<p>Proteases participate in various aspects of different biological processes to serve purposes as diverse as nutrition and biological controls, all of which are effected by the cleavage of peptide bonds. Nevertheless, proteolysis is an irreversible process because there is no known biological repair mechanism for a broken peptide bond. To ensure that proteases act only beneficially, proteolytic activities are regulated by different control mechanisms, one of which is composed of the natural protease inhibitors. The inhibitors neutralize the proteases by complex formation. The interactions between inhibitors and proteases in vitro have been well documented whereas information on the in vivo behaviours of protease-inhibitor complexes is limited at present. α₂M-protease complexes have been shown to disappear from the circulation extremely rapidly while the clearance of α₁AT-protease complexes is comparatively slow. Thus, it seems, probable that different elimination pathways may exist for different types of protease-inhibitor complexes. The purpose of this study is to examine the in vivo behaviours of some AT Ill-protease complexes, with an attempt to elucidate a probable elimination pathway for such complexes.</p> <p>Complexes of rabbit, human and rat AT III with rabbit plasmin and of rabbit AT III with rabbit or bovine thrombin were formed from radioactively-labelled proteins and separated from the uncomplexed reactants by preparative electrophoresis. In certain cases, unlabelled proteins were complexed and the complexes were labelled after they had formed. The in vivo behaviour of the complexes was studied in rabbits by the measurement of plasma radioactivity. The integrity of the complexes after injection into the rabbits was monitored by gel filtration of selected plasma samples on Sephadex G-200. Affinity chromatographic columns, such as Sepharose-AT III, -trypsin, -heparin and -lysine were used for the isolation and study of the various components. The stability of AT III-protease complexes under different in vitro conditions was examined by gel filtration and polyacrylamide gel electrophoresis. As a comparison, the complex of rabbit α₁AT with rabbit plasmin was also studied. α₁AT-plasmin complex was separated from uncomplexed materials by affinity chromatography on a Sepharose-lysine column.</p> <p>The AT III-protease complexes tested were eliminated considerably more slowly than α₂M-protease complexes. The speed of elimination of AT III-protease complexes resembled that of α₁AT-protease complexes. All five combinations of AT III-protease complexes dissociated soon after injection into rabbits, though to variable extents. However, rabbit AT III-plasmin complexes were stable for at least 6 h at 37°C in vitro. Post-complex AT III and post-complex plasmin appeared to have been altered as compared to the corresponding native proteins. Thus post-complex AT III lost more than 80% of its affinity for Sepharoseheparin and this form of the inhibitor no longer bound to Sepharosetrypsin (80% of the control AT III bound to conjugated trypsin). However, post-complex AT III was eliminated only marginally faster than the native AT III and it retained its reactivity with antibodies raised against normal AT III. Post-complex plasmin did not bind to Sepharose-AT III or -Trasylol but about 86% of its ability to bind to Sepharose-Iysie was preserved. Whether the above observations for AT III-protease complexes in rabbits hold true in other species, remains to be established.</p> / Master of Science (MS)

