<p>The Ames Assay is a battery of tests which measures the mutagenicity of chemical substances. In order for its results to be relevant to human health, Ames Assay mutagenicity must relate to human cancer in a biologically credible and comprehensive way. It must also correlate with other measures and attributes of carcinogenicity. The evidence presented demonstrates its biologic credibility, but illustrates the improbability of its being a comprehensive measure of all attributes of carcinogenesis. Agreement between the Ames Assay and animal cancer studies, other short-term bioassays, human carcinogens, and chemical structures predictive of carcinogens is presented. The range of possible predictive accuracy for the Ames Assay is calculated from these agreement evaluations. A hypothetical model is developed to test whether the Ames Assay is a sufficiently valid predictor of human carcinogenesis regardless of the extremes of this range. It is not sufficiently valid in the lower half of the range. So more work must be done to define precisely its accuracy.</p> <p>The problems of applying the range of accuracy of a laboratory test of pure substances to biologic and environmental samples are explored. Unsolved problems in collection and analysis of airborne samples are identified. A study design is presented which minimizes the chance of bias in collecting airborne samples. The limitations of the laboratory procedure and the problems of sample collection are brought together in two continuous flow schemes. Together these demonstrate the unsolved biologic and biostatistical problems in validating the Ames Assay.</p> / Master of Science (MS)

<p>A retrospective cohort analytic study will be conducted to compare the incidence of abnormal reproductive outcomes of members of the Canadian Society of Laboratory Technologists with that of members of the Canadian Physiotherapy Association in order to identify exposures which may pose a threat to the reproductive process.</p> <p>Members of each of the organizations will be mailed a questionnaire requesting demographic and health information. A second questionnaire requesting detailed information about reproductive outcomes will be sent to those subjects who have experienced pregnancies in the previous five years. Additional mailings by conventional mail and special delivery wil be used to increase the response rate. Subjects who have given birth to stillborn children or children with congenital abnormalities and a random sample of those who have had miscarriages will be asked to provide written consent for their physician to be contacted to provide confirmatory information from their medical record.</p> <p>The analyses will compare rates of abnormal reproductive outcomes in total and by specific type between laboratory technologists and physiotherapists controlling for age, parity, smoking status, infection, X-ray, drug and alcohol exposure during pregnancy.</p> / Master of Science (MS)

<p>Hereditary muscular dystrophy of the chicken is a primary defect of muscle which is expressed specifically in the fast twitch, focally innervated glycolytic muscle, while slow tonic, multiply innervated oxidative muscles are spared disease characteristics (for review, Cosmos et. al., 1979b). Since motor nerves influence the characteristics of skeletal muscles, and since slow tonic muscles in the chicken do not express dystrophic phenotypes, it was decided to replace the motor innervation of the fast twitch posterior latissimus dorsi (PLD) muscle with the motor innervation of the slow tonic anterior latissimus dorse (ALD) muscle within a dystrophic chicken, in order to alter the fate of the dystrophic fast twitch muscle. Selected mechanical, histochemical and structural properties of the ALD and PLD muscles of normal (White Leghorn) and dystrophic (Storrs line) chickens 15-800 days ex ovo were compared to determine which of these properties were altered as a result of the disease, and to provide baseline data for the analysis of the cross reinnervation experiments. ALD muscles of dystrophic chickens exhibited normal phenotypes, i.e. slow tonic isometric contraction in response to nerve stimulation in vivo, acid and alkaline stable myosin ATPase activity, "en grappe" innervation, weak phosphorylase (Pase), strong succinic dehydrogenase (SDH) enzymic activities and peripheral location of nuclei. PLD muscles of dystrophic genotype demonstrated structural and histochemical alterations but retained contraction and relaxation times characteristic of fast twitch muscle of normal genotype. Further, they exhibited focal "en plaque" innervation and alkaline stable myosin ATPase activity similar to that of normal PLD muscles. The abnormalities identified in the dystrophic PLD included the following: lower muscle weight, abnormal size and shape of fibres, increased number of internal nuclei, abnormal Pase and SDH enzymic activities and lower twitch and tetanic tensions. The surgical cross reinnervation between the ALD nerve and the PLD muscle was performed at hatching. The muscles were examined at various time intervals postoperatively using the criteria established in the study of the unoperated muscles. Self reinnervation of PLD muscles by their own nerves, and cross reinnervation with normal chickens served as control experiments. Fibres of both normal and dystrophic PLD muscles which had been successfully cross reinnervated acquired mechanical, structural and histochemical properties characteristic of the ALD muscle. Thus, muscles of both genotype responded similarly to the ALD nerve. These results established that the PLD muscle of dystrophic chicken is able to accept the new innervation, and is as capable of responding to the influence of the ALD nerve as is the normal PLD. Furthermore, the successfully cross reinnervated fibres are spared disease characteristics. The present cross reinnervation experiment demonstrates, for the first time, an experimental manipulation which interferes with the expression of phenotypic characteristics of dystrophy during development ex ovo. The results support the rationale underlying the present study: the PLD muscle of dystrophic genotype is unable to respond appropriately to the demand from its own motor nerve to complete successfully the transition from embryo to adult metabolism, thus expressing the dystrophic phenotypes. However, when this request is removed by cross reinnervating the PLD muscle with the ALD nerve, the dystrophic phenotype is not expressed. These findings strongly suggest that regardless of the time during development when slow tonic characteristics are achieved, i.e. either during development in ovo or by surgical manipulation ex ovo, slow tonic fibres are spared dystrophic phenotypes.</p> / Doctor of Philosophy (PhD)

