<p>There is growing evidence that factors other than cell-surface recetors are involved in regulating the sensitivity of cells to external signals. In particular, G proteins have been implicated in the increased sensitivity of numerous receptor systems under a variety of conditions (Mishra et al., 1997). The goal of this research project was to determine the role of G proteins in mediating dopamine receptor supersensitivity in Parkinson's disease. Prelimary studies of G protein levels in human post-mortem brain tissue proved inconclusive, due to the limited availability and variability of tissue samples. Subsequent studies in the 6-hydroxydopamine (6-OHDA) rat lesion model of Parkinson's disease revealed that stimulatory G protein levels are persistently elevated following denervation (Marcotte et al., 1994). These G proteins are presumably coupled to dopamine D₁ receptors, which show clear evidence of supersensitivity despite apparently normal receptor levels. This result supports the hypothesis that G proteins are involved in the maintenance of dopamine receptor supersensitivity (Marcotte and Mishra, 1997). Stimulatory G proteins acutely following MPTP mouse model, with decreased stimulatory G proteins acutely following MPTP treatment, and increased stimulatory G proteins after long-term recovery (Marcotte et al., 1998a). Although the significance of these findings is unclear, they provide additional support for the hypothesis that G proteins are modulated in response to dopaminergic denervation. Attempts to measure functional changes in stimulatory G protein activity in the rat striatum proved unsuccessful, consistent with the available literature. Specifically, neither the GTPase nor a specific GTP binding assay was able to consistently detect stimulatory G protein activity following dopamine D₁ receptor stimulation. To provide direct evidence for the role of Golf in mediating dopamine receptor supersensitivity, Golf antisense oligonucleotides were administered to 6-OHDA lesioned rats. Intrastriatal infusion of Gold antisense, but not control sense oligonucleotides, specifically reduced apomorphine-induced rotational behaviour and Gold levels. The effects of Golf antisense infusion were at least partially reversible, supporting a specific antisense mechanism of action. However, one of the control oligonucleotides, Golf missense, consistently reduced rotational behaviour and G protein levels in a non-specific fashion. This effect was dose- and sequence-dependent, and may be due to a non-specific binding to other nucleotides or proteins (Marcotte and Mishra, 1998). Taken together, these studies support the hypothesis that stimulatory G proteins are involved in mediating dopamine D₁ receptor supersensitivity. Further characterization of the effects of in vivo antisense oligonucleotides may provide more definitive conclusions regarding the role of G proteins in mediating this phenomenon.</p> / Doctor of Philosophy (PhD)

<p>Thrombin is a multifunctional serine protease that plays a central role in hemostasis. Unlike related serine proteases of the hemostatic system, thrombin is unique in that is has both procoagulant and anticoagulant activities. Structural features defined by X-ray crystallographic studies of thrombin provide a molecular basis for the enzyme's specificity. These features include the active site cleft and two anion-binding electropositive exosites located on opposite poles of the thrombin molecule. What is less evident from crystallographic studies is the thrombin's flexibility and its capacity to undergo conformational changes upon ligand binding to the exosites. These studies were undertaken to explore different but interrelated aspects of thrombin regulation. The first goal of this thesis was to determine how prothrombin fragment 2 (F2), a prothrombin activation fragment, binds to thrombin and modulates its activity. Cocrystallographic studies have shown that the interaction F2 with thrombin involves the formation of salt bridges between the kringle inner loop of F2 and anion-binding exosite II of thrombin. When F2 binds to thrombin, it has been shown to evoke conformational changes at the active site and at exosite I of the enzyme. Using plasma, recombinant, and synthetic F2 peptides (F2, rF2, and sF2, respectively) we have further localized the thrombin binding domain on F2. F2, rF2(1-116), rF2(55-116), and sF2(63-116), all of which contain the kringle inner loop (residues 64-93) and the acidic C-terminal connecting peptide (residues 94-116), bind to thrombin-agarose. In contrast, analogues of the kringle inner loop, sF2(63-90), or