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Concanavalin A, provoked a 35-fold increase in the rate of proliferation of rat thymocytes. Insulin (10-6M), and insulin-like growth factor I (10-10M) approximately doubled the rate of DNA synthesis. Both of these structurally related molecules acted through the type I insulin-like growth factor receptor. The sequential addition of Concanavalin A and insulin, promoted a much greater proliferative response than to either of the two agonists alone. Insulin also increased the uptake of glucose and amino acids by the cells. Glucose uptake was enhanced at insulin concentrations of 10-6M and 10-10M. Amino acid uptake was more strongly affected at the higher concentration. Insulin-like growth factor I (10-11M) also enhanced amino acid uptake. The effects of insulin on metabolism were mediated by both insulin and type I insulin-like growth factor receptors. These effects were greatly enhanced after a pre-treatment with Concanavalin A. Concanavalin A provided a primary mitogenic signal to the cells. Amongst the responses was an increased expression of insulin and/or type I insulin-like growth factor receptors. The consequent enhanced cellular sensitivity to these agonists, enabled them to facilitate the passage of the cells through the cell cycle by: i) providing a secondary mitogenic signal, and ii) promoting the uptake of raw materials and energy substrates. The initiation of DNA synthesis and passage through the cell cycle was thus punctuated by the sequential expression of various cell surface receptors. This regulated cellular sensitivity, enabling them to react in a precisely orchestrated fashion to hormones and other molecules in their environment. The intracellular mechanism of insulin action remains an enigma. Although the presence of extracellular calcium was essential for insulin stimulation of amino acid uptake and DNA synthesis, the cation did not subserve a direct mediator function. Insulin promoted an increase in intracellular pH, which was mediated by the Na+/H+ antiport. Other mechanisms were probably also involved in mediating the full cellular response to insulin.

The effects of haem limitation and iron restriction on cells of non typable Haemophilus influenzae were investigated. Haem limitation was achieved by adding concentrations of haem to growth media which resulted in substantial decreases in final cell yields. Iron restriction was achieved by substituting protoporphyrin IX (PPIX) for haem in the growth medium and adding an iron chelator to the system. The effect of these nutrient limitations on a) outer membrane composition, and b) respiratory systems of non typable H.influenzae was investigated. Several of the strains examined produced new PPIX-specific outer membrane proteins when cultured utilising PPIX as a porphyrin source. The immune response of patients with bronchiectasis to outer membrane antigens of H.influenzae cultured under iron-restricted conditions was analysed by ELISA and immunoblotting techniques. ELISA analysis revealed that individuals with severe bronchiectasis had high titres of antibodies directed against H.influenzae OMs in both serum and sputum. Immunoblotting with homologous serum showed that where PPIX-specific OMPs were produced they were antigenic and were recognised by patients' serum. This suggested that these H.influenzae OMPs may be expressed in vivo. Additionally, the development of the immune responses to non typable H.influenzae outer membrane antigens was investigated using a rat lung model. Bacteria encased in agar beads were inoculated intratracheally into rat lungs, infection was established, and the immune response monitored for 6 weeks. The animals developed antibodies to PPIX-specific OMPs during the course of infection, providing further evidence that H.influenzae express these novel OMP antigens when growing in vivo. Studies in vitro on respiratory systems of phenotypically altered H.influenzae showed that bacteria grown utilising PPIX as a porphyrin source, or under conditions of iron-restriction produced ten fold fewer cytochromes than cells grown in nutrient excess, while haem limited H.influenzae produced no detectable cytochromes. Respiration of various substrates was depressed in haem limited and in PPIX-grown cultures as compared with cells grown in nutrient excess.

