Preface: The general indications that the pineal gland might be involved in homeostasis, and more specifically the evidence suggesting a role in amelioration of seizure states warranted further investigation . No reports had examined a possible link between anticonvulsant drug administration and pineal gland function, and few enabled any type of presumption to be made as to possible effects. This study was an attempt to evaluate in which ways anticonvulsant drugs might alter pineal gland indole metabolism, with a view to increasing understanding of the role of the pineal in modulation of epileptic discharges. In order to make the study as meaningful as possible extensive preliminary investigations were necessary. Pharmacokinetic determinations gave an indication of tissue concentrations of the drugs, which could then be related to observed effects. As far as possible, where existing information was lacking, the catalytic behaviour of the various enzymes was characterised in order to explain any observed effects at a molecular level. An attempt was also made to characterise the regulatory mechanisms controlling indole metabolism, again in order to define the pharmacological effects exerted by the drugs used. The complexity of the system made it impossible to suggest a single uniform regulatory hypothesis, although some significant observations were made. Finally, the studies involving the anticonvulsant drugs were conducted on intact animals, isolated organs and individual enzymes in an attempt to determine whether the observed effects were occuring at a molecular, local or central level.

The effects of di-2 ethylhexyl phthalate (DEHP) over periods of 3 days to 9 months were examined in both male and female rats using biochemical, histological and ultrastructural techniques. Responses occurred in a characteristic order. Initial effects included : changes in the. distribution of lipid in the liver, proliferation of hepatic peroxisomes and induction of laurate hydroxylases. More slowly developing changes were (1) hypertrophy of the hepatocytes, (2) centrilobular loss of glycogen and (3) a fall in glucose-6-phosphatase activity. After 9 months of treatment, accumulation of lipid-loaded lysosomes were observed. All these changes were observed in rats treated with 1000 or 200 mg/kg/day of DEHP, but rats treated with 50 mg/kg/day of DEHP did not show the later changes. In addition to these sustained alterations, two transient changes were observed. Male rats, treated with 1000 mg/kg/day of DEHP showed changes in the biliary system as shown by electron microscopy, by examination of bile flow, bile enzymes and proteins. Furthermore, a burst of mitosis occurred immediately after commencement of treatment of DEHP, the magnitude and time of which was dose-dependent. Changes in female rats were qualitatively similar, but quantitatively smaller, than in male rats. Mature male rats treated for 3 or 13 days with either DEHP or the hypolipidaemic drugs fenofibrate or clofibrate, showed similar changes to young rats with the exception of the mitotic burst which did not occur in these animals. The initial short term effects of DEHP and the straight chain analogues, di-n-octyl phthalate (DN0P) and di-n- hexyl phthalate (DNHP) in vivo and their respective esters, MEHP and MNHP, were reproduced in cultured hepatocytes. There was induction of peroxisomal enzymes in response to treatment with DEHP or MEHP but little or no induction after treatment with DNOP, MNOP or MNHP. Accumulation of lipid was seen after 24 hours of treatment With MEHP, MNOP and MNHP. However, the mitotic burst was not reproduced in cultured hepatocytes treated with MEHP, neither was there a fall in glucose-6-phosphatase activity. There was no increase in H[2]O[2] production either in vitro in cultured hepatocytes treated with MEHP, or in vivo as measured by catalase compound I in perfused liver from rats treated with DEHP in the diet. There was no evidence for mutagenic activity of DEHP or MEHP in the Ames Test. Treatment of isolated hepatocytes with MEHP, straight chain phthalate esters or clofibrate, resulted in early marked pertubations in lipid metabolism, namely, an increase in production of neutral fats and generally in fatty acid oxidation. This may explain the increased storage of lipid, in the liver. There was a marked difference in response between hepatocytes isolated from fed and fasted rats, the latter being more sensitive to all compounds. Indeed with fed rats there was, in some cases, a slight decrease in fatty acid oxidation. The effects on lipid metabolism were observed at concentrations which produced peroxisome proliferation in cultured cells.

