<p>Washed human platelet suspensions were used to study platelet-surface interactions in tubes at wall shear rates between 80 s⁻¹ and 640 s⁻¹ and exposure times up to 900 s. Radiolabeled platelets were used to measure platelet accumulation and release from accumulated platelets as a function of time, shear rate, distance from the inlet of a tube, surface composition and drug treatments of platelets. These empirical data were used in a calculation procedure based upon diffusion and convection, designed to yield the maximum interfacial fluid concentration (IFC) for each of the materials which are liberated from platelets during platelet accumulation upon surfaces. Substances such as AMP, ATP, serotonin, pyrophosphate, PGG₂ and PGH₂ were found not to be present in sufficient quantity to produce IFC's which could affect platelet aggregation. A second set of materials, von Willebrand factor, fibronectin, and calcium had IFC's less than the concentrations normally found in plasma. A third group, ADP, PGD₂, and TA₂ had IFC's close to those known to affect platelet aggregation. These last materials, along with materials formed by a vessel wall or in plasma are most likely to determine the rate of thrombus growth on subendothelium or on a blood-contacting biomaterial.</p> <p>As well, epi-fluorescent video microscopy was used to monitor platelet-surface interactions with different surfaces giving different spectrums of results.</p> <p>[table removed]</p> <p>Aspirin, sulfinpyrazone, indomethacin and PGE₁ treatment of platelets inhibited aggregate formation on a collagen surface but not platelet adhesion. Heparin, hirudin, imipramine, mepacrine, adenosine triphosphate, creatine phosphate/creatine phosphokinase treatment of platelets had no or little effect on adhesion, release or aggregate formation. Modification of platelet functions using pharmacological and suspension modification techniques demonstrated mural aggregate formation was independent of release of adenosine diphosphate by adherent platelets but dependent on prostaglandin and thromboxane formation.</p> / Doctor of Philosophy (PhD)

<p>Galanin, a 29 amino acid polypeptide, is widely distributed in enteric nerves of the gastrointestinal tract (GIT) in mammals. Previous functional studies demonstrated that galanin may function as neurotransmitter and neuromodulator in GIT. In canine small intestine, galanin induces TTX-insensitive inhibition of small intestinal circular muscle motility in vivo and in vitro, suggesting a direct smooth muscle action of galanin (Fox et al, 1986). Intraarterial infusion of galanin inhibits vasoactive intestinal polypeptide release from isolated perfused canine small intestinal circular muscle, indicating a neuronal action of galanin (Fox et al, 1988). We used ¹²⁵I-porcine galanin as a ligand to study the galanin receptors in the circular muscle and deep muscular plexus from the canine small intestine. The separation, purification and characterization of nerve and muscle membranes were carried out using the technique developed in our laboratory by Ahmad et al (1988). Specific binding sites for galanin were found in both nerve and smooth muscle membranes. The galanin receptor on synaptosomes showed some similar characteristics to that on smooth muscle membranes: (1) The equilibrium binding analysis showed a high affinity and high capacity binding sites in nerve (Kd = 1.1 nM, Bmax = 244 fmol/mg) and in muscle (Kd = 0.58 nM, Bmax = 389 fmol/mg); (2) The specific binding of ¹²⁵I-galanin was inhibited by galanin or N-terminal galanin fragments (galanin 1-16, 1-15, 1-11), but not inhibited by C-terminal fragment galanin 15-29, suggesting a crucial role of the N-terminal region of the galanin molecule in receptor recognition. Computer analysis suggested a two-site model of galanin receptor; (3) The receptor-bound ¹²⁵I-galanin was only partially dissociated by addition of excess (1 μM) unlabelled galanin. However, in the presence of a GTP analog, GTPγS, the dissociation of bound ¹²⁵I-galanin was accelerated and completed, implicating involvement of G proteins in binding of galanin to a two-site receptor. This was supported by the observation that GTPγS abolished the high affinity galanin binding site, leaving only a low affinity binding site in competition studies; (4) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cross-linked galanin-receptor