<p>Crohn's disease is characterized by altered epithelial physiology including ion secretion and disrupted epithelial barrier. It has been suggested that the pathophysiology and epithelial damage in Crohn's disease may be due to inappropriate immune reactions. The purpose of my studies was to examine the role of cells of the monocyte/macrophage lineage in mediating epithelial physiology. Confluent monolayers of the T84 colonic epithelial cell line were co-cultured with human monocyte/macrophages (peripheral blood monocytes [PBM], or lamina propria mononuclear cells [LPMC]) from controls or patients with crohn's disease ± the activating agents, bacterial lipopolysaccharide (LPS) and/or the bacterial tripeptide, f-met-leu-phe (fMLP). Monolayers were then removed and epithelial secretory (baseline short circuit current [Isc] and ΔIsc to 10-5 forskolin) and barrier transepithelial resistance [TER]) were measured 48h later in the Ussing chambers. Epithelial functional parameters were unchanged after co-culture with non-activated PBM. Addition of activating agent (LPS or LPS/fMLP) significantly increased Isc and reduced TER with PBM from both control and Crohn's disease. No pathophysiology occurred after co-culture with control LPMC ± LPS. However, non-activated LPMC from patients with Crohn's disease spontaneously induced changes in T84 physiological parameters similar to those produced by activated PBM. A non-pathogenic control strain of E. coli , added to the luminal compartment of the culture well, induced comparable changes in epithelial physiology in the presence of PBM. The role of Tumor Necrosis Factor α (TNFα) in the regulation of epithelial secretory and barrier functions was examined. TNFα production was significantly increased by activated PBM and non-activated LPMC from Crohn's disease. The epithelial abnormalities could be successfully abrogated by inclusion of a neutralizing antibody to TNFα. These studies show that monocyte/macrophages are potent immune cells which may mediate some of the pathophysiology in inflammatory disorders of the gut. TNFα is a key factor mediating the monocyte/macrophage induced epithelial pathophysiology.</p> / Doctor of Philosophy (PhD)

<p>Fibrinolysis is the process by which blood clots are solubilized. For the purposes of intravascular fibrinolysis, tissue-type plasminogen activator (t-PA) catalyzes the rate-limiting step by converting the zymogen, plasminogen (Pg), to the active enzyme, plasmin. Plasmin then degrades the fibrin meshwork, generating soluble fibrin degradation products. Because the catalytic efficiency of Pg activation by t-PA is higher in the presence of fibrin than that in the presence of fibrinogen(Fg), t-PA is designated a fibrin-specific Pg activator. Despite this designation, t-PA causes systemic plasminemia and fibrinogenolysis when given to patients. Recently, it was demonstrated that the fibrin-specificity of t-PA is compromised because (DD)E, a major soluble degradation product of crosslinked fibrin, stimulates Pg activation to the same extent as fibrin. This project was initiated to gain a better understanding of how (DD)E compromises the fibrin-specificity of t-PA by characterizing the interactions of t-PA and Pg with fibrin, (DD)E and Fg. t-PA binds to fibrin through two classes of sites, one high affinity, finger-dependent site, and one low affinity, kringle-dependent site. In contrast, t-PA binds (DD)E and Fg solely through its second kringle domain, because these interactions are blocked by lysine or its analogues. Both t-PA and Pg bind (DD)E with affinities similar to those for fibrin, thereby explaining why (DD)E stimulates t-PA-mediated Pg activation to the same extent as fibrin. t-PA primarily binds to carboxy-terminal lysines on (DD)E because the affinity of t-PA for (DD)E is reduced 160-fold when (DD)E is exposed to carboxypeptidase B (CPB) or the active form of thrombin activatable firbinolysis inhibitor (TAFIa), an endogenous CPB-like enzyme. In contrast, the affinity of Pg for (DD)E is reduced only 2- to 4-fold when (DD)E is exposed to CPB or TAFIa, suggesting that Pg binds primarily internal lysine residues on (DD)E. t-PA and Pg have weak affinity for Fg, thereby explaining why Fg is a poor stimulator of Pg activation by t-PA. These studies demonstrate that the fibrin-specificity of t-PA is compromised by its kringle-dependent interactions with (DD)E and, to a lesser extent, Fg. The limited fibrin-specificity of t-PA has prompted the development of Pg activators with greater selectivity for fibrin. Two such agents are the activator isolated from the saliva of the vampire bat (b-PA), and a bioengineered t-PA variant, TNK-t-PA. b-PA is structurally distinct from t-PA in that b-PA lacks a lysine-binding kringle. TNK-t-PA was designed to have a longer half-life than t-PA, resistance to inhibition by plasminogen activator inhibitor-1, and increased fibrin-specificity over t-PA. When the fibrin-specificities of t-PA, b-PA, and TNK-t-PA were compared, the hierarchy of fibrin-specificity (b-PA > TNK-t-PA > t-PA) correlated with the ratio of their activity in the presence of fibrin relative to (DD)E. Whereas all activators have similar activities in the presence of fibrin, they are distinguished by their activity in the presence of (DD)E. b-PA, the most fibrin-specific, exhibits minimal stimulation by (DD)E, whereas t-PA, the least fibrin-specific, exhibits the greatest stimulation by (DD)E. Stimulation by (DD)E, in turn, reflects the affinity of the activator for (DD)E. b-PA does not bind (DD)E, presumably because it lacks a lysine-binding kringle. t-PA binds (DD)E with high affinity, whereas TNK-t-PA has ∼9-fold lower affinity for (DD)E than t-PA. These studies highlight the importance of (DD)E in compromising the fibrin-specificity of t-PA. Furthermore, they suggest that the fibrin-specificity of t-PA or t-PA variants could be improved by abolishing their lysine-binding properties.</p> / Doctor of Philosophy (PhD)

