<p>Moderate to high intensity exercise generates lactate and hydrogen ions which accumulate in both blood and muscle and are thought to contribute to the development of fatigue. Alteration in the extracellular pH has been shown to influence the appearance of lactate in plasma, acidosis decreasing and alkalosis increasing blood lactate concentration. The present research was designed to explore the potential mechanisms by which acid-base alterations influence lactate production and the associated metabolic changes in exercise. Normal subjects took part in two studies. Acidosis induced by ingestion of ammonium chloride and alkalosis via ingestion of sodium bicarbonate were compared to control during rest and exercise. Needle biopsies of the vastus lateralis muscle, blood flow and arterial and femoral venous blood samples were taken. Acidosis resulted in decreased lactate production and efflux, secondary to decreased glycogenolysis from reduced transformation of glycogen phosphorylase to its active form and the lack of accumulation of positive allosteric modulators. Lower glycogen utilization resulted and was associated with an increase in free fatty acid (FFA) utilization from intramuscular stores. Pyruvate dehydrogenase activity was lower. Alkalosis had opposite effects: greater lactate production, efflux, glycogenolysis, pyruvate dehydrogenase activity and glycogen utilization, with lower FFA utilization. However, the increased glycogenolysis resulted from allosteric activation of glycogen phosphorylase through increases in the concentration of its positive modulators adenosine monophosphate and substrate free inorganic phosphate. These studies conclude that alterations in lactate production result entirely from a mismatch in the catalytic rates of glycogen phosphorylase and pyruvate dehydrogenase. Additionally, these studies identified for the first time the mechanisms responsible for the complex interplay of regulatory enzyme activity, fuel utilization and the importance of acid-base homeostasis in its control.</p> / Doctor of Philosophy (PhD)

<p>Endothelin-1, the most potent endogenous vasoconstrictor peptide known, plays a key role in regulating coronary artery vascular tone. Reactive oxygen species generated during cardiac ischemia-reperfusion cause several types of damage to cardiovascular tissues, in particular to the intracellular Ca2+ -regulating mechanisms. This thesis explores the endothelin-1 receptor types in coronary artery smooth muscle and their signal transduction pathways and the effects of reactive oxygen on them . Endothelin-1 mediated contraction of de-endothelialized pig coronary artery rings. There are two types of endothelin receptors known: ETA and ETB . Using ETA and ETB receptors selective agonists and antagonists, the contraction mediated by ETB -receptors was approximately 20% and the remaining was due to ETA receptors. Ca2+ pools mobilized by the two receptors were similar except that the ETB receptor activation utilized more of the intracellular Ca2+ pool than the ETA activation. 125 I-ET-1 binding to microsomes isolated from smooth muscle of this artery also showed ETA and ETB binding sites with most binding occurring at the ETA sites. Thus, the endothelin-1 induced vasoconstrictor response in pig coronary artery smooth muscle involves both ETA and ETB -receptors with ETA being predominant. Pretreating the artery with hydrogen peroxide inhibited the subsequent contraction upon ETA or ETB receptor activation. However, the ETB -mediated contraction was significantly (p < 0.05) sensitive to peroxide (IC50 = 0.3 ± 0.08 mM) than the ETA receptor mediated contraction (IC50 = 1 ± 0.3 mM). Pretreating smooth muscle cells cultured from pig coronary artery with 0.3 mM hydrogen peroxide inhibited the endothelin-1 induced increase in [Ca2+ ] i by more than 95%. Thus the exposure to reactive oxygen damaged the ETB receptor mediated contractions preferentially, possibly as a result of a Ca2+ -independent component in ETA receptor mediated contractions. ATP-dependent azide-insensitive oxalate-stimulated Ca2+ -uptake in permeabilized smooth muscle cells cultured from pig coronary artery exhibited the expected kinetic properties of the sarcoplasmic reticulum (SR) Ca 2+ -pump. Under optimum conditions, inositol 1,4,5 trisphosphate (IP 3 ) released up to 65% of 45 Ca2+ -loaded into the SR. Pretreating the cells with hydrogen peroxide or superoxide did not affect the IP3 dependent Ca2+ -release but inhibited the Ca2+ -uptake in the SR. Peroxide was equipotent in inhibiting 45 Ca2+ -loading into IP3 -sensitive and IP 3 -insensitive Ca2+ pools but superoxide inhibited loading only into the IP3 -sensitive pool indicating that the SR Ca 2+ pump in vascular smooth muscle cells is heterogenous. These results indicate that both ETA and ETB receptors are involved in ET-1 mediated contraction in smooth muscle pig coronary artery, with similar Ca2+ utilization pathways but the ETA receptors may also induce contraction in part by a Ca2+ -independent mechanism. The peroxide pretreatment damages the SR Ca2+ pump and this leads to a diminished contraction by endothelin-1, with the exception of the Ca2+ -independent mechanism(s) associated with ET A receptor activation which may be resistant to peroxide. This Ca 2+ -independent mechanism provides a potential therapeutic target for diseases where ETA plays a major role. The second major finding is the heterogeneity of SR Ca2+ pool which can also be used in designing pharmacological agents specific to a distinct component of the SR Ca2+ pool.</p> / Doctor of Philosophy (PhD)

<p>An ovarian carcinoma culture model is the focus of this investigation based on a unique association between an elevated mitochondrial membrane potential and resistance to the chemotherapeutic agent cisplatin. The model consists of the 2008 parental cell line, the C13* subline that has acquired stable resistance to cisplatin and the RH4 revertant line that has regained sensitivity to cisplatin following selection with the anti-mitochondrial agent rhodamine 123. The cumulative data for resistance mechanisms that operate in this model suggest that increased tolerance to damage is a major determinant of the overall cytotoxic response to cisplatin. We have adopted the general hypothesis that mitochondria play a role in the mediation of resistance to cisplatin, and have furthered the investigation of these cells in terms of their energetic and redox balance characteristics. No significant differences exist between 2008, C13* and RH4 cells in terms of glycolytic capacity but when assayed for mitochondrial function both the cisplatin-resistant C13* and the cisplatin-sensitive RH4 cells have a significantly reduced capacity for mitochondrial respiration. This common characteristic indicated that alterations in energy production do not influence resistance, but perhaps other associated mitochondrial activities like reactive oxygen species production could have an impact. Measurement of extracellular hydrogen peroxide (H2 O2 ) production revealed a significant increase in the C13* versus 2008 cell population, which is the result of contributions by multiple intracellular sources including mitochondria and potentially novel flavoproteins. The exact relationship between increased H2 O2 production and cisplatin resistance is not yet defined but one major implication is the evidence for extracellular H2 O2 as a required autocrine growth factor for various cultured cell lines including 2008 and C13* cells. The pro-proliferation role of H2 O2 suggests it could influence the balance of survival versus death effector signals that may have an impact on the threshold of apoptosis initiation in these cells.</p> / Doctor of Philosophy (PhD)

<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)