The steroid hormone progesterone is considered indisPensible for pregnancy - it is the 'hormone of pregnancy' as described by corner (1928). It is generally agreed that the role of progesterone in all species is to Prepare the endometrium for pregnancy and to contribute to the maintenance of myometrial quiescence from implantation to the end of Pregnancy. There is however, a conflict of opinion concerning its role in pregnancy termination. In some species, for example the sheep, there is considerable evidence that progesterone provides an important link between fetal cortisol and the control of uterine activity. But in human pregnancy the evidence is conflicting. The present study investigates the role of progesterone in the initiation of uterine activity in; - late pregnant ehtes, - early human pregnancy and - the luteal phase of the human menstrual cycIe. The study was designed following the development of a 3 B-nyaroxysteroid dehydrogenase (3 B-ttso) inhibitor (Epostane) by Sterling Winthrop Research. In vitro, Epostane acts comPetitively to inhibit the conversion of pregnenolone to Progesterone. This provides a method of investigating the role of Progesterone in the classical manner - depressing the rate of secretion.

The series of studies comprising this thesis was developed to answer a number of key inter-related questions in regard to eye banking and corneal transplantation in New Zealand. The source and management of donor tissue procured by the New Zealand National Eye Bank (NZNEB) was analysed. Significant trends were identified with respect to donor demographics, donor procurement source, improved donor tissue processing and storage, decreased biological contamination, and increased utilization of corneal tissue. Current trends and ethnicity differences in indications for penetrating keratoplasty (PKP) were investigated. Keratoconus was identified as the most common indication for PKP in New Zealand, accounting for a significantly higher proportion of PKPs than other published reports. Keratoconus was the most common indication for PKP throughout all ethnicity groups and was particularly common in the Maori and Polynesian populations. Significant trends were identified including an increase in the number of PKPs for regraft and Fuchs’ endothelial dystrophy and a decrease for aphakic or pseudophakic bullous keratopathy and viral keratitis. Survival and visual outcome following PKP in New Zealand was investigated using univariate and multivariate analysis. Several independent risk factors were identified that influenced outcome of PKP. Active inflammation at PKP, pre-existing vascularisation, pre-operative glaucoma, small or large graft size, intra-operative complications, episodes of reversible rejection and a pre-operative diagnosis of regraft, trauma or infection resulted in a significantly decreased survival rate. Advancing recipient age, active inflammation at the time of PKP, pre-existing vascularisation, pre-operative glaucoma, episodes of reversible rejection, bullous keratopathy, trauma and non-infective keratitis were associated with poor visual outcome. Patient characteristics, indications, surgical details, and outcome of paediatric keratoplasty were analysed. Acquired non-traumatic indications accounted for the majority of paediatric keratoplasties in New Zealand. This study highlighted keratoconus as a particularly common indication for paediatric keratoplasty when compared to other countries. Survival and visual outcome was better for acquired compared to congenital indications. The effects of corneal parameters on the measurement of endothelial cell density (ECD) in the normal eye were analysed. Corneal thickness appears to be negatively correlated to ECD in the normal cornea for all age groups. Corneal diameter is correlated to ECD measurement in children but not in adults. Corneal curvature was not significantly correlated to ECD measurement, but this needs further investigation. Confocal microscopy and slit scanning topography were used to analyze endothelial morphology and function in the short and long term following PKP. The results of this study are in concordance with other published reports that have identified an accelerated loss of endothelial cells and more rapid development of abnormal endothelial cells in transplanted corneas compared to normal corneas.

In New Zealand, rhegmatogenous retinal detachment (RRD) is recognised as a serious and potentially blinding disorder but little is known about the prevalence, risk factors, management and outcomes for treatment for our population. This thesis attempts to investigate these issues and part of that documentation involved a clinical review of those individuals presenting with RRD. That survey was performed over a 16 month interval enabling the annualised rate for individuals presenting with a new RRD to be determined. The prevalence was found to be 11.8 per 100,000. The risk was age-related with the incidence of RRD increasing for each decade up until the age of 70 years. Men had a slightly greater risk of RRD, and high myopes (greater than 6 dioptres) accounted for approximately 1/3 of the presentations. A history of cataract surgery was also noted to be a significant risk factor for RRD. A subsequent investigation documented in this thesis determined the rate of RRD following cataract surgery using phaco-emulsification techniques was 1%. The risk for pseudo-phakic patients was inversely related to age. The initial survey revealed approximately 2/3 of the patients presented with macula-off RRDs. While individually many of these patients did well, as a group the functional improvement following surgery was limited and less than 1/3 of eyes achieved LogMAR 0.3. It was not always apparent what factors negatively impacted on the functional prognosis but certainly those individuals requiring more than one surgery tended to fare worse. The impact of a poor visual outcome was not directly assessed in this thesis but it is likely those individuals do suffer in terms of visual functioning and quality of life issues. In New Zealand there are a number of agencies that care and support visually impaired persons but there is inadequate data to assess and benchmark treatment and rehabilitation. If this could be achieved for patients with RRD then those barriers which potentially restrict successful outcomes might provide useful insight for other individuals with visual impairment.

