<p>pRb, p107 and p130 are structurally and functionally related polypeptides which comprise the retinoblastoma family of proteins. All three proteins are found in complexes with several cell cycle-regulating proteins, including cyclins and cyclin-dependent kinases (cdk's) which are thought to regulate the function of the pRb family members through phosphorylation. In vivo, p130 is observed in cyclin A/cdk2 and cyclin E/cdk2 complexes but not in complexes containing D-type cyclins and cdk4. This thesis examines these observations by identifying regions within the p130 sequence required for cyclin interactions. In vitro binding studies determined that D-type cyclin interactions require the majority of the "pocket domain" of p130. These interactions are disrupted upon phosphorylation of p130 by the cyclin D-associated kinase cdk4. Additional in vitro binding studies determined that a short sequence within the "spacer region" of p130 is required for interactions with cyclins A and E. This sequence contains an "RRL" motif which is present in several other cyclin A and cyclin E binding proteins. In vivo, the amino terminus of p130 is required to stabilize p130 interactions with cyclins A and E and this may be a result of inhibition of cdk2-associated kinase activity. Taken together, these results suggest that stable complexes containing p130 and cyclins A and E are not disrupted by phosphorylation of p130, perhaps because p130 inhibits the kinase activity of cdk2. In contrast, p130 interactions with D-type cyclins are disrupted in vivo because of cdk4-mediated phosphorylation of p130. This analysis concludes that D-type cyclin interactions with p130 are structurally and functionally distinct from interactions between p130 and cyclins A and E and these differences may be important in the regulation of p130 during the cell cycle.</p> / Doctor of Philosophy (PhD)

<p>Drug resistance limits the clinical efficacy of many cancer treatment modalities. Mutations, as a result of genomic instability within tumours, are thought to generate subpopulations of tumour cells which are less responsive to cytotoxic agents. This thesis investigates the hypothesis that drug resistance is associated with alterations in the mitochondria of cancer cells and these mitochondrial alterations provide a means to restore drug sensitivity in drug-resistant cell populations. A human ovarian carcinoma model was used to further characterize mitochondrial changes associated with cisplatin resistance (Chapter 1). It was observed that mitochondria in a cisplatin-resistant variant, C13*, accumulated and retained more of a lipophilic cation, rhodamine 123 (Rh123), compared to its parental line, 2008. This extended mitochondrial retention of lipophilic cations rendered C13* cells sensitive to another lipophilic cation, dequalinium chloride (Deca), compared to 2008 cells. Moreover, combinations of Deca and cisplatin induced synergistic cell kill in both cell types. These data suggest that disruption of mitochondrial function may sensitize tumour cells, including cisplatin-resistant tumour cells, to the cytotoxic effects of cisplatin. An association between mitochondrial alterations and resistance to another type of cancer therapy was also examined (Chapter 2). Photofrin II-mediated photodynamic therapy (PDT) is thought to destroy tumour cells, in vitro, by disrupting mitochondria. It was observed that a variant resistant to Photofrin II-mediated PDT, RIF-8A, contained mitochondria that differed structurally and functionally compared to its parental line, RIF-1. This was the first report to associate mitochondrial changes with PDT resistance. Similarities between RIF-8A mitochondria and C13* mitochondria at the ultrastructural level, suggested that RIF-8A may be resistant to cisplatin compared to RIF-1 cells. This hypothesis was supported by the observations that RIF-8A cells were cross-resistant to cisplatin (Chapter 3). These data indicate that mitochondrial characteristics may modulate the sensitivity of tumour cells to certain cytotoxic agents. Since cisplatin's cytotoxicity is believed to be mediated through its interactions with nuclear DNA, the mechanisms through which mitochondria influence cisplatin sensitivity, are unclear. Several of the steps involved in the repair of cisplatin-DNA lesions require ATP hydrolysis. Therefore, an increase in mitochondrial activity may augment the production of ATP, which can then be used for DNA repair. A host cell reactivation (HCR) assay indicated that C13* cells did not have an enhanced capacity to repair cisplatin-damaged DNA compared to either 2008 or RH4 cells suggesting that changes in the mitochondrial membrane