Doctor of Philosophy / Department of Anatomy and Physiology / David C. Poole / The purpose of this dissertation is to expand our knowledge on the physiological effects of the ubiquitous signaling molecule nitric oxide (NO). Focus is given to the impacts of the nitrate (NO[subscript]3[superscript]-) nitrite (NO[subscript]2[superscript]-) NO pathway on skeletal muscle vascular and metabolic function during exercise. The NO[subscript]3[superscript]--NO[subscript]2[superscript]--NO pathway has garnered tremendous research interest due to its ability to upregulate NO bioavailability independently of NO synthase (NOS) function and thus impact the metabolic responses to exercise. Chapter 2 demonstrates that NO[subscript]3[superscript]- supplementation via beetroot juice (BR) augments the skeletal muscle vascular responses to exercise. Five days of BR supplementation resulted in a significantly higher skeletal muscle blood flow (BF) and vascular conductance (VC) during exercise when compared to control. The increases in BF and VC were preferentially directed to muscles and muscle portions comprised predominantly of fast twitch fibers. Furthermore, exercising blood [lactate] was reduced, suggesting improved metabolic control. In chapter 3, BR resulted in a slower fall in the microvascular PO[subscript]2 (PO[subscript]2[subscript]m[subscript]v, the soul driving force for blood myocyte O[subscript]2 flux) during the crucial rest-contraction transition thereby preserving the pressure head needed to move O[subscript]2 from the capillary into the myocyte. Chapter 4 examines the effects of BR on fast vs. slow twitch muscles in which BR raised the PO[subscript]2[subscript]m[subscript]v during the steady state of muscle contractions in fast but not slow twitch muscles, likely due to the lower PO[subscript]2[subscript]m[subscript]v at rest and throughout muscle contractions within these tissues. Chapter 5 investigates the effects of direct arterial NO[subscript]2[superscript]- infusion on skeletal muscle BF and VC during exercise in rats with NOS blockade via N[superscript]G-nitro-L arginine methyl ester. NO[subscript]2[superscript]- infusion restored MAP and VC to levels observed in healthy control animals (with intact NOS function) highlighting the potential for a NO[subscript]2[superscript]- based therapy to positively impact vascular function in those with compromised NOS function such that is evident in many prevalent diseases. These results provide crucial mechanistic insight into the improved exercise tolerance observed in humans following NO[subscript]3[superscript]- supplementation whilst also challenging our current understanding of NO’s role in physiology and pathophysiology.

Doctor of Philosophy / Department of Anatomy and Physiology / Thomas J. Barstow / The primary aim of this dissertation was to describe the changes in microvascular hematocrit, as total[hemoglobin+myoglobin] (T[Hb+Mb] measured with near-infrared spectroscopy (NIRS), during post-occlusive reactive hyperemia (PORH). Mechanisms of reactive hyperemia within skeletal muscle were also explored. The investigation detailed in Chapter 2 of this dissertation found that the differing time courses of the kinetic responses of both oxy- and deoxy[Hb+Mb], are related to changes in T[Hb+Mb]. We also determined that adipose tissue thickness had no effect on a purely temporal analysis of NIRS data. In Chapter 3 we observed that brachial artery reactive hyperemia preceded changes in T[Hb+Mb] during reactive hyperemia. Assuming that myoglobin remained constant, we posited that changes in T[Hb+Mb] must reflect alterations in red blood cell concentration in the microvasculature, i.e., microvascular hematocrit. In Chapter 4 comparisons were made between brachial artery blood flow, cutaneous and skeletal muscle flux and T[Hb+Mb]. The conduit artery response was faster than the microvascular responses in all tissues. Within skeletal muscle, time to peak and the time constant for the on-kinetics were faster in T[Hb+Mb] compoared with intramuscular flux as measured with intramuscular laser-Doppler. We observed no differences in temporal responses between cutaneous and intramuscular measures and suggested that in a purely temporal analysis the cutaneous microvasculature could serve as an analog for the skeletal muscle microvasculature. Finally, in Chapter 5 we found that prostaglandin inhibition with ibuprofen altered the initial T[Hb+Mb] response during PORH without impacting cutaneous flux or brachial artery blood flow. Chapter 5 also discussed that the addition of a wrist cuff to our standard instrumentation prevented the accumulation of T[Hb+Mb] during the occlusion period.