The somatotrophic axis plays a vital role in the hormonal regulation of growth and intermediary metabolism. It encompasses the regulation of pituitary growth hormone (GH) secretion from the pituitary gland, the actions of GH on peripheral tissues via interactions with specific growth hormone receptors (GHR) and subsequent endocrine, paracrine and autocrine events, many of which are mediated via the insulin-like growth factor (IGF) system and its regulators. There are multiple effectors and points of regulation within the axis, including feedback loops, functioning to maintain homeostasis of the organism in physiological and pathophysiological situations. This thesis focuses on a number of studies of GH action and the GH receptor and its regulation, and subsequent processes mediated via the IGF system. Specific aims include further understanding the role of GH and the GHR in fetal and early life, exploring the interaction of other hormones (particularly placental lactogen and somatostatin) with the somatotrophic axis and examining the somatotrophic axis in a rapidly growing tissue (the antler) which does not appear to express the GHR. The ontogenic and GH regulation of the hepatic GHR (as reflected by hepatic bovine GH (bGH) specific binding) and serum GH binding protein (GHBP) were studied in the pig. Marked age-related increases were seen in serum GHBP and hepatic bGH binding, and both were increased by recombinant porcine GH treatment in infant and pubertal animals. Serum IGF-I correlated significantly with serum GHBP and hepatic bGH specific binding. Serum GHBP levels reflected major changes in the hepatic GHR, but not closely in pubertal animals, suggesting some differential regulation. Also, the low levels of hepatic GHR in the infant pig were inducible by GH, suggesting GH responsiveness and a role for GH in early life. In further exploring the role of GH in early life, GH (but not IGF-I) administration to neonatal dwarf rats was found to have small but significant somatogenic effects on growth, serum IGF-I and body composition, with an associated decrease in hepatic bGH specific binding. These studies support a role for GH and GH responsiveness in the neonatal rat. This concept was able to be explored indirectly in the human by studying birth weight and early growth in GH-deficient infants. These infants were short at birth with relative adiposity, and had impaired longitudinal growth in the first year of life, suggesting some GH-dependence of growth in fetal life and early infancy. In investigating the perinatal changes in the GHR and serum IGF-I levels, post-mature fetuses were found to have much lower hepatic bGH specific binding than neonatal lambs of the same post-conceptional age, suggesting that these increases relate to events at parturition, and not to post-conceptional age or intrinsic timing. Thus, while evidence is emerging for a significant role of GH and the GHR in fetal life, major induction of these somatotrophic axis components appears to be inhibited until after birth. Since placental lactogen (PL) has been suggested as having an important role in fetal growth and metabolism, the binding properties and somatogenic properties of ovine PL (oPL) were explored. Studies in dwarf rats demonstrated somatogenic effects of oPL which were in some instances greater than those of bGH. Receptor binding studies in rat and sheep livers consistently showed greater potency of oPL, although with detailed displacement studies in sheep showing parallel changes in oPL and bGH binding over a range of developmental stages. This and other supporting evidence suggest that oPL may interact with the GHR or a closely related receptor, although the possibility of a distinct oPL receptor cannot be conclusively discounted. While the GHR is clearly of major importance in the regulation of growth in many tissues, no GHRs were demonstrated in deer antler by autoradiography or radioreceptor assays. Specific binding sites were identified for IGF-I and IGF-II, with properties suggestive of the type 1 and type 2 IGF receptors. Thus, endocrine IGF-I is proposed to have a prominent role in antler growth, although local IGF production and action is also likely to be important. Finally, in exploring other potential regulators of the GHR, the possibility of direct effects of somatostatin on the GHR was examined, since somatostatin has been suggested to influence the peripheral somatotrophic axis by reducing GH-induced IGF-I expression. Octreotide administration was associated with decreased IGF-I expression, yet with increases in hepatic GHR expression, suggesting that the suppressive effects of octreotide on IGF-I metabolism are not mediated via downregulation of GHR expression, but more likely by direct effects on IGF-I expression. The studies in this thesis have furthered the understanding of some aspects of the role and regulation of GH and the GHR in the somatotrophic axis. Many questions remain to be answered on the complicated role played by these systems in the regulation of growth and metabolism. / Whole document restricted, but available by request, use the feedback form to request access.

This thesis explores whether low birth weight affects glucose homeostasis and other aspects of the metabolic syndrome in twin children. The key parameter studied is insulin resistance, and whether insulin resistance is also associated with abnormalities in blood pressure or other aspects of the metabolic syndrome. This thesis is a comparison of twins to singletons, rather than being a study of these traits within twin pairs. The fetal origins hypothesis suggests that low birth weight ultimately is associated with adult onset diseases namely coronary heart disease, glucose intolerance and hypertension. All twins to a degree are born prematurely and with low birth weight. It is unclear whether their metabolism in later life reflects this, or alternatively reflects their uniqueness as twins irrespective of birth weight. This thesis reviews how adaptation for their unique fetal life has affected in particular, glucose homeostasis in twins. Insulin resistance has been consistently identified prior to the onset of both type 2 diabetes mellitus and hypertension and is also the primary metabolic abnormality persisting from programming of the undernourished fetus. Both small-for-gestational-age and prematurely born infants are insulin resistant when examined in mid-childhood. It has been postulated that this represents an attempt of the fetus to salvage itself from a state of inadequate nutrition. Twins when examined in this thesis are also shown to be insulin resistant, or to have a reduction in insulin sensitivity. This insulin resistance was independent of low birth weight and prematurity, and reflected a unique twin effect. Examing blood pressure precisely revealed that twins had increased night-time blood pressure, a feature also seen in a variety of pre-hypertensive states. However, there was no association between low birth weight and any 24 hour blood pressure monitoring parameter in twins. Twins also had elevated leptin levels but reduced TNF-alpha levels in twins irrespective of birth weight or prematurity. Twins have unique metabolic profiles which are not correlated with low birth weight, and twins should be considered an exception to the fetal origins hypothesis. / Whole document restricted, but available by request, use the feedback form to request access.

