<p>Patients presenting to the emergency department (ED) with chest pain suggestive of acute coronary syndrome (ACS) often wait long hours before a decision on their care is made. The recommended blood test to aid in diagnosing myocardial infarction (MI) is cardiac troponin I (cTnI) or cardiac troponin T (cTnT). However, other biomarkers representing acute processes and diseases related to ACS might also be useful for early identification. To that end, I evaluated whether a biomarker panel at presentation could improve the diagnostic performance of identifying patients at high risk for MI or any other related cardiac outcome as compared to using cardiac troponin alone. The patient population consisted of 102 patients who presented to the ED with chest pain. Sixteen biomarkers measured in serum obtained at presentation were ranked via receiver-operating-characteristic (ROC) curve analysis for a composite cardiac outcome within the first 72 hours following presentation to the ED. The top four biomarkers (soluble fms-like tyrosine kinase, Creatinine, monocyte chemoattraction protein-1, and NT-pro brain natriuretic peptide) were used to construct the panel test. The ROC derived cutoffs for each of the biomarkers were used to characterize abnormal concentrations with an overall biomarker score incorporating all 4 biomarkers used to classify patients that were either positive or negative for the biomarker panel. When used in conjunction with high-sensitivity cardiac troponin, the panel’s sensitivity and specificity were 100% (95%CI: 75-100%) and 54% (95%CI: 43-65%), respectively. This represented an improvement compared to high-sensitivity cardiac troponin I (hs-cTnI) or hs-cTnT alone which had a sensitivity/specificity of 92% (95%CI: 64-100%)/57% (95%CI: 46-68%) and 85% (95%CI: 55-98%)/55% (95%CI: 44-66%), respectively. In summary, a 4-biomarker blood-based panel used in conjunction with cardiac troponin at ED presentation may identify patients at risk for MI or related outcomes in the short term.</p> / Master of Science (MSc)

<p>ER stress in the kidney is associated with proteinuria. Clinical studies have linked proteinuria with the progression of chronic kidney disease (CKD) at all stages of GFR decline. We hypothesized that treatment with a chemical chaperone, 4-phenylbutyrate (4-PBA), would reduce the severity of CKD and proteinuria in salt sensitive hypertension. The differences in renal pathology between salt sensitive and insensitive hypertension when animals were fed an 8% NaCl (HS) diet were assessed. The Dahl salt sensitive (Dahl S) rat was used as a model of salt sensitive hypertension, while the spontaneously hypertensive rat (SHR) was used as a model of salt insensitive hypertension. The myogenic response of the arcuate artery was studied to determine whether the differences in renal pathology between these models of hypertension was due to an effect of salt on myogenic constriction. Myogenic constriction displayed salt sensitivity in the Dahl S as there was a significant reduction in blood vessel constriction with increasing intralumenal pressures. Myogenic constriction was reduced, but not completely abolished in the SHR with high salt (HS), providing a possible explanation of why this model of hypertension does not develop an equivalent level of renal damage with blood pressure increase as the Dahl S rat. ER stress induction with tunicamycin in arcuate arteries from normotensive animals resulted in an attenuation of the myogenic response. Myogenic constriction was protected from tunicamycin induced ER stress with 4-PBA. 4-PBA treatment (1g/kg/day) in HS fed Dahl S ameliorated proteinuria, renal intratubular protein casts, and renal fibrosis. This correlated with a protection of myogenic constriction and integrity of the glomerular filtration barrier. This suggests that myogenic constriction of the renal vasculature is an important mechanism to protect against salt sensitive hypertension-induced proteinuria. Further, that high salt feeding may inhibit this protective mechanism by inducing ER stress in the renal blood vessels.</p> / Master of Science (MSc)

<p>Successful pregnancy involves the accumulation of non-T, FcR-positive cells at the implantation sites of syngenic allogeneic of CTL (cytotoxic T cells) in vitro and in vivo by blocking the response to interleukin 2. It has been shown that xenogeneic embryos can be gestated successfully if enveloped in the trophoblast genotypically compatible with the pseudopregnant recipient (Rossant et al., 1982, J. Emb. Exp. Morph., 69: 141). We have recently demonstarted that the trophoblast plays a critical role in the localization of the deciduaassociated suppressor cell in pseudopregnant mice. We now show that supernatants genereated from trophoblast cell cultures and day 9.5 ectoplacental cone cultures were successful in the recruitment of the non-T, granulated suppressor cell which sediments at 3 ± 0.05 mm/h. These results suggest that the trophoblast elaborates a factor(s) which plays an important role in the accumulation of the deciduaassociated suppressor cells in the decidue which may protect the anti-genic fetus from maternal immue rejection.</p> / Doctor of Philosophy (PhD)