complexes from synaptosomes and smooth muscle membranes revealed a similar radioactive band at an approximate molecular mass of 50,000 dalton. Although, the neuronal and muscular galanin receptors have the similar properties, studies on characterizing the G proteins involved in these two groups of receptors using bacterial toxins indicated that galanin-receptor interaction in nerves involves a pertussis toxin-sensitive G protein, while the receptor in smooth muscle plasma membranes is coupled to a cholera toxin-sensitive, pertussis toxin-insensitive G protein. We conclude that, in canine small intestine, galanin may act as a neurotransmitter and/or neuromodulator by interacting with a specific receptor subtype coupled by distinct G proteins on smooth muscle membrane as well as synaptosomes.</p> / Doctor of Philosophy (PhD)

<p>Cytotoxic T lymphocytes (CTL) are important in controlling a number of viral infections including those caused by members of the herpesvirus family. Many studies have focussed on the phenotype of human CTL specific for herpes simplex virus (HSV) although few studies address their specificity. HSV glycoproteins B (gB) and D (gD) can serve as target antigens for CD4⁺ and CD8⁺ anti-HSV CTL clones generated by repeated stimulation of peripheral blood mononuclear cells (PBMC) with HSV antigens or HSV-infected cells. In the present study, the presence of gB- and gD-specific CTL in polyclonal cultures of anti-HSV CTL stimulated with a single in vitro exposure to HSV-1 was examined using autologous EBV-transformed B-lymphoblastoid cell lines (LCL) infected with recombinant vaccinia virus or adenovirus vectors encoding gB (vgB11, AdgB) or gD (vgD52, AdgD). Of six HSV seropositive donors tested, only one individual generated CD4⁺ and CD8⁺ T cells capable of lysing LCL infected with HSV, vgB11 or vgD52. Anti-HSV CTL from the remaining five HSV seropositive donors lysed HSV-infected LCL only. Therefore, gB and gD can serve as target antigens for polyclonal cultures of human anti-HSV CTL although the majority of the bulk CTL response in most patients is directed at HSV antigens other than gB and gD. LCL infected with recombinant adenoviruses were not recognized by gB- and gD-specific CTL due to the restricted expression of the inserted gene product. Since LCL infected with recombinant adenoviruses were not lysed by human anti-HSV CTL, human fibroblasts, which are permissive to adenovirus infection, were chosen as potential target cells. However, human fibroblasts infected with HSV-1 (HSV-FB) were not lysed by human anti-HSV CTL or human allo-antigen specific CTL (allo-CTL). HSV-FB were not only resistant to lysis by human CTL, but exposure of CTL to HSV-FB rendered the CTL unable to lyse its normally sensitive target cell. Studies concerning the mechanism of this inhibition suggested that there were two distinct mechanisms of inhibition of CTL lysis. The first involved FB infected with HSV-1 for 2 hours and required the expression of ICP4, an immediate-early protein of HSV-1, but not infectious virus or virus-induced shut-off of host protein synthesis. The second mechanism of inhibition occurred later in the HSV replication cycle and involved the infection of CTL via cell-to-cell spread of HSV-1 from FB infected for 20 hours. The elucidation of mechanisms involved in HSV-induced immunosuppression may foster the development of preventative or therapeutic strategies aimed at controlling these pathogens in humans.</p> / Doctor of Philosophy (PhD)

<p>Human hemoglobin (Hb) is composed of two pairs of globin chains, each of them contains a heme group. One pair of globin chain is encoded by a gene on the street arm of human chromosome 16 within the α-globin gene cluster and another pair encoded by a gene on the short arm of human chromosome 11 within the β-globin gene cluster. During human embryonic and fetal development, there are orderly changes of Hbs from embryonic to fetal, and later to adult Hbs, which are referred to as hemoglobin switching. These alterations are due to sequential activation of the globin genes in the order of their locations in chromosomes. Embryonic α-globin chains are present in abundance during the first five to six weeks of gestation. Subsequently, ζ-globin chains are supplanted by the expression of α-globin chains (Peschle et al 1985). In infants older than 3 months of age and