potential do not influence the repair of cisplatin-DNA lesions. Independent observations have implicated the proto-oncogene, c-fos, as a potential regulator of mitochondrial activity and/or cisplatin sensitivity. This hypothesis was examined in three different model systems (Chapter 5). In both instances where there was an overexpression of the c-fos gene there was no corresponding increase in mitochondrial membrane potential. Similarly, in C13* cells which have an elevated mitochondrial membrane potential compared to 2008 cells, there was no significant difference in either c-fos mRNA or protein levels. Cells that overexpressed c-fos were resistant to cisplatin however, reducing c-fos expression did not sensitize cells to cisplatin. These data suggest that alterations in both mitochondria and c-fos expression may modulate cisplatin sensitivity but these two characteristics are not interdependent.</p> / Doctor of Philosophy (PhD)

<p>Bone is a common site of metastatic involvement. The mechanisms by which bone metastasis occurs are partially elucidated. Interactions between tumor cells and the bone microenvironment are being scrutinized, since their unravelling could lead to novel therapeutic approaches targeting bone metabolism additionally to tumor cells. This thesis summarizes experimental efforts addressing the role of metastatic cells in directly causing local osteolysis. Traditionally it has been thought that tumor cells, once in the bone, can only cause bone degradation by paracrine activation of osteoclasts. This work demonstrates that cancer cells are capable of degrading bone matrix and that their matrix metalloproteinases are partially responsible for this effect. The evidence reviewed here provides in vivo observations suggesting that this may occur during late "osteoclast-independent" phases of experimental bone metastasis by murine B16/F1 melanoma cells. In vitro documentation of bone and bone-related matrix degradation was obtained using these metastatic murine cells. A similar approach with human cell lines obtained from bone metastases demonstrated that these cell lines produce osteolysis in vitro. Since type I collagen is predominant in bone matrix, the potential involvement of tumor-derived matrix metalloproteinases was addressed. These studies showed that, in addition to murine B16/F1 melanoma cells, human prostate PC-3 adenocarcinoma, SK-N-SH neuroblastoma and Hs696 adenocarcinoma cells all produce these enzymes. Neutralizing their activity abrogated matrix degradation by melanoma cells. Induction of enzyme activity correlated with increase in the degradative ability of these cells. Additionally, B16/F1 and PC-3 cells degraded type I collagen, further implicating collagenases as mediators of these effects. These findings are of potential clinical use since they put forward the possibility of using inhibitors of matrix metalloproteinases, already in clinical trials for other neoplastic conditions, in addition to the currently used antineoplastic and antiosteoclastic approaches, for the prevention and treatment of bone metastases.</p> / Doctor of Philosophy (PhD)

<p>Use of multi-nutrient supplements for preterm infants fed mother's milk is common practice in neonatal intensive care units to provide sufficient amounts of minerals and other nutrients. The research delineated in this thesis investigated calcium, magnesium, zinc and iron bioavailability from supplemented mother's milk for preterm infants. Multi-nutrient supplementation to mother's milk had the benefit for preterm infants of achieving short-term growth rates parallel to the fetus of similar gestational age without reducing the dietary bioavailability of calcium, magnesium and zinc. In the infant-piglet model it was demonstrated that the addition of calcium and phosphorus to the diet in similar proportions as to preterm infants fed fortified mother's milk did not reduce iron bioavailability. Multi-nutrient supplementation to mother's milk did not result in better short- or long-term growth, greater whole body bone mineral content or better zinc status in comparison to supplementation with calcium and phosphorus alone. If it is, however, desirable for the preterm infant to attain short-term growth rates similar to the intrauterine fetus of the same post-menstrual age, future investigations should address the impact of multi-nutrient supplementation on the bioavailability of other minerals important for growth and development. Long term outcomes of whole body bone mineral, lean and fat mass and zinc status appeared to be determined by post-hospital discharge nutrition rather than nutrition in early neonatal life. Growth and body composition of