<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)

<p>In order to establish a model of arenavirus infection of monocytes, human peripheral blood monocytes (PBM), human promyelocytic HL60 cells and human THP-1 promoncytic cells were infected with Pichinde virus (PV). PV replication was analyzed using a variety of assays which detected viral antigen, RNA and progeny virus. While human PBM were susceptible to PV infection and replication, HL60 cells did not support PV replication, even when cells were induced to differentiate to monocytes with the phorbol ester, PMA. THP-1 cells supported high levels of PV replication only when cells were exposed to PMA. THP-1 cells not treated with PMA supported lower levels of PV replication. Infection of PMA treated THP-1 cells by PV was dependant on protein kinase C (PKC) activation and host cell transcription.</p> <p>The restriction of PV replication in untreated THP-1 cells was characterized further. Experiments with lysosomotropic compounds demonstrated that equal amounts of PV were bound and internalized by both THP-1 cells and PMA treated THP-1 cells. These studies also indicated that PV enters THP-1 cells by endocytosis into acidic vesicles. The expression of PV specific RNAs in PMA treated and untreated THP-1 cells were also examined. PV SRNA genomes, antiger omes, GPC mRNA, NP mRNA and L RNAs were expressed at higher levels in PMA treated THP-1 cells versus untreated THP-1 cells. Degradation of input viral S RNA could not account for the reduction of PV RNA replication in the untreated THP-1 cells. Increasing the multiplicity of infection of untreated THP-1 cells with PV was only able to partially overcome the restriction of virus multiplication. This suggested that the restriction of PV replication in the THP-cells occurred later than the initial binding and penetration stages but at, or just prior to, primary transcription of viral mRNAs. These studies supported a role host cell factors and a dependence on the activation or differentiation state of the THP-1 cell in order to support PV replication.</p> <p>In order to gain further insight into the mechanisms utilized by PV to initiate transcription and replication, the 5' termini of PV S RNA genomes, antigenomes, GPC mRNA and NP mRNA were characterized. All termini sequenced had at least one extra nontemplated base. In clones that contained a single extra nucleotide, this was invariably a G nucleotide. Clones containing single nontemplated G nucleotides were only the derived from PV infected total cellular RNA. The 5' termini of NP and GPC mRNAs had on average 4-8 nontemplated bases. In addition, on genomic sense RNAs the base a -1 did not appear to be conserved. These data have important implication with respect to the mechanisms of PV transcription and replication initiation and are discussed in the context of two possible models.</p> / Doctor of Philosophy (PhD)

<p>In animal models it has been demonstrated that stimulation of muscle glycogenolysis is associated with the activation of phosphorylase b (phos b) to phosphorylase a (phos a). In similar experiments in rat skeletal muscle, we found that sclatic nerve stimulation increased glycogenolysis in the first 15 sec. of stimulation in close correlation with an increase in the phos a activity. However by 30 sec. the phos a had returned to resting levels although glycogenolysis continued at a stimulated rate.</p> <p>We did a series of experiments in human subjects to determine whether activation of phos b to phos a was essential in the activation of skeletal muscle glycogenolysis. Six normal male subjects underwent maximal voluntary isometric contraction of the quadriceps for 60 sec. Muscle needle biopsies were obtained at 0,10,20 and 60 sec. after onset of contraction. The rate of muscle glycogenolysis increased from less than 2.05 ± .35 at rest to 17.5 ± 0.7 umol/g during contraction, calculated from the increase in muscle lactate concentration. Enzyme analysis in the same biopsies revealed that the phos a: total phosphorylase ratios remained unchanged (0.05 ± 0.006). In separate experiments 5 normal male subjects exercised on a cycle ergometer at 66% of their VO₂ max. work load until exhaustion. After 40 min. exercise at 66% VO₂ max. load the muscle lactate concentration rose from 1.1 ± 0.005 umol/g at rest to 11.6 ± 0.8 umol/g. When subjects were exercised at 90% VO₂ max. until exhaustion; the change in muscle lactate was taken as a semi-quantitative reflection of the rate of muscle glycogenolysis. The change in the rate of music lactate production increased from 0.2 umol/g/min at the end of 66% work load, to 1.2 umol/g/min at exhaustion, indicating that glycogenolysis was stimulated. The phos a: total phosphorylase ratio and the active to total phosphorylase b kinase ratio remained unchanged at the end of 40 min. exercise and at exhaustion, from the resting value. We conclude that activation of phosphorylase (phos b to phos a conversion) is not an absolute prerequisite for stimulating glycogenolysis in skeletal muscle.</p> / Master of Science (MS)

<p>Hypertension is a chronic affliction which has a significant economic and health impact on Canadian society. Efforts to control hypertension are likely to produce significant returns, if the programs are effective in treating the hypertensive population. Before research efforts can be profitably directed at determining the most efficient method of achieving hypertension control, the state of, and deficiences in, the present health care system must be identified. The best approach to obtaining the information needed to evaluate the present system, is through a special-purpose, population-based survey.</p> <p>The proper methodologic design of a population survey requires the use of probability sampling procedures. In addition, the blood pressure should be measured at several visits, using a standardized procedure. Examination of the literature reveals that no study satisfies all of the basic standards.</p> <p>It is possible to identify six steps that must be followed if hypertension control is to be achieved. These steps form a conceptual model that can provide the basis of a general measurement strategy that can be used to assess the degree of hypertension control in a specific community.</p> <p>This measurement strategy is used to develop a survey design to measure the degree of hypertension control in the Province of Ontario. A specially-created interview team will examine 3,850 individuals, located in selected geographic areas across the Province. Blood pressure will be measured using a Hawksley Random-zero Sphygmomanometer, at a maximum of three separate visits. Questionnaires will be developed to obtain valid information about health knowledge, attitudes and beliefs. The survey design will permit regional comparisons.</p> / Master of Science (MS)

<p>The role of testosterone in follicular development and ovulatIon has been investigated by immunizing female rabbits to testosterone-3-bovine serum albumin (T-3-BSA). The intent of this procedure was to render any endogenously produced testosterone inactive by means of its high affinity binding to specific antibody.</p> <p>In order to determine the time course of the effects of this procedure three groups of 12 - 6 control, 6 experimental - immaturely immunized rabbits were sacrificed at 5, 8 and 11 weeks after immunization. The effects observed at 11 weeks were compared with a group of 10 - 6 control, 4 experimental - maturely immunized rabbits sacrificed after the same length of immunization. In addition a group of 11 - 5 control, 6 experimental - immaturely immunized animals were mated to a proven male 14 weeks after immunization.</p> <p>Animals were boosted regularly and the antiserum titer followed weekly. Blood samples were taken weekly for the determination of FSH and LH levels and the total and percent bound testosterone and estradiol. After sacrifice the ovaries and uteri were removed for histology. Follicular development was examined and the maximum follicular diameter ≥ 1.0 mm and ≥ 1.5 mm was recorded. In mated animals the number of corpora lutea per ovary were counted.</p> <p>SignifIcant antiserum titers to testosterone were observed in all experimental rabbits by 5 weeks of immunization. Over the 14 weeks of the experiment values ranged from 1:700 to 1:52,000.</p> <p>In control rabbits serum testosterone values were less than 0.5 ng/ml while values in experimental rabbits rose steadily over the length of the experiment (group values ranged from 0.6 ± 0.1 ng/ml to 2.1 ± 0.9 ng/ml, 2.2 ± 0.7 ng/ml to 5.7 ± 0.8ng/ml and 4.4 ± 1.2 ng/ml to 6.8 ± 1.2 ng/ml at 5,8 and 11 weeks respectively). Testosterone binding in control animals remained in the 93% to 95% range throughout the experiment while in experimental animals testosterone binding increased significantly to approximately 99% at 5 weeks of immunization and remained constant thereafter.</p> <p>Estradiol values in control rabbits remained relatively constant throughout the ewperiment (group range: 50.7 ± 5.4 pg/ml to 81.8 ± 4.9 pg/ml). A consistent and significant increase in estradiol occurred in experimental animals (group values ranged from 69.5 ± 15.4 pg/ml to 88.5 ± 8.7 pg/ml, 74.5 ± 9.8 pg/ml to 118.5 ± 13.1 pg/ml and 81.6 ± 6.9 pg/ml to 157 ± 30.3 pg/ml at 5,8 and 11 weeks after immunization. Prior to immunization estradiol binding in experimental rabbits was not different from control values (range : 83% to 88%). There was a significant increase in estradiol binding by 5 weeks of immunization in the experimental groups (range: 89.4% to 94.5%). By 11 weeks estradiol binding increased to as much as 97.9%.</p> <p>Follicular development was abnormal in T-3-BSA immunized rabbits. The ovaries contained numerous large cystic and hemorrhagic follicles. At 8 weeks experimental ovaries contained more follicles ≥1.0 mm (27.2 ± 3.1 versus 15.3 ± 2.9, p < 0.01) and ≥1.5 mm (5.83 ± 1.7 versus 0.42 ± 0.2, p < 0.005). At 11 weeks there were more follicles ≥ 1.5 mm in both immature (5.0 ± 0.9 versus 0.92 ± 0.31, p < 0.001) and mature (7.25 ± 0.92 versus 4.17 ± 0.74, p < 0.05) experimental ovaries. Increased vascularization, some thecal cell hypertrophy, and marked interstitial cell hypertrophy were characteristic of the experimental ovaries. There was also a significant increase in the number of ovulations in the experimental rabbits (8.0 ± 2.1 versus 4.4 ± 1.9, p < 0.005).</p> <p>Immunization of female rabbits to testosterone is a useful tool for the study of hormone interactions in the regulation of follicular development and ovulation. In addition it may serve as a useful model for the study of the processes involved in the development of cystic ovaries.</p> / Master of Science (MS)

