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Hepatic glucose-6-phosphatase (G6Pase) catalyses the final step in blood glucose production by the liver. It is a multicomponent system: the catalytic subunit is on the luminal surface of the endoplasmic reticulum membrane; there are transport proteins for glucose-6-phosphate, inorganic phosphate and glucose across the ER membrane; and there is a calcium-binding stabilising protein associated to the catalytic subunit. In fasted and diabetic humans (and rat models) kinetic analysis has shown that the capacity of the glucose-6-phosphate transport protein is the rate-limiting step in G6Pase activity, making this protein vital in controlling hepatic glucose output. However, deficiency of any part of this system will lead to hypoglycaemia and other possible metabolic upsets (type 1 glycogen storage diseases). The structure and known regulation of the G6Pase system are reviewed in the introduction to this thesis. The aims of the work presented here are to investigate the human glucose-6-phosphatase system by studying adult patients newly diagnosed with abnormalities of the G6Pase system, and tissues not previously proven to contain G6Pase in healthy adults thereby improving understanding of the enzyme, its regulation and physiological role and to look for a tissue more accesible than liver in which to study human G6Pase activity. A unique series of eight adult patients each with an abnormality of hepatic G6Pase (two with previously unrecorded defects) is presented and the features of these cases are discussed with reference to the existing literature on type 1 glycogen storage diseases. The cases demonstrate how difficult it can be to prove hypoglycaemia in adults; the diversity of presenting symptoms and signs; the use of a screening test (blood glucose response to a 1mg intramuscular dose of glucagon) for such patients; and the benefits of developing reliable assays for the protein components of the G6Pase system. This series of patients also give further clues to the physiological role of glucose-6-phosphatase in extra-hepatic tissues and the regulation of the hepatic G6Pase system. The diagnosis and subsequent follow-up of the above patients would have been eased by being able to study a more accessible tissue than liver. Intestinal mucosa and neutrophils have been described as abnormal in G6Pase deficiencies. Therefore G6Pase activity was sought in these tissues from normal adult humans.

A series of nicotine gums was made to investigate the effect of formulation variables on release of nicotine from the gum. Using a directly compressible gum base, in comparison to Nicorette® the gums crumbled when chewed in vitro, resulting in a faster release of nicotine. To investigate the effect of altering the gum base, the concentration of sodium salts, sugar syrup, the form of the active drug, the addition sequence and the incorporation of surfactant into the gum, the traditional manufacturing method was used to make a series of gum formulations. Results showed that the time of addition of the active drug, the incorporation of surfactants and using different gum base all increased the release of nicotine from the gum. In contrast, reducing the concentration of sodium carbonate resulted in a lower release. Using a stronger nicotine ion-exchange resin delayed the release of nicotine from the gum, whilst altering the concentration of sugar syrup had little effect on the release but altered the texture of the gum.

The calcitonin gene-related peptide (CGRP) receptor is an unusual G protein-coupled receptor (GPCR) in that it comprises the calcitonin receptor-like receptor (CLR), receptor activity modifying protein 1 (RAMP1) and the receptor component protein (RCP). The RAMP1 has two other homologues – RAMP2 and RAMP3. The endogenous ligand for this receptor is CGRP, a 37 amino acid neuropeptide that act as a vasodilator. This peptide has been implicated in the aetiology of health conditions such as inflammation, Reynaud’s disease and migraine. A clear understanding of the mode of activation of this receptor could be key in developing therapeutic agents for associated health conditions. Although the crystal structure of the N-terminal extracellular domain (ECD) of this receptor (in complex with an antagonist) has been published, the details of receptor-agonist interactions at this domain, and so ultimately the mechanism of receptor activation, are still unclear. Also, the C-terminus of the CLR (in the CGRP receptor), especially around the presumed helix 8 (H8) region, has not been well studied for its role in receptor signalling. This research project investigated these questions. In this study, certain residues making up the putative N-terminal ligand-binding core of the CLR (in the CGRP receptor) were mapped out and found to be crucial for receptor signalling. They included W69 and D70 of the WDG motif in family B GPCRs, as well as Y91, F92, D94 and F95 in loop 2 of CLR N-terminus. Also, F163 at the cytoplasmic end of TM1 and certain residues spanning H8 and associated C-terminal region of CLR were found to be required for CGRP receptor signalling. These residues were investigated by site-directed mutagenesis where they were mutated to alanine (or other residues in specific cases) and the effect of the mutations on receptor pharmacology assessed by evaluating cAMP production, cell surface expression, total cell expression and aCGRP-mediated receptor internalization. Moreover, the N-terminal ECDs of the CLR and RAMPs (RAMP1, RAMP2 and RAMP3) were produced in a yeast host strain (Pichia pastoris) for the purpose of structural interaction study by surface plasmon resonance (SPR). Following expression and purification, these receptor proteins were found to individually retain their secondary structures when analysed by circular dichroism (CD). Results were analysed and interpreted with the knowledge of the secretin family receptor paradigm. The research described in this thesis has produced novel data that contributes to a clearer understanding of CGRP receptor pharmacology. The study on CLR and RAMPs ECDs could be a useful tool in determining novel interacting GPCR partners of RAMPs.

