Les acides aminés sont des molécules importantes pour la synthèse des protéines, la fourniture de substrats énergétiques ou la production de facteurs de régulation de l'activité cellulaire. L'ornithine aminotransférase {OAT} est une enzyme clef du métabolisme des acides aminés qui relie l'arginine, un acide aminé indispensable à la synthèse protéique au cours de la croissance, et le glutamate, la glutamine et l'a-cétoglutarate, qui peuvent être transformées en glucose. Nous nous sommes intéressés à la caractérisation biochimique de l'activité de l'OAT et à ses variations chez la souris, en utilisant deux contraintes physiologiques, le développement postnatal et l'exposition au froid. Au cours d'une première étude, nous avons caractérisé une activité OAT fonctionnelle dans tous les organes étudiés. Nous avons démontré l'existence d'un dimorphisme sexuel de l'activité, en faveur des femelles, dans la plupart des organes. Dans une seconde étude, nous avons montré des variations organe et sexe spécifiques de l'activité OAT au cours du développement postnatal en réponse aux besoins spécifiques en acides aminés : une régulation de l'expression de l'OAT par la testostérone et le changement de régime alimentaire lors du sevrage ; une contribution potentielle importante et insoupçonnée du muscle squelettique au métabolisme de l'ornithine. Enfin, nous avons mis en évidence que l'exposition au froid augmente ou diminue l'activité OAT de façon tissu-spécifique en liaison avec les besoins énergétiques de l'organisme. Nos résultats montrent que l'activité OAT est modulée dans les différents organes en fonction des besoins en acides aminés et en substrats. Cela suggère un rôle important du métabolisme des acides aminés dans les réponses adaptatives des organismes à leur environnement / Amino acids are important molecules for protein synthesis and energetic substrates supply. Ornithine aminotransferase {OAT} is a key enzyme of amino acids metabolism linking arginine, an essential amino acid for protein synthesis during growth, and glutamate, glutamine and alpha-ketoglutarate which could be converted into glucose. We principally focused on biochemical characterization of OAT activity and its variations in the mouse using two physiological constraints, postnatal development and cold exposition. During a first study, we characterized a functional OAT activity in all the males and females mice organs studied. We showed the existence of a sexual dimorphism of OAT activity in almost all the organs studied, with a higher OAT activity in females. ln a second study, we showed that the variations of OAT activity were organ and sex specific during the postnatal development in response to the specific needs in amino acids during the growth. We linked these variations to the regulation of OAT expression by testosterone and the change of the diet during weaning. We demonstrated an important and unsuspected contribution of skeletal muscles in pups' ornithine metabolism. Finally, we showed that cold exposition modulates OAT activity in a tissue specific way, in relation with energetic needs and the multiple roles of the substrates generated by the metabolic pathways of OAT. Our results show that in the different organs, OAT activity is modulated in function of amino acids needs, energetic substrates or detoxification needs of the organism during the different metabolic constraints. This is suggesting an important role for the amino acids metabolism in adaptive responses of organisms to their environment

Orientadores: José Wilson Magalhães Bassani, Rosana Almada Bassani / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Elétrica e de Computação / Made available in DSpace on 2018-08-24T11:00:56Z (GMT). No. of bitstreams: 1 Oshiyama_NataliaFerreira_D.pdf: 4721295 bytes, checksum: 5ed8a9a173462afb13315f133bf426f8 (MD5) Previous issue date: 2014 / Resumo: O potencial de ação (PA), variação do potencial elétrico através da membrana (Em), é gerado por fluxos iônicos através de canais e transportadores, cuja função e expressão pode ser alterada por hormônios, neurotransmissores, drogas e toxinas. Trata-se de um sistema complexo, para o qual os modelos computacionais constituem ferramenta importante de estudo. No presente trabalho, foi desenvolvido um modelo de PA e transporte de Ca2+ em células ventriculares de ratos neonatos, para o que foi necessário medir a concentração intracelular de Na+ ([Na+]i) e a corrente de Na+ (INa) em cardiomiócitos isolados, sobre as quais há pouca informação na literatura, e as correntes de Ca2+ (ICa), transiente de saída (Ito) e retificadora tardia (IK) de K+, além do próprio PA para melhorar a precisão do modelo. Medições em miócitos de ratos adultos foram realizadas para comparação. Foi observada menor excitabilidade das células de ratos neonatos, o que poderia ser explicado por um deslocamento da curva de ativação de INa de ~10 mV para a direita, i.e., a ativação dos canais de Na+ ocorreu em Em menos negativos e numa faixa mais ampla de Em em miócitos de neonatos do que em células de adultos. Outra diferença encontrada foi com relação à densidade de INa, ~2 vezes maior em células de neonatos. O maior influxo de Na+ poderia causar um aumento da [Na+]i durante a atividade em células de recém-nascidos, que foi confirmado