O presente estudo tem por objetivo avaliar as características clinicopatológicas, imunoistoquímicas e moleculares de 513 casos brasileiros de tumor estromal gastrintestinal, incluindo o estudo da expressão imunoistoquímica de CD117, CD34, proteína DOG1, actina de músculo liso, desmina, proteína S-100, antígeno Ki-67, proteína p53, molécula de adesão CD44v3, receptor para fator de crescimento epidérmico, proteína HER2 e receptor para fator de crescimento derivado de plaqueta alfa, além da análise molecular de mutações envolvendo os genes KIT e receptor para fator de crescimento derivado de plaqueta alfa e da pesquisa de amplificação dos genes receptor para fator de crescimento epidérmico e HER2 por hibridização \"in situ\" fluorescente. As características clínicas e morfológicas dos casos estudados não foram diferentes das referidas pela literatura. Houve expressão de CD117 (KIT) em 95,7% dos casos. A proteína DOG1 foi expressa em 85,9% dos tumores, sendo capaz de diagnosticar 20% dos casos com ausência de expressão de CD117 (KIT). O índice de proliferação celular determinado pelo antígeno Ki-67 foi superior nos casos classificados como de alto risco para comportamento biológico agressivo segundo critérios do \"National Institutes of Health\". A expressão da proteína p53 esteve restrita aos casos classificados como de alto risco. Não se observou expressão imunoistoquímica da molécula de adesão CD44v3. A proteína receptora para fator de crescimento epidérmico foi expressa em 84,4% dos casos, porém não se observou superexpressão da proteína HER2. A expressão imunoistoquímica da proteína receptora para fator de crescimento derivado de plaqueta alfa foi observada em 94,4% dos casos estudados, não apresentando relação com o tipo de mutação encontrado. As mutações do gene KIT foram as mais frequentemente observadas, principalmente do éxon 11. Mutações do gene receptor para fator de crescimento derivado de plaqueta alfa foram observadas em 8,1% dos casos, sendo que 54,5% dos casos com ausência de expressão imunoistoquímica de CD117 (KIT) apresentavam mutações nesse gene. A hibridização \"in situ\" fluorescente não demonstrou amplificação dos genes receptor para fator de crescimento epidérmico e HER2 nos tumores estromais gastrintestinais. / The present study presents the clinicopathologic, immunohistochemical and molecular genetic features of 513 Brazilian cases of gastrointestinal stromal tumor, including the immunohistochemical expression of CD117, CD34, DOG1 protein, smooth muscle actin, desmin, S-100 protein, Ki-67 antigen, p53 protein, CD44v3 adhesion molecule, epidermal growth factor receptor, HER2 protein and platelet derived growth factor receptor alpha, the mutation analysis of KIT and platelet-derived growth factor receptor alpha genes, and the investigation of amplification of HER2 and epidermal growth factor receptor genes by fluorescence \"in situ\" hybridization. The clinicopathologic features of Brazilian gastrointestinal stromal tumors do not differ from those published from other countries. CD117 (KIT) was expressed by immunohistochemistry in 95.7% of cases. DOG1 protein was expressed in 85.9% of tumors, being able to establish the diagnosis of GIST in 20% of those cases that were negative for CD117 (KIT). Ki-67 proliferation index was higher in those cases classified as high-risk of aggressive behavior by the National Institutes of Health consensus approach. The immunohistochemical expression of p53 protein was restricted to cases classified as high-risk of aggressive behavior. The adhesion molecule CD44v3 was not expressed in any of the cases. The epidermal growth factor receptor protein was overexpressed in 84.4% of cases, and HER2 protein was not expressed in all cases. The platelet-derived growth factor receptor alpha protein was detected by immunohistochemistry in 94.4% of tumors, and there was no relationship between its expression and the mutation status. KIT mutations were the most frequent, mainly of exon 11. Plateletderived growth factor receptor alpha mutations were found in 8.1% of the cases, and 54.5% of those cases that were negative for CD117 (KIT) had mutations in platelet-derived growth factor receptor alpha gene. Fluorescence \"in situ\" hybridization revealed no amplification of epidermal growth factor receptor and HER2 genes in gastrointestinal stromal tumors.