<p>Idiopathic thrombocytopenic purpura (ITP) is a common autoimmune disorder characterized by the premature destruction of autoantibody-sensitized platelets. For many years, elevated platelet-associated IgG (PAIgG) was thought to be a diagnostic characteristic of ITP. However, observations that PAIgG is increased in both immune and non-immune thrombocytopenia, questioned the clinical significance of PAIgG measurements, and the mechanism(s) by which IgG accumulates in platelets. The origin of IgG on the surface of platelets of patients with non-immune thrombocytopenia, is not known. Additionally, it is not clear why these IgG-sensitized platelets escape destruction by phagocytic cells of the reticuloendothelial system. In this investigation, possible biological explanations for the elevated PAIgG in adult patients with ITP and non-immune thrombocytopenia, were examined. Traditionally, laboratory investigation of ITP, has focused on the development of serologic assays to measure anti-platelet autoantibodies. As an alternative investigative approach, ultrastructural techniques were used to evaluate platelets from patients with ITP and non-immune thrombocytopenia. Using these techniques, it was possible to localize IgG in platelets, and determine the immunomorphologic characteristics of PAIgG in patients with immune and non-immune thrombocytopenia. The results of these investigations demonstrated, that apart from differences in numbers, ITP platelets and platelets from patients with non-immune thrombocytopenia are morphologically identical to normal platelets and show no evidence of structural abnormalities. Additionally, elevated total PAIgG levels in thrombocytopenic patients are not attributable to alterations in the physical characteristics of platelets, including increased platelet size or increased numbers of storage granules per platelet, but reflected quantitative abnormalities in the pool of exogenous a-granule proteins. Immunomorphologic characteristics of PAIgG in patients with ITP and non-immune thrombocytopenia differ. In patients with ITP, elevated PAIgG is observed both within and on the surface of platelets. In patients with non-immune thrombocytopenia, elevated PAIgG was observed within but not on the platelet surface. In these platelets, IgG is not elevated on the platelet surface and, consequently, platelets were not prematurely cleared by phagocytic cells of the reticuloendothelial system. These results further explain why patients with non-immune thrombocytopenia have a normal platelet lifespan. Demonstrations that immunomorphologic characteristics of surface PAIgG, in platelets from patients with non-immune thrombocytopenia change over time suggests that in vitro leakage of α-granule IgG onto the surface of platelets may account for the origin of surface PAIgG in these patients. Immunomorphologic characteristics of surface PAIgG in patients with immune and non-immune thrombocytopenia suggest that rim patterns of surface PAIgG in ITP reflect the binding of autoantibodies to glycoprotein targets on the platelet surface whereas, "beaded necklace" patterns of surface PAIgG in non-immune thrombocytopenia reflect IgG exocytosis from internal platelet storage pools in vitro . In summary, ultrastructural techniques have been useful for the study of early structural events leading to the pathogenesis of disease and more recently have been utilized in studies of platelet pathology. Whereas current measurements of PAIgG do not provide good diagnostic or prognostic value, ultrastructural patterns of IgG distribution in ITP platelets may in themselves, or in conjunction with other laboratory measures, provide additional information about a common thrombocytopenic disorder.</p> / Doctor of Philosophy (PhD)