pela medição de [Na+]i. No entanto, não houve aumento significativo quando ICa e o trocador Na+/Ca2+ (NCX) foram inibidos, o que indica que o aumento da [Na+]i se deve mais ao efluxo de Ca2+ via NCX do que ao influxo pelos canais de Na+ do sarcolema. Além disso, observou- se maior duração do PA em miócitos de neonatos, que poderia ser explicada pela menor densidade observada de correntes repolarizantes (Ito e IK). No entanto, não foi detectada diferença entre idades na densidade de ICa. Dados de simulações mostraram que o retículo sarcoplasmático (RS) é a principal fonte do Ca2+ ativador da contração e que a liberação fracional de Ca2+ do RS nos ratos neonatos é menor que nos adultos, confirmando dados experimentais deste laboratório. Portanto, o modelo poderá ser utilizado para predizer possíveis alterações eletrofisiológicas dos cardiomiócitos de ratos neonatos em diferentes condições. / Abstract: The action potential (AP), a change in electrical potential across the membrane (Em), is generated by ionic fluxes through channels and transporters, of which function and expression may be affected by hormones, neurotransmitters, drugs and toxins. Computational models constitute an important tool for the study of this highly non-linear and complex system. In this work, a model of AP and Ca2+ transport in ventricular cells of neonatal rats was developed. It was necessary to measure the intracellular Na+ concentration ([Na+]i) and the Na+ current (INa), for which information in the literature is scarce, and the Ca2+ current (ICa), as well as the outward transient (Ito) and delayed rectifier (IK) K+ currents, in addition to the AP itself, to improve the accuracy of the model. Measurements from adult rat myocytes were also made in order to compare these developmental phases. It was observed that neonatal rat cells are less excitable, which could be explained by a ~10 mV shift to the right of the channel activation curve, i.e., Na+ channels activation occured at less negative Em value and over a higher range of Em compared to adult cells. On the other hand, INa density was twice as great as that in adults. This might promote increase in [Na+]i during activity in cells from newborns, which was confirmed by measurement of [Na+]i. Nonetheless, significant Na+ accumulation was suppressed when ICa and the Na+ / Ca2+ exchanger (NCX) were inhibited, which indicates that the increase in [Na+]i probably depends more on Ca2+ efflux via NCX than on the influx through sarcolemmal Na+ channels. The longer AP duration in neonatal myocytes could be explained by the lower density of the repolarizing currents (Ito and IK). However, age-dependent difference in ICa density was not observed. Simulation data agreed with experimental data from this laboratory regarding the sarcoplasmic reticulum (SR) as the main source of Ca2+ during excitation-contraction coupling and the lower SR fractional release in neonatal than in adult myocytes. In conclusion, the present model may be used to predict possible electrophysiological alterations in developing cardiomyocytes under different conditions. / Doutorado / Engenharia Biomedica / Doutora em Engenharia Elétrica

O estado nutricional influencia o crescimento gástrico, e enquanto a proliferação celular é estimulada pelo jejum em filhotes, ela é inibida em ratos adultos. A corticosterona também age no desenvolvimento, e seus efeitos são regulados pela corticosterone binding globulin (CBG) e receptores de glicocorticóides (GR). Para investigar se a atividade da corticosterona responde ao jejum e como possíveis mudanças poderiam controlar o ciclo celular epitelial gástrico, nós avaliamos diferentes parâmetros durante a progressão do jejum em ratos de 18 e 40 dias de vida pós-natal. A restrição alimentar induziu um aumento de corticosterona no plasma em ambas as idades, mas apenas em filhotes o binding da CBG se elevou após o jejum curto, permanecendo alto até o final do tratamento. O jejum aumentou a atividade transcricional do GR na mucosa gástrica e os níveis protéicos, porém o efeito foi mais pronunciado em adultos. Além disso, observamos que nos filhotes, o GR é principalmente citoplasmático, enquanto em animais adultos, o receptor é acumulado no núcleo durante o jejum. As proteínas HSP 70 e HSP 90 foram diferencialmente reguladas, e podem contribuir para a estabilidade do GR no citoplasma em filhotes, e para o trânsito de GR para o núcleo em animais adultos. Quanto ao ciclo celular epitelial, observamos que em filhotes, ciclina D1 e p21 aumentaram durante o jejum, enquanto em ratos adultos, a ciclina E diminuiu e a p27 aumentou muito. Assim, nós demonstramos que a atividade da corticosterona é diferencialmente regulada pelo jejum em filhotes e ratos adultos, e que as variaçõe observadas poderiam atenuar um possível efeito supressor durante o desenvolvimento pós-natal. Sugerimos que este mecanismo pode estimular a proliferação celular e possibilitar o crescimento da mucosa gástrica durante condições nutricionais adversas. / The nutritional status influences gastric growth, and interestingly, whereas cell proliferation is stimulated by fasting in suckling rats, it is inhibited in adult animals. Corticosterone takes part in the mechanisms that govern development, and its effects are regulated in particular by