breast-fed premature infants in the first year of life followed a different pattern in comparison to preterm infants fed a standard term formula. Therefore, future investigations should establish appropriate references for growth and body composition derived from breast-fed term infants in order to evaluate these outcomes in preterm infants fed mother's milk. The research described in this thesis has contributed to the concept that moderate nutrient and mineral intakes from mother's milk supplemented with a multi-nutrient fortifier or from mother's milk alone with only supplemental calcium and phosphorus may be appropriate for the healthy preterm infant even though current nutrient recommendations are not met. Based on the information provided in this thesis, functional responses to mineral nutrition should be considered in setting nutrient recommendations for preterm infants.</p> / Doctor of Philosophy (PhD)

<p>The fusion of cellular and viral membranes induced by herpes simplex virus type 1 (HSV-l) is essential for many stages of the replication cycle including virus penetration into host cells, nucleocapsid envelopment, virus egress and transmission of virus from infected to uninfected cells. Wild-type infections also induce low levels of cell-cell fusion, and syncytial mutants of HSV cause cells to fuse extensively. Previous studies have demonstrated that a large fraction of syncytial viruses contain mutations in the HSV-l UL53 gene, indicating that UL53 likely plays a central role in HSV -induced membrane fusion. Consequently, the objectives of this thesis are as follows: 1) Identify and characterize the UL53 gene product produced by wild-type and syncytial strains of HSV -I, and 2) Investigate the role of this protein in HSV -induced membrane fusion and in HSV-l replication. A single 40-kDa protein containing unprocessed, high-mannose N-linked oligosaccharides was detected in cells infected with wild-type and syncytial strains of HSV -I. This protein was designated gK, the ninth HSV -l glycoprotein to be described. gK is unique among HSV -1 glycoproteins since all other HSV glycoproteins examined to date exist as two proteins species; an immature protein with unprocessed oligosaccharides and a mature fonn containing complex N-linked oligosaccharides and O-linked oligosaccharides acquired during post-translational processing in the Golgi apparatus. The gK protein also exhibited signs of heat induced aggregation, an attribute which is commonly observed in proteins spanning the membrane multiple times. Low levels of gK were expressed in HSV -infected cells relative to HSV glycoproteins with a direct role in membrane fusion. which is more consistent with a regulatory role for gK in the membrane fusion process. Wild-type gK. but not other HSV glycoproteins. inhibited cell-cell fusion induced by HSV -1 strains encoding a mutant form of gK (gKˢʸⁿ). As such. the recessive phenotype displayed by gKˢʸⁿ may be symptomatic of a loss-of-function mutation, which disrupts a regulatory function governing membrane fusion. Glycoprotein K was not detected in HSV-l virions and immunofluorescence microscopy demonstrated that gK is not transported to the surfaces of cells infected with either wild-type or syncytial HSV. Instead, gK accumulates in the perinuclear and nuclear membranes of cells, and is unlikely to reach the Goigi apparatus because gK oligosaccharides remained sensitive to endoglycosidase H, These findings are in contrast to the behaviour of all other HSV glycoproteins described to date, which are expressed on the cell surface and incorporated into the virion envelope. Therefore gK must influence membrane fusion indirectly. since the protein is absent from the virion and does not reach the plasma membrane. Furthermore, these results imply that syn mutations in gK induce fusion between the surface membranes of HSV -infected cells by deregulating some aspect of virus replication. Resident proteins are maintained within intracellular compartments (eg. Goigi membranes) by one of three mechanisms: retrieval signals. retention through oligomerization, or retention through membrane thickness. Increasing gK protein synthesis by 10-20 fold did not overcome the block in cell surface transport, and this property is typical of membrane proteins maintained within intracellular compartments by retention signals. Furthermore, gK expressed in the absence of other HSV proteins by using a recombinant adenovirus vector, exhibited the traits and subcellular distribution of gK expressed in HSV -infected cells. These observations indicate that gK contains the targeting signals responsible for gK retention in the endoplasmic reticulum (ER) and