<p>The role of RNA and protein synthesis in follicular testeterone production has been investigated in the rabbit ovarian follicle in vitro.</p> <p>lsolated follicles were incubated (usually 2 hours) with labeled amino acids or uridine plus LH, cyclic AMP or cyclic GMP alone, or together with various metabolic inhibitors.</p> <p>In dose response studies, testosterone production was stimulated at all concentrations of LH (0.1 to 10 μg/ml) tested (p < .05), with optimal stimulation occurring at 5 μg LH/ml (p < .01). However, a significant uptake of ³H-leucine into follicular protein occurred only at > 2.5 μg LH/ml (p < .01), with optimal stimulation occurring at 5 and 10 μg LH/ml. Cyclic AMP (5 and 10 mM) enhanced both testosterone production and the uptake of ³H-leucine into follicular protein (p < .01). Lower cyclic AMP concentrations were ineffective. Neither LH nor cyclic AMP had any effect on the incorporation of labeled uridine into follicular RNA.</p> <p>In time course studies, testosterone was stimulated within 15 minutes (p < .01), in the presence of LH (5 μg/ml) or cyclic AMP (5 mM). The incorporation of ³H-leucine also increased with time in both LH and cyclic AMP treated follicles, compared to controls, but a significant difference was observed only after 90 and 60 minutes, respectively (p < .01). However, electrophoretic fractionation and radio autographic examination of total follicular proteins after exposure, of LH and ³⁵S-methionine for 15, 60 and 120 minutes showed no apparent difference in the distribution of protein bands when compared to controls.</p> <p>Actinomycin D (20, 80 and 160 μg/ml) together with LH (5 μg/ml) inhibited the incorporation of ³H-uridine into follicular RNA by 79, 85 and 86%, respectively (p < .01). At these concentrations, no inhibitory effect on LH-induced testosterone production was observed. Paradoxically, Actinomycin D (1 μg/ml) enhanced LH-induced testosterone production above that elicited by LH alone.</p> <p>Cycloheximide (20 and 10 μg/ml) inhibited LH-induced testosterone production by 64 and 57% (p < .01), as well as the uptake of ³H-leucine into follicular protein by 94 and 93%, respectively. However, cycloheximide (1 μg/ml) did rot inhibit LH-induced testosterone production, yet inhibited ³H-leucine incorporation by 81.7%. Similarly, puromycin (40 μg/ml) inhibited LH-induced testosterone production by 66%, and the uptake of ³H-leucine into protein by 74% (p < .01). However, puromycin (10, 1 or 0.1 μg/ml) did not inhibit LH-induced testosterone production, yet ³H-leucine incorporation was inhibited by 58, 37 and 31%, respectively (p < .01).</p> <p>The methylxanthines, theophylline (25, 10 and 1 mM) and MIX (5 and 0.5 mM) had no synergistic effects with cyclic AMP on follicular testosterone production. However, these methylxanthines inhibited the incorporation of ³H-uridine (35 to 68%) and ³H-amino acids (45 to 69%) into follicular RNA and protein.</p> <p>Cyclic GMP (25, 10 and 1 mM) had no stimulatory effect on follicular testosterone production or the uptake of protein. However, cyclic GMP (25 and 10 mM) significantly enhanced the uptake of ³H-uridine into follicular RNA (p < .01).</p> <p>These data collectively suggest that de novo RNA and protein synthesis are not required for acute LH-induced testosterone production in the rabbit follicle.</p> / Doctor of Philosophy (PhD)

<p>The origins of the immunoglobulin-containing cells in the intestinal, respiratory, mammary, and genital tissues were studied in mice, rats, and guinea pigs by using an adoptive lymphocyte transfer method. Proliferating lymphocytes (lymphoblasts) were obtained from various donor lymphoid sources and radiolabelled in vitro with either ³H-thymidine or ¹²⁵-deoxyuridine. Radiolabelled lymphoblasts were then injected into the circulation of recipient animals. Twenty-two - 24 h later, various tissues taken from recipient animals were examined for the presence of radiolabelled donor cells by either radiocounting or autoradiographic techniques. Furthermore, radiolabolled B-cells containing different immunoglobulin isotypes were identified in mouse tissues by a combination of immunofluorescent staining with autoradiographic procedures. It was found possible to greatly accelerate this procedure by the use of liquid scintillation fluid so that ³H-thymidine-labelled cells could be identified in less than 24 h as opposed to 4-6 wks in the absence of scintillation fluid.