<p>This study is a partial evaluation of a program of foster care for chronic psychiatric patients (Residential Homes for Special Care) in 41 homes in Hamilton and surrounding area, Ontario.</p> <p>A review of the development of foster care and of controversies over its value, as well as a review of the existing evaluative literature is included.</p> <p>An attempt was made to use adequacy of neuroleptic maintenance is chronic schizophrenic residents as a tracer condition for evaluation of quality of care given by primary care physicians.</p> <p>The objectives of the program were examined and operating definitions evolved for measures to evaluate some of these objectives. These included a field worker's rating of the quality of hostesses and an audit of prescription written by attending physicians for adequacy of neuroleptic maintenance, occurrence of drug interactions and prescribing frequency.</p> <p>The cohort studied was all 178 residents first admitted to foster care in the area in the years 1966 to 1969. These residents were followed for a three year period using existing health records. Data were complete on every resident for all variables excepts some drug prescription measures.</p> <p>Home factors such as rural location and inferior quality of hostesses were important determinants of rehospitalisation. Residents first placed in rural homes had many more rehospitalisations than those first placed in urban homes, although there were no significant differences in the age, sex, number and duration of previous psychiatric hospitalisation or diagnosis between these groups.</p> <p>Resident factors such as younger age, female sex and shorter duration of previous psychiatric hospitalisation were significant determinants of discharges from the program.</p> <p>The prescription audit measures did not predict outcome, but examination of the prescriptions did reveal 17 patients on prolonged tricyclic antiderressant therapy, 10 for more than four years.</p> / Master of Science (MS)

<p>It has been repeatedly demonstrated that radiation induces chromosome aberrations in mouse oocytes and spermatocytes. However, the results of previous studies, in which the frequency of aberrations recovered from irradiated oocytes and spermatocytes were compared, are conflicting (reviewed by L. B. Russell, 1962, L. B. Russell, 1968). The development of new techniques for making chromosome preparations from oocytes (Edwards, 1965; Tarkowski, 1966) and spermatocytes (Evans et al., 1964) has made it possible to reinvestigate the radiosensitivities of spermatocytes and oocytes.</p> <p>In the present study, the frequency of chromosome aberrations recovered from irradiated dictyotene oocytes was compared to the frequencies of aberrations recovered from irradiated pachytene and diplotene spermatocytes.</p> <p>Oocytes and spermatocytes were collected from mature mice one day and five days after exposure to a single acute dose of 300R ϒ-radiation. At the time of irradiation, all of the oocytes were in dictyotene while the spermatocytes collected one day and five days post-irradiation were in diplotene and pachytene, respectively. The average number of oocytes collected from irradiated mice was no different from the average number collected from controls. Although the ability of oocytes collected one day post-irradiation to mature in vitro (58.0%) was not affected, significantly fewer (53.6%) of the oocytes cultured five days post-irradiation matured in vitro. Since the frequency of abnormal cells vas the same in oocytes cultured one day and five days post-irradiation, it is unlikely that oocytes with chromosome aberrations were selectively inhibited from maturing in vitro.</p> <p>The frequencies of chromosome aberrations in dictyate oocytes cultured one day and five days after irradiation were not significantly different from the frequency of aberrations in diplotene spermatocytes (20.7%). However, significantly more chromosome aberrations (32.0%) were recovered fraa irradiated pachytene spermatocytes than from either dictyate oocytes or diplotene spermatocytes. Some variation in the relative frequencies of aberrations was found.</p> / Master of Science (MS)

<p>The discovery of Q-banding made it possible to identify every member of the human chromosome complement. Spontaneous abortions known to have a better than average chance of presenting with a chromosome anomaly were selected for cytogenetic analysis. A highly successful technique for culturing chorion from these specimens was developed. Direct chromosome preparations were obtained from the coverglasses on which the fibroblasts were subcultured. The characteristics of each specimen were noted as that as the information pool gives, it may be possible to define abortion syndromes.</p> <p>Fifty-nine per cent of the specimens selected were abnormal. These abnormalities included trisomies 2, 8, 14, 16 and 22, triploidy and tetraploidy. Vesicular villi, maternal age over 40 and conception coincident with maternal ingestion of contraceptives were found to be excellent forcasters of chromosome anomalies. Only one embryo, 69, XXY, in which congenital malformations could be identified was collected during the duration of this project. A possible polymorphism in a non-heterochromatic region of chromosome 17, in a 16-trisomy specimen, was noted.</p> <p>Heteromorphic bands have made it possible to distinguish between the members of a homologous pair of chromosomes 3, 4, 13, 14, 15, 21 and 22. A study of such markers in a twin abortus allowed for speculation on the zygosity of the embryos. Similar polymorphisms were used to determine whether the extra set of chromosomes in two triploid abortuses was of maternal or maternal origin. Distribution of these marker chromosomes was also used to determine when in meiosis the event of abnormal development occurred. The point was stressed, that some cell-to-cell variability does occur in these heteromorphic Q-bands and that great care must be taken in distinguishing maternal and paternal marker chromosomes, before the distribution of these parental chromosomes can be used to make statements about abnormal development events.</p> <p>Heteromorphic bands have made it possible to distinguish between the members of a homologous pair of chromosomes 3, 4, 13, 14, 15, 21 and 22. A study of such markers in a twin abortus allowed for speculation on the zygosity of the embryos. Similar polymorphisms were used to determine whether the extra set of chromosomes in two triploid abortuses was of maternal or paternal origin. Distribution of these marker chromosomes was also used to determine when in meiosis the event of abnormal development occurred. The point was stressed, that some cell-to-cell variability does occur in these heteromorphic Q-bands and that great care must be taken in distinguishing maternal and paternal marker chromosomes, before the distribution of these parental chromosomes can be used to make statements about abnormal developmental events.</p> / Master of Science (MS)