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Introduction The objectives of the present study were twofold: 1) to investigate the cross-sectional and longitudinal accuracy of percentage body fat (%fat) estimates from laboratory methods and field methods in a sample of mainly overweight and obese adolescents; and 2) to examine the use of each method to assess the effectiveness of the Carnegie International Weight Loss Camp (CIC) intervention. ABSTRACT / / Methods The laboratory methods: dual-energy X-ray absorptiometry (OXA), air displacement plethysmography (AOPSiri. AOPLoh), total body water (TBW73, TBWLoh) , threecompartment mineral model (3CMin) and three-compartment total body water model (3CrBw), and the field methods: skinfold thicknesses (SKFs), bie-electrical impedance analysis (BIA) and four-compartment bie-electrical impedance model (4CB1A) were evaluated against a criterion four-compartment model (4CLoh). 76 adolescents participated in the cross-sectional study, age (mean ± SO) 14.0 ± 1.6 y, body mass index 30.0 ±6.7 kg.m-2 and %fat (4C model) 36.9 ± 11.9%. 13 children attending the CIC (campers), 14 overweight comparison group children (OWCG) and 8 normal weight comparison group children (NWCG) participated in the longitudinal and intervention evaluation study. Results Cross-sectional analysis of laboratory methods, in all subjects, revealed mean percentage body fat (%fat) determined by 3CrBW (37.0 ± 11.8%) and 3CMin (36.8 ± 12.9%) were within ± 0.5% of that determined by 4CLoh (36.9 ± 11.9%). %Fat determined by AOPsiri (38.0 ± 12.4%), AOPLoh (36.0 ± 12.8%), TBW73 (35.8 ± 11.7) and TBWLoh (37.6 ± 11.4%) were within between ± 0.7% and ± 1.2% of that determined by 4CLoh, whilst OXA overestimated %fat by 3.6%. Considering individual agreement 3CrBW revealed the lowest 95% limits of agreement (± 1.1%) followed by values between ± 3.6% and ±7.2% for all other methods. Cross-sectional analysis of field methods, in all subjects, revealed mean %fat determined by 4CBIA (36.2 ± 12.8%) and 1 BIA prediction equation were within ± 0.5% of that determined by 4CLoh %fat (35.9 ± 12.5%). %Fat determined by 3 BIA prediction equations were within between ± 0.8% and ± 1.8% of that determined by 4CLoh , whilst all other methods differed by between ± 2.4% and ± 6.0%. Considering individual agreement 4CB1A revealed the lowest 95% limits of agreement (± 3.2%) and SKFs the greatest (± 15.6%). The 95% limits of agreement for all other BIA prediction equations ranged from ± 7.9% to ± 12.8%. Longitudinal analysis of laboratory methods, by group, revealed a less than ± 1% mean difference to 4CLoh %fat delta values in all cases except by 3CMin, TBW73 and TSWLoh in Campers, 3CMin in the OWCG and DXA in the NWCG. Considering individual agreement the lowest 95% limits of agreement were produced by 3CrBW.(± 0.6% to ±1.0%) and the highest by DXA (%fat ±4.2% to ±5.9). Longitudinal analysis of field methods, by group, revealed a less than ± 1% mean difference to 4CLoh %fat delta values in all cases except 2 BIA prediction equations and SKFs in Campers, but a greater than ± 1% mean difference in all cases in the OWCG and NWCG with the exception of SKFs in the NWCG. In all cases the lowest 95% limits of agreement were produced by 4CB1A (%fat ± 1.8% to ± 6.2%). Evaluation of the CIC intervention revealed that 3CrBW was the only method to show agreement with the 4C model findings of a significant difference between each group. Discussionl Conclusion The present study demonstrates that a number of methods were able to provide accurate mean but not individual cross-sectional and longitudinal body composition estimates compared to a criterion 4C 'model. In general laboratory methods provided more accurate estimates than field methods. In addition, the study highlights 3CTBW as an accurate alternative to 4C' model analysis in lean, overweight and obese adolescents when very accurate individual estimates are required. Similarly, when evaluating changes in an intervention compared to control groups using only a small number of participants the 3CrBW model is the most accurate altemative method. Findings from the present study also highlight caution is warranted when interpreting data, both cross-sectionally and longitudinally, without careful attention to and understanding of t~e theoretical underpinnings of the method being used.

