Abnormalities in adrenal steroid production have been implicated in certain forms of hypertension. Mutations in the CYP11B1 gene which result in complete loss of 11-hydroxylase function cause 11-hydroxylase deficiency and hypertension due to abnormally high levels of mineralocorticoid, DOC. Mutations have been identified which destroy aldosterone synthase 18-hydroxylase activity or 18-oxidase activity or both, resulting in lack of aldosterone. Structure-function studies have identified aldosterone synthase residues specifically involved in 18-hydroxylation and 18-OHDOC production respectively. Analogous mutations in the human CYP11B2 gene in exons 3 and 4 which result in amino acid substitutions, E136D and K251R have been shown to increase aldosterone production. In essential hypertension adrenal steroids have been implicated as a contributing factor in some cases and it is possible that mutations in aldosterone synthase and 11-hydroxylase may be responsible in part for abnormalities in steroid production. The studies reported in this thesis have investigated some of the residues which may be responsible for the special properties of these enzymes and also the effects of potential inhibitors on enzyme steroid production in vitro. This thesis presents new studies on the relationship between structure and function of aldosterone synthase and 11-hydroxylase. Artificially induced changes, some relatively conservative and distant from centres of known functional importance, have been shown to alter activity significantly. A number of variations from consensus sequences of these enzymes have been identified in subjects with essential hypertension; whether these affect enzyme activity in such a way as to explain the clinical observations of mild 11-hydroxylase deficiency or suppressed renin or whether they might be used as diagnostic markers remains to be evaluated.

Mechanisms underlying relaxation in response to inhibitory NANC nerve stimulation and putative neurotransmitters of these nerves have been examined in the guinea-pig internal anal sphincter (IAS) and compared with those in the bovine retractor penis muscle (ERP) and guinea-pig taenia caeci. Two types of techniques were employed. One which measured the effects of nerve stimulation and drugs on electrical membrane properties where intracellular microelectrode and simultaneous mechanical recording techniques were used. Drugs, for example ATP or cromakalim were applied by perfusion in the Krebs' solution, microinjection into the bath, or by hydrostatic pressure ejection. A second method assessed the underlying biochemical changes accompanying relaxation by measuring alterations in second messenger systems, for example cyclic AMP and cyclic GMP using radiomimmunoassay techniques. Electrical events were clearly an important accompaniment to mechanical inhibition in the IAS. Field stimulation (single pulse and 5 pulses at 5, 10 and 20 Hz; 0.5ms; supramaximal voltage) produced large inhibitory junction potentials of up to 15mV in amplitude which accompanied relaxation of 80% of muscle tone. Indeed, hyperpolarising electrotonic current passed into the IAS produced relaxation. The neurotransmitter which is released by field stimulation of the inhibitory nerves is probably ATP since exogenous application of purine by hydrostatic pressure ejection (5.8x10-4M; 10-55ms) produced a dose-dependent hyperpolarisation. The membrane potential change was similar in size, rate of decline and duration to the ijp. Neither hyperpolarisation nor relaxation could be achieved with the P2x-purinergic agonist, betaMeATP (10-5-10-3M) or the P2-purinergic agonist adenosine (10-3M) thus ATP was acting on the P2y-purinergic receptor. Inhibitory NANC neurotransmission was not peptidergic since VIP (10-7-10-5M), bradykinin (10-3M), neuropeptide Y (10-5M), bombesin (10-5M), leu-enkephalin (1.8x10-4M), met-enkephalin (1.8x10-5M), somatostatin (10-6-10-3M) and substance P (7.6x10-6 - 7.6x10-4M) each had no effect on the membrane potential of the IAS. There is also evidence that stimulation of -adrenoceptors by isoprenaline (10^-9 - 10^-5M) produced relaxation which was accompanied hyperpolarisation of the IAS. In all cases where hyperpolarisation and relaxation are associated in the IAS, the mechanism underlying the electrical change appeared to be an