<p>In chickens afflicted with hereditary muscular dystrophy, the two major types of muscle present respond dissimilarly to the disease process: fast twitch glycolytic muscles possess and express the dystrophic gene overtly during ex ovo development while the genotypically dystrophic slow tonic muscles fail to express dystrophic phenotypes. Therefore, in chickens, muscular dystrophy is muscle fibre type specific.</p> <p>The primary goal of this thesis was to experimentally alter the genetically dystrophic slow tonic musle in an attempt to induce this muscle to express dystrophic phenotypes. Since motor nerves influence the phenotypes of skeletal muscles, it was decided to replace the motor innervation of a slow tonic muscle with that of a fast twitch muscle within a-dystrophic chicken. The surgical cross-reinnervation between the ALD muscle and the 'fast' nerve was performed at hatching by transposing the right ALD muscle to the left side of the back in order to prevent selective self-reinnervation by the severed ALD nerve. The experimental muscles were examined at various time intervals from 2 to 104 weeks postoperatively and compared to age-matched ALD muscles in genetically normal chickens. In addition, denervation of ALD muscles, with and without transposition served as control experiments.</p> <p>Selected histochemical and structural properties of unoperated ALD and fast twitch posterior latissimus dorsi (PLD) muscles of normal and dystrophic chickens were compared between 2 and 32 weeks ex ovo to provide criteria for the analysis of muscles cross-reinnervated by 'fast' nerves and to determine which of these parameters were altered as a result of the dystrophic process. Normal and dystrophic ALD muscles exhibited similar phenotypes: acid and alkaline stable myosin ATPase activity, 'en grappe' endplates, weak glycolytic and strong oxidative capacities, and peripheral location of nuclei. Furthermore, the growth rate, size, and shape of fibres in the normal and dystrophic ALD muscles were similar. In contrast, the myosin ATPase and innervation patterns of normal and dystrophic PLD muscles differed from those of ALD muscles: PLD fibres of either genotype exhibited alkaline stable, acid labile myosin ATPase activity and focal 'en plaque' innervation. Normal and dystrophic PLD muscles also exhibited different phenotypes: dystrophic muscles had a lower muscle weight, abnormal size, shape, and growth rate of fibres, increased number of scattered nuclei, add abnormal glycolytic and oxidative capacities.</p> <p>The results presented from this work indicate that the genetically dystrophic ALD muscles respond differently to cross-reinnervation than do normal ALD muscles. The cross-reinnervated muscles in normal birds demonstrated all of the characteristics of an unoperated ALD muscle with the exception of the presence of isolated groups of fibres exhibiting a fast twitch type of myosin ATPase response. This result suggests that the principle response of normal ALD muscles to a foreign 'fast" nerve is one of resistance to alteration. In contrast, data from the experimental ALD muscles of dystrophic genotype support the conclusion that these muscles are dramatically remodelled after cross-reinnervation. An augmented regenerative response within the dystrophic muscles resulted in hyperplasia which, in turn, led to a complete restructuring of these muscles. Therefore, the present cross-reinnervation experiments demonstrate, for the first time, that a phenotypically normal muscle of dystrophic genotype can be induced to express a dystrophic characteristic: an augmented regenerative response after an experimentally-induced injury. It is important to note that the initial induction of this dystrophic phenotype was demonstrated in the absence of the foreign 'fast' nerve and seemed to be due to the response of this muscle to the severe injury imposed upon the muscle during the inital operation.</p> / Doctor of Philosophy (PhD)