<p>In the work described in this theses, hormonal and physiological responses to exercise were investigated in women in relation to the menstrual cycle. Alterations in estradiol, progesterone, luteinizing or follicle stimulating hormones with exercise may interfere with the delicate hormonal balance which is necessary for regular ovulatory menstrual cycles. At rest, estradiol and progesterone have effects on carbohydrate and lipid metabolism, on pulmonary and cardiovascular function and on the plasma concentrations of a number of other hormones, all of which may also be important during exercise. As wide fluctuations in resting levels of the ovarian steroids occur during a normal menstrual cycle, they may influence exercise performance or the physiological adaptations to exercise at different times in the cycle.</p> <p>To investigate these issues, nine subjects were studied during cycle ergometer exercise at three exercise intensities on two occasions in the menstrual cycle. Tests were conducted when resting levels of estradiol and progesterone were low, in the early follicular phase, and when the concentrations of both hormones were elevated, in the mid-luteal phase. Separation into the two phases was confirmed by hormone analysis.</p> <p>For the first time, it has been shown that in women, exercise is associated with an increase in the plasma levels of estradiol, progesterone, luteinizing hormone and follicle stimulating hormone. The response of the ovarian hormones is more marked in the luteal phase while the gonadotropins are affected only in the follicular phase. The magnitude of the increase in all four hormones is related to the intensity of exercise. These alterations in ovarian and gonadotropic hormone levels with exercise may possibly help to explain the occurrence of menstrual, irregularities in women engaged in strenuous physical activity.</p> <p>In the luteal phase, at a time when resting levels of estradiol and progesterone are higher, exhaustive exercise was maintained for longer than in the follicular phase. The difference was associated with a lower blood lactate concentration in the luteal phase, in the absence of differences in heart rate, oxygen uptake or cardiac output. This suggests that the effect of the menstrual cycle on the capacity to exercise is exerted at a local level in exercising muscle rather than through changes in oxygen delivery. A final study in this thesis showed an increased epinephrine response to exercise in the follicular phase. Further studies are required to investigate the part played by epinephrine in modulating the biochemical responses to exercise.</p> <p>Exercise results in increases in the plasma levels of estradiol, progesterone, luteinizing and follicle stimulating hormones that are dependent on both the intensity of exercise and the phase of the menstrual cycle. In addition, there is an improvement in performance of high intensity exercise in the luteal phase, in association with lower levels of plasma lactate. The mechanisms which underlie these findings remain to be elucidated.</p> <p>With the exception of the catecholamine assays, the work reported in this thesis was performed by the author. This includes the remaining hormone assays, lactate analysis, exercise tests and calculation of the data and most of the statistical analysis.</p> <p>Portions of this work have been published and/or presented previously:</p> <p>Jurkowski, J.E., N.L.Jones, W.C.Walker, E.V.Younglai and J.R.Sutton Ovarian hormonal responses to exercise. J.Appl.Physiol:Respirat,Environ. Exercise Physiol. 44:109-114.1978.</p> <p>Jurkowski, J.E.,N.L.Jones, J.R.Sutton, C.J.Toews Exercise performance and blood lactate levels in relation to the menstrual cycle. Med Sci Sports 9:70,1977. (abstract)</p> <p>Jurkowski, J.E.,J.R.Sutton.and P.M.Keane Effect of the menstrual cycle on the plasma catecholamine response to exercise in normal females. Canadian J.Appl.Sport Sci. 3:194,1978. (abstract)</p> / Master of Science (MS)

<p>Cytomegalovirus (CMV), one of the herpes viruses, is a common infectious agent among immunosuppressed renal transplant patients. Although CMV infection is usually mild or asymptomatic among general population, it can result in critical illness, allograft rejection and even death among transplant patients. This thesis designs experiments for the study of CMV with respect to its incidence, morbidity and mortality as well as its effects on the cellular immunity among renal transplant and hemodialysis patients. The principles of incidence study are outlined, the concept of sensitivity, specificity and predictive value of a test explained, and the difference between the laboratory and epidemiologic usage of these terminologies are discussed. The sources of error in Lymphocyte Stimulation Test as an instrument to assess cell-mediated immunity are discussed. The complexity of morbidity study is also discussed. Sample size determinations for the incidence study, morbidity as well as cellular immunity studies are discussed. Statistical analysis of the data (when available) is discussed.</p> / Master of Science (MS)

<p>A. A comprehensive and in depth review of the increased permeability of the vascular wall in thrombocytopenia was expedited. As part of this review, the following areas were covered: i. irradiation induced thrombocytopenia and the vascular wall defect; ii. anti-platelet serum induced thrombocytopenia and the vascular wall defect; iii. the endothelium and current theories on the role of platelets in supporting endothelial cell homeostasis; iv. the direct effect of irradiation and of anti-platelet sera on endothelium cell function and vitability; v. current models of explaining the endothelial cell changes and the escape of erythrocytes from the macroscopically intact capillaries and venules in thrombocytopenia; vi. a comparative analysis of the two thrombocytopenia models.</p> <p>B. Although both qualitative and quantitative studies of the spontaneous extravasation of erythrocytes in irradiation induced thrombocytopenia have been made in the past, only qualitative electron microscopic studies have beeh reported up until now for the anti-platelet serum thrombocytopenic model. In the present work, we established such a quantitative estimate by studying the changes of the cellular content of the thoracic duct lymph in anti-platelet serum rendered thrombocytopenic animals. The level of those changes was lower than the level of corresponding changes reported by others in the irradiation thrombocytopenic model, and for equally severe thrombocytopenia. The possible reasons for this difference were evaluated and discussed.</p> <p>C. The experimental technique developed here to study the vascular wall thrombocytopenic changes has certain advantages that renders it a useful tool in the study of platelet functions and in the evaluation of drugs claimed to have an anti-purpuric effect. One of the main attractions of this model is that it distinguishes between red blood cells extravasated through direct bleeding during the cannulation operations and spontaneously extravasated red blood cells post-operatively and post-APS. This is simply achieved by infusing the tagged RBC long after any possible bleeding has ceased and using those tagged RBC as a measure of RBC content in lymph. The animals are rendered thrombocytopenia post the infusion of the tagged RBCs. Furthermore, the model has been shown to be reversible and platelets can be transfused and survive into the thrombocytopenic animals shortly after the anti-platelet serum infuslon into the same animals.</p> / Master of Science (MS)

