Dextran-coated ultra-small, superparamagnetic, iron oxide particles (USPIO) have been proposed as magnetic resonance (MR) lymph node contrast agents. This thesis analyzed the topographic distributions of the pelvic and inguinal lymph nodes and quantified their spatial relations with the adjacent vascular system. We hypothesized that USPIO would facilitate identification of normal lymph nodes in a manner superior to that afforded by computed tomography or unenhanced MR, but using current clinically available scanners would be unlikely to identify microscopic nodal metastases. We have constructed a high quality nodal atlas describing probability distributions for lymph node number, size and position. Using this model, we then defined a generic three-dimensional nodal clinical target volume and a means of accurate delineation of this volume in a three-dimensional representation. This is the most quantitative assessment of the pelvic and inguinal lymphatics to date and will help to improve the successful targeting of lymph nodes for radiotherapy.

Dextran-coated ultra-small, superparamagnetic, iron oxide particles (USPIO) have been proposed as magnetic resonance (MR) lymph node contrast agents. This thesis analyzed the topographic distributions of the pelvic and inguinal lymph nodes and quantified their spatial relations with the adjacent vascular system. We hypothesized that USPIO would facilitate identification of normal lymph nodes in a manner superior to that afforded by computed tomography or unenhanced MR, but using current clinically available scanners would be unlikely to identify microscopic nodal metastases. We have constructed a high quality nodal atlas describing probability distributions for lymph node number, size and position. Using this model, we then defined a generic three-dimensional nodal clinical target volume and a means of accurate delineation of this volume in a three-dimensional representation. This is the most quantitative assessment of the pelvic and inguinal lymphatics to date and will help to improve the successful targeting of lymph nodes for radiotherapy.

Quoique très immature à la naissance, l’opossum grimpe de l’orifice urogénital de la mère jusqu’à une tétine à laquelle il s’accroche pour poursuivre sa maturation. Des informations sensorielles sont nécessaires pour que l’animal atteigne la tétine et que le réflexe d’attachement soit déclenché. Une modalité sensorielle envisagée est l’olfaction. Or, des expériences physiologiques effectuées au laboratoire sur des préparations in vitro suggèrent que le système olfactif central est trop immature pour influencer les comportements moteurs. Une étude immunohistochimique employant un marqueur de maturité axonique (NF200) a montré une absence de marquage dans le cerveau antérieur, à l’exception d’un mince faisceau reliant les bulbes olfactifs aux régions caudales du cerveau. L’implication de l’olfaction dans les comportements du nouveau-né est donc peu probable, mais la présence de ce faisceau est intrigante et l’étude de son développement est approfondie dans le présent travail. Le développement du faisceau exprimant NF200 est décrit de la naissance jusqu’à la fin de la 2e semaine postnatale, âge auquel le marquage NF200 n’est plus observé à ce niveau. Il est aussi montré que le trajet de ce faisceau se superpose à celui de fibres nerveuses exprimant GnRH1, une neurohormone exprimée par des neurones hypothalamiques chez l’adulte. Les résultats indiquent que ce faisceau est le nerf terminal et pourrait servir de voie pionnière pour la croissance des fibres GnRH1. Aucun marquage NF200 dans le cortex olfactif n’est observé avant P15, supportant l’idée que le système olfactif n’influence pas les comportements de l’opossum nouveau-né. / While quite immature at birth, the opossum is nevertheless able to crawl from the urogenital opening to a mother's nipple to which it attaches to pursue its development. Sensory information are required to guide the newborn to the nipple and induce attachment. Olfaction is one of the sensory modalities often proposed. However, recent physiological experiments in the laboratory using in vitro preparations suggest that the olfactory system is too immature to influence the newborn behaviors. Furthermore, an immunohistochemical study using a marker of axonal maturity (neurofilament 200kDa, NF200) has shown that the prosencephalon is nearly devoid of mature fibers except for a thin fascicle running from the olfactory bulbs to more caudal areas of the brain. Olfaction is thus unlikely to guide the locomotion of the newborn, but the presence of this fascicle is intriguing and its development is studied in the present thesis. This fascicle is described from the day of birth to the end of the second postnatal week, when NF200 labeling is no more visible in this region. It is also shown that this fascicle superpose with fibers expressing GnRH1, a neurohormone characterizing hypothalamic neurons in the adult. The results indicate that this fascicle is the terminal nerve, and might serve as a pioneer pathway to GnRH1 fibers cells. Until P15, the olfactory cortex was devoid of NF200 projections, supporting that the olfactory systems is too immature to influence the behavior of newborn opossums.