Master of Science / Department of Kinesiology / Thomas Barstow / Previously in our laboratory, capillary blood flow (QCAP) kinetics were found to be significantly slower than femoral artery (QFA) kinetics following the onset of knee extension exercise. If the increase in QCAP does not follow a similar time course to QFA, blood must be flowing into the leg but not to the working muscle. One possible explanation for this discrepancy is that blood flow also increases to the nonworking lower leg muscles. Purpose: To determine if cuffing below the knee alters the kinetics of QFA and QCAP during knee extension exercise, and provide insight into the potential mechanisms controlling the rapid increase in QFA. Methods: Subjects performed a ramp max test to determine the work rate at which gas exchange threshold (GET) occurred. At least four constant work rate trials in each condition were conducted at work rates eliciting ~80% GET. Trials were performed with and without below knee occlusion. Pulmonary gas exchange, near-infrared spectroscopy, QFA and mean arterial pressure (MAP) measurements were taken. Muscle oxygen uptake (VO2m) and deoxy[hemoglobin + myoglobin] were used to estimate QCAP. Conductance (C) was calculated (QFA/MAP) and the percent change from baseline at 60s into exercise was calculated to indicate a time course of change. Results: There was no significant difference between the uncuffed and cuffed conditions (P>0.05). The mean response times (MRT) of QFA were 18.7 ± 14.2s (uncuffed) and 24.6 ± 14.9s (cuffed). QCAP MRTs were 51.8 ± 23.4s (uncuffed) and 56.7 ± 23.2s (cuffed), which were not significantly different from the time constants (τ) of VO2m (39.7 ± 23.2s (uncuffed) and 46.3 ± 24.1s (cuffed)). However, the MRT of QFA was significantly faster (P<0.05) than the MRT of QCAP and τVO2m. τVO2m and MRT QCAP were significantly correlated. The QFA and C percent increase from baseline at 60s were significantly different from MAP but not from each other. Conclusion: Cuffing below the knee did not significantly change the kinetics of QFA, QCAP or VO2m. Estimated QCAP kinetics tracked VO2m following exercise onset, while changes in QFA appeared to be primarily driven by an increase in C, not an increase in MAP.

Doctor of Philosophy / Department of Anatomy and Physiology / Thomas J. Barstow / The physiological mechanisms determining the tolerable duration of exercise dictate human physical accomplishments across all spectrums of life. Despite extensive study, these specific mechanisms, and their dependence on oxygen delivery and oxygen utilization, remain, a certain extent, undefined. The purpose of this dissertation was to test the overarching hypothesis that muscle contraction characteristics (i.e., intensity of contraction, muscle contraction-relaxation duty cycle, etc.) alter oxygen delivery and oxygen utilization, which directly influence the power-duration relationship and fatigue development, and therefore, exercise tolerance. To accomplish this, specific interventions of altered muscle contraction-relaxation duty cycle and blood flow occlusion were utilized. In the first investigation (Chapter 2), we utilized low and high muscle contraction-relaxation duty cycles to alter blood flow to the active skeletal muscle, demonstrating that critical power (CP) was reduced with the high muscle contraction-relaxation duty cycle due to a reduction in blood flow, while the curvature constant (W’) was not altered. The second investigation (Chapter 3) utilized blood flow occlusion to show that CP was reduced and W’ increased for blood flow occlusion exercise conditions compared to control blood flow exercise conditions. The final investigation (Chapter 4) utilized periods of blood flow occlusion during and post-exercise to reveal greater magnitudes of peripheral and central fatigue development during blood flow occlusion exercise compared to control blood flow exercise. Moreover, this investigation demonstrated that W’ was significantly related to the magnitude of fatigue development. Collectively, alterations in oxygen delivery and oxygen utilization via muscle contraction characteristics and blood flow occlusion directly influence CP and the magnitude of fatigue development. However, W’ does not appear to be influenced by manipulations in oxygen delivery and oxygen utilization, per se. Rather, W’ may be determined by the magnitude of fatigue accrued during exercise, which is dependent upon oxygen delivery and oxygen utilization. The novel findings of the investigations presented in this dissertation highlight important physiological mechanisms that determine exercise tolerance and demonstrate the need for interventions that improve oxygen delivery and oxygen utilization in specific populations, such as those with chronic heart failure or chronic obstructive pulmonary disease, to improve exercise tolerance.

