<p>Atopic asthma is immunologically-driven condition characterized by reversible bronchoconstriction and Th2-polarized eosinophilic airways inflammation. As such, the development of Th2 responses is a key step in the pathogenesis of the disease. We utilised a murine model of respiratory mucosal sensitization to examine the roles of different cytokines in the generation of polarized immune-inflammatory responses in the airways. Mice were sensitized to ovalbumin (OVA) by daily aerosol exposure in the context of airway expression of GM-CSF, resulting in a Th2-polarized eosinophilic inflammatory response in the airways, reminiscent of asthma; the addition of IL-12 to the airway microenvironment deviated this response toward the Th1 phenotype. We analyzed expression of key Th1-and Th2-associated genes in the lymph nodes during Th1- and Th2-polarized sensitization, and showed that cytokine expression compartmentalized to the respiratory tract can have a profound impact on the nature of developing immune responses in the draining lymph nodes. Next, we investigated the necessity for IL-4 during GM-CSF-driven respiratory mucosal sensitization. We analyzed a variety of Th2-associated factors, including transcription factors, cytokines, chemokine receptors, and cell surface markers, and found that IL-4 was not necessary for Th2-polarization in this model. Finally, we examined whether IL-10 played a rol in mediating Th2 polarization, and observed that mice treated with anti-IL-10 antibodies or genetically deficient in IL-10 showed impaired development of Th2-polarized immune-inflammatory responses. This research highlights the importance of the cytokine microenvironment of the airways in determining the nature of the ensuing immune-inflammatory response, and defines some of the molecular requirements for the polarization of Th responses during respiratory mucosal sensitization in vivo.</p> / Doctor of Philosophy (PhD)

<p>Shortened platelet survival observed in individuals with thromboembolic vascular disease could be due to platelet consumption or turnover in thrombi or platelet turnover on the damaged vessel wall. The object of this study was to examine the relationship between:</p> <p>a) platelet consumption and exchange in thrombi and platelet survival</p> <p>b) platelet accumulation and turnover on the injured vessel wall and platelet survival.</p> <p>Indwelling catheters were inserted into rabbit aortae to induce repeated vessel injury and macroscopic chrombus formation. Radiolabelled platelets monitored platelet participation in thrombosis and platelet accumulation on the injured vessel wall. Morphological studies assessed the thrombus characteristics and the cellular events on the vessel wall.</p> <p>During the first 24 hours following the insertion of indwelling aortic catheters, thrombi formed, attained their maximum size and maintained a constant weight thereafter. ⁵¹Cr-labelled platelet incorporation into thrombi following the growth, decreased by about 50% by 3 days. Loss of radioactivity from the thrombus during the time when thrombus weight remained constant indicated platelet lysis, phagocytosis or loss of whole platelets from the thrombus.</p> <p>Both morphological studies and studies using platelets doubly labelled with ¹²⁵I and ⁵¹Cr demonstrated that some of the platelets initially incorporated into thrombi lysed. Studies of changes in platelet density as well as studies showing thrombi retained some capacity to accumulate circulating platelets indicated that platelets exchanged in thrombi and some of the platelets that had participated in thrombus formation returned to the circulation as less dense platelets.</p> <p>The consumption and exchange of platelets in thrombi during the acute phase of growth correlated with maximal shortening of platelet survival time. When platelet survival studies were carried out 1 week after insertion of the catheter (at a time when thrombus growth had ceased and platelet accumulation into thrombi was decreased) platelet survival continued to be shortened. At this time, the extent of platelet accumulation on the injured vessel wall was similar to that observed at the time of insertion of the catheter suggesting platelet interaction with the injured vessel wall influenced platelet survival.</p> <p>Studies using long and short catheters demonstrated that short catheters induced comparatively less vessel injury than long catheters but thrombus formation was significantly increased. Platelet survival studies carried out at 3 days after insertion of long or short catheters (when thrombus growth had ceased) indicated that the short catheters had no significant effect on platelet survival whereas long catheters continued to shorten platelet survival time.</p> <p>In conclusion, extensive thrombus formation with evidence of platelet exchange in thrombi is associated with significant shortening of platelet survival time. However, this effect is detectable only during the acute phase of thrombus formation. In contrast, repeated vessel injury in association with continuing platelet turnover on the vessel wall can shorten platelet survival time.</p> <p>Therefore, shortened platelet survival time is a manifestation of acute thrombus formation, repeated vessel injury or both.</p> / Doctor of Philosophy (PhD)