increase in K^+ conductance. Apamin (4.5 x 10^-6M) which blocks certain Ca^2+-mediated K^+ channels, antagonised the electrical and mechanical responses produced by field stimulation and ATP. Similarly, TEA (8x10^-2M), which blocks most K^+ channels, antagonised the hyperpolarisations and relaxations produced by field stimulation, ATP and isoprenaline. Indeed, the K^+ channel activator cromakalim (10^-9-10^-5M) produced hyperpolarisation and relaxation of the IAS suggesting that an increase in K^+ conductance is important in the mediation of mechanical inhibition of the IAS. Relaxation of the IAS was also produced without a significant change in membrane potential by altering the levels of cyclic nucleotides within the smooth muscle cells of the IAS. Forskolin (10^-9-10^-5M), which activates adenylate cyclase with a subsequent increase in cyclic AMP, relaxed the IAS. Similarly, sodium nitroprusside (10^-9-10^-4M) - a cyclic GMP phosphodiesterase inhibitor, and 8-bromo-cyclic GMP (10^-4M) each increased cyclic GMP and produced relaxation of the IAS. Direct measurement of cyclic nucleotide levels of the IAS showed that field stimulation (80 pulses at 8H_Z; 0.5ms; supramaximal voltage) and ATP (10^-4) elevated the cyclic AMP and cyclic GMP contents of the IAS. All other stimuli which produced slow, prolonged electrical and mechanical changes increased the level of only one cyclic nucleotide. Isoprenaline (10^-4M), cromakalim (10^-5M) and forskolin (10^-5) increased cyclic AMP content while sodium nitroprusside (10^-5M) increased the cyclic GMP content. Further investigation of other second messenger systems involved in relaxation of the IAS showed that increase in inositol phosphate turnover was not associated with stimulation of inhibitory P_2y-purinoceptors by ATP (10^-2M) in the IAS. However, an increase in inositol phosphate accumulation was produced by noradrenaline (10^-4M) and associated with contraction. A method was devised to measure the intraluminal pressure changes of the internal anal spincter in the anaesthetised guinea-pig using a Millar pressure transducer. Using this method the in vitro results were largely confirmed by this in vivo study. Basal intraluminal sphincter pressure was increased by noradrenaline acting on -adrenoceptors and decreased by isoprenaline acting on -adrenoceptors, ATP on P_2y-purinoceptors and 2-chloroadenosine on P_1-purinoceptors.

This thesis describes approaches to further understand the structure and enzymology of D-Ala-D-Ala ligases (DDL) and those ligases with altered second substrate specificity, which confer glycopeptide antibiotic resistance. DDL is an essential enzyme in the biosynthetic pathway of the bacterial cell wall peptidoglycan. The approaches described are based primarily on the previous transition state mimic; VanA and EcDdlB, co-crystal structures. Active site mapping of VanA by site directed mutagenesis yielded VanA mutants that were expressed and purified for kinetic studies. The active site mapping of VanA supports the predictions about catalysis from the X-ray structure of VanA, but failed to identify a catalytic base. Additionally, the first enzymatic characterisation of a VanD ligase was performed. This analysis revealed that VanD4 ligase is a less efficient D-Ala-D-Lac ligase, and selected for a wider variety of second substrates in comparison to VanA. EcDdlB was expressed in E.coli, purified and crystallised. Three structures of EcDdlB have been solved to 1.4-1.7Å resolutions, representing a product inhibition complex, a ternary complex, and a D-cycloserine inhibited complex. Based upon the latter EcDdlB structure, we propose a novel suicide-substrate mechanism for the Dcycloserine mediated inhibition of DDLs, involving phosphorylation of D-cycloserine. Future studies will validate this mechanism. Finally, contributions towards rational design of EcDdlB and VanA inhibitors have been made. This work has significantly increased knowledge in the field of DDL enzymes, progressing towards the exploitation of these targets for antibiotic development. The deposition of PDB files of three new EcDdlB structures will provide the tools for further rational based drug design campaigns against EcDdlB. The novel mechanism of Dcycloserine inhibition may also have implications for the further development of inhibitors of DDLs.