<p>Ovulation is the result of the positive feedback action of estrogen, either alone or in combination with progesterone, and the integrated sensory input from the external environment onto the neural circuits controlling gonadotrophin secretion. During the dioestrus II in the 4-day cycling rat the ovaries secrete an increasing quantity of estrogen which stimulates neurons in the preoptic area (POA) to trigger the surge of luteinizing hormone releasing hormone into the capillaries of the hypophysical portal plexus. However, the mechanisms whereby estrogen stimulates this increased activity are, as yet, unknown.</p> <p>The binding of estrogen to specific target areas within the brain may induce changes in the metabolic function of neurons or it may directly or indirectly affect the electrical properties of neuronal membranes. The latter possibility has been tested by observing the affects of an intravenous (i.v.) injection of estrogen on the spontaneous activity and responsiveness to iontonphoretically applied putative neurotransmitters of identified preoptic neurons.</p> <p>Experiments I and II were undertaken in order to establish certain parameters of the experimental design. Experiment I demonstrated that halothone anaesthesia inhibits ovulation in the cycling rat by an action on the neuroendocrine circuits which control gonadotrophin secretion. In experiment II measurements were made of the plasma concentration of estrogen following various doses of an i.v. injection of estrogen or progesterone would stimulate an increased secretion of LH in estrogen primed ovariectomized rats. Measurements of serum LH concentrations one and five hours after the injection showed there was no stimulation of LH under these conditions.</p> <p>Experiment III was undertaken to identify neurons in the POA which are thought to implicated in a neuroendocrine circuit controlling gonadotrophin secretion and which may be target neurons for the positive feedback action of estrogen. Cells in the POA, which receive an input from the stria terminals have been identified by stimulating this pathway at its point of convergence in the amygdala. Antidromic stimulation techniques were employed to identify cells in the POA which make a direct connection with the basal hypothalamus.</p> <p>Experiment IV constitutes the major objective of the research, which was to thought to be implicated in a neuroendocrine circuit controlling gonadotrophin secretion and which may be target neurons for the positive feedback action of estrogen. Cells in the POA which receive an input from the stria terminalis have been identified by stimulating this pathway at its point of convergence in the amygdala. Antidromic stimulation techniques were employed to identify cells in the POA which make a direct connection with the basal hypothalamus.</p> <p>Experiment IV constitutes the major objective of the research, which was to investigate 1) the sensitivity of antidromically identified preoptic neurons to iontophoretically applied putative neurotransmitters; the cells were inhibited by both the cataecholamines, dopamine (DA) and norepinephrine (NE) but were unresponsive to acetylcholine applied iontophoretically; b) to test the possible membrane effects of estrogen on those antidromically identified cells. Two indices of a change of the excitability of preoptic neurons were used (i) the effects of an i.v. injection of estrogen on the rate of spontaneous activity and (ii) changes in the response of a neuron to electrophoretically applied DA or NE following the injection of estrogen or progesterone in primed and unprimed animals. The results of the first series of experiments demonstrated that estrogen can decrease the rate of spontaneous activity in identified preoptic units. Some cells showed a long lasting depression in their spontaneous activity following the steriod injection while others showed a short term decrease in the rate of unit discharge five minutes after estrogen administration. In the second series of experiments no changes were observed in the dose response curves of iontophoretically applied DA or NE thirty minutes after the steroid injection in either group of animals. The negative finding could be due to the technical limitations of this type of experiment or to the fact that estrogens do not affect the response of neurons to synaptic inputs in which these amines may function as neural transmitters.</p> / Doctor of Philosophy (PhD)

