<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)

<p>Platelet aggregation plays an important role in both the formation of a hemostatic plug and in the generation of thrombus. The mechanism of platelet aggregation has not been fully elucidated. It is conceivable that changes in the synthesis, breakdown or rate of turnover of some of the constituents of the platelet membrane may be important in the mechanisms of aggregation. Recent experimental evidence in other mammalian tissues has implicated the closed circle conversion between MPI and PA as part of the membrane receptor-effector interaction process, and a possible role permeability toward of TPI-DPI interconversion in the regulation of membrane permeability toward Na⁺ and K⁺. Platelets have been recognized to be a useful model for these studies because of its abundance in phosphoinositides and easiness of obtaining pure platelet suspensions. Previous studies showed that addition of ADP to ³²P-labeled washed platelets caused an increase in radioactivity in PA in 2 sec, DPI in 30 sec and MPI in 2-3 min. TPI-DPI interconversion was suspected but consistent changes in ³²P-radioactivity in TPI was not detected. Similar changes were observed in platelets in response to thrombin stimulation. In none of the studies in other tissues all the metabolic pathways for inositol lipids were studied simultaneously. The aims of the present experiments were to investigate TPI-DPI interconversion and MPI metabolism during platelet aggregation and the release reaction caused by ADP, ionophore A23,187 and thrombin. Since these stimuli are considered to act on platelets by different mechanisms, their effects on the platelet phosphoinositide metabolism were compared. Furthermore, experiments were carried out with unstimulated platelets labeled with ³²PO₄ to find out if there vere differences in the patterns of ³²PO₄ incorporation into the major platelet phospholipids (PC, PE, PS) under in vitro and in vivo conditions, as claimed by former investigators.</p> <p>Using improved methods for separation of phosphoinositides, it was found that the pattern of ³²P-incorporation into the major phospholipids in platelet suspension was similar to that of in vivo platelets when the incubation was carried out for many hours. Therefore, the phosphate moiety in all phospholipids turn over; but the phosphate in the major phospholipids turn over much more slowly than that in the phosphoinositides.</p> <p>During ADP-induced platelet aggregation, hydrolysis of TPI to DPI was measurable at 60 sec, with a loss of ³²PO₄, ¹⁴C-arachidonic acid and ³H-inositol from TPI. This hydrolysis was abolished by AMP, an inhibitor of ADP-induced aggregation. Resynthesis of TPI occurred during platelet deaggregation, with the ³²PO₄ radioactivity of this compound being restored to the control level. With A23,187-induced aggregation and release, hydrolysis of TPI did not occur. While there was a significant amount of MPI converted into PA, the majority of MPI appeared to be hydrolysed to fatty acid and lyso MPI. Using platelets labelled with ¹⁴C-arachidonic acid, ³H-glycerol, ³H-inositol, ³²PO₄ and phosphorus assay, it was found that thrombin caused a decrease in TPI (-5.64 ± 1.55%, P < 0.005) as early as 9 sec when platelet shape change was maximal, ³²P-content was also decreased. Resynthesis of TPI was measurable at 60 sec. Most of the MPI was metabolised via the 1,2-diacylglycerol pathway. The the amount of PA was increased. A small amount of the MPI was converted to polyphosphoinositides or lyso MPI and free fatty acid. These experiments have shown a close relationship between changes in phosphoinositide metabolism and platelet aggregation and the release reaction.</p> <p>These principal changes are: (1) increased TPI- DPI interconversion; (2) increased conversion of MPI to l,2-diacylglycerol PA and MPI; (3) with the release reaction conversion of MPI to lyso MH and free fatty acids.</p> / Doctor of Philosophy (PhD)

<p>The goal was to determine the physiological significance of pyruvate dehydrogenase (PDH) activation in muscle. The PDH activity was measured in small samples of muscle obtained by needle biopsy from normal volunteers. In heart PDH was measured using an isolated perfused rat heart preparation. The PDH activity was measured by determining ¹⁴Co₂ production from pyruvate-1-¹⁴C using whole tissue homogenates.</p> <p>The total PDH (PDHt) activity in human skeletal muscle was 302 ± 10 nmol/g/min. with about 40 ± 3% in the active form (PDHa). With aerobic exercise (60% VO₂max) more than 88% of PDH was converted to PDHa within 5 min. Aerobic training did not increase the resting muscle PDHt activity (untrained 304 ± 4, trained 309 ± 8 nmol/g/min,) but it increased the PDHa from 124 ± 9 nmol/g/min. to 215 ± 15 nmol/g/min.</p> <p>Muscle immobilization by arm cast for 6 weeks decreased the PDHa from 112 ± 4 to 28 ± 4 nmol/g/min. but it had no effect on the PDHt activity. Exercise following immobilization decreased the rate and the extent of PDH activation while exercise after weight training increased the extent of PDH activation.</p> <p>Starvation for 24 and 48 hr. in rat heart decreased PDHa from 2768 ± 73 to 1286 ± 109 and 920 ± 149 nmol/g/min. respectively.</p> <p>In the perfused heart PDH was fully activated by epinephrine (1.3 μg/ml), insulin (1 mU/ml) and high (>0.2 mM) pyruvate concentration. PDH activation was not mediated by cyclic AMP since insulin activated PDH but did not change the heart cyclic AMP concentration. Propranolol prevented epinephrine from activating PDH. Octanoic acid decreased the proportion of PDH in the active form.</p> <p>In the heart a detailed study of the relationship between the rate of heart pyruvate oxidation and PDH activation was undertaken. In all experimental conditions where the rate of pyruvate oxidation was changed, parallel changes in the PDHa activity was seen. The correlation between PDH activation and rate of heart pyruvate oxidation was greater than 0.92. These findings suggest that interconversion of PDHa and PDHɨ probably control the rate of heart pyruvate oxidation. A similar correlation was not seen in skeletal muscle during exercise. The calculated rate of muscle pyruvate oxidation was several fold greater than the extent of PDH activation. Although the activation of PDH regulates pyruvate oxidation in both heart and skeletal muscle, in heart this is achieved through the interconversion of active and inactive PDH whereas in skeletal muscle allosteric conversion of the active form of PDH plays a greater part.</p> / Doctor of Philosophy (PhD)

<p>Proteolysis of short-lived cellular proteins was investigated in early- and late-passage cells from normal donors and in cells from donors with the premature aging diseases of progeria and the Werner syndrome. Studies were conducted using intact cells or crude cell extracts to measure the degradation of cellular proteins pre-labeled for 1 hour with ³H- and ¹⁴C-Iabeled phenylalanine.</p> <p>The purpose of these studies was:</p> <p>1. To determine whether aging cells (late-passage normal, progeria and Werner) show increased proteolysis compared to early-passage normals as would be predicted from the Orgel hypothesis.</p> <p>2. To investigate the effect of growth state and inhibitors on proteolysis in these cells.</p> <p>3. To identify specific cell fractions or proteins which undergo increased proteolysis in aging cells.</p> <p>4. To measure proteolysis of cellular proteins in extracts of cells, using both endogenous cellular proteases and added exogenous proteases. This was to probe the role of cellular proteins as substrates for prot eases in elevated proteolysis in aging cells.</p> <p>Proteolysis was increased in late-passage normal, progeria and Werner cells compared to early-passage normal cells. This was found in growth and at confluence. The chymotrypsin inhibitor TPCK reduced the elevated proteolysis in freshly plated late-passage and progeria cells but did not have this effect on cultures growing logarithmically or those at confluence. Increased proteolysis of soluble (post-microsomal) proteins may be occurring in progeria cells but SDS-polyacrylamide gels failed to show increased turnover of any protein bands.</p> <p>Protease activity in cell extracts could only be demonstrated at acid pH with endogenous cellular proteases. This proteolysis did not preferentially degrade proteins which have short half-lives in vivo including those containing the amino acid analogue canavanine. Proteins from early- and late-passage normal cells showed no difference in acid optimum proteolysis. However, proteins from progeria cells showed less proteolysis than normal controls. This was observed for labeled progeria proteins with proteases from extracts of normal or progeria cells. Paradoxically, proteins from progeria cells were more susceptible to labeled in the trypsin than normal controls but when proteins were labeled in the presence of TPCK no difference in trypsin susceptibility was observed.</p> <p>The finding indicate:</p> <p>1. Increased proteolysis is a feature of cellular aging.</p> <p>2. Proteolysis in aging cells during growth shows greater elevation compared to early-passage normals, than that seen at confluence.</p> <p>3. In progeria, cellular proteins have altered susceptibility to both endogenous and exogenous proteases.</p> <p>4. The inhibitor TPCK can, under certain conditions, reduce increased proteolysis in late-passage normal and progeria cells.</p> <p>Therefore, it is concluded that increased proteolysis occurs in aging cells and that increased activity of TPCK-sensitive proteases may in some circumstances be responsible. Thus, altered control of the proteolytic apparatus and not abnormal proteins may be causing increased proteolysis in aging cells.</p> / Doctor of Philosophy (PhD)

