<p>The aim of the present study was to characterize the pituitary GnRH binding site in the rabbit and investigate its possible role in sexual maturation of the female rabbit. A radioligand binding assay was established and the presence of specific ¹²⁵I-DAla⁶EA binding sites in the anterior pituitary gland of the rabbit was demonstrated. ¹²⁵I-DA1a⁶EA binding was saturable, specific, displaceable, reversible, correlated with increasing tissue concentrations and susceptible to physiological manipulation. Significant ¹²⁵I-DA1a⁶EA binding was not present in the rabbit ovary suggesting that GnRH or GnRH-related peptides are not directly involved in the control of luteal function of the rabbit. 12S1_DAla6EA binding indicated the presence of two binding sites in the female adult rabbit pituitary; a high affinity, low capacity site (Kd= 0.3-0.4 nM; Bmax = 100-200 fmol/mg protein) and a lower affinity, high capacity site (Kd= 30 nM; Bmax=5-8000 fmol/mg protein). Ontogeny of ¹²⁵I-DA1a⁶EA binding in the female rabbit (40 to 120 days of age) did not show a correlation between binding site number and serum LH. In addition, the net serum LH response in female rabbits to a subcutaneous injection of DA1a⁶EA (10ng, 100ng, 1μg per kg body weight) was not significantly different between animals 40, 75 and 120 days of age. This suggests that a decrease in pituitary responsiveness to GnRH is not associated with sexual maturation in the female rabbit. Results indicate that factors other than and/or in addition to GnRH binding site number, such as post-receptor events play a role in gonadotropin secretion in the female rabbit.</p> / Doctor of Philosophy (PhD)

<p>Roberts syndrome (RS) is a rare autosomal recessive developmental disorder that is characterized by severe pre- and post-natal growth retardation, tetraphocomelia and various craniofacial abnormalities. In some patients with Roberts syndrome (RS+) but not others (RS-), RS is associated with a cytogenetic abnormality which presents as an unusual "puffing" of the constitutive heterochromatin and nucleolar organizing regions. The decondensed appearance of the constitutive heterochromatin suggests an alteration in chromatin structure in RS+. Two main questions were addressed in this study; (1) are altered levels of DNA methylation associated with the constitutive heterochromatin abnormality of RS+ chromosomes and (2) is the timing of DNA replication of constitutive heterochromatin, relative to the rest of the genome, altered in RS+ lymphoblast cells? Levels of methylated cytosine residues of RS+ and RS- heterochromatin DNA, assessed using MspI and HpaII isoschizomer restriction enzyme digestion and Southern blot hybridization with a repetitive probe (D15Z1) to the heterochromatin DNA, were found to be significantly lower (p = 0.048) in RS+ fibroblast cell strains compared to control and RS- cell strains. In addition to this, D15Z1 DNA methylation levels differed significantly (p ≤ 0.05) at early and late passage in fibroblast cell strains of all three groups, suggesting that decreased levels of DNA methylation were associated with an in vitro aging effect. The chromosome replication patterns of the constitutive heterochromatin regions of chromosomes 1, 9, 16 and Y were investigated using terminal 5-bromodeoxyuridine labelling and an S phase subclassification system. A significant delay (p < 0.001 for male cell lines; p<0.05 for female cell lines) was observed in the timing of RS+ constitutive heterochromatin. These results suggest that the cytogenetic abnormality of the constitutive heterochromatin of RS+ cells is associated with alterations in DNA conformation and synthesis.</p> / Doctor of Philosophy (PhD)