<p>The muscle of the pregnant uterus transforms into an active and reactive tissue at term which is necessary for expulsion of the fetus and termination of pregnancy. Gap junctions develop between the smooth muscle cells of the uterus immediately prior to term and disappear after delivery. Gap junctions may be providing the structural basis for improved communication between myometrial cells during labor thereby leading to the synchronized coordinated muscle activity required for effective labor. Although gap junctions appear to have significant implications in the termination of pregnancy, the regulation of their presence in the myometrium is not completely understood but may depend upon hormonal changes that occur prior to parturition.</p> <p>In the studies reported in this thesis, quantitative thin section electron microscopy showed that high doses of estradiol stimulated, whereas progesterone when administered with estradiol, suppressed the presence of myometrial gap junctions in non-pregnant animals. The steroid hormones regulate the presence of gap junctions by controlling the synthesis of the junction proteins probably through their receptor mechanism. In addition, high doses of estradiol may selectively stimulate the synthesis of a prostaglandin which may be required for the development of gap junctions. Prostaglandins appear to be involved in junction regulation since inhibitors of cyclo-oxygenase potentiated estradiol stimulation of gap junctions. Moreover, treatment of pregnant animals with estradiol resulted in the presence of numerous myometrial gap junctions and abortion. These results demonstrate that the steroid hormones and prostaglandins regulate the presence of myometrial gap junctions and that their presence in the myometrium may be a requirement for the occurrence of term as well as preterm labor.</p> <p>Cell-to-cell communication via gap junctions is also believed to be involved in the control of cellular and tissue growth. The myometrium grows dramatically throughout pregnancy to accommodate the growing fetus. Moreover, the steroid hormones, in particular estradiol, stimulates growth of the uterus and prostaglandins also appear to be involved in the process. Since the hormones and prostaglandins regulate uterine growth and the presence of myometrial gap junctions (see above), the relationship between the two events was further evaluated in this thesis. Studies on non-pregnant animals after various hormonal treatments and in untreated pregnant animals demonstrated that regulation of uterine growth (of which the myometrium is a major component) and the presence of gap junctions in the myometrium are dependent upon the hormonal environment. The results suggest a complex interaction between the steroid hormones, a product of the cyclo-oxygenase and/or lipoxygenase pathway for control of gap junctions and myometrial growth. Myometrial growth may occur because of the lack of gap junctions but the cessation of growth of this tissue does not depend on the presence of the junctions. The development and presence of gap junctions, however, may be partially dependent upon myometrial growth.</p> / Doctor of Philosophy (PhD)

<p>Saunders and his associates reported that during submaximal neuromuscular blockade, vital capacity was reduced to a greater extent than would have been predicted according to the reduction in the maximal inspiratory pressure in a simple in a simple mechanical model (J. Appl. Physiol. 44:589,1978). Disproportionate respiratory muscle weakness and a different recruitment pattern for different voluntary efforts were proposed as possible explanations.</p> <p>This issue was addressed by measuring the rectified electromyogram of the diaphragm, intercostal, scalenes, and sternomastoid muscles during maximal and submaximal static inspiratory manoeuvres. Two studies were performed. The control series of experiments showed that at very low levels of static pressure, only the diaphragm was activated. Further increases in pressure then recruited the intercostal/accessory muscles, in some subjects more than in others. There was generally an orderly increase in the rectified EMG in all muscles with elevations in pressure. For particular levels of pressure to be produced at lung volumes above the relaxed end-expiratory position, the EMG was increased. Compared to the maximum EMG elicited with maximal static pressures or full inspirations, resting ventilatory requirements still leave a large myo-electric reserve.</p> <p>In the submaximal neuromuscular block study, the mean maximal inspiratory pressure was decreased from 103 to 39 cm H20, but only two of the five subjects behaved in the manner observed by Saunders and his associates. There was no clear evidence that the diaphragm was less affected than the other muscles. In many cases however, a submaximal level of pressure was achieved by augmented EMG in all muscles. It is suggested that curare interferes with the conversion of electrical events into whole muscle tension and/or that the impaired chest wall volume compartments are inefficient at producing static pressure.</p> / Doctor of Philosophy (PhD)