<p>Nature mammalian myofibres are multinucleated elongate cells in which the nuclei are regularly distributed along the entire length of the cell. The multinucleated condition arises by the fusion of mononucleated myoblasts to form myotubes during primary development. A fundamental and essentially unexplored problem in muscle cell biology is the question of the nuclear-cytoplasmic relationship in muscle fibres. Although local membrane specializations occur, most notably in the region of innervation, it is not known if each myonucleus controls the biochemical functions of a local territory comprised of the immediately adjacent muscle cytoplasm, organelles, and membrane, or if the gene products of every myonucleus are uniformly distributed in the fibre. An understanding of this aspect of the organization of muscle cells could help solve other problems in the biology of skeletal muscle; for example, what is the significance of X-chromosome mosaicism in the expression of muscle disease problems in the biology of skeletal muscle; for example, what is the significance of X-chromosome mosaicism in the expression of muscle disease in carriers of X-linked myopathies? Which myonuclei are the targets of neurotrophic influences on muscle?</p> <p>In this study I have taken advantage of the fact that in mouse chimaeras two genotypically distinct types of myonuclei are often incorporated into single myofibres skeletal muscle cells in vivo. Mouse chimaeras were produced by the aggregation of embryos homozygous for different alleles for both the monomers of the cytoplasmic enzyme glucosephosphate isomerase (GPI-1) and the monomers of the nuclear-coded mitochondrial matrix protein malic enzyme (MOD-2). Therefore, mosaic myofibres contained both Gpi-1ª/Gpi-1ª, Mod-2ª/Mod-2ª myonuclei and Gpi-1ᵇ/Gpi-1ᵇ, Mod-2ᵇ/Mod-2ᵇ myonuclei.</p> <p>The distribution of the electrophoretic variants of GPI-1 was measured at various points along the length of mosaic muscle fibres. Changes in the isozyme composition along the length of a cell would indicate that the enzyme remains localized in cytoplasmic territories. A uniform isozyme profile would result if the products of individual myonuclei are homogeneously distributed. The results presented here indicate that the intracellular distribution of GPI-1 isozymes does not change along the length of mosaic myofibres in both adult and young mouse chimaeras. Therefore, the products of each myonucleus are widely distributed in skeletal myofibres.</p> <p>Functional GPI-I is an oligomeric enzyme composed of two monomers. Analyses of the proportions of GPI-I dimer types in mosaic myofibres reveal that the homogenous distributions of GPI-I isozymes arise by the widespread distribution of precursors of GPI-I (monomers or monomer precursors such as messenger RNA) before dimer formation; dimers are not initially assembled in local territories around each myonucleus and then widely distributed.</p> <p>The investigation was extended to the question of whether individual mitochondrion in skeletal myofibres have access to nuclear-toded proteins encoded by one or by multiple myonuclei. Evidence was obtained that indicates that both types of monomers of nuclear-coded mitochondrial malic enzyme (MOD-2) are present in an individual mitochondrion of a mosaic myofibre.</p> <p>The results indicate that the gene predicts of different myonuclei are widely distributed both before the assembly of cytoplasmic oligomeric proteins and before the incorporation of proteins into mitochondria.</p> <p>Taken together, the results obtained in this study constitute direct evidence that mammalian skeletal myofibres are functional syneitia in which the gene products of many myonuclei are distributed throughout each cell. Problems and possibilities regarding the mechanisms of distribution and the nature of the gene products distributed are discussed.</p> / Doctor of Philosophy (PhD)