Preterm birth remains a major challenge in obstetrics. It complicates up to 13% of all pregnancies and accounts for approximately 80% of neonatal mortality and morbidity. Bacterial Vaginosis (BV) is associated with a 1.4-fold increased risk of preterm birth. Due to ineffectiveness of antibiotics in preventing preterm labour, probiotics have been proposed to serve as an alternative for treatment of BV and prevention of preterm birth. The objectives of this thesis were to determine 1) the effect of Lactobacillus rhamnosus GR-1 (L. rhamnosus GR-1) supernatant on cytokine profile and prostaglandin (PG)-regulating enzyme expression in lipopolysaccharide (LPS)-stimulated human chorion and placental trophoblast cells from human placentae, 2) the potential signaling pathways through which lactobacilli act and 3) the potential role of immune and placental trophoblast cells in initiating a response to LPS and L. rhamnosus GR-1 treatments. Primary cultures of human placental trophoblast cells were pre-treated with lactobacilli supernatant and then with LPS. In addition, immune cells were removed from cell suspensions using a magnetic purification technique to determine their role in modulating cytokine levels. The expression of pro- and anti-inflammatory cytokines and prostaglandin-regulating enzymes was then determined. We found sex-specific differences in the ability of LPS to increase the output of TNF-α, IL-10, and PTGS2. We also showed that L. rhamnosus GR-1 is able to act through the JAK/STAT and MAPK pathways to increase IL-10 and G-CSF, and independently down-regulates PTGS2 and TNF-α and up-regulates PGDH. The increase in G-CSF and PGDH were only observed in women carrying a female fetus. L. rhamnosus GR-1 may serve as an alternative to antibiotics in preventing some infection/inflammation-mediated cases of preterm birth.

The mammalian central nervous system maintains a delicate balance between neuronal excitation and inhibition. Conventional synaptic inhibition is mediated through the transient activity of postsynaptic γ-aminobutyric acid (GABA) at type A GABA (GABAA) receptors. A subset of GABAA receptors is also located outside of inhibitory synapses. These extrasynaptic receptors generate a tonic inhibitory conductance in response to low concentrations of extracellular GABA. Tonic inhibition broadly suppresses neuronal activity and regulates many vital processes such as sleep, consciousness and memory formation. This thesis examines the physiological effects of tonic inhibition at the cellular level and in the behaving animal. This thesis also explores whether gabapentin, a commonly used sedative, anxiolytic, and analgesic drug, enhances tonic GABAergic inhibition. I hypothesize that: (1) tonic GABAA receptor activity reduces the intrinsic excitability of neurons; (2) the activity of tonically active GABAA receptors in spinal pain pathways attenuates nociception; and (3) tonic inhibition can be upregulated by gabapentin. The results show that a tonic inhibitory current generated by α5 subunit-containing GABAA (α5GABAA) receptors reduces the excitability of hippocampal pyramidal neurons excitability by increasing the rheobase, but does not change the gain of action potential firing. A similar shunting inhibition is present in spinal cord lamina II neurons that is generated by δ subunit-containing GABAA receptors. The activity of these receptors in spinal nociceptive pathways reduces acute thermal nociception and may constrain central sensitization in a behavioural model of persistent pain. Finally, gabapentin increases a tonic inhibitory current in cultured hippocampal neurons independent from changes in the expression of α5GABAA receptors or in the concentration of GABAA receptor ligands. The results of this thesis demonstrate that tonically active GABAA receptors play an important role in the regulation of neuronal activity and nociception, and that tonic inhibition represents a novel target of therapeutic drugs.

