<p>Herpes Simplex Virus DNA displays a number of unusual features which have been the subject of intense scrutiny in a number of laboratories. The genome is composed of two segments, each of which is flanked by inverted repeats. These segments invert freely with respect to each other generating equisolar quantities of four different isomers. This phenomenon, called segment inversion, was reputed to be the result of a site specific recombination mechanism operating on the terminal repeat, the 'a' sequence, which is part of the inverted repeats flanking each segment. The 'a' sequence was also implicated as the cleavage/packaging signal utilized by the virus to process viral DNA concatemers. The underlying mechanism of this process was believed to be a double strand break at a specific site between two 'a' sequences. The models of HSV maturation were deficient, however, in explaining several phenomena, namely the tendency of the 'a' sequence to accumulate tandem iterations of itself, the asymmetric distribution of these tandem iterations to one end of the genome, but not to the other, and the ability of defective genomes, which do not have tandemly iterated 'a' sequences, at least initially, to be efficiently packaged. I have shown that the "a" sequence actually contains two signals for cleavage/packaging, not one, that the cleavage occurs it specific distances from these signals, not in specific sequences, and that the cleavage mechanism results in a duplication of the cleavage signal and flanking DNA. Furthermore, I have determined that the 'a' sequence is not a target for site specific recombination, and that there is better evidence to support the idea that segment inversion is accomplished by a number of related, but independent mechanisms, including generalized recombination.</p> / Doctor of Philosophy (PhD)

<p>Cytotoxic T-lymphocytes (CTL) are important in resistance to HIV-1 infection, and modulate disease progression through various effector functions. Conventional CTL recognizing antigenic peptides in the context of major histocompatibility (MHC) class I molecules eliminate virally-infected cells, and have been shown to modulate the course of disease progression. Other functions of CD8⁺ lymphocytes include non-lytic suppresion of viral transcription and replication, and induction of programmed cell death in activated CD4⁺ cells. In the work presented here, the interactions of CD8⁺ lymphocytes with activated CD4⁺ cells were examined. Lysis of activated CD4⁺ cells was unique for CTL derived from HIV-1 infeced persons, and was not restricted by MHC molecules. The interaction was dependent on target cell activation, correlated with cell proliferation, and was independent of viral replication. Furthermore, the lytic process was biochemically consistent with apoptosis, and resulted in nuclear fragmentation in the target cells. In vitro susceptibility to infection by primary HIV-1 isolates was assessed in a cohort of couples consisting of an infected and an uninfected, but exposed, partner. These findings were correlated with the genetic integrity of the HIV-1 co-receptors, and with the presence of CTL specifically directed against the partner's primary isolate. Individuals with partial or complete resistance to infection were identified, as well as some individuals that had CTL recognizing the partner's viral isolate. The latter results suggested that cellular immune responses comprise a major component of resistance to HIV infection, and that they influence resistance even in individuals lacking the main HIV-1 co-receptor. The later studies were extended by examining the in vitro infectivity of CD4⁺ cells from a group of highly exposed, but persistently uninfected, Kenyan sex workers. No barrier to infection of CD4⁺ lymphocytes with primary Kenyan HIV isolates was identified, however, CD8⁺ cells from these subjects were able to efficiently limit viral replication in autologous CD4⁺ cells. In summary, the broadly different CTL effector functions described in these studies illustrate the importance of the cellular immune system in resistance to HIV infection, and in modulating the disease pathogenesis.</p> / Doctor of Philosophy (PhD)