The aims of this thesis were to investigate certain aspects of the regulation of osteogenesis by BMPs and their physiological inhibitors; to determine whether the balance between the two could have an effect on fracture repair; and to suggest strategies for using this information to provide orthopaedic surgeons with better ways to treat recalcitrant fractures . BMP-2 and its inhibitor chordin were produced endogenously during the in vitro osteogenic differentiation of human mesenchymal stem cells (MSCs) in response to dexamethasone. The addition of exogenous BMP-2 increased the rate of osteogenic differentiation of the MSCs. Knockdown of the BMP inhibitor chordin led to an increase in the rate of osteogenic differentiation of human MSCs, secondary to an increase in the bioavailability of BMP-2. These results raise the possibility that the impaired healing of delayed- and non-unions reflects alterations in the ratios of BMPs to their inhibitors. They further suggest that it may be possible to increase the rate of osteogenesis, and thus improve fracture repair in vivo, by down-regulating the endogenous production of BMP inhibitors. To evaluate these possibilities further, the presence of BMP-2 and -14, as well as their inhibitors chordin and noggin, were investigated by semi-quantitative immunohistochemistry in human fracture biopsies. The expression of BMP-2, BMP-14, chordin and noggin during fracture repair was demonstrated in areas of cartilage and bone formation. Levels of expression of these proteins were compared between healing and non-healing human fractures. There was a decreased relative expression of BMP-2 and BMP-14, compared to the BMP inhibitors, in the non-healing fractures v/s the healing fractures. This suggests an imbalance between BMPs and their inhibitors in fractures that do not heal. These data indicate novel ways to reduce rates of non-union by the local application of BMP-2 or BMP-14, or the blocking of BMP antagonists. Fracture healing may also be impaired by drugs used clinically. The antibiotic tobramycin, commonly used to treat or prevent infections of bone, was shown to reduce the osteogenic potential of human MSCs in vitro. An in vivo study was carried out to determine whether a clinically relevant dose of tobramycin would impair fracture repair induced by BMP-2. The biomechanical and radiological properties of the repair tissue, induced by BMP-2 in a rat femoral defect model, were not affected by the presence of tobramycin. Overall, this thesis demonstrates that the balance between BMPs and their inhibitors has a major influence on the rate of fracture repair and that this balance is altered in non-healing fractures. The data suggest novel, clinically relevant, biological strategies for enhancing bone healing.

Prion diseases are a group of fatal neurodegenerative disorders that include Creutzfeldt-Jakob Disease (CJD), Bovine Spongiform Encephalopathy (BSE) and scrapie, which are all associated with the misfolding of the cellular form of the prion protein, PrPC, into the disease associated isoform, PrPSc. This thesis investigates two properties of PrP that may influence the misfolding process; (i) the normal folding mechanism of PrP and (ii) the interactions of PrP with lipid membranes. Firstly, equilibrium folding experiments investigate whether the folding pathway of PrP is influenced by a disease modulating mutation, Q167R, which confers disease resistance. The unfolding of PrPWt is compared to PrPQ167R by monitoring fluorescence and circular dichroism of folding sensitive tryptophan mutants. The results show that the mutation significantly destabilises the protein, which can be rationalised from high resolution structures of PrP. Furthermore, comparison of the folding of mouse and hamster PrP highlights dramatic differences between their folding pathways, which may contribute to the species barrier that is observed in prion disease transmission. The second part of the thesis studies the influence of membrane environments on prion conversion. Firstly, the interaction between PrP and lipid membranes composed of POPC (a zwitterionic phospholipid) and POPS (an anionic phospholipid), are investigated through fluorescence, circular dichroism and centrifugation binding assays. The results show that PrP interacts peripherally with POPC membranes, without significant changes in protein structure. In contrast, high affinity binding to POPS membranes, results in membrane penetration and an increase in β-sheet structure. Furthermore, cryo-electron microscopy reveals that the PrPmembrane interaction disrupts the native vesicle structure and results in the formation of membrane junctions. Finally the morphology and mechanism of growth of prion aggregates on supported lipid bilayers are studied through atomic force microscopy, which shows how the phospholipid content of membranes directs prions down alternative aggregation pathways.

Beside the well-known role of CRH and CRH-related peptides in controlling HPA axis, the peptides have been implicated as important mediators various physiological processes including reproduction, endocrinology of pregnancy, energy homeostasis. Diverse functions of CRH and UCNs are governed via activation of two types of CRH receptors, R1 and R2. Since tissue sensitivity to agonists is determined by the availability of receptor in the plasma membrane and how fast the signal is terminated, one of goals of this project was to investigate the cellular expression of CRH-R1 variants (α, β, d and β/d), and internalization characteristics of the receptors following homologous and heterologous activation of the receptor. Also, I investigated the structural and functional characteristics of the CRH-R1d receptor, a splice variant that contains deletion of 14 amino acids within the 7th TMD. The co-expression studies utilizing HEK293 cells expressing CRH-R1d and CRH-R2β demonstrated attenuation of CRH-R2β mediated cAMP production and MAPK activation in the presence of CRH-R1d. This could be of potential importance in human peripheral tissues which express both types of CRH receptors, such is uterus. Additionally, I investigated the signalling and internalization characteristics of CRH-R2β and explore the possible link between the CRH-R2 internalization and MAPK activation. The analysis of the spatio-temporal characteristics of MAPK activation revealed important differences between CRH-R1 and R2 mediated signalling cascades. Immunofluorescence analysis demonstrated that activation of both type of CRH receptors led to a recruitment of β-arrestin to the plasma membrane; however the internalization pathways of the receptors were different. Since human pregnancy is associated with changes in the myometrial CRH-R variant expression profile and functional activity, as a part of the study, I characterised the effect of IL-1β (an important mediator of the onset of labour) on the regulation of CRHR1 gene expression and the functional properties of the CRH-R. Data showed that IL-1β can potentially target CRH-R1 gene transcription and splicing mechanisms of the CRHR1 gene; these interactions appeared to involve two members of the MAPK family of proteins, ERK1/2 and p38 MAPK and NF-κB activation. Interestingly, increased CRHR1 gene transcription and generation of receptor splice variants was not associated with increased CRH-R protein levels and CRH signalling activity. Furthermore, the signalling characteristics of CRH-R activated by UCN-II (CRH-R2 specific agonist) were investigated. The data showed that activation of CRH-R2 did not lead to cAMP production which is associated with the quiescent state of uterus, but the MAPK signalling cascade was activated, which have been implicated in mediating pathways that promote contractility. During the course of this project biological role of CRH receptors was investigated in T37i cells. RT-PCR analysis showed the presence of CRH-R1 and R2 mRNA in mice brown adipose tissue and T37i cells. Immunofluorescence and western blot analysis demonstrated the presence of CRH-Rs in T37i cells. The functional capacity of adipose CRH-Rs to activate adenylyl cyclase and MAPK signalling cascade was assessed. Low concentration of agonists (close to receptors Kd=1 nM) stimulated activation of adenylyl cyclase/cAMP/PKA signalling cascade resulting in lipolysis. However; higher concentration (10-10 nM) of agonists activated MAPK signalling cascades.

1. Energy homeostasis requires the co-ordination of several metabolic fluxes at the systemic level among various peripheral organs and the central nervous system. A breakdown of these metabolic fluxes can lead to hyperglycaemia. An explicit representation of this complex dynamical system can aid the interpretation of diagnostic tests and help formulate therapeutic interventions. Hence, mathematical models could constitute a valuable clinical tool in the management of hyperglycaemia associated with diabetes. However, a very large range of such models is available, making a judicious choice difficult. To better inform this choice, the most important models published to date are presented in a uniform format, discussing similarities and differences in terms of the decisions faced by modellers. We review models for glucostasis, based on the glucose-insulin feedback control loop, and consider extensions to long-term energy balance, dislipidæmia and obesity. 2. Whole-cell patch-clamp recording techniques were used in isolated hypothalamic brain slice preparations to investigate and compare the electrophysiological properties of arcuate nucleus (ARC) neurones from fed and fasted rats, including a fed control group housed as fasted animals. Subthreshold active conductances were differentially expressed in ARC neurones including: anomalous inward rectification (Ian), A-like transient outward rectification (IA), time and voltage-dependent inward rectification (Ih) and T-type calcium-like conductance. Significant differences in active and passive subthreshold membrane properties of ARC neurones were observed between the three groups, including: changes in magnitude of IA and Ih, action potential duration, membrane time-constant (tau), neuronal input resistance and spontaneous activity. Furthermore, both housing and fasting conditions affected electrophysiological properties of rat ARC neurones, suggesting both stress and fasting can modify electrophysiological properties of ARC neurones. 3. The effects of intracellular adenosine triphosphate (ATP) on neuronal excitability of ARC neurones were investigated and compared between fed and fasted rats. This was performed by manipulating extracellular glucose levels from 2.0 to 0.0 mM whilst intracellular ATP was manipulated by changing the levels in the patch pipette solution (0.0, 1.0, 2.0, 5.0 and 10.0 mM). The level of ATP required to maintain resting membrane potential and glucose-sensing capability of ARC neurones was determined. Data from this study suggests 1.0 mM and 5.0 mM ATP for fasted and fed rats, respectively, were appropriate levels for maintaining electrophysiological and glucose-sensing integrity of these neurones. Hence levels of or sensitivity to ATP appears subject to modulation depending on the energy status of organism. 4. Glucose-sensing neurones and associated underlying mechanisms in ARC neurones in both fed and fasted states were studied. Extracellular glucose levels (2.0 – 0.2 – 0.5 – 1.0 – 2.0 – 5.0 mM) were manipulated with appropriate intracellular ATP levels determined as outlined above. Three types of glucose-sensing neurone were identified: glucose-excited (GE) neurones, glucose-inhibited (GI) neurones and glucose-rapidly adapting (GRP) neurones. The proportions of these three groups of neurones varied between fed and 24-hour fasted rats. Changing energy status, fasting, also appeared to affect the sensitivity of glucose-sensing neurones, i.e. the threshold levels of glucose they detect. GE neurones operated through ATP-sensitive potassium (KATP) channel-dependent mechanisms in fed and fasted rats and through a chloride-dependent mechanism in fed rats. GI neurones detect changes in glucose levels through chloride and/or non-selective cation conductance-dependent mechanisms. Finally a potential mechanism of GRA neurones may be through transient opening of chloride conductances. Further work is required to confirm the ionic mechanisms of these glucose-sensing neurones. 5. Analysis of responses observed in ARC glucose-sensing neurones was also examined using a mathematical approach. An empirical sigmoid response function was assumed to describe the time course of the transition in neuronal activity and the integral of this function was fitted to cumulative action potential numbers to characterise the response dynamics. Four parameters were estimated for each transition, employing the least-squares criterion; the activity prior to and at the end of each change in extracellular glucose concentration, the half sigmoid time before the neuronal activity changes following a step change in extracellular glucose and the duration of the transition. Statistical tests revealed a statistically detectable difference in response delay of GE neurones following step changes in glucose levels between fed and 24-hour fasted rats. In addition, in fed rats, statistical test revealed that GE neurones required a significantly shorter delay time in changing their activity but longer change-over times than GI neurones. In 24-hour fasted rats, only the difference in activity of neurones at the start and at the end of glucose application was found to be statistically significant difference between GE and GI neurones. Further work is required to confirm these data.

Understanding the mechanisms through which proteins acquire their three dimensional structure is currently one of the most challenging tasks in structural biology. The formation of native disulfide bonds is an important step in the post-translational modification and folding of many proteins, helping to stabilise their structure. Protein disulfide isomerase (PDI) is a folding enzyme that catalyses thiol-disulfide exchange. As well as forming disulfide bonds in newly synthesised proteins, PDI also catalyses the rearrangement of intramolecular disulfides. The mechanisms through which PDI binds to substrate proteins are still not well understood. In this study, interactions are examined between PDI and a model substrate protein, bovine pancreatic trypsin inhibitor (BPTI). Since PDI functions primarily as a folding enzyme its natural substrates will be unfolded or partly folded proteins. Here, recombinant BPTI constructs were prepared that represented different stages along the folding pathway of this small protein: unfolded, partly folded and natively folded BPTI. A variety of biophysical techniques were then used to characterise each BPTI construct, both in isolation and in the presence of PDI. NMR spectra obtained at 5°C, including hydrogen deuterium exchange experiments, demonstrated the unfolded, partly folded and natively folded nature of each construct at low temperatures. The addition of PDI to each BPTI construct showed that, even at sub-stoichiometric concentrations, both the unfolded and partly-folded substrate proteins showed line broadening. In contrast, line broadening of natively folded BPTI required much higher concentrations of PDI. NMR was also used to observe the effects of differently folded BPTI substrates binding to PDI. Focus was on the key bb’x binding region of PDI. Perturbations were observed even at low concentrations of unfolded and partly-folded substrate, whereas much larger concentrations were required for the natively folded protein. However, detailed investigations into the specific regions of binding suggest that the same key sites were involved at all stages of folding. Contrary to expectations, this small full length protein showed little binding to regions beyond the key b’ domain. The binding affinities between PDI and each BPTI substrate were estimated using surface plasmon resonance (SPR). As expected, PDI has a greater binding affinity to unfolded BPTI compared to the partly folded construct, with least affinity to the natively folded protein. However, the difference in affinity between unfolded and partly folded constructs was relatively small. This is the first study to investigate the structural interaction of PDI with a partly folded, full length protein substrate. It is hoped that the findings of this study will contribute to a general understanding of oxidative protein folding in the endoplasmic reticulum (ER).

The introduction of antibacterial drugs in the middle of the last century heralded a new era in the treatment of infectious disease. However the parallel emergence of antibiotic resistance and decline in new drug discovery threatens these advances. The development of new antibacterials must therefore be a high priority. The biosynthesis of the bacterial cell wall is the target for several clinically important antibacterials. This extracellular structure is essential for bacterial viability due to its role in the prevention of cell lysis under osmotic pressure. Its principal structural component, peptidoglycan, is a polymer of alternating N-acetyl-glucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc) residues crosslinked by peptide bridges anchored by pentapeptide stems attached to the MurNAc moieties. The biosynthesis of peptidoglycan proceeds in three phases. The first, cytoplasmic, phase is catalysed by six enzymes. It forms a uridine diphosphate (UDP) bound MurNAc residue from UDP-GlcNAc and attaches the pentapeptide stem. This phase is a relatively unexploited target for antibacterials, being targeted by a single clinically relevant antibacterial, and is the subject of this thesis. The Streptococcus pneumoniae enzymes were kinetically characterised and in silico models of this pathway were developed for this species and Escherichia coli. These models were used to identify potential drug targets within each species. In addition the potentially clinically relevant interaction between an inhibitor of and feedback loops within this pathway was investigated. The use of direct parameter estimation instead of more traditional approaches to kinetic characterisation of enzymes was found to have significant advantages where it could be successfully applied. This approach required the theoretical analysis of the models used to determine whether unique parameter vectors could be determined. Such an analysis has been completed for a broad range of biologically relevant enzymes. In addition a relatively new approach to such analysis has been developed.

PLD catalyses hydrolysis of phosphatidylcholine (PtdCho) to produce phosphatidic acid (PtdOH) and choline. PtdOH is a second messenger responsible for a multitude of cell processes, ranging from cytoskeletal rearrangement to cell proliferation. Antigenic stimulation of RBL-2H3 mast cells and growth factor stimulation of endothelial HeLa cells results in PLD-dependent exocytosis and endocytosis, respectively. A novel fluorescent PtdCho (fPtdCho) was used to label both cell lines and Bligh-Dyer lipid extraction of fPtdCho-labelled RBL-2H3 cells showed the lipid was intact post-labelling. fPtdCho co-localised up to 50% with the lysosomal marker LysoTracker Red in RBL- 2H3 cells, and was not secreted in response to antigenic stimulation as recorded using real-time confocal microscopy. Primary alcohol treatment of fPtdCho-labelled RBL- 2H3 cells altered fPtdCho-labelling to diffuse from punctate distribution, suggesting PLD-generated PtdOH is responsible for retention of punctate fPtdCho staining. PLD isoforms 1b and 2a were labelled with Cherry (a red fluorescent protein) and transiently expressed in fPtdCho-labelled HeLa cells. Localisation was assessed using FRET by FRAP technology in live cells and showed that substrate and lipase were in close proximity. These findings will facilitate future development of a live real-time in vivo PLD assay. Furthermore, localisation of PLD and its activator Rac1 was assessed at rest and in EGF-stimulated HeLa cells in real-time. This showed co-localisation between PLD and Rac1 following stimulation. The fluorescent PtdCho was also used to develop a novel real-time in vitro PLD assay, monitoring fPtdCho metabolism at two second intervals. This in vitro assay is more sensitive than traditional end-point assays and will help clarify the relative rate of PLD activation in response to small G-protein activators and other co-factors in real-time.