corticosterone binding globulin (CBG) and glucocorticoid receptor (GR). To investigate whether corticosterone activity responds to fasting and how possible changes might control gastric epithelial cell cycle, we evaluated different parameters during the progression of fasting in 18- and 40-d-old rats. Food restriction induced higher corticosterone plasma concentration at both ages, but only in pups did CBG binding increase after short and long-term treatments. Fasting also increased gastric GR at transcriptional and protein levels, but the effect was more pronounced in 40-d-old animals. Moreover, in pups, GR was observed in the cytoplasm, whereas in adults, it accumulated in the nucleus after the onset of fasting. HSP 70 and HSP 90 were differentially regulated, and might contribute to the stability of GR and to the high cytoplasmic levels in pups and elevated shuttling in adult rats. As for gastric epithelial cell cycle, whereas cyclin D1 and p21 increased during fasting in pups, in adults, cyclin E slowly decreased concomitant with higher p27. In summary, we demonstrated that corticosterone function is differentially regulated by fasting in 18- and 40-d-old rats, and such variation might attenuate any possible suppressive effects during postnatal development. We suggest that this mechanism could ultimately increase cell proliferation and allow regular gastric growth during adverse nutritional conditions.

AMP-activated protein kinase (AMPK) is an important metabolic sensor in eukaryotic organisms and it plays an important role in regulating energy homeostasis, at both the cells and the whole organism. AMPK controls glucose and lipid metabolism by direct stimulation of enzymes or by long term stimulation of the gene expression of energy metabolism. Skeletal muscles significantly contribute to the total body weight and metabolic rate and to the maintenance of glucose homeostasis. Due to the ability of the muscle to increase energy expenditure to 95% of whole-body energy expenditure, could be the proper development and programming of metabolism in the early postnatal period crucial for the further development of the organism in adulthood. Early postnatal development leads to substantial changes in energy requirements of the body and this suggests the significant involvement of AMPK in this period. The aim of this thesis was to study the activity and expression of isoforms of the catalytic subunit of AMPK in skeletal muscle during early postnatal development of both mouse strains A/J and C57BL/6 that differ in the development of diet-induced obesity. The next task was to analyze the expression of selected genes involved in energy metabolism - GLUT4, PGC-1α and UCP3 that AMPK regulates. It was found that the...

Les modèles murins de la Sclérose Latérale Amyotrophique (SLA) ont permis des avancées dans la compréhension des mécanismes pouvant conduire à la mort sélective et progressive des motoneurones (Mns) mais ils présentent des disparités dans la sévérité et le décours temporel de la maladie. Parmi les hypothèses avancées figurent des modifications des propriétés intrinsèques des motoneurones conduisant à des modifications de l’excitabilité et de l’homéostasie du calcium intracellulaire et à la mort du motoneurone.Nous avons donc étudié les propriétés électrophysiologiques des Mns lombaires de souris SOD1G85R et SOD1G93A, deux modèles à faible nombre de copies du gène humain muté, durant les deux premières semaines postnatales afin d’identifier d’éventuelles anomalies pré-symptomatiques précoces. Nos travaux ont été réalisés sur deux préparations in vitro de moelle entière isolée et de tranches de moelle épinière. Les Mns mutants présentent, sur les deux types de préparations, une altération des propriétés du potentiel d’action se traduisant par un allongement de la durée associée à une diminution des vitesses maximales de dépolarisation et repolarisation et une réduction d’amplitude. Ces altérations apparaissent entre P2-P5 dans les Mns SOD1G85R et entre P6-P10 dans les Mns SOD1G93A et suggèrent une diminution de la densité des canaux sodiques et potassiques associés au potentiel d’action. Nous avons aussi observé sur des tranches de moelle épinière entre P6-P10 que le gain de fréquence des Mns SOD1G85R diminue et celui des SOD1G93A augmente sans aucune modification des densités des courants entrants persistants sodiques et calciques. On note également que, sur tranches de moelle épinière, les Mns SOD1G93A présentent un potentiel de repos diminué. En présence d’une surcharge calcique extracellulaire, les propriétés membranaires des Mns SOD1G85R entre P6-P10 sont moins affectées que celles des Mns témoins. Les effets différentiels de cette surcharge peuvent être dus à des modifications différentes de la dépendance au voltage des canaux voltage-dépendants et/ou à la modulation de certains types de canaux activés par le calcium extracellulaire. Une arborisation dendritique plus ramifiée que celle de Mns témoins, comparable à celle précédemment décrite dans les Mns SOD1G85R, a été observée dans les Mns SOD1G93A à P8-P9 avec des altérations du potentiel d’action citées plus haut et une réduction de la rhéobase. Ces altérations morphologiques et électriques pourraient indiquer des modifications de cinétiques et/ou de densités de canaux sur des sites différents dans ces Mns. Nos travaux montrent donc, d’une part que les mutations SOD1G85R et SOD1G93A induisent dans ces deux modèles murins des altérations des propriétés des Mns lombaires comparables mais décalées dans le temps et d’autre part que certaines altérations semblent être spécifiques à une mutation SOD1 donnée. / The SOD1 murine models of Amyotrophic Lateral Sclerosis (ALS) allowed major progress in the understanding of mechanisms which could lead to a selective loss of motoneurons (Mns), but these models display differences in the severity and time course of the disease. Changes in intrinsic properties of motoneurons may induce changes in excitability and intracellular calcium homeostasis leading to motoneuron death.Therefore, we studied electrophysiological properties of lumbar Mns from SOD1G85R and SOD1G93A mice, low expressor lines, during the first two postnatal weeks in order to identify possible early presymptomatic abnormalities. Our studies were carried out on two in vitro preparations: the whole isolated spinal cord and acute spinal cord slices. Mutant Mns display, in the two preparations, a modified action potential characterized by an increased duration due to a decrease of the maximal speeds of depolarisation and repolarisation and a reduction of the spike amplitude. These alterations appeared between P2-P5 in SOD1G85R Mns and between P6-P10 in SOD1G93A Mns and suggest a decrease of the density of sodium and potassium channels related to action potential. We also showed on spinal cord slices between P6-P10 that the gain of frequency decreases for SOD1G85R Mns and increases for SOD1G93A Mns without any change in the density of persistent inward sodium or calcium currents in these different mutant Mns. We observed also that the resting membrane potential of SOD1G93A Mns on spinal cord slices is decreased. The membrane properties of SOD1G85R Mns between P6-P10 were less susceptible to changes in presence of an extracellular calcium overload. Differential effects of this extracellular calcium overload on membrane properties of WT and SOD1G85R Mns could be due to different alterations of the potential dependence of voltage-gated channels and/or to the modulation of some types of channels sensitive to extracellular calcium. An over-branching of dendritic arborization, similar to that previously described in SOD1G85R Mns, was observed in SOD1G93A at P8-P9 with the above-mentioned action potential alterations and a weak rheobasic current. These morphogical and electrical changes could indicate together alterations of kinetics and/or density of channels on different sites on these Mns. In conclusion, our work shows on one hand that SOD1G85R and SOD1G93A mutations induce similar alterations of lumbar Mns properties but time-shifted in these two murine models and on the other hand that some alterations seem to be specific to a given SOD1 mutation.

Ce travail avait pour objet d’étudier les propriétés d’horloge et de synchronisation de la peau, un modèle potentiel d’horloge périphérique. L’activité rythmique a été analysée par bioluminescence en temps réel, sur des explants de peau abdominale et des fibroblastes dermiques primaires, isolés à partir de rats transgéniques Per1-luciférase. Nous avons montré que des explants de peau présentent une activité rythmique soutenue en culture, indiquant une importante synchronisation interne dans le tissu. Cette synchronisation se manifeste au cours du développement post-natal à partir de 1 mois et augmente jusqu’à 6 mois, avant de décroître, laissant place à des rythmes altérés à l’âge de 2 ans. Nous avons aussi établi que les fibroblastes dermiques présentent la propriété de compensation thermique commune à toutes les horloges circadiennes, et qu’ils sont potentiellement synchronisables par la mélatonine puisque celle-ci augmente leur amplitude en culture. Nous avons aussi préparé un vecteur lentiviral exprimant le gène rapporteur luciférase sous le contrôle du promoteur du gène horloge Bmal1, un nouvel outil pour compléter l’étude des rythmes dans les cellules de la peau. / This work aimed to investigate the skin as a potential model of peripheral clock by characterizing its rhythmic and synchronization properties. Circadian activity was examined in abdominal skin explants and fibroblasts derived from Per1-Luciferase transgenic rats by real-time recording of bioluminescence. First, the skin clock was characterized from early postnatal to old age. Low amplitude oscillations appeared at 1 month only and their robustness increased until 6 months. In 1-2 year-old rats, skin circadian rhythms showed decreasing amplitude and abnormal cycles. Primary fibroblasts derived from the skin at the same ages demonstrated similar pattern of clock activity. Temperature compensation, an intrinsic clock feature, was shown the first time in skin and primary fibroblasts. Secondly, we demonstrated a phase-dependent effect of melatonin to increase the amplitude of oscillations in skin primary fibroblasts, indicating it displays a synchronising role in the circadiansystem. Finally, to facilitate our studies on the multioscillatory skin tissue, we constructed a lentivirus carrying a Bmal1-luciferase reporter, to measure clock genes activities in human skin cells.