The primary object of this thesis was to investigate aspects of empirically-measured, anaesthesia-induced electroencephalographic (EEG) changes that could be explained by network models of cortical interactions. The thesis consists of a collection of various theoretical and clinical papers in three sections: (a) A background section summarizing previous relevant published works on the molecular actions of anaesthetic agents, and the origin and problems with the acquisition of the EEG. (b) A second section dealing with the development of various theoretical / computer models of general anaesthetic action. (c) A third section of various clinical and EEG studies of anaesthesia and sleep. These were done to confirm and clarify some of the theoretical results from the models. However there are many different ways of numerically capturing the information contained within the EEG. Because this problem needed to be overcome before the primary aim of the thesis could be accurately handled; the clinical studies tended to be diverted into this secondary "signal-acquisition" aspect of the EEG analysis in anaesthesia and sleep. Because the thesis has a constellation of different interlocking threads of investigation, I have summarized the various themes in the following table. (A) Background Section The relevant conclusions from the background section were: 1) The transition from aesthesia to anaesthesia involved the whole animal, and thus may not be entirely measurable by the EEG. In contrast the transitions from consciousness to coma - and even more specifically - from mnesis to amnesis were predominantly observable by changes in the activity of the cortex and in the resultant EEG signal. 2) The EEG signal arose largely from the summation of coherent post-synaptic potentials in the cortical pyramidal cells. These were, in turn, mainly controlled by cortical layer-l modulation - which was subject to ascending neuromodulatory systems. 3) The main artifacts in the EEG signal were eye-movements, blinks, and frontalis EMG. 4) The Bispectral index (BIS) consisted of three subcomponents. The first two (BetaRatio and SynchFastSlow) probably reflected the loss of high-frequency activity that is associated with the transition from consciousness to unconsciousness. 5) The state of the midbrain reticular formation projecting via the non-specific thalamocortical connections can profoundly alter the oscillatory state of the cortex and thence the EEG. Direct or indirect actions of general anaesthetic agents on these structures may be a major cause of the observed EEG changes. These changes are qualitatively similar to the changes observed during natural sleep. 6) Although there was variation between different agents, the most consistent action of commonly-used general anaesthetic agents was to prolong the fast inhibitory postsynaptic potential> 50% by GABAergic potentiation. This action was consonant with the observed EEG effects of anaesthesia - which are (1) an increase in EEG spectral power and frequency followed by, (2) a decrease in dominant frequency and overall power with increasing concentrations: the so-called "biphasic response" There were also a number of more specific features: namely loss of alpha band activity, an initial increase in relative beta activity, followed by a shift to lower frequencies, spindles - and in deep anaesthesia - burst suppression patterns. Other contributions to anaesthetic action may include: (1) inhibition of NMDA receptor function, (2) reduction of nicotinic acetylcholine modulation, and (3) a direct hyperpolarizing action by opening potassium channels. Anaesthetic agents disrupted cortical function at lower doses than those required for inhibition of brain stem function. (B) Theoretical Models The fact that the cerebral cortex consisted of, effectively, a two-dimensional interconnecting neural network was of overriding importance. All the theoretical models were constrained by this topological fact, and showed a remarkable degree of similarity. Regardless of whether the model was continuous (appendix 2) or discrete (cellular automata), some robust and consistent features emerged: 1) Some form of abrupt decrease in information flow within the network occurred when the interaction between the network elements was interrupted greater than certain critical threshold amount - the order/disorder phase transition. 2) This critical point was manifest as (i) a large change in the simulated mean soma potential (the order parameter), (ii) divergence of the spectral power of the simulated pseudoEEG signal, and (iii) decrease in frequency content (in the cellular automaton models). Unfortunately the order parameter for the EEG is probably unmeasurable in clinical practice. The first derivative is measurable, but not completely reliable as a proxy order parameter. 3) All models required some form of noisy external (subcortical) input to drive them. Variations in this input could explain some of the anomalies observed in the clinical section of the thesis. (C) Clinical Papers The clinical papers were a series of papers recording the clinical data, which then influenced the development of the theoretical models. The single most popular clinical measure of anaesthetic depth in present practice is the Bispectral Index (BIS). Many of these studies explore the strengths and weaknesses of this index in various clinical situations. The main disadvantage of the BIS was that crucial aspects of its formulation are not in the public domain for commercial reasons. We attempted to separate out the changes in the various subcomponents of the BIS in patients under general anaesthesia, and natural sleep. 1) In the first paper we described how the EEG changed during induction and recovery from general anaesthesia with propofol and isoflurane. We compared EEG vs heart rate variability changes as monitors of anaesthetic depth. This study served to evaluate a clinically accepted measure of anaesthetic depth, as well as gaining practical EEG experience in collecting experimental data, both using the ASPECT EEG monitor, and in devising the best clinical methods of quantifying levels of consciousness. The main results were that the BIS was relatively reliable in steady-state conditions, and better than the raw 95% spectral edge frequency, and the approximate entropy of the EEG. 2) Auditory recall and response-to-command during recovery from propofol anaesthesia. In this paper we were able to collect data in a very controlled situation, somewhat different from that pertaining to clinical practice. From this we were able to identify separate components of consciousness and mnesis and how they correlated with EEG changes. We were also able to compare frontal and parietal EEG data. We established that there is a clear divergence of spectral power around the time of loss-of-consciousness when the propofol was administered as a slow infusion. This is good evidence supporting the basis of the theoretical models. This experiment also established that it is possible for a patient to be conscious, not paralyzed, but not responding to verbal command. 