Perinatal inflammation contributes to neurodevelopmental diseases, and animal models have revealed that the inflammatory response within the central nervous system is age dependent. It remains unclear what intrinsic and/or extrinsic factors are responsible for this variation. Here, my aim was to discover the mechanisms responsible for the age-dependent changes in the inflammatory response of the brain by injecting interleukin-1 (IL-1β) into the brain of mice at postnatal day (P)7, P14, P21 or into adult mice. A "window of susceptibility" was found at P14, which was associated with marked neutrophil recruitment and blood-brain barrier (BBB) breakdown, in response to a low dose of IL-1β. Evaluation of cytokine, chemokine, and adhesion molecule mRNA transcripts failed to reveal any specific increases in basal or reactive expression following the injection of IL-1β at P14. The extrinsic hepatic acute phase response (APR) was evaluated, but, once again, there was no evidence that an altered APR might account for the enhanced inflammatory response at P14. Indeed, an inverse relationship between the magnitude of the leukocyte recruitment to the brain and the APR was discovered. Enhancement of the APR with intravenous IL-1β after injection of a low dose of IL-1β into the brain was found to reduce the number of neutrophils and BBB permeability in the brain. While no molecular changes seem to account for the presence of the "window of susceptibility", a population of Iba-1⁺ large, flattened and irregular perivascular cells was discovered within the P14 brain, that may contribute to the increased leukocyte recruitment at P14. Although variations in the brain inflammatory response with development were not fully account for, my results highlight the importance of the systemic inflammatory response on the outcome of acute brain injury and suggest that the systemic APR might be manipulated therapeutically to protect the brain in the perinatal period.

The name of William Harvey is imortal, and it is fitting that a quotation form his epoch-making 'De Motu Cordis et Sanguinis' should preface this thesis. the discoverer of the circulation did not fall to point out the difference between foetal and postnatal conditions of the heart and great vessels. Harvey, however, was not particularly concerned with the problems of the foetal circulation, and devoted only a passing glance to the subject, using foetal conditions to illustrate his general argument about the circulation of the blood. The subsequent progress of though on the subject of foetal circulation has been admirably set out in the first chapter of Barclay, Frankin, and Prichard's book 'The Foetal Circulation', published in 1944, but there is no doubt that the major advance since Harvey's time is represented by the cine-radiographic observations made by the authors of this book on the foetal lamb. They provided, for this species, a convincing and complete picture of the pattern of the foetal circulation, together with the change brought on by allowing the foetus to breathe and by severing the umbilical cord, thus simulating the event of birth.

The purpose of the present thesis was to examine and quantitatively study key postnatal events in the developing and mature human cerebral cortex within the context of the systematic variation in the laminar structure of the cortex that underlies connectivity patterns and cortical function. To address this, we expanded upon previous work completed by LeRoy Conel and performed extensive analysis of Cresyl-Violet, Cajal, and Golgi-Cox images of various Von-Economo’s Areas for five age groups in order to see where variations in dendritic tree arborization, which is visualized by Golgi-Cox staining, pruning, which is visualized by Cresyl Violet staining, and cytoskeletal changes, which are visualized by Cajal staining, may lie. The results of this analysis showed that across all age groups and cortical types, there are consistently high levels of dendritic tree arborization, cytoskeletal changes, and cell body density changes in all layers across younger age groups, followed by slight declines in older age groups, following the trend of rapid gray matter expansion during the first two years of postnatal life before shifting towards white matter growth. These levels were also the most exaggerated in cortical layers III and V of Agranular, Dysgranular, Eulaminate II, and Eulaminate III areas, suggesting that cortical layers III and V of these areas change the most rapidly during the key events of postnatal development that Conel sought to analyze in his staining experiments. We also found that in layers III and V, Golgi-Cox values increased, reflecting the growth of dendritic trees while Cresyl-Violet and Cajal values decreased, reflecting decreases in cell body density, allowing for increased space between cells, highlighting a complementary inverse relationship that is seen between neurite outgrowth and cell body density. Since postnatal developmental processes in the cortex are largely driven by transcription factors that regulate chromatin expression well into adulthood, we also examined how certain epigenetic modifications in the nuclei of neurons in the adult cortex may explain for variations seen between areas in the analysis of Conel’s images. To address this, we analyzed populations of H4K12 and SC35 expressing pyramidal neurons in layers III and V of Area 46 and Area 32 in order to further understand how patterns of acetylation and RNA splicing may play a role in the rapid changes of layers III and V seen in Eulaminate II and Dysgranular areas. In the case of SC35, higher levels of splicing were seen in layer III of Area 32 and layer V of Area 46, suggesting that splicing centers within the neurons of these layers and areas are more organized. In the case of H4K12, similar levels of acetylation were observed in layers III and V of Area 46 whereas higher levels were observed in layer V of Area 32, indicating a certain degree of regulation of gene expression within these areas. The results of this study demonstrate variations in the timing of the major events of postnatal development across layers in different structural types of cortical areas that correspond to limbic, multimodal, and highly specialized unimodal areas and the inverse relationship seen between neurite outgrowth and changes in cell body density. Additionally, these results demonstrate the impact that patterns of RNA splicing and acetylation may have on these events and can be utilized to identify disruptions in developmental disorders such as autism. / 2024-09-21T00:00:00Z

