<p>Calcium (Ca2+ ) for contraction of smooth muscle comes from two sources: release of Ca2+ from an internal site, the sarcoplasmic reticulum (SR), and influx of Ca2+ from the extracellular space across the plasma membrane (PM). We hypothesize that caveolae, flask-shaped invaginations of the PM, are the protected source of Ca2+ . This thesis provides biochemical evidence that supports the hypothesis of caveolae involvement in Ca 2+ handling. Caveolae were isolated from canine tracheal smooth muscle by detergent treatment of PM-derived microsomes. Immunoprecipitation experiments confirmed the presence of calsequestrin and calreticulin in caveolae. Antibodies to caveolin coimmunoprecipitated caveolin with calsequestrin and calreticulin. These experiments also indicated that at least some of the associated calsequestrin and calreticulin are located on the cytoplasmic face of each caveola, since no part of the caveolin protein crosses into the luminal side of each caveola. Immunohistochemistry of fixed tracheal smooth muscle cryosections confirmed that the PM Ca2+ pump, nNOS, and caveolin were all located on the cell periphery, while the SR Ca2+ pump is located deeper in the cell. Based on the results presented here and our previous results of contractility experiments, a model of Ca2+ handling for airway smooth muscle is proposed. This is an extension of the superficial buffer barrier hypothesis first proposed by van Breemen (1986). In our model, caveolae provide the physical basis for the junctional space between the PM and SR. Ca2+ can move into the lumen of the caveolae via the PM Ca2+ pump, and be released from caveolae into the junctional space via L-type Ca 2+ channels. Calsequestrin and calreticulin, located on the cytoplasmic face of caveolae, may act as a physical barrier facilitating the direct refilling of the closely associated SR with Ca2+ . Similarly, nitric oxide, produced by the nNOS located on the caveolae, may inhibit release of Ca 2+ by the SR, thereby enhancing refilling. The Ca2+ in the lumen of the caveolae may be retained by calsequestrin, calreticulin, or another unidentified Ca2+ binding protein located in the caveolae lumen. (Abstract shortened by UMI.)</p> / Doctor of Philosophy (PhD)

<p>In recent years attention has been drawn to the possible role of iron in a number pathological conditions including ischemia/reperfusion, halogenated aryl hydrocarbon toxicity as well as hereditary and transfusion-dependent iron overload. Although several different mechanisms may be operating in each of these situations, one of the chemical processes thought to be involved is lipid peroxidation (LP). LP is the free radical-mediated, oxidation of polyunsaturated fatty acids which has the potential to cause membrane, protein and nucleic acid damage. Using the non-invasive technique of measuring whole-body ethane and pentane production as an index of in vivo LP in mice, it is shown here that the ferric-NTA complex (Fe³⁺-NTA) is the most potent stimulus of this process yet described. Fe³⁺-NTA was found to be lethal to mice at doses above 5 mg Fe³⁺/kg body weight with a dose of 7.5 mg Fe³⁺/kg, giving rise to a greater than 750-fold rise in the rate of ethane and pentane production 30 min following treatment of mice.</p> <p>Liver and kidney were identified as likely sites of alkane origination. Radioactive iron (presented as ⁵⁹Fe³⁺-NTA) was concentrated primarily in the liver, and, to a lesser extent, in the kidney. In addition, estimation of products of lipid peroxidation with the 2-thiobarbituric acid (TBA) test confirmed liver and kidney as locations where Fe³⁺-NTA-stimulated LP had occurred in mice treated with the iron complex. Further work, therefore, examined the mechanism by which Fe³⁺-NTA promotes LP in liver tissue.</p> <p>Isolated rat hepatocytes were found to be susceptible to Fe³⁺-NTA-dependent LP when the process was monitored by measuring the formation of ethane and TBA reacting compounds or emission of low level chemiluminescence. LP and iron association with cells was found to be temperature dependent, both processes being inhibited by incubation at 4°. Subcellular fractionation of rat liver indicated that Fe³⁺-NTA was capable of promoting LP in both mitochondrial and microsomal (primarily, endoplasmic reticulum) only in the presence of reduced pyridine nucleotides (NADH or NADPH). In this, Fe³⁺-NTA was similar to previous reports regarding the ferric-adenosine diphosphate complex (Fe³⁺-ADP).