<p>Allergic diseases, including asthma, result from airway inflammatory responses directed against ubiquitous antigens-allergens. The detailed immunological mechanisms underlying the development of allergy (allergic sensitization) have not been fully elucidated. Our understanding of the complexity of cellular interactions underlying allergic inflammation originates mainly, but not exclusively, from studies in experimental animal models. The studies presented in this thesis utilize experimental mouse models of anti-geninduced allergic airways inflammation in order to investigate 1) the events occurring during sensitization (primary immune responses) and following re-challenge (secondary immune responses) in two immunologically important sites: lungs and lymph nodes following experimental allergen (ovalbumin; OVA) exposure; 2) the role of the secondary lymphoid organs vs. lungs in elicitation of immune responses to allergen; 3) the importance of two major costimulatory pathways - CD28/B7 and ICOS/B7RP-1 - in the generation of allergic airways inflammation. Findings presented here indicate that introduction of antigen leads to vigorous T and B cell activation in the draining lymph nodes. Such activation translates into the acquisition of a Th2 phenotype, an important step in the generation of allergic sensitization. Considering the multitude of changes occurring in the draining lymph nodes, the importance of lymph nodes during sensitization was investigated in mice devoid of lymph nodes - Iymphotoxin (l deficient mice. The study demonstrated the absolute requirement of lymphoid organs, either lymph nodes or spleen in generating of Th2-type inflammatory responses. Finally, studies on CD28 and B7RP-1 deficient mice indicated that, whereas the CD28/B7 pathway is necessary for the establishment of allergic airway inflammation, the ICOSIB7RP-1 pathway is redundant. The data presented in this thesis identifies several important aspects by which the immune system generates efficient allergic airway inflammation. As we suspect that new-sensitization occurs after each exposure to allergens. information in this thesis may provide insights into novel therapeutic strategies.</p> / Doctor of Philosophy (PhD)

<p>In view of the importance of maintaining normal mobility in the aging population, the function of two opposing groups of leg muscles, the ankle dorsiflexors and plantarflexors, was studied in a sample of 111 healthy men and women aged 20 to 100 yr. Three major questions were asked in this investigation: (1) To what extent does muscle strength decline with age? (2) Can descending motor pathways be optimally utilized by the elderly for activating lower motor neurons? (3) Does the time-course of muscle contraction become prolonged with age?</p> <p>Summarized results were:</p> <p>1. Maximal voluntary isometric strength (MVC) of the dorsiflexor and plantarflexor muscles showed a general pattern of decreased values after the fifth decade in both sexes. A similar decline of approximately 13% per decade was observed in the two muscle groups. As the plantarflexor muscles produced 4 times more torque than the dorsiflexor muscles in young adults, the absolute loss of strength was much greater for plantarflexor MVC.</p> <p>2. The majority of subjects at all ages were able to utilize their descending motor pathways optimally for full muscle activation.</p> <p>3. Contraction time and one-half relaxation time of the isometric twitch were prolonged with increased age in both muscles.</p> <p>4. An additional observation was that flexibility of the ankle joint was reduced with increased age, although considerable rotation of the ankle was still possible in the oldest subjects.</p> <p>Evidence from analysis of the compound muscle action potentials, peak twitch torques and muscle cross-sectional areas supported the conclusion that the decrease in strength with aging was due to a loss of excitable muscle mass. It was hypothesized that fat and connective tissue replaced muscle in the elderly. The findings of this study add to our knowledge about the aging process and its influence on neuromuscular function. It is also anticipated the results will be useful in geriatric clinics and for planning programs aimed at the prevention and rehabilitation of neuromuscular disability in the elderly population.</p> / Doctor of Philosophy (PhD)