Derivation of lung epithelial cells from stem cells remains a challenging task, due in part to a lack of understanding of the molecular mediators driving commitment of endoderm to an early lung lineage. Reciprocal signalling between the lung mesenchyme and epithelium is crucial for proper differentiation and branching morphogenesis to occur. We hypothesized that the combination of signalling pathways comprising early epithelial-mesenchymal interactions and the 3-D spatial environment are required for induction of embryonic and induced pluripotent stem cells (ESC and iPSC, respectively) into a lung cell phenotype with the hallmarks of the distal niche. Aggregating early lung mesenchyme with endoderm-induced ESC and iPSC resulted in differentiation to an NKX2.1 and pro-SFTPC positive lineage. The differentiating cells organized into tubular structures and became polarized epithelial cells. Ultrastructure analysis revealed precursors of lamellar bodies, and Sftpb mRNA expression was detected. Quantification of the differentiation using an Nkx2.1-reporter ESC line revealed that 80% were committed to an early lung lineage, a vast improvement over what has previously been published. The FGF growth factor family comprises well-known mediators of growth and differentiation during the development of many organs, including the lung. We found that FGF2 signalling through the FGFR2iiic receptor isoform was mediating the commitment of the stem cells to an early lung epithelial phenotype, as defined by NKX2.1/proSFTPC expression. FGF7 signalling through the FGFR2iiib receptor was found to be important for the maturation and morphogenesis of the NKX2.1/proSFTPC positive lineage, but did not play a role in the initial commitment. The addition of FGF2 to endoderm-induced ESC or iPSC in the absence of mesenchyme was able to commit the cells to an NKX2.1-positive lineage, but no proSFTPC was detected. Furthermore,the cells did not become polarized and no longer organized into tubular structures. These findings suggest that while FGF2 is important for initial commitment, additional mesenchyme components including matrix proteins, supporting cell lineages and other growth factors are crucial for an efficient differentiation to an early lung epithelial cell lineage.

Synaptic transmission requires the fusion of neurotransmitter containing vesicles with the neuron's plasma membrane in a temporally restrictive manner. In Drosophila, this challenge is accomplished in part by the SNARE protein neuronal-Synaptobrevin (n-Syb). The juxtamembrane region of this molecule, linking the cytosolic SNARE motif and transmembrane region, is hypothesized to play a functional role in facilitating membrane fusion. This short, 10 amino acid, segment contains numerous charged residues and one conserved tryptophan residue. Its short rigid structure may be important in transducing force during SNARE complex assembly. Tryptophan residues, common in membrane proteins, are often observed at the membrane-water interface. It was hypothesized that this conserved tryptophan residue was important for anchoring and positioning n-Syb in the membrane. Proteins produced with tryptophan mutated were tested for anchoring and stability in a membrane model using NMR spectroscopy. Experiments testing depth of insertion using exposure to oxygen, a paramagnetic species, and exchange with deuterium demonstrated that tryptophan anchored n-Syb in the membrane. To test a potential functional role for the juxtamembrane region of n-Syb in synaptic transmission, a reverse genetic approach was employed. Wild-type and mutant P-element clones were made using the genomic sequence of n-syb including the endogenous promoter. n-Syb was found to be expressed, integrate and orient correctly in the membrane of Drosophila S2 cells. Transgenic Drosophila, produced via P-element transformation, were also found to produce transgenic protein. Transgenic expression of wild-type n-syb was found to restore an n-syb hypomorphic mutant from severe motor impairment and limited lifespan to wild-type levels. Synaptic transmission was assessed in 3rd instar larval preparations of mutant and wild-type transgenics. Mutation of the tryptophan residue and insertion of a short flexible linker were both found to inhibit synaptic transmission, while insertion of a long flexible linker was not.