</p> <p>In depth comparison of the promotion of microsomal LP by Fe³⁺-NTA and Fe³⁺-ADP suggested a common biochemical mechanism, central to which is the reduction of the ferric complex to ferrous. This led to an examination of the initiation of microsomal peroxidation by "free" ferrous ions. In contrast to NADPH/Fe³⁺-NTA-dependent LP which is very rapid, Fe²⁺ addition to microsomal suspensions caused LP only after a variable lag period. This delay could be reduced or abolished by simultaneous addition of Fe³⁺ or by conditions which would accelerate the oxidation of Fe²⁺ to Fe³⁺. In contrast, the delay Iength was increased by antioxidants which acted by hydrogen donation or one electron transfer indicating that the delay period represents time required for the formation of a species capable of initiating microsomal LP. Furthermore, additional Fe²⁺ was also found to increase the delay length. It is proposed that the mechanism behind the delayed initiation of LP is one electron transfer from excess Fe²⁺ to an as yet unidentified initiating species (possibly and Fe²⁺-O₂-Fe³⁺ complex) thus quenching it. Lipid peroxidation is then initiated following consumption of the surplus Fe²⁺. The lack of a delay in NADPH/Fe³⁺-complex dependent LP would be due to the rapid formation of an optimal ratio of Fe²⁺ : Fe³⁺; however, the mechanism of initiation of LP would appear to be similar.</p> <p>The work presented in this thesis demonstrates the remarkable potential of small amounts of iron, when presented appropriately, to stimulate in vivo lipid peroxidation on a massive scale and with apparently lethal consequences. Furthermore the common mechanism of initiation of LP by iron complexes and the observation that one electron reduction prevents the initial step(s) in LP may prove invaluable in the development of antioxidant drugs for the prevention of iron-dependent cellular damage.</p> / Doctor of Philosophy (PhD)

<p>Giardia muris is an intestinal parasite of mice. It has a simple life cycle and is non-invasive. Therefore, G. muris infection provides a model to study immune mechanisms that operate at mucosal surfaces. Immunocompetent mice eliminate primary G. muris infections. T cell-dependent humoral immune mechanisms are involved in this process.</p> <p>The CBA/N mouse bears an X-linked immunodeficiency gene (Xid), the expression of which results in defective B cell maturation and consequent impairment of certain humoral immune responses. The antibody responses of CBA/N mice are particularly defective in certain isotypes and specificities.</p> <p>CBA/N mice fail to eliminate G. muris. The major focus of this dissertation was an attempt to elucidate the basis for chronic giardiasis in this strain.</p> <p>Cellular reconstitution experiments showed that the ability to eliminate G. muris was transferred to CBA/N mice with lymphoid cells from immunocompetent, CBA/Ca mice. Reconstitution required prior irradiation of recipient mice, and was not effective with semi-purified B cells and T cells. These results indicate that conventional B cells and T cells are insufficient, and that another cell type is also required. This cell may be the Lyl+ B cell.</p> <p>CBA/N mice make quantitatively deficient serum IgG antibody responses to G. muds infection. Providing CBA/N mice with this antibody failed to induce elimination of the parasite, thus this isotype defect was ruled out as the cause of their susceptibility to chronic giardiasis.</p> <p>Analysis of G. muris antigen recognition failed to reveal a specificity defect in the antibody response of CBA/N mice. However, a glycolipid component of G. muris bound serum IgM from CBA/J and BALB/c mice, but not serum IgM from CBA/N mice. These results indicate a possible structural defect in IgM from CBA/N mice.</p> <p>Although unable to eliminate primary G. muris infection, drug-cured CBA/N mice are resistant to reinfection. These results indicate that the immune mechanisms that mediate elimination of G. muris are different from those that mediate resistance to reinfection.</p> / Doctor of Philosophy (PhD)