<p>A broad overview of the irritable bowel syndrome (IBS), with an emphasis on theoretical problems, is followed by a comparative psychophysiological study of normal subjects, psychoneurotic patients and patients with IBS. All subjects had a psychiatric interview and psychometric testing followed by measurement of activity in the rectosigmoid colon during baseline, neutral interview, stressful interview and following a meal or neostigmine (0.5 mg I.M.). Myoelectrical activity was recorded from two intraluminal bipolar suction electrodes (4 cm apart); motor activity was recorded from two intraluminal strain gauges at the same sites. The number and duration of contractions per minute were determined by inspection of the motor activity record; the frequency content of both the motor and myoelectrical records was determined using a Fast Fourier Transform (FFT) computer assisted method. Measurements were also made of severity of pain following varying degrees of distension of the rectosigmoid colon, serum gastrin and motilin levels and heart rate responses. The two patient groups were psychologically similar to each other and more disturbed than normals both at psychiatric interview and on psychometric measures of anxiety, depression and neuroticism. The IBS patients were significantly different from the neurotics only on the lie scale of Eysenck Personality Inventory (p < .05). The ratings of pain to distension of the sigmoid colon were not significantly different in the three groups (p >.05). The ISS group did not differ significantly from the neurotic group on any of the physiological measures (p >.05); during baseline the ISS group had a greater number and duration of contractions than the normals at one recording site (p <.05). We conclude that previous reports of physiological characteristics unique to ISS have resulted from a failure to control for relevant psychological characteristics and from recording and analysis deficiences. A behavioral model, capable of accounting for both the experimental findings and observed clinical features, is described.</p> / Doctor of Philosophy (PhD)

<p>The differentiation of any cell leads to the synthesis of certain proteins which are responsible for its specific function. In the nervous system this includes such functions as the conductance of action potentials, synaptic transmission and the establishment of specific connections. The developmental biology of proteins unique to the nervous system is therefore of great importance when studying the relationship of proteins to physiological function.</p> <p>Although some neural specific components of adult chicken brain and their ontogeny in the chicken embryo have been previously described by others, they have not been extensively characterized. The present investigation was undertaken in order to enumerate, characterize and study the embryological appearance of antigens specific to the adult chicken brain that can be demonstrated by rabbit anti-brain sera. Particular interest was given to the isolation and purification of one such neural specific antigen in hopes of further elucidating its role in adult function and embryonic development.</p> <p>Antiserum produced in response to a saline soluble extract of adult chicken brain (ABE), when absorbed with adult liver, serum and kidney extracts, yielded a neural specific antiserum (AABS). This polyvalent antiserum was capable of demonstrating at least eight adult neural specific antigens within ABE during immunoelectrophoret analysis. The sequential appearance of seven of these antigens during embryonic development (D2 to D11 of incubation) was established. An eighth antigen could not be detected within the embryonic brain extracts and was assumed to be associated with posthatching neural development. By immunochemical criteria, the majority of the neural specific antigens, but not all, were demonstrated to be restricted to avian brain. Similarly the majority of these antigens, but not all, were demonstrated to be protein in nature since reactions with trypsin and chymotrypsin resulted in loss of antigenicity.</p> <p>Separation of the antigens within ABE, based on their net ionic charge and molecular weight, was attempted with each resulting fraction being analyzed by immunoelectrophoresis. One neural antigen (CNA-1), possessing alpha-1 globulin mobility was isolated from the other neural specific antigens. This thesis further describes a procedure developed for the purification of CNA-1, utilizing ammonium sulphate fractionation, ion exchange chromatography, gel filtration chromatography and preparative polyacrylamide gel electrophoresis. Highly sensitive and quantitative immunochemical techniques (crossed and fused rocket immunoelectrophoresis) were employed in monitoring the purification steps.</p> <p>Studies on the purified antigen revealed it to be homogeneous by a number of criteria, having a native molecular weight of 65,000 daltons as determined by molecular exclusion chromatography and possessing two identical subunits (MW 30,000) as determined by SDS polyacrylamide gel electrophoresis. The structure of CNA-1 was concluded to be protein on the basis of its sensitivity to trypsin and chymotrypsin.</p> <p>Immunochemical studies utilizing a monovalent antiserum to CNA-1 revealed the protein to be avian specific and not present in brain extracts of mammalian species. Also both a high (> 1,500,000 daltons) and low (65,000 daltons) molecular weight component which shared the CNA-1 antigenic determinant were observed, indicating possible aggregation of the protein during the isolation procedure.</p> <p>CNA-1 was first detectable by the seventh day of incubation and continued to accumulate during the period of embryonic neural development in which both neurogenesis and synaptogenesis are known to occur. Within the adult chicken cerebellum, CNA-1 was specifically localized to the cell surfaces of particular neuronal cell types (i.e. Purkinje cells, granule cells and neurons of the deep cerebellar nuclei) by immunohistochemical techniques. Glial elements were not labelled.</p> <p>Organ specific brain antigens have been described by different investigators. The antigen described within this thesis appears to be completely different from all others described in the literature as revealed by a study of its physical and chemical properties. The function and role of CNA-1 on the cell surface remains to be elucidated. Its localization on specific cell types suggests that it may be associated with inhibitory synaptic function. For this reason, further study of CNA-1 may prove to be important in unravelling some of the questions concerning cell-cell interaction and neuronal specificity during development of the central nervous system.</p> / Doctor of Philosophy (PhD)

<p>There are some data human epidemiological studies which suggest that radiation has a small but significant effect in causing aneuploid gametes. Alberman et al. (1972) suggested that much of this radiation occurred more than ten years before the conception of the abnormal child. Mice have been used to study experimentally radiation effects on chromosome segregation. Radiation induces nondisjunction in several strains, including (C₃HxICR/Swiss)F₁ females. The present study was designed to investigate the possibility of repair of the mechanisms which results in nondisjunction following irradiation. Female (C₃HxICR/Swiss)F₁ mice were randomized into 4 experimental groups at 6-8 weeks of age. Some were irradiated at 3 months with 20R gamma rays from a ¹³⁷Cs source. Young mice were sacrificed at 3 months of age, within 24-48 hours of irradiation. Others were irradiated then housed until 9 months old. At the time of sacrifice ovaries were removed and the oocytes obtained were cultured to the metaphase II stage. Chromosomes were then analyzed. Oocytes from 974 mice were studied. The frequency of spontaneous nondisjunction in oocytes from young mice was quite low (0.2%) and this increased significantly after irradiation to 1.6% (p=0.001). The spontaneous frequency of nondisjunction in oocytes increased with age from 0.2% to 1.2%. In contrast the nondisjunction frequency in oocytes from irradiated-aged animals was 0.0%. In other words, following irradiation at a young age, no aneuploid oocytes were recovered. This is significant different from the frequency in control aged animals, p=0.002. The mechanism by which radiation causes nondisjunction is not known. Possibilities include damage to the centromere region or radiaton could act by causing premature or accelerated terminalization of chiasma. The disappearance of aneuploid oocytes in irradiated-aged animals in this study suggests that in addition to causing nondisjunction immediately there is a delayed effect somewhere between 48 hours and 6 months which results in an elimination of abnormal cells. This experimental finding in mice is quite different from epidemiological information from humans. It provides an avenue for further research into agents causing chromosome abnormalities and the factors involved in germ cell selection and survival.</p> / Doctor of Philosophy (PhD)