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13533号 / 論医博第2273号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 斎藤 通紀, 教授 遊佐 宏介 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

GABAA receptor binding is transiently increased in rat whisker barrels during the second postnatal week, at a time when neurons in the developing rat cortex are vulnerable to excitotoxic effects. To test whether these GABAA receptors might serve to protect neurons from excessive excitatory input, polymer implants containing the GABAA receptor antagonist bicuculline were placed over barrel cortex for a 4-day period in young (postnatal days 8 - 12) and adult rats. In the cortex of young, but not adult rats, the chronic blockade of GABAA receptors resulted in substantial tissue loss and neuron loss. The greater loss of neurons in young rats supports the hypothesis that a high density of GABAA receptors protects neurons from excessive excitatory input during a sensitive period in development.

The lumbar motor activity in isolated spinal cords of 72 postnatal Balb/C mice aged 2, 5, 10 and 21 days (PN2-21) was electroneurographically recorded (ENG) via bilateral ventral roots following treatment with three different concentrations (25, 100 and 200 pM) of the neurotransmitter, 5-hydroxytryptamine (5-HT), i.e., serotonin, to determine its effects on spinal pattern generation.

La carence précoce en donneurs de méthyles (folates et vitamine B12) entraîne une hyperhomocystéinémie ainsi que des troubles cognitifs et de la coordination motrice chez les ratons de 21 jours. Ces troubles ont été reliés à une altération de la neurogenèse dans l'hippocampe et le cervelet, mécanisme étroitement relié à la neurostéroïdogenèse (NSG), définie par la synthèse locale de stéroïdes dans le cerveau. L'objectif principal de ce travail a été de déterminer si ces troubles étaient associés à une altération de la NSG. Ce travail a conduit à plusieurs résultats marquants, montrant notamment l'effet délétère de la carence gestationnelle en donneurs de méthyles sur la voie stéroïdogénique dans le cervelet, région cérébrale particulièrement vulnérable en période post-natale. Ceci s'est traduit par un effondrement du contenu en estradiol et autres neurostéroïdes, consécutivement à la diminution de l'expression de StAR, TSPO et de l'aromatase, ainsi que des récepteurs aux estrogènes. Ces effets délétères sont associés à une atteinte de la voie de signalisation dépendante de l'AMPc associée à l'activation du récepteur LHR, et la diminution de facteurs de transcription impliqués dans la régulation des gènes de la NSG, notamment SF-1, dont la présence dans les cellules de Purkinje du cervelet n'avait jamais été décrite auparavant. Cette réponse à la carence se ferait selon un dimorphisme sexuel, puisque seules les jeunes femelles nées de mères carencées étaient affectées. Enfin, ces études ont permis la mise en évidence d'altérations fonctionnelles associées aux dommages cellulaires et moléculaires observés dans les bulbes olfactifs (BO), se traduisant par une diminution des fonctions de la discrimination olfactive. Ceci pourrait contribuer au retard de croissance associé au poids corporel réduit mesuré chez les animaux carencés / Early methyl-donor deficiency (MDD, folate and vitamin B12) produces hyperhomocysteinemia, cognitive and motor disorders in 21 days-old rat pups. These disorders are linked to an alteration of neurogenesis in the cerebellum and the hippocampus, which is closely linked to neurosteroidogenesis, defined as the local synthesis of steroids in brain. The aim of this work was to verify if these troubles are associated with an altered neurosteroidogenesis. This work led to several striking results, showing in particular the deleterious effects of the methyl donor deficiency on the steroidogenic pathway in the cerebellum, particularly vulnerable during postnatal development. This was confirmed by the reduced contents of estradiol and other neurosteroids, as a result of the decrease of the expression of StAR, TSPO and aromatase, as well as the estrogen receptors. These deleterious effects are associated to the impairment of AMPc dependent signaling pathway and the decrease of transcription factors, notably SF-1, which the presence in the cerebellar Purkinje cells had never been described previously. This response to the deficiency would be made according to a sexual dimorphism, since only the young deficient females were affected. Finally, these studies allowed us to show functional changes associated with the cellular and molecular damage observed in the olfactory bulbs, being traduced by a decrease of the olfactory discrimination. This could contribute to the growth retardation associated with the reduced body weight of the deficient animals