nuclear envelope. Although the process controlling gK retention is still uncertain, gK oligomeric structures were observed in cells overexpressing the gK protein, suggesting that the fonnation of oligomeric assemblies may contribute to gK retention in the ER. In addition, sequence analysis identified a tyrosine-based motif (YTK[FILM]) conserved by the gK homologs of eight different alphaherpesviruses, which may have the potential to act as an ER retrieval signal. To further investigate the role of gK in membrane fusion and HSV replication, a gK-negative mutant (F-gKβ) was constructed. Since gK was found to be essential for virus replication, F-gKβ was propagated on complementing cells which can express gK. F-gKβ produced normal plaques characterized by rounded cells when plated on complementing cells, and F-gKβ produced syncytia on cells expressing low levels of gK. In contrast, F-gKβ formed microscopic plaques (consisting of 3-6 infected cells) on gK-negative cell lines and these plaques were reduced by 10² to 10⁶ in number. In the absence of gK, large quantities of unenveloped caps ids accumulated in the cytoplasm of HSV-infected cells and virus panicles did not reach the cell surface. The few enveloped panicles that were assembled displayed a reduced capacity to enter cells and initiate infection. Overexpression of gK also caused defects in virus egress, although virions accumulated in the perinuclear space, and large multilamellar membranous structures juxtaposed with the nuclear envelope were observed. Together these results demonstrate that gK regulates or facilitates HSV egress from cells. In addition, these findings raise the possibility that defects in gK may influence cell-cell fusion by altering the cell surface transport of viral particles, or other viral andlor cellular components which govern the fusion process.</p> / Doctor of Philosophy (PhD)

<p>This thesis examines the roles of nerve growth factor (NGF) in developing and mature dorsal root ganglion neurons, especially in the context of reparative nerve growth mechanisms in the adult peripheral nervous system (PNS). Peripheral nerve injury evokes two types of growths in the periphery, regeneration and/or collateral sprouting. However, these growth processes differ in a number of respects. Axonal regeneration is triggered by the injury and occurs independently of NGF whereas collateral sprouting is evoked and sustained by an increase in a target-derived signal, NGF. Indeed, cutaneous denervation is shown to result in a significant and prolonged increase in the level of NGF mRNA compared to the level in innervated skin. In addition, NGF mRNA is demonstrated to be expressed in not only the distal nerve pathways but also in non-nerve associated epidermally- and dermally-located cells. These findings indicate that an increase in NGF synthesis is associated with the increased availability of NGF in denervated skin, and that cutaneous nerves play a role in regulating NGF synthesis. Other findings in this thesis serve to strengthen the distinction between the injury-induced nerve growth responses. mRNA expression of the two NGF receptors, p75ᴺᴳᶠᴿ and trkA, are shown to increase in undamaged DRG neurons whose axons are sprouting into denervated skin, a proposed response that is most-likely related to the increased availability of target-derived NGF based on the findings that (i) NGF mRNA is increased in denervated skin, and (ii) polyclonal anti-NGF antiserum blocks the increase in the mRNA level of at least p75ᴺᴳᶠᴿ. In contrast to these findings, there was little or no change in receptor mRNA levels in regenerating neurons, consistent with the observations that NGF does not play a role in this process. Finally, here it is demonstrated that the NGF-driven collateral sprouting is severely impaired in adult rats that had received daily injections of anti-NGF serum during the first 2 weeks of postnatal life. However, after nerve crush the same nociceptive axons regenerated normally. Since NGF expression levels was found to be normal in innervated and denervated skin, NGF insufficiency was not responsible for the impairment in sprouting. Moreover, the possibility of a permanently defective axoplasmic transport was eliminated since sensory thresholds and axon calibers were also normal. Lastly, the finding that systemically-injected NGF promoted nociceptive sprouting, implies that the neurons had not become NGF-insensitive, nor had they reached a sprouting "ceiling". Indeed, the difference between the effects of exogenous and endogenous NGF is understandable if the latter accesses only the nerve terminals, and if the terminals' ability to take up NGF is defective. Thus, the