Die Dissertation beschäftigt sich mit den möglichen Gründen für die divergente evolutionäre Entwicklung von Beuteltieren und plazentalen Säugetieren nach der Kreide/Tertiär-Grenze. Eine Erklärung könnten ihre unterschiedlichen Reproduktionsstrategien sein. Während die Beuteltiere sehr embryonale Jungtiere nach einer kurzen Tragzeit gebären, bringen Plazentalier deutlich weiter entwickelte Jungtiere nach einer relativ langen Tragzeit zur Welt. Die Aufrechterhaltung eines stabilen Metabolismus und thermoregulatorische Fähigkeiten der Jungtiere bieten einen großen Vorteil für die Anpassungsfähigkeit an ungünstige Umweltbedingungen, wie sie für die K/T-Grenze vermutet werden. Aus diesem Grund untersucht diese Studie den strukturelle Entwicklungsgrad der Lunge und die metabolischen Fähigkeiten von neonaten Marsupialia und Plazentalia. Histologische, ultrastrukturelle und kalorimetrische Untersuchungen erfolgten in einer integrativen Studie. Basierend auf den Ergebnissen wurde eine Grundplanrekonstruktionen der Neonaten von Marsupialia und Plazentalia durchgeführt. Als Vertreter für nesthockende Plazentalia wurde der Goldhamster (Mesocricetus auratus), die Moschusspitzmaus (Suncus murinus) und das Belangeri Spitzhörnchen (Tupaia belangeri) untersucht. Das Wildmeerschweinchen (Cavia aperea) und die Kurzohr-Elefantenspitzmaus (Macroscelides proboscideus) repräsentieren die nestflüchtenden Plazentalia. Als Marsupialia wurden die Hausspitzmaus-Beutelratte (Monodelphis domestica) und das Tammar-Wallaby (Macropus eugenii) untersucht. Die Ergebnisse bestätigen die starken Unterschiede im Entwicklungsgrad der Neonaten und in der postnatalen Entwicklung zwischen Marsupialia und Plazentalia. Die neonatalen Lungen von M. auratus und S. murinus befinden sich im späten “terminal air sac”-Stadium und weisen viele kleine Atemkammern von 50-80 µm Durchmesser auf. Die Alveolenbildung erfolgt bei M. auratus und S. murinus bereits im Alter von zwei beziehungsweise vier Tagen. Bei T. belangeri, C. aperea und M. proboscideus sind Alveolen bereits zum Zeitpunkt der Geburt vorhanden. Im Gegensatz dazu, befinden sich die Lungen der neonaten Beuteltiere M. domestica und M. eugenii im frühen “terminal air sac”-Stadium mit wenigen großen Atemkammern von 300-400 µm im Durchmesser. Die postnatale Lungenentwicklung erfolgt sehr langsam und die Alveolenbildung startet mit 28 Tagen bei M. domestica und mit 65 Tagen bei M. eugenii. Die Metabolismusmessungen ergaben, daß Marsupialia mit einer niedrigen Metabolismusrate geboren werden und den Adultmetabolismus erst spät in der postnatalen Entwicklung erreichen. Einhergehend mit der weit entwickelten Lungenstruktur weisen die Plazentalia hohe Metabolismusraten zur Geburt auf und erreichen den Adultmetabolismus innerhalb der ersten Lebenswoche. Die Ergebnisse zeigen, dass Jungtiere der Plazentalia, im Vergleich zu jungen Marsupialia, eine höhere Widerstandskraft gegen Umweltschwankungen haben, was als ein evolutiver Vorteil der Reproduktionsstrategie der Plazentalia unter ungünstigen Klimabedingungen interpretiert werden kann. / This project deals with the possible reasons for the evolutionary differentiation between marsupial and placental mammals after the K/T-event. One explanation could be their different reproductive patterns. Marsupialia bear virtually embryonic young after a brief gestation period. In contrast, many eutherians bear anatomically advanced, highly precocious young after a relatively long gestation period. A stable metabolism and thermoregulatory abilities of the young are considered to offer a large adaptive advantage in a changing environment, how it is presumed for the K/T-boundary. Therefore this study determines the developmental stage and the respiratory efficiency of the lungs of marsupial and placental young. Histological, ultrastructural and calorimetric investigations were carried out in an integrated study and from the results morphotype reconstructions of the marsupial and placental neonates were carried out. As representatives for altricial Placentalia, the Golden hamster (Mesocricetus auratus), the Musk shrew (Suncus murinus), and the Belanger’s tree shrew (Tupaia belangeri) were examined. Furthermore the Guinea pig (Cavia aperea) and Short-eared elephant shrew (Macroscelides proboscideus) as typical precocial Placentalia were included. The Marsupialia were represented by the Grey short-tailed opossum (Monodelphis domestica) and the Tammar wallaby (Macropus eugenii). The results confirm clear differences in the developmental degree of the neonates and the postnatal development between marsupial and placental mammals. The newborn lungs of the altricially born placentals M. auratus and S. murinus are at the late terminal air sac stage with numerous small air sacs of 50 - 80 µm in diameter. Alveoli are formed shortly after birth at the age of 2 days in M. auratus and at the age of 4 days in S. murinus. In T. belangeri and in the precocially born C. aperea