The aim of this study was to identify and measure areas of physiology relevant to the initiation of secondary metabolism and spore formation. It was discovered that the growth kinetics of Saccharopolyspora erythraea in a liquid culture environment were dependent on the type of nutrient limitation the organism was exposed to. Sacc. erythraea was able to produce erythromycin and formed structures which had properties similar to those of spores (designated 'sonic resistant units' in this thesis) in a nitrogen limited medium but, would produce erythromycin but no 'sonic resistant units' in a carbon limited medium. This resulted in two liquid culture systems, one which supported the formation of 'sonic resistant units' and one which did not, and thus enabled the comparison of the conditions which resulted in sporulation or secondary metabolism. The measurement of the rate of DNA, RNA and protein synthesis showed distinct profiles of synthesis in both media. Measurements of these rates in Streptomyces hygroscopicus showed similar profiles in the same media suggesting that the behaviour of the macromolecular synthesis rates in different nutrient limiting conditions might be applicable to streptomycetes in general. A change in macromolecular synthesis rate was implicated in inducing secondary metabolism. Antibiotics were used to modulate these rates in an attempt to induce secondary metabolism. Results from these experiments suggested the possible role of the intracellular concentration of charged and uncharged tRNA in induction of antibiotic synthesis. This relationship implies an analogy between the stringent response and the physiology of the induction of secondary metabolism. The formation of spores ('sonic resistant units') did not appear to be induced by changes in macromolecular synthesis rate or changes in tRNA levels or ratio. However, it was evident that the rates of macromolecular synthesis increased during the production of spores and this implied that it is the action of some intracellular agent that is inducing macromolecular synthesis to fuel the spore forming process.

Methyl methanesulphenate has been prepared labelled with radioactive carbon and its metabolism studied in rat, mouse and rabbit. The distribution of the drug after injection has been determined in the rat. Quantitative studies in this species have shown that less than as % of the injected drug is excreted or exhaled in 24 hours. Assay of tissue levels of radioactivity after injection has confirmed that the majority of the drug remains bound within the animal body. In the rat, the principal metabolic reaction has been shown to take place in the liver with glutathione. The S-methylglutathione formed is excreted in bile and this intermediate was the source of the urinary metabolites. In the urine, conjugates of S-methylcyateine and S-methylthioglycollic acid, together with small quantities of radioactive urea, have been recognized. The main metabolite has not been identified but appears to be a substituted guanidine compound. The possible significance of these results of these results has been discussed in terms of sell biochemistry.

The literature on collagen has been reviewed, with especial emphasis on structural aspects of collagen and the collagenolytic effects of enzymes. The proteolytic enzyme of Streptotmyces griseus (Pronase) has been examined and shown to consist of four fractions, each having proteolytic activity. The collagenolytic effect of the above enzyme and its fractions, and to a lesser extent of other enzymes has been examined by a variety of methods, both physical and chemicals using both soluble and insoluble collagen as substrates. Pronase has been shown to have a collagenolytic effect on collagen in both the soluble and insoluble states, and the products of collagenolysis have been examined. The action of Pronase has been shown to result in peptide bond cleavage only in the terminal regions of the collagen molecule, resulting in the liberation of small peptides and free amino acids, while leaving the body of the molecule intact. Fractionation of Pronase-treated collagen after denaturation showed that the three constituent chains of the collagen molecule had been separated, indicating that the enzyme is also affecting lateral cross linkages. An extract of sisal has been shown to have a proteolytic effect using gelatine as substrate, but a very slight collagenolytic effect, which is limited to collagen in solution. Insoluble collagen has been shown to contain a small quantity of non-protein-nitrogen material which can be removed by physical methods, and which consists of free amino acids, small peptides and a carbohydrate component.

The liver has a major role in the metabolism of cholesterol, being the main site of lipoprotein assembly and degradation and the only tissue where the metabolism of cholesterol to bile acids occurs. This provides the major pathway for the removal of cholesterol from the body. The results described in this thesis concern the use of specific enzyme inhibitors (58-035, Azacholesterol, Mevinolin) to determine the intracellular use of different sources of cholesterol in monolayers of rat hepatocytes. In particular, the fates of newly synthesized cholesterol from mevalonic acid and cholesterol derived from HDL2 were investigated. Incubation of hepatocyte monolayers with 58-035 resulted in the inhibition of esterification. In the presence of mevalonic acid as a cholesterol source, 58-035 stimulated bile acid synthesis. Azacholesterol inhibited bile acid synthesis, had no effect on cholesterol synthesis, and in the presence of mevalonic acid, stimulated secretion of cholesterol by the hepatocytes; it had no effect on cholesterol esterification. Mevinolin inhibited cholesterol synthesis and as a result inhibited esterification. HDL2, in the presence of mevinolin, was used as a cholesterol source. It stimulated bile acid synthesis and cholesterol esterification. Addition of 58-035 to the system resulted in the inhibition of both esterification and bile acid synthesis. Overall, the results indicated that different intracllular pools of free cholesterol exist and that the inter-relationships of these pools give a complex pattern of flux of intracellular cholesterol between various pathways in the rat hepatocyte.