exogenous NGF may work entirely through regions of the neuron that are outside this functional "compartment". To this end, although NGF uptake must have been adequate to account for the healthy state of the neurons, the terminals apparently failed to take up enough of the increased NGF produced in the collateral sprouting paradigm either to evoke sprouting, or to cause the usual upregulation of p75ᴺᴳᶠᴿ in the neurons of the dorsal root ganglion (DRG); there was, however, enough increased uptake to bring about the usual upregulation of trkA. This last finding suggests that neurotrophin deficiencies induced by adverse conditions during development might permanently compromise the organism's ability to mount neurotrophin-based reparative nerve growth in response to neuronal loss or peripheral nerve injury.</p> / Doctor of Philosophy (PhD)

<p>The interaction of platelets with damaged vessel walls plays a role in the development of atherosclerosis and its thromboembolic complications. The platelets that adhere to injured vessels release a growth factor that promotes the migration of medial smooth muscle cells through the internal elastic lamina into the subendothelial connective tissue where they proliferate and form a neointima. If a vessel is exposed to repeated or continuous injury the process continues and ultimately a frank atherosclerotic plaque develops. A plaque contains not only smooth muscle cells, but large quantities of intracellular and extracellular lipid and cholesterol. These plaques can lead to distrubed flow, and the development of thrombi, particularly in regions where abnormal flow patterns caused further endothelial cell injury or desquamation. The purpose of the work reported in this thesis was to examine the factors influencing the response of vessels to injury, since our understanding of these should contribute to our knowledge concerning the processes involved in atherosclerosis and its complications. Previous studies have examined the responsiveness of normal vessels of young animals to a single injury and may therefore have little relevance to injury of previously damaged or diseased vessels. The present experiments were therefore designed to produce conditions likely to be found in older vessels that have been exposed to previous injury. In these experiments rabbit aortae were de-endothelialized with a balloon catheter and a smooth muscle cell-rich neointima was allowed to develop over a 7 day period. The response of this surface to a second injury with a balloon catheter was examined and compared with the response of normal vessels to a single injury. The initial response to a second injury was very different from the response to a single injury. Whereas a monolayer of platelets accumulates after de-endothelialization, following injury of a smooth muscle cell-rich neointima, large platelet-fibrin thrombi form on the injured surface. The platelet-fibrin thrombi are largely oriented in the direction of blood flow. Despite the fact that coagulation is activated (the thrombin that is generated leads to fibrin formation) the number of platelets (measured using platelets prelabelled with ⁵¹Chromium) that accumulates on the injured aortic surface is similar to the number that accumulates on a de-endothelialized aorta, and the surface that is initially very reactive to circulating platelets rapidly loses its ability to attract fresh platelets. The reasons for the non-reactivity of normal endothelium and loss of reactivity of both de-endothelialized vessels and vessels subjected to a second injury were explored. Although it has been speculated that the PGI₂ produced by normal vessels or injured vessels could account for the lack of interaction of platelets with these surfaces, this did not seem to be so, since inhibition of PGI₂ production by treatment of vessels in vitro or in vivo with aspirin did not lead to platelet accumulation on undamaged vessels or enhance platelet accumulation on injured vessels. Similarily, products of the lipoxygenase pathway that have been implicated as responsible for the non-thrombogenicity of vessels did not appear to be responsible for the lack of accumulation of platelets on these surfaces, since inhibitors of lipoxygenase such as ETYA (15-hydroxy-5, 8, 11, 13-eicososatetraenoic acid) or NDGA(nordihydroguiaretic acid) did not influence the number of platelets that accumulated on the vessels under the conditions tested. To determine if factors other than platelets could account for the lack of vessel wall reactivity, platelet accumulation in vivo on de-endothelialized vessels or on vessels with an injured neointima was prevented by treating animals with injections of dipyridamole or with constant infusions of PGI₂. It was observed that 6 to 8 hours of these treatments are required for the vessels to become non-reactive; shorter durations of treatment do not allow the surface to develop its non-thrombogenic properties. Thus, the loss of reactivity of injured vessels do not require platelet adherence to the injured surface. In addition, it can be inferred that plasma factors or material from circulating red blood cells does not contribute to this effect. It may be that the loss of reactivity that develops over a 6 to 8 hour period may be attributable to a factor(s) elaborated by injured vessels and this is an avenue worthy of exploration.</p> <p>Several general conclusions can be reached based on the observations in this study. First, since injured vessels rapidly lose their reactivity to circulating platelets, repeated or continuous vessel injury is probably required for the development of severe arterial disease. Second, since activation of coagulation plays major role in the response to injury of previously injured vessels, treatment with anticoagulants could be useful therapy in some forms of arterial thrombosis. Finally, since injured vessels lose their reactivity to platelets even when the initial interaction of platelets with the injury site is prevented, it may prove useful to administer drugs that inhibit platelet adherence to damaged vessels for short periods only, for example, after coronary bypass surgery or transluminal angioplasty. This would allow "passivation" of the surface, prevent the effects on smooth muscle cell proliferation of growth factors released from adherent platelets, and reduce the vessel wall thickening that could compromise flow to the tissues.</p> / Doctor of Philosophy (PhD)

<p>With the advent of radioligand binding assays, central nervous system dopamine receptors have been well characterized in their membrane bound state. These receptors have been grouped into D-1 and D-2 subclasses on the basis of their relationship to the enzyme adenylate cyclase and affinities for dopamine agonists and antagonists. The dopamine D-2 receptor is considered relevant to the behavioral and pharmacological effects of neuroleptic drugs. The studies presented in this dissertation describe a successful method of solubilization of bovine striatal membrane bound dopamine D-2 receptor. The solubilized receptor exhibited typical pharmacological characteristics to that of membrane bound dopamine D-2 receptor. The rank order potency of agonists and antagonists to displace [³H]spiroperidol binding was the same as those observed with the membrane bound receptor. Analysis of the [³H]spiroperidol/agonist competition curves and the [³H]NPA binding revealed the retention of high and low affinity states of dopamine D-2 receptor in the solubilized preparation. This study demonstrated for the first time, a successful affinity chromatography method for the purification of dopamine D-2 receptor. One cycle affinity purification resulted in a 2000-fold enrichment of dopamine D-2 receptor activity with a recovery of 12% from the membrane-bound state and a specific activity of 169,600 fmol/mg protein (assayed with [³H]spiroperidol). The order of potency of D-2 agonists (N-propylnorapomorphine > N0434 > apomorphine > dopamine) and antagonists (spiroperidol > (+)-butaclamol > domperidone) with a purified preparation was found to be similar to that of the membrane bond or solubilized dopamine D-2 receptor. The adsorption of receptor to the affinity matrix was biospecific as pre-incubation of the solubilized preparation with D-2 receptor agonists or antagonists blocked retention of receptor activity. Elution of receptor was also biospecific as dopaminergic drugs were effective in eluting the bound receptor. Affinity purified preparations should be useful in producing monoclonal antibody to dopamine D-2 receptor and also prove to be important in understanding the molecular events from receptor drug binding to final response.</p> / Doctor of Philosophy (PhD)