<p>The primary focus of this research was to investigate the possible influence of biological sex, specifically the sex chromosomes and hormones, on level of cognitive abilities and pattern of hemisphere specialization in humans. Normal males, and females are known to differ in level of cognitive abilities, with males typically having a higher level of spatial ability than females, and females achieving higher scores on tests of several aspects of linguistic ability. A growing body of research indicates that the sexes may also differ in degree of hemisphere specialization, with males being more lateralized than females. It has been suggested that degree of hemisphere specialization for spatial and linguistic processing may be related to the level of these cognitive abilities. The specific hypothesis of this research was that since the sexes differ in cognitive abilities and perhaps in hemisphere specialization, then perhaps the sex chromosomes and hormones may play a role in determining patterns of neural organization and cognitive abilities.</p> <p>This hypothesis was explored by comparing a group of eight Turner syndrome subjects to normal control groups of both females and males. Turner syndrome individuals were studied because they do not have a normal second X chromosome as do normal females or the Y chromosome of normal males. Thus they are genetically different from both normal males and females. Nor do Turner syndrome individuals have normal sex hormone production. Thus the possible role of these biological variables could be studied. However, on the basis of this syndrome alone, the relative importance of these factors cannot be disentangled. The study of Turner syndrome also allows one to rule out environmental factors since Turner syndrome individuals phenotypically resemble normal females. Previous research has indicated that TS individuals have a deficit in spatial ability compared to normal females.</p> <p>Male and female control subjects were matched with the Turner syndrome subjects on aspects of both Verbal and Performance IQ. Each group had a mean age of approximately 17 years.</p> <p>A battery of tests measuring spatial ability, linguistic ability and hemisphere specialization was administered to the eight subjects in each of the three groups. A trend toward bilateral representation for linguistic processing was observed in the Turner syndrome group. Right hemisphere specialization for the processing of nonverbal stimuli appeared to be normal.</p> <p>With regard to cognitive abilities, the Turner syndrome subjects were found to perform at a normal level on many tests such as a phonetic reading test, tests of verbal intelligence, and tests of some aspects of spatial ability, compared to normal males and females. However, their performance was found to be deficient on some aspects of a test of three-dimensional perception, a word recognition reading test, a written word fluency test and a coding or digit symbol test. It is suggested that a common element required in the performance of these tests on which Turner syndrome individuals were deficient may be some aspects of visuospatial perception and memory which become particularly deficient when there are time constraints.</p> <p>Given the cognitive differences and the tendency toward a difference in hemisphere specialization between the Turner syndrome and normal subjects, it is suggested that either the lack of a second X chromosome or the lack of normal sex hormones, or both, play a role in determining the level of cognitive abilities and pattern of hemisphere specialization in normal humans. The evidence from this and previous studies suggests that the absence of a normal second X chromosome alone may underlie the differences between Turner syndrome subjects and normal females.</p> <p>Some of the methodological problems encountered in this type of research are also discussed.</p> / Master of Science (MS)