<p>Th~ thermoregulatory response to exercise in the heat, especia!ly sweating rate, differs between children and adults. This project investigated the effect of physical maturation on the thermoregulatory response to exercise (50% V02mc.x) in the heat (42°C 20%RH) among circum-pubertal boys, using a mixed cross-sectional, longitudinal design. Subjects were initially divided into three groups, based on Tanner (pubic hair) staging: 16 pre-pubenal (PP, Tanner 1), 15 mid-pubertal (MP, Tanner 2,3,4) and 5 Iatepubertal (LP, Tanner 5). The thermoregulatory response was observed every 6 months for a period of 18 months (4 sessions). Thirty of the 36 boys completed the four sessions. During each session, the exercise task :onsisted of three 20-min bouts of cycling and 10 min rest periods. Measurements included rectal and skin temperatures and hean rate continuously, VO:! at the midpoint of the second bout, forearm blood flow (FBF) upon entry to the climatic chamber and immediately following each exercise bout, sweat collection during each bout, photography of sweat drops after bouts 1 and 2, and whole body sweating rate. Blood samples before and after exercise in the heat were analyzed for hormonal, lactate and electrolyte concentrations. Body temperatures tended to be higher among LP and FBF was higher among PP. However, the rate of heat exchange and heat storage per kg body weight were similar among groups. Sweating rate per body surface area and per gland were higher among LP compared to PP. This was accompanied by lower sweat lactate concentrations during the initial stages of exercise, hi~her lactate excretion rate per gland. ~l')wer activated sw~at gland population density, and higher mean sweat drop area. Aldosterone and prolactin concentrations increased during exercise in the heat in all groups. The increase in prolactin was greater among LP compared to PP. Longitudinal observations tended to support cross-sectional findings. It is concluded that, in boys a) heat exchange and heat storage are similar among preto late-pubescents exercising in dry heat; b) physical maturation is characterized by enhanced sweating rate per body surface area and per gland, and this may be associated with increased sweat gland anaerobic metabolism; c) physical maturation may potentiate the increase in prolactin but not the increase in aldosterone during exercise in the heat.</p> / Doctor of Philosophy (PhD)

<p>Cell-cell adhesion is an important and an initiating event in thrombosis, inflammation and metastasis. Recent data suggest that these adhesion events are mediated by cell adhesion receptors and/or ligands present on blood cells, the endothelium and the basement membrane underlying the endothelium. This thesis hypothesizes that endogenous lipoxygenase metabolites regulate blood cell-vessel wall interactions. The following data, as presented in this thesis, support this hypothesis. First, the intracellular level of 13-HODE in endothelial cells, inversely correlated with platelet, tumor cell and leukocyte adhesion. Second, intracellular 13-HODE levels and/or 13-HODE:HETE ratios in blood cells and in tumor cells also inversely correlated with cell adhesivity. Third, the level of 13-HODE in basement membranes and in artificial grafts, inversely correlated with platelet adhesion. Fourth, 13-HODE co-localized with the vitronectin receptor (VnR) in resting endothelial cells. Following stimulation, 13-HODE and the VnR dissociated, and the VnR relocated to the periphery surface of the endothelial cell. Fifth, the increased peripheral expression of the VnR on stimulated endothelial cells mediated platelet adhesion. Finally modulation of the lipoxygenase pathway markedly influenced experimentally-induced thrombosis. In summary, there is an abundance of data presented in this thesis which suggest that monohydroxy lipoxygenase metabolites regulate blood cell-vessel wall adhesion. This regulation appears to be a result of lipoxygenase metabolites influencing adhesion receptor/ligand recognition. Therefore, it is a reasonable expectation that agents which modulate the lipoxygenase pathway may also be useful in the prevention and treatment of thrombosis, inflammation, and metastasis.</p> / Doctor of Philosophy (PhD)

<p>A phosphotyrosyl-protein phosphatase, designated pTPIII, was purified to near homogeneity from soluble extracts prepared from 11-day old chicken embryo bodies. SDS-PAGE analysis suggested that this pTP activity correlated with the presence of a 58 kd protein band in affinity purified enzyme preparations. The purified protein exhibited biochemical characteristics in common with a phosphotyrosyl-protein phosphatase purified from human placental protein extracts, pTP1B (Tonks et al., 1988a; Pallen et al., 1991), and thus may represent the chicken homolog of this protein. Two partial cDNA clones encoding phosphotyrosyl-protein phosphatases were also isolated. Both code for members of the transmembrane class of tyrosine specific protein phosphatases, and thus probably bear no relation to the pTPIII and pTPI activities purified from soluble protein extracts. One cDNA appeared to encode the COOH-terminus of the chicken homolog of LAR, while the second seemed to code for the chicken homolog of PTP-zeta. Two cDNAs encoding PTP-zeta were isolated, however the evidence presented suggested that both clones were likely derived from incompletely processed nuclear RNAs.</p> / Doctor of Philosophy (PhD)