La cellule cardiaque requiert un apport énergétique conséquent qui exige une production et un transfert énergétiques efficaces. Si la production de l’énergie dépend essentiellement des propriétés intrinsèques des mitochondries, il semblerait que l’efficacité du transfert d’énergie du site de production vers les sites consommateurs (ATPases) pourrait être liée à l’architecture spécifique du cardiomyocyte qui conduit à une organisation spatiale singulière des structures internes (mitochondries, réticulum sarcoplasmique, myofilaments). Pour comprendre ce qui lie la cytoarchitecture, la compartimentation cellulaire et la fonction contractile, il a été entrepris d’étudier l’architecture cellulaire et la signalisation énergétique de cardiomyocytes au cours du processus de maturation de la cytoarchitecture et dans un modèle présentant une désorganisation des structures intracellulaires. La première partie de ce travail, réalisée durant le développement postnatal de la souris, a permis de démontré qu’il existe une synchronisation parfaite entre la mise en place de la cytoarchitecture et la maturation fonctionnelle du transfert d’énergie par canalisation directe des nucléotides adényliques entre les mitochondries et les ATPases. Si cette étude apporte un élément qui tendrait à démontrer l’implication de l’architecture cellulaire dans l’efficacité des transferts d’énergie, elle a également mis en avant la maturation très précoce de l’énergétique cellulaire. La mitochondrie faisant partie intégrante de cette architecture et étant modelée par des mécanismes de fusion et de fission, la deuxième étape de ce travail de thèse a consisté à étudier l’implication de la morphologie mitochondriale dans l’énergétique du cardiomyocyte. Il a ainsi été montré que, chez la souris, la diminution d’expression de la protéine OPA1, impliquée dans la fusion mitochondriale, conduit à des perturbations de la morphologie mitochondriale qui n’affectent pas la fonction intrinsèque mitochondriale mais qui altèrent le système de canalisation directe entre les mitochondries et les ATPases des myofilaments. De manière générale, ces résultats démontrent clairement une dépendance des transferts d’énergie à l’architecture cellulaire spécifique de la cellule musculaire cardiaque. / The cardiac cell function requires a large amount of energy and therefore needs a high efficiency of energetic production and energetic transfer. While the energy production depends on the intrinsic properties of the mitochondria, it appears that the efficiency of energetic transfers from the main producers (mitochondria) to consumers (ATPases) could be related to the specific architecture of the cardiomyocyte, which ensures a unique spatial organization of internal structures (mitochondria, sarcoplasmic reticulum, myofilaments). In order to reveal the role of mitochondrial network organization in cardiac energy metabolism, we studied the cellular architecture and the energetic signalling of cardiomyocytes in the process of maturation of the cytoarchitecture and in a model which exhibits a perturbation of the mitochondrial dynamics. The first part of this work, which was performed during postnatal development of the mouse, showed the perfect synchronisation between the establishment of the cytoarchitecture and the maturation of the transfer of energy by direct channelling of adenine nucleotides between mitochondria and ATPases. While this study provides an element which would demonstrate the involvement of cellular architecture in the efficiency of energy transfer, it also highlighted the very early maturation of the energetic system of the cell. Knowing that the mitochondria are an integral part of the cell architecture and that the mitochondrial network is controlled by fusion and fission mechanisms, the second step of this work consisted in investigating the involvement of mitochondrial dynamics in cardiomyocyte energetics. Our work has shown that a decrease in expression of OPA1, a protein responsible for mitochondrial fusion, leads to disruption of mitochondrial morphology which does not affect intrinsic mitochondrial function but affects the direct channelling of ATP and ADP between mitochondria and ATPases of the myofilaments. Overall, these results clearly demonstrate that energy transfer in cardiomyocytes strictly depends on specific cellular architecture.

