The aim of this thesis is to further our understanding of neuronal nAChRs from a pharmacological and molecular viewpoint. Research described in this thesis focuses on numerous aspects of neuronal nAChRs; allosteric modulators, insect nAChRs and chaperone proteins. Allosteric modulators of nAChRs are ligands that alter the receptor’s responsiveness to agonists via sites that are separate from the agonist‐binding site (the orthosteric site). Positive allosteric modulators (PAMs) increase the receptor's sensitivity to agonist acetylcholine whereas negative allosteric modulators (NAMs) decrease sensitivity. Using molecular and electrophysiological techniques on the α7 nAChR, studies have been conducted with three PAMs (ivermectin, NS‐1738 and PNU‐120596) and one NAM (methanandamide). Chimeric receptor constructs and site‐directed mutagenesis studies, together with ligand docking simulations, have highlighted the importance of the transmembrane region of the α7 nAChR for modulation by these ligands. The second topic covered by this thesis is insect nAChRs and the molecular chaperone RIC‐3 (resistance to cholinesterase inhibitors); a protein that facilitates folding and assembly of nAChRs. In the past, recombinant expression of insect receptors has proved largely unsuccessful and in some (but not all) circumstances co‐expression with RIC‐3 has alleviated the problems. This research is aimed at investigating the influence of alternatively spliced isoforms of Drosophila melanogaster RIC‐3 on the maturation of a variety Drosophila recombinant nAChR subunits. Lastly, ongoing work is also described on the cloning of insect nAChR subunits from pest species Frankliniella occidentalis (western flower thrip), which has developed resistance to the insecticide spinosad.