the C-terminal connecting peptide, sF2(92-116), do not bind. Thus, contrary to predictions from the crystal structure, the C-terminal connecting peptide as well as the kringle inner loop are involved in the interaction of F2 with thrombin. F2 and sF2(63-116) bind saturably to fluorescently labelled-active-site-blocked-thrombin with Kd values of 4.1 and 51.3 μM, respectively. The affinity of sF2(63-116) for thrombin increases about 5-fold (kd=10 μM) when Val at position 78 is substituted with Glu. F2 and sF2(63-116) bind to exosite II on thrombin because both reduce the heparin-caalyzed rate of thrombin inhibition by antithrombin - 4-fold. In contrast, only F2 slows the uncatalyzed rate of thrombin inactivation by antithrombin. Like F2, sF2(63-116) induces allosteric changes in the active site and exosite I of thrombin because it alters the rates of thrombin-mediated hydrolysis of chromogenic substrates and displaces fluorescently-labelled hirudin₅₄₋₆₅ from active-site-blocked thrombin, respectively. Both peptides also prolong the thrombin clotting time of fibrinogen in a concentration-dependent fashion reflecting their effects on the active site and/or exosite I. The different functional changes evoked by F2 and sF2(63-116) likely reflect additional contacts if F2 relative t the smaller sF2(63-116) and suggest that ligand binding to various subdomainds within exosite II may have different effects on thrombin function. The important implication of these findings is that distinct allosteric effects evoked by ligand binding to subdomainds of exosites may contribute to the diversity of thrombin function at the molecular level. The activity of thrombin is also regulated by blood-borne protease inhibitors. The second goal of this work was to gain insight into the mechanism by which thrombin is inactivated by heparin cofactor II (HCII), a serine protease inhibitor (serpin) in plasma that selectively inhibits thrombin in a reaction that is accelerated ≥1000-fold by glycosaminoglycans (GAGs) such as dermatan sulfate (DS) and heparin. Current thinking is that GAG binding to HCII disrupts ionic bonds between the amino-terminal acidic domain and the GAG-binding domain of HCII, thereby permitting the acidic domain to interact with exosite I on the thrombin. Based on this allosteric activation model, we predicted that substitution of basic residues in the GAG-binding domain of HCII with neutral ones would mimic the catalytic effect of GAGs. Compared with wild-type recombinant HCII expressed in BHK cells (wt rHCII), mutation of Arg¹⁸⁴, Lys¹⁸⁵, Arg¹⁹², Arg¹⁹³ (Mut C) or Arg¹⁸⁴, Lys¹⁸⁵, Arg¹⁸⁹, Arg¹⁹², Arg¹⁹³ (Mut D) reduced the affinity for heparin-Sepharose and increased the uncatalyzed rate of thrombin inactivation ~130-fold (from 4.6 x 10⁴ M⁻¹ min⁻¹ to 6.2 x 10⁶ and 6.0 x 10⁶ M⁻¹ min ⁻¹, respectively). Furthermore, unlike wt rHCII or plasma-derived HCII (pHCII), neither heparin nor dermatan sulfate increased the rate of thrombin inhibition by Mut C or Mut D. The increased basal rate of thrombin inhibition by these mutants reflects displacement of their amino-terminal acidic domainds because (a) they inhibit ϒ-thrombin at a 65-fold slower rate than α-thrombin, (b) the exosite 1-binding fragment hirudin-(54-65) decreases the rate of thrombin inhibition, and (c) deletion of the amino-terminal acidic domain (-del74) of Mut D reduces the rate of thrombin inhibition ~ 100-fold. To determine whether GAG-mediated bridging of thrombin to HCII contributes to accelerated thrombin inhibition, we compared the catalytic effects of longer heparin or dermatan sulfate chains with those of shorter chains. Heparin chains comprised of 30 or more saccharide units produced an ~5-fold greater increase in the rate of thrombin inhibition by pHCII, wt rHCII, and wt-del74 than heparin chains comprised of 20 or fewer saccharide units. In contrast, dermatan sulfate and a low molecular weight fragment of dermatan sulfate stimulated thrombin inhibition by pHCII and wt rHCII to the same extent, and neither agent affected the rate of thrombin inhibition by wt-del74. Our findings support the concept that heparin and dermatan sulfate activate HCII by releasing the acidic amino-terminal domain from intramolecular connections with the GAG-binding domain. Since both GAGs produce ≥ 1000-fold increase in the rate of thrombin inhibition by HCII, our observation that only heparin serves as a template raises the possibility that dermatan sulfate induces more extensive allosteric changes than heparin. In addition to regulation by serpins, thrombin function is also modulated by its incorporation into forming