<p>In response to an enteric infection, the host mobilizes inflammatory and immune cells to combat the invading pathogen. Studies suggest that physiologic tissues such as smooth muscle are also recruited to aid in host defense, and that this recruitment depends on the hosts immune response. The purpose of this thesis was to use the intestinal phase of a primary Trichinella spiralis infection in mice to study the accompanying changes in smooth muscle contraction, including their role in host defense and their immunological basis. Naive mice were infected with the nematode parasite T. spiralis . Infection caused a significant increase in intestinal longitudinal muscle contraction in response to carbachol in vitro . Testing several mouse strains, we found that the strains that developed the greatest increases in muscle contraction also expelled the infection the most rapidly. Two phases of increased contraction were identified, the early phase when muscle contraction first increases, and the persistent phase, when the increased muscle tension was sustained until at least day 21 post-infection. We also found that infected mice lacking T cells, and specifically CD4 +ve T cells, exhibited an attenuated increase in both phases of muscle contraction. We also tested the role of the Th2 cytokines, interleukins 4 and 5, which are produced by CD4 +ve T cells during a nematode infection. We found that during infection, IL-5 deficient mice developed a normal early phase of increased muscle contraction, but were significantly impaired in the persistent muscle response. We also examined the role of IL-4, through gene transfer. Following surgery, we infected the intestinal surface with either control adenoviral vectors, or a virus encoding the cytokine IL-4. IL-4 overexpression significantly increased intestinal muscle contraction, while the control virus had no effect. In summary, my studies show that intestinal longitudinal muscle function is subject to immunomodulation, specifically by the actions of CD4 +ve T cells, and through the cytokine mediators IL-4 and 5.</p> / Doctor of Philosophy (PhD)

<p>The electrical pacemaker slow wave is responsible for the generation of anally propagating phasic contractions underlying the peristaltic motor activity of the gastrointestinal musculature. Yet, the cellular origins of the slow wave and mechanisms of the slow wave regulation or generation still remain unresolved and constituted primary goals of the current thesis. As described in detail in Chapters Three-Six, spontaneously genetic knock out mice with genetic mutations affecting the structure (W / Wν mice), expression (Wbd / Wbd mice), or the ligand (Sl / Sld mice) of the kit tyrosine kinase receptor were shown to lack both the network of interstitial cells of Cajal associated with the myenteric plexus and the slow wave activity in the small intestine, hence, supporting the proposed role of the interstitial cells of Cajal as pacemaker cells responsible for the slow wave generation. In the absence of the slow wave, the mutant musculature was either electrically quiescent or showed action potentials in regular or irregular patterns as recorded with a standard microelectode technique. The observed action potentials were also clearly distinguished from the slow waves by their shape and pharmacological sensitivities to L-type Ca2+ channel and K+ channel blockade. The mechanisms of the slow wave generation and regulation are addressed in Chapters Seven-Nine. The data indicate that the slow wave generation involves primarily Na+ and Ca2+ conductances not mediated by TTX- or mexiletine-sensitive Na+ channels, gadolinium sensitive nonselective cation channels, or L-type Ca2+ channels. Cl- channels may be also involved in the regulation but not in the slow wave initiation. Pharmacological agents acting on cytosolic Ca2+ , SR Ca2+ ATPase, and intracellular Ca 2+ release mechanisms support the role of intracellular Ca 2+ release mechanisms, sensitive to IP3 , in the regulation of the slow wave frequency and amplitude. Furthermore, activation of the Ca 2+ induced Ca2+ release (CICR) mechanism leads to depolarization not mediated predominantly by chloride channels nor likely by KCa channels. The CICR may be also involved in the regulation of the slow wave. These experiments importantly identify intracellular metabolic pathways that may potentially lead to the development of therapeutic approaches aimed at treating certain gastrointestinal motor disorders by modifying the slow wave frequency or amplitude.</p> / Doctor of Philosophy (PhD)