3) Electroencephalographic measures of depth of anaesthesia: the importance of the gamma band and the electromyogram signal. This study was concerned with different technical aspects of EEG signal acquisition and processing. It suggested that usually the EMG is not a significant problem, and that the gamma (40-60Hz) frequency band is important in distinguishing the awake from the anaesthetized state. 4) The Bispectral Index: A Measure of Depth of Sleep? Because the EEG changes of general anaesthesia are very similar to those of sleep, this study was a simple observational study collecting some information about changes in the BIS with natural sleep. 5) The Bispectral Index and Sleep Stage: A Polysomnographic study. In this study we formally compared the BIS with a full polysomnogramnographic sleep staging. It confirmed the results of the previous study, and demonstrated that the subcomponents of the BIS (the BetaRatio and the SynchFastSlow) changed in a manner very similar to those observed under general anaesthetic. Conclusions The observed changes in the EEG on induction of anaesthesia can be explained by changes in relatively simple theoretical network models. These changes can be reliably reduced to univariate Parameters

The primary object of this thesis was to investigate aspects of empirically-measured, anaesthesia-induced electroencephalographic (EEG) changes that could be explained by network models of cortical interactions. The thesis consists of a collection of various theoretical and clinical papers in three sections: (a) A background section summarizing previous relevant published works on the molecular actions of anaesthetic agents, and the origin and problems with the acquisition of the EEG. (b) A second section dealing with the development of various theoretical / computer models of general anaesthetic action. (c) A third section of various clinical and EEG studies of anaesthesia and sleep. These were done to confirm and clarify some of the theoretical results from the models. However there are many different ways of numerically capturing the information contained within the EEG. Because this problem needed to be overcome before the primary aim of the thesis could be accurately handled; the clinical studies tended to be diverted into this secondary "signal-acquisition" aspect of the EEG analysis in anaesthesia and sleep. Because the thesis has a constellation of different interlocking threads of investigation, I have summarized the various themes in the following table. (A) Background Section The relevant conclusions from the background section were: 1) The transition from aesthesia to anaesthesia involved the whole animal, and thus may not be entirely measurable by the EEG. In contrast the transitions from consciousness to coma - and even more specifically - from mnesis to amnesis were predominantly observable by changes in the activity of the cortex and in the resultant EEG signal. 2) The EEG signal arose largely from the summation of coherent post-synaptic potentials in the cortical pyramidal cells. These were, in turn, mainly controlled by cortical layer-l modulation - which was subject to ascending neuromodulatory systems. 3) The main artifacts in the EEG signal were eye-movements, blinks, and frontalis EMG. 4) The Bispectral index (BIS) consisted of three subcomponents. The first two (BetaRatio and SynchFastSlow) probably reflected the loss of high-frequency activity that is associated with the transition from consciousness to unconsciousness. 5) The state of the midbrain reticular formation projecting via the non-specific thalamocortical connections can profoundly alter the oscillatory state of the cortex and thence the EEG. Direct or indirect actions of general anaesthetic agents on these structures may be a major cause of the observed EEG changes. These changes are qualitatively similar to the changes observed during natural sleep. 6) Although there was variation between different agents, the most consistent action of commonly-used general anaesthetic agents was to prolong the fast inhibitory postsynaptic potential> 50% by GABAergic potentiation. This action was consonant with the observed EEG effects of anaesthesia - which are (1) an increase in EEG spectral power and frequency followed by, (2) a decrease in dominant frequency and overall power with increasing concentrations: the so-called "biphasic response" There were also a number of more specific features: namely loss of alpha band activity, an initial increase in relative beta activity, followed by a shift to lower frequencies, spindles - and in deep anaesthesia - burst suppression patterns. Other contributions to anaesthetic action may include: (1) inhibition of NMDA receptor function, (2) reduction of nicotinic acetylcholine modulation, and (3) a direct hyperpolarizing action by opening potassium channels. Anaesthetic agents disrupted cortical function at lower doses than those required for inhibition of brain stem function. (B) Theoretical Models The fact that the cerebral cortex consisted of, effectively, a two-dimensional interconnecting neural network was of overriding importance. All the theoretical models were constrained by this topological fact, and showed a remarkable degree of similarity. Regardless of whether the model was continuous (appendix 2) or discrete (cellular automata), some robust and consistent features emerged: 1) Some form of abrupt decrease in information flow within the network occurred when the interaction between the network elements was interrupted greater than certain critical threshold amount - the order/disorder phase transition. 2) This critical point was manifest as (i) a large change in the simulated mean soma potential (the order parameter), (ii) divergence of the spectral power of the simulated pseudoEEG signal, and (iii) decrease in frequency content (in the cellular automaton models). Unfortunately the order parameter for the EEG is probably unmeasurable in clinical practice. The first derivative is measurable, but not completely reliable as a proxy order parameter. 3) All models required some form of noisy external (subcortical) input to drive them. Variations in this input could explain some of the anomalies observed in the clinical section of the thesis. (C) Clinical Papers The clinical papers were a series of papers recording the clinical data, which then influenced the development of the theoretical models. The single most popular clinical measure of anaesthetic depth in present practice is the Bispectral Index (BIS). Many of these studies explore the strengths and weaknesses of this index in various clinical situations. The main disadvantage of the BIS was that crucial aspects of its formulation are not in the public domain for commercial reasons. We attempted to separate out the changes in the various subcomponents of the BIS in patients under general anaesthesia, and natural sleep. 