<p>Lack of compliance with therapeutic regimens is an important cause of inadequate or incomplete medical care.</p> <p>For the purposes of furthering knowledge about problems of compliance, this thesis first surveys issues of compliance as reported in the current scientific literature and then proceeds with the development of specific strategies to improve compliance and finally with the development of a research design for testing these strategies in a controlled clinical fashion among a cohort of medical patients newly initiated into therapy.</p> <p>The compliance-intervention strategies include, first, special techniques in patient education, utilizing potent behavior-oriented teaching materials, second, a flexible, opportunistic approach to fitting medical appointments and medication-taking into a patient's existing rituals and daily routine, a process here termed "tailoring", and, third, a behavior modification paradigm which reinforces prescribed behavior.</p> <p>Hypertension has been chosen as a disease appropriate for the testing of the strategies because of its high prevalence and its known harmful effects, because of tho existence of efficacious treatments for it, and because of the small proportion of its victims who are receiving adequate treatment, whether for lack of detection of the condition or lack of compliance with its therapy.</p> <p>A steel mill (Dominion Foundries and Steel Company) in Hamilton, Ontario, has been selected as an ideal study site for several reasons. First, the Company is owned by its employees and this has led to an exceptionally stable employee group. Second, it has an active and cooperative employee health service. Thirdly, the health service staff has become concerned about the problem of untreated hypertension through its periodic health assessment program and the high prevalence of hypertension among employees lost from active duty through vascular disease.</p> <p>At the time of this writing the project has been funded through the Medical Research Council and is just getting underway.</p> / Doctor of Philosophy (PhD)

<p>Antithrombin (AT) and heparin cofactor II (HCII) are the predominant inhibitors of thrombin in plasma. They belong to the se rine p[barbelow]rotease in hibitor, or serpin, family of proteins and they inhibit their target proteases through a mechanism that is unique to this family of molecules. AT and HCII provide an ideal substrate on the reactive centre loop and subsequently form 1:1 stoichiometric inhibitory complexes with their target proteases through a mechanism that results in major conformational changes in these serpins. While thrombin acts as a target protease with these serpins, neutrophil elastase (NE), another serine proteases, reacts with AT and HCII within the reactive centre loop. Unlike thrombin, however, the interaction of NE with AT and HCII results in cleavage and inactivation of these serpins, without forming inhibitory complexes with NE. The aim of this thesis was to analyze the effects of mutagenesis of the NE cleavage sites within AT and HCII to determine the limits of amino acid substitutions that would permit AT and HCII to retain function but be less susceptible to inactivation by NE. Analyses of proteins expressed in a cell-free expression system and then in COS-1 mammalian cell culture were used to study the effects of mutagenesis at P4 and then at P4 and P5 in rabbit and human AT. While charged and polar amino acid substitutions severely reduced the function of AT, substitution of the bulkiest residue, tryptophan, at P4 and then at P5 and P5 had minimal effects on the thrombin-inhibitory activity of AT. However, the susceptibility to NE cleavage did not appear to be affected by any substitutions that were made in AT. Analysis of amino acid substitutions in bacterially-derived HCII P6 variants demonstrated a similar flexibility in amino acid composition at the site of NE cleavage, with respect to the ability to inhibit thrombin, although substitution of polar and bulky residues within the reactive centre loop of this serpin increased its resistance to NE inactivation. These results demonstrate that the amino acid composition of the reactive centre loop of AT and HCII is flexible to allow the maintenance of thrombin-inhibitory activity, although some limitations do exist. The effects of residue substitutions within the reactive centre loop on the susceptibility to NE cleavage are less clear, but the presence of bulky amino acids at the primary NE cleavage site, at least in HCII, appears to reduce the proteolytic activity of this protease. The production of an NE-resistant thrombininhibitory serpin provides the basis for the possible development of "hardened serpins" which might be of therapeutic importance in the future.</p> / Doctor of Philosophy (PhD)

<p>Central dopaminergic systems have been implicated in CNS disorders such as schizophrenia and Parkinson's disease. The characteristics of dopamine (DA) receptors has been well studied using a variety of pharmacological and biochemical techniques. DA receptor function is known to be modulated by the endogenous tripeptide pro-leu-gly-NH2 (PLG). A combination of novel pharmacological (guanosine-5' -O -(3[ 35 S]thio)triphosphate; [35 S]GTPγS binding) and molecular biological (differential display mRNA by RT-PCR; ddPCR) techniques were used to examine the function of endogenously expressed D2 DA-receptors in the striatum. Studies were undertaken to increase our understanding of the mechanism of action of PLG and its role in D2 receptor regulation and signal transduction. Measurement of antagonist effects in the absence of D2 receptor stimulation revealed that basal [35 S]-GTPγS binding was significantly decreased by haloperidol, butaclamol and chlorpromazine but not clozapine, sulpiride and spiperone. Thus haloperidol, butaclamol and chlorpromazine acted as negative antagonists; while clozapine, sulpiride and spiperone acted as silent antagonists. The role of PLG in D2 receptor stimulation of Gi G-proteins was examined using the [35 S]-GTPγS binding technique. The possible effect of PLG on NPA-stimulated [35 S]GTPγS binding was assessed at both maximal (1 μM NPA) and submaximal (0.1 and 0.03 μM NPA) levels of D2 receptor stimulation. It was found that PLG did not significantly alter [35 S]-GTPγS binding in bovine striatum either in the absence or presence of D2 receptor stimulation; indicating that PLG does not alter the rate of GDP:GTP exchange in the Giα G-protein subunit. Haloperidol and clozapine have distinct pharmacological profiles and have differential effects on many systems in the brain. This likely accounts for the tendency toward the development of extra pyramidal side effects (EPS) with the use of haloperidol but not clozapine. In an attempt to elucidate the mechanism of action of PLG, ddPCR was utilised to discover genes that are regulated by protracted treatment with PLG (20 mg/kg, i.p. for 28 days). In the present study, I have attempted to further the understanding of D2 receptor coupling to G-proteins in the striatum with respect to agonist efficacy and negative versus silent antagonism. I have also determined that PLG does not modulate D2 receptor signal transduction by altering the rate of GDP:GTP exchange in Gi. Furthermore, I have used the [35 S]-GTPγS binding technique to compare the effects of typical and atypical neuroleptic treatment on D2 receptor coupling to Gi. (Abstract shortened by UMI.)</p> / Doctor of Philosophy (PhD)

<p>There is a high rate of pregnancy failure in humans. The greatest loss occurs at the time of implantation or immediately after the embryo has implanted. Up to 50-60% of this loss can be attributed to embryonic chromosomal abnormalities. The absence or anomalous amounts of physiologic factors which are necessary for implantation and early embryonic development may be the cause of pregnancy failure. Since the embryo is foreign, it is specifically necessary to explore the role of rejection and failure of mechanisms that suppress rejection at implantation.</p> <p>The murine system has been used to investigate the identity of immunosuppressive molecules produced during the process of preimplantation embryo development. Supernatants from mouse in vitro fertilized (IVF) embryo cultures can suppress in vitro lymphocyte proliferation stimulated by the mitogen concanavalin A. Medium conditioned by incubation with mouse epididymal spermatozoa alone were even more inhibitory to mitogen stimuhued lymphocyte proliferation. Thin layer chromatography detected the polyamines spermine in sperm, and spermidine as well as spermine in IVF embryo culture supernatants. Evidence was obtained that these were possibly the ill vitro molecules that were immunosuppressive and were likely produced by the embryo. The diamine putrescine was also detected but was not immunosuppressive.</p> <p>The conclusion from the studies suggest that polyamines may have a role in vivo in suppressing uterine immune response thereby assisting the embryo in the process of implantation. Failure of embryos to produce sufficient amounts of polyamines perhaps due to chromosome abnormalities, may explain failure of embryo implantation. As well, the failure of IVF embryos to produce adequate quantities of polyamines which are known to be essential for cell proliferation, could lead to embryo division arrest. In this broad sense polyamines may be viewed as "growth factors" i.e. defined molecules essential for cell division.</p> / Doctor of Philosophy (PhD)