<p>For the development of many cell types, terminal differentiation and continued cell cycle progression are incompatible processes. Rb, p107 and p130 comprise a gene family encoding transcriptional regulators that act within a complex network to control exit from, and progression through the cell cycle. The distinct requirements for p130 and p107 were assessed in vivo using homologous recombination in embryonic stem (ES) cells. p130 expression is ubiquitous, although the level of expression varies between tissues. As well, its induction during neuronal cell differentiation is consistent with p130 function accompanying terminal differentiation. p130 deficiency was incompatible with embryo survival in the hybrid 129Sv;Balb/c genetic background. The mutant embryos died between E11-13 and exhibited striking delays in growth and development at 10.5 dpc with specific deficits in neurogenesis, somitogenesis and cardiogenesis. p130 appears to be required for the maintenance and survival of specific cell types, most notably neuronal cells. The data indicate that the p130 gene is essential for normal development, but in a strain-specific manner. On a hybrid 129Sv;C57B1/6 background, the p130 mutants exhibited no phenotype. The p107 mutants were viable and fertile, indicating that p107 function was adequately compensated by other proteins during development, potentially Rb and p130. The p107-/- mice did however exhibit a postnatal growth deficit and a diathetic myeloid proliferative disorder. The accelerated proliferation and cell-cycle kinetics of p107-/- EF indicated that p107 functions, in part, to regulate cyclin expression and cell cycle progression. p107-/- myoblasts also exhibited accelerated prliferation and aberrant in vitro differentiation. Lastly, the p107-/- phenotype was also dependent on the genetic strain, indicating the presence of modifying genes. The mice produced in these studies can be assessed for genes that modify the phenotypes in these different strains, potentially revealing epistatic relations to p107 and p130. Although both striking and subtle cell-specific phenotypes were exhibited, these experiments strongly reconfirm that functional overlap and compensation exist within the Rb family. The overlapping expression patterns and apparent functional relations indicate that p130, p107 and Rb regulate transitions in a concerted manner during cell proliferation, and cell cycle exit and entrance.</p> / Doctor of Philosophy (PhD)

<p>The spontaneously hypertensive rat (SHR) was used as a model of human essential hypertension. The overall hypothesis was that hypertrophy of the smooth muscle layer of small muscular arteries in essential hypertension results in greater contractility of these vessels that then results in elevated total peripheral resistance and higher blood pressures. Elevated total peripheral resistance and small artery hypertrophy are well documented in the SHR, however, it remains unknown if these changes are the cause of result of elevated blood pressure. For this reason, we have focused our studies on young SHR during the initiation of hypertension to attempt to separate cause from effect. Studies were done to determine when SHR's blood pressure begins to differ from its normotensive control the Wistar-Kyoto rat (WKY). We found that blood pressure began to diverge between SHR and WKY at four weeks of age. Structural and functional differences between small muscular arteries from the mesenteric vascular bed of 4-week old SHR and age matched WKY controls were studied using a new morphometric protcol involving confocal microscopy and a pressurized artery myograph. Arteries from SHR had a larger medial volume, increased number of smooth muscle cell layers, but similar lumen size when compared with WKY in the maximally relaxed condition. Functional studies showed that SHR arteries contracted more in response to stimulation by KC1 and norepinephrine, resulting in significantly smaller lumen size in these vessels as compared to WKY. We concluded that structural and functional differences in SHR arteries were primary changes which may contribute to the development of hypertension. Further studies were conducted to determine if a differential incidence of apoptosis during the development of SHR and WKY arteries contributes to the structural differences. One to two week old animals were used for these studies since at this time the structure was similar between the strains. To measure the incidence of apoptosis, we used both DNA laddering and end labeling. It was found that SHR had a significantly decreased incidence of apoptosis over WKY. The cellular nature of the medial layer hypertrophy in SHR at 4-weeks was also assessed. Numerical density of smooth muscle cell nuclei in the medial layer was measured with a three dimensional disector method under confocal microscopy. We found that the numerical