<p>A. The involvement of central catecholaminergic neurons in tonic gonadotrophin release in male rats was investigated.</p> <p>(i) Animals were given a single intraventricular injection of 6-hydroxydopamine hydrochloride (6-ODHA; 170 μg free base) dissolved in 0.001 N HCl. Plasma LH and FSH were measured by double antibody radioimmunoassay. LH in the 6-OHDA treated animals was significantly reduced after one hour and remained consistently lower for eight hours. The controls, after a transient elevation, showed no significant change. No difference in LH concentrations was found between experimental groups sampled at two days or later. FSH in treated animals showed less consistent difference from the controls.</p> <p>(ii) Animals given an intraventricular injection of 6-OHDA dissolved in different vehicles, with varied pH and osmolarity, gave similar LH and FSH results which suggests the quinone derivative of 6-OHDA is effective in producing the catecholaminergic impairment.</p> <p>(iii) Administration of 6-OHDA dissolved in different vehicles, with varied pH and osmolarity, gave similar LH and FSH results which suggests the quinone derivative of 6-OHDA is effective in producing the catecholaminergic impairment.</p> <p>These findings indicate the LH release in the male rat may be controlled (or at least modulated) by a central adrenergic mechanism.</p> <p>B. The involvement of the ventral noradrenergic ascending pathway in tonic gonadotrophin release was investigated. Bilateral injection of 6-hydroxydopamine hydrochloride given into the preoptic area of the medial forebrain bundle significantly reduced plasma LH at all times tested. Plasma FSH was not significantly different. These results suggest that tonic LH release is modulated by an extrahypothalamic mechanism.</p> <p>C. The hypothalamic catecholamine content was pharmacologically manipulated with various drugs.</p> <p>(i) An intraperitoneal injection of protriptyline was given before 6-OHDA in order to selectively reduce the hypothalamic content of dopamine. Plasma LH, but not FSH was significantly reduced 3 hours after 6-OHDA in animals pretreated with protriptyline compared to controls. Neither plasma nor testicular testosterone were significantly different between groups.</p> <p>(ii) Animals given DL-threo-dihydroxyphenylserine showed a significant increase in plasma LH (previuosly decreased by 6-OHDA) compared to controls. DL-threo-dihydroxyphenylserine selectively restores the noradrenaline content in the hypothalamus.</p> <p>(iii) γ-Butyrolactone blocks the central release of dopamine and produces anaesthesia resembling deep sleep. No consistent significant differences were found in either plasma LH or FSH at any of the times or doses tested in animals given either γ-butyrolactone or a metabolite, γ-hydroxybutyric acid, compared to the control group receiving saline injection alone.</p> <p>(iv) 6-hydroxydopa which selectively reduces brain noradrenaline content, had no effect on plasma LH and FSH.</p> <p>The above findings give further support to the concept of modulation of tonic gonadotrophin release by an extrahypotalamic noradrenergic mechanism.</p> / Doctor of Philosophy (PhD)

<p>Cellular transformation by human adenoviruses requires the expression of two early transcription units, E1A (0 to 4.5% of the genome) and E1B (4.5 to 11.2%). The products of both genes are required to disrupt normal cellular growth control and morphology. Early region 1A products alone can induce cellular immortalization, but establishment of fully transformed cells requires cooperation with E1B. Early region 1B produces two unrelated proteins, 496R and 176R, each of which can act independently with E1A to induce cellular transformation.</p> <p>176R is a membrane associated protein whose molecular role in transformation is not known. Anti-peptide sera specific for the amino- and carboxy-termini of 176R were produced. These two sera were used for further purification and biochemical characterization of the protein. The 176R migrated as two species termed 18.5 and 19K on SDS-PAGE. The generation of the two species was not due to proteolysis, nor any post-translational modification investigated.</p> <p>Two post-translational modifications of 176R were identified: phosphorylation and acylation. The phosphorylation site was mapped to serine residue 164, and a mutant virus in which this amino acid was altered, pm2204, was produced by oligonucleotide-directed mutagenesis. Characterization of pm2204 as well as an E1 plasmid carrying this mutation suggested that phosphorylation is not important for the biological activity of 176R. 176R was also found to contain covalently bound palmitate and myristate. Both tryptic and chymotryptic peptide mapping as well as CNBr cleavage were used to localize the acylation site. These experiments suggested that multiple acylation sites exist, and at least one site was localized to the amino terminal region by tryptic peptide mapping. The linkage of fatty acid to 176R was o found to be via an amide, or an unusually stable ester bond to an internal amino acid. Acylation of 176R likely facilitates its membrane association, but it was not possible to test this hypothesis without precise mapping of the acylation site(s) and construction of specific mutants.</p> / Doctor of Philosophy (PhD)