The enteric nervous system (ENS) constitutes a gut-intrinsic network of interconnected ganglia which control multiple aspects of gastrointestinal activity, including motility, secretion and blood flow. Most enteric neurons and glia are derived from a relatively small population of neural crest (NC) cells which originate primarily from the vagal NC and migrate ventromedially to invade the foregut mesenchyme. Within the gut microenvironment, ENS progenitors receive signals that allow them to survive, proliferate, differentiate and migrate extensively, giving rise to a uniformly distributed population of enteric neurons and glia. So far, most developmental studies have explored the behaviour of ENS progenitors within the gut wall, but little is known about the properties of NC cells prior to foregut invasion. To explore the spatiotemporal dynamics of ENS development, we conditionally inactivated Geminin (Gem), a mouse gene with fundamental roles in genome integrity and cell fate decisions. Gem deletion from pre-enteric NC cells results in extensive DNA damage followed by P53-mediated apoptotic cell death and intestinal aganglionosis. In contrast, ablation of Gem from enteric NC cells has minimal effects on ENS development. To determine whether ENS lineages display a stage-specific sensitivity to generic genotoxic stress, we monitored the in vivo response of NC cells to γ-irradiation at different developmental stages. While both pre-enteric and enteric NC cells where characterised by robust DNA damage response, pre-enteric NC cells displayed a far greater apoptotic response relative to their enteric counterparts. These experiments reveal a previously unknown spatiotemporal sensitivity of NC cell lineages to genotoxic stress and provide an experimental paradigm for understanding the mechanisms by which DNA damage from genetic or environmental insults leads to congenital ENS deficits. Our studies allow us to draw general conclusions as to how DNA damage and fundamental developmental processes are integrated in vertebrate embryos.

The liver plays an important role in drug toxicity, in part through its significant expression of drug-metabolising enzymes. For this reason, liver cells are often used in toxicity screening. Chronic liver injury is also a growing health concern, but its study in-vivo is limited by the severity of the bile duct ligation (BDL) animal model. In investigating ways to replace animals as a source of liver cells for toxicity screening and reduce and refine the BDL model, rat AR42J-B-13 (B-13) cells have been examined as an alternative to primary hepatocytes and chemical alternatives to BDL have been examined respectively. B-13 cells were readily converted by glucocorticoid treatment to hepatocyte-like (B-13/H) cells, which expressed functional CYP1A and CYP3A sub-families whose expression could be induced in response to prototypical inducers. CYP2B1 could be induced at mRNA but not protein level. The CYP1A2 gene in B-13 cells was disrupted/non-functional, however stable introduction of human CYP1A2 showed B-13 cells could be humanised and used for assessment of bioactivation-dependent genotoxins. Drug transporter mRNA expression was low in B-13 and B-13/H cells, but HNF4α overexpression enhanced transporter mRNA expression and function in B-13/H cells. The chronic administration of methapyrilene and α-naphthylisothiocyanate in rats and mice respectively, caused liver injury qualitatively equivalent to that seen after BDL but without the associated mortality or severity seen with BDL. These data suggest that B-13/H cells could be used as an alternative to primary hepatocytes for drug toxicity screening. Chronic administration of methapyrilene and α-naphthylisothiocyanate could be used as alternative less severe models of periportal fibrosis in rats and mice respectively. Use of B-13/H cells would reduce the number of animals required for hepatocyte derivation and through refinement of the BDL model of periportal fibrosis, fewer rodents would be subject to the associated complications and high mortality rate of BDL.

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No description available.