Abstract Cardiac remodeling is defined as changes in the size, shape and function of the heart, caused most commonly by hypertension-induced left ventricular (LV) hypertrophy and myocardial infarction (MI). It is characterized by changes in cellular and extracellular compartments regulated by e.g. neurohumoral and inflammatory factors. In the present study the expression of novel, load induced factors, thrombospondin (TSP)-1 and -4, matrix Gla protein (MGP), tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fn14, was investigated during cardiac remodeling. Their expression in the heart was characterized using experimental models of pressure overload, hypertensive hypertrophy and MI, and the effect of hypertrophic agonists and cellular stretch was studied in vitro. The effect of beta-blocker treatment on TSP expression was also examined. TSP-1 and -4 were rapidly upregulated in response to pressure overload, and the induction of TSP-4 gene expression was attenuated in hypertrophied heart. After MI, TSP-1 and -4 mRNA and TSP-1 protein levels were increased, and the induction was attenuated by metoprolol. TSP-1 and -4 expression correlated with natriuretic peptide expression and LV remodeling after MI. In hypertensive hypertrophy, only TSP-4 expression decreased after metoprolol treatment and was correlated with LV remodeling. MGP gene expression was increased in response to pressure overload and MI both in the early and late phase of cardiac remodeling. MGP protein levels were increased in the acute phase of post-MI remodeling and in hypertensive hypertrophy. In vitro, angiotensin II increased MGP gene expression in myocytes and fibroblasts, whereas expression decreased in response to mechanical stretch. In response to increased cardiac load Fn14 expression was upregulated both acutely and chronically while TWEAK expression remained relatively constant. Fn14 localized mainly to fibroblasts in the inflammatory area while TWEAK localized to myocytes and endothelial cells. In myocytes, Fn14 expression was induced by hypertrophic agonists and mechanical stretch in contrast to stabile or decreased TWEAK expression. This study provides new insights into the expression of the studied novel factors in cardiac remodeling. The distinct expression of TSPs in pressure overload and post-MI suggests that TSP-1 and -4 may have unique roles in the remodeling process. The results also imply that MGP is part of the common gene program of hypertrophic remodeling in vivo and contributes to the molecular basis of cardiac hypertrophy. Finally, the study demonstrates differential regulation of TWEAK and Fn14 expression in the heart and emphasizes the importance of Fn14 as a mediator of TWEAK/Fn14 signaling and as a potential target of therapeutic interventions.

Diameters of microvessels undergo continuous structural adaptation in response to hemodynamic and metabolic stimuli. To ensure adequate flow distribution, metabolic responses are needed to increase diameters of vessels feeding poorly perfused regions. Possible modes of metabolic control include release of signaling substances from vessel walls, from the supplied tissue and from red blood cells (RBC). Here, a theoretical model was used to compare the abilities of these metabolic control modes to provide adequate tissue oxygenation, and to generate blood flow velocities in agreement with experimental observations. Structural adaptation of vessel diameters was simulated for an observed mesenteric network structure in the rat with 576 vessel segments. For each mode of metabolic control, resulting distributions of oxygen and deviations between simulated and experimentally observed flow velocities were analyzed. It was found that wall-derived and tissue-derived growth signals released in response to low oxygen levels could ensure adequate oxygen supply, but RBC-derived signals caused inefficient oxygenation. Closest agreement between predicted and observed flow velocities was obtained with wall-derived growth signals proportional to vessel length. Adaptation in response to oxygen-independent release of a metabolic signal substance from vessel walls or the supplied tissue was also shown to be effective for ensuring tissue oxygenation due to a dilution effect if growth signal substances are released into the blood. The present results suggest that metabolic signals responsible for structural adaptation of microvessel diameters are derived from vessel walls or from perivascular tissue.

