Systemic sclerosis (SSc) is a rare connective tissue disease affecting skin and internal organs. The pathogenesis of SSc is multifactorial and includes autoimmunity, inflammation and vasculopathy. Pulmonary arterial hypertension (PAH) is among the most serious of SSc complications and is characterized by augmented vasoconstriction, neointimal remodeling and occlusion of small arteries in lung. Elevated pulmonary arterial blood pressure and volume overload in the right heart eventually lead to death from heart failure. Pre-existing elevated pro-fibrotic signaling, systemic vasculopathy and chronic inflammation are additional factors that likely contribute to the more severe PAH manifestation in SSc patients, whose response to existing therapies is suboptimal. The aim of my thesis research was to investigate the pathological role of altered transcriptional regulation of endothelium in pulmonary vasculature, as well as testing potential novel therapies for SSc-PAH in vivo. I found that endothelial downregulation of GATA6 promotes increased production of reactive oxygen species (ROS) by suppressing enzymatic machinery responsible for ROS clearance. Increased ROS production triggered ER stress and inflammation, exacerbating endothelial dysfunction and vascular injury both in vitro and in vivo. Another discovery is that simultaneous depletion of two ETS-family factors, ERG and FLI1 synergistically activates interferon signaling in pulmonary endothelial cells and promotes inflammation in lung in vivo. Based on observed pathological contribution of oxidative stress and inflammation to vasculopathy and fibrosis I tested an anti-oxidative and anti-inflammatory agent- dimethyl fumarate (DMF) in mouse models of PAH, as well as lung and skin fibrosis. DMF efficiently ameliorated increased pulmonary artery pressure and vascular remodeling, as well as fibrotic changes in lung and skin. Mechanistically, I found that DMF promotes a proteasomal, βTRCP-dependent degradation of pro-fibrotic mediators TAZ/YAP, β-catenin and Sp1. In conclusion, I characterized new elements of pathological mechanism that promote vasculopathy and fibrosis, as well as provided an insight into anti-inflammatory and anti-fibrotic DMF therapy. Importantly, elucidating the novel mechanism of DMF action and recognizing the pathological role of Hippo and Wnt signaling in fibrosis might help to design more specific and effective pharmacological intervention in SSc-PAH patients.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20975号 / 医博第4321号 / 新制||医||1026(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 川上 浩司, 教授 鈴木 茂彦, 教授 開 祐司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24494号 / 医博第4936号 / 新制||医||1063(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 村川 泰裕, 教授 小杉 眞司, 教授 松田 文彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Adult exposure to high fat diet (HFD) has been linked to increased risk of musculoskeletal, cardiovascular, and metabolic diseases; however, the contribution of gestational HFD to elevated oxidative stress (OS), perinatal cardiovascular, skeletal, and metabolic dysfunction as well as long-term effects on adult offspring are incompletely understood. Pathophysiologic mechanisms linking gestational HFD, OS, and insulin resistance to perinatal development and adult-onset chronic diseases are explored in the present study, and maternal antioxidant (quercetin) is offered as a potential preventive dietary supplement to reduce fetal and maternal sequelae of HFD. Female C57BL/6 mice were fed "cafeteria-style" HFD (including 32.1% saturated fat to mimic a typical fast food menu) with or without quercetin for one month prior to conception, and throughout gestation. HFD dams developed gestational diabetes with significantly increased placental OS and vasculopathy. Neonates were smaller at birth than age-matched controls, and surviving offspring developed type 2 diabetes, hypertension and osteoporosis during adulthood, despite having been fed healthy diet throughout their postnatal life. Additional measures of bone using three-dimensionally reconstructed computed tomographic image analysis (microCT) revealed microarchitectural changes of bone at birth, and at 6 and 12 months postnatally. Fetuses from HFD dams displayed diminished bone mineral density (BMD) and disrupted endochondral and intramembranous ossification with significantly shortened distal limb lengths, as compared to offspring of standard rodent chow dams. Skeletal malformation persisted into adulthood despite the fact that both control and HFD offspring were fed conventional rodent chow throughout postnatal life. The offspring gestationally exposed to HFD showed significant decreased femoral BMD at 6 months of age and dysregulation of distal femoral trabecular architecture at 12 months of age, indicating development of osteoporosis. We were able to reduce incidence of placental vasculopathy, fetal maldevelopment and adult-onset type 2 diabetes, hypertension and osteoporosis with concurrent maternal quercetin supplementation during pregnancy. Collectively, these data suggested that maternal HFD increases placental OS and vascular damage during pregnancy, which are associated with fetal malformation and elevated adult-onset multisystemic chronic diseases. Maternal quercetin supplementation must be further explored as a potential dietary intervention for improved placental integrity, fetal development and lifelong health. / Ph. D.

No description available.

Coronary atherosclerosis is by far the most frequent cause of ischemic heart disease. Intravascular ultrasound (IVUS) along with virtual histology (VH) is a useful tool for quantification of coronary plaque buildup and provides new insights into the diagnosis of coronary disease. Rupture of vulnerable plaque causing acute coronary syndromes, coronary remodeling maintaining lumen size and plaque phenotype revealing pathological severity are among the most important topics related to atherosclerosis. In this thesis, variations of IVUS-VH-derived thin-cap fibroatheroma (TCFA) definitions are proposed to evaluate the plaque rupture, which is further analyzed in a layered manner; statins effects on coronary remodeling are comprehensively assessed with the implementation of automated IVUS segmentation and registration of IVUS pullbacks based on baseline and 1-year followup datasets; plaque phenotypes are determined and analyzed morphologically and compositionally on segmental basis using the same serial datasets. In addition, our research involves another important coronary disease — coronary allograft vasculopathy (CAV) which is a frequent complication of heart transplantation (HTx). Another intra-coronary imaging modality — intravascular optical coherence tomography (IVOCT) for quantifying CAV is involved. We present an optimal and automated 3-D graph search approach for the simultaneous IVOCT multi-layer segmentation by transforming the 3-D segmentation problem into finding a minimum-cost closed set in a weighted graph. Furthermore, a computer-aided just-enough-interaction refinement method is proposed to help achieve fully satisfactory 3-D segmentation of IVOCT images. We believe this is the first work that provides a fast, efficient and accurate solution for IVOCT multi-layer assessment in the context of CAV. The major contributions of this thesis are: (1) Proving that IVUS-VH-derived TCFA prevalence may be overestimated, and elucidating the potential loss of plaque material during rupture, (2) providing a comprehensive understanding of remodeling in the context of both changing the remodeling direction and changing the remodeling extent, and demonstrating the statin therapy effects on remodeling across patients, based on automated segmentation of IVUS images and registration of serial data, (3) showing that the pathological intimal thickening is the most active plaque phenotype in terms of plaque composition changes and plaque vulnerability progression, and (4) developing and validating a method for multi-layer 3-D segmentation of IVOCT images within a novel interactive environment.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21002号 / 医博第4348号 / 新制||医||1027(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 大森 孝一, 教授 山田 亮, 教授 Shohab YOUSSEFIAN / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

No description available.

Les interférons de type I (IFN I) sont des cytokines antivirales aux propriétés puissantes. L’induction, la transmission et la résolution de la réponse immunitaire engendrée par les IFN I est minutieusement régulée. Le concept d’interféronopathie de type I, récemment individualisé par notre équipe, repose sur l’hypothèse que certaines pathologies seraient secondaires au déséquilibre de ces voies de signalisation complexes et à la sécrétion excessive et inappropriée d’IFN I. L’inhibition de celle-ci par des thérapeutiques ciblées permettrait de valider cette hypothèse, si les symptômes allégués s’amélioraient, voire disparaissaient. Ce travail de thèse s’est initialement concentré sur la caractérisation clinique et biologique des interféronopathies monogéniques et polygéniques, et secondairement sur l’identification moléculaire de nouvelles mutations du gène TMEM173 à l’origine de l’interféronopathie liée à STING, également appelée SAVI (STING associated vasculopathy with onset in infancy), syndrome auto-inflammatoire associant une atteinte sévère cutanée et pulmonaire. De nouvelles techniques ont permis la sélection de patients présentant une augmentation de l’IFN I en comparaison à des contrôles sains : la signature IFN I, qPCR de 6 gènes stimulés par l’IFN (IFN stimulated genes – ISGs) et le dosage d’IFN alpha sérique ou plasmatique par méthode du SIMOA (single molecule array) permettant la détection de molécules d’IFN de l’ordre du femtogramme (10-18g). Ces méthodes nous ont ainsi permis d’élargir le spectre clinique phénotypique des interféronopathies de type I, initialement considéré comme essentiellement neurologique. Les patients atteints du syndrome d’Aicardi-Goutières, première interféronopathie monogénique décrite, présentaient les signes suivants : dystonie, spasticité, décalage des acquisitions, calcifications intra-cérébrales et anomalies de la substance blanche. Cependant, l’utilisation systématique de nos méthodes de criblage associée à l’avènement des technologies de séquençage à haut débit (next generation sequencing – NGS) a permis de révéler un phénotype plus large, caractéristique des interféronopathies de type I : sur le plan cutané (engelures, vascularite nécrosante des extrémités, sclérodermie), pulmonaire (pneumopathie interstitielle isolée ou non), musculo-squelettique (arthralgies, arthrites, arthropathie de Jaccoud, myalgies et myosites), ophtalmologique (glaucome), néphrologique (néphropathies lupiques), gastro-entérologique (maladies inflammatoires chroniques intestinales précoces), associées à de l’auto-immunité ou un déficit immunitaire inconstants. Notre méthode de sélection nous a notamment permis d’identifier des patients présentant de manière variable des signes cardinaux de SAVI et une de trois nouvelles mutations activatrices dans une région spécifique du gène TMEM173 (codant pour STING). Ces mutations circonscrivent une région de la protéine à ce jour encore jamais impliquée dans le contrôle de la voie de l’IFN I. STING est une protéine du réticulum endoplasmique qui agit comme adaptateur cytosolique de senseurs intracellulaires d’ADN viral dans une voie de signalisation de l’IFN I. STING active TBK1 (TANK-binding kinase) et permet la transcription des IFN I par la phosphorylation d’IRF3. La Janus Kinase 1 (JAK1) et la tyrosine kinase 2 (TYK2) sont activées suite à la stimulation des récepteurs de l’IFN I et phosphorylent les facteurs de transcription STAT1 et STAT2, conduisant à l’expression de nombreux ISGs. Les analyses génétiques, de conformation tridimensionnelle, sur un modèle cellulaire in vitro (HEK293T) et ex vivo sur cellules mononuclées périphériques des patients nous ont ainsi permis de mettre en évidence pour ces mutations un caractère constitutionnellement activé, indépendant de la liaison au ligand cGAMP, mais transmettant ce signal à travers la voie d’aval par TBK1. (...) / Type I interferons (IFN I) are antiviral cytokines with potent properties. Hence, the induction, transmission and resolution of the immune response generated by IFN I is tightly regulated. The concept of the type I interferonopathies, recently formulated by our team, rests on the assumption that some diseases arise from a disturbance of this complex signalling pathway, leading to excessive and inappropriate IFN I secretion. On this basis, targeted therapeutics should improve or cure features of such type I interferonopathies, thereby providing a validation of the underlying hypothesis. This PhD project initially focused on the clinical and biological characterisation of monogenic and polygenic interferonopathies, and secondarily on the molecular identification of novel mutations in the gene TMEM173 causing the interferonopathy called STING associated vasculopathy with onset in infancy (SAVI), an auto-inflammatory syndrome with severe cutaneous and pulmonary features. Our selection of patients in comparison to healthy controls was made possible through the use of novel screening tools: IFN signature (qPCR of 6 IFN stimulated genes – ISGs), and measurement of IFN alpha protein levels in serum or plasma (SIMOA-single molecule array - enabling the detection of molecules of IFN in the femtogram [10-18g]) range. In this way, we have been able to expand the phenotypic spectrum of the interferonopathies, which was initially considered as primarily neurological. Patients with Aicardi-Goutières syndrome (AGS), the first described of the monogenic interferonopathies, exhibit dystonia, spasticity, developmental delay, intra-cranial calcifications and white matter abnormalities. However, the systematic use of our interferon screening assays, plus the advent of next-generation sequencing technology, has revealed a much broader set of features relevant to this novel disease grouping – involving the skin (chilblains, necrotising vasculitis, scleroderma), lungs (isolated lung interstitial disease or associated with other signs), musculoskeletal system (joint pain, arthritis, Jaccoud’s arthropathy, muscle pain and myositis), eyes (glaucoma), kidneys (lupus nephritis) and gastro-intestinal tract (early inflammatory bowel disease), as well features of autoimmunity and immunodeficiency. Using our screening assays enabled us to identify three patients variably exhibiting the core features of SAVI, all of whom were found to harbour distinct novel activating mutations in STING. These mutations highlight a protein domain not previously implicated in the control of IFN I signalling. STING is an endoplasmic reticulum protein, acting as a cytosolic adaptor of intracellular sensors of viral DNA in the type I IFN signalling pathway. STING activates TANK-binding kinase (TBK1), allowing transcription of IFN I through phosphorylation of IRF3. Janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2) are activated following stimulation of the IFN I receptor, leading to phosphorylation of the transcription factors STAT1 and STAT2 and the subsequent induction of a large number of ISGs. Genetic analysis, conformational studies, an in vitro cellular model (HEK293T) and ex vivo experimental data (using patient peripheral blood mononuclear cells - PBMCs) enabled us to confirm the constitutive activating nature of these variants, and show that this activation did not require binding with cGAMP, but was dependent on signalling through TBK1. Ruxolitinib, a JAK1/2 inhibitor, could antagonise this constitutive activation ex vivo. These results indicate a promising therapeutic approach in such patients, and more widely in the monogenic, and perhaps even, polygenic, interferonopathy context.