<p>Transient embryonic antigens have been described in many systems in development. Although their function has not yet been elucidated their detection in the embryo but not in the adult state suggests a direct relationship with embryogenesis. The present investigation was undertaken in order to characterize and study a transient antigen (TEA) in chick embryo brain extracts.</p> <p>TEA was identified by using specific antiserum prepared against 9 day embryonic chick brain extract. The antiserum was first absorbed with adult serum, liver and kidney extracts to remove non-neural antibodies. Then adult brain extract was added to remove antibodies directed against adult neural antigens. This antiserum (TAS) was then considered to be both embryo and neural specific. However, subsequent studies demonstrated TEA in extracts of 9 day embryonic liver, kidney and serum in concentrations similar to that found in 9 day brain extracts. TEA was therefore not specific to neural tissue as initially considered.</p> <p>TEA demonstrated anodal migration in an electric field, similar to an alpha-globulin at pH 8.6. Ontogenic studies using immunoelectrophoresis and the quantitative technique of rocket immunoelectrophoresis were performed. TEA was present by 2 days incubation, and an initial peak at 4 days was noted. From day 6 on, TEA accumulated until a maximum concentration was reached at 12 days incubation. TEA levels then decreased until it could no longer be detected in 20 day embryo or adult brain extracts.</p> <p>Molecular exclusion chromatography revealed the molecular weight of TEA to be 73,000 daltons. Isoelectric focusing demonstrated the isoelectric point at pH 4.8. The antigenic site of the TEA molecule was considered to be proteinaceous on the basis of its sensitivity to pronase; however it was not hydrolysed by either trypsin of chymotrypsin.</p> <p>Based on the ontogenic pattern and the physical and chemical characteristics it was concluded that TEA was a chick alpha-fetoprotein. The possible role of TEA in embryonic development and the mechanisms of its regulation were also discussed.</p> / Master of Science (MS)

<p>Methods for the production of hybrid cell lines (hybridomas) secreting monoclonal antibodies against specific antigenic determinants have recently been developed (Kohler G. and Milstein C., Nature 256, 495, 1975). Monospecific antibodies to Herpes simplex virus (HSV) antigens would greatly facilitate the analysis of HSV type-specific determinants, be of use in molecular studies of virus-cell interactions and could have potential applications in immunization against HSV infections. To this end, spleen cells from HSV2-immunized BALB/c mice have been fused to a BALB/c derived HGPRT myeloma line (Sp 2/0 Ag-14) and the resulting hybrids selected in HAT medium. From 13 successful fusions, 102 hybrid populations secreting antibodies recognizing antigenic determinants specified on HSV2-infected cells have been identified by FITC-immunofluorescence, an ELISA method or a ¹²⁵I-protein A binding assay. Ten of the positive hybrids have been cloned by limited dilutions to generate 124 monoclonal lines reacting specifically with HSV-infected but not with mock-infected cells. High titres of anti-HSV specific antibodies have also been detected in two ascitic fluids recovered from tumors induced in mice by injection of positive hybridomas. Preliminary data on the characterization of two hybridomas in terms of the subclass of immunoglobulins they secrete and the specificities of antibody they define has also been obtained.</p> / Master of Science (MS)

<p>The response of the intrapulmonary artery (IPA) to a variety of endogenous lung amines and prostaglandins (PG) was examined to determine if a differential sensitivity to these vasoactive substances existed between segments taken from two sites on the artery. Longitudinal strips of proximal (PIPA) and distal (DIPA) segments of the left lower lobar intrapulmonary artery were taken from rabbit lungs and isometric tension measured during superfusion abt 37°C with physiological saline. Full or partial dose-response curves were obtained for 5-Hydroxytryptamine (5HT), Histamine (HIS), Norepinephrine (NE), Isoproterenol (IsoP), Arachidonic Acid (AA), PGA₁, PGB₁, PGB₂, PGE₁, PGE₂ and PGF₂α. In addition to pharmacological studies, length-tension properties of the segments utilized were examined and a qualitative analysis of smooth muscle content and orientation was undertaken. All prostaglandins elicited contractile effects, of varying magnitudes, at high doses. Prostaglandins A₁, E₁, and E₂ produced little or no contractile responses or slight relaxant activity in unstimulated PIPA and DIPA segments at low doses. 5HT contracted both PIPA and DIPA segments in a dose dependent manner, however, proximal segment maximal effects and sensitivity were significantly greater than those of the distal segment. Both PIPA and DIPA segments contracted to HIS and maximal effects were similar both segments. Mepyramine (10ˉ⁹M) antagonized contractile responses to HIS. In the presence of 10ˉ⁷M mepyramine, HIS produced dose dependent relaxation of precontracted PIPA and DIPA segments. Cimetidine (10ˉ⁵M) antagonized this relaxation indicating that HIS relaxant effects are mediated by H₂-HIS receptor stimulation in both segments. PIPA segments contracted in response to NE while the DIPA segment responded poorly or not at all suggesting a paucity of alpha adrenoreceptors in distal segments. IsoP produced dose dependent relaxation, that was antagonized by propranolol, of precontracted PIPA and DIPA segments. The dose related contractile response to AA was similar in both PIPA and DIPA segments. These studies indicate that regional differences exist in the response of rabbit IPA to some agonists.</p> / Master of Science (MS)