Endogenous glucocorticoids and serotonin have been implicated in the pathophysiology of depression, anxiety and schizophrenia. This thesis investigates the potential of downregulating expression of central Type II glucocorticoid receptors (GR) both in vitro and in vivo, with empirically-designed antisense oligodeoxynucleotides (ODN), to characterise GR modulation of 5-HT2A receptor expression using quantitative RT-PCR, Western blot analysis and radioligand binding. The functional consequence of GR downregulation is also determined by measuring 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane hydrochloride (DOI) mediated 5-HT2A receptor specific headshakes. Using a library of random antisense ODN probes, RNAse H accessibility mapping of T7-primed, in vitro transcribed GR mRNA revealed several potential cleavage sites and identified an optimally effect GR antisense ODN sequence of 21-mer length (GRAS5). In vitro efficacy studies using rat C6 glioma cells showed a 56% downregulation in GR mRNA levels and 80% downregulation in GR protein levels. In the same cells a 29% upregulation in 5-HT2A mRNA levels and 32% upregulation in 5-HT2A protein levels was revealed. This confirmed the optimal nature of the GRAS5 sequence to produce marked inhibition of GR gene expression, and also revealed GR modulation of the 50-HT2A receptor subtype in C6 glioma cells to be a tonic repression of receptor expression. The distribution of a fluorescently-labelled GRAS5 ODN was detected in diverse areas of the rat brain after single ICV administration, although this fluorescence signal was not sustained over a period of 5 days. However, fluorescently-labelled GRAS5 ODN, when formulated in polymer microspheres, showed diverse distribution in the brain which was maintained for 5 days following a single ICV administration. This produced no apparent neurotoxic effects on rat behaviour and hypothalamic-pituitary-adrenal (HPA) axis homeostasis.

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Disturbances in electrolyte homeostasis are a frequent adverse side-effect of the administration of aminoglycoside antibiotics such as gentamicin, and the antineoplastic agent cis-platinum. The aims of this work were to further elucidate the site(s) and mechanism(s) by which these drugs may produce disturbances in the renal reabsorption of calcium and magnesium. These investigations were undertaken using a range of in vivo and in vitro techniques and models. Initially, a series of in vivo studies was conducted to delineate aspects of the acute and chronic effects of both drugs on renal electrolyte handling and to select and evaluate an appropriate animal model: subsequent investigations were focused on gentamicin. In a study of the acute and chronic effects of cis-platinum administration, there were pronounced acute changes in a variety of indices of nephrotoxic injury, including electrolyte excretion. Most effects resolved but there were chronic increases in the urinary excretion of calcium and magnesium. The renal response of three strains of rat (Fischer 344, Sprague-Dawley (SD), and Wistar) to a ranges of doses of gentamicin was also investigated. Drug administration produced substantially different responses between strains, in particular marked differences in calcium and magnesium excretion. The results suggested that the SD rat was an appropriately sensitive strain for use in further investigations. Acute infusion of gentamicin in the anaesthetised SD rat produced rapid, substantial increases in the fractional excretion of calcium and magnesium, while sodium and potassium output were unaffected, confirming previous results of similar experiments using F344 rats. Studies using lithium clearance measurements in the anaesthetised SD rat were undertaken to investigate the effects of gentamicin on proximal tubular calcium reabsorption. Lithium clearance was unaffected by acute gentamicin infusion, suggesting that the site of acute gentamicin-induced hypercalciuria may not be located in the proximal tubule.

The bioequivalence of sustained release theophylline formulations, marketed in the United Kingdom, has been investigated in relation to the co-administration of food in both single dose and steady state volunteer studies. The effect of food on pharmacokinetic parameters and their clinical relevance was researched. Experimentation using drug induced modification of gastric motility to ascertain the component influences of the rate of gastric emptying on the absorption of theophylline from sustained release formulations was conducted. Prolongation of time to maximum plasma theophylline concentration by food reported in the literature and its clinical importance was investigated in once daily compared with twice daily administration of sustained release theophylline formulations and smoking habit. The correlation between saliva and plasma theophylline concentrations as a means of developing a non-invasive sampling techniques was examined. Data obtained from in vitro dissolution studies was compared with in vivo results. This thesis has shown no significant differences occurred in the pharmacokinetic parameters measured between sustained release formulations available in the United Kingdom. The investigations into the influence of food on prolongation of time to maximum plasma theophylline concentration and other measured pharmacokinetic parameters demonstrated no important pharmacokinetic or clinical effects. Smoking adults taking sustained release theophylline formulations had similar drug clearances to those reported in the literature for smokers taking plain uncoated theophylline formulations.