Une fois activées, les cellules dendritiques (DCs) migrent dans les organes lymphoïdes ou elles exercent leur rôle de cellules présentatrices d’antigène professionnelles. Elles sont capables d’activer et d’induire la différenciation des lymphocytes T naïfs en différentes sous-populations de lymphocytes T auxiliaires. L’ajustement de la réponse lymphocytaire T au type d’inflammation est assuré par les DCs à deux niveaux. Premièrement, grâce à leur plasticité, les DCs adaptent leur comportement en fonction de la combinaison de signaux issus du microenvironnement inflammatoire. Deuxièmement, il existe différentes sous-populations de cellules dendritiques ayant de différentes spécialisations fonctionnelles.Mon travail de thèse s’est concentré sur l’étude de la diversité des réponses des sous-populations de cellules dendritiques humaines suite à la stimulation par la cytokine lymphopoïetine stromale thymique (TSLP). Cette cytokine est secrétée par les cellules épithéliales et la peau au cours de l’inflammation. La TSLP active principalement les DCs myéloïdes, induisant celles-ci à secréter les chimiokines inflammatoires CCL17 et CCL22. Les DCs activées par la TSLP (TSLP-DCs) induisent une réponse inflammatoire de type Th2, et sont impliquées dans le développement de l’allergie. La comparaison systématique de la réponse à la TSLP par les sous-populations de DCs du sang, BDCA1+ et BDCA3+, nous a permis de montrer que ces deux sous-populations sont activées par la TSLP. Toutefois, nos résultats montrent que la TSLP, en synergie avec le TGF-β, induit la différenciation des DCs BDCA-1+ en cellules de Langerhans, mais pas celle des DCs BDCA-3+. De plus, la TSLP induit la migration cellulaire et la sécrétion de chimiokines seulement chez la sous-population de DCs BDCA-1+. Des analyses complémentaires des mécanismes impliqués dans la migration des DCs BDCA-1+ en réponse à la TSLP révèlent que, d’une part la TSLP est indispensable à l’induction de la migration et d’autre part, qu’un récepteur de chimiokines, sensible à la Toxine Pertussique serait impliqué. Au final, nos résultats révèlent (i) de nouvelles capacités des DCs en tant que cellules précurseurs, (ii) de différentes propriétés fonctionnelles des sous-populations de DCs en réponse à la stimulation par la TSLP, (iii) et démontrent la complexité des mécanismes impliqués dans la migration des DCs induite par la TSLP. / Once activated, Dendritic Cells (DCs) migrate to the lymphoid organs and exert their role as professional antigen presenting cells. They are able to induce the activation and differentiation of naïve T cells into different types of T helper cells. The T cell response must be suited to the type of inflammation. This is ensured by DCs at two levels. First DCs are functionally plastic. This means that their behavior is subdued to the integrated signals coming from the inflammatory microenvironment. Secondly, the DC population is diverse. Indeed, different DC subsets have different functional specializations. My thesis was focused on the differential response of human DC subsets to Thymic stromal lymphopoietin (TSLP). This cytokine is secreted by inflamed skin and epithelia, and strongly activates myeloid DCs. The TSLP-activated DCs secrete the inflammatory chemokines CCL17 and CCL22, prime an inflammatory Th2 response, and have been involved in the pathogenesis of allergic inflammation. By systematically comparing the response of human blood BDCA-1+ and BDCA-3+ DCs to TSLP stimulation we found that both of these DC subsets get activated by TSLP. However TSLP synergizes with TGF-β to induce the differentiation of blood BDCA-1+ and not BDCA-3+ DCs into Langerhans Cells. Moreover, TSLP induces cell migration and chemokine secretion only on the blood BDCA-1+ subset. Further analysis of the mechanisms implicated in TSLP-induced DC migration revealed that TSLP is required to induce DC migration, but this effect is dependent on the expression of a PTX-sensitive chemokine receptor. Overall our results reveal new precursor capacities of blood DC subsets, different functional properties of blood DC subsets stimulated by TSLP and highlight intricate mechanisms underlying TSLP-induced DC migration.