<p>Rheumatoid arthritis is a chronic, systemic inflammatory disease. It can be treated with the gold(I) based drug, Myochrysine. Much research has been done in the past, in an attempt to elucidate the mechanism of action of this drug. To date, the mechanism remains a mystery. It is known however that Myochrysine inhibits the action of inflammatory cells. Myochrysine has been shown to inhibit phagocytosis, chemotaxis and the respiratory burst in mononuclear phagocytes and polymorphonuclear cells; T and B cell proliferation; interleukin 2 secretion from T cells; interleukin 2 receptor expression on T cells; as well as thrombin stimulated platelet aggregation and release. All of these inflammatory cell functions are mediated by a biochemical pathway involving receptor-G-protein mediated phospholipase C activation which results in Ca²⁺ mobilization and protein kinase C activation. It is proposed that Myochrysine acts to relieve rheumatoid arthritis by interfering with some point(s) in this biochemical pathway in inflammatory cells. Through the use of the platelet as a model system, it has been shown that Myochrysine inhibits the collagen, ADP, and U46619 induced platelet aggregation and serotonin release; that Myochrysine inhibits collagen induced myosin light chain and pleckstrin phosphorylation; that the inhibitory action is fast and reversible; that the inhibitory effect is not dependent on the presence of albumin; that the inhibitory effect does not involve cyclic nucleotide increases and that Myochrysine does not inhibit TPA induced pleckstrin phosphorylation or A23187 induced myosin light chain phosphorylation. More importantly, it is shown that Myochrysine inhibits platelet activation in a manner very similar to inhibition of platelet activation by other sulfhydryl reacting agents, including cell impermeant compounds. The data presented are consistent with the action of Myochrysine being at a membrane surface sulfhydryl group, most probably at a common structurally important sulfhydryl group within platelet receptors involved in the activation of the protein kinase C/Ca²⁺ mobilization pathway. Data are also presented which indicate that both the gold(I) moiety and the thiomalate ligand of Myochrysine can inhibit platelet function.</p> / Doctor of Philosophy (PhD)

<p>In platelets, the activators of guanylyl cyclase used were sodium nitroprusside (SNP) and SIN-1 (the active metabolite of molsidomine). PGE$\sb1,$ a functional analogue of prostacyclin (PGI$\sb2),$ and adenosine were the activators of platelet adenylyl cyclase studied. Changes in cyclic ($\sp3$H) nucleotides were measured in platelets prelabelled with ($\sp3$H) guanine and ($\sp3$H) adenine. Incubation of labelled platelets with SNP or SIN-1 caused inhibitions of platelet function which were associated with large dose-dependent increases in ($\sp3$H) cGMP and 1.4 to 3.0-fold increases in ($\sp3$H) cAMP. However, addition of either SNP or SIN-1 with PGE$\sb1$ or adenosine, at concentrations of the latter that had little effect alone, caused much larger increases in ($\sp3$H) cAMP and greatly enhanced the inhibition of platelet function. Experiments with an adenylyl cyclase inhibitor, 2$\sp\prime,$5$\sp\prime$-dideoxyadenosine (DDA), suggested that these synergistic interactions depend on an enhanced accumulation of cAMP. Hemoglobin inhibited all nitrovasodilator-mediated effects. Cilostamide, a selective inhibitor of platelet cGMP-inhibited cAMP phosphodiesterase (cGI-PDE), had effects identical to those of SNP, suggesting that the actions of the latter depend on inhibition of the same enzyme. Also, the results obtained with M&B 22,948, a cGMP-selective PDE inhibitor, strongly suggest that the increases in platelet ($\sp3$H) cAMP caused by nitrovasodilators, in the presence or absence of activators of adenylyl cyclase, are mediated by the inhibition of cAMP breakdown of cGMP. To determine if a similar synergism is seen in vascular smooth muscle (VSM), the interactions between isoproterenol and either nitrovasodilators or cAMP-PDE inhibitors were studied. A fast and sensitive prelabelling method was established for the measurement of cyclic nucleotides in cultured vascular smooth muscle cells (VSMC) from rat aorta. Using this method, agonist-induced changes in the cyclic nucleotide levels of two different aortic VSMC lines were studied. (Abstract shortened by UMI.)</p> / Doctor of Philosophy (PhD)

<p>The effect of sex hormones on pineal melatonin production has been examined. The diurnal profile in urinary aMT6s excretion, which is believed to represent pineal melatonin production and 24 hour sex hormone excretion was determined by RIA in male and female rats at age of 3 weeks, 2 months, 8 months, 14 months and 20 months. A concomitant decrease in metabolite excretion corrected for body weight was found with increasing age together with a highly significant sex difference in aMT6s output at 3 weeks, 2 months and 8 months of age. Increase of body weight with age is an important factor responsible for the age-related alteration while the difference in sex hormone milieu between male and female animals could be one explanation for the sex difference in aMT6s excretion in younger rats. The effect of castration on the diurnal profile in urinary aMT6s excretion was observed in both male and female rats at 3 weeks, 2 months and 8 months of age. Castration caused a fall in urinary aMT6s excretion in 3 week old female rats but increased the metabolite output in both 2 month and 8 month old female rats. In male rats castration reduced aMT6s excretion at 2 months of age but there was no significant effect at 3 weeks or 8 months of age. In addition, urinary aMT6s excretion varied with the stages of the rat oestrous cycle; the lowest excretion was observed at proestrus corresponding with the highest 24 hour urinary oestradiol excretion. Sex hormone treatment was performed on 2 month old castrated male and female rats. Both testosterone treatment on male castrated rats and oestradiol on female castrated rats abolished the effects of castration on aMT6s excretion. Administration of oestradiol to male rats and testosterone to female rats decreased and increased urinary aMT6s excretion, respectively. Castration had no significant effect on the half life of melatonin elimination from plasma either in 2 month old male and female rats or 6 month old male rats. The effect of castration and sex hormone treatment on the pineal response to isoproterenol (ISO) was also investigated in 2 month and male and female rats. Castration increased aMT6s excretion in female rats but reduced aMT6s output in male animals in both time-course and dose-dependence studies. Changes in serum and pineal melatonin response to ISO with castration and sex hormone treatment were examined by an Elisa. Consistently, castration in female rats increased pineal and serum melatonin responses to ISO and oestradiol treatment to castrated female rats blocked the elevation, whereas in male rats castration decreased the responses and testosterone treatment to castrated male rats abolished the reduction. The effect of sex hormones on pineal beta-adrenergic receptors were studied in 2 month old rats. The beta-adrenergic receptor binding was determined by using $\sp3$H-dihydroalprenolol (DHA) in single pineal glands. Castration in female rats increased the receptor density compared with oestradiol treated and sham-operated animals. By contrast, the receptor concentration in castrated male rats was lower than testosterone treated and sham-operated male animals. The results of the present study demonstrate that sex hormones are involved in the regulation of pineal melatonin production; in 2 month old rats, oestradiol has an inhibitory while testosterone exerts a stimulating effect. The effects do not result from the alteration of circulating melatonin metabolism. One of the mechanisms is that sex hormones alter the pineal response to adrenergic stimulation and act on the pineal beta-adrenergic receptors.</p> / Doctor of Philosophy (PhD)

<p>Human adenoviruses can induce tumors in rodents through the action of the products of two viral genes, termed E1A and E1B. E1A produces multiple transcripts as a result of differential splicing, however, only the products of the 13S and 12S mRNAs appear to play a role in oncogenicity and cell transformation. These mRNAs encode proteins of 289 and 243 residues that are identical except for the presence of a unique 46-amino internal sequence. Each of these species migrates in polyacrylamide gels as at least two electrophoretically separable forms which differ in apparent molecular mass by about 4 kDa. Previous studies had indicated that differential phosphorylation is largely responsible for this shift in gel migration. The objective of this thesis was to identify the phosphorylation events responsible for this mobility shift, and to determine the functional significance. These studies focussed on phosphorylation sites towards the amino terminus of E1A proteins. Mutants were generated by site-directed mutagenesis in which known phosphorylation sites at Ser89 and Ser96 were altered to alanine residues. Characterization of these and other mutants suggested that phosphorylation at Ser89 was largely responsible for the shift in gel migration of the E1A proteins, and may regulate phosphorylation at Ser96. Removal of the phosphorylation site at Ser89 was found to have no significant effect on the ability of E1A proteins either to transactivate E3 expression or to repress SV-40 enhancer activity, but it did reproducibly reduce E1A-mediated transforming activity by about three fold. These results suggested that phosphorylation at Ser89 may be of some regulatory significance. Ser89 as well as Ser219 were shown to be phosphorylated in vitro by the cell cycle protein p34^(cdc2Hs) which had been purified by immunoprecipitation using specific antiserum. In addition, E1A proteins were found to be most highly phosphorylated during mitosis, the period in the cell cycle of maximal p34^(cdc2Hs) activity. These results suggested that p34^(cdc2Hs) or a related protein kinase phosphorylates E1A proteins at these two sites in vivo.</p> / Doctor of Philosophy (PhD)

<p>The acquired immune deficiency syndrome (AIDS) is an incurable disease caused by infection with the human immunodeficiency virus (HIV). In the eight years following identification of HIV as the cause of AIDS, little progress has been made in understanding the complex pathogenesis of AIDS or in developing effective therapies to forestall disease progression. All current strategies to treat HIV infection assume that disease progression is directly related to infection of and replication in CD4⁺ lymphocytes by HIV. Recent theoretical and experimental studies have raised the possibility that the pathogenesis of AIDS depends upon indirect effects of HIV infection; namely the induction of deleterious immune responses. In this study, we examined the effects of HIV infection on cytotoxic T lymphocyte (CTL) activity. Three aspects of CTL activity in HIV infection were examined: the role of anti-HIV CTL in suppressing viral replication and disease progression; the role of autoimmune CTL in CD4⁺ lymphocyte depletion in HIV infection; and changes in the CTL repertoire in HIV infection and the relationship of these changes to progressive CD4⁺ lymphocyte depletion. Although a possible role for circulating anti-HIV CTL in suppressing HIV replication in vivo was seen, there was no evidence that these CTL protect against CD4⁺ lymphocyte depletion. This suggests a dissociation between viral replication and CD4⁺ lymphocyte depletion. Autoimmune CTL that lysed uninfected activated CD4⁺ lymphocytes were found in HIV-infected individuals. The natural history of CD4⁺ lymphocyte depletion suggested a relationship between this CTL activity and in vivo depletion of CD4⁺ lymphocytes. Autoimmune CTL had a conventional phenotype, but were not HLA-restricted. The characteristics of these CTL suggested a mechanism of CD4⁺ lymphocyte depletion involving recognition of CD4⁺ T cell receptor (TCR) idiotypes by the autoimmune CTL. Skewing of the CTL repertoire was also demonstrated in HIV infection. The increased incidence of skewing of TCR variable domain (V) gene usage observed in parallel to CD4⁺ lymphocyte depletion is consistent with an interdependence of the selective expansion of CTL and depletion of CD4⁺ lymphocytes. Results of this thesis suggest that autoimmune CTL play a role in the pathogenesis of AIDS and are rational targets for novel therapeutic approaches to the treatment of HIV infection.</p> / Doctor of Philosophy (PhD)