</p> <p>In mice and rats, mesenteric lymph node (MLN) lymphoblasts showed a propensity to selectively localize in the cut mucosa. Careful examination of other mouse tissues revealed that MLN lymphoblasts were present, not only in the intestinal lamina propria, but also beneath the mucosal epithelia of the respiratory and genital tracts, the mammary glands and in the MLN. In these mucosal tissues approximately 60% of these cells contained IgA and 25% contained IgG. In peripheral lymph noded (PLN), a few labelled MLN cells were observed and 40% of these contained IgG, whereas only 8% were of tho IgA isotype. The preference of MLN to populate mucosal sites was clear from the results.</p> <p>In marked contrast, when labelled PLN cells were adoptively transferred, the majority returned to their sites of origin and contained IgG. Of the small number of labelled PLN cells found in mucosal tissues, approximately equal percontages (30%) of IgA- and IgG-containing cells were soon.</p> <p>Dividing cells prepared from bronchial (mediastinal) lymph nodes (BLN) showed a propensity to localize in the lungs rather than in the intestine or lymph nodes. However, tho predominant immunoglobulin content of these donor cells in tho gut, lungs, and MLN was IgA. Thus, the BLN made a quantitatively minor, but presumably significant, contribution to the IgA plasmacytes found in the gut lamina propria; in the lungs, BLN made a quantitatively major contribution to population of IgA-containing cells residing in the lung mucosa.</p> <p>It was concluded that, in rodents, the MLN was a major contributor of the immunoglobulin-containing celIs beneath the mucosal epithelia of the gut, lungs, cervix, and vagina and gestational mammary glands. Thus, lymph nodes draining two mucosal surfaces, though differing in IgA plasmacyte precursor content, both possessed cells destained to localize in mucosal tissues. Furthermore, there was organ specificity for the distribution of these cells; those immediately derived from the lymph nodes draining the bowel tended to return to the bowel, whereas those from the lungs tended to return to the lungs.</p> <p>To further explore the properties of MLN lymphoblasta, the influence of the mouse estrous cycle on MLN lymphoblast localization in the cervix and vagina was investigated. Compared to proestrus and estrus, a 2-fold reduction in the number of MLN lymphoblasts localizing in the diestral cervix and vagina was observed. Detection of the immunoglobulin isotype of these cells suggested that this reduction was restricted to the IgA plasmacyte progenitor population, i.e., the major B-lymphocyte subpopulation entering these sites. No significant changes in B-lymphoblast subpopulation lodging in the small intestine were observed over the course of the estrous cycle. Moreover, although the small intestine more than doubled in wet weight by late gestation, this increased quantity of gut tissue did not appear to compete for a finite number of donor MLN lymphoblasts. It was concluded that changes in the sex hormone status of mice may influence selective localization of MLN lymphoblasts in sex hormone target tissues.</p> <p>Since subpopulations of cells can often be separated from each other on the basis of cell size, some preliminary studies were done to purify, on the basis of size, subpopulations of mouse MLN lyphoblasts. These results indicated that the labelled cells being transferred were all large in size, likely lymphoblasts, and that it was possible to use this technique to obtain donor cell populations highly enriched in the proportion of labelled cells. Some evidence was obtained to suggest that the MLN contained a minor population of lymphoblasta with a propensity to localize in the spleen.</p> <p>To examine the possibility that some lymphocytes in the gut mucosa may themselves have a predisposition to localize in other mucosal tissues (assuming that they were able to migrate), lymphocytes were mechanically prepared from the lamina propria of the guinea pig small intestine. A subpopulation of these cells incorporated ¹²⁵I-deoxyuridine and localized (within 24 h after adoptive transfer) in the gut mucosa in a manner similar to transferred MLN lymphoblasts and different from either PLN or Peyer's patch lymphoblasts. It was concluded that a portion of the lymphocytes seen in the gut mucosa were similar in migration characteristics to cells found in the MLN.</p> <p>The results of these studies support the concept of a common mucosal immunologic system in which different mucosal surfaces are linked by migrating IgA plasmacyte precursors originating primarily in the lymphoid tissue associated with the intestinal and respiratory tracts. s of these studies support the concept of a which different mucosal surfaces migrating IgA plasmacyte precursors originating primarily in the lymphoid tissue associated with the intestinal and respiratory tracts.</p> / Doctor of Philosophy (PhD)