in normal adults, ζ-globin chains are not detected by sensitive radioimmunoassays. However, exceptions were observed in a subgroup of α-thalassemia-1 carriers with a deletion of two adjacent α-globin genes while the ζ-globin gene in cis to the deletion remains intact, e.g., (--^(SEA)/) α-thalassemia-1. In adult carriers with this --^(SEA) deletion, embryonic ζ-globin chains are persistently present at a low level in their erythrocytes (Chung et al 1984, Chui et al 1986). In order to understand the mechanism leading to ζ-globin gene expression in the adult carriers with the --^(SEA) deletion, we constructed stable mouse-human somatic cell hybrids with APRT deficient murine ertythroleukemia cells bearing human chromosome 16 with either the --^(SEA) deletion or the normal α-globin gene cluster. The results showed that the human ζ-globin gene was constituitively expressed at higher levels in hybrids containing human chromosome 16 with --^(SEA) deletion than that in hybrids with the normal human chromosome 16. These observations indicate that the DNA sequences within the deletion may have a role in regulating ζ-globin gene expression. Moreover, expression of the human ζ-globin gene in cis to the deletion was readily induced by DMSO and HMBA but not butyrate, in parallel with the induction of endogenous mouse α-globin gene expression. These observations are consistent with the hypothesis that ζ-globin gene expression in the adult carriers is linked to the (--^(SRA)) deletional human chromosome 16. The ζ-globin gene in the cis to the --^(SRA) deletion can be activated by adult regulatory factors in the mouse erythroleukemia cells. It is conceivable that the ζ-globin gene is normally suppressed in the adult erythroid cells by the DNA sequences within the --^(SRA) deletion.</p> / Doctor of Philosophy (PhD)

<p>In intense exercise the maintenance of muscle contraction and metabolism is critically dependent on the regulation of intracellular homeostasis. The present studies have examined the physiological and biochemical effects of three factors which influence this regulation at rest and in exercise; these factors are the strong ions, the weak ions (weak acid and base electrolytes), and carbon dioxide (CO₂).</p> <p>This thesis describes three series of experiments which were designed to demonstrate the importance of ion regulation in the maintenance of muscle performance and metabolism in intense exercise. The purposes of these studies were three-fold: (1) to quantify the intracellular composition of strong ions of fast- and slow-twitch skeletal muscles at rest and at the end of intense exercise; (2) to determine the relative contributions of strong ions, weak acids and bases, and CO₂ to the total intracellular ionic composition muscle at rest and in exercise; and (3) to demonstrate the between intracellular ion regulation, muscle metabolism and muscle performance during intense exercise.</p> <p>The first series of experiments examined the ionic and metabolic composition rat hindlimb muscles (white gastrocnemius, WC; plantaris, PL; red gastrocnemius, RG; soleus, SL) sampled at rest and following 4.5 min of intense swimming exercise. Intracellular ionic status is dependent upon the PCO₂ strong ion difference ([SID]), and the total concentration of weak acids and bases ([ATOT]) of the intracellular fluids. PCO₂ was held constant at various levels and intracellular strong ion concentrations r were measured using instrumental neutron activation analysis. [ATOT] is a pooled term representing all of the intracellular weak electrolytes and cannot be directly measured. A method for determining [ATOT) indirectly from measurements of pH, [SID] and PCO₂ muscle homogenates was formulated and is described. Intracellular [SID) was found to be significantly higher in fast twitch WG (161 mEq/l) than in slow twitch SL (137 mEq/l); this was primarily due to a higher [K+] in resting WG. Muscle [ATOT] averaged 190 mEq/l at rest, and there was little difference between muscles. Intramuscular pH was determined using three methods: the distribution of the weak acid DMO, from pH measurements of muscle homogenates, and was calculated from the independent variables [SID], [ATOT}, and PCO₂. Corresponding to its higher [SID], the WG had a significantly higher intracellular pH at rest (6.94) than SL (6.72).</p> <p>The second series of experiments examined changes in extra- and intra-cellular ion and metabolite concentrations, and quantified the ion and metabolite fluxes between muscle and blood at rest and during intense electrical stimulation using an isolated perfused rat hindlimb preparation. These studies confirmed that K⁺ and La⁻ leave the muscle cells during contraction and that Na⁺ and Cl⁻ enter, and that these ionic disturbances cause the increase in intracellular [H⁺j associated with the decrease in muscle performance. With stimulation, the major changes affecting intracellular ion status and metabolism were large increases in intracellular lactate concentration ([La⁻li) and [Na⁺]i, and a marked reduction in [K⁺]i. These changes were greatest in WG, a highly glycolytic muscle, and least in SL, a predominantly oxidative muscle; the rise in [La⁻]i accounted for 67% and 50% of the fall in [SID] in WG and SL, respectively. Correspondingly, the largest changes in ion status occurred in WG. The high initial [SID]i in resting we prevented excessive increases in [H⁺] and protein ionization state during muscle contraction.</p> <p>The third series of experiments examined the effects of extracellular metabolic and respiratory alkalosis on muscle performance, metabolism and ion regulation, and provided an opportunity to test the hypothesis that changes in intracellular ionic status will affect the regulation of metabolism. With alkalosis, compared to controls, there were no differences with respect to performance and metabolism, however, the rate of La⁻ efflux from muscle was significantly increased and [La⁻]i was significantly reduced. Extracellular alkalosis was also associated with increased fluxes of Na⁺ from perfusate into muscles, resulting in large increases in [Na⁺] at rest and during stimulation; K⁺ efflux in alkalotic hindlimbs was significantly reduced, compared to controls, during stimulation. A theory is proposed whereby exercise-induced changes in intracellular [La⁻], [K⁺] and [Na⁺] exert direct effects on the ionized state of intracellular proteins and on metabolic regulation during exercise.</p> / Doctor of Philosophy (PhD)

<p>Previous work has demonstrated that resistance exercise may increase dietary protein (PRO) requirements to a varying degree. Much of these discrepancies probably arose from differences in training intensity, classification of exercise, habituation to a training stimulus, and methodological considerations such as: 1. inadequate dietary adaptation periods; 2. failure to measure routes of nitrogen (N) loss (i.e., sweat); 3. failure to measure the dietary PRO (nitrogen (N)) content; 4. collection periods too short to measure an impact of exercise (i.e., delay in N excretion due to dehydration); and 5. the use of measurements insensitive to short-term alterations in PRO metabolism (i.e., lean body mass estimate from skinfolds to assess changes in muscle mass over a week of treatment). In addition, there is a paucity of information quantitating the influence of acute and chronic resistance exercise on PRO turnover. The overall purpose of this thesis was to examine N metabolism and the PRO requirements of resistance athletes, under well controlled conditions, with three discrete, but related studies. The first investigation was a randomized, cross-over study designed to determine the dietary PRO requirements of young men performing intensive body building resistance exercise during the early stages of training (novice) and to determine whether the consumption of excessive amounts of PRO had an ergogenic (work enhancing) effect upon muscle mass/strength gains. Twelve inexperienced, young, male volunteers received either a PRO supplement (total PROIN=2.62 g·kg⁻¹·d⁻¹) or a carbohydrate (CHO) supplement (total PROIN=1.35 g·kg⁻¹·d⁻¹) each for a period of 4 weeks during intensive (1.5 h·d⁻¹, 6 d·wk⁻¹) circuit weight training. Nitrogen balance (NBAL) measurements taken over the last 3 days on each diet were used to determine that the PRO intake (PROIN) for zero NBAL was 1.43 g·kg⁻¹·d⁻¹ (+1SD=1.62 g·kg⁻¹·d⁻¹ (recommended intake)). Pre- and post-training measurements of strength and estimates of muscle mass (density, creatinine excretion, and biceps muscle N content) were not different between diet treatments in spite of a significant training effect (increased strength and lean body mass) due to the exercise programme. It was recommended that young males performing body building-type resistance exercise require a dietary PROIN of 1.62 g·kg⁻¹·d⁻¹ during the first 2 months of training and that there are no greater increases in strength or muscle mass by consuming PRO in excess of this amount. The acute effects of resistance exercise upon leucine oxidation and whole body PRO synthesis (WBPS) were studied using stable isotope methodology in the second study. L-[1-¹³C] leucine was used as a tracer to calculate these variables in 6 healthy, fed, male athletes in response to a 1 h bout of circuit-set resistance exercise. The measurements were performed prior to, during and for 2 h after exercise and corrections were made for the background ¹³CO₂/¹²CO₂ breath enrichment and bicarbonate retention factor (c). Results demonstrated significant increases in the background ¹³CO₂/¹²CO₂ breath enrichment at 1 and 2 h after exercise and in c during exercise. At 15 min after exercise c was significantly lower than at rest. There were no effects of exercise on leucine oxidation, WBPS, nor the rate of appearance of endogenous leucine or total leucine flux. We concluded that circuit-set resistance exercise did not affect the measured variables of leucine metabolism. In addition, large errors in calculating leucine oxidation and WBPS during resistance exercise can occur if background ¹³CO₂/¹²CO₂ breath enrichment and c are not accounted for. In the final study, leucine kinetics and NBAL were used to determine the dietary PRO requirements of sedentary (S) and resistance trained (BB) subjects. Each subject was randomly assigned to each of 3 dietary PROIN (LP=0.86; MP=1.40; HP=2.40 g PRO·kg⁻¹·d⁻¹) for a total period of 13 days. Over the last 3 d, NBAL measurements were completed and on the last day WBPS and leucine oxidation were determined from L-[1-¹³C] leucine turnover. Regression analysis of the NBAL data was used to determine the PROIN for zero NBAL for S=0.69 g·kg⁻¹·d⁻¹ and BB=1.41 g·kg⁻¹·d⁻¹ and a recommended intake (ZERO intake +1 SD) for S=0.89 g·kg⁻¹·d⁻¹ and BB=1.76 g·kg⁻¹·d⁻¹. For BB the LP diet did not provide adequate PRO and resulted in an accommodated state (↓ WBPS vs MP and HP), the MP diet resulted in a state of adaptation as evident by the increase in WBPS (vs LP) and no increase in leucine oxidation (vs LP), while the HP diet did not result in increased WBPS compared to the MP diet but leucine oxidation did increase significantly indicating a nutrient overload. For S the LP diet provided adequate PRO and increasing PROIN did not increase WBPS. Leucine oxidation increased for S on HP diet. Taken together, these results indicated that the MP and HP diets were nutrient overloads for S. There were no effects of dietary treatment on indices of lean body mass (creatinine excretion, body density) for either group. Overall, this research provided evidence that the PRO requirements of resistance athletes are greater than for sedentary individuals and are above current Canadian and U.S. recommended daily PROIN for young, healthy males. The second study demonstrated that acute resistance exercise did not affect whole body leucine turnover and, in contrast to endurance exercise, there was no significant increase in leucine oxidation during exercise. In addition, this study demonstrated that changes occur in c and background breath enrichment during and after resistance exercise that can have an impact on leucine oxidation measurements. The final study also demonstrated that the positive NBAL that occurs at high PROIN does not appear to represent a physiological entity (i.e., no increase in WBPS or lean mass) but is probably an error in the NBAL method.</p> / Doctor of Philosophy (PhD)

<p>Comparative studies on the regulation of secretion from<br />dense and α-granules of electropermeabilized human platelets<br />indicated the involvement of three distinct factors, Ca²⁺, protein<br />kinase C (PKC) and an unidentified GTP-binding protein (GE). Any<br />two of these factors must be present for marked secretion from<br />dense or α-granules to occur. Thus, Ca²⁺ was not essential to the<br />exocytotic mechanism and the combination of phorbol ester (a PKC<br />activator) and a metabolically-stable GTP analogue (GTP[S]) could<br />produce marked Ca²⁺-independent secretory response. Ca²⁺ appeared<br />to have a more modulatory role, enhancing both the rate and extent<br />of secretion. Secretion did not correlate with phospholipase C (PLC) activity or with the accumulation of 1,2-diacylglycerol (DAG), both of which required Ca²⁺ and were inhibited by phorbol ester. The Ca²⁺-independent activation of phospholipase A₂ was shown for the first time in platelets, but moderate to complete inhibition of this enzyme had no effect on secretion or other hospholipase activities. Only the activation of phospholipase D, assayed by the formation of [³H]phosphatidic acid (PA) and<br />[³H]phosphatidyl-ethanol (PEt), in the absence and presence of ethanol, respectively, correlated with secretion under all<br />conditions tested. BAPTA and analogues, known inhibitors of Ca²⁺--independent secretion in other permeabilized cells, caused parallel dose-dependent inhibitions of PLD activity and secretion. PKC activity detected by the phosphorylation of its major endogenous substrate, pleckstrin, was enhanced by GTP[S] apparently by stimulating the formation of PA, as well as of DAG in the presence of Ca²⁺. An optimal dose of the protein kinase inhibitor staurosporine could not block secretion produced by GTP[S] in the presence of Ca²⁺, suggesting an additional role for PLD activation, independent of protein phosphorylation. However, although correlations between PLD activity and secretion were also been in intact platelets, this enzyme is known to account for only 10 - 20% of the total PA formed in intact thrombin-stimulated platelets .. This PA may be different from that formed via the<br />PLC - DAG kinase pathway and therefore the nature and subcellular<br />localization of this PA will have to be investigated in order to<br />establish whether PLD has a major role in secretion. The possible<br />existence of an alternate GTP[S]-stimulated effector protein<br />cannot be excluded.</p> / Doctor of Philosophy (PhD)

<p>Immediate hypersensitivity (allergy) is a very common disorder, which may<br />develop after exposure of an individual to an antigen. Intestinal hypersensitivity<br />to luminal antigens has been postulated as a possible cause or triggering<br />mechanism in the pathogenesis of ulcerative colitis, Crohn's disease, eosinophilic<br />gastroenteritis, celiac disease and peptic ulcer. The mechanism by which<br />individuals became sensitized is not known but naturally occurring adjuvants<br />(bacteria and their products) may be crucial for the development of<br />hypersensitivity. In my studies, I investigated in the role of pertussis toxin, a<br />product of Bordetella pertussis, in intestinal hypersensitivity, since pertussis<br />vaccine containing attenuated bacteria was used previously for the induction of<br />anaphylaxis in experimental animals. Pertussis toxin has the enzymatic activity<br />of ADP-ribosyltransferase. which can block the function of some G proteins,<br />important elements in the transduction of signals into the cell. I sensitized rats<br />or mast cell-deficient mice with ovalbumin (OVA) plus recombinant wild type<br />pertussis toxin (wPT) as adjuvant. The reaction to secondary antigen challenge<br />was evaluated 14 days later by determining changes in short-circuit current (Isc, indication of net ion transport) in small intestinal segments mounted in Ussing chambers. Other parameters measured included evaluation of antibody levels in the circulation and histological enumeration of mast cells in the intestinal mucosa.</p> <p>Sensitization of rats with OVA and wPT resulted in enhancement of the<br />jejunal ion secretory response to secondary antigen challenge as compared to<br />rats sensitized with OVA. Injection of doses of wPT as low as 10 ng with OVA<br />caused significantly elevated secondary responses to OVA, while 50 ng wPT<br />resulted in the maximal response. The responses to OVA challenge were<br />enhanced 18.7 fold when OVA was added to the luminal side of the tissues<br />while on serosal side this effect was enhanced 2.2 fold in sensitized rats. The<br />induction of hypersensitivity by wPT adjuvant was dependent on the enzymatic<br />activity of wPT. since the enzymatically inactive mutant (mPT) did not influence<br />responses to secondary antigen challenge. This suggests that ADP-ribosylation<br />of G proteins is involved in the elevation of hypersensitivity by wPT.</p> <p>Two classes of antigen-specific antibodies, IgE and IgG₂s., were measured<br />in the circulation of sensitized rats. The levels of both antibody isotypes were<br />increased at day 14 in wPT but not mPT plus OVA injected rats, which indicates<br />that enzymatically active wPT influences antibody production. The role of<br />particular classes of antibodies was examined by passive sensitization of naive<br />animals with serum from actively sensitized rats. Ion transport responses to<br />secondary antigen challenge of intestine were present in those animals.<br />Blockage of IgE receptors (with myeloma IgEI prior to passive sensitization<br />showed that the response to OVA was totally abolished. This finding shows that<br />in this model of hypersensitivity. IgE antibodies are crucial for the response to antigen. Experiments on mast cell-deficient mutant mice revealed that mast cells<br />are a necessary element for the responses to antigen. Additionally, the number<br />of stainable mast cells in rat intestinal mucosa was significantiy increased by<br />40% on day 14 in wPT but not mPT plus OVA injected rats. This indicates that<br />wPT-induced increases in mast cell numbers are partially responsible for elevated<br />hypersensitivity responses to antigen. Experiments with the neural toxin,<br />tetrodotoxin (TTX), showed that responses to luminal (but not serosal) OVA<br />were neurally regulated in that these responses to OVA were diminished by 66%<br />after treatment of the tissues with TTX.</p> <p>Evaluation of tne responses to OVA at different days after primary<br />sensitization with OVA plus wPT revealed that the responsiveness to antigen<br />appeared between day 3 and 7 after sensitization, and it was still present on day<br />228. In rats sensitized with OVA, the responsiveness to OVA was highest on<br />day 7 and than it decreased very rapidly, showing no responses on day 56.<br />Evaluation of IgE levels in rats demonstrated a similar pattern: the IgE levels were<br />detectable for a long time in wPT plus OVA treated rats but only on day 7 in<br />OVA treated rats. This suggest that elevated IgE levels caused by wPT may<br />account for the long lasting hypersensitivity responses.</p> <p>My data showed that wPT is a very potent adjuvant for hypersensitivity<br />reactions and that the mechanism involves elevation of antigen-specific<br />antibodies, increases in the number of mucosal mast cells and enhanced neurally-mediated<br />antigen uptake.</p> / Doctor of Philosophy (PhD)

<p>Ryanodine, a neutral alkaloid, is a widely used pharmacological tool in the studies<br />of muscle excitation-contraction coupling. The specific binding sites for ryanodine have<br />been identified to exist on sarcoplasmic reticulum (SR) in both skeletal muscle and<br />cardiac muscle. The ryanodine receptor has been purified from different tissue types. The purified ryanodine receptor from skeletal muscle was found to be identical with foot<br />structure, a protein spanning the gap between t-tubule and SR membrane. The ryanodine<br />receptor was also found to have Ca²⁺ channel activity, a Ca²⁺-induced Ca²⁺ release channel.<br />In functional studies with isolated SR vesicle or single channel, ryanodine was found to<br />have dual effects on Ca²⁺ channel. At lower concentrations, ryanodine locked Ca²⁺ channel<br />in the open state, but fully closed the channel at higher concentrations. In smooth muscle,<br />ryanodine was also found to affect intracellular Ca²⁺ movement in the functional studies<br />using intact tissue. However, direct evidence was not available for the presence of<br />ryanodine receptor in smooth muscle before this research was carried out.</p> <p>In the present study, a high affinity binding site was found located on SR<br />membranes of rat vas deferens (RVD) smooth muscle (Kd=5.6 nM, Bmax=435 fmol/mg).<br />The ryanodine receptor in smooth muscle shared many similarities with that of skeletal<br />muscle, but was not identical. The time required for [³H]ryanodine binding to microsomal<br />fraction was about two hours to reach a steady state, and [³HJryanodine could be dissociated from its binding site by 20 fold dilution. However, the dissociation was very<br />slow and incomplete when initiated by excess ryanodine. The [³H]ryanodine binding in<br />smooth muscle was Ca²⁺ dependant. The affinity was increased with increased Ca²⁺<br />concentrations, but the Bmax was unchanged. The [³H]ryanodine binding also increased<br />with higher ionic strength and higher osmolarity, but later has less effect. Many factors<br />that affect Ca²⁺-induced Ca²⁺ release (CICR) channel activity were also found to affect [³H]ryanodine binding in my study; e.g., both Mg²⁺ and ruthenium red inhibited binding<br />and caffeine potentiated it, especially in the presence of a low Ca²⁺ concentration. In the<br />present study, I also showed that varied levels of [³H]ryanodine binding site existed among different smooth muscles. This variation was not correlated with the density of innervation or the SR content of different smooth muscles.</p> <p>In dog mesentery artery smooth muscle, a low affinity binding site (Kd=269 nM) was also identified, in addition to the high affinity binding site.</p> <p>In the present study, I also tried to show functional effect of ryanodine on the<br />CICR channel using subcellular membrane vesicles from vas deferens. Ryanodine, at<br />higher concentrations, inhibited oxalate-stimulated Ca²⁺ uptake, an effect which was observed as early as 5 minutes after uptake was initiated. This inhibitory effect was partially additive to that of cyclopiazonic acid (CPA), a potent SR Ca²⁺ pump inhibitor,<br />when this agents were used at submaximal concentrations. However, when CPA was used at a maximal concentration, ryanodine had no additional effect. Ryanodine at concentrations of 10⁻⁹ to 5x10⁻⁴ M, did not significantly change the Ca²⁺ release rate. In Ca²⁺ release experiments, no functional Ca²⁺ channel was observed, but the data are consistent with an inhibition of the SR Ca²⁺ pump at high ryanodine concentrations.</p> <p>My study provided the first direct evidence ofthe existence ofryanodine receptors located on smooth muscle SR which may represent a CICR channels. Since another type of Ca²⁺ channel, IP₃-induced Ca²⁺ release channel (IICR), has also been identified in different smooth muscles, the varied levels of ryanodine binding site observed in the present study may be correlated to the ratio of occurrences of the two types of Ca²⁺ channel. This suggests that different excitation-contraction coupling mechanisms may exist in different tjpes of smooth muscle depending on different expression of these two channels. The present study also suggests that ryanodine, at higher concentrations, may inhibit the Ca²⁺ pumps located on SR. Suitable conditions for membrane isolation and Ca²⁺ transport experiment must be defmed to restore a functional Ca²⁺ release channel in smooth muscle, in order to study the functional effect of ryanodine on this channel.</p> / Doctor of Philosophy (PhD)

<p>A strategy to investigate the effect of two exercise modes upon patients with chronic airflow obstruction (CAD) is developed. The difficulties in defining and diagnosing the various pathological entities covered by the umbrella term CAO are discussed. Following a review of the published studies of endurance exercise in the before mentioned patient population a clinical problem is identified. Cycle ergometer exercise and voluntary hyperventilation were the two modalities chosen to be investigated. A 2² factorial design is selected in order that both modalities may be efficiently studied, singly and in combination, with the inclusion of a placebo exercise group.</p> <p>A statistical method is depcribed for measuring agreement between two technicians conducting a test identifying the diagnostic inclusion criteria. An additional criterion for entry into the study will be inclusion of only those patients who are particularly likely to maintain the randomly assigned maneuver. This will be determined by the response to carried out a pre-experimental sequence of three weekly test events carried out current to a CAO stabilization period. The intensity of the exercise will be established using a standardized progressive exercise test and a maximum sustainea ventilatory capacity procedure. The choice of the three dutcomes was based upon a more total definition of rehabilitation. The three primary outcomes are endurance as measured by both a twelve minute walking test and a progressive multistage treadmill test. The patients' perception of their social, emotional and physical function in response to the exercise regimen is additionally measured using a health index questionnaire.</p> / Doctor of Philosophy (PhD)