Le transporteur de Na+/ acide monocarboxylique sensible à l’ibuprofène (SMCT1) est exprimé dans la membrane apicale de plusieurs épithélia. Son rôle physiologique dans la glande thyroïde reste cependant obscur mais on présume qu’il pourrait agir comme un transporteur apical d’iode nécessaire pour la synthèse des hormones thyroïdiennes. Récemment, on a montré que SMCT1 possède un courant de fuite anionique sensible à [Na+]e qui permettrait de transporter l’iode de façon électrogénique. Cependant, un efflux d’iode sensible à l’ibuprofène, mais indépendant de la [Na+]e a été aussi observé sur des cultures primaires des thyrocytes porcins, suggérant un autre mécanisme de transport d’iode par SMCT1. Ce travail vise à comprendre les caractéristiques de ce genre de transport en utilisant comme modèle d’expression les ovocytes de Xenopus laevis. Les résultats obtenus des essais de captation d’iode radioactif montrent que SMCT1 présente un transport d’iode sensible à l’ibuprofène de l’ordre de 30nmol/ovocyte/h. Si ce transport est non saturable en iode (0-100 mM), il nécessite du Na+ dans la solution externe. En effet, le remplacement du Na+ extracellulaire par le NMDG inhibe complètement le transport. En outre, on s’est intéressé à exclure la possibilité de différents artefacts. En ayant trouvé que la grande majorité de l’iode radioactif se trouve dans la partie soluble de l’ovocyte, on exclut une liaison non spécifique de l’iode à la membrane cellulaire. Cependant, une bonne proportion de l’iode transporté pourrait être liée à des protéines à l’intérieur de l`ovocyte. En effet, on observe une réduction du transport d’iode dans les ovocytes exprimant SMCT1 de 81,6 ± 2 % en présence de 2 % BSA dans la solution extracellulaire. Également, on écarte la possibilité que le transport d’iode soit le résultat de la surexpression de protéines de transport endogènes dont les canaux chlore. Le transport d’iode semble spécifique à l’expression de SMCT1 et de manière intéressante à l’expression d’un autre transporteur de monocarboxylates, MCT1. L’analyse de l’ensemble des essais, y compris le fait que l’amplitude du transport observé est 20 fois plus grande que celle du courant de fuite nous mène à proposer que SMCT1 puisse transporter l’iode de façon électroneutre. Cependant, le mécanisme par lequel ceci est accompli n’est pas évident à identifier. L’utilisation d’un autre modèle cellulaire serait surement utile pour répondre à cette question. / Ibuprofen sensitive, Sodium Monocarboxylate Transporter (SMCT1) is expressed in the apical membrane of diverse epithelia. Its physiological role in the thyroid remains however unknown, but it has been proposed that SMCT1 could act as an apical iodide transporter required for the main function of the gland: the thyroid hormone synthesis. We previously reported that SMCT1 exhibit a [Na+]e sensible anionic leak current that could account for the electrogenic transport of iodide. However, an iodine efflux sensitive to ibuprofen but independent of [Na+]e, was also observed in primary cultures of porcine thyrocytes, suggesting another mechanism of iodine transport mediated by SMCT1. This work aims to understand the characteristics of this type of transport using Xenopus laevis oocytes as an SMCT1 expression system. By realising 125I uptakes, we found that SMCT1 transports iodide in an ibuprofen sensitive manner (30nmol/oocyte/h). While nonsaturable uptake iodide kinetics were observed, SMCT1 iodide transport was Na+ dependent as shown by the transport reduction when the [Na+]e is replaced by NMDG. The possibility of artifacts, such as non specific binding and the overexpression of endogenous proteins, was analysed. By observing that the vast majority of the radioactive iodide is found in the soluble portion of the oocyte, we excluded non-specific binding of iodide to the cell membrane. However, it is believed that most of the iodide entering the cell is not free and must be bound to some intracellular proteins. Indeed, there is a significant reduction of SMCT1-mediated iodide transport when 2% BSA is present at the extracellular solution. Furthermore, the lack of iodide transport when overexpressing other proteins than SMCT1, precludes the possibility of an overexpression of endogenous transport proteins like chloride channels for example. In fact, the transport of iodide appears to be specific to the expression of SMCT1 and interestingly of another monocarboxylate transporter MCT1. The analysis of all trials, including the fact that the amplitude of the observed transport is 20 times larger than the leak current lead us to propose that SMCT1 can carry iodide in an electroneutral manner. However, the mechanism by which this is accomplished is not easy to identify and future experiments will be necessary to determine whether this transport is observed in other SMCT1 expression systems.