<p>Benzodiazepines (BZ) are a class of drugs that are extensively used for their anxiolytic, anticonvulsant and sedative properties. The therapeutic actions of these drugs may be mediated through the central-type BZ receptors that are linked to the GABA receptor complex (BZ/GABA). When administered in large doses, the pineal hormone, melatonin, can interact with BZ receptors. The pharmacological actions of melatonin, similar to those listed above for BZs, appear to be mediated primarily by BZ/GABA receptors, although other BZ receptors may be involved. Recently, pharmacological doses of melatonin were found to decrease apomorphine-induced rotations in 6-hydroxydopamine lesioned rats. The main objective of this thesis was to determine the mechanism(s) underlying the antidopaminergic effect of melatonin as well as BZs using the 6-hydroxydopamine lesion model of dopaminergic supersensitivity. It was hypothesized that the antidopaminergic action of BZs and melatonin was due to the ability of these drugs to either enhance GABAergic activity (since GABA can suppress striatal dopaminergic activity) or block cyclic AMP (cAMP) production since dopamine enhances cAMP formation in the striatum. The major findings may be summarized as follows: 1) Clonazepam, a central-type BZ agonist, the melatonin blocked apomorphine-induced turning in lesioned animals; 2) intrastriatal injection of the GABA antagonist, bicuculline, caused a significant reduction in the antidopaminergic effect of clonazepam and melatonin; 3) the peripheral-type BZ antagonist, PK 11195, attenuated the antidopaminergic effect of these drugs but with much less potency than bicuculline; 4) the combination of bicuculline and PK 11195, injected directly into the striatum, completely blocked the antidopaminergic action of clonazepam or melatonin; 5) PK 11195 also blocked BZ-induced inhibition of cAMP production which is involved in striatal dopaminergic function. Therefore, in addition to a GAVAergic mechanism, inhibition of a cAMP pathway may be a secondary mechanism in the antidopaminergic action of clonazepam and melatonin. In studying the effects of BZs on the cAMP pathway, a significant increase in the inhibitory effect of diazepam on adenylate cyclase (AC) activity was observed in striatal membranes from lesioned animals. Further studies indicated that this sensitization to the inhibitory effect of diazepam on AC activity may involve upregulation of BZ receptors as well as enhanced functional coupling of these receptors to inhibitory G proteins. Taken together these findings indicate that the antidopaminergic effect of clonazepam and melatonin in lesioned animals involved at least two distinct mechanisms: 1) a predominant GABAergic action and 2) possibly the suppression of cAMP production in the striatum.</p> / Doctor of Philosophy (PhD)

<p>The first objective of this thesis was to investigate the targets of cyclic GMP (cGMP) action in platelets. Proteins that bind cGMP were first detected by photoaffinity labelling with [³²P]cGMP and subsequently identified by molecular, pharmacological and immunological criteria. Since cGMP was already known to exert major effects in platelets through the cGMP-inhibited phosphodiesterase family (PDE3) (Maurice and Haslam, 1990a), an additional objective was to explore the molecular basis of the unique properties of this enzyme by cloning and mutagenesis studies. A photolabelling technique using [³²P]cGMP was modified to permit the rapid detection of cGMP-binding proteins in crude platelet extracts. Five labelled proteins (110, 80, 55, 49 and 38 kDa) were detected in platelet supernatant and four (80, 65, 49 and 38 kDa) in platelet membranes. The sensitivity of photolabelling to PDE3 inhibitors and specific immunoprecipitation established that the 110 kDa photolabelled species was a product of the PDE3 gene family. In addition, the 80 kDa species was identified as cGMP-dependent protein kinase (PKG) by similar methods. Interestingly, cyclic AMP (cAMP) greatly enhanced the labelling of the 80 kDa protein, suggesting the existence of a novel co-operative interaction between cAMP and cGMP. This study also detected a previously unknown 65 kDa cGMP-binding protein in platelet membranes. Since both cAMP and cGMP inhibited labelling of this protein, it may represent a novel target for both cyclic nucleotides in platelets, possibly a subunit of a cyclic nucleotide-gated (CNG) ion channel. The inhibitory effects of variouus compounds on the photolabelling of PDE3 were quantitated by [³²P]cGMP. Thus, concentration-dependent inhibition of photolabelling of PDE3 was observed with trequinsin (IC₅₀ = 13 ± 2 nM), lixazinone (IC₅₀ = 22 ± 4 nM), milrinone (IC₅₀ = 56 ± 12 nM), cilostamide (IC₅₀ = 70 ± 9 nM), siguazodan (IC₅₀ 117 ± 29 nM) and 3-isobutyl 1-methylxanthine (IBMX) (IC₅₀ = 3950 ± 22 nM). The effects of these phosphodiesterase inhibitors on iloproststimulated cAMP accumulation in intact platelets were also investigated. Discrepancies between the abilities of these compounds to inhibit photolabelling of PDE3 and to increase platelet cAMP accumulation are probably related to differences in the rates of entry of the individual inhibitors into the intact platelet. The general applicability of this photolabelling technique and its value in the detection of novel cGMP-binding proteins in crude cell extracts was demonstrated in rat tissues. Distinctive photolabelling patterns were observed in different rat tissues and the 110 kDa protein found in human platelets was replaced by a 115 kDa species in rat platelets. To clarify the molecular mechanisms by which cGMP and inhibitory drugs modulate PDE3 activity, an attempt was made to define the roles of different PDE3 domains in the action of the enzyme. To that end, the C-terminal half of platelet PDE3 was cloned, identified as a product of the PDE3A gene and expressed as an active enzyme in E.coli. Further deletion mutants were generated by removing either an additional 100 amino acids from the N-terminus or the 44 amino acid insert, characteristic of members of the PDE3 family, from the catalytic domain. Site-directed mutagenesis of the 44 amino acid insert was also conducted to explore the function of this region. Kinetic analysis of these mutant enzymes demonstrated that the deletion of N-terminal sequences from PDE3 was accompanied by progressively lower Km values and by an increased Vmax for cGMP relative to that for cAMP. Thus, N-terminal sequences exert modulatory effects on cGMP hydrolysis. Deletion of the 44 amino acid insert abolished enzyme activity, as did site-directed mutagenesis of putative β-turns located at the N- and C-termini of the insert. Mutation of a cluster of negatively charged residues in the insert did not have major effects on the hydrolysis of cAMP or cGMP. The results suggest that this insert is required to preserve an effective catalytic domain structure in PDE3. In conclusion, the major targets of cGMP in platelets were shown to include not only PKG, but also PDE3A and an unidentified membrane protein, possibly a CNG ion channel. This thesis has also identified some of the functionally and structurally important domains within PDE3A.</p> / Doctor of Philosophy (PhD)

<p>The role of visual-spatial and short-term memory factors in reading and arithmetic disabilities was explored in 30 children with a reading disability: 41 with an arithmetic disability; and 70 who were achieving normally in reading, spelling, and arithmetic. All of the children had a PPVT-R score 80 and/or a WISC-R Block Design score 9. Percentile cut-off scores on the WRAT were used to classify the children into the three achievement or groups. The children were divided into three age groups (7-9, 10-11, 12-14) in order to study possible differences in cognitive performance between each disability group and the normal comparison group as a function of age. Two experimental arithmetic (Mixed Drill, Missing Symbols) and two experimental language tests (McGill Decoding Words and Nonwords), plus 4 visual-spatial tasks (Block Design, Rey-Osterreith CompIex Figure (Copy), Yerkes Blocks, Left-Right Orientation), and 5 short-term memory measures (Rey-Osterreith Complex Figure (Memory), Rey-Davis Form Board, Numerical Square, Digit Span, Phonemic - Confusability Rhyming and Nonrhyming Letters) were, administered to the children. The scores of the reading disabled children, compared to those of the normally achieving children in the same age category differed significantly in all three age groups on the following measures: McGill Decoding Words and Nonwords, Mixed Drill and Missing Symbols, Rey-Osterreith Complex Figure (Copy version), Digit Span, Phonemic - Confusability, and Numerical Square. In addition, the scores of the reading disabled children in the 7-9 age group on the Block Design and Rey-Osterreith (Memory version) tests, and in the 10-11 and 12-14 groups on the Rey-Davis 3-dimensional task, were significantly lower than those of the normally achieving children. There were no differences between the two achievement groups on the Yerkes Blocks and Left-Right Orientation tests. Considering the arithmetic disabled children in the 7-9 and 10-11 age groups, their scores differed significantly from those of the normally achieving children on the Yerkes Blocks