Doctor of Philosophy / Department of Anatomy and Physiology / Thomas J. Barstow / Peripheral arterial disease (PAD) is a manifestation of the systemic disease of atherosclerosis that results in arterial stenoses of the lower extremities. Patients with PAD demonstrate slowed dynamics of pulmonary oxygen uptake (VO2 kinetics) following the onset of exercise and a profound reduction in peak oxygen uptake and work capacity. However, whereas the primary pathophysiology of PAD results from the lower extremity hemodynamic limitation, there are abnormalities distal to the arterial stenoses in PAD-affected skeletal muscle that may also contribute to the impaired exercise responses. Thus, the potential contributions of abnormal muscle metabolism versus local circulatory defects in the PAD exercise impairment remains unclear. In this context, the purpose of the dissertation was to advance our understanding of the abnormal pulmonary VO2 kinetics in PAD and characterize the local muscle deoxygenation responses during the rest-exercise transition exercise in health and PAD. The present series of investigations were designed to: 1. localize the abnormal pulmonary VO2 kinetics in PAD to the affected lower extremities, 2. characterize the kinetics of calf muscle deoxygenation during walking in PAD and healthy subjects, 3. describe muscle deoxygenation kinetics in relation to exercise work rate and blood flow in PAD and health, and 4. evaluate the effect of arterial revascularization on pulmonary VO2 kinetics in PAD. These investigations revealed a persistent abnormality in muscle oxygen utilization in PAD-affected skeletal muscle that was not associated with the severity of hemodynamic compromise. In particular, we observed slowed pulmonary VO2 kinetics in PAD only during exercise of the PAD-affected skeletal muscles. Moreover, muscle deoxygenation kinetics following the onset of walking and lower intensity calf exercise were prolonged in PAD subjects while leg blood flow responses were normal. However, at higher work rates, PAD muscle deoxygenation kinetics accelerated, demonstrating a work rate and presumably blood flow dependence. Lastly, arterial revascularization tended to improve, but not consistently normalize, pulmonary VO2 kinetics in PAD subjects. Thus, these investigations demonstrate abnormal oxygen uptake kinetics in PAD and provide evidence that local abnormalities of the affected skeletal muscle may contribute to the abnormal VO2 kinetics and exercise intolerance of patients with PAD.

Master of Science / Department of Anatomy and Physiology / Mark L. Weiss / Wharton's jelly is a non-controversial source of mesenchymal stromal cells. Isolation of the cells is non-invasive and painless. The cells have been shown to have a wide array of therapeutic applications. They have improved symptoms when transplanted in a variety of animal disease models, can be used in tissue engineering applications to grow living tissue ex vivo for transplantation, and can be used as drug delivery vehicles in cancer therapy. The cells have also been shown to be non-immunogenic and immune suppressive. This thesis focuses on optimizing isolation protocols, culture protocols, cryopreservation, and characterization of cells in different growth conditions. Results from the experiments indicate that isolation of cells by enzyme digestion yields cells consistently, a freezing mixture containing 90% FBS and 10% DMSO confers maximum viability, and the expression of mesenchymal stromal cell consensus markers does not change with passage and cryopreservation. The results of the experiments also show that cells grow at a higher rate in 5% oxygen culture conditions compared to 21% oxygen culture conditions, serum does not have an effect on growth of the cells, serum and oxygen do not have effects on the expression of mesenchymal stromal cell consensus markers and the cells are stable without nuclear abnormalities when grown in 5% oxygen and serum free conditions for six passages after first establishing in serum conditions.