La Fibrose Kystique, causée par des mutations du canal CFTR, mène à la dysfonction du transport des fluides et des ions causant la déshydratation du liquide de surface des voies aériennes et ainsi une défaillance de la clairance mucocilliaire. Ce défaut entraine l’accumulation et l’épaississement du mucus au niveau des bronches qui devient alors un environnement idéal pour le développement d’infections chroniques et d’inflammation qui sont associées à la destruction progressive de l’épithélium chez les patients Fibrose Kystique. Même si leur rôle dans les processus lésionnels est très bien connu, l’impact de médiateurs inflammatoires sur la capacité de réparation ne l’est cependant pas. L’objectif de ma maitrise était donc d’étudier la régulation des mécanismes de réparation de l’épithélium bronchique sain et Fibrose Kystique par le facteur de nécrose tumoral (TNF)-alpha, une cytokine pro-inflammatoire cruciale dans l’initiation et la propagation de la réponse inflammatoire chez les patients FK. À l’aide d’un modèle de plaies mécaniques, nous avons montré que le TNF-alpha stimule la réparation de l’épithélium bronchique sain (NuLi-1) et Fibrose Kystique (CuFi-1). De façon surprenante, l’exposition chronique au TNF-alpha augmente cette stimulation tout comme le taux de migration cellulaire pendant la réparation. Cette augmentation de réparation semble être médiée par l’activation de la métalloprotéinase MMP-9, la relâche d’EGF par les cellules épithéliales et ainsi l’activation de la voie d’EGFR. De plus, l’activation de la réparation par le TNF-alpha semble aussi impliquer l’activation des canaux K+, dont nous avons démontré le rôle important dans la réparation. Contrairement à son effet sur la migration cellulaire et sur la réparation, le TNF-alpha diminue la prolifération cellulaire. En somme, en plus de son rôle dans les processus lésionnels, le TNF-alpha semble avoir un rôle complexe dans les processus de réparation puisqu’il stimule la migration et ralentit la prolifération cellulaire. / Cystic fibrosis (CF) pathology, caused by mutations of cftr gene, leads to ion and fluid transport dysfunction that results in mucus thickening and accumulation in the airways. This mucus accumulation promotes bacterial infection and airway inflammation associated with progressive airway epithelial damage in CF patients, unfortunately leading to respiratory failure. However, the effect of inflammatory products on the repair capacity of respiratory epithelia is unclear. Thus, the objective of my project was to study the regulation of normal and CF bronchial epithelial repair mechanisms by tumor necrosis factor-alpha (TNF)-alpha, a major component of inflammation initiation and propagation in CF. With a wound healing model, we observed that TNF-alpha stimulated the non-CF (NuLi-1) and CF (CuFi-1) bronchial wound healing rate. Surprisingly, chronic exposure to TNF-alpha enhanced this stimulation as well as the migration rate during repair. This wound healing rate stimulation by TNF-alpha seems to be due to metalloproteinase MMP-9 activation, EGF shedding by epithelial cells and subsequent EGFR transactivation. Furthermore, we recently reported a crucial relationship between the EGF response and K+ channel function, both controlling bronchial repair. We now show that TNF-alpha wound healing stimulation also implicated KvLQT1 and KATP currents activation. In contrast to its effect on cell migration, TNF-alpha downregulate cell proliferation. Thus, in addition to its recognized role in the inflammatory response leading to epithelial injury, TNF-a could exert complex actions on repair mechanisms of CF airway epithelia by upregulating cell migration while downregulating proliferation.