N6-methyladenosine (m6A) is the most ubiquitous post-transcriptional RNA modification and has an important role in determining the fate of mRNA transcripts. Among the key proteins of the m6A pathway are methyltransferases (METTL family enzymes), demethylases (FTO, ALKBH family enzymes), and m6A binding proteins (e.g., YTHDF family) which recognize RNA sequences depending on the amount and localization of m6A in target transcripts and subsequently influence the fate of mRNA transcripts. The role of methyltransferases and demethylases is to provide a dynamic balance of m6A levels and possibly to convey mechanisms of specificity for these so-called epitranscriptomic marks, which are not yet fully understood. The main objective of this work was to determine the relative changes in the expression of key m6A pathway proteins during early postnatal development and adulthood in the rat brain. We found that the level of expression of key m6A pathway proteins decreases from birth to adulthood, with the exception of a transient increase between postnatal days 10 and 18. During this period, we also found significant changes in the expression of respiratory chain complexes. However, further research is needed to provide evidence of a mechanistic link between the m6A pathway and brain energy homeostasis during...

Orientador: Raquel Fantin Domeniconi / Resumo: O estado nutricional materno desempenha papel crucial na saúde e no bem-estar do feto. Alterações testiculares, prostáticas e espermáticas foram observadas em animais adultos, cujas mães sofreram restrição de proteína. No entanto, não há trabalhos com este modelo experimental que enfoquem o desenvolvimento, padrão de vascularização e seus reflexos na expressão de proteínas no epidídimo. O objetivo deste estudo é investigar o padrão das respostas morfológicas, imuno-histoquímicas e expressão de proteínas do epidídimo da prole de ratos Wistar de mães submetidas à restrição proteica no período de gestação e de lactação. Fêmeas prenhes foram alocadas nos grupos experimentais normoproteico (NP) e hipoproteico (HP) que receberam, respectivamente, dietas contendo 17% e 6% de proteínas durante gestação e lactação. Após o desmame, os ratos receberam dieta padrão para roedores até as idades de 21, 44 e 120 dias pós-natais (DPN) quando foram eutanasiados. Os epidídimos foram coletados e processados segundo técnicas histológicas, imuno-histoquímicas e de western blotting. Nos filhotes HP, o tamanho reduzido e baixo peso observados ao nascimento se mantiveram até o DPN 120, acompanhados da redução dos órgãos do sistema genital masculino para todas as idades analisadas. A dieta hipoproteica materna diminuiu os níveis séricos de testosterona nos animais no DPN 44, aumentou os níveis de aldosterona nos animais no DPN 21 e não alterou os níveis de estradiol em nenhuma das idades. Nos animais ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Maternal nutrition status plays a crucial role in the health and well-being of the fetus. Changes of testes and prostate as well as spermatic disorders were observed in adult animals whose mothers were subjected to protein restriction. However, there are no studies with this experimental model that focus the development and the vascular pattern of epididymis, as well as their reflexes in the expression of proteins of the epididymis. The aim of this study is to investigate the pattern of morphometric, immunohistochemical and protein expression of the epididymis of the Wistar rat offspring whose mothers were subjected to a low-protein diet during gestation and lactation. Pregnant females were divided into normoprotein (NP) group and low-protein (LP) group that received, respectively, diets containing 17% and 6% of protein during gestation and lactation. After weaning, the LP and NP male pups received the standard diet for rodents until the ages of 21, 44 and 120 days (PND), when they were euthanized. The epididymides were collected and processed according to histological, immunohistochemical and western blotting techniques. In the LP offspring, the smaller body size and low weight observed at birth remained until the PND 120, as well as the reduction of the male genital system organs weight for all analyzed ages. Maternal low-protein diet decreased testosterone serum levels in animals at PND 44, increased aldosterone serum levels in animals at PND 21 and did not altered estradi... (Complete abstract click electronic access below) / Doutor