La myélofibrose primitive (MFP) est un néoplasme myéloprolifératif chromosome Philadelphie négatif rare, mais de pronostic sévère. Elle se caractérise par une prolifération clonale et une mobilisation des cellules souches et progéniteurs hématopoïétiques (CSH/PH) de la moelle osseuse vers la rate et le foie. Cette anomalie de l’hématopoïèse est associée à une pathologie du stroma (myélofibrose, ostéosclérose et néoangiogenèse). L’existence d’anomalies moléculaires de la CSH/PH telles que les mutations de Jak2, Mpl, TET2 ou CALR ne permet pas à elle seule d’expliquer la physiopathologie de la maladie. Les résultats obtenus dans le laboratoire suggèrent que le microenvironnement médullaire au sein des niches hématopoïétiques et en particulier les cellules stromales mésenchymateuses (CSM), participe vraisemblablement à cette dérégulation de l’hématopoïèse, favorisant le développement du clone pathologique. Cependant, aucune preuve tangible d’une altération des CSM médullaires n’a été jusqu’à présent apportée.Dans ce travail, nous avons isolé les CSM de la moelle de patients atteints de MFP et réalisé une caractérisation « complète » de ces cellules : prolifération, phénotype, soutien de l’hématopoïèse, sécrétome, transcriptome, miRNome et capacités de différenciation. Nos résultats ont permis de dégager un faisceau d’arguments en faveur d’une dérégulation de leur différenciation ostéoblastique (DOB). (i) Les cytokines BMP2, RANTES, PDGF, TGF-β1, VEGF et Il-6 sont significativement produites en plus grande quantité par ces cellules. (ii) L’étude du transcriptome a révélé une expression significativement différente d’un ensemble de gènes impliqués dans la DOB tels que RUNX2, DLX5, TWIST1 et NOGGIN. (iii) De nombreux micro-ARN, dont certains sont connus pour être impliqués dans la DOB comme miR-210 ou dans le nichage des cellules souches hématopoïétiques comme miR-34a, sont dérégulés à l’état basal et au cours de cette DOB. (iv) Enfin, l’étude de leurs capacités de différenciation ostéoblastique in vitro et in vivo chez la souris immunodéprimée est en faveur d’une augmentation de ces capacités. Nous avons étudié l’impact du TGF- β1 dans cette DOB. Nous avons mis en évidence que les CSM de malades présentent un état basal d'activation de la voie de signalisation pSmad significativement augmenté, confirmant l’expression endogène de TGF-β1. En utilisant des inhibiteurs spécifiques du récepteur de type I au TGF- β, nous avons montré l’implication de cette cytokine dans les altérations de la DOB. En conclusion, notre travail montre pour la première fois que les CSM des malades de MFP sont anormales et ce indépendamment de la stimulation par le clone hématopoïétique pathologique, suggérant la présence d'anomalies constitutives ou acquises. Ces anomalies impliquent deux acteurs majeurs de la pathologie : le TGF-β1 et l'ostéogenèse. / Primary myelofibrosis (PMF) is a Philadelphia-negative myeloproliferative neoplasm, rare but associated with a poor prognosis. Its features are a clonal proliferation and an egress of hematopoietic stem cells (HSC) from bone marrow to spleen. These abnormalities of hematopoiesis are in relation with a pathological stroma (myelofibrosis, osteosclerosis and neoangiogenesis). Molecular abnormalities present in HSC partially explain the physiopathology of the disease. Results from our lab suggest that the bone marrow micro-environnement, especially mesenchymal stromal cells (MSC), are involved in the deregulation of hematopoiesis, promoting the clonal cells. However, there is no strong evidence of bone marrow MSC alterations reported for now.In our study, we isolated MSC from bone marrow of patients suffering from PMF and performed a broad characterization: proliferation, phenotype, hematopoiesis supporting capacities, secretome, transcriptome and miRNome analysis. Our results highlight arguments in favor of a deregulation of their osteogenic capacities. (i) Cytokines NMP2, RANTES, PDGF, TGF-β1, VEGF and Il-6 were significantly overproduced by MSCs. (ii) Transcriptome analysis revealed a specific signature involving genes participating in osteogenic differentiation such as RUNX2, DLX5, TWIST1 and NOGGIN. (iii) Many micro-RNAs, some know to be involved in osteogenic differentiation regulation, as mir-34a, are deregulated in MSCs and in MSC-derived osteoblasts. (iv) Finally, study of their osteogenic potency in vitro and in vivo in nude mice showed an increasing of their osteogenic potency. We studied the impact of TGF-β1 in this process and showed that PMF MSCs showed a basal expression of Smad pathway significantly increased as compared to control. Using specific inhibitor of TGF-β1 receptor, we demonstrated the implication of this cytokine in the osteogenic impairment.To summarize, our work shows for the first time that MSCs from PMF patients are abnormal, independently from stimulation by clonal cells, suggesting intrinsic abnormalities. These abnormalities involve two main factor of the disease: TGF-β1 and osteogenesis.

