<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)

<p>Breast cancer research has focused on a number of key molecular events suspected to play critical roles in the establishment and progression of the human disease. For example, the c-ErbB2 receptor is amplified or overexpressed in approximately 30% of all human breast cancer cases, its overexpression inversely correlating with a positive patient prognosis. In addition, the estrogen receptor is a key marker in assessing patient prognosis, the progression of the disease state, and is by far the most utilized target to treat breast cancer with approximately 100 million hours of clinical experience with the antiestrogen, Tamoxifen. While these two receptor systems play critical roles in breast cancer progression, the activation of downstream signaling pathways and its consequences on cellular function are unclear. Interestingly, the c-Src tyrosine kinase has been identified to play a role in the modulation of both the c-ErbB2/Neu and the estrogen receptor. Here I will describe a role for c-Src in modulating c-ErbB2/Neu receptor activation and its associated ability to transform at the molecular level, as well as a role for c-Src in estrogen receptor mediated mammary gland development. Significantly, the association of c-Src to the c-ErbB2/Neu receptor plays an important role in the direct and specific activation of the receptor. Furthermore, the loss of c-Src within the mammary gland negatively impact its development, this delay being mediated via the estrogen receptor. Taken together, these studies demonstrate the importance of c-Src in two receptor systems that have been heavily implicated in development and disease, the estrogen receptor and the c-ErbB2/Neu. In both cases, c-Src plays a critical role in receptor activation, coordinating downstream events impinging on reproductive development and in mammary gland transformation. The understanding of the molecular crosstalk between these receptor systems within the mammary gland may provide insight into potential translational therapies in clinical research.</p> / Doctor of Philosophy (PhD)

<p>Mammalian cells have the ability to endogenously generate and metabolize hydrogen peroxide (H₂O₂). This H₂O₂ can interact with protein and lipid components of intracellular signal transduction pathways to regulate cell behaviour. It has been demonstrated in our laboratory and by others that many types of cells, including transformed cells, require H₂O₂ for efficient growth in culture. Excess H₂O₂ has long been identified as a source of oxidative stress that can mediate cell growth arrest and death. However, the addition of the H₂O₂ scavenger catalase to culture media or the overexpression of intracellular catalase can block proliferation. This effect is reversed by inactivation of the enzyme or upon co-incubation of cells with H₂O₂ and H₂O₂-generating sources such as glucose oxidase. Thus, at non-toxic levels, H₂O₂ appears to act as a cellular growth factor. The purpose of this work is to help elucidate molecular mechanisms underlying this H₂O₂-regulated aspect of cell physiology. The effects of extracellular H₂O₂ level manipulation upon the activities of the HER-2/Neu receptor tyrosine kinase, mitogen-activated protein kinases (MAPKs), and stress-activated protein kinases (SAPKs) are discussed. The c-Jun-NH₂terminal kinase 1 (JNK1), a member of the SAPK family, is involved in several diverse aspects of cellular functioning including apoptosis and transformation. The JNK1 signal has also been implicated as a cell sensor of redox (reducing/oxidizing) stress. We have observed that the growth-inhibitory effect of both high level H₂O₂ treatment and H₂O₂-scavenging catalase treatment is accompanied by a transient increase in JNK1 activity. To determine the importance of this response in growth regulation, the JKN1 signal wa stably altered in SK-OV-3 human ovarian adenocarcinoma cells by the expression of ectopic JNK1 (HA-JNK1). Levels of HA-JNK1 protein expression correlated with increases in basal c-Jun phosphorylation in a dose-dependent manner. Transient expression of HA-JNK1 potentiated cell growth arrest by catalase activity, however with stable expression a degree of resistance to this response was observed. Resistance was accompanied by a lowered endogenous production of H₂O₂. Transient HA-JNK1 expression also reduced H₂O₂ generation, and this effect was reversed by the JNK inhibitor SP600125. These resultts indicate that the JNK1 stress response contributes to the inhibition of proliferation by catalase treatment, possibly via additional reductions in environmental H₂O₂ caused by a lowered endogenous production. Stable amplification of the JNK1 pathway leads to cellular adaptation to its signal, resulting in a diminished reliance upon basal H₂O₂ levels for efficient growth. These data contribute to the understanding of the mechanisms involved with the overexpression and/or hyperactivity of JNKs observed in certain cancers.</p> / Doctor of Philosophy (PhD)

<p>Background: peritoneal dialysis (PD) is a valuable therapy for end-stage renal disease. Changes in the peritoneal transport properties, including increased solute transport and ultrafiltration (UF) dysfinction are main clinical limitations of PD. The capillary wall of peritoneal blood vessels plays a key role as a barrier to solute transport. Therefore, neovascularization of the peritoneal tissues is a main determinant in the functional changes in the peritoneal membrane. I hypothesize that ultrafiltration of dysfunction is caused by increased peritoneal vascularization with a concomitant increase in glucose transport from peritoneal cavity and rapid loss of the ultrafiltration gradient. Further, I hypothesize that this increased vascular surface area is induced by profibrotic and inflammatory cytokines such as transforming growth factor (TGF) β, interleukin (IL) 1β, and tumour necrosis factor (TNF) α through upregulation of angiogenic cytokines such as vascular endothelial growth factor (VEGF). Methods: I used adenovirus mediated gene transfer of cytokines such as TGFβ1, il-1β, and TNFα to the peritoneal of rats and studied the effects of histology, angiogenesis, gene regulation, solute transport, and UF. I also used an animal model of daily peritoneal exposure to dialysate solution. The angiogenic and fibrogenic response of the peritoneum to this model was detailed, along with the effect of overexpression of angiostatin and decorin by adenovirus mediated gene transfer Results: Adenovirus mediated gene transfer of TGFβ1 led to increased peritoneal fibrosis and angiogenesis which persisted to 28 days. These histologic changes were associated with increased solute transport of glucose and decreased UF. In vitro and in vivo, TGFβ1 appeared to up regulate expression of VEGF. Inflammatory cytokines such as TNFα and IL-1β both induced fibrosis, angiogenesis, and peritoneal membrane dysfunction. The kinetics of the response was quite different. TNFα induced a very transient response with a complete resolution of changes by 21 days after adenovirus infection. The response induced by IL-1β was more sustained. I hypothesize that the strong expression of tissue inhibitor of metalloproteinase after IL-1β treatment may explain the prolonged fibrogenic response after IL-1β. Daily exposure to dialysate also induced a strong fibrogenic and angiogenic response in the peritoneum. The anti-angiogenic agent angiostatin, when overexpressed using adenovirus mediated gene transfer, reduced the vascularization and improved the ultrafiltration dysfunction. Decorin, a proteoglycan that binds and inactivates TGFβ, reduced peritoneal fibrosis, but did not alter the angiogenic response, nor did it improve peritoneal membrane function. Discussion: These experiments have clarified the role of angiogenesis in UF dysfunction. The correlation between blood vessel density and ultrafiltration, along with the improvement in UF after treatment with angiostatin, is compelling evidence that vascularization of the peritoneal membrane causes UF dysfunction. There is a close associate between fibrosis and angiogenesis in the perotineum. Fibrosis appears to be necessary for a prolonged angiogenic response. Further work is required to identify the factors leading to the interaction between fibrosis and angiogenesis. The role of the interstitium and lymphatics in solute transport and UF dysfunction needs to be better defined. Finally, anti-angiogenic therapies need to be studied in these models of peritoneal membrane dysfunction and eventually applied to patients on PD to improve the quality and duration of this therapy.</p> / Doctor of Philosophy (PhD)

<p>Enhanced transepithelial antigen transport in intestine of sensitized rats was previously demonstrated using horseradish peroxidase (HRP) as a model antigen. In senitized rats, transepithelial antigen transport was rapid (< 2 min) and of greater magnitude (three-fold) compared to controls. In my project, the essential components involved in the enhanced antigen transcytosis were investigated using mice with genetic deletions for IL-4 and CD23/FcεII. Increased transepithelial antigen transport was demonstrated in actively sensitized IL-4⁺/⁺ mice, but not IL-4⁻/⁻ mice, in which the phenomenon paralleled the expression of CD23 protein on enterocytes. Passively sensitized mice (both IL-4⁺/⁺ and IL-4⁻/⁻ mice) displayed greater antigen transport after transfer of immune serum unless the serum was first depleted of IgE or IL-4. IL-4 added to cultures of epithelial cells upregulated expression of CD23 mRNA. Finally, this augmented antigen uptake system was inhibited by luminal anti-CD23 and was absent in sensitized CD23⁻/⁻ mice. RT-PCR showed that cultured cells expressed only the b isoform of CD23. Sequencing revealed classical and alternative CD23b transcripts lacking exon 5 (b∆5) or 6 (b∆6), in which all forms were translated into functional IgE receptors. Endocytosis of the b∆5 and b∆6, but not the classical CD23b protein, was observed after binding with saturating anti-CD23, suggesting continuous endocytosis of the alternative forms that agrees with their intracellular localization at steady state. Classical CD23b proteins were expressed on the cell surface and only endocytosed upon antigen-induced IgE cross-linking of the receptor. Taken together, the results demonstrated that IgE/CD23 mediates enhanced transepithelial transport of antigen in sensitized mouse intestine, and that IL-4 plays a major regulatory role. We identified the expression of classical and alternative CD23b transcripts in intestinal epithelial cells, and demonstrated taht the translated proteins display distinct endocytic functions. We concluded that antigen binding to epithelial CD23/IgE facilitates its entry into the body resulting in intestinal anaphylaxis.</p> / Doctor of Philosophy (PhD)