<p>Early in the development of the embryo, cells lose their ability to differentiate into any adult type of cell and become determined. A cell which has achieved a certain degree of differentiation can ultimately reproduce to form only a limited number of cell types along the original line of differentiation: for example, a primitive mesenchymal cell can give rise to only connective tissues such as muscle or bone.</p> <p>Apart from the primordial germ cells (those cells which later give rise to oocytes in the female and spermatocytes in the male), all cells have begun to specialize, with a consequent narrowing of their potential, very early in gestation.</p> <p>Teratomas are neoplasms which contain many kinds of tissues which are foreign to the organ in which they arise. There is controversy over whether these tumours arise from the growth of cells which have escaped early developmental controls, or whether they arise from the proliferation of the only multipotential cell in the body, the germ cell. Rather than following their normal path of differentiation to oocytes or spermatocytes, they might fulfil their potential to form almost any kind of tissue. Thus tumours composed of skin, bone, muscle, nervous tissue, and glands are found in adult testes and ovaries.</p> <p>Since the germ cells arise in the embryonic yolk sac and travel up the umbilical cord to the developing gonads, there is a theory that these multipotential cells may occasionally become lost and much later, often in adulthood, begin their proliferation in sites along the midline: at the base of the spine, in the chest and throat.</p> <p>The experiments in this thesis explore an animal model in which teratomas can be produced by the grafting of 7½-day mouse embryos to the gonads of adult hosts. According to the literature the tumour which develops as a result of this operation is identical to those arising spontaneously in a highly inbred strain of mouse. Those experimental tumours which contain areas of undifferentiated tissues can be transplanted to other hosts; sometimes retaining their embryonic nature for many generations.</p> <p>Whole embryos develop as teratomas in up to 80% of grafts to male hosts. The embryos can be cut in such a way that one half contains extra-embryonic material (that which eventually forms the placenta) and also the yolk sac containing the germ cells, and the other half which contains only sonatic tissue.</p> <p>Grafts of the somatic parts result in teratomas in 74% of the testes, a rate similar to tumours from whole embryos, whereas grafts of the extra-embryonic parts containing germ cells resulted in only 1 small teratoma out of 35 grafts.</p> <p>In this experimental model, it has been demonstrated that the primordial germ cells of the embryo are not responsible for the development of the neoplasms, but rather it is the somatic cells which are giving rise to these tumours. One must ask whether the controls which govern normal differentiation may become faulty and allow somatic cells to retain or regain their pluripotential nature making it possible, when conditions are right, to proliferate to form the "monstrous" tumour, teratoma.</p> / Doctor of Philosophy (PhD)

<p>Repeated electrical stimulation of certain brain sites in animals leads to the progressive development of clinical seizures, a phenomenon known as kindling. The basic neuronal mechanisms underlying this effect may be similar to those involved in human epilepsy, but are not well understood. We have examined the properties of single neurones and populations of neurones in hippocampal slices derived from rats kindled in the hippocampus, fimbria/fornix, perforant path, or amygdala, and compared them with those of unstimulated animals.</p> <p>At the neuronal population level, tissue from kindled rats did not display an increased tendency to generate spontaneous epileptiform activity. This as also the finding in ionic substitution experiments, where the levels of extracellular potassium were increased as high as 9 mEq/1. Electrical stimulation of afferents to the CA1 region evoked synaptically-mediated population responses which were similar to those of controls, except for tissue derived from rats kindled in the fimbria or in the amygdala. In these experiments, there was a tendency for multiple population spikes to appear in the responses to stimulation and this may reflect an increased excitability of the neuronal population.</p> <p>Intracellular studies of hippocampal CA1 neurones from amygdala-kindled rats revealed that the basic activity and passive membrane properties did not differ from those of controls. However, the amplitude of the afterhyperpolarization following synaptic activation was increased in these neurones. In addition, the response to intracellular injection of depolarizing current was altered in some neurones, observed as an increased action potential discharge during the initial phase of depolarization.</p> <p>These findings are discussed in terms of an apparent immunity of the hippocampus towards the development of chronic epileptogenic properties, and the ionic and membrane mechanisms which may be responsible for the observed differences between kindled and unkindled tissue.</p> / Doctor of Philosophy (PhD)