density of medial smooth muscle cells was significantly less in SHR than WKY, and the number of smooth muscle cells was significantly less in SHR than WKY, and the number of smooth muscle cells was similar between the strains. The smooth muscle cell length from SHR was significantly longer than WKY. We concluded that increased smooth muscle cell length in prehypertensive SHR is responsible for their increased medial volume. These studies have shown that medial layer hypertrophy due to smooth muscle cell lengthening in the small muscular arteries of SHR which increases their contractile ability, occurs at the initiation of hypertension. This evidence demonstrates that structural and functional changes in these SHR arteries can not be the result of increased blood pressure but may be a factor causing hypertension by increasing the total peripheral resistance in these animals.</p> / Doctor of Philosophy (PhD)

<p>Food allergies are a significant clinical problem, with symptoms including diarrhea, vomiting, or systemic anaphylaxis. To elicit allergic reactions, antigens must first cross the intestinal epithelium. The purpose of my studies was to examine macromolecular transport across intestinal epithelium, and the effect of sensitization and immune activation on transepithelial antigen transport. Rats were sensitized to a model protein antigen, horseradish peroxidase (HRP) by injection with adjuvants. Intestinal segments were removed and mounted in Ussing chambers for the study for transepithelial movement of HRP. Electon microscopy analysis of HRP transport showed that specifically sensitized rats transported HRP across the epithelium in greater amounts, and more rapidly, than naive controls or rats sensitized to an irrelevant antigen. After the hypersensitivity response, there was a significant increase in HR flux across the intestinal epithelium in HRP sensitized, but not control rats. This was accompanied by an opening of the epithelial tight junctions to allow paracellular flow of antigen. Sensitized mast cell deficient rats also had an enhanced initial uptake of antigen, but did not develop a non-specific decrease in epithelial barrier function. The role of interleukin-4 (IL-4) in the regulation of transepithelial antigen transport was examined. Treatment of human epithelial monolayers with IL-4, or with serum from atopic patients, caused a significant increase in transepithelial transport of HRP. Antibodies against IL-4 abolished the effect of atopic serum on transepithelial HRP transport. Electron microscopy analysis showed an increase in both transcellular and paracellular HRP transport. These studies show that transepithelial transport of antigen is profoundly altered by sensitization, and that mast cells and interleukin-4 enhance the delivery of antigen across the intestinal epithelium.</p> / Doctor of Philosophy (PhD)

<p>Cancer of the cervix is the second most common form of cancer in women worldwide. The natural history of cervical cancer is thought to involve sequential changes from varying grades of precursor intraepithelial lesions called CIN. However, little is known of the risk factors that can predict the oncogenic potential of a particular CIN lesion. Based on their preferential occurrence in cervical cancers and their potential oncogenic properties, the human papillomaviruses (HPV) particularly the genotypes 16, 18, 31, 33, 35 and 42 have been implicated in the etiology of invasive cervical cancer. However, these viruses could occur as either secondary pathogen of cancer or as predictor of those CIN lesions that are likely to progress to invasive disease. The hypothesis that was verified in this study was that HPV 16,33 and 18 are likely to be predictive of CIN lesions that progress to invasive cancer.</p> <p>First of all, in order to characterize the prevalent type of HPV in the target study population of B.C., a cross-sectional study was conducted and the presence of specific HPV types ascertained by the tissue in situ hybridization. The frequency of HPV types 16, or 33, was found to vary with the severity of the CIN grades, in contrast to the frequency of HPV types 6/11 and 18 that segregated independently of the CIN grades.</p> <p>Next, a case-control study was undertaken to verify the main hypothesis of HPV being predictive of CIN lesion progression to invasion. It was reasoned that the particular HPV would occur at higher frequency in CIN biopsies of cervical cancer cases than in CIN biopsies of noncases (controls). A total of 47 cases and 94 controls were enrolled from patients registered by the Cancer Control Agency of the Province of British Columbia. A case was defined as a post-pubertal woman with invasive disease and who had a CIN diagnosis at least two years prior to the invasive disease. For each case an attempt was made to enrol two control matched on grade of CIN and year of diagnosis. On each subject attempt was made to gather demographic informations that are known to be associated with cervical cancer. The HPV probes that were used included HPV 16/33 and HPV 6/11. The relative frequency of occurrence of specific HPV in the preinvasive biopsies of cases and controls were as folow: HPV 16/33 occurred in 10.6% of controls and 12.8% of cases; HPV 18 was found in 3.2% of controls and 8.7% of cases and HPV 6/11 in 2.2% of controls and 8.7% of cases. Conditional Chi-square analysis showed that the difference in the proportions of HPV positivity between cases and controls was compatible with sampling variation. Hence, with a statistical power approximately 60%, it was concluded that particular HPV could not be predictive of CIN lesions progression in the sample of population that was studied.</p> <p>However an excess risk for incurring cervical cancer, by being exposed to particular HPV at the CIN stage, was noted. The relative risk for HPV 16/33 was 2.34, [95% CI 0.70 to 7.66]; for HPV 18 was 2.45, [95% CI 0.22 to 27.80]; for HPV 6/11 2.19, [95% CI 0.39 to 12.42] or for all HPV combined was 1.87, [95% CI 0.55 to 6.28].</p> <p>Interestingly, a comparison of the frequency of HPV occurrences in the case-control study with that in the cross-sectional study revealed a lower rate of HPV positivity in the case-control component. This could possibly be due to a cohort effect.</p> / Doctor of Philosophy (PhD)

<p>Rat transferrin (RTf) was used to study the interaction of transferrin' (Tf) with the liver, with special reference to the çlycan of Tf.</p> <p>Iron uptake from Tf by cells is believed to occur by Tf receptor (TfR)-mediated endocytosis. Results from the present studies revealed that over a large range of competing diferric RTf concentrations, a constant percentage of iron is uptaken from RTf by the liver in vivo, and by hepatocytes in vitro. Hepatocytes were found to contain ~129,000 TfR/cell with ~40% expressed on the surface as estimated by both a polyclonal anti-TfR antiserum, produced as part of this thesis work, and a monoclonal anti-TfR antibody. On the basis of this estimate it was calculated that TfRs are not present in sufficient quantities to account for the observed uptake of iron. Studies of uptake and release of polyvinyl-pyrrolidone (PVP), RTf, and iron (as diferric Tf) were carried out in suspended hepatocytes. These studies demonstrated that the most likely mechanism to account for the results and to explain the iron uptake is "mixed-type" pinocytosis. The uptake of iron was found to be modulated by the type of glycan on RTf.</p> <p>On the basis of glycan microheterogeneity, at least six subforms of RTf are found to exist in rat plasma. These subforms are RTf-1, RTf-2·and RTf-3 as resolved by concanavalin A; ~20% of each is fucosylated and ~80% non-fucosylated. (The presence of fucose was found to have no measurable effect on catabolic rate, plasma iron disappearance or iron donation to liver in vivo or hepatocytes in vitro.) The slalylated subforms have different half-lives (RTf-1>RTf-2>RTf-3) with RTf-1 being significantly longer than RTf-3. Comparison of plasma iron disappearance and rates of iron donation to liver in suggested a trend (RTf-l>RTf-3>RTf-2) which was reproduced and found to be significant in studies with hepatocytes: iron uptake by hepatocytes from RTf-1 and RTf-3 could be competitively inhibited by an excess of the homologous subform. Desialylation of the subforms (RAsTfs) significantly reduced the half-lives and altered the order (RAsTf-3>RAsTf-1>RAsTf-2) with RAsTf-3 being significantly longer than RAsTf-2. The desialylated subforms were superior donors of iron to the liver in vivo. Studies to explain the enhanced rate of iron delivery by RAsTf, discounted the possibility of differing rates of iron release, but allowed postulation of a synergistic dual receptor mechanism. Results from experiments with hepatocytes in vitro supported the proposed mechanism. It is concluded that subtle differences in glycan structure can result in functional differences between Tf subforms.</p> / Doctor of Philosophy (PhD)