test; as did those in the 10-11 and 12-14 age groups on the Block Design, Rey-Osterreith (Copy version), and Phonemic - Confusability (Rhyming letters) tasks. In addition, the groups differed significantly at the 7-9 and 12-14 age levels on the Rey-Osterreith (Memory) and Digit Span tests; and on the Rey-Davis 3-d and Numerical Square tasks at the 12-14 age level. There were no significant differences on the Left-Right Orientation test. The results suggest that while the performance of the reading disabled children in general is essentially normal on some measures of spatial visualization and spatial orientation; it is poor on other tasks measuring language, arithmetic, visual-motor skills, and short-term memory for language stimuli. In addition, only the younger (7-9) reading disabled children appear to have problems involving aspects of visual-spatial abilities (i.e., visual-perceptual, and spatial organization skills), and difficulties in memory for complex visual figures. The results pertaining to the arithmetic disabled children at all three age levels suggest that they have a problem in spatial visualization, and a complex memory difficulty which particularly involves visual materials. The data also point to a problem involving spatial organization and visual-motor skills which is evident after the age of 9. These findings highlight the importance of chronological age and developmental factors in learning disabilities research. They also point to the need to consider both the maturational lag and deficit positions in explaining the complex pattern of cognitive problems in both reading and arithmetic disabilities. In addition, the results indicate the possibility of a predominant involvement of the left cerebral hemisphere in reading disabilities, and a primary role of the right hemisphere in arithmetic disabilities.</p> / Doctor of Philosophy (PhD)

<p>Regular, coordinated contractions of uterine smooth muscle are thought to facilitate the delivery of the fetus(es) at parturition. The development of synchronous activity at term follows the development of many, large gap junctions between uterine smooth muscle cells. The objectives of this thesis were to test the hypothesis that the formation of gap junctions improves direct intercellular communication between the muscle fibers and to determine whether the functional properties of the junctions are regulated. Increased intercellular communication in uterine tissues may facilitate synchronous activity during labor and the regulation of such cell-cell interactions by modulating the function of the gap junctions may participate in the control of uterine contractility during pregnancy and parturition.</p> <p>A technique was developed to study the diffusion of a small radiolabelled glucose analog, 2-deoxyglucose, through small strips of myometrium. Tissues with many gap junctions from rats in labor demonstrated a significantly greater redistribution of tracer compared to muscle removed from days 17-20 pregnant and days 2-3 post partum animals not in labor which had few junctions. This movement of tracer was shown to be the result of intracellular and direct, cell-to-cell diffusion. Thus, there is evidence for improved intercellular communication in the myometrium during parturition when gap junctions are present.</p> <p>The extent of intercellular communication in the parturient myometrium was reduced by elevating the intracellular concentrations of Ca⁺⁺ and cyclic AMP in the absence of a change in the extent of gap junctions. More significantly, however, reduced communication was shown in tissues exposed to specific agents which are thought to play a role in the regulation of pregnancy and parturition. Thus, in addition to providing for the increase in gap junctions in the myometrium at term, the hormonal alterations which precede and accompany labor may also regulate the functional properties of these cell-to-cell membrane channels.</p> / Doctor of Philosophy (PhD)