thrombi. Despite being catalytically active, fibrin-bound thrombin is protected from inactivation by inhibitors, notably antithrombin (AT)/heparin. The resistance of fibrin-bound thrombin to inactivation by AT is thought to reflect formation of a productive ternary thrombin-fibrin-heparin complex in which thrombin is protected from inactivation by AT. The anchoring of thrombin in a productive ternary complex is mediated by thrombin's exosites, fibrin via exosites I and heparin via exosite II. It has been proposed that productive ternary complex assembly is dependent on binary interactions between thrombin-heparin, thrombin-fibrin, and heparin-fibrin. Unlike heparin, DS inhibitors soluble and fibrin-bound thrombin equally well, however the explanation for this phenomenon is unclear. The third goal of this work was to determine why fibrin-bound thrombin is susceptible to inactivation by the HCII/DS complex but not by the AT/heparin complex. The results of this study indicate that, unlike heparin, DS does not promote the formation of a productive ternary thrombin-fibrin-DS complex. This concept is supported by three lines of evidence. First, in the presence of fibrin monomer (Fm), thrombin is protected from inhibition by HCII/heparin, but not by HCII/DS as quantified by protease inhibition assays under pseudo first-order conditions. Second, DS does not promote the binding of radiolabeled active site-blocked thrombin (¹²⁵I-FPR-thrombin) to fibrin. In contrast, heparin augments ¹²⁵I-FPR-thrombin binding to fibrin in a concentration-dependent manner. Third, DS does not interact with fibrin and binds to thrombin with a 22-fold lower affinity than heparin (Kd values of 2.6 μM and 117 nM, respectively). These results reveal that, although exosite I and exosite II of thrombin can be ligated by fibrin and DS, respectively, productive ternary complex does not occur because DS is unable to bridge thrombin to fibrin. These findings indicate that all three binary interactions are essential for productive ternary complex formation. We also examined the protective effect of the thrombin-fibrin-heparin complex on thrombin inhibition by Mut D. Whereas Fm alone has little effect on the uncatalyzed rate of thrombin inhibition by Mut D, addition of heparin decreases the rate of thrombin inhibition by Mut D ~ 30 fold (from 6.0 x 10⁶ M⁻¹ min⁻¹ to 2.1 M⁻¹ min⁻¹). Furthermore, in the presence of Fm, heparin causes a dose-dependent decrease in the DS-catalyzed rate of thrombin inhibition by HCII. These observations reveal that the protective effect of heparin results from the anchorin of thrombin in a productive thrombin-fibrin-heparin complex in which exosite I is inaccessible to the amino-terminus of HCII. Collectively, these studies illustrate different modes of regulating thrombin function, all of which are intricately interrelated. The remarkable diversity of thrombin activity allows thrombin to serve multiple functions in highly controlled processes in hemostasis.</p> / Doctor of Philosophy (PhD)

<p>A method for the preparation of human prothrombin fragment 1.2 (F1.2) from freshly clotted plasma was developed. Addition of exogenous F1.2 to citrated nonnal human plasma prolonged prothrombin time and activated partial thromboplastin time by 0.9 and 2.4 s/μM Fl.2, respectively. Delayed thrombin generation was not attributed to inhibition of tissue factor activity nor inhibition of factor X activation but to interference with phospholipid interactions in the prothrombinase complex. In mice, 500 μg (14 μM) of F1.2 gave 100% protection from tissue factor lethality, whereas 6 units (3 μM) of heparin was required for 100% protection. Either dose of F1.2 or heparin protected mice from thrombin-induced death. However, 500 μg of F1.2 was not effective in protecting mice from lethal effect of factor Xa and cephalin, while 0.75 units of heparin prevented such lethality. These findings demonstrate that human prothrombin F1.2 has anticoagulant and antithrombotic properties.</p> / Doctor of Philosophy (PhD)