1) In the first paper we described how the EEG changed during induction and recovery from general anaesthesia with propofol and isoflurane. We compared EEG vs heart rate variability changes as monitors of anaesthetic depth. This study served to evaluate a clinically accepted measure of anaesthetic depth, as well as gaining practical EEG experience in collecting experimental data, both using the ASPECT EEG monitor, and in devising the best clinical methods of quantifying levels of consciousness. The main results were that the BIS was relatively reliable in steady-state conditions, and better than the raw 95% spectral edge frequency, and the approximate entropy of the EEG. 2) Auditory recall and response-to-command during recovery from propofol anaesthesia. In this paper we were able to collect data in a very controlled situation, somewhat different from that pertaining to clinical practice. From this we were able to identify separate components of consciousness and mnesis and how they correlated with EEG changes. We were also able to compare frontal and parietal EEG data. We established that there is a clear divergence of spectral power around the time of loss-of-consciousness when the propofol was administered as a slow infusion. This is good evidence supporting the basis of the theoretical models. This experiment also established that it is possible for a patient to be conscious, not paralyzed, but not responding to verbal command. 3) Electroencephalographic measures of depth of anaesthesia: the importance of the gamma band and the electromyogram signal. This study was concerned with different technical aspects of EEG signal acquisition and processing. It suggested that usually the EMG is not a significant problem, and that the gamma (40-60Hz) frequency band is important in distinguishing the awake from the anaesthetized state. 4) The Bispectral Index: A Measure of Depth of Sleep? Because the EEG changes of general anaesthesia are very similar to those of sleep, this study was a simple observational study collecting some information about changes in the BIS with natural sleep. 5) The Bispectral Index and Sleep Stage: A Polysomnographic study. In this study we formally compared the BIS with a full polysomnogramnographic sleep staging. It confirmed the results of the previous study, and demonstrated that the subcomponents of the BIS (the BetaRatio and the SynchFastSlow) changed in a manner very similar to those observed under general anaesthetic. Conclusions The observed changes in the EEG on induction of anaesthesia can be explained by changes in relatively simple theoretical network models. These changes can be reliably reduced to univariate Parameters

This volume contains an introduction to the topic of pertussis followed by twelve papers on pertussis arising from research conducted while I was a research fellow at St. George's Hospital Medical School from 1985 to 1988. Paper 1 provides a qualitative description of the serum antibody responses to several Bordetella pertussis antigens. It demonstrates that the serum antibody response to a specific B. pertussis antigen, following either natural infection or vaccination, is dependent both on the antigen and on the subject. Some B. pertussis antigens appear to consistently evoke strong serum antibody responses following either natural infection or vaccination, while other B. pertussis antigens appear to consistently fail to evoke any significant serum antibody response. Other B. pertussis antigens induce an antibody response in a variable proportion of subjects. Paper 2 describes the serum IgG, IgA and IgM responses to B. pertussis antigens in patients with pertussis. These antibody responses are compared with those in the family contacts of patients with pertussis and with those in infants immunised with a whole cell pertussis vaccine. It demonstrates that both pertussis and pertussis vaccination produce marked serum antibody responses to three B. pertussis antigens. In contrast, family contacts who are exposed to a patient with pertussis, but who themselves fail to develop disease, have a minor antibody response to the same antigens. Immunity to disease in these family contacts is associated with high titres of antibody to B. pertussis antigens at the time of exposure to infection. Paper 3 demonstrates the serum antibody responses to B. pertussis antigens in two subjects immunised with new acellular pertussis vaccines. The antibody responses are compared with those in subjects who had been immunised with a whole cell pertussis vaccine, and with those in subjects who had suffered natural infection. The new acellular pertussis vaccines are shown to induce antibody responses solely to two purified B. pertussis antigens, and not to other potential contaminating antigens. Paper 4 demonstrates that the IgA antibody response in nasal mucus in patients with pertussis is less pronounced than the serum antibody responses. It also demonstrates that titres of antibody in nasal mucus are not especially strongly correlated with immunity to pertussis in family contacts. Paper 5 compares the serum antibody responses to B. pertussis antigens in breast fed infants with pertussis with those in bottle fed infants with pertussis. It also compares the levels of IgA to B. pertussis antigens in breast milk from mothers of infants with pertussis with the levels in breast milk from mothers of healthy infants. This paper provides further evidence that B. pertussis infection does not produce an especially strong secretory immune response. Paper 6 provides evidence that B. pertussis infection does not produce a significant impairment of immunoglobulin synthesis. Paper 7 compares the responses of peripheral blood lymphocytes, from patients with pertussis and two control groups, to in vitro stimulation with B. pertussis antigens. This study is the first to show that B. pertussis infection induces cell mediated immune responses. Paper 8 describes the respiratory physiology of six infants with severe pertussis. It demonstrates that abrupt severe hypoxaemia may be due to prolonged apnoea or may occur despite continued breathing movements and respiratory airflow. It postulates that these findings are due to a ventilation perfusion mismatch secondary to alveolar atalectasis caused by a defect in lung surfactant synthesis, secretion or function. Paper 9 describes the effects of B. pertussis infection on the ciliary function and electron microscopic appearances of human nasal epithelial cells. These findings are extended by similiar investigations perfomed on human nasal epithelial cells exposed to B. pertussis toxins in vitro. Paper 10 attempts to explain the large-scale epidemiology of pertussis by a simple mathematical formula. This formula is then used to derive an estimate of the proportion of the population susceptible to pertussis. Finally the relationship between the incidence of pertussis and the level of vaccine uptake is illustrated using data from several countries during the last century. Paper 11 describes the small-scale epidemiology of pertussis and demonstrates that pertussis is usually transmitted by patients with clinical disease rather than by persons with either atypical disease or asymptomatic infection. Paper 12 briefly discusses the use of erythromycin and other antimicrobial agents in the treatment and prophylaxis of pertussis.

Background Nitric oxide (NO) was well known to be a component of air pollution, often in the form of nitrogen dioxide (NO2). However its importance in biological systems altered dramatically with the discovery in 1987 that it was the 'endothelial-derived relaxing factor'. Since then there has been an explosion of research on NO demonstrating that this gaseous molecule was a widespread physiological mediator and was simultaneously recognised as a vital component of immune function contributing to macrophage-mediated cytotoxicity. NO was therefore a key molecule in modulating inflammation, including airway inflammation. The aim of this thesis was: 1. To adapt a NO chemiluminscence analyser from measuring airway pollution to measuring exhaled air in human subjects. 2. To measure NO levels in exhaled air in adult subjects. 3. To evaluate whether altering measurement parameters altered the levels of NO obtained. 4. To adapt this technique from adults to measure exhaled NO in children. 5. To compare levels of NO from healthy children to groups of asthmatic children on either bronchodilator therapy only, or on regular inhaled corticosteroids. 6. To compare the levels of NO in a pilot group of asthmatic children before and after commencement of inhaled corticosteroids. Methods A Dasibi Environmental Corporation Model 2107 chemiluminescence analyser was adapted specifically requiring a reduction in response time, which was achieved by modification of the circuitry and re-routing of the analogue signal directly to a chart recorder, achieving a reduction of the response time by 80%. Addition of a number of analysers allowed the measurement of exhaled NO, carbon dioxide (CO2), mouth pressure and flow for each exhalation from total lung capacity. Twenty adult subjects (in total) were then studied looking at direct (NO, CO2, mouth pressure) versus t-piece (with the addition of flow) measurements making five exhalations from total lung capacity, at 3-minute intervals (direct/t-piece/direct or t-piece/direct/t-piece in series). The area of NO under the curve versus the peak of the NO trace was compared and the exhalation pattern of NO versus CO2 was compared. Measurement conditions were altered to evaluate the effect of individual parameters on the exhaled NO result. This included separately assessing different expiratory flows, different expiratory mouth pressures, the effect of a high versus a low background NO level and the effect of drinking water (of varying temperatures) prior to exhalation. Healthy control children were then enrolled to the study from a local school (Park Walk Primary School) and compared with asthmatic children enrolled from outpatient clinics at the Royal Brompton Hospital. The asthmatic children were further divided into those on bronchodilator treatment only and those on regular inhaled corticosteroid therapy. NO was also measured before and two weeks after commencing inhaled corticosteroid therapy in previously steroid-naive asthmatics. Results It was possible to modify a chemiluminescence analyser to enable measurement of exhaled NO. In 12 healthy subjects (mean age 32 years, 6 males) peak direct NO levels were 84.8 parts per billion (ppb) (standard error of the mean (SEM) 14.0ppb), significantly higher than 41.2ppb (SEM 10.8ppb) measured via the t-piece system. The exhaled NO rose to an early peak and plateau while the CO2 levels continued to rise to peak late in the exhalation. The mean times to peak NO levels were 32.2 seconds (s) (direct) and 23.1s (t-piece), which was significantly different from the mean times to peak CO2 levels at 50.5s (direct) and 51.4s (t-piece, p<0.001). At peak NO level, the simultaneous CO2 level of 4.9% (SEM 0.47%, direct) and 5.2% (SEM 0.18, t-piece) were significantly lower than the peak CO2 achieved of 5.8% (SEM 0.21%, direct, p<0.001) and, 6.2% (SEM 0.28, t-piece, p<0.001). There was no difference between repeat direct or t-piece measurements. With regard to varying measurement conditions, the mean peak concentrations of NO decreased by 35ppb (95% confidence intervals 25.7-43.4) from a mean of 79ppb (SEM 15.4ppb) at an expiratory flow rate of 250mls/min to 54.1ppb (SEM 10.7ppb) at 1100mls/min. The mean peak concentration of NO did not change significantly when mouth pressure was increased in eight of ten subjects, although in two it did decrease in the highest pressure. The mean NO concentration with machine and subjects sampling from a low NO reservoir was 123ppb (SEM 19.4), which was an increase from results obtained before at 81.9ppb, SEM 10.2ppb, p=0.001 95%, CI -19.9 to -62.7) and after at 94.2ppb(SEM 18.3ppb, p=0.017, 95% CI 6.0-5.18) sampling with high ambient NO levels. The mean peak NO concentration decreased from 94.4ppb (SEM 20.8) to 70.8ppb (SEM 16.5, p=0.002 95% CI 12.9 -33.1) with water consumption. In 39 healthy pre-pubertal children with a mean age of 9.9 years (range 9-11 years, 23 girls) the mean direct exhaled No level was 49.6ppb (SD 37.8ppb, range 11.5-197.2ppb) compared with mean exhaled No via t-piece sampling of 29.2ppb (SD 27.1ppb, range 5.1-141.2ppb). There was no significant difference between boys and girls for either the direct or the t-piece recordings. In comparison with normal children, 15 asthmatic children on bronchodilator therapy only had much higher levels of exhaled NO at 126.1ppb via the direct system (SD 77.1ppb, p<0.001) and 109.5ppb via the t-piece system (SD 106.8ppb, p<0.001). In 16 asthmatics on regular inhaled corticosteroids the mean peak exhaled levels were significantly lower at 48.7ppb via the direct method (SD 43.3ppb, p<0.001) and via the t-piece system at 45.2ppb (SD 45.9ppb, p<0.01). There was no difference between the normal children and the asthmatic children who were on regular inhaled corticosteroids (p=0.9 direct, p=0.2 t-piece).There was no significant difference in CO2, mouth pressure, duration of expiration and expiratory flows between the three groups or between the two methods (direct and t-piece). In six asthmatic children the mean peak exhaled NO levels fell from a medium peak level of 124.5ppb to 48.6ppb when measured before and two weeks after commencement of inhaled corticosteroids on treatment. Discussion This research showed it was possible to modify an NO chemiluminescence analyser to enable measurement of exhaled NO in adult and paediatric subjects. Furthermore, it was possible to measure both healthy and asthmatic children. There were significant differences between the exhalation pattern of NO and CO2 suggesting that NO was produced in the airways, not at alveolar level, unlike CO2. The measurement of exhaled NO required a standardised approach as exhaled NO levels decreased with increasing expiratory flow, when measuring at a time of high ambient NO concentration, and with consumption of either hot or cold water immediately preceding exhalation (such as might be given if a subject was coughing). The findings with expiratory mouth pressure were less certain, with a difference seen in only two of ten subjects. The levels of exhaled NO measured in children aged 9-11 years were lower than that measured in the adult subjects. There was no difference between boys and girls, or with other parameters such as having a personal history of atopy, a family history of atopy, or the presence of a smoker or furry pets within the house-hold. These findings may have altered with increased numbers in this group and could possibly be a type two statistical error. The results of exhaled NO in asthmatic children on bronchodilator therapy only were significantly elevated compared to both normal children and asthmatic children treated with regular inhaled corticosteroids. The exhaled NO level also fell significantly by two weeks following the commencement of inhaled corticosteroid treatment in steroid-naive asthmatic children. These results suggested that the methods of measuring exhaled NO required standardization and that it could potentially be a non-invasive measure of airway inflammation to follow - particularly in children with asthma who were commencing inhaled steroid treatment.

Objectives This thesis had the following objectives: 1) To assess the existing evidence base for the effectiveness of tubal flushing as a treatment for infertility (Section II, Chapters 4 and 5). 2) To assess current practice and prior beliefs amongst Australasian fertility specialists concerning the role of tubal flushing as a treatment for infertility (Section III, Chapter 6). 3) To investigate the possible mechanism of the fertility enhancing effect of the oil soluble contrast medium lipiodol, and specifically whether there is an effect on the endometrium (Section IV, Chapter 7). 4) To generate definitive evidence from a randomised controlled trial for the effectiveness of lipiodol flushing as a treatment for infertility (Section V, Chapters 8 and 9). 5) To evaluate the adoption of lipiodol flushing as an innovative treatment into clinical practice (Section VI, Chapter 10). Methods The work undertaken in this thesis was based on prospective study protocols using the following research methodologies: Systematic reviews and meta-analyses of treatment efficacy to meet objective 1. Two types of structured survey questionnaires with a Bayesian analysis to meet objective 2. A randomised animal study involving 60 Swiss white mice, combined with genital flushing procedures under anaesthesia, some of which involved microsurgical techniques, followed by genital tissue harvesting, tissue preparation and immunohistochemistry studies to meet objective 3. An open, parallel group, single centre, randomised controlled trial involving 158 women with unexplained and endometriosis-related infertility to meet objective 4. A survival analysis of women in the lipiodol flushing randomised trial to meet objectives 4 and 5. A prospective observational study of the first 100 women to undergo lipiodol flushing in clinical practice to meet objective 5. A clinical hysterosalpingogram procedure to meet objective 4 and 5. Results 1) Eight randomised controlled trials involving 1,971 women were identified and included in the systematic review. Tubal flushing with oil soluble contrast media versus no intervention was associated with a significant increase in the odds of pregnancy (Peto odds ratio [OR] 3.57, 95% confidence interval [CI] 1.76 to 7.23) but there were no data for live birth. There were no data from RCTs to assess tubal flushing with water-soluble media versus no intervention. Tubal flushing with oil soluble contrast media was associated with a significant increase in the odds of live birth versus tubal flushing with water soluble contrast media (OR 1.49, 95% CI 1.05 to 2.11) but the odds of pregnancy showed no significant difference (OR 1.24, 95% CI 0.97 to 1.57) and there was evidence of statistical heterogeneity for these two outcomes. The addition of oil soluble contrast media to flushing with water soluble contrast media (water plus oil soluble contrast media versus water soluble contrast media alone) showed no significant difference in the odds of live birth (OR 1.06, 95% CI 0.64 to 1.77) or pregnancy (OR 1.16, 95% CI 0.78 to 1.70). 2) Nineteen Australasian fertility specialists returned survey questionnaires. Eighteen of the 19 specialists believed that lipiodol flushing was more likely to be beneficial than harmful. The most widely held prior belief, reflected in both textual and numerical responses, was that lipiodol was likely to produce a small beneficial response. The credible limits of this belief were compatible with a reasonable fertility benefit, as more than 50% believed that a 1.5-fold increase in pregnancy rate was plausible. The two surveys found that a 1.2-fold or 1.4-fold increase in pregnancy rate was the median expected level of benefit at which clinicians would have been inclined to recommend lipiodol flushing to their patients (combined range 1.1 to 2.3-fold). Individual and collective equipoise was justification to proceed with a definitive randomised controlled trial. 3) The mean number of cluster determinant (CD) 205+ uterine dendritic cells decreased significantly in mice following lipiodol treatment compared to sham treated and saline treated mice, particularly in endometrial and subendometrial tissues. The mean number of CD1+ uterine dendritic cells increased significantly following lipiodol treatment compared to shamtreatment. No significant differences were found in the mean number of total leukocytes or macrophages in the murine uterus between the three treatment groups. 4) Six month follow up of the randomised trial of 158 women showed that lipiodol flushing resulted in a significant increase in pregnancy (48.0% versus 10.8%, RR 4.44, 95% CI 1.61-12.21) and live birth (40.0% versus 10.8%, RR 3.70,.95% CI 1.30-10.50) rates versus no intervention for women with endometriosis (n=62), although there was no significant difference in pregnancy (33.3% versus 20.8%, RR 1.60, 95% CI 0.81-3.16) or live birth (27.1% versus 14.6%, RR 1.86, 95% CI 0.81-4.25) rates for women with unexplained infertility without confirmed endometriosis (n=96). Survival analysis up to 24 months showed a significant benefit in overall pregnancy rate following lipiodol treatment (hazard ratio 2.0, 95% confidence interval [CI] 1.3 to 3.2) for the combined endometriosis and unexplained infertility populations. Amongst women with endometriosis, the benefit in pregnancy rate seen in the first 6 months following lipiodol (hazard ratio 5.4, 95% CI 2.1 to 14.2) was not present at 6 to 24 months following lipiodol (hazard ratio 0.6, 95% CI 0.2 to 2.1). There was a more consistent effect of lipiodol on fertility throughout the 24 month follow up amongst women with unexplained infertility (hazard ratio 2.0, 95% CI 1.1 to 3.5). 5) Six month follow up in the observational study of 100 women undergoing lipiodol flushing as an innovative treatment in clinical practice showed an overall pregnancy rate 30% and live birth or ongoing pregnancy rate 27% six months after the procedure. For women under 40 years old, a 32% pregnancy rate and 25% live birth or ongoing pregnancy rate was seen in women with unexplained infertility; a 50% pregnancy rate and 47% live birth or ongoing pregnancy rate was seen in women with endometriosis. Of women aged 40 years and older, the pregnancy rate was 13% and the live birth or ongoing pregnancy rate was 13%. The pregnancy rates included those occurring after additional interventions, such as intrauterine insemination and in-vitro fertilisation, accounting for 12 of the 30 pregnancies. There were no treatment complications. Conclusions The conclusions of this thesis are as follows. Lipiodol flushing is a simple, inexpensive, effective fertility treatment, which carries a very low chance of complications and no increased chance of multiple pregnancy. Lipiodol treatment is particularly effective in the short term for women with endometriosis who have normal patent fallopian tubes. The fertility benefit from lipiodol treatment lasts longer for women with pure unexplained infertility than for women with endometriosis. The level of benefit from lipiodol treatment for women with unexplained and endometriosis-related infertility is of sufficient magnitude to convince most fertility specialists surveyed that it is a worthwhile treatment to offer routinely in clinical practice; however complex factors govern the implementation of an innovative fertility treatment. Observational study of the first 100 women to undergo lipiodol treatment in clinical practice has provided further evidence of the efficacy and safety of this approach. Uterine dendritic cell changes following lipiodol flushing in mice suggest that the mechanism of the fertility enhancing effect might be an immunobiologic effect on the endometrium that could improve the receptivity of the endometrium (rather than a mechanical tubal flushing effect), although this hypothesis requires further exploration in women.