Orientador: Leonardo Perez Faverani / Coorientadora: Roberta Okamoto / Banca: Daniela Ponzoni / Banca: Ellen Cristina Gaetti Jardim / Resumo: O objetivo deste estudo foi avaliar um novo método de texturização por PEO com incorporação de Ca e P na superfície do Ti-6Al-4V em ossos de baixa densidade, por meio de avaliação in vitro, ex-in vivo e in vivo, em função de parâmetros topográficos e reparacionais. 57 ratas Wistar (Rattus novergicus), sendo 38 ratas com 6 meses de idade (Grupos OXV - submetidas à ovariectomia e SHAM - cirurgia fictícia) e 19 ratas senis (18 meses de idade: Grupo SENIL), foram divididas para realização do estudo ex-in vivo (n=9) e in vivo (n=48). Os grupos para análise ex-in vivo foram submetidos à eutanásia e os fêmures foram removidos e transportados em meio de cultura contendo meio essencial mínimo modificação alfa (α- MEM) suplementado com 500 µg/mL de gentamicina e 3 µg/mL de fungisona. As células-tronco mesenquimais de medula óssea (CTMs-MO) dos fêmures, foram isoladas e cultivadas em meio de crescimento para manterem-se como CTMs. Após alcançar a subconfluência, as células foram cultivadas em 3 superfícies de discos de Ti-6Al-4V, grupo CONTROLE (superfície usinada) grupo AC (superfície tratada por Ataque Ácido e Jateamento) e grupo PEO (superfície tratada por Oxidação de Plasma Eletrolítico com associação de Cálcio e Fosforo). Para avaliação das respostas celulares foram realizados ensaios de viabilidade celular, expressão gênica de marcadores osteoblásticos, imunolocalização de sialoproteina óssea (BSP) e osteopontina (OPN), atividade da fosfatase alcalina (ALP) e formação de matriz mi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to evaluate a new PEO texturing method with Ca and P incorporation on the Ti-6Al-4V surface in low bone density, by means of in vitro, ex vivo and in vivo evaluation through topographic and repairment parameters. 57 Wistar rats (Rattus novergicus), being 38 at 6 months of age (OXV Groups - submitted to ovariectomy and SHAM surgery) and 19 senile rats (18 months of age: SENIL Group) were divided into three subgroups: ex-in vivo (n = 9) and in vivo (n = 48). The Groups for ex-in vivo analysis were euthanized and femurs were removed and transported in culture medium containing minimal alpha modification (α- MEM) medium supplemented with 500 μg / ml gentamicin and 3 μg / ml fungizone. The mesenchymal stem cells from bone marrow (MSC-M) of the femur were isolated and cultured in growth medium to remain as MSCs. After reaching the subconfluence, the cells were grown on 3 surfaces of Ti-6Al-4V discs, CONTROL group (machined surface) group AC (surface treated by etched-acid) and PEO group (surface treated by Electrolytic Plasma Oxidation with the association of Calcium and Phosphorus). Cell viability assays, gene expression of osteoblastic markers, bone sialoprotein (BSP) and osteopontin (OPN), alkaline phosphatase (ALP) activity, and mineralized matrix formation were performed to evaluate cellular responses. Data were submitted to ANOVA 1 factor test or Kruskal-Wallis test (P <0.05). In the groups for the in vivo study, after 90 days, an implant was i... (Complete abstract click electronic access below) / Mestre

O presente estudo tem por objetivo avaliar as características clinicopatológicas, imunoistoquímicas e moleculares de 513 casos brasileiros de tumor estromal gastrintestinal, incluindo o estudo da expressão imunoistoquímica de CD117, CD34, proteína DOG1, actina de músculo liso, desmina, proteína S-100, antígeno Ki-67, proteína p53, molécula de adesão CD44v3, receptor para fator de crescimento epidérmico, proteína HER2 e receptor para fator de crescimento derivado de plaqueta alfa, além da análise molecular de mutações envolvendo os genes KIT e receptor para fator de