Over the past five years the role of epigenetic modifiers in brain development has become increasingly evident. In this regard, Snf2l, a homolog of the chromatin remodeling protein ISWI, was shown to have enriched expression in the brain and be important for neuronal differentiation. Mice lacking functional Snf2l have hypercellularity of the cerebral cortex due to increased cell cycle re-entry. In this thesis I demonstrate the effects of Snf2l-ablation on cortical progenitor cells including increased proliferation and cell cycle deregulation, the consequence of which is a delay in neuronal migration and altered numbers of mature cortical neurons. This phenotype arises from increased expression of Foxg1, a winged-helix repressor expressed in the forebrain and anterior optic vesicle. Moreover, genetically reducing its overexpression rescues the Snf2l-ablated phenotype. Snf2l is bound directly to a promoter region of Foxg1 suggesting that it acts as a repressive regulator in vivo and is an important factor in forebrain differentiation.

Obese pregnant women have increased risk of a number of pregnancy complications, including poor maternal health, fetal growth restriction (FGR) and fetal demise. The success of pregnancy is dependent on precise regulation of the immune response within the utero-placental environment. Rats as a model for human related pregnancy complications are beginning to be widely used because of the similarities between these species in terms of trophoblast invasion and spiral artery remodeling. However our knowledge of immune cells and cytokine localization in the rat utero-placental tissue relating to these processes is limited. Therefore our first aim was to characterize the immune cell populations, such as uterine natural killer (uNK) cells, neutrophils and macrophages in the rat utero-placental unit at two crucial gestational ages relevant to trophoblast invasion and spiral artery remodeling, gestational day (GD) 15 and GD18. In addition, we characterized the cytokine distribution of TNFα, IFNγ and IL-10 in the utero-placental tissue at both above mentioned gestational ages. Our study has demonstrated co-localization of TNFα and IFNγ with uNK cells in the perivascular region of the spiral arteries in the rat mesometrial triangle. Neutrophils were localized at the maternal fetal interface and in the spiral artery lumen of the rat mesometrial triangle at both gestational ages. TNFα and IL-10 demonstrated a temporal change in the localization from GD15 to GD18 which coincides with the leading edge of trophoblast invasion into the mesometrial triangle. The results of the current study furthers our knowledge of the localization and temporal expression of uterine immune cells and relevant cytokines, and provides a base to research the function of these immune cells and cytokines during rat pregnancy as a model to study human pregnancy and complications related to immune functions. Since obesity is associated with a peripheral and systemic pro-inflammatory state in humans, our second objective was to investigate whether maternal obesity could alter the utero-placental and systemic immune response in the rats. To characterize maternal obesity induced changes in uterine immune state we used pregnant rats fed a control diet (normal weight; CD) or a high fat diet (obese; HFD) at GD15 and GD18. We performed immunohistochemistry to localize TNFα and IL-10, and quantified the levels of TNFα, IL-1β and IL-10 in the uterine tissue by immunoassay. To assess the systemic immune state, circulating levels of pro-inflammatory cytokine MCP-1 were assessed by immunoassay. We demonstrated an increased concentration of the pro-inflammatory marker TNFα and a reduced anti-inflammatory IL-10-positive cell distribution in the rat mesometrial triangle in response to a HFD. In addition increased circulating MCP-1 was observed in the HFD-fed dams at both gestation ages. HFD induced obesity in our rat model leads to an increase in uterine and systemic pro-inflammatory markers. These markers have demonstrated the potential to alter utero-placental development. Pregnancy complications such as FGR and fetal demise have been shown to be associated with impaired placental development as a result of altered trophoblast invasion and aberrant maternal spiral artery remodeling. Therefore, our third aim was to compare these parameters between the CD-fed rats and HFD-fed rats at GD15 and GD18. Early trophoblast invasion was increased by approximately 2-fold in HFD-fed dams with a concomitant increase in the expression of matrix metalloproteinase-9 protein, a mediator of tissue remodeling and invasion. By late gestation reduced trophoblast invasion was observed in HFD-fed dams. Furthermore, we also observed in late gestation significantly higher levels of smooth muscle actin surrounding the uterine spiral arteries of HFD-fed dams, suggesting impaired spiral artery remodeling. We also determined the impact of human serum from obese mothers on trophoblast invasion. We compared the invasion of HTR-8/SVneo cells treated with pooled first-trimester serum from obese women with or without fetal growth restriction vs. cells treated with serum from normal-weight women with or without fetal growth restriction. First-trimester serum from obese pregnant women reduced invasion of the trophoblast cell