<p>For a wide variety of visual tasks, subjects perform more poorly when stimuli are oriented obliquely than when they are oriented vertically or horizontally. It is generally believed that this 'oblique effect' has two manifestations. One is meridional anisotrophy, i.e., lower acuity for obliquely oriented stimuli than for vertical or horizontal stimuli. The second is poor oblique discrimination; i.e., greater difficulty discriminating between mirror-image oblique stimuli than between vertical and horizontal ones, even when the stimuli are above the threshold of acuity. This thesis explores whether these two phenomena are in fact both manifestations of the same effect.</p> <p>Chapter One provides a detailed survey of studies showing meridional anisotrophy and considers the nature of its etiology and development. Chapter Two provides a review of the existing literature on the discrimination problem as evidenced in young children. The author argues that while meridional anisotrophy and poor oblique discrimination by young children appear to be related phenomena, it is possible that both are not manifestations of the same effect. The author tested this possibility by comparing the developmental course of poor oblique discrimination with the know developmental course of meridional anisotrophy.</p> <p>Chapters Three through Six present four experiments in which the author tested young infants' ability to discriminate between mirror-image oblique stripes with their ability to discriminate between "vertical-and horizontal ones, with the stripes above the threshold of acuity. In one experiment six week olds were tested; in the other three, 17-18 week old infants were tested using different variants of the habituation procedure.</p> <p>The major finding of the research is that neither 6 week nor 17-18 week old infants, both of whom are known to show meridional ranisotrophy, show evidence that a mirror-image oblique discrimination is more difficult than a discrimination between a vertical and horizontal. Furthermore, modification of the testing procedure, so that it more closely mimics a task in which children show difficulty discriminating mirror-image oblique lines, does not appear to affect 17-18 week olds discrimination performance.</p> <p>In the final chapter, the author discusses the implications of the research findings. The author concludes that since infants known to show meridional anisotrophy do not show poor oblique discrimination, then it is probable that these two phenomena are not both manifestations of the same effect nor do they appear to be generated by the same underlying mechanism. The author also discusses the implications of the research findings for two cognitive theories which have been advanced to explain how children process obliquely oriented stimuli.</p> / Doctor of Philosophy (PhD)

<p>The effects of strength training and immobilization upon neuromuscular function in man were investigated. The selected measures of neuromuscular function were voluntary strength and a number of electrophysiological measurements, including motor unit counts, motor nerve conduction velocity, reflex potentiation, and the contractile properties of isometric twitch contractions.</p> <p>Three kinds of experiments were conducted. First, subjects participated in training and immobilization experiments. Training, which consisted of maximal isometric and concentric muscle contractions and weight lifting, was performed three times per week over a period of 10-20 weeks. Relative disuse of selected muscle groups was achieved by immobilizing the elbow, wrist and thumb joints in a cast for 3-6 weeks. Second, measurements were made in selected groups of athletes (weight lifters, gymnasts, cyclists, sprinters, cross country skiers) to provide cases of long term training. Third, measurements were made on control subjects, whose results were compared to those of the athletes.</p> <p>Training resulted in an increase in voluntary strength. As cases of long term training, voluntary strength was enhanced in weight lifters and cyclists. Immobilization resulted in a decrease in voluntary strength.