1. The life cycle of this rosette aquatic plant was discussed in detail. 2. Anatomical differences between terrestrial and submerged forms of the rosettes were found. Terrestrial rosettes were induced very rapidly by exposure of previously submerged rosettes to air. Laminal development was greatly reduced in the leaves and roots of the terrestrial rosettes. 3. Two main aspects of the life cycle were considered in detail propagation of the rosettes; and leaf elongation. 4. Propagation: In nature the rosettes have the ability to propagate by (a) stolon formation or (b) by a less efficient means where the rosette elongates vertically. (a) Experimental evidence presented indicates that stolon initiation is controlled by gibberellic acid, although cytokinins may be involved. In nature, stolons from similar rosettes may be of varying lengths, and so the elongation phase of stolon development was considered. It was found that gibberellic acid may bring about a protraction of this elongation phase of growth of the stolon, at the expense of the development of the terminal plantlet. Ethylene supplied as ETHREL E inhibited further elongation of the stolons, and furthermore, promoted accumulation of starch. Starch grains in stolons of rosettes treated with gibberellic acid were limited to a central ring of cells. Some radial growth in response to supplied ETHREL E was noted. Stolons may persist in some cases, thus interconnecting many rosettes, but in some cases the stolons senesce. Evidence presented above suggests that gibbarellic acid and ethylene may control senescence. Where gibberellins levels are low in the stolons, accumulated ethylene will rapidly effect the senescence of the stolon, thus making the rosetts independent. (b) Where rosettes are partially and repeatedly buried by sediment, they have the ability to perennate themselves vertically by means of an elongated stem axis. Various rooting levels on the same rosette reflect the sequential nature of the deposition of the sediment. This was found experimentally where rosettes were grown under different conditions of deposition of sediment, and rosettes similar to those experimentally induced were found on a silt fan, near the inflow in Loch Drumore, near Glenshee, in Perthshire. The ability of the plant to propagate so rapidly by stolon formation on eroded shores, or by altering its rooting level and then forming stolons, explains to a great extent the ubiquity of this species throughout Great Britain, and thus implicitly explains the persistence of this species through many of the stages in hydrosere formation.

The sensitivity of Notch signaling pathway to metabolism has been proposed in several recent studies but it is unclear if the changes in fly diet composition can directly lead to changes in Notch phenotype in vivo. In this work we show that activity of Notch pathway is sensitive to amino acids via the TOR pathway. In addition we will test a candidate protein as a sensor for amino acid level in the fat body of Drosophila melanogaster but also independently in other tissues.

During aging, a decline in metabolism appears to be associated with altered hippocampal function. Thus, 1-13C-glucose and 2-13C-acetate were employed to assess neural metabolism in the dentate gyrus and CA1 of F344 rats with respect to aging and cognitive status. The results obtained when 1-13C-glucose was used as the metabolic substrate suggest that glucose metabolism may not be altered in neural tissue itself with respect to aging or a decline in hippocampal function. In contrast, the use of 2-13C-acetate, a substrate that is preferentially metabolized by astrocytes, revealed significantly increased astroglial metabolism associated with age and preserved hippocampal function. Specifically, greater 2-13C-acetate incorporation into glutamine and glutamate was observed in the dentate gyrus and CA1 of aged rats that performed similar to young rats in the Morris water maze task. Since glutamate is the primary excitatory neurotransmitter of the hippocampus and plays a central role in synaptic plasticity, the mechanism proposed to underlie learning and memory, these finding were taken to represent an adaptive metabolic response by astroglia in the aged hippocampus. Subsequently, astroglia metabolism could potentially be a future target of therapeutic strategies for age-related cognitive decline.

No description available.