Temporomandibular disorders (TMD) are a family of prevalent chronic pain disorders affecting masticatory muscles and/or the temporomandibular joint. There is no unequivocally recognized peripheral aetiology for idiopathic TMD. The central nervous system (CNS) may initiate and/or maintain the pain in idiopathic TMD due to sustained or long-term nociceptive input that induces maladaptive brain plasticity, and/or to inherent personality-related factors that may reduce the brain's capacity to modulate nociceptive activity. The main aim of this thesis is to determine whether there are structural neural abnormalities in patients with TMD, and whether these abnormalities are related to TMD pain characteristics, or to neuroticism. The specific aims are to delineate in TMD: (1) gray matter (GM) brain abnormalities and the contribution of pain and neuroticism to abnormalities; (2) the contribution of abnormal brain GM aging in focal cortical regions associated with nociceptive processes; and (3) abnormalities in brain white matter and trigeminal nerve and the contribution of pain. In groups of 17 female patients with TMD and 17 age- and sex- matched controls, magnetic resonance imaging revealed that patients with TMD had: (1) thicker cortex in the somatosensory, ventrolateral prefrontal and frontal polar cortices than controls, (2) cortical thickness in motor and cognitive areas that was negatively related to pain intensity, orbitofrontal cortical thickness that was negatively correlated to pain unpleasantness, and thalamic GM volume correlated to TMD duration, (3) an abnormal relationship between neuroticism and orbitofrontal cortical thickness, (4) abnormal GM aging in nociceptive, modulatory and motor areas, (5) widespread abnormalities in white matter tracts in the brain related to sensory, motor and cognitive functions, (6) reduced trigeminal nerve integrity related to pain duration, and (7) abnormal connectivity in cognitive and modulatory brain regions. In sum, this thesis demonstrates for the first time abnormalities in both peripheral nerve and CNS in patients with TMD.

Temporomandibular disorders (TMD) are a family of prevalent chronic pain disorders affecting masticatory muscles and/or the temporomandibular joint. There is no unequivocally recognized peripheral aetiology for idiopathic TMD. The central nervous system (CNS) may initiate and/or maintain the pain in idiopathic TMD due to sustained or long-term nociceptive input that induces maladaptive brain plasticity, and/or to inherent personality-related factors that may reduce the brain's capacity to modulate nociceptive activity. The main aim of this thesis is to determine whether there are structural neural abnormalities in patients with TMD, and whether these abnormalities are related to TMD pain characteristics, or to neuroticism. The specific aims are to delineate in TMD: (1) gray matter (GM) brain abnormalities and the contribution of pain and neuroticism to abnormalities; (2) the contribution of abnormal brain GM aging in focal cortical regions associated with nociceptive processes; and (3) abnormalities in brain white matter and trigeminal nerve and the contribution of pain. In groups of 17 female patients with TMD and 17 age- and sex- matched controls, magnetic resonance imaging revealed that patients with TMD had: (1) thicker cortex in the somatosensory, ventrolateral prefrontal and frontal polar cortices than controls, (2) cortical thickness in motor and cognitive areas that was negatively related to pain intensity, orbitofrontal cortical thickness that was negatively correlated to pain unpleasantness, and thalamic GM volume correlated to TMD duration, (3) an abnormal relationship between neuroticism and orbitofrontal cortical thickness, (4) abnormal GM aging in nociceptive, modulatory and motor areas, (5) widespread abnormalities in white matter tracts in the brain related to sensory, motor and cognitive functions, (6) reduced trigeminal nerve integrity related to pain duration, and (7) abnormal connectivity in cognitive and modulatory brain regions. In sum, this thesis demonstrates for the first time abnormalities in both peripheral nerve and CNS in patients with TMD.

L’inhibition est nécessaire à la génération d’outputs coordonnés entre muscles antagonistes lors de la locomotion. Une baisse de la concentration neuronale en ions chlorure au cours du développement des mammifères conduit à l’émergence de l’inhibition. Cette baisse repose sur l’équilibre entre deux cotransporteurs cation-chlorure, KCC2 et NKCC1. KCC2 expulse Cl- de la cellule alors que NKCC1 pompe Cl- dans la cellule. L’opossum Monodelphis domestica naît dans un état très immature. Le seul comportement locomoteur qu’il présente à la naissance consiste en des mouvements rythmiques et alternés des membres antérieurs pour grimper le long du ventre de la mère vers une tétine. Les membres postérieurs sont des bourgeons immobiles dont le développement est en grande partie postnatal. Pour cette raison, cette espèce constitue un modèle idéal pour l’étude du développement locomoteur. Afin d’étudier les mécanismes conduisant à l’émergence de l’inhibition durant le développement moteur, nous avons décrit l’expression développementale de KCC2 et NKCC1 chez l’opossum postnatal par immunohistochimie au niveau des renflements spinaux. Les motoneurones et afférences primaires ont été identifiés en utilisant un marquage rétrograde au TRDA. Le marquage pour KCC2 et NKCC1 est détecté dans la moelle épinière ventrale dans la matière grise et blanche présomptive dès la naissance, ce qui suggère que l’inhibition serait déjà mise en place avant la naissance, permettant subséquemment l’alternance des membres antérieurs observée chez les nouveau-nés. L’expression développementale de KCC2 et NKCC1 suit des gradients ventrodorsal et médiolatéral, tels qu’observés chez les rongeurs (rats et souris). Le patron mature d’expression de ces cotransporteurs est observé aux alentours de la 5ème semaine postnatale lorsque la locomotion de l’opossum est mature. Enfin, entre la naissance et P5, les dendrites exprimant KCC2 au niveau de la corne dorsale sont retrouvées en apposition aux afférences primaires ce qui suggère un rôle de KCC2 dans la formation des circuits sensori-moteurs. / Inhibition is necessary to generate coordinated outputs between antagonistic muscles during locomotion. Inhibition is set by a lowering in neuronal chloride concentration during mammalian development. This lowering relies on the proper balance between two cation-chloride cotransporters, KCC2 and NKCC1. KCC2 extrudes Cl- out of the cell while NKCC1 pumps Cl- into the cell. The opossum Monodelphis domestica is born at a very rudimentary stage of development. Newborn opossums show rhythmic and alternate movements of the forelimbs as they crawl on the mother's belly to a nipple. The hindlimbs are immobile paddle-shaped buds and their development is mostly postnatal. Thus, this species is an ideal model to study motor development. In order to investigate the mechanisms involved in the appearance of inhibition during motor development, we described the developmental expression of KCC2 and NKCC1 on postnatal opossums using immunohistochemistry in the spinal cord enlargements. Motoneurons and primary sensory afferents were identified using retrograde labeling with TRDA. Immunolabeling for both KCC2 and NKCC1 is detected in the ventral spinal cord in the presumptive grey and white matter from birth on, suggesting that the development of inhibition begins before birth, enabling the alternate movements exhibited by the newborns. The developmental expression of KCC2 and NKCC1 follows ventrodorsal and mediolateral gradients, similar to those demonstrated in rodents (rats and mice). The mature pattern for both cotransporters is observed around the 5th week at a time when opossums demonstrate mature patterns of locomotion. Between birth and P5, dendrites expressing KCC2 in the dorsal horn superimpose on the primary afferents, suggesting a role for KCC2 in the establishment of sensorimotor circuits.

La transplantation d’îlots chez des sujets diabétiques permet la normalisation de leur glycémie mais nécessite l’utilisation d’immunosuppresseurs. Afin d’éliminer l’utilisation de ceux-ci, une capsule d’alginate capable d’immunoprotéger l’îlot a été proposée. Cependant, un problème persiste : la survie de l’implant est limitée. Deux moyens afin d’améliorer ce facteur seront présentés dans ce mémoire: l’utilisation d’alginate purifié et la co-encapsulation des îlots avec des cellules canaliculaires pancréatiques. La première étude rapporte un aspect nouveau : les effets directs de l’alginate non-purifié, versus purifié, sur la survie d’îlots encapsulés. Ceci est démontré in vitro sur la viabilité à long terme des îlots, leur fonction et l’incidence de leur mort cellulaire par apoptose et nécrose. Ces investigations ont permis de conclure que l’alginate purifié permet de maintenir à long terme une meilleure survie et fonction des îlots. De plus, cette étude ajoute un autre rôle aux contaminants de l’alginate en plus de celui d’initier la réaction immunitaire de l’hôte; celle-ci étant indirectement reliée à la mort des îlots encapsulés. La deuxième étude consiste à déterminer les impacts possibles d’une co-encapsulation d’îlots de Langerhans avec des cellules canaliculaires pancréa-tiques. Les résultats obtenus démontrent que cette co-encapsulation n’améliore pas la survie des îlots microencapsulés, par des tests de viabilité et de morts cellulaires, ni leur fonction in vivo testée par des implantations chez un modèle murin immmunodéficient. Pour conclure, la survie des îlots encapsulés peut être améliorée par la purification de l’alginate mais reste inchangée lors d’une co-encapsulation avec des cellules canaliculaires pancréatiques. / Islets transplantation can normalize glycaemia in diabetic patients but only with the use of immunosuppressive drugs. The elaboration of an alginate microcapsule to immunoprotect the islets has been developed to overcome the use of those harmful drugs. However, one problem still subsists: the limited survival of the transplant. Two different aspects to overcome this problem will be discussed in this thesis: the use of purified alginate and the co-encapsulation of islets with pancreatic duct cells. The first study investigated a new proposition: the direct effects of non-purified alginate, compared to the purified one, on the survival of encapsulated islets. This was demonstrated by in vitro studies on the islets long-term viability, function and the incidence of their death by apoptosis and necrosis. These investigations helped us to conclude that purified alginate can maintain a better long-term survival and function of encapsulated islets. This investigation also demonstrated that alginate contaminants have a direct influence on encapsulated cells besides their role in immune cell activation; which have an indirect implication in the encapsulated islets death. The second study investigated the possible effects of pancreatic duct cells when co-encapsulated with islets of Langerhans. The results showed no significant effects on the viability of co-encapsulated islets, by viability and cellular death assays, and neither on their function in vivo tested with implantations in a mouse immmunodeficient model. To conclude, alginate purification appeared to improve the survival of encapsulated islets while pancreatic duct cells failed to do the same.

L’inhibition est nécessaire à la génération d’outputs coordonnés entre muscles antagonistes lors de la locomotion. Une baisse de la concentration neuronale en ions chlorure au cours du développement des mammifères conduit à l’émergence de l’inhibition. Cette baisse repose sur l’équilibre entre deux cotransporteurs cation-chlorure, KCC2 et NKCC1. KCC2 expulse Cl- de la cellule alors que NKCC1 pompe Cl- dans la cellule. L’opossum Monodelphis domestica naît dans un état très immature. Le seul comportement locomoteur qu’il présente à la naissance consiste en des mouvements rythmiques et alternés des membres antérieurs pour grimper le long du ventre de la mère vers une tétine. Les membres postérieurs sont des bourgeons immobiles dont le développement est en grande partie postnatal. Pour cette raison, cette espèce constitue un modèle idéal pour l’étude du développement locomoteur. Afin d’étudier les mécanismes conduisant à l’émergence de l’inhibition durant le développement moteur, nous avons décrit l’expression développementale de KCC2 et NKCC1 chez l’opossum postnatal par immunohistochimie au niveau des renflements spinaux. Les motoneurones et afférences primaires ont été identifiés en utilisant un marquage rétrograde au TRDA. Le marquage pour KCC2 et NKCC1 est détecté dans la moelle épinière ventrale dans la matière grise et blanche présomptive dès la naissance, ce qui suggère que l’inhibition serait déjà mise en place avant la naissance, permettant subséquemment l’alternance des membres antérieurs observée chez les nouveau-nés. L’expression développementale de KCC2 et NKCC1 suit des gradients ventrodorsal et médiolatéral, tels qu’observés chez les rongeurs (rats et souris). Le patron mature d’expression de ces cotransporteurs est observé aux alentours de la 5ème semaine postnatale lorsque la locomotion de l’opossum est mature. Enfin, entre la naissance et P5, les dendrites exprimant KCC2 au niveau de la corne dorsale sont retrouvées en apposition aux afférences primaires ce qui suggère un rôle de KCC2 dans la formation des circuits sensori-moteurs. / Inhibition is necessary to generate coordinated outputs between antagonistic muscles during locomotion. Inhibition is set by a lowering in neuronal chloride concentration during mammalian development. This lowering relies on the proper balance between two cation-chloride cotransporters, KCC2 and NKCC1. KCC2 extrudes Cl- out of the cell while NKCC1 pumps Cl- into the cell. The opossum Monodelphis domestica is born at a very rudimentary stage of development. Newborn opossums show rhythmic and alternate movements of the forelimbs as they crawl on the mother's belly to a nipple. The hindlimbs are immobile paddle-shaped buds and their development is mostly postnatal. Thus, this species is an ideal model to study motor development. In order to investigate the mechanisms involved in the appearance of inhibition during motor development, we described the developmental expression of KCC2 and NKCC1 on postnatal opossums using immunohistochemistry in the spinal cord enlargements. Motoneurons and primary sensory afferents were identified using retrograde labeling with TRDA. Immunolabeling for both KCC2 and NKCC1 is detected in the ventral spinal cord in the presumptive grey and white matter from birth on, suggesting that the development of inhibition begins before birth, enabling the alternate movements exhibited by the newborns. The developmental expression of KCC2 and NKCC1 follows ventrodorsal and mediolateral gradients, similar to those demonstrated in rodents (rats and mice). The mature pattern for both cotransporters is observed around the 5th week at a time when opossums demonstrate mature patterns of locomotion. Between birth and P5, dendrites expressing KCC2 in the dorsal horn superimpose on the primary afferents, suggesting a role for KCC2 in the establishment of sensorimotor circuits.