alveoli are already present at birth. In contrast, the lungs of the newborn marsupials M. domestica and M. eugenii are at the early terminal air sac stage with few large air sacs of 300 – 400 µm in diameter. The postnatal lung development proceeds very slowly in marsupials and alveoli are not present before the age of 28 days in M. domestica and 65 days in M. eugenii. The metabolic investigations demonstrate that Marsupialia have a low metabolism at birth and achieve the adult metabolism late in the postnatal development. All examined Placentalia showed the same pattern of oxygen consumption. Corresponding to their advanced differentiation of the lungs they also exhibit high metabolic abilities at birth and reach the adult metabolism during the first week of life. During this critical period placental young have a higher resistance against certain environmental stresses than marsupial young and this could mean an evolutionary advantage of the placental “reproductive strategy” under suboptimal climatic conditions.

La vue est sans doute le plus important des sens. L'anatomie, l'histologie et la physiologie de l'œil normale chez les espèces de laboratoire adultes ont généralement été bien documentées. Cependant, les références décrivant les caractéristiques histomorphologiques du développement oculaire postnatal chez les animaux de laboratoire demeurent sporadiques et incomplètes. L'évaluation de tissus oculaires provenant d'animaux immatures peut être nécessaire lors d’études précliniques juvéniles de toxicité effectuées dans le but d’évaluer l'innocuité de médicaments destinés à la population pédiatrique. En effet, les données découlant d'études précliniques réalisées avec des animaux matures ne sont pas toujours jugées appropriées pour évaluer la toxicité d'un composé lorsqu'il est administré à des enfants. Cependant, la rareté des références histologiques sur le développement postnatal chez les animaux, ainsi que l'absence courante de témoins appariés selon l'âge pour les animaux sacrifiés de façon précoce lors d’études juvéniles, peuvent rendre difficile l'analyse des structures de l’œil en développement. Ainsi, l'objectif de cette thèse était de fournir des connaissances histologiques et immunohistochimiques (IHC) sur le développement oculaire postnatal, de la naissance au stade prépubère, chez le rat Sprague Dawley (SD), cochon domestique (DP), cochon miniature Göttingen (MP) et chien Beagle (BG). Les résultats de cette thèse ont démontré l'immaturité marquée de toutes les structures oculaires chez les rats SD et les chiens BG à la naissance et pendant la période postnatale. À la naissance, la rétine chez ces espèces altriciales était encore composée de la couche neuroblastique externe fœtale, et plusieurs étapes cruciales de la rétinogenèse, mises en évidence avec l’IHC, se sont produites lors des premières semaines de vie. D’autres évidences d'immaturité oculaire chez ces espèces incluaient la stratification de l’épithélium cornéen lors de l'ouverture des paupières et la présence de vestiges de la vascularisation hyaloïde. En revanche, les yeux des DP et MP, considérés comme une espèce précoce, étaient davantage développés à la naissance, néanmoins, d'importants changements de morphogenèse ont été observés lors de la période postnatale. Par exemple, la rétine du cochon néonatal présentait des photorécepteurs peu développés. Chez toutes les espèces examinées, la prolifération cellulaire et l'engagement des cellules dans le cycle cellulaire, mis en évidence avec Ki-67 et/ou PHH3, étaient prédominants dans la majorité des structures oculaires en développement. L'apoptose, démontrée avec l'IHC contre caspase-3 activé et/ou l'histochimie TUNEL, s’est avérée une caractéristique histologique clé à des âges précis de la rétinogenèse chez les rats SD et les chiens BG. Ce changement était aussi notable dans l'épithélium immature du cristallin du rat SD, ainsi que dans les vestiges hyaloïdes chez toutes les espèces. Enfin, des évidences d’activation non-apoptotique de caspase-3 ont été observées dans différents types cellulaires chez toutes les espèces. Les connaissances présentées dans cette thèse pourront servir de référence pour les pathologistes devant évaluer des structures oculaires en développement dans le cadre d'études précliniques de toxicité. Par ailleurs, les résultats de cette thèse ouvrent la voie pour des investigations plus approfondies sur le développement oculaire, particulièrement chez le chien et le cochon, qui pourront servir à des recherches futures en ophtalmologie pédiatrique. / Vison is arguably the most important of senses. The normal anatomy, histology, and physiology of the eye in mature laboratory species have generally been well documented, particularly in rodents. However, references addressing the histomorphological features of the postnatal ocular development in laboratory animals, notably in nonprimate large animal models, remain sporadic and incomplete. From