</p> <p>Training had no effect upon motor unit counts and the motor unit counts in the weight lifters were normal; however, the gymnasts exhibited reduced motor unit counts in distal but not proximal muscles. It was hypothesized that injury to the nerves at the wrist and ankles was responsible for the reduced motor unit counts in the gymnasts. Immobilization had no effect upon motor unit counts.</p> <p>Neither training nor immobilization caused a change in motor nerve conduction velocity in relation to the control condition; however, in one group of subjects, there was a small though significant difference between the greater post training and the lesser post immobilization values. Nerve conduction velocity was greater in weight lifters and gymnasts than in controls.</p> <p>Reflex potentiation increased following training, providing new evidence in support of the hypothesis that adaptation occurs within the nervous system in response to training. In agreement with the above finding was the enhanced reflex potentiation in the weight lifters. Immobilization caused a decrease in reflex potentiation, indicating that the nervous system is involved in the adaptation to relative disuse as well as to training.</p> <p>Muscle (triceps surac) twitch tension and contraction time were greater in weight lifters than in controls. In this same muscle, short term training resulted in an increase in twitch half relaxation-time. These findings represent the first report of a slowing of muscle contraction in response to training.</p> <p>In conclusion, the present investigation provided new evidence indicating adaptation within both the muscle and the nervous system in response to strength training and within the nervous system in response to immobilization.</p> / Doctor of Philosophy (PhD)

<p>The purpose of the studies reported in this thesis was to obtain data concerning the embryonic origin and formation of the pectoralis major muscle of the chicken. This muscle is used extensively in studies on muscle development because it is large; readily available, and is composed almost entirely of one muscle fiber type. Moreover, it is the largest muscle to be affected by hereditary muscular dystrophy in the line of chickens afflicted with this disease. Information concerning its embryonic origin could be used for in vivo studies on the early development of both normal and dystrophic muscles.</p> <p>Previous investigations into the embryonic origin od skeletal muscle in several classes of vertebrates have resulted in controversy. Some investigators have concluded that all skeletal muscles arise from the myotomal layer of the somites. Others have cited evidence to show that some muscles, including the pectoralis major muscle of the chick, are derived from the somatopleuric mesoderm adjacent to the somites.</p> <p>In the present investigation, interspecific chimaeras have been used to study the problem. Whole somites, somite halves, or limb-buds were grafted from quail to chick embryos between 2 and 3 days in ovo. After further development, the chimaeras were fixed, embedded in paraffin, sectioned, and stained using the Feulgen reaction for chromatin. This procedure permitted the identification of those structures that were derived from the grafted quail tissue.</p> <p>The observations in this study have led to the following conclusions:</p> <p>The pectoralis major muscle arises from the dorsal halves of somites 16-21 of the 2-day in ovo chick embryo. These somites also give rise to all other wing and wing-associated muscles of the shoulder and thorax. Each somite plays a specific role in the development of these muscles. The cells that ultimately form the pectoralis and other brachial muscles migrate from the somites into the lateral mesoderm between 2 and 2.5 days in ovo. The myotomal layers of the somites do not appear until 2.5 days in ovo and do not contribute to the formation of the brachial muscles.</p> / Doctor of Philosophy (PhD)