<p>Indirect calorimetry was used to examine energy expenditure at rest and dietary-induced energy expenditure in normal-weight and overweight volunteers, whose daily energy intakes were similar. The influences of exercise, exercise training, and insulin on dietary-induced energy expenditure were also assessed. At rest energy expenditure was 70.3 kcals/hour in the overweight volunteers, 7.4 kcals/ hour greater than in the normal-weight volunteers. Consumption of a mixed meal of nearly 1000 kcals elicited similar increases in energy expenditure in the normal-weight and overweight volunteers of 10.8 kcals/hour and 12.5 kcals/hour respectively. The results refute the suggestion that a deficiency in energy expenditure at rest or in dietary-induced energy expenditure could be responsible for the greater propensity towards obesity in the overweight volunteers.</p> <p>When exercise preceded the meal, the oxygen debt of exercise summated with the dietary-induced energy expenditure in the normal-weight group. The post-prandial response in the overweight group was similar whether or not exercise preceded the meal, even though the metabolic response to exercise alone was not impaired in the overweight group.</p> <p>Six volunteers (four normal-weight and two over-weight) completed an exercise training programme, and increased their fitness level. However, dietary-induced energy expenditure in response to the 1000 kcal meal was similar before and after training.</p> <p>Hyperinsulinaemia in six normal-weight, non-diabetic volunteers did not influence the post-prandial energy response, although the infusion of insulin and glucose itself elicited an obligatory increase in energy expenditure.</p> <p>The work described in the thesis thus suggests that in mildly overweight individuals there is no evidence that reductions in post-prandial energy expenditure account for their excess weight, but that the lack of a further increase in post-prandial energy expenditure following exercise may do so. The results of the thesis indicate that exercise training does not alter post-prandial energy expenditure, and that hyperinsulinaemia does not influence post-prandial energy expenditure.</p> / Doctor of Philosophy (PhD)

<p>The relationship between fibre structural adaptation to strength and endurance training and the fibre physiological adaptations to these training procedures has been unexplored in humans. Methodological difficulties in fibre identification have prevented these investigations since traditional fibre classification techniques utilize various enzymes which are inactivated during fixation for electron microscopy. However, myoglobin is unaffected by glutaraldehyde fixation.</p> <p>In this study, structural and functional properties of the triceps surae were studied to determine the effects of endurance and strength training on: 1. the relationship between the fibre volume of sarcoplasmic reticulum and transverse tubules (SR) to the time to peak torque (TPT) of the isometric twitch; and 2. the relationship between fibre volume of mitochondria to muscle fatiguability. Needle biopsies were obtained from the gastrocnemii and soleus muscles and fibre types were classified for electron microscopy analysis on the basis of their myoglobin content. Electron micrographs were taken from the interior of 35 type I and 35 type II fibres of each muscle and were analyzed blindly by a stereological short-line test. Contractile properties were obtained from the isometric twitch in the triceps surae complex and separately from the gastrocnemii and soleus.</p> <p>Structural and contractile properties were examined in two subject groups: 1. a cross-sectional group made up of 6 subjects each of active controls, strength athletes and endurance athletes (N=18); and 2. a longitudinal training group (N=7) whom, in a unilateral training model, exercised one leg with a strength protocol and the other leg with an endurance protocol for 16 weeks.</p> <p>The results indicated that TPT was greater (p<.05) following chronic strength vs. endurance training (119.0 vs. 95.3 ms respectively) but TPT was decreased (p<.01) by 24% and 16% following short-term strength and endurance training respectively. The fibre volume of SR was not altered by strength or endurance training in either cross-sectional or longitudinal training groups. Resistance to fatigue at an absolute load was increased by 1.7 fold after short-term strength training and by 3.5 fold after short-term endurance training. Mitochondria volume was unaffected by either training protocol in the gastrocnemii but lower (p<.05) in type I fibres of the soleus after short-term strength (5.76%) vs. short-term endurance training (7.26%).</p> <p>It was concluded that functional adaptation to strength or endurance training may occur independent of fibre organelle volume adaptation to these training programs.</p> / Doctor of Philosophy (PhD)