a veterinary toxicologic pathology perspective, the evaluation of ocular tissues from immature animals may be needed during different types of preclinical juvenile animal toxicity studies conducted to assess the safety of xenobiotics on the pediatric population. Data from preclinical studies conducted in mature animals are often not deemed appropriate to evaluate the toxicity of a drug when administered to children, thus warranting the need to use juvenile animals. However, the paucity of histological references describing the postnatal development of laboratory animals, along with the common lack of age-matched controls when animals are unexpectedly sacrificed (or found death) early during juvenile studies, can render the analysis of developing ocular structures challenging. Thus, the objective of this thesis was to provide comprehensive histological and immunohistochemical (IHC) knowledge on the postnatal ocular development, using several age timepoints from birth to the peripubertal stage, in the Sprague Dawley (SD) rat, domestic pig (DP), Göttingen minipig (MP) and Beagle (BG) dog. Overall, the results from this thesis demonstrated the marked immaturity of all ocular structures in SD rats and BG dogs at birth and during the postnatal period. Notably, the retina at birth in these altricial species still contained the highly proliferative fetal outer neuroblastic layer, and critical retinogenesis events, highlighted with IHC, occurred rapidly during the first few weeks of life. Other noteworthy features of ocular immaturity in these species included the corneal epithelial stratification happening around the time of eyelid opening, the presence of hyaloid vascular remnants, and the globally poorly developed eye anterior segment. Contrastingly, the eyes of the DP and MP, considered a precocial species, were appreciably more developed at birth, although important ocular morphogenesis changes still occurred after birth. Importantly, the neonatal pig retina presented poorly developed cone and rod photoreceptors. In all examined species, cellular proliferation and the engagement of cells in the cell cycle, highlighted by Ki-67 and/or PHH3 IHC, were prominent in nearly all developing ocular structures for variable periods of time. Physiologically occurring apoptosis, highlighted by cleaved-caspase-3 IHC and/or TUNEL histochemistry, was a key histological feature of retinogenesis at specific age timepoints in SD rats and BG dogs, and was notable in the SD rat immature lens epithelium, as well as in regressing hyaloid vasculature remnants of all species. Lastly, evidence of nonapoptotic activation of caspase-3 was observed in different ocular cell types in all species. The information presented in this thesis will hopefully serve as general reference material for pathologists evaluating immature ocular structures in the context of preclinical toxicity studies. Moreover, the results pave the way for more in-depth investigations of specific ocular developmental events in nonprimate large animal models that may be useful for pediatric ophthalmology translational research.

Intestinal cylindrical growth peaks in mice a few weeks after birth, simultaneously with crypt fission activity. It nearly stops after weaning and cannot be reactivated later. Transgenic mice expressing Cd97/Adgre5 in the intestinal epithelium develop a mega-intestine with normal microscopic morphology in adult mice. Here, we demonstrate premature intestinal differentiation in Cd97/Adgre5 transgenic mice at both the cellular and molecular levels until postnatal day 14. Subsequently, the growth of the intestinal epithelium becomes activated and its maturation suppressed. These changes are paralleled by postnatal regulation of growth factors and by an increased expression of secretory cell markers, suggesting growth activation of non-epithelial tissue layers as the origin of enforced tissue growth. To understand postnatal intestinal growth mechanistically, we study epithelial fate decisions during this period with the use of a 3D individual cell-based computer model. In the model, the expansion of the intestinal stem cell (SC) population, a prerequisite for crypt fission, is largely independent of the tissue growth rate and is therefore not spontaneously adaptive. Accordingly, the model suggests that, besides the growth activation of non-epithelial tissue layers, the formation of a mega-intestine requires a released growth control in the epithelium, enabling accelerated SC expansion. The similar intestinal morphology in Cd97/Adgre5 transgenic and wild type mice indicates a synchronization of tissue growth and SC expansion, likely by a crypt density-controlled contact inhibition of growth of intestinal SC proliferation. The formation of a mega-intestine with normal microscopic morphology turns out to originate in changes of autonomous and conditional specification of the intestinal cell fate induced by the activation of Cd97/Adgre5.