<p>Peripheral nerve injuries have a devastating impact on muscle function due to the distance the axons must regenerate to reinnervate the target muscle. By the time the axons reinnervate the muscle, it has strophied and lost itself receptiveness resulting in impaired function. The objective of this study was to answer the following questions: 1. Do sensory axons delay muscle atrophy following denervation? 2. Does the protective effect observed by sensory axons translate into restored muscle function? 3. Do sensory axons preserve the architecture of the distal nerve sheath and provide a favourable regeneration substrate? 4. Do sensory axons influence the trophic effect on denervated muscle to maintain its "receptiveness" to reinnervation? Following tibial nerve transection, rats were assignedd to one of the following groups: (1) saphenous to distal tibial nerve neurorrhaphy (Nerve-to-Nerve sensory protected (SP)); (2) saphenous to gastrocnemius neurotization (Nerve-to-Muscle sensory protected (SP) in the absence of the distal nerve sheath); (3) Unprotected controls (tibial nerve transection) or, (4) immediate common peroneal to tibial nerve neurorrhaphy (Immediate Repair with a motor nerve). The unoperated contralateral leg of treated animals served as a control. After a 6-month denervation period followed by motor reinnervation, ultrastructure, histology, morphometrics of nerve and muscle were assessed and muscle function was measured. Specimens of distal tibial nerve in the Nerve-to-Nerve (SP) group were superior to Unprotected controls shown by a significant increase in axon density, a significant decrease in collagen area, and improved axon-to-Schwann cell coupling. These features are characteristic of the original neural environment and reflect sustained neural integrity. Although axon number in the Nerve-to-Nerve (SP) group was similar to the Nerve-to-Muscle (SP) group, improved regeneration was evident in the Nerve-to-Nerve (SP) group shown by several axons at various stages of myelination, in a "normal" one-to-one association with a Schwann cell. The Nerve-to-Nerve (SP) group also displayed a significant increase in mean axon area than the Nerve-to-Muscle (SP) group. Gastrocnemius muscle specimens from both sensory protected groups displayed less collagenization and fat deposition than Unprotected control muscle, similarity in mean total muscle fibre size, and evidence of reinnervation (fibre type grouping) among regions of "mosaicism" suggesting a preservation of normal muscle features. Fast twitch fibres predominated in both sensory-protected groups (60% to 40%) as in normal muscle. Unprotected controls contained no fast twitch fibres and the total muscle fibre area of this group was significantly smaller than all other experimental groups. The man area of fast twitch fibres in Nerve-to-Muscle (SP) group was significantly larger than the Nerve-to-Nerve (SP) group was suggesting a possible trophic influence on fast twitch muscle fibre area. Mean compound muscle action potential amplitude in the Nerve-to-Nerve (SP) group was significantly higher than the Nerve-to-Muscle (SP) and Unprotected control groups. Although the Nerve-to-Muscle (SP) group demonstrated a significantly higher isometric contractile twitch force than Unprotected controls, this as only slightly increased possibly due to the unusually high values for the Unprotected controls. These findings suggest that a feasible method to optimally diminish the denervation changes in muscle is to preserve the architecture of the distal nerve and concomitantly maintain the trophic influence on the muscle fibres.</p> / Doctor of Philosophy (PhD)

<p>The overall objective of this study was to investigate the physiological and trophic interactions that can occur between sensory nerves and their targets or end organs, specifically those between cutaneous mechanosensory axons and the epidermal Merkel cells. Previous work has shown that in salamander skin, the Merkel cell-neurite complex forms the morphological basis of the rapidly-adapting, low-threshold touch receptor (Cooper and Diamond, 1977; Parducz et al., 1977), while in mammalian species, it is associated with slowly-adapting mechanoreceptors. The present investigation has been carried out using Xenopus frogs (Xenopus laevis), in which the Merkel cells are located around the visible openings of the cutaneous gland ducts. A voltage-controlled mechanical stimulator of 10 μm tip diameter was used to compare the mechanosensory thresholds when the stimulator was applied directly over the gland openings ("on" locations) to those when the stimulator was located between the openings. The most sensitive points were always the "on" ones, and the results indicated that these represented a single population of rapidly-adapting, low-threshold touch receptors. Therefore, the locations of these mechanoreceptors coincided with the positions of the epidermal Merkel cell-neurite complexes, suggesting the latter have a mechanosensory function in Xenopus laevis.</p> <p>An attempt was made to clarify the role of the Merkel cell in the mechanosensory process and in the trophic interactions believed to take place between the Merkel cells and the sensory nerves; this was done by following the development of mechanosensitivity when sensory nerves grow into nerve-free skin and observing whether there was any correlation with the appearance of the morphological features characteristic of the Merkel cell-neurite complex. These studies involved monitoring of the reinnervation of denervated skin, and the innervation of new skin that had regenerated in place of a portion previously excised. Merkel cells were shown to be present in both situations by using the fluorescent dye quinacrine as a marker for the Merkel cells and by EM examination. The development and maintenance of the Merkel cells seemed to be independent of nerves; they survived denervation, and they appeared in regenerated skin even in a totally denervated limb. Ingrowing sensory nerves eventually contacted these Merkel cells, which thus act as targets for these nerves. Preliminary results suggest that recovery of discrete low-threshold touch spots requires that contacts occur between the nerve endings and the Merkel cells. The mechanosensitivity develops gradually, however, concomitant with the gradual maturation of the Merkel cell-neurite complexes.</p> / Doctor of Philosophy (PhD)

<p>The addition of dopamine to homogenates or slice preparations from rat caudate nucleus results in a 100-150% increase in tissue cAMP levels. Since protein kinases are the only known physiological receptors for cAMP, it has been hypothesized that the response of the postsynaptic neurons to dopamine may be mediated in part through the phosphorylation of specific membrane proteins. This study examines the effects of dopamine on cAMP and its associated protein kinase in caudate nucleus.</p> <p>Caudate tissue contains a Type II protein kinase that is activated by cAMP, and that catalyzes the phosphorylation of several synaptic membrane proteins. Dopamine, however, did not appear to enhance the phosphorylation of these proteins either in broken cell or intact preparations, although increases in cAMP could be demonstrated under similar experimental conditions.</p> <p>Destruction of postsynaptic neurons using the neurotoxin kainic acid significantly reduced cAMP-dependent protein kinase activity. Destruction of presynaptic nerve terminals, on the other hand, had no effect on the activity of this enzyme system. However, this procedure was associated with an increase in dopamine receptor sensitivity as measured by an increase in dopamine-mediated turning behaviour. This behavioural response was not abolished by intrastriatal administration of kainic acid, although this technique reduced dopamine-sensitive adenylate cyclase and cAMP-dependent protein phosphorylation by 80-90%.</p> <p>The administration of chronic haloperidol also produced behavioural supersensitivity, as well as increases in the number of postsynaptic dopamine receptors, but this was not associated with comparable increases in the activity of dopamine-sensitive adenylate cyclase, or in cAMP-dependent protein phosphorylation. These studies therefore do not support a role for cAMP or cAMP-dependent protein kinases in the mediation of postsynaptic dopamine receptor supersensitivity.</p> <p>Rat caudate nucleus also contains a number of proteins that are phosphorylated by Ca⁺⁺-dependent protein kinases. Substrates for calmodulin-dependent and independent protein kinases were identified and characterized on the basis of their solubilization properties and response to neuroleptic drugs. Dopamine enhanced the phosphorylation of two of these proteins in rat striatal slices. The effects of dopamine on protein phosphorylation could be distinguished from those of depolarizing agents such as KCl or veratridine, and were not mimicked by 8-bromo-cAMP.</p> <p>The present studies demonstrate that increases in cAMP produced by dopamine have no measurable effect on cAMP-dependent protein phosphorylation. Although these data do not definitely preclude a role for cAMP-dependent protein kinases in the regulation of postsynaptic function, they indicate that the physiological relevance of dopamine-mediated increases in cAMP should be re-examined.</p> / Doctor of Philosophy (PhD)

<p>This work encompasses two primary studies utilizing peripheral quantitative computed tomography (PQCT) in the assessment of bone characteristics at the distal radius. PQCT enables separate analyses of cortical, trabecular and total bone density, content and geometry. Further analysis of the pQCT image with specialized software permits in vivo trabecular structure assessment. The first study is a two-year prospective trial evaluating the effects of five different medication regimens (hormone replacement therapy (HRT), etidronate , alendronate, etidronate plus HRT and alendronate plus HRT) used for the treatment of osteoporosis in 123 postmenopausal women with low bone mass. Results of analyses on baseline data found that trabecular structure indices could significantly discriminate between osteoporotic and osteopenic groups of women. Longitudinal data at the lumbar spine (LS) and femoral neck (FN) for women taking any medication ('Any-Tx') showed similar results to those of large clinical trials. For example, the 'Any-Tx' group gained significant bone mineral density (BMD) over baseline at 2 years at the LS (5.5%, p<0.001) and FN (1.6%, p<0.05). Women on combination therapy gained significantly more BMD than women taking one medication over 2 years at the LS and FN. Data suggests that alendronate plus HRT may produce the largest BMD gains at the LS and FN at 2 years. Longitudinal pQCT data for total, cortical and trabecular compartments at the distal radius in the control group over the 2 years suggest endocortical resorption and trabecular thinning with age. PQCT data in the 'Any-Tx' group show gains in total and cortical bone density and cortical content, but losses in trabecular density, content and area. This suggests that anti-resorptive medications promote endocoritcal apposition and reduce intracortical porosity. The second study evaluated five different clinical measurement tools in the prediction of in vitro failure load at the distal radius and found that cortical content measured by pQCT was a significantly better predictor than ultrasound or digital x-ray radiogrammetry.</p> / Doctor of Philosophy (PhD)