<p>Asthma is a condition of the airway characterized by 1) a reversible increase in airway resistance; 2) airway hyperresponsiveness; and 3) airway inflammation (Juniper et al., 1981; O'Byrne, 1986; Boushey and Fahy, 1995). Defects of the airway epithelium have been suggested to play a role in the pathogenesis of asthma (Goldie et al., 1986; Knight et al., 1994), and loss of the epithelium is associated with increased reactivity of the underlying smooth muscle (Jongejen et al., 1991; Candenas et al., 1992). It has been proposed that the airway epithelium releases one or more factors which inhibit smooth muscle contraction, termed the epithelial derived inhibitory factor (Tschirhart et al., 1987; Fernandes et al., 1989; Ullman et al., 1991). The inhibitory prostaglandin PGE₂ has been demonstrated to be released from the epithelium (Barrel and Bigby, 1995) and to modulate airway smooth muscle contraction (Braunstein et al., 1988; Abela and Daniel, 1995). In patients with asthma, inhalation of an allergen may result in a biphasic response consisting of an early asthmatic response and a late asthmatic response (Dolovich et al., 1989; Sterk et al., 1993). The late asthmatic response is an indirect measure of allergen-induced airway inflammation (Dolovich et al., 1989; Sterk et al., 1993) and an animal model of the late asthmatic response is produced by inhalation of the allergen Ascaris suum by dogs (Sasaki et al., 1987). in the studies presented in this thesis, the release and actions of PGE₂ from canine airway epithelium, both under unstimulated conditions and following inhalation of the Ascaris suum antigen, were examined. Tracheal and bronchial tissues were excised and studied in the organ bath where contractile responses to agonists and electrical field stimulation, as well as PGE₂ release were measured. Finally, a potential mechanism by which PGE₂ may effect smooth muscle relaxation was examined. In unstimulated animals, PGE₂ was released from tracheal epithelium and inhibited smooth muscle contraction. This release of PGE₂ was dependent upon electrical field stimulation, and was not blocked by the addition of neurotoxins. In the antigen model, tracheal PGE₂ release was increased from animals that inhaled antigen but did not develop late airway hyperresponsiveness comapred to animals that inhaled vehicle or inhaled allergen and did develop a late response. The release of PGE₂ was not dependent on field stimulation. Demonstration of in vitro hyperresponsiveness of the tracheal smooth muscle was dependent on removal of the epithelium. Bronchial smooth muscle from the antigen model did not demonstrate in vitro hyperresponsiveness, even when hyperresponsiveness was observed in vivo. There was an increase in the basal release of PGE₂ from the bronchi of animals that were hyperresponsive in vivo. PGE₂ increases intracellular cAMP concentrations (Madison et al., 1989; Coleman et al., 1994). Our investigations in tracheal smooth muscle demonstrated that cAMP does not lower intracellular Ca²⁺ and cause relaxation of airway smooth muscle by stimulation of the sarcoplasmic reticulum Ca²⁺ pump. The results indicate that PGE₂ was released from airway epithelium and modulated smooth muscle contraction. Alterations in the release of PGE₂ were demonstrated in an animal model of asthma, as PGE₂ played a protective role in the development of airway hyperresponsiveness. Modulation of PGE₂ may be a possible therapy for the treatment of asthma and prevention of the late asthmatic response.</p> / Doctor of Philosophy (PhD)

<p>Human adenoviruses are known to transform rodent cells in culture and these cells are tumorgenic when injected into new born animals. It has been well established that the early region 1 (El) of human adenovirus type 5 is necessary and sufficient for oncogenic transformation. The El region is comprised of two transcription units known as E1A (0 to 4.5% of the genome) and E1B (4.5 to 11.2%), each of which produces multiple species of mRNAs and polypeptides. E1A is also required to activate the transcription of other viral early regions. In the present study anti-peptide sera were used to identify and characterize these viral proteins.</p> <p>Anti-peptide sera specific for the amino- and carboxy-termini of E1A were raised and these two sera precipitated an identical set of four major polypeptides of 52, 50, 48.5, and 45K and two minor species of 37.5 and 35K. Studies using E1A mutant viruses also revealed that 52, 48.5, and 37.5K polypeptides are derived from the 1.1 kb mRNA, and the 50, 45, and 35K species from the 0.9 kb mRNA of E1A. These sera were also used to identify polypeptides that are associated with E1 proteins. A set of five cellular polypeptides consisting of >250K, 105K (doublet), 68K, and 65K species were found to co-precipitate with E1A proteins under various conditions and the nature of this association was investigated using the anti-peptide sera as well as an E1A-specific monoclonal activity.</p> <p>Antisera against synthetic peptides corresponding to the both termini of E1B 58K were also raised and used to identfy 58K from wild-type and mutant-infected cells. It had previously been shown that protein kinase activity was associated with 58K. To ask if protein kinase activity was intrinsic to this viral protein several conventional methods were used to purify 58K and the results suggested that such activity may be intrinsic to this viral protein.</p> <p>The anti-peptide sera were used to purify El proteins. A simple purification procedure using these sera and their corresponding synthetic peptides was developed and highly purified 58K and E1A proteins were obtained. Attempts were made to study protein kinase activity using these purified El proteins, however, the results were inconclusive and it was not possible to unequivocally determine if kinase activity was intrinsic to them.</p> / Doctor of Philosophy (PhD)

<p>These studies were undertaken to characterize cytotoxic cell responses to Pichinde virus (PV; a member of the arenavirus family) in various strains of inbred mice. Emphasis was placed on examining the relationship between cells with natural killer (NK) activity and H-2 restricted, virus-specific cytotoxic T lymphocytes (CTL) that are detected in the spleens of inbred mice after infection with PV.</p> <p>Primary i.v. inoculation of mice with PV resulted in augmented spleen NK activity that peaked at 3- 4 days after infection. This NK response was followed by an H-2 restricted, virus-specific CTL response that peaked 3 days later. Rechallenge of PV-primed mice with homologous virus resulted in a slight but significant increase in spleen NK activity 1 day after reinfection, and this was followed 3 days later by peak CTL activity. Thus, memory cell-mediated immune responses appeared more rapidly after secondary in vivo challenge with PV. Furthermore, the temporal kinetic relationship between virus-induced NK and CTL responses was maintained after both primary and secondary infection with PV, which suggested that virus-induced NK cells may represent pre-CTL.</p> <p>To investigate the relationship between these two cytotoxic cell populations, expression of lineage specific cell-surface antigens on virus-induced NK and CTL effectors was examined. NK cells induced after primary and secondary infection with PV were found to rapidly acquire the pan-T cell marker Thy-1, which was expressed on mature anti-viral CTL. In addition, asialo-GM 1 (a glycolipid which has been considered a marker of NK cells) appears to be expressed on PV-specific CTLp; treatment of PV-primed spleen cells with a polyclonal rabbit antiserum to this marker plus complement prior to secondary in vitro restimulation with PV-infected macrophages prevented the generation of secondary CTL responses to PV. Furthermore, multiple i.v. injections of this antiserum were able to abrogate the in vivo generation of both NK and CTL responses after primary or secondary infection with PV.</p> <p>Secondary NK and CTL responses were generated in mice that had been pretreated with cyclophosphamide (CY), suggesting that memory cell-mediated immune responses can be reactivated in vivo without undergoing cell division. In contrast, treatment with CY before primary infection delayed the appearance of virus-induced NK activity and abrogated the generation of H-2 restricted, virus-specific CTL. Rechallenge of these CY-treated, NK-primed mice resulted in the rapid generation of a secondary NK response that was not followed by either a primary or secondary CTL response. This long-term block in CTL generation was not due to the establishment of a persistent PV infection. Memory CTL generation could be restored by secondarily coinfecting mice with PV and a second arenavirus such as Tacaribe virus (TV) or lymphocytic choriomeningitis virus (LCMV), or by injection of interleukin 2 (IL 2)-containing supernatants after rechallenge with PV. To demonstrate that IL 2 was the responsible lymphokine in these supernatants, highly purified IL 2 was added to in vitro cultures of spleen cells from CY-treated, PV-primed mice. In the presence of PV-infected syngeneic macrophages, addition of purified IL 2 resulted in a dose dependent restoration of H-2 restricted, anti-PV CTL activity. In addition, the CTL precursor frequency of CY-treated, PV-primed mice appeared to be markedly reduced compared to that in normal PV-primed mice. Thus, the long-lasting block in the ability to generate PV-specific memory CTL appears to be due to both a lack of helper T cell activity a significant reduction in the number of CTLp. Furthermore, these results suggest that priming the NK compartment is sufficient to prime for a memory CTL response, provided helper factors such as IL 2 are supplied.</p> / Doctor of Philosophy (PhD)

<p>Two inbred strains of Syrian hamster have been shown to display genetically determined differences in resistance to infections with the arenavirus, Pichinde virus (PV). After intraperitoneal injection, the virus grows to higher titres in the spleens of the susceptible strain, MHA, than in the spleens of the resistant strain, LSH. Preliminary studies examining the basis of susceptibility demonstrated that resistance or susceptibility to the virus did not lie in an inherent difference in target cells to become infected, but suggested that there was a quantitative difference in target cells between the two strains of hamster. The following experiments were conducted in attempts to verify the hypothesis that MHA hamsters are susceptible to infection with Pichinde virus because they possess larger numbers of a splenic lymphocyte that serves as a target cell for virus replication and that also functions as an effector cell of nonspecific cytotoxicity.</p> <p>The spleens and thymi of the high NK strain were found to display greater cellularity than those of the low NK strain. Additionally, thymocytes from MHA hamsters were found to proliferate to a greater extent than those of LSH hamsters in response to ConA-induced conditioned medium or purified interleukin 2 plus mitogen. As well, splenocytes from MHA hamsters showed high levels of lymphokine-activated killer cell (LAK) activity after culture in conditioned medium or interleukin 2. In both the thymus and the spleen, this difference in responsiveness was due to increased numbers of precursor cells responding to lymphokines in MHA organs. When lymphokine production was assessed, it was found that cells from the the high responder, MHA, synthesized less interleukin 2 than cells from LSH hamsters. Interleukin 1 production was equal in the two strains. These results led to the hypothesis that the susceptible hamsters contain immature lymphocytes, possibly because of the reduced interleukin 2 production. Increased numbers of relatively immature cells could account for the increased cellularity of lymphoid organs in these animals, and in the spleen, these cells may be responsible for increased NK activity, increased numbers of LAK precursors, and serve as target cells for PV replication.</p> <p>Splenic cytotoxic cells in the hamster were characterized. Endogenous NK cells, virus-induced NK cells and LAK were all plastic nonadherent cells, as were the precursors for LAK. All populations expressed an antigen homologous to the murine Thy 1.2. Endogenous NK cells and virus-induced NK cells were similar in the expression of an asialo GMl homologue; both were reduced by 50% by treatment with this antiserum plus complement. LAK precursors and LAK effectors were negative for this marker, suggesting that LAK arise from the asialo GM1 negative component of endogenous NK activity. Treatment of hamsters with anti-asialo GM1 serum also reduced splenic NK activity in normal and virus-infected hamsters by 50%.</p> <p>Cells infected with virus were characterized using the same criteria. A preferential infection of nonadherent cells was not evident before day 3 of infection, although MHA spleens already contained twice as much virus as LSH spleens. Both asialo GM1 negative and positive cells were infected. Culture of infected splenocytes in interleukin 2 induced high LAK, but failed to select out a population enriched for infectious centres compared to culture in medium alone, where no cytotoxic activity was evident. However, susceptible MHA hamsters with reduced NK activity after treatment with anti-asialo GM1 serum did display less virus at day 1 after infection with PV, but by day 3, virus loads were at control levels. Treatment with anti-asialo GM1 serum had no effect on the mortality of either strain of hamster. Treatment with purified interleukin 2 slowed mortality of MHA hamsters, although it did not do so by reducing viral replication. In total, t~ese data indicate that cells expressing an antigen detected by anti-asialo GMl serum that mediate some ~K ~ctivity can serve as target cells for PV replication, but these cells alone are not responsible. for the susceptibility of MHA hamsters • • Cytotoxic activity and infected centres could be dissociated, also suggesting that the jnitial hypothesis was incorrect. It could be that some other cell is the relevant target, that MHA hamsters may b~ susceptible because of increased number~ of all splenocyt~s, or that some .factor other than the presence or absence of a target cell accounts for their susceptibility.</p> / Doctor of Philosophy (PhD)

<p>Food restriction has profound effects on various endocrine axes and on amine metabolism. In the present study, the effect of reduced food availability on pineal and serum indole was determined in adult male Wistar rats. Under a lighting regimen of 14 h light and 10 h dark, 3 weeks of 50% food restriction led to a reduction in 24 h mean serum tryptophan and serum serotonin levels but an increase in serum melatonin levels. The duration of the night-time melatonin rise was increased secondary to an earlier rise of both pineal and serum melatonin. Such changes in circulating melatonin may account for the gonadal regression observed in underfed animals. This pineal-gonadal interaction was further investigated after animals were subjected to shortened photoperiod or after pinealectomy. Shortened photoperiod failed to influence either the serum melatonin profile or the undernutrition-related gonadal regression. Pinealectomy, however, was able to reverse though incompletely the gonadal regression in underfed animals. When the pineal responsiveness to beta-adrenergic stimulation was determined in food restricted animals, both the time course and the dose responses were altered. The changes in pineal and serum melatonin post-stimulation, however, were atypical of either a sub- or supersensitive pineal gland.</p> <p>Based on the present study, food availability proves to be another factor that can influence pineal activity. Its effect on the pineal, however, depends on the duration of food restriction and the environmental light/dark cycle.</p> / Doctor of Philosophy (PhD)

<p>Disabled children are increasingly being integrated into the regular school environment. Poor attitudes of able-bodied peers are a major obstacle to the social success of this process. Our knowledge about the determinants of attitudes and methods of improving attitudes has been hampered by the poor quality of available attitude measures. This thesis describes a new measure to overcome this problem.</p> <p>The Chedoke-McMaster Attitudes Toward Children with Handicaps (CATCH) scale is a 36-item self-report measure of children's expressed attitude along three dimension; affective response, behavioral intent, and cognitive understanding. CATCH is intended for children in grades four to eight. Over 800 children have been involved in the testing of the first and then the revised draft of CATCH.</p> <p>Children had no difficulty completing the scale. The items related to their everyday experiences. Descriptive aspects of CATCH were good. Total scores varied from 53 to 143 (possible interval of 0 to 144). The total sample mean was 99.1 with a standard deviation of 16.1. The measure was reliable with a coefficient alpha of .90 for the total score.</p> <p>Factor analysis of CATCH revealed three factors accounting for 83% of the variance. Factor one and factor three were a mixture of affective and behavioral items and accounted for 62% and 6% of the variance, respectively. Factor two contained primarily cognitive items and accounted for 15% of the variance.</p> <p>The construct validity of CATCH was established by testing its ability to discriminate groups based on previously hypothesized differences. Girls had significantly more positive attitudes than boys (104.0 versus 94.5; p=.001). Children who had a friend who was disabled; or who had contact with a disabled child in the last week had significantly better attitudes than children without disabled friends or contact. The mean CATCH score for friend was 106.2 versus 95.6 for no friend. For contact the mean score was 108.1 versus 97.0. Children who volunteered for a buddy program or who had previously taken part in a buddy program with a disabled child had significantly higher CATCH scores.</p> <p>CATCH was used as the primary outcome measure in a randomized controlled trial of a buddy program between able-bodied and disabled children. A criterion score improvement occurred significantly more frequently in buddies than controls (43% versus 18%) p=.05). This intervention also appeared to have a significant effect on parental attitudes with buddy parents having a significant improvement compared to control parents.</p> <p>These results demonstrate that CATCH is both a reliable and valid measure of children's expressed attitudes. While the limitations of this measure are recognized. CATCH will be extremely valuable in the study of attitude determinants and in the evaluation of interventions to improve attitudes.</p> / Doctor of Philosophy (PhD)