<p>The acidosis accompanying short-term maximal exercise has been quantified and the mechanisms contributing to its control examined. Maximal exercise lasting 30 s was performed on a constant-velocity cycle ergometer. In 3 subjects, acid-base changes were examined across the working quadriceps femoris muscle after arterial and femoral venous catheterisation (Part A). The acid-base changes across the inactive forearm muscle were examined in 6 subjects following arterial and deep forearm venous catheterisation. Gas exchange was measured breath-by-breath during exercise and recovery (Part B). Muscle biopsies were taken from the quadriceps femoris muscle in 6 subjects and analysed for intracellular strong ion changes using neutron activation analysis (Part C).</p> <p>The intracellular acid load was due to both increased CO₂ production and strong anion production; the muscle [lactate] increased to 30 mmol/kg w.w. after 30 s exercise. The CO₂ and strong ion concentration contributed 25% and 75%, respectively, to the increase in intracellular [H⁺]. The weak acid concentration was assumed not to change during exercise and recovery. CO₂ and strong ions were removed from the intracellular fluid during recovery.</p> <p>Initially CO₂ output from the muscle reduced the intracellular PCO₂; the femoral venous PCO₂ increased to 105 mm Hg. The increased CO₂ flux to the lungs increased the CO₂ elimination from the body; the CO₂ output increased to 3060 ml/min by the end of exercise. The lungs were effective in removing the excess CO₂ delivered to them as the arterial PCO₂ was less than resting levels throughout recovery. Elimination of excess CO₂ from muscle was complete by 3 min recovery.</p> <p>Strong ion exchange occurred more slowly; lactate disappeared at a rate of 2 mmol/kg w.w./min. Immediately after exercise the intracellular-femoral venous [lactate] gradient was 40 mmol/l and favoured diffusion of lactate into the circulation. Approximately 55-60% of the lactate diffused from the muscle, the remaining lactate was oxidised or converted to glycogen. Lactate was taken up by the inactive forearm muscle; the v-a [lactate] difference was approximately 4.5 mmol/l. Only about 45% of the lactate taken up by the inactive tissue was oxidised, the remaining lactate was metabolised to other metabolic end points. Lactate uptake by inactive tissue reduced the anion concentration of the body and increased the strong ion difference across the inactive tissue. Recovery of acid-base balance is not complete until all the lactate has been removed from the body.</p> / Doctor of Philosophy (PhD)

<p>Ribonucleotide reductase catalyzes the first unique step in DNA synthesis by reduction of all four ribonucleotides to the corresponding deoxyribonucleotides. Herpes simplex virus (HSV), which codes for at least three enzymes of DNA metabolism (thymidine kinase, DNA polymerase and DNAase) was found to induce a novel ribonucleotide reductase activity upon infection of mammalian cells. The HSV-2 induced reductase was purified essentially free of the endogenous cellular enzyme and found to differ from the cellular reductase in several of its biochemical properties, most notably in its resistance to allosteric inhibition by dTTP and dATP (Huszar and Bacchetti, 1981). In addition, a rabbit antiserum was prepared (Rl serum) which was capable of specifically immunoprecipitating the HSV-2 induced reductase, thus demonstrating that the induced and cellular enzymes could also be immunologically distinguished (Huszar et al., 1983). Further experiments established that Rl serum cross-reacted with two monoclonal antibodies, both specific for HSV-2 polypeptides of approximately 144,000 and 38,000 daltons, which were capable of either immunoprecipitating the HSV-2 induced reductase (H11 antibodies) or directly neutralizing it in solution (Bg7 antibodies) (Huszar et al., 1983).</p> <p>These data demonstrate that either one or both of the HSV-2 144,000 and 38,000 dalton polypeptides are associated with viral ribonucleotide reductase activity. Based on the mapping of these polypeptides (Anderson et al., 1981; Docherty et al., 1981; Galloway et al., 1982a), these data also locate the coding sequences for at least a component of the enzyme between .56 - .60 map units on the viral genome within DNA sequences associated with cell transformation. The identification of viral DNA sequences coding for, and of viral polypeptides associated with, the HSV-2 ribonucleotide reductase will facilitate studies on the relevance of the enzyme to viral replication, latency and cell transformation.</p> / Doctor of Philosophy (PhD)