<p>The acute inflammatory response is an organism's immediate reaction to injury. In mammals the response is facilitated by an abundance of molecules circulating in the plasma. These molecules are responsible for clotting the blood and chemotaxis of inflammatory cells. Polymorphonuclear leukocytes and monocytes usually comprise the cell infiltrate during acute inflammation.</p> <p>Within several hours of a localised injury and the start of the acute inflammatory response, systemic indications of the response are detectable. A number of plasma glycoproteins, collectively referred to as the Acute Phase Proteins, change in concentration during acute inflammation. Acute Phase Proteins are synthesized primarily by the liver parenchymal cells, the hepatocytes. The molecules which induce hepatocytes to adopt Acute Phase Protein synthesis (hepatocyte-stimulating factors) arise from the inflammatory macrophages at the site of inflammation. The phenomena of the Acute Phase Protein changes constitutes the Acute Phase Protein Response. The predictable nature of the Acute Phase Protein Response, during inflammation caused by noxious agents, has led to the speculation that the response may serve as a manifestion of infectious diseases.</p> <p>Certain intestinal-dwelling nematodes elicit an inflammatory response in the small bowel of their host. Nippostrongylus brasiliensis and Trichinella spiralis are two such parasites of rodents. It was important for an understanding of the nature of intestinal inflammation, to establish whether the Acute Phase Protein Response occurred in animals harboring the parasites.</p> <p>Following a subcutaneous injection, N. brasiliensis larvae pass through the rat host's lungs before reaching and maturing in the intestine. Acute Phase Protein changes were detected in infected rats at the time at which the worms were passing through the lungs and following the establishment of the adults in the intestine. Infective T. spiralis must be ingested and subsequently mature in the small intestine. Adults live, and give birth to larvae, while living in an intracellular compartment comprised of intestinal epithelial cells. No Acute Phase Protein Response was detected during infection of rats by T. spiralis.</p> <p>An infection by T. spiralis did not inhibit the Acute Phase Protein Response due to a second stimuli, and macrophage secretion of factors relevant to the Acute Phase Protein Response, from sites other than the intestine, was normal. An Acute Phase Protein Response was detected in animals which harbored concurrent N. brasiliensis and T. spiralis infections. No Acute Phase Protein Response was detected in rats into which mature N. brasiliensis larvae were transferred, in order to eliminate the lung stage of infection. It was concluded that the lung phase of an infection by N. brasiliensis was important in the genesis of the Acute Phase Protein Response during intestinal inflammation. Furthermore, it was concluded from these studies that inflammation in the rat intestine does not lead to the Acute Phase Protein Response.</p> <p>When the intestinal cells of normal rats were examined for the production of factors which may induce the Acute Phase. Protein Response, it was shown that constitutive secretion of the relevant molecules occurred. Secretion of these factors increased during infection of rats by N. brasiliensis but declined during infection by T. spiralis. A rat intestinal epithelial cell line was used to demonstrate that these cells, in addition to macrophages, secrete molecules important in the regulation of the Acute Phase Protein Response. It was concluded that T. spiralis likely inhibited the constitutive hepatocyte-stimulating factor activity by infecting epithelial cells of the intestine.</p> <p>The pathology that the rodent infections elicit are good models of inflammation for the study of mechanisms of the acute inflammatory response of the intestine.</p> / Doctor of Philosophy (PhD)

<p>The acute phase response in mammals to tissue injury or infection is characterized by a number of systemic effects including fever, neutrophilia and increases in serum levels of liver-derived (synthesized by hepatocytes) acute phase proteins. Soluble mediators or cytokines released by cells of the monocyte/macrophage lineage have been implicated in the initiation of the increased synthesis of protein by hepatocytes and include Hepatocyte Stimulating Factor (HSF) and Interleukin-1 (IL-l). The nature of HSF and IL-1 and their activities in inducing acute phase protein synthesis in vitro by primary cultures of rat hepatocytes and by human Hep-G2 cells was examined. Human peripheral blood monoocyte (PBM) -derived HSF showed different characteristics than IL-1 upon separation by chromatography and gel electrophoresis. HSF strongly stimulated some acute phase proteins (rat α₂-macroglobulin and α₁-cysteine protease inhibitor, human fibrinogen and α₁-antichymotrypsin) whereas Il-1 strongly stimulated human α₁-acid glycoprotein.</p> <p>Human PBM derived HSF showed biochemical similarities to another cytokine, Interferonβ₂ (IFNβ₂), that had previously been cloned from fibroblasts and from T-lymphocytes. HSF and 1FNβ₂ showed immunological similarities on the basis of antibody binding and activity inhibition assays. Cloned IFNβ₂ from T-cells showed potent inducing activity of acute phase protein synthesis and stimulated maximally the same proteins that did HSF. Both human fibroblast cultures and PBM cultures secreted HSF activity and possessed mRNA species that hybridized to a cDNA probe for IFNβ₂. These data suggest that human PBM derived HSF and human IFNβ₂. These data suggest that human PBM derived HSF and human IFNβ₂ are identical and that fibroblasts and T-lymphocytes as well as monocytes are capable of releasing Hepatocyte Stimulating Factor/lnterferonβ₂.</p> / Doctor of Philosophy (PhD)