line HTR8/SVneo compared to serum from normal-weight pregnant women. Taken together, the results of this study suggest that maternal obesity can negatively influence crucial utero-placental development processes resulting in the poor pregnancy outcomes and increased fetal demise. To summarize, the HFD increased the pro-inflammatory marker TNFα which was associated with altered trophoblast invasion profiles and impaired vascular remodeling. These disturbances in utero-placental development were also associated with decreased birth weights (indication of FGR) and increased rates of stillbirths in our obese rat model. In conclusion, we have made progress in defining the influence of maternal obesity (HFD) on utero-placental development. The importance of these studies is evident since FGR represents a leading cause of perinatal morbidity and mortality. Furthermore, FGR fetuses have an increased risk of becoming obese in their lifetime as a result of fetal programming, therefore resulting in the propagation of a transgenerational obesity cycle. Therefore by understanding the mechanisms by which maternal obesity influences utero-placental development leading to FGR, we may be able to impact short term morbidity and prevent the programming of obesity in future generations. In addition, characterization of maternal obesity’s influence on utero-placental development will also help in the search for therapeutics or intervention strategies to help optimize fetal growth and improve pregnancy outcomes in obese women.

The packaging of genomic DNA into nucleosomes creates a barrier to transcription which can be relieved through ATP-dependent chromatin remodeling via complexes such as the switch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complex. The SWI/SNF complex remodels chromatin via conformational or positional changes of nucleosomes, thereby altering the access of transcriptional machinery to target genes. The SWI/SNF complex does not possess intrinsic DNA binding ability, and therefore its recruitment to target loci requires interaction with DNA-associated transcription factors. The Cdx family of homeodomain transcription factors (Cdx1, Cdx2 and Cdx4) are essential for a number of developmental programs in the mouse. Cdx1 and Cdx2 also regulate intestinal homeostasis throughout life. Although a number of Cdx target genes have been identified, the basis by which Cdx members impact their transcription is poorly understood. We have found that Cdx members interact with the SWI/SNF complex and make direct contact with Brg1, a catalytic member of SWI/SNF. Both Cdx2 and Brg1 co-occupy a number of Cdx target genes, and both factors are necessary for transcriptional regulation of such targets. Finally, Cdx2 and Brg1 occupancy occurs coincident with chromatin remodeling at certain of these loci. Taken together, our findings suggest that Cdx transcription factors regulate target gene expression, in part, through recruitment of Brg1-associated SWI/SNF chromatin remodeling activity.

Chromatin remodeling enzymes are instrumental for neural development as evidenced by their identification as disease genes underlying human disorders characterized by intellectual-disability. In this regard, the murine Snf2h and Snf2l genes show differential expression patterns during embryonic development, with a unique pattern in the brain where Snf2h is predominant in neural progenitors, while Snf2l expression peaks at the onset of differentiation. These observations led me to investigate the role of Snf2h and Snf2l in brain development by using conditionally targeted Snf2h and Snf2l mice. I selectively ablated Snf2h expression in cortical progenitors, cerebellar progenitors, or postmitotic Purkinje neurons of the cerebellum, while Snf2l was deleted in the germline. I found that Snf2h plays diverse roles in neural progenitor expansion and postmitotic gene expression control, while Snf2l is involved in the precise timing of neural differentiation onset. Gene expression studies revealed that Snf2h and Snf2l co-modulate the FoxG1 and En1 transcription factors during cortical and cerebellar neurogenesis, respectively, to precisely control the transition from a progenitor to a differentiated neuron. Moreover, Snf2h is essential for the postmitotic neural activation of the clustered protocadherin genes, and does so by functionally interacting with the matrix-attachment region protein Satb2. My neurobehavioral studies also provided insight into how Snf2h loss in cerebellar progenitors results in cerebellar ataxia, while Snf2h loss in cortical progenitors, or in postmitotic Purkinje neurons of the cerebellum, resulted in learning and memory deficits, and hyperactive-like behavior. Molecularly, Snf2h plays an important role in linker histone H1e dynamics and higher order chromatin packaging, as evidenced by loss of chromatin ultrastructure upon Snf2h deletion in progenitor and postmitotic neurons. I further demonstrated that Snf2h loss in a neuronal cell culture model results in reduced H1e deposition, and that overexpression of human SNF2H or SNF2L upon Snf2h knockdown rescues this biochemical dysfunction. My experiments suggest that Snf2h and Snf2l are regulatory nucleosome remodeling engines that co-modulate the gene expression programs necessary for proper brain development, maturation and function.

Du fait de l'augmentation de la durée de vie de la population et de la fréquence de certains types d'accidents, la problématique de l'évolution des articulations et du vieillissement osseux devient primordiale, ce qui implique de nombreuses opérations chirurgicales nécessitant une pose d'implant. Bien que ces interventions soient réalisées depuis longtemps de façon routinière, il subsiste des risques d'échec chirurgical pouvant avoir des conséquences dramatiques pour le patient. Malgré les évolutions apportées, le taux d'échec implantaire est encore important car les phénomènes mis en jeu restent mal compris du fait de leur complexité et de leur nature multi-échelle. Un des aspects essentiels de la réussite des ces opérations concerne l'ostéointégration de l'implant, c'est-à-dire la capacité de l'os à se régénérer autour de ce corps étranger en l'intégrant durablement. La réussite chirurgicale dépend en premier lieu des caractéristiques physiques de l'interface os-implant transmettant les efforts biomécaniques qui jouent un rôle majeur dans le remodelage osseux. Ces travaux de recherche s'inscrivent dans le cadre du développement d'une approche expérimentale multimodale pour la caractérisation de l'interface os-implant afin d'évaluer l'impact de la reconstruction osseuse sur la réponse mécanique de l'implant. La première partie met en œuvre une approche multiphysique utilisant un modèle animal dédié. Des pastilles en titane sont implantées in vivo sur la partie proximale du tibia du lapin pendant différents temps de cicatrisation. Une technique de nanoindentation permet de mesurer les propriétés mécaniques de l'os néoformé à l'échelle microscopique. La technique de diffusion micro-Brillouin permet de mesurer la vitesse ultrasonore de l'os néoformé à la même échelle. Les résultats obtenus à partir de ces deux techniques permettent de déterminer la différence de densité volumique entre l'os mature et l'os néoformé aux différents temps de cicatrisation considérés. Dans la seconde partie, un dispositif ultrasonore destiné à l'étude de la stabilité d'implants dentaires en titane est présenté. La réponse ultrasonore obtenue en mode échographique est sensible aux propriétés du matériau (os, biomatériau) au contact de l'implant. Premièrement, la réponse ultrasonore d'implants dentaires insérés dans un substitut dentaire bioactif (silicate tricalcique) et sollicités par un protocole de fatigue mécanique in vitro est mesurée. Pour cela, un banc de fatigue mécanique simulant la mastication a été développé. Deuxièmement, ce même dispositif ultrasonore est utilisé pour déterminer in vitro la stabilité primaire d'un implant dentaire placé dans un os bovin. Troisièmement, une étude in vivo utilisant un modèle animal (lapins) a permis de mettre en évidence l'effet du temps de cicatrisation sur la réponse ultrasonore de l'implant. Ce dispositif ultrasonore permet de quantifier la stabilité primaire et secondaire d'un implant dentaire. Les phénomènes de propagation ultrasonore dans l'implant sont modélisés en utilisant des techniques de simulations numériques par éléments finis. Les simulations montrent le potentiel de la technique pour suivre les variations de plusieurs paramètres déterminants pour l'ostéointégration de l'implant dans des conditions contrôlées / Due to the increase of life duration and to the frequency of certain types of accidents, the problematic of the evolution of joints and aging bone has become crucial, leading to an important number of surgical interventions requiring implant placement. Although these interventions are carried out routinely in the clinic, there are still risks of surgical failure, which induce dramatic consequences for the patient. Despite the evolution of the surgical strategies, the implants failure rate remains important because the phenomena involved are not well understood due to their complexity and to their multi-scale nature. One of the main determinants of the success of these surgical interventions lies in the implant osseointegration, that is to say the ability of bone tissue to regenerate around the implant integrating the implant in a sustainable manner. The surgical success depends primarily on the physical characteristics of the bone-implant interface transmitting the biomechanical efforts, which play a major role in bone remodeling. The approach carried out in the present research consist in developing a multimodal experimental approach to characterize the biomechanical properties of the bone-implant interface in order to assess the impact of bone remodeling around the implant on the mechanical response of the implant. In the first part, a multiphysical approach is carried out using a dedicated animal model. Coin-shaped titanium implants are implanted in vivo on the proximal part of the tibia of rabbits during different periods of healing time. A nanoindentation device is used to measure the mechanical properties of the newly formed bone at the microscopic level. A micro-scattering Brillouin device is employed to estimate the ultrasonic velocity of newly formed bone at the same scale. The results obtained with both techniques are used to determine the difference of bone mass density difference between mature bone tissue and newly formed bone tissue for different healing times.In the second part, an ultrasonic device aims at investigating the stability of titanium dental implants. The ultrasonic response is measured in echographic mode and is shown to be sensitive to the properties of the material (bone, biomaterial) in contact with the implant. Firstly, the evolution of the in vitro ultrasonic response of dental implants inserted into a bioactive dental substitute (tricalcium silicate based cement) and loaded using a mechanical protocol stress is assessed. To do so, a mechanical fatigue bench simulating chewing motions was developed. Secondly, the same ultrasonic device is used to determine in vitro the primary stability of an implant placed into bovine bone tissue. Third, an in vivo study using an animal model (rabbit) investigates the effect of healing time on the ultrasonic response of the implant. The ultrasound device is used to quantify the primary and secondary dental implant stability. The phenomena of ultrasonic propagation in the implant are modeled using techniques of numerical simulations by finite elements. The simulations show the potential of the technique to monitor changes in several key parameters for osseointegration of the implant under controlled conditions

La malposición dentaria influye en la estética y funcionalidad de los pacientes. Ante esta situación, es posible realizar la corrección mediante el tratamiento ortodóntico fijo; sin embargo, a la consulta, acuden pacientes con características clínicas tales como corticales vestibulares reducidas, reabsorciones radiculares y posiciones de las piezas dentarias no compatibles con el hueso alveolar, los cuáles limitan la posibilidad de alcanzar los objetivos del tratamiento ortodóntico. A pesar de esto, es posible complementar el tratamiento ortodóntico con procedimientos periodontales para optimizar el movimiento dental y reducir efectos adversos en los tejidos de soporte. Este trabajo reporta el tratamiento ortodóntico realizado en seis meses de un paciente de sexo femenino de 25 de edad complementado con corticotomía e injerto óseo para corregir el apiñamiento de las piezas dentarias sin efectos adversos de los tejidos de soporte. Es recomendable contar con un análisis tomográfico previo al tratamiento para poder evaluar correctamente la topográfica del hueso alveolar, de tal forma que se puedan identificar factores que puedan alterar la estabilidad del tratamiento ortodóntico. / Dental malposition influences the aesthetics and functionality of patients. In this situation, it is possible to make the correction by means of fixed orthodontic treatment; However, at the consultation, patients with clinical features such as reduced vestibular cortices, root resorption and positions of teeth not compatible with alveolar bone, which limit the possibility of achieving the objectives of orthodontic treatment. Despite this, it is possible to complement orthodontic treatment with periodontal procedures to optimize dental movement and reduce adverse effects on supportive tissues. This work reports the orthodontic treatment performed in six months of a 25-year-old female patient supplemented with corticotomy and bone graft to correct the crowding of the dental pieces without adverse effects of the support tissues. It is advisable to have a tomographic analysis prior to treatment in order to correctly assess the topography of the alveolar bone, so that factors that can alter the stability of the orthodontic treatment can be identified. / Trabajo académico

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21953号 / 医博第4495号 / 新制||医||1037(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 柳田 素子, 教授 斎藤 通紀, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18880号 / 医博第3991号 / 新制||医||1008(附属図書館) / 31831 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 篠原 隆司, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM