<p>A large number of studies have identified that bronchoconstricting agents have increased potency and effect following inhaled ozone, but the contribution of bronchodilatory mediators to attenuation of the airway hyperresponsiveness observed following inhaled ozone remains unclear. The purpose of this investigation was to identify the contribution of nitric oxide synthase (NOS) and cyclooxygenase (COX) to the regulation of ozone-induced airway hyperresponsiveness. These mediators are derived largely from the airway epithelium and ozone exposure is likely, through epithelial damage, to alter the effect of NOS and COX in the airway. Using an ozone exposed guinea pig model of airway hyperresponsiveness, the in vivo responses to inhaled histamine were compared to responses in sham treated animals. Administration of the NOS inhibitor L-NAME (5120 μg/mL) increased the histamine sensitivity after ozone treatment. The non-selective COX inhibitor indomethacin (10 mg/kg) also increased the histamine sensitivity after ozone treatment. The COX-2 selective inhibitor DFU (1 or 10 mg/kg) caused a 2-fold leftward shift after ozone exposure, similar to that observed with indomethacin. The thromboxane antagonist, SQ 29,548 (1 mg/kg i.p.) attenuated the histamine responsiveness in a time dependent manner. The combination of COX and NOS inhibition produced a histamine-induced airway hyperresponsiveness greater than that observed with either NOS or COX inhibition alone. Pre-administration of dexamethasone inhibited the ozone-induced histamine hyperresponsiveness at all time points. Biochemical measurements of NOS activity identified an increase in NOS enzyme activity following ozone. Bronchoalveolar lavage revealed an inflammatory cell profile that did not correlate to the in vivo airway hyperresponsiveness. In vitro tissue bath experiments identified the presence and biological activity of NOS in the guinea pig trachea. Liquid chromatographic detection of nitric oxide metabolites and Western blot detection of COX-2 and NOS isoforms did not provide reliable data. This thesis demonstrates that histamine airway responsiveness following ozone changes rapidly and that both NOS and COX are upregulated a few hours after ozone exposure and, through functional antagonism, modulate ozone-induced histamine airway hyperresponsiveness.</p> / Doctor of Philosophy (PhD)

<p>Mitochondria play a critical role in the provision of energy to individual cells through the synthesis of ATP. The relationship between mitochondrial energy production and cell cycle progression has been explored in this work with the intention of defining the fuel requirements of the cell cycle engine. Because mitochondrial physiology and cell division proved to be intimately linked, we also examined the feasibility of disrupting mitochondrial properties in order to alter cell cycle dynamics. We show that the rate of utilization of mitochondrially-derived ATP fluctuates throughout the cell cycle and that stages where ATP levels are lowest correspond to stages most sensitive to pharmacologic inhibition of mitochondrial function. These particular transition points comprise theoretical "energetic checkpoints" of cell cycle progression. It is here that the biochemical events that are sensitive to changes in the cellular energy status will determine whether the cell continues through its cycle or pauses until a favourable energetic balance is restored. The increase in the number of cells in the G1 component of the cell cycle that results from antagonizing mitochondrial function is accompanied by an augmented proportion of persistently active Retinoblastoma protein (Rbp). Failure to inactivate this tumour suppressor protein, whose role it is to brake cell cycle progression in response to suboptimal conditions, does not result from an upregulation of the "classical" G1 inhibitory proteins but rather does so secondary to a decrease in the availability of a critical G1 cyclin. This sensitive regulatory protein, cyclin D, is essential for the activation of kinases responsible for the initial phosphorylation of Rbp, which renders it inactive and allows passage out of the G1 component of the cell cycle. This is the first work to report cell cycle-specific periods of increased ATP utilization which correlate with checkpoints through which a cell will not pass if its energetic balance is sufficiently disrupted. We also demonstrate that changes in mitochondrial function elicit changes in the core proteins of the cell cycle machinery suggesting some intracellular link between the energy balance within the cell and the proteins responsible for cell cycle progression. Finally this report confirms previous suggestions that mitochondria represent viable targets for altering the division characteristics of a cell population, particularly in the context of the altered mitochondrial phenotype of many tumour cells.</p> / Doctor of Philosophy (PhD)

<p>The interaction of circulating coagulation factor VII with cell surface associated tissue factor (TF) is critical for the initiation of blood coagulation, and appears to play a significant role in several other biological processes. Thus, an understanding of the mechanism of this interaction should provide valuable insight into the development of these important processes. The first epidermal growth factor-like domain (EGF-1) of factor VII is the principle contact site with TF and possesses the structural elements characteristic of the ubiquitous EGF-like motif. The aim of this thesis was to investigate the relationship between the conformational structure of the EGF-1 domain in factor VII and its function in the initiator complex. Recombinant generation and analysis of a naturally occurring factor VII EGF-1 mutant (N57D) indicated a protein deficient in its cellular secretion, as well as possessing compromised procoagulant activity and TF binding capability. Using structural investigative techniques, this mutation was found to compromise the appropriate conformation of the EGF-1 domain via the loss of a previously undescribed, structurally-important intermolecular hydrogen bond, highlighting the delicate balance between conformation and function in this domain. Once developed, these structural investigative techniques allowed further investigation, indicating the EGF-1 domain adopted a conformation specific to the "active state" of factor VIIa and requiring the presence of calcium and the adjacent Gla domain. Once activated, occupation of the active site of factor VIIa with substrate-like inhibitors resulted in further conformational changes to the EGF-1 domain, indicating the presence of a novel active site occupant-specific allosteric linkage between the EGF-1 domain and the active site. This allosteric link also appeared to affect the functional processes of TF binding and enzymatic activity of the factor VIIa molecule. Overall, this study has contributed new knowledge on the relation of structural elements of the factor VII EGF-1 domain to the important factor VIIa functional processes of TF binding and enzymatic activity. As well, the study indicates a novel role for the EGF-1 domain in the unique co-factor mediated specificity observed for factor VIIa.</p> / Doctor of Philosophy (PhD)

<p>Inflammatory conditions of the gut, such as ulcerative colitis and Crohn's disease are associated with an alteration in intestinal motility. The mechanisms underlying altered motor function is unknown, but may be the result of alteration in smooth muscle and/or enteric nerve function. Recent studies using Trichinella spiralis (T.spiralis) infection of rats, as model of intestinal inflammation, have shown that the accompanying inflammatory response in these animals is associated with an altered enteric nerve function. These changes included a suppression of noradrenaline and acetylcholine release and an increased substance P content within the neuromuscular layer of the inflamed jejunum. Furthermore, there was an expression inflammatory cytokines within the mucosa and the deeper muscular layers of the jejunum within 12 hrs from the onset of T.spiralis infection. This elevation in cytokine expression occurred prior to the changes in myenteric neurotransmitters. It is therefore possible that inflammatory cytokines within neuromuscular layer cause a change in neurotransmitter release and content, and thus contribute to the altered gut motility observed in the nematode-infected rats.</p> <p>Addition of exogenous IL-1β or TNFα to isolated longitudinal musclemyenteric plexus (LM-MP) preparations caused a suppression of stimulated noradrenaline release, which was tiem and concentration dependent. This suppressive action was biphasic in manner, displayign an early protein synthesis independent effect and a delayed protein synthesis dependent effect. Furthermore, the delayed suppressive action of these cytokines on noradrenaline release were dependent on prostanoid synthesis. On examination of a putative direct interaction between these cytokines and adrenergic nerves using a nerve varicosity preparations, only IL-1β was found to suppress the evoked noradrenaline release. This suppressive action by IL-1β was time- and concentration-dependent, and in part, mediated by prostanoids. Although incubation of TNFα alone with the varicosities was unable to induce a response, the presence of TNFα did potentiate IL-1β-induced suppression of noradrenaline release. The conclusions drawn from this study are that TNFα causes a suppression of noradrenaline release from myenteric nerves which is evident only in a multicellular preparation and ilkely involves intermediary cells and their products including prostaglandins and/or thromboxanes. This contrasts with IL-1β, which in addition to an indirect effect, also suppressed noradrenaline release by directly interacting with adrenergic nerve terminals.</p> <p>IL-1β also caused an increase in substance P content within the myenteric plexus. The increase in this neuropeptide was time and concentration dependent, and could be depleted by scorpion venom. Immunohistochemical studies indicated that substance P was only found present within myenteric nerves. The IL-1β-induced increase in substance P was considered to be due to increased synthesis, since cycloheximide prevented the cytokine-stimulated increase substance P content induced by IL-1β. Moreover, the increase in substance P content was mediated by prostanoid synthesis, but not nerve growth factor. The conclusion drawn from these experiments is that IL-1β stimulates the synthesis of substance P within intrinsic nerves of the myenteric plexus.</p> <p>A putative role of endogenous IL-1 in the alteration of neurotransmitters in the T.spiralis model of intestinal inflammation was examined using a selective IL-1 receptor antagonist (IL-1ra). Treatment of the animals with IL-1ra prior to T.spiralis infection attenuated the suppressed noradrenaline release and increased substance P content observed in the inflamed LM-MP preparations. Therefore the results fro these experiments and those preformed using exogenous IL-1β and TNFα support and notion that inflammatory cytokines alters myenteric neurotransmitters, resulting in a disruption of enteric nerve function and thereby contributing to alterations n intestinal motility.</p> / Doctor of Philosophy (Medical Science)

<p>Gender differences in substrate utilization have been observed with females having higher lipid and less carbohydrate oxidation during endurance exercise. This thesis consists of a series of three studies conducted with the overall objective of advancing our understanding of gender differences in substrate metabolism and the mechanism behind these differences. The first and second studies investigated the effect of endurance training on whole-body substrate, glucose and glycerol utilization muscle enzyme adaptations in males and females. Carbohydrate utilization was lower following endurance training when tested at the same absolute exercise intensity. Glucose rate of appearance (Ra) and metabolic clearance rate (MCR) were lower following training. Females had a lower glucose MCR later in exercise as compared to males. Regardless of training status, females had a higher glycerol Ra as compared to males suggesting a greater whole-body lipolysis. Gender differences in lipid utilization were not accompanied by gender differences in maximal enzyme activities, which increased following endurance training for both genders. The third study investigated the effect of 17-β-estradiol on whole-body substrate, glucose and glycerol utilization during endurance exercise in males. The administration of 17-β-estradiol resulted in a lower MCR during exercise as compared to placebo. 17-β-estradiol also resulted in a higher plasma glucose concentration during exercise, but had no effect on substrate oxidation, glycerol turnover or plasma glycerol and FFA concentrations at rest or during endurance exercise. It can be concluded that females have a higher whole-body lipolysis and oxidize a greater proportion of fat. The glucose kinetic data from the training study together with the data from the 17-β-estradiol study suggested that females used less hepatic glucose during endurance exercise. Although alterations in glucose kinetics were found following the administration of 17-β-estradiol to males further research is required to unravel the mechanism(s) behind the gender difference in substrate metabolism.</p> / Doctor of Philosophy (PhD)

<p>Are the outcomes of rheumatoid arthritis patients improved when their family physicians are provided with information gathered by specially trained physiotherapists?</p> <p>Two physiotherapists were selected and trained in detailed objective techniques of evaluation and problem identification, leading to a clear and succinct report designed to assist the family physician in treatment decisions. From June 1974 to January 1977, a randomized controlled trial was conducted to assess the effect of these reports on the outcomes of women with rheumatoid arthritis treated in the community by their family physicians.</p> <p>On admission to the trial, patients were randomly allocated to two groups: one experimental group which was assessed according to modern quantitative techniques of evaluation and, following communication of results to family physicians, were treated at home by the trained physiotherapists; and a control group which was assessed according to traditional techniques of evaluation and, following, communication of results to family physicians were treated at home by traditional physiotherapists. All subjects were interviewed initially, at four, and at 12 months by independent assessors who used a standardized prestructured questionnaire designed to measure the level of joint inflammation, functional capacity, compliance and mood.</p> <p>In association with this triaI, a pooled index was developed converting live separate outcome measures into a single prespecified variable, to measure treatment differences.</p> <p>The author's unique contribution to this trial related to: development, formulation and execution of the experimental design, and more specifically, the use of independent assessors in rheumatology, allocation strategies, processing of intakes, supervision and control of data gathering procedures and the introduction of modifications in the design following the protest and during the experiment period that made this trial feasible.</p> <p>This is a "compound" thesis, focussing on the present state of knowledge and rationale for the research, the original design and modifications introduced following the pretest and during the experimental period. The rationale and effects of these modifications will be discussed in detail.</p> / Master of Science (MS)

<p>This thesis is a report of the evaluation of an industrial alcoholic rehabilitation program. The evaluation covered the years 1970 - 1974 inclusive and all those referred to the program were included in the sample.</p> <p>Since employed problem drinkers are identified by deteriorating job performance, the variables selected for investigation were directly related to job performance. Absenteeism in four categories and visits to the industrial medical clinic were compared for three years; the year before referral, the year of treatment and the year after referral. Number of accidents sustained, both occupational and non-occupational and number of suspensions incurred were compared for the year before referral and the year after referral.</p> <p>Information was collected on age, marital status, number of years employed in the industry and diagnosis of one or more selected alcohol-related conditions. Data were collected on the number of appointments scheduled and kept to determine compliance and its' relationship to outcome.</p> <p>Those win refused to enter the program are described by mean age and mean number of years worked in the industry. Those who dropped out of the program before completing one full year post-referral are described by mean age, marital status, number of dependents, mean number or years worked in the industry and the mean number of months between referral and drop-out.</p> <p>The evaluation demonstrated that the program has a 78.8% success rate. The data obtained led to the formulation of hypotheses suitable for testing in future controlled trials.</p> / Master of Science (MS)

<p>Hyperkinetic children and their treatment are currently the subjects of heated discussions among medical persons, parents, teachers, government officials, the press and a concerned public. The focus of these discussions is often the appropriateness or inappropriateness of stimulant medications for hyperkinetic children.</p> <p>The first part of this thesis contains a review of a substantial portion of the literature on hyperkinesis including : definitions and diagnosis of hyperkinesis, prevalence of hyperkinetic symptoms and of hyperkinesis, characteristics of children considered hyperkinetic, drug treatments for hyperkinesis, and non-drug treatments for hyperkinesis. The major purpose of the literature review is to examine all available studies of the effectiveness of methylphenidate for hyperkinesis and all available studies of the effectiveness of non-drug treatments for hyperkinesis. This review indicates that methylphenidate is the most effective drug treatment tested and that behaviour modification or operant conditioning is the best tested, effective non-drug treatment for hyperkinesis. The relative effectiveness of methylphenidate and behaviour modification alone or in combination for the treatment of hyperkinesis is not known.</p> <p>In the literature review, Tables are provided which summarize information gleaned from an extensive selection of publications. These Tables include : A) Estimates of the Prevalence of Hyperkinetic Symptoms and of the Hyperkinetic Disorder in Children; B) Some Characteristics which Distinguish Children Diagnosed as Hyperkinetic from Normal Children; C) Some Characteristics which Distinguish Children Diagnosed as Hyperkinetic from Neurotic or Normal Children; D) A Summary of Studies of Methylphenidate for Hyperkinesis; E) The Effectiveness of Methylphenidate versus Placebo for Hyperkinesis : measures on which M and P have differed significantly in therapeutic effectiveness; F) The Effectiveness of Methylphenidate versus other Active Drugs for Hyperkinesis; G) A Summary of Studies of Non-Drug Treatments for Hyperkinesis; H) Evaluation of Miscellaneous Non-Drug Treatments for Hyperkinesis, and I) The Effectiveness of conditioning in the Treatment of Hyperkinesis.</p> <p>The second part of this thesis is a research proposal which has been developed on the basis of the current state of knowledge pertaining to the treatment of Hyperkinesis. The proposed research is designed to determine which of three very promising treatments for hyperkinesis has the greatest effectiveness and fewest side-effects: methylphenidate, behaviour modification, or methylphenidate plus behaviour modification. The protocol covers the following areas : Rationale for the study; Selection criteria for hyperkinetlc children; Collection of the sample of children; Sample size required; Pre-treatment assessments; Assignment to treatments; Methylphenidate treatment at .7mg/kg/day; Behaviour modification program including 8 sessions; Methylphenidate and behaviour modification in combination; Post-treatment assessments; Data analysis and budget for the study. This protocol could be adapted for use by other investigators interested in the area.</p> / Master of Science (MS)

<p>The energy required for the performance of light exercise is largely supplied by the aerobic oxidation of fat and carbohydrate. As the intensity of exercise increases, the percentage of energy derived from anaerobic processes, predominantly the breakdown of muscle glycogen to lactic acid, is increased proportionally. Evidence suggests that the increase in anaerobic metabolism influences the mobilization of free fatty acids from adipose tissue. Studies have shown that lactate inhibits FFA mobilizltion in dogs. In man, it was found that plasma FFA are reduced during heavy exercise and remain low as long as plasma lactate is increased. However, when lactate is increased under these conditions, the pH is reduced.</p> <p>To separate the effects of pH and lactate in the blood, subjects performed three identical exercise tests with artificially induced changes in blood pH prior to two of the studies. Ammonium chloride and sodium bicarbonate were ingested over a three hour period prior to exercising to produce an acidosis and alkalosis, respectively. Calcium carbonate was given as a placebo for the control study. In all subjects, the studies were performed in a random order, in a fasted state and were separated by at least 72 hours. Arterialized venous blood was sampled from a vein in the hand.</p> <p>The maximum power output of each subject was measured by a progressive exercise test to exhaustion on a cycle ergometer. During the studies, they exercised at 33% and 66% of their maximum power output for 20 minutes each, followed by 90% until exhaustion.</p> <p>This protocol resulted in a significant separation in both blood pH and exhaustion times at the 90% workload. The mean exhaustion times were 4 min., 2.5 min., and 6.5 min. for the control, acidosis and alkalosis studies, respectively.</p> <p>The plasma lactate concentrations were also significantly different. The lowest lactate levels were found during acidosis and the highest during alkalosis.</p> <p>Plasma glycerol and FFA concentrations were similar during the control and alkalosis studies, but significantly lower during acidosis. The lowest levels of glycerol and FFA were found when the pH was lowest, but plasma lactate was also lowest.</p> <p>These results suggest that changes in pH are more important than changes in lactate, per se, in the inhibition of FFA mobilization during severe exercise. In addition, it appears that acidosis causes either a decreased rate of production of lactic acid, a decreased rate of efflux of lactate from muscle, or an increased utilization of lactate by other tissues.</p> <p>Although the mechanisms cannot be determined from this study, the results suggest that acidosis inhibits glycolysis as well as FFA mobilization, reducing the anaerobic production of energy. This presents no serious problem during moderate exercise, but seriously impairs performance of heavy work.</p> / Master of Science (MS)

<p>The in vitro metabolism of testosterone, progesterone and androstenedione was studied in liver, testes, hypothalamus and cortex incubations of normal BALB/c♂, BALB/c♀ and a number of sex-mutant mice carrying the testicular feminization (Tfm) and sex-reversal (sxr) genes.</p> <p>The metabolism of testosterone was studied in liver homogenates of BALB/c♂ , Tfm (o⁺)⚥ and Tfm (oʰᵛ)⚥. Androstenedione was identified as the major metabolite of testosterone, in all incubations, indicating the presence of 17β-hydroxysteroid dehydrogenase activity. Comparison of the percentage conversions of testosterone to androstenedione suggested that of the three genotypes studied, the activity of 17β-hydroxysteroid dehydrogenase was lowest in the Tfm (oʰᵛ) liver.</p> <p>Similarly, the metabolism of testosterone and progesterone was studied by double-labelled tracer experiments using testes minces from BALB/c⚥ , Tfm (o⁺)⚥ and Tfm (oʰᵛ)⚥ mice. The conversion of progesterone to testosterone was greatest in BALB/c♂ incubations and least in the Tfm (o⁺) incubations. These data suggest that the relative activities of the 17-ketoreductase for the conversion of androstenedione to testosterone are in the order: BALB/c♂ > Tfm (oʰᵛ) ⚥ > Tfm (o⁺).</p> <p>Progesterone metabolism was studied in testis incubations of normal male (BALB/c♂), testicular-feminized, sex-reversed (Tfm+(oʰᵛ) Blo/+++, sxr/+ ♂) and sex-reversed (+Ta++/+++, sxr/+♂) mice. Comparison of the amounts of androstenedione and testosterone formed from P suggests that differences are present in the relative activity of the testis 3-ketoreductase. The relative activity is in the order BALB/c♂ > +Ta++/+++, sxr♂ > Tfm+(oʰᵛ) Blo/+++, sxr♂.</p> <p>The metabolism of androstenedione was also studied in testes incubation of Tfm (o⁺)⚥ and Tfm (oʰᵛ)⚥ mice. The formation of testosterone was similar in all incubations. A number of unidentified metabolites of androstenedione were detected in the Tfm (oʰᵛ)⚥ incubations which were not present in the Tfm (o⁺) incubations.</p> <p>Finally, the aromatization of testosterone was studied in hypothalamus and cortex incubations of normal BALB/c♂, BALB/c♀, and testicular feminized Tfm (o⁺)⚥ and Tfm (oʰᵛ)⚥mice. A new, rapid method was developed for studying aromatization based toluene-sodium hydroxide partitioning and a novel estrogen methylation procedure known as extractive alkylation. Using this method the formation of estrone and estradiol was detected. The conversion of testosterone to estradiol by hypothalamus minces was about 1.5 times greater in normal BALB/c♂ than BALB/c♀ and about the same as in Tfm (o⁺) and Tfm (oʰᵛ). Total aromatization by cortex minces was about 30-50% that of the hypothalamus. The major metabolite of testosterone in normal BALB/c ♂ and BALB/c ♀ incubations was estrone while in the Tfm (o⁺) and Tfm (oʰᵛ) incubations it was estradiol-17β.</p> <p>The studies confirm the work of others which suggests that the metabolism of steroids is altered in the liver and testis of the Tfm (o⁺)/Y⚥ mouse. The present work extends these observations to include a number of other mice carrying the Tfm and sxr genes. Using the extractive alkylation technique aromatization was detected in Tfm hypothalamus and although the overall quantitative yields of estrogen from testosterone were similar to normals, qualitative differences were evident which may be significant to the explanation of altered hypothalamic-pituitary gonadotrophin relationships in Tfm (o⁺) and Tfm (oʰᵛ) mice.</p> / Master of Science (MS)