<p>The studies presented in this thesis are the first attempts to compare in a comprehensive manner the basic electrical and mechanical properties of the two muscle layers of the small intestine of the rabbit, a species that showed electrical control activity (ECA). The activities of the two muscle layers were distinctly different. Cells in the longitudinal muscle layer (LM) were spontaneously active with action potentials occurring on every control potential (CP). Similarly, muscle strips dissected along the long axis of LM (LS) contracted spontaneously at the same frequencies as the ECA. Cells of the circular muscle layer (CM) usually did not exhibit spontaneous spiking activity although ECA was also present.</p> <p>The characteristics of the ECA of the two muscle layers from the same muscle strips were similar in terms of amplitude, frequency, and their response to temperature change and external electrical stimuli. How the ECAs of the two muscle layers interact was investigated in light of the hypothesis that LM is the site of origin of ECA and that the ECA in CM is the result of electrotonic spread from LM (Bortoff, 1961, 1976; Connor, Kreulen, Prosser & Weigel, 1977). This hypothesis was tested directly in this study by measuring electrotonic coupling between the two muscle layers. It was found the there was little electrotonic interaction between muscle layers. Therefore, the result of this study is not consistent with the existing model in regard to the origin of the ECA.</p> <p>Study of the control of muscle function by the intrinsic nerves also showed drastic differences between the two muscle layers. LM was innervate by cholinergic excitatory nerves and possibly by an inhibitory neural system. In CM, three types of neural excitatory events were identified in addition to the powerful non-adrenergic inhibitory nerves. Besides the familiar cholinergic excitatory nerves, a tetrodotoxin-resistant component and an excitatory response that emerged only after prolonged repetitive stimulation was also observed. The neurotransmitters for these two excitatory neural systems remain to be identified.</p> <p>The results of this study indicate that the properties of the two muscle layers of the small intestine are very different. Nonetheless, normal physiological function of the intestine requires good coordination of the two muscle layers. The exact role of the individual layers in motility is not well defined. How these two muscle layers each with its separate neural, hormonal and local control mechanisms interact to produce the final intestinal motility pattern will be a challenging problem in the future.</p> / Doctor of Philosophy (PhD)

<p>Human muscles are composed of different percentages of two major fibre types which have specific metabolic and physiological properties. Previous studies the metabolic differentiation of these fibres during foetal development did not agree as to when differentiation occurred. They did however agree that differentiation was completed at full term based on criteria which had not been validated.</p> <p>Developmental studies in a number of different mammalian species have shown that both metabolic and physiological properties change postnatally. Preliminary studies by this author suggested that postnatal differentiation may also occur in human muscles.</p> <p>A detailed study of how and when fibre type differentiation occurs during development should contribute to an understanding of the mechanisms controlling differentiation. In addition, the results of normal development are essential for determining the involvement of neuromuscular disorders in foetal and postnatal muscle.</p> <p>The objective of this thesis was to study the metabolic differentiation of fibres during foetal and postnatal development.</p> <p>Five limb muscles, the soleus, triceps, biceps, vastus lateralis, gastrocnemius, and in some cases the diaphragm were excised at autopsy from 21 foetuses ranging in age from 25-43 weeks gestation. The same limb muscles were examined in five infants between one and 20 months, three children between 6-8 years and four adults aged 18-28 years. Multiple sites were taken from each muscle, mounted cross-sectionally and frozen rapidly in isopentane at -160°C. Serial sections were cut at 10 μm and the following reactions run, m-ATPase pH 10.3, 10.0, 4.58, 4.28, NADH-TR, SDH, α-GPD, Rna-Dna, gomori trichrome and H&E. One longitudinal sample was taken from the motor end plate zone of each foetal muscle for examination of motor nerves and end plates by an acetylcholinesterase reaction. Fibre type distributions were determined in three areas per site, from 35 mm. colour slides of the m-ATPase pH 10.3 reaction.</p> <p>In order to determine the number of sites it was necessary to sample per muscle, the between site variability of fibre type distributions was studied in four different muscles from four adults. Fifteen sites were sampled and a mean of 15,000 fibres counted per muscle. Distributions between and within sites showed greater variation (p<0.01) than could be expected from a homogeneously distributed muscle. Between site standard deviations ranged from 6.63% to 8.12% type II fibres in the different muscles. At least two and up to five sites need to be sampled to reliably estimate the fibre type distribution of adult muscle.</p> <p>The results of the developmental study suggest that the process of metabolic differentiation can be described in four stages. During stage 1, i.e. 6-20 weeks, all muscle fibres were shown by previous studies to have the same histochemical properties. During stage 2, i.e. 20-30 weeks, the m-ATPase pH 10.3, 4.3 and NADH-TR reactions could classify all fibres as either type I or type II. Most muscles had approximately 7% type I distributions and weak α-GPD and SDH reactions between 25-30 weeks. During stage 3, i.e. 30-43 weeks, a type IIC fibre could also be distinguished. At full term, type I fibres had increased to approximately 35%, type II fibres decreased to 51% from 93%, and 15% were type IIC. The activities of the metabolic enzymes had also greatly increased. On this evidence the increase in type I fibres is thought to be due to a conversion of type II to type I fibres, with type IIC fibres being in a transitional stage of conversion.</p> <p>Based on postnatal increases in the percentage of type I fibres in different muscles and decreases in type IIC fibres; differentiation was not completed at full term. The soleus had the greatest postnatal increase in type I fibres from 30% at term up to 70-80% in the children and adults. The results suggested that differentiation may be completed by the end of the first year of life.</p> <p>This study has led to a better understanding of the normal process of differentiation, the results of which are helpful in the clinical investigation of neonates and infants thought to have neuromuscular disorders.</p> / Doctor of Philosophy (PhD)

<p>Pichinde virus, a member of the arenaviridiae, causes a fatal disease when injected intraperitoneally into the inbred MHA strain of hamsters, but not in other strains. The fatal infection is associated with an inability to limit virus replication, and death appears to be a consequence of the virus-induced cytopathic effect within the reticuloen-dothelial system. The purpose of the current studies was to determine whether susceptibility to the lethal Pichinde virus infection was genetically acquired, and to obtain an understanding of the basis for this susceptibility.</p> <p>Studies on survival and the ability to limit viremia following Pichinde virus infection in F₁ and back-cross progeny gave the results expected if a single autosomal dominant gene or linked genes were responsible for each of these phenotypes.</p> <p>Initial Studies on the basis for the susceptibility of MHA hamsters to fatal Pichinde virus infections were designed to test the hypothesis that this strain was unable to mount a cell-mediated immune response against the virus. However, MHA hamsters were able to limit Pichinde virus replication and they survived the infection when the virus was inoculated by the footpad route. Furthermore, footpad-immunized MHA hamsters survived a normally lethal intraperitoneal challenge of Pichinde virus. These observations suggested that susceptible MHA hamsters were able to produce a protective immune response when the virus was given by this route.</p> <p>Since cells of the reticuloendothelial system appeared to be a major target for Pichinde virus replications in vivo, a search for a target cell difference within the spleens of susceptible and resistant hamsters were undertaken. No difference in the ability of various spleen cell fractions from susceptible or resistant hamsters to support Pichinde virus growth in vitro could be demonstrated. However, the spleens of MHA hamsters which had been infected with Pichinde virus in vivo contained 10-fold more virus-producing cells at three days after infection than did spleens of resistant hamsters. The majority of the virus-producing cells in MHA hamster spleens were associated with the non-adherent fraction which sedimented at a rate typical of lymphocytes. In contrast, spleen cells from the resistant LSH strain of hamsters appeared to be deficient in this population. These observations support the hypothesis that the susceptible strain of hamsters had a spleen target cells for Pichinde virus replication which the resistant strain lacked.</p> <p>Interestingly, the putative target cell was observed to co-purify with a cell population which mediated in vitro cytotoxicity against syngenetic or allogeneic tumour target cells. The cytotoxic effector cell was shown to be a non-adherent, non-phagocytic, small- to medium-sized cell which lacked detectable surface immunoglobulin. The cytotoxic activity was labile at 37°C, but was not abrogated by pretreatment with amonium chloride. Thus, this hamster effector cells resembled the natural killer (NK) lymphocyte which has been described in several species. Susceptible MHA hamsters exhibited high levels of endogenous NK activity, and this cytotoxicity was further augmented by Pichinde virus infection. In contrast, the resistant LSH strain showed lower levels of endogenous cytotoxicity, and Pichinde virus infection did not induce the same magnitude of increase in activity. These results support the hypothesis that susceptible MHA hamsters have an additional splenic target cell for Pichinde virus replication which the resistant strain lacks, and are consistant with the possibility that his target cells is in fact the NK cell.</p> / Doctor of Philosophy (PhD)

<p>Phenylketonuria (PKU) is an inherited disorder of amino acid metabolism in which the conversion of L-phenylalanine (Phe) to L-tyrosine (Tyr) is greatly diminished. This metabolic block results in high blood Phe levels with low to normal Tyr levels and is usually accompanied by mental retardation unless the disease is treated by dietary restriction of Phe early in life. In recent years it has become apparent that many of the non-PKU offspring of PKU women are damaged in utero by their mother's disease. Approximately 90% of the non-PKU children of PKU mothers are mentally retarded, over 50% are microcephalic and suffer intrauterine growth retardation, while nearly 40% have some congenital malformation. The risk to the offspring of women with hyperphenylalaninemia (hyperphe) probably decreases as the maternal Phe level decreases, althought the "safe" level of maternal hyperphe is not yet well defined. It does appear however, that the developing offspring of women with Phe levels of at least 15 mg/100 ml face substantial risk of in utero damage. Attempts to prevent offspring damage by treatment of pregnant hyperphe women with dietary restriction of Phe have had only limited success. There are still many unanswered questions regarding the management of maternal hyperphe and these will become even more pressing as increasing numbers of PKU women who were success fully treated as children reach reproductive age. Since clinical data are limited attempts have been made to establish experimental animal models of maternal hyperphe.</p> <p>Early animal studies produced maternal hyperphe by administering large doses of Phe to pregnant animals. Although some of these studies suggested associations between maternal hyperphe and behavioral and biochemical abnormalities found in the offspring, most were confounded by maternal hypertyrosinemia, an abnormality never found in PKU. More recent approaches have utilized the Phe hydroxylase inhibitor p-chlorophenylalanine (pCPA) in order to prevent Tyr elevation following Phe administration. Experiments using both Phe and pCPA administration have more closely mimicked the biochemical characteristics of PKU, however methodological problems have limited the interpretation of many of these studies. In addition, the work with combined Phe and pCPA treatment has been restricted to the rat, a species in which significant differences from human prenatal development exist. The prenatal development of the guinea pig is more similar to man, especially with respect to the brain, and a study was therefore undertaken to determine the suitability of the guinea pig as a possible model of maternal hyperphe. Initially guinea pigs were injected with various regimens of Phe and pCPA. Blood Phe was transiently elevated to levels comparable to those found in PKU patients, however even following very high doses of both Phe and pCPA, Phe fell to near-normal levels within 10 hours of the Phe injection. The blood Phe response of the animals injected with pCPA suggested that the Phehydroxylase inhibition induced by pCPA in guinea pigs is of a much shorter duration than has been reported in rats. Since Phe does not fall to normal levels at any time in untreated PKU, other methods of administering pCPA and Phe were evaluated. Studies were undertaken in which both Phe and pCPA were incorporated into test diets and then fed to guinea pigs. This approach resulted in hyperphe comparable to that associated with significant risk to the human fetus which could be maintained for many weeks in both pregnant and nonpregnant animals. The optimum dietary pCPA supplement was determined by maximizing hepatic Phe hydroxylase inhibition and plasma Phe concentration. The amount of pCPA required to maximally decrease Phe hydroxylase activity in guinea pigs is considerably more than is needed in the rat. Indirect evidence was obtained which suggests that this species difference may be due, at least in part, to rapid excretion of pCPA as p-chlorophenylpyruvic acid by the guinea pig. The appropriate dietary Phe supplement was determined by monitoring plasma Phe and Tyr levels as well as food intake and weight gain in animals fed test diets supplemented with pCPA and various amounts of Phe. Additional work demonstrated that plasma Phe levels remained elevated for at least a 12 hour period during a single day and that ascorbic acid is needed for guinea pigs to efficiently metabolize Tyr.</p> <p>When stable maternal hyperphe was induced by feeding pregnant guinea pigs appropriate test diets, abortion occurred and was found to be related to pCPA, even in the absence of substantial hyperphe. Further study of the effects of pCPA and hyperphe during early pregnancy was undertaken by feeding guinea pigs test diets from day 1 of pregnancy and collecting embryos on gestation day 17. Only7. Only pCPA was associated with embryonic death, however malformed embryos were significantly associated with maternal hyperphe, even in the absence of pCPA administration. The relationship between maternal hyperphe and malformed embryos had not been previously demonstrated in animals and it may have relevance to the high frequency of congenital defects found in offspring of PKU women. Evidence of embryonic developmental retardation was also found and hyperphe may be causally related to this abnormality as well. Both Phe and Tyr were found to be actively transported to the early embryo and this transport of Phe might be related to its teratogenicity. The embryo toxicity of pCPA limits the utility of the current approach. Use of newer Phe hydroxylase inhibitors in pregnant guinea pigs may prove informative.</p> / Doctor of Philosophy (PhD)

<p>Little is known about the potential toxicity of neuroleptics administered during pregnancy, although they are known to cross the placenta and reach the fetal brain. The neuroleptic, haloperidol, is often the drug of choice for treatment of psychotic and affective disorders and sometimes as an adjunct during labour. Hence it is frequently administered to women of childbearing age and during pregnancy. Haloperidol is a potent antagonist of dopamine (DA) receptors in the brain. The question raised is "Does the administration of haloperidol during the period of DA neuron synaptogenesis (third trimester in the rat) interfere with the normal development of DA neurons and post-synaptic receptors? If so does this alter subsequent brain DA function as reflected in behaviour and hormonal regulation in the offspring?" Haloperidol or vehicle (control) were administered to timed-pregnant rats on gestation days 15 to 21. At birth, all offspring were fostered by untreated lactating dams. The effects of this treatment on three DA systems, (nigrostriatal, mesolimbic and tuberoinfundibular systems) in the brains of offspring were assessed on postnatal day 25 using morphological, pharmacological, biochemical and behavioural analyses. The results indicated that haloperidol treatment during the third trimester produced a deficit in the growth of nigrostriatal DA neurons and decreased postsynaptic DA receptors without affecting the postsynaptic enzyme, adenylate cyclase. Offspring of haloperidol rats showed locomotor hyperactivity even after reaching adulthood. They also showed an abnormal increase in prolactin secretion when challenged with haloperidol on postnatal day 25. There was a significant sex difference in behavioural and hormonal responses to haloperidol, with female offspring showing greater sensitivity. These data demonstrate that treatment with haloperidol for even a short period during pregnancy can produce long term, perhaps even permanent alterations in morphological, pharmacological, behavioural and hormonal development of brain DA neurons, in the absence of overt teratogenic effects. Subtle defects in offspring brain development may pre-dispose them to later difficulties associated with abnormal DA function, especially in situations of stress. These findings emphasize the need for further investigation of the potential toxicity of neuroleptics, especially during prenatal development.</p> / Doctor of Philosophy (PhD)