Le système endocannaboïde (eCB) est constitué des ligands, des récepteurs – les plus étudiés étant les récepteurs CB1 et CB2 – et les enzymes de synthèse et de dégradation. Les ligands étant lipophiles, ils ne sont pas encapsulés dans des vésicules, ce qui place les enzymes de synthèse et de dégradation dans une position de régulateurs clés. Plusieurs études démontrent une participation du système eCB à des processus de développement dans le système nerveux central (SNC). La rétine est un modèle important pour l’étude de ces processus car elle contient plusieurs types cellulaires bien connus, dont le patron de développement est clairement établi. Pour l’instant très peu est connu sur l’expression du système eCB durant le développement rétinien. C’est dans ce cadre que les patrons d’expression du récepteur CB1 et de l’enzyme de dégradation FAAH ont été étudiés pendant le développement rétinien postnatal chez le rat. Pour identifier les types cellulaires exprimant ces protéines, des co-marquages ont été accomplis pour le récepteur CB1 ou FAAH et des marqueurs des types cellulaires rétiniens. À P1, les cellules ganglionnaires, amacrines, horizontales et mitotiques expriment le récepteur CB1. Les cellules ganglionnaires et amacrines cholinergiques sont FAAH-positives. Au cours du développement, certains types cellulaires démontrent une expression transitoire de ces deux protéines, suggérant une implication du système eCB dans les processus de développement. Nos données démontrent également une importante expression du système eCB dans la rétine adulte, ce qui soutient l’hypothèse de son implication dans la réponse rétinienne. En bref, des études fonctionnelles in vitro sur des rétines de non-mammifères ont révélées que le récepteur CB1 modulait la réponse des cônes et des cellules bipolaires. Malgré la récente démonstration de sa présence dans la rétine, il n’existe pas de d’étude sur le rôle du récepteur CB2 dans la rétine. Dans cette thèse, les conséquences fonctionnelles de l’élimination des récepteurs CB1 ou CB2 ont été évaluées chez des souris transgéniques. Les réponses rétiniennes ont été enregistrées par électrorétinographie chez des souris cnr1-/- (CB1R-KO) et cnr2-/- (CB2R-KO). Nos données suggèrent une implication différente pour chaque récepteur dans la formation de la réponse rétinienne / The endocannabinoid (eCBs) system is composed of the ligands, the receptors - the most studied are CB1R and CB2R – and the synthesizing and degradative enzymes. The lipophilic ligands are not stored in vesicles, thereby placing the synthesizing and degradative enzymes as key regulators of the receptor function. The eCB system is thought to participate to developmental processes in the central nervous system (CNS). The rodent retina is a valuable model to study CNS development, as it contains well identified cell types with established developmental timelines. Very little is known about the distribution of this neuromodulationsystem in the developing retina. In this thesis, the expression patterns of CB1R and eCB degradative enzyme FAAH were investigated in the rat retina during postnatal development. To identify the cells expressing these proteins, co-stainings were carried out for CB1R or FAAH and retinal cell type markers. At P1, CB1R was expressed in ganglion, amacrine, horizontal and mitotic cells, whereas FAAH was present in ganglion and cholinergic amacrine cells. In the course of development, both CB1R and FAAH were transiently expressed in some cell type, suggesting a role of the eCB system in developmental processes. Furthermore, our data demonstrated an important expression of both proteins in adult animals, supporting the hypothesis that the eCB system is involved in retinal functions. Briefly, functional in vitro studies on non-mammalian retinae have revealed an effect of CB1R on cone photoreceptors and bipolar cells response. Despite the recent demonstration of CB2R mRNA and protein presence in the retina, there are no data on CB2R functional role in retina have been published. In this thesis, the consequences of removing either CB1R or CB2R from the retina of transgenic mice were evaluated. Retinal response was recorded by electroretinogram in cnr1-/- (CB1R-KO) and cnr2-/- (CB2R-KO) mice. This data suggests that both receptors are involved in shaping the retinal response to light and they have different roles in this process.