<p>Conclusions about the effects of pregnancy in women - how they feel about being pregnant and their attitudes to the unborn child - have been based almost exclusively upon observations of primigravidas. It is claimed that a satisfying and/or more stressful than later pregnancies. The research for this thesis suggests that some of these claims are mistaken.</p> <p>This thesis examines the similarities and the differences between a group of primigravidas and a group of second pregnancy multigravidas on a range of maternal attitudes during pregnancy. The predictions were based on findings from a pilot study conducted at McMaster University Medical Centre. An interview was designed to elicit the women's thoughts and feelings about their expected infant and about themselves as mothers.</p> <p>Primigravidas and multigravidas were found to be equally positive about the coming baby and equally anxious about their capacities as mothers. The primigravidas reported significantly more anxiety about the welfare of the expected infant and the multigravidas reported significantly more conflict and negative feeling. The common and unique features of a first and a second pregnancy are discussed. The findings suggest that new adaptations and family real realignments accompany the birth of each child.</p> <p>A third sample of women was examined using the same measures. Comparisons were made between a group of multigravidas who had lost an infant by stillbirth or neonatal death and a group of multigravidas without a history of infant loss. Women who had previously lost an infant were found to be still mourning the death of the first infant and less invested in a relationship with the expected infant. The thesis discusses the effects of infant loss upon women and makes recommendations for the clinical management of bereaved mothers.</p> / Doctor of Philosophy (PhD)

<p>Breathlessness may be defined as the conscious awareness of respiratory muscle effort. As with any skeletal muscle it is to be expected that the sense of effort increases as the pressure generated by this muscle increases as well as the velocity and extent of shortening. The purpose of this study was; 1. to quantify the intensity of breathlessness during exercise and respiratory loading; 2. to isolate the contributions of inspiratory muscle pressure to breathlessness; 3. to see if extent of shortening, velocity of shortening, frequency (fb), and duty cycle (Ti/Ttot) contribute to the intensity of breathlessness independently. The intensity of inspiratory muscle pressure was quantified by measurement of mouth pressure (Pm) as well as the estimated esophageal pressure (Pes), the extent of shortening by tidal volume (Vt), and the velocity of shortening by inspiratory flow (Vi). Six normal subjects underwent eight incremental (100 kpm/min/min) exercise tests on a cycle ergometer to maximum capacity. The first and last test were unloaded and the intervening tests were performed with external added resistances and elastances presented in random order. The resistances and elastances were selected to provide a wide range inspiratory pressures, tidal volumes, and flows. The inspiratory resistive loads (33, 57, 73 cm H2 O/1/s) were used mainly to vary the flow (functional velocity of shortening of inspiratory muscles). The inspiratory elastic loads (21, 41, 52 cm H2O/1) were used mainly to vary the tidal volume (functional extent of shortening). At rest and at the end of each min during exercise the subjects estimated the intensity of breathlessness (Y) by selecting a number ranging from 0-10 (Borg psychophysical scale), 0 indicating no appreciable breathlessness and 10 the maximum tolerable sensation.</p> <p>When the velocity was altered (resistive loading study) breathlessness was significantly related to inspiratory pressure (p<0.0001), peak inspiratory flow (p<0.0001), frequency of breathing (p<0.01) and duty cycle (p<0.01). When the extent of shortening was altered (elastic loading study) breathlessness was significantly related to inspiratory pressure (p<0.001), tidal volume (p<0.001), and frequency of breathing (p<0.001).