crescimento derivado de plaqueta alfa e da pesquisa de amplificação dos genes receptor para fator de crescimento epidérmico e HER2 por hibridização \"in situ\" fluorescente. As características clínicas e morfológicas dos casos estudados não foram diferentes das referidas pela literatura. Houve expressão de CD117 (KIT) em 95,7% dos casos. A proteína DOG1 foi expressa em 85,9% dos tumores, sendo capaz de diagnosticar 20% dos casos com ausência de expressão de CD117 (KIT). O índice de proliferação celular determinado pelo antígeno Ki-67 foi superior nos casos classificados como de alto risco para comportamento biológico agressivo segundo critérios do \"National Institutes of Health\". A expressão da proteína p53 esteve restrita aos casos classificados como de alto risco. Não se observou expressão imunoistoquímica da molécula de adesão CD44v3. A proteína receptora para fator de crescimento epidérmico foi expressa em 84,4% dos casos, porém não se observou superexpressão da proteína HER2. A expressão imunoistoquímica da proteína receptora para fator de crescimento derivado de plaqueta alfa foi observada em 94,4% dos casos estudados, não apresentando relação com o tipo de mutação encontrado. As mutações do gene KIT foram as mais frequentemente observadas, principalmente do éxon 11. Mutações do gene receptor para fator de crescimento derivado de plaqueta alfa foram observadas em 8,1% dos casos, sendo que 54,5% dos casos com ausência de expressão imunoistoquímica de CD117 (KIT) apresentavam mutações nesse gene. A hibridização \"in situ\" fluorescente não demonstrou amplificação dos genes receptor para fator de crescimento epidérmico e HER2 nos tumores estromais gastrintestinais. / The present study presents the clinicopathologic, immunohistochemical and molecular genetic features of 513 Brazilian cases of gastrointestinal stromal tumor, including the immunohistochemical expression of CD117, CD34, DOG1 protein, smooth muscle actin, desmin, S-100 protein, Ki-67 antigen, p53 protein, CD44v3 adhesion molecule, epidermal growth factor receptor, HER2 protein and platelet derived growth factor receptor alpha, the mutation analysis of KIT and platelet-derived growth factor receptor alpha genes, and the investigation of amplification of HER2 and epidermal growth factor receptor genes by fluorescence \"in situ\" hybridization. The clinicopathologic features of Brazilian gastrointestinal stromal tumors do not differ from those published from other countries. CD117 (KIT) was expressed by immunohistochemistry in 95.7% of cases. DOG1 protein was expressed in 85.9% of tumors, being able to establish the diagnosis of GIST in 20% of those cases that were negative for CD117 (KIT). Ki-67 proliferation index was higher in those cases classified as high-risk of aggressive behavior by the National Institutes of Health consensus approach. The immunohistochemical expression of p53 protein was restricted to cases classified as high-risk of aggressive behavior. The adhesion molecule CD44v3 was not expressed in any of the cases. The epidermal growth factor receptor protein was overexpressed in 84.4% of cases, and HER2 protein was not expressed in all cases. The platelet-derived growth factor receptor alpha protein was detected by immunohistochemistry in 94.4% of tumors, and there was no relationship between its expression and the mutation status. KIT mutations were the most frequent, mainly of exon 11. Plateletderived growth factor receptor alpha mutations were found in 8.1% of the cases, and 54.5% of those cases that were negative for CD117 (KIT) had mutations in platelet-derived growth factor receptor alpha gene. Fluorescence \"in situ\" hybridization revealed no amplification of epidermal growth factor receptor and HER2 genes in gastrointestinal stromal tumors.

Hui, Fan Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 153-181). / Abstract also in Chinese. / Acknowledgements --- p.ii / Abstract --- p.iii / Abbreviations --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xv / List of Tables --- p.xv / List of Figures --- p.xvi / Major Conference Presentations --- p.xix / Publications in Preparation --- p.xxii / Chapter Chapter 1 --- Study Background --- p.1 / Chapter 1. --- Introduction --- p.2 / Chapter 1.1. --- Spinal Deformities --- p.2 / Chapter 1.1.1. --- Treatment --- p.2 / Chapter 1.2. --- Spinal fusion --- p.4 / Chapter 1.2.1. --- Gold Standard of Spinal Fusion --- p.4 / Chapter 1.2.2. --- Decortication in Spinal Fusion --- p.4 / Chapter 1.2.3. --- Autograft in Spinal Fusion --- p.4 / Chapter 1.2.4. --- Local Factors Influencing Spinal Fusion --- p.5 / Chapter 1.2.5. --- Ultimate Goals of Spinal Fusion --- p.7 / Chapter 1.2.6. --- Limitations of Spinal fusion --- p.7 / Chapter 1.3. --- Alternatives of Different Components for Enhancing Spinal Fusion / Chapter 1.3.1. --- Bone Graft Substitute --- p.9 / Chapter 1.3.2. --- Bioactive Factors --- p.15 / Chapter 1.4. --- Limitations of the Alternative Methods in Spinal Fusion Enhancement --- p.19 / Chapter 1.4.1. --- BMPs --- p.19 / Chapter 1.4.2. --- Gene Therapy --- p.20 / Chapter 1.4.3. --- Biophysical Stimulation --- p.20 / Chapter 1.5. --- Recent Methods in Enhancing Spinal Fusion --- p.21 / Chapter 1.5.1. --- Low Intensity Pulsed Ultrasound --- p.21 / Chapter 1.5.2. --- Mesenchymal Stem Cells in Spinal Fusion --- p.24 / Chapter 1.6. --- Conclusion --- p.26 / Chapter Chapter 2 --- "Hypothesis, Objectives and Plan of Study" --- p.29 / Chapter 2. --- "Hypothesis, Objectives and Plan of Study" --- p.30 / Chapter 2.1 --- Study Hypothesis --- p.31 / Chapter 2.2 --- Study Objectives --- p.31 / Chapter 2.3 --- Plan of Study --- p.32 / Chapter 2.3.1 --- For First Objective --- p.32 / Chapter 2.3.2 --- For Second Objective --- p.32 / Chapter 2.3.3 --- For Third Objective --- p.33 / Chapter Chapter 3 --- In vitro Study of Effect of Low Intensity Pulsed Ultrasound on Mesenchymal Stem Cells --- p.34 / Chapter 3.1. --- Introduction --- p.35 / Chapter 3.2. --- Materials and Methods --- p.36 / Chapter 3.2.1. --- Experimental Animal --- p.36 / Chapter 3.2.2. --- Materials and Reagents --- p.36 / Chapter 3.2.2.1. --- Dulbecco，s Modified Eagle Medium (DMEM) --- p.36 / Chapter 3.2.2.2. --- Phosphate Buffered Saline (PBS) --- p.37 / Chapter 3.2.2.3. --- Osteogenic Medium (OS) --- p.37 / Chapter 3.2.2.4. --- Alkaline Phosphatase (ALP) Buffer --- p.37 / Chapter 3.2.2.5. --- ALP Substrate Buffer --- p.38 / Chapter 3.2.2.6. --- MTT Stock Solution --- p.38 / Chapter 3.2.2.7. --- MTT Working Solution --- p.38 / Chapter 3.2.2.8. --- Lysis buffer --- p.38 / Chapter 3.2.2.9. --- Alkaline Phosphatase (ALP) Working Reagents --- p.39 / Chapter 3.2.3. --- Isolation of Bone Marrow Derived Mesenchymal Stem Cells (BM derived MSCs) --- p.39 / Chapter 3.2.4. --- In vitro Low Intensity Pulsed Ultrasound Treatment --- p.40 / Chapter 3.2.4.1. --- In vitro LIPUS Devices --- p.40 / Chapter 3.2.4.2. --- Treatment Procedure and Experimantal Groupings --- p.40 / Chapter 3.2.5. --- Effect of LIPUS on Cell Viability and Osteogenesis in bone marrow derived-MSCs --- p.41 / Chