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Investigation into the molecular basis of diabetes-induced erectile dysfunction was carried out on corpus cavernosum tissue from control and streptozotocin (STZ)-induced diabetic rats. Increased expression of endothelial nitric oxide synthase (eNOS) and increased nitrosative stress was found in the diabetic penile tissue. Diabetic neuropathy is associated with a failure in axonal regeneration. In vitro axon growth, guidance and regeneration model systems were used to study both the intrinsic nature of diabetic DRG neurones to grow and respond to guidance cues, but also to study the supportive properties of the diabetic nerve environment for axon regeneration. Cyclic Adenosine-3’5-Monophosphate (cAMP) signalling is compromised in diabetic DRG neurones. Increasing cAMP in diabetic neurones reduced the deficit in neurite growth and reversed the aberrant response of diabetic neuronal growth cones to the cAMP-dependent trophic factor, nerve growth factor (NGF). Diabetic neurones display reduced expression of Exchange protein activated by cAMP (Epac), a downstream effector of cAMP. Specifically activating Epac also rescued the abnormal turning responses of diabetic DRG neurones. Impaired axonal regeneration in diabetes has been, in part, attributed to delayed Wallerian degeneration. Using the cryoculture bioassay, it was shown that during regeneration the peripheral nerve environment, of both control and diabetic rats are equally supportive for axon growth. In summary, this is the first study to imply that specific signalling mechanisms, involving the cAMP-Epac pathway, may be compromised in neurones from diabetic rats, which contribute towards reduced neurite growth and abnormal responses to axon guidance cues.

In this thesis we show that Saturn’s magnetopause and bow shock oscillate with a period near that of planetary rotation that we term the ‘magnetospheric period’. In our first study, using two Cassini orbits as exemplars, we demonstrate that Saturn’s magnetopause oscillates at the magnetospheric period in response to changes in the magnetospheric pressure. The change in internal pressure required to produce such oscillations, which typically correspond to a ~10% change in the boundary radius, is estimated to be ~30-40% of the unperturbed background pressure. In our second study we develop a simple theoretical model of motion through an oscillating planar boundary that is equally applicable to the magnetopause and the bow shock. In our third study we use data from ~40 Cassini orbits to conduct a first systematic investigation of the magnetospheric period magnetopause oscillations. We show that boundary oscillation events are highly organized by the phase of the magnetic oscillations in the ‘core’ region of the magnetosphere. When radial propagation is accounted for, the phase of maximum outward boundary displacement is found to be directly related to the phase of the density maximum in the Enceladus torus. The boundary oscillation amplitude is estimated to be ~1.2 Saturn radii, but is occasionally as great as ~4-5 Saturn radii. In our fourth study we use data from 35 Cassini orbits that crossed both the magnetopause and the bow shock to provide first evidence for magnetospheric period bow shock oscillations. We find that the oscillations are significantly organized by the phase of the ‘core’ magnetosphere magnetic oscillations, though the degree of organization is less than for the magnetopause. The bow shock and magnetopause are found to oscillate approximately in phase within a phase uncertainty of about ± 25° and to have similar oscillation amplitudes.

This study was initiated by the need to develop change agent skills in a group of consultants from a large government organisation facing major challenge from a competitive market. In order to develop a program, a group of mature and experienced change agents, with around 20 years experience in a variety of different organisations in each case, was asked, through the medium of an unstructured interview, to tell the story of their own growth and development. A model of 12 factors was developed, based on recent studies of leadership, the researcher's own career experience and an analysis of a well known practitioner's biography. This model was used to analyse the content of the interview material. The study found that there were a small number of factors which distinguished the development of this group from that of leaders who had been the subject of earlier studies. These were, firstly, the primary influence of the mother as role model. for both men and women. Secondly, the influence of early confrontations with established systems of power being employed in abusive ways, which developed a commitment to preserve the freedom of individuals to participate in decisions which affect their lives. Finally, a belief in the critical part ethics, integrity and the continual practice of learning for self-awareness, plays in maintaining a sustainable career as a change agent. The conclusions for a program of development for change agents are, firstly, that prospective participants should self assess their personal experiences from childhood, their attitudes to learning and the maintenance of an integrated self as an essential for mediating between stakeholder groups. Secondly, the influence of practical experience far outweighs that of schooling or academic studies. Thirdly, change agents employ what Bateson describes as type III learning, more than any particular theoretical approach, when designing organisational interventions. The study concludes that further research into the nature of the attachment bonds formed by change agents in childhood is needed to better understand the psychological dynamics which support excellent and sustainable practice.

Dental caries is a dynamic process rather than a diagnosis. It is therefore inappropriate to cite an exact 'threshold' at which interventive operative dental care becomes the 'correct' treatment option. This thesis explored this problem by applying decision theory to one diagnostic test for caries - namely the bitewing radiograph. Fifteen 'mock' dentitions were assembled, using materials of similar radiodensity to oral structures. A number of cariously involved teeth were included in these dentitions. Radiographs were taken using a standardised technique to ensure that the quality and density of the simulated radiographs were as similar as possible to those taken 'in vivo'. Twenty dental practitioners each made 360 treatment decisions based on these bitewing radiographs. Subsequently the teeth were serially sectioned, examined histologically, and the extent of any caries in each tooth was recorded and correlated with the visual appearance of the tooth surfaces. The dentists' decisions were then subjected to Receiver Operator Characteristic (ROC) and Kappa analysis. The work described also explored the value placed on various tooth-states by the population. By using the available literature and the results of the two studies described above, a decision tree was constructed which permitted examination of the expected values of treatment decisions. This decision analysis allowed recommendations to be made concerning the setting of treatment thresholds. The first part of the study showed that the microscopic, radiographic and visual appearances of a tooth were not always directly related to one another. The analysis of the dentists' decisions indicated that individual practitioners have differing views about the importance of sensitivity and specificity, and therefore have different thresholds at which they institute treatment. These values and attitudes influenced the treatment decisions made to a greater extent than the dentists' views about the depth of lesion needing restoration. The decision analysis indicated that, for the sample populations of dentists and patients investigated, the detectability of caries on bitewing radiographs was not great enough to warrant their routine use as diagnostic tools. The treatment thresholds held by the group of dentists in the study were inappropriate in relation to the utilities for dental health held by the population examined. This study has highlighted the importance of three methods of evaluating diagnostic tests. It has shown that the values dentists place on the outcomes of their decisions affect their treatment thresholds more profoundly than lesion depth. Therefore restorative treatment thresholds should be reviewed and appropriate training given so that dental diagnostic skills are improved and clinical decisions made according to the values that patients hold.

My work is an attempt to create a visual metaphor for my personal attitude towards life which stems not only from intuition, but also from my continued interest in literature, history, and philosophy. I have tried to clarify my intuitive feelings by drawing upon other points of view which seem to resemble my own and to question personal opinions when others seem more reasonable. Consequently, my work is the visual product of a great deal of mental speculation and the imagery often has a close relationship with the thinking that inspires it.

This thesis describes the construction and analysis of a cosmid library specific for the human Y chromosome. The purpose of preparing such a library is to provide a source of ordered overlapping clones for use in the identification of genes, analysis of chromosome structure, and perhaps, as a starting point for the sequencing of the chromosome. Approximately 2,000 cosmid clones containing inserts derived from human DNA have been identified in a library prepared from a somatic cell hybrid containing the Y chromosome as the only human material. These clones have been analysed in two principal ways. Firstly, hybridisation to various characterised DNA fragments has identified cosmids containing known repeat elements and single copy DNA sequences which have been previously assigned to regions of the chromosome by deletion mapping. Secondly, a unique 'fingerprint' has been produced for each clone; band patterns of Hint I digestion products separated by polyacrylamiae gel electrophoresis have been analysed by computer to detect overlapping clones. A significant proportion of the chromosome is represented in this way.

Intracellular microelectrodes were used in conjunction with ion substitution, agonists and inhibitors of known transport processes and diuretic hormone (DH), to investigate the mechanisms whereby ions cross the basal and apical cell membranes of the Malpighian tubules of Locusta migratoria, and their endocrine control. Values for basal, apical and transepithelial potentials in control saline were -70.4 + 0.9 mV, -76.1 ± 1.5 mV and +5.5 ± 1.3 mV (lumen positive), respectively (n=73).Ion substitution experiments, involving K(^+), Na(^+) and CI(^-) in the bathing medium, indicated that the basal membrane is more permeable to K(^+) than Na(^+) and CI(^-). Ion flux studies suggest that CI(^-) entry across the basal membrane is not by direct cotransport with K(^+), nor Na(^+). Some CI(^-) entry also appears to be stimulated under conditions of high K(^+)(_o). Crude corpus cardiacum (CC) preparations with DH activity effected ca. 150% increase in tubular fluid secretion above basal secretion in vitro, and ca. 106% and 335% increase in net transepithelial Na(^+) and Cl(^-) movement from bathing medium to lumen above basal flux, respectively. Treatment of tubules with CC extract also resulted in a significant increase in levels of the intracellular second messengers, cAMP and the Ca(^2+)-mobilizing Inositol 1,4,5- trisphosphate. Ion substitution experiments using dibutyryl cAMP suggest that cAMP stimulates an apical cation pump, whilst having no effect on Cl(^-) conductance. Cytochemical localization and membrane separation techniques used in conjunction with biochemical analyses indicate the presence of (Na(^+)+K(^+))-ATPase on the basolateral membranes, and HCO(_3)- stimulated ATPase on both the basolateral and apical membranes. The results referred to above are discussed, and a hypothetical model is proposed to describe the endocrine control of ion movements across the two cell membranes mediated by changes in intracellular cAMP and Ca(^2+).

The characteristics of photosynthesis and inorganic carbon accumulation were investigated in two species of marine intertidal macroalgae: Porphyxa umbilicalis and Ulva lactuca. Both species have a similar mid-intertidal location, although P. umbilicalis grows on bare rock faces while U. 1actuca-is found submersed-in rock pools. The similar morphology of the species allows a direct comparison of both the gas exchange and the light response characteristics. For P. umbilicalis-the low light compensation and saturation points are comparable to the light requirements of shade adapted terrestrial plants. Photosynthetic capacity is greater-in seawater although the efficiency of light utilization is no higher than that in air. In the summer population, however, the photosynthetic response is reduced. In the absenceof-any corresponding effect on the the light requirements, this-can be related in part to seasonal variation in the-total chlorophyll concentration. Even after acclimation the summer-population can be photoinhibited by periods of excess PAR. P. umbilicalis is capable of using both HC03 and C02 as a substrate for photosynthesis. The maximum photosythetic capacity is greater in air, and corresponds to a higher affinity-for'C02. The gas exchange characteristics are 02-insensitive indicating low photorespiratory activity in this species. The-suppression of RuBPc-oxygenase may be attributable, to-an effective method of inorganic carbon uptake. The mechanism in P. umbilicalis involves the diffusive uptake of C02, supplied directly or by the Carbonic Anhydrase (CA) dependent catalysis of HC03-, external to the plasma membrane. An intracellular CA maintains the rate of transfer of inorganic carbon to the site C02 fixation in the chloroplast. U. 1actuca shows very similar light response characteristics to those of P. umbilicalis. The low light-requirements are the same as for the summer population, while the decrease in the Vmax of the response is again independent of an effect on the photosynthetic efficiency. The change in the capacity corresponds to a seasonal variation in the total chlorophyll content and corresponding ratio of chlorophyll a: b. Photoinhibition of the photosynthetic response significantly alters the characteristics of the light response, even though this species has the ability to maintain efficient rates of carbon fixation. In U. 1actuca the mechanism of inorganic carbon accumulation involves the direct uptake of both HC03- and C02, independent of any external CA activity. C02 will diffuse across the plasma membrane, while HC03- must be actively transported. Internal CA activity modifies the photosynthetic capacity of this species although more than one possible location is proposed for this enzyme. The difference in the characteristics of photosynthesis and inorganic carbon accumulation may be determined by the specific habitat of the two species. P. umbilicalis, more often exposed, shows a greater photosynthetic efficiency in air and a higher affinity for C02. In contrast, the photosynthetic system in U. 1actuca appears to be adapted to longer periods of submersion. In relation to the inorganic carbon accumulating mechanism, there is evidence that those marine intertidal macroalgae more often exposed have a greater affinity for C02, dependent to a degree on the activity of an external CA.

The aim of this project was to understand the molecular mechanisms underlying movement disorders and to identify new genes involved in neurodegeneration. By genetic crossing two mutations identified in the Shakin' Stevens mouse, were separated into individual mouse strains. Both have provided important new insights into two pathways characteristic of neurodegeneration: The autophagy-lysosome pathway, a protein degradation pathway in the Nymphe mouse (nym/nym), and lipid metabolism defects in the Jabber mouse (jab/+). The nym homozygous mouse was identified as a novel mouse model of the lysosomal storage disorder mucolipidosis II (MLII) homozygous for a patient mutation in the GNPTAB gene. The novel mouse model of MLII more fully recapitulates the human pathology than the previously described Gnptab knock-out mouse. Histological analysis of the brain revealed for the first time progressive neurodegeneration in the cerebellum with severe Purkinje cell loss. In addition, based on similar features between Niemann-Pick type C disease and MLII, the nym homozygous mice were treated with 2-hydroxypropyl-β-cyclodextrin, a drug previously reported to rescue Purkinje cell death in a mouse model of NPC disease. No improvement in brain pathology was observed, demonstrating that cerebellar degeneration is not primarily triggered by loss of Npc2 function. The jab mouse model, harbouring a mutation in the small GTPase Arl1, was characterised as a novel model of chylomicron retention disease, a lipoprotein deficiency, and offered the opportunity to study a novel gene involved in the maintenance of lipid homeostasis and neurodegeneration, specifically demyelination in the peripheral nervous system. The jab mutation in Drosophila presented with characteristic features of the jab mouse, highlighting the functional conservation of Arl1 and validated the use of Drosophila for further studies. Interestingly, together with constitutively active and inactive mutants, pathology in lipid droplet formation and defective insulin signalling was associated with defective Arl1 function. Using Drosophila will enable further studies into Arl1's role in tissue specific function and help elucidate molecular pathway leading to demyelination. Thus, this work has highlighted the value of a phenotype-driven approach to investigate novel neurodegenerative pathways and their amenability to therapy.

Sleep and circadian rhythm disturbance has been widely observed in neurological disorders although the mechanistic basis for this association remains unknown. In order to understand this association further, a combination of rest/activity and molecular profiling was carried out on mouse models of Parkinson’s Disease (PD), Lysosomal Storage Disorders (LSDs) and schizophrenia. Data from rest/activity behavioural screening of new BAC (Bacterial Artificial Chromosome) transgenic PD models displayed scotophase hyperactivity and decreased fragmentation patterns. Interestingly, rest/activity profiles of LSD models displayed possible core clock defects under constant conditions (Hexb-/-) and potential re-entrainment deficits following a 6hr phase advance (Npc1-/-). Together these data suggest new associations between disruptions in rest/activity cycles and neurodegeneration. The blind-drunk (Bdr) mutant is a mouse model of synaptosomal-associated protein (Snap)-25 exocytotic disruption that displays schizophrenic endophenotypes and phase advanced rest/activity cycles. Despite identification of phase advanced expression of signalling neuropeptides (e.g. arginine vasopressin) in the Bdr suprachiasmatic nucleus (SCN), the underlying mechanisms regulating circadian disruption in this model remain elusive; therefore, label-free shotgun proteomics was carried out over 24 hours to elucidate potential post-transcriptional pathways. A number of novel circadian patterns of protein expression were identified including myristoylated alanine-rich C-kinase substrate (MARCKS) which exhibited a robust phase advanced expression profile. This study has identified novel SCN post-transcriptional mechanisms that may link schizoaffective disorder biomarkers to dysfunctional rest/activity cycles.

Characterized by the severe progressive wastage of skeletal muscle, Duchenne muscular dystrophy (DMD) is a crippling X-linked recessive disease that is caused by the absence of the protein dystrophin. This thesis aimed to critically evaluate the potential of different therapeutic options to combat this disease. Utrophin is a paralogue of dystrophin. The Fiona mouse is an mdx (dystrophin-deficient) transgenic mouse that overexpresses the full-length utrophin protein in skeletal muscle, and various studies have shown that it does not display a dystrophic phenotype. However, these studies have only been performed on sedentary mice. In this work it is demonstrated that utrophin’s protective effect is partially diminished after a sustained period of exercise-induced stress, highlighting for the first time a functional difference between dystrophin and utrophin. This thesis also presents results of two mdx mouse drug trials testing the ameliorative effects of the administration of the drugs GW501516 and C1100, which show that treatment with both drugs results in partial amelioration of the dystrophic phenotype. GW501516 administration results in a beneficial fast-to-slow fibre type switch and an in vivo increase in utrophin protein levels. We have also shown that C1100 treatment results in a significant increase in utrophin A promoter activity in vitro, and the mechanism of action of this drug on this promoter has been deciphered. The global dysregulation of microRNAs in skeletal muscle of mdx and dko (dystrophin- and utrophin-deficient) mice was evaluated by microarray analysis to identify microRNAs involved in the dystrophic pathological cascades. The results of detailed expression analyses of miR-31, miR-206 and miR-503 are presented, and two therapeutically-relevant predicted targets of miR-503 were validated. Overall, this thesis evaluates the potential of different and possibly complementary therapeutic options to combat DMD.