<p>Sera raised in rabbits against adult rat kidney has long been known to be teratogenic when administered to pregnant rats at a time when the embryos are in the gastrulation stage. The main hypothesis postulated on the mode of action of teratogenic kidney antiserum is that the antibodies directly interfere with placental development and function; thus resulting in a nutritional deficit to the embryo. A nutritional deficit is believed to interfere with embryonic morphogenesis. To date no investigations have been performed on placental function and development.</p> <p>In the present investigation, histological and histochemical techniques were used to study the effects of kidney antiserum administered to the pregnant rat on the development and function of the fetal placental structures.</p> <p>The results of this study have demonstrated that both the visceral yolk sac and chorio-allantoic placentae were adversely affected. The effects of kidney antiserum on the visceral yolk sac is consistent with an interpretation of an inhibition of nutrient uptake. The development of the chorio-allantoic villi and the allantoic vasculature was markedly retarded. The effects of kidney antiserum on the fetal placental structures suggest that the embryo undergo a nutritional deficit.</p> <p>Glycogen and protein content of the maternal placenta was also investigated. The glycogen content was significantly lower in experimental rats as compared to controls. However, this difference was not noted until 72 hours after the administration of the kidney antiserum. This was a time when the embryos were not very susceptible to teratogenic antiserum insult.</p> <p>Localization of kidney antibodies was also performed. The antibodies were demonstrated to be present within the embryo proper, amnion, parietal yolk sac, chorion and allantois.</p> <p>Numerous Feulgen positive necrospherules were also observed within the embryo proper within 12 hours after the administration of kidney antiserum.</p> <p>The observations of the present study is thus consistent with the interpretation that the cause of kidney antiserum teratogenesis is an effect of kidney antibodies on placental function and development resulting in a nutritional deficit to the embryo and/or is a direct effect of kidney antibodies on the integrity of the cells of the embryo proper.</p> <p>Both control and experimental trophoblast shells contained islets of cells. These islets, not previously reported in the literature, contained mitotic figures. It is suggested that these islets are the proliferation sites of the trophoblast shell.</p> / Doctor of Philosophy (PhD)

<p>Infection by Pichinde virus, a member of the arenavirus group, was studied in vivo in Syrian hamsters, and in vitro in cell culture. Emphasis in the in vivo studies was placed on the mechanism of resistance to virus infection in adult hamsters. Two hamster strains were found to differ in their susceptibility to lethal Pichinde virus infection. LVG/Lak hamsters were found to be 100% susceptible to low doses of Pichinde virus during the first 6 days of life, but after 8 days of life, mortality was uncommon. Peak serum virus titers in animals infected at 3 days of life were 4 logs greater than in animals infected at 12 days. MHA/Lak hamsters, in contrast, were found to be susceptible to lethal virus infection both as newborns and as adults. Peak serum virus titers of greater than 10⁸ PFU/ml were observed 8 days after infection of adult MHA hamsters compared with less than 10³ PFU/ml in their LVG counterparts. Cultured primary kidney cells and peritoneal macrophages from either hamster strain supported Pichinde Virus replication equally well in vitro. Antibody levels measured by complement-fixation test were similar at 13, 21, and 30 days after infection. Cyclophosphamide immunosuppression, administered 3 days after infection, rendered adult LVG animals susceptible while slightly increasing the mortality among MHA hamsters. These findings suggest that immunologic factors maturing early in life in LVG hamsters and deficient in MHA hamsters limit Pichinde virus infection. The relationship of these observations with previously reported arenavirus diseases is discussed.</p> <p>The antigenic structure of Pichinde virus was examined. Lysates of BHK₂₁ cells, infected with Pichinde virus and harvested 48-96 hours after infection contained virus-specific antigens detectable by complement-fixation (CF) test. Immunodiffusion analysis of the lysates demonstrated two antigens which differed in their properties of heat and proteolytic enzyme resistance. The antigen accounting for the major proportion of the CF antigen activity was a heat stable, pronase resistent protein of 20-30,000 molecular weight estimated by gel filtration. The minor antigen was heat labile and susceptible to proteolysis. Antiserum prepared against partially purified major antigen, yielded a band of identity when tested against antiserum prepared against virus "cores" obtained by NP-40 solubilization of purified Pichinde virus. Disruption of purified virus by treatment with 1% NP-40 and 50 ug/ml RNase liberated 2 soluble antigens which identified with those demonstrated in lysates of infected cells. The liberated antigenic activity was shown to contain 3 of the 4 polypeptides found in the virion. These findings suggested that the antigens detectable by CF and immunodiffusion were components of the virion core. Major antigen derived from infected cells was purified by rate-zonal sedimentation, isoelectric focusing and gel filtration. Two low molecular weight polypeptides were demonstrable in the purified antigen.</p> <p>Since multiple segments of RNA exist in the Pichinde virion, it was of interest to determine, whether antigen synthesis and virus production could be dissociated in the infected cells. In Vero cells infected by Pichinde virus, antigen on the cell surface, and production of infectious virus shut down 48-96 hours after infection, whereas antigen detectable in the cytoplasm of infected cells appeared stable for over 5 days. In BHK₂₁ cells, actinomycin D (AD) at dosage levels of 1-4 ug/ml reduced virus yields by greater than 95% while reducing antigen detectable by CF by only 30%. The quantity of antigen produced was independent of AD dosage within the range tested. Both the major and the minor antigens were identified in lysates of AD treated cells. The observed decrease in infectious virus production could not be attributed to increased cell associated virus or to greater production of defective interferring particles. Surface antigen, demonstrable by immunofluorescence and by antibody binding assay was found to accumulate of infected cells incubated with AD. These findings suggest that Pichinde virus replication in AD treated BHK₂₁ cells is blocked at the level of virus maturation.</p> / Doctor of Philosophy (PhD)

<p>Changes in motor control processes associated with advancing age have been the focus of considerable research interest. However, the effect of age on the processes involved in motor skill acquisition have received little attention. In this thesis, two experiments are reported that examine the differences in motor learning between young and older normal adults. The strategy was to evaluate the age-related effects on learning of knowledge of results (KR) variables. The learning task in both experiments was a multisegment task with spatial/temporal requirements. The KR variables evaluated were, (1) random and blocked KR schedules, and (2) different schedules of relative frequency of KR. In general, while the findings indicated large performance differences between age groups, there was no difference between age groups in how the KR variables were used to learn the task. The results suggest that despite declines in performance that accompany age, there seems to be no decline in the capabilities of older adults to use these KR variables to acquire and retain this motor skill.</p> / Doctor of Philosophy (PhD)

<p>Partial denervation of a shared target tissue leads to 'collateral' sprouting of the remaining nerve fibres supplying the same region. The mechanisms responsible for this widespread phenomenon are unknown, but it has generally been believed that products from the degenerating fibres constitute the sprouting stimulus. Recently it was shown that a similar sprouting of intact nerves occurred in the salamander hind-limb when the axoplasmic transport in one of the neighbouring nerves was blocked by colchicine. This block did not, however, appear to interfere with the functioning of the treated nerve. On this evidence it was proposed that nerves release factors which are concerned in the regulation of their peripheral fields. The experiments of this thesis test this hypothesis further. In particular this study quantitatively examines the organization of the cutaneous mechanoreceptors of the salamander hindlimb, and the effects of various nerve treatments on this organization.</p> <p>A mechanical prodder of 10-50μ tip diameter was used to determine the sensory threshold of the skin at selected spots. The mechanoreceptors were found to be all rapidly-adapting. An analysis of the distribution of their thresholds across the skin points to the existence of a single population of mechanosensory endings of similar threshold which are fairly uniformly distributed in the plane of the skin. A simple model, based on the results, indicates that these receptors have receptive field sizes of about 50μ, and are spaced approximately 150-200μ apart. Direct inspection of data from systematically surveyed skin areas gave findings which were consistent with these estimates. These quantitative results made it possible to establish that the blocking of fast axoplasmic transport does not affect the sensitivity or distribution of the individual mechanosensory endings of the treated nerve. Nevertheless new sprouts appeared from the adjacent nerves, and it seemed that there was a tendency for these to grow preferentially to the site of the still-functioning endings of the treated nerve. Most significantly, after partial denervation of the skin the number of newly sprouted endings quantitatively matched the number of endings lost by nerve section.</p> <p>It was concluded that there is a control mechanism which continuously regulate the density of the skin innervation. This control system probably involves the mutual interaction of substances continuously secreted from the target tissue, which cause nerves to sprout, and factors released from the nerve endings which offset the effects of the growth stimulus. The nerve field density reflects an equilibrium state between the neural and tissue influences.</p> / Doctor of Philosophy (PhD)