</p> <p>The results indicated that the perceived magnitude of breathlessness is closely related to the pressure generated by the inspiratory muscles and the shortening pattern of these muscles as reflected in Vt, Vi, Fb, and Ti/Ttot. The results also indicated that the contribution of these factors to the intensity of breathlessness differs quantitatively between loaded and unloaded breathing. Thus, in normal unloaded breathing the velocity and degree of shortening are important factors contributing to breathlessness during exercise; with resistive loading the inspiratory pressure, the velocity, and the duty cycle are important; with elastic loading the inspiratory pressure, the extent of shortening, and the frequency are important.</p> <p>The major contributions of these studies were in quantifying the intensity of breathlessness, and defining both the factors contributing to breathlessness and the relative importance of each.</p> / Doctor of Philosophy (PhD)

<p>Despite recent interest in developing novel microparticle (MP)-based antigen delivery systems for mucosal vaccination, existing MP formulations possess a number of liabilities potentially precluding their widespread use. Nevertheless, there is sufficient evidence to suggest that if immunogenically stable vaccine material can be incorporated into biocompatible/biodegradable MPs and delivered to various mucosal surfaces, this may be an effective way to elicit protective systemic and mucosal immunity. To overcome difficulties associated with existing MP formulations, the present work describes the development of a novel polymergrafted starch MP system which is capable of entrapping a wide variety of soluble antigens under mild conditions and which will elicit efficacious immune responses following intragastric (i.g.) or intranasal (i.n.) administration. The novel MP delivery technology was developed using starch, a well-studied, biologically acceptable and non-toxic material when given orally or parenterally. Antigen-containing starch MP were subsequently grafted with a hydrophobic silicone [3-(triethoxysilyl)-propyl-terminated polydimethylsiloxane, TS-PDMS] which, it was hypothesized, would protect microentrapped antigen from the deleterious environment found in the gastrointestinal (GI) tract, facilitate MP uptake by M cells overlying mucosae-associated lymphoid tissues (MALT) and/or act as an adjuvant or immunopotentiator. Moreover, following selective transport into mucosal inductive sites, antigen-containing MPs should stimulate the generation of robust antigen-specific disseminating mucosal and circulating humoral immune responses. In the work reported here, it was demonstrated that antigen-containing TS-PDMS-grafted MPs could be fabricated under mild conditions, allowing for the successful entrapment of a variety of protein and peptide antigens without any demonstrable loss in immunogenicity. Furthermore, it demonstrated that under acidic conditions, protein release from MPs was retarded when MPs were grafted with TS-PDMS. Although protein release from TS-PDMS-grafted MPs compared to ungrafted MPs was hindered, microentrapped protein was still released from TS-PDMS-grafted MPs, thus demonstrating that the MP composition allowed for entrapped proteins to readily learn from the MP matrix. Overall, these observations suggested that TS-PDMS-grafted MPs could serve as an efficacious MP-based mucosal vaccine delivery system. To be considered an attractive alternative to current mucosal MP vaccine delivery vehicles, TS-PDMS-grafted MPs should incite both local mucosal and systemic humoral immunity following mucosal administration of low doses of microentrapped antigen. The studies in this report clearly demonstrated that oral immunization with very low doses of TS-PDMS-grafted MPs simulated both systemic and mucosal humoral immune responses. Indeed, the adjuvanticity of TS-PDMS-grafted MPs seems to have arisen via a unique physicochemical relationship occurring between protein antigen and silicone in the starch matrix. This led to a predominantly Th2-type immune response following oral MP administration of relatively small amounts of microentrapped antigen. Furthermore, serum antibody titres were augmented after an oral or systemic boost, suggesting that the initial immunization protocol stimulated serum antibody memory capability, an advantage when developing successful mucosal immunization strategies. Surprisingly, systemic