<p>2-Azidoadenosine has previously been shown to be a potent activator of adenylate cyclase in human platelet particulate fractions. In the present study, the activation of adenylate cyclase was enhanced by: (a) reducing Mg²⁺ concentration in the assay mixture, (b) adding excess adenosine deaminase during the incubation, and (c) using rabbit platelets instead of human platelets. Under these optimal conditions a 200% increase in adenylate cyclase activity was achieved with 10 μM 2-azidoadenosine.</p> <p>2-Azidoadenosine was stable in aqueous buffer systems in the absence of light. At pH 7.4, 2-azidoadenosine exists as two tautomers, the azide form and the tetrazole form which were shown to interconvert in a first order fashion with a half-life of 15.3 min at 22°C. These two tautomers appear to be equipotent in their activation of platelet particulate fraction adenylate cyclase. However, on irradiation at 313 nm only the azide form appears to be photolysed to a mixture of products, presumably via a nitrene intermediate.</p> <p>Although the photolysis of 2-azidoadenosine in the presence of platelet particulate fraction produced activation of platelet adenylate cyclase, evidence was obtained that this was not the result of photoaffinity labeling. The individual effects of pre-exposure to 2-azidoadenosine and irradiation at 313 nm were additive.</p> <p>Activation of adenylate cyclase by 2-azidoadenosine added to the enzyme assay was reduced if the particulate fraction had been exposed to UV radiation. The same reduction in activation by 2-azidoadenosine added to the assay was seen when the prior irradiation of the enzyme was carried out in the presence of 2-azidoadenosine.</p> <p>The ³H covalently bound to the platelet particulate fraction upon irradiation in the presence of 2-azido[³H] adenosine was shown to be radiation dependent. However, pre-irradiation of 2-azido-[³H] adenosine generated a product that covalenty labeled platelet particulate fraction as effectively as irradiation of 2-azido[³H]-adenosine in the presence of particulate fraction. This indicates that affinity labeling rather than photoaffinity labeling had occurred. Some inhibition of this labeling was produced by the addition of unlabeled 2-azidoadenosine, 2-chloroadenosine and theophylline. However, neither adenosine nor N⁶-cyclohexyladenosine caused inhibition of labeling when present during photolysis, suggesting that adenosine receptors were not detected. Inhibition of labeling by dipyridamole and coformycin suggested reactions with adenosine transport proteins and adenosine deaminase, respectively. Further studies with various combinations of ligands are required to determine the nature of sites labeled by the photolysis products of 2-azido[³H]adenosine.</p> / Master of Science (MS)

<p>The genetic region which exerts a major influence on graft rejection is known as the major histocompatibility complex or MHC. The MHC in man, the HLA system, includes at least five distinct multi-allelic loci, HLA-A,-B,-C,-D and -DR. The HLA system possesses three fundamental characteristics: it is highly polymorphic; its alleles are in linkage disequilibrium with one another as well as with other loci; and the HLA antigens demonstrate extensive cross-reactivity.</p> <p>A number of previous reports have established associations between certain HLA-B antigens and juvenile-onset insulin-dependent diabetes mellitus (JOD). In this investigation we have studied the distribution of HLA antigens in patients with diabetes mellitus with a special emphasis on HLA antigens belonging to the DR locus. We observed a significant increase in the incidence of HLA-DR4 in JOD patients as compared to its frequency in the normal controls. The present data also confirm earlier reports of the increased incidence of HLA-B15, -B40 and -CW3 in the patient group. Further analyses of the data showed that the primary association of JOD was with HLA-DR4 and the associations with the HLA-B and -C loci antigens were secondary in nature.</p> / Master of Science (MS)