<p>A PROTOCOL FOR THE DESIGN OF A RANDOMIZED CLINICAL TRIAL IN A FAMILY PRACTICE SETTING IS DESCRIBED. THE TRIAL IS DESIGNED TO TEST IF TWICE A DAY (B.I.D.) ANTIBIOTICS PRODUCED BETTER COMPLIANCE THAN THE STANDARD FOUR TIMES A DAY (Q.I.D.) REGIMENS. THE DISEASE MODELS OF STREPTOCOCCAL PHARYNGITIS AND UNCOMPLICATED URINARY TRACT INFECTION ARE USED BECAUSE OF THEIR SIMILARITY AND BECAUSE THEY ARE COMMON IN THE FAMILY PRACTICE SETTING. COMPLIANCE MEASURES INCLUDE URINE ASSAY FOR ANTIBIOTIC PILL COUNTS AND DROP OUT RATES. COMPLIANCE WILL BE ANALYZED IN RELATIONSHIP TO THE TYPE OF REGIMEN, SIDE EFFECTS AND DISEASE OUTCOME.</p> / Master of Science (MS)

Caspersson (1970) discovered that each chromosome pair has a characteristic banding pattern when chromosomes are stained with the fluorescent dye, quinacrine. With this dye there are certain chromosome regions which are polymorphic. These regions can be used as markers in the study of the inheritance of chromosome anomalies. The purpose of this study was to determine, using chromosome markers, the parental origin of the extra chromosomes found in triploid and D, G trisomic spontaneous abortuses. Polymorphisms in the chromosomes of parents of 15 triploid and 12 trisomic abortuses were compared with those of their abortus to determine which parent donated the extra chromosome(s). The distribution of the markers was used to determine whether the error occurred during meiosis I or meiosis II or, in the case of triploids, to distinguish between a meiotic error and dispermy. Of the 15 triploids examined, 7 were informative as to the origin of the extra set of chromosomes. In 3 of these there was failure to extrude to second polar body during oogenesis. In 3 other cases it was impossible to distinguish between an error during meiosis of spermatogenesis and dispermy. The other informative case definitely arose by dispermy. Mechanisms for the origin of triploidy were discussed, in particular, aging of gametes. Only 1 of the 12 trisomies examined was informative. This was a trisomy 22 in which the extra 22 was from the mother but it was not possible to distinguish between non-disjunction during meiosis I and meiosis II. Possible mechanisms for the production of trisomies were discussed including maternal irradiation, autoimmune processes, possible endocrine factors and drugs.</p> <p>A number of technical factors which influence the appearance of polymorphic regions were also discussed. / Master of Science (MS)