antigen challenge failed to boost antigen-specific sera igA titres following i.g. immunization with microentrapped or soluble antigen. These results suggest Peyer's patch (PP)-stimulated igA lymphocytes migrate to mucosal lymphoid compartments following i.g. MP administration, while antigen-specific PP-stimulated IgC plasmacytes have the propensity to migrate to both mucosal and systemic lymphoid compartments. In addition to stimulating specific serum antibody responses, i.e. immunization with TS-PDMS-grafted or ungrafted MPs resulted in specific sIgA responses in the gut; this is in contrast to soluble antigen which was incapable of inciting specific intestinal immunity following i.g. administration. Thus, compared to soluble antigen, TS-PDMS-grafted MPs have immunopotentiating activity when delivered orally. The studies outlined in this work strongly suggest that i.g. administration of TS-PDMS-grafted MPs stimulated mucosal immunity via PP. Following i.g. immunization with a low dose of antigen-containing MP, but not soluble antigen, specific proliferation of PP cells was observed. Lymphocyte proliferation was subsequently observed in mesenteric lymph node (MLN) and splenic tissue. In contrast, antigen-specific lymphocyte proliferation was not observed in gut lamina propria (LP) lymphocytes following i.g. immunization with MPs, thus suggesting that MP-induced immunity was incited by MP uptake and processing solely by PP. It was shown also that i.n. immunization with low doses of microentrapped, but not soluble, antigen evoked robust circulating specific IgG responses, indicating that TS-PDMS-grafted MPs could enhance the immunogenicity of an i.n. administered soluble antigen. Indeed, i.n. immunization with microentrapped protein stimulated greater levels of specific sera IgG than was observed after i.g. MP administration with equal amounts of microentrapped antigen. However, unlike i.g. TS-PDMS-grafted MP immunization, antigen specific IgA was not detected in local mucosal secretions or sera following i.n. immunization with microentrapped antigen, thereby suggesting that i.n. immunization expresses unique immune responses compared to those evoked after comparable i.g. immunization protocols. Although that nasal-associated lymphoid tissue (NALT) is considered to be the equivalent of Waldeyer's ring in humans, the exact process of generating immune responses when NALT is exposed to antigen is not clear. The present work describes the development of a novel, rapid and precide method for isolating NALT from mice to study its immune function and cell populations. Following the development of this precise NALT isolation technique, the pathway of i.n. administered TS-PDMS-grafted MPs was examined. It was demonstrated that i.n. immunization with low doses of microentrapped, but not soluble protein, evoked robust circulating IgG responses via NALT cell activation. Numerous antigen-specific spot-forming cells (SFCs) were observed in the NALT and later the posterior cervical lymph nodes (pCLN) and spleen (SPL), confirming the selective drainage of NALT cells exclusively to the pCLN following i.n. administration of a particulate antigen. Although B cell activity was observed in the pCLN following i.n. MP administration, no specific IgA was detected in any lymphoid tissue examined or in nasal secretions following i.n. immunization with TS-PDMS-grafted MPs. This suggested that either pCLN in mice are not intermediate in evoking sIgA responses in the nasopharynx or that TS-PDMS-grafted MPs are incapable of stimulating this arm of the murine immune system. However, since it was previously demonstrated that i.g. immunization with comparable doses of TS-PDMS-grafted MPs evoked intestinal IgA responses, the inability to detect antigen-specific IgA in nasal secretions probably reflects differences between murine NALT and PP and not a unique inability of TS-PDMS-grafted MPs to evoke secretory immunity in the nose. Thus, the present work describes the successful development of a novel polymer-grafted starch MP system which is capable of entrapping a wide variety of soluble antigens under mild conditions and which can elicit efficacious immune responses both orally and nasally, via MALT activation.</p> / Doctor of Philosophy (PhD)

<p>Classical force-velocity studies by A. V. Hill demonstrated that there was an optimal velocity for maximal power output of isolated muscles and human movements. Thus to study muscle performance during maximal dynamic exercise it is important to measure mechanical power output at several constant velocities of movement. At the start of this work no instruments were available to measure maximal power during isokinetic movements over a wide range of velocities. For this reason a cycle ergometer (CVE) was developed which restricted the crank velocities to chosen upper limits, despite maximal efforts by the subject.</p> <p>Measurements were obtained in male subjects of maximal peak torque generated over 81% of the functional range. There was a consistent inverse linear relationship between peak torque, and crank velocity, and the results were reproducible from day to day. Considerable inter-subject variability in peak torque was accounted for only partly by differences in thigh muscle volume. Maximal peak power occurred at various crank velocities ranging from 120 to 160 rpm; differences in muscle fibre types may have contributed to the variation observed. Maximal power occurred when the force equalled 0.3 to 0.4 of the predicted maximal isometric tension, in agreement with Hill's studies.</p> <p>Torque, work and power were also measured during 30 s of maximal effort at 60, 100 and 140 rpm. Increases in crank velocity were associated with both a higher initial power, and a greater rate and extent of decline in power, but total work was similar. The greater decline at faster velocities may reflect differences in energy metabolism, or motor unit activation. In addition to defining the effects of velocity on maximal power output in healthy young subjects, the studies showed the CVE to be a sensitive, reliable instrument, with potential applications to the assessment of human muscle function in health and disease.</p> / Doctor of Philosophy (PhD)

<p>Many tumors express antigens that are capable of being recognized by effector cells of the immune system. However, these tumors persist in immunocompetent hosts. The mechanism by which immune effector cells are rendered unresponsive to these antigens is unknown. Previous data from our laboratory demonstrated the use of adenoviral vectors constructed to express single cytokines (IL-2 and IL-12) in mediating regression of tumors in a mouse mammary adenocarcinoma model. The ability of these molecules to enhance the immune response to effectively regress these tumors was hampered by toxicities associated with these factors. To over come this toxicity and enhance the efficacy of IL-2, double recombinant adenoviral vectors expressing the co-stimulatory molecules B7-1 or B7-2 with IL-2 were constructed to allow for the expression of both immuno-modulatory molecules from the same cell. An alternative approach to enhancing the efficacy of these factors involved the use of double recombinant adenoviral vectors expressing combinations of the chemokine lymphotactin with IL-2 and IL-12. Mammary tumor cells from transgenic mice expressing polyoma middle T (PyMT) or an oncogenic form of the proto-oncogene Neu (Neu 8142) were transplated into syngeneic FVB/N recipients to establish the subcutaneous tumor models. Intra-tumoral injection of adenoviral vectors constructed to express murine B7-1 or B7-2 and IL-2 resulted in regression of PyMT or Neu (8142) tumor bearing mice, superior to that observed for the single cytokine or co-stimulatory molecule expressing vectors. Cured mice were shown to have generated systemic immunity to a subsequent challenge with fresh tumor cells and tumor specific cytotoxic T lymphocyte activity could be detected. Intra-tumoral injection of adenoviral vectors constructed to express lymphotactin IL-2 or IL-12 demonstrated complete regression in a small number of PyMT tumor bearing mice. None of these single cytokine or chemokine expressing vectors was capable of inducing regression of Neu (8142) tumors. In contrast, administration of vectors expressing either a combination of lymphotactin and IL-2 or lymphotactin and IL-12 resulted in an increased incidence of complete regression in both tumor models. All regressed mice demonstrated systemic protection and anti-PyMT CTL. The activity of these vectors in the Neu tumor model is different and suggests some variance in the immunogenicity of the two tumor models. These results demonstrate the ability of double recombinant vectors expressing the co-stimulatory molecules (B7-1 or B7-2) or lymphotactin in combination with IL-2 or IL-12 to enhance the anti-tumor effects of either cytokine alone. These vectors demonstrated no associated toxicities, due to the reduced levels of cytokine that are needed to maximally co-operate with the co-stimulatory or chemokine molecules. Development of this type of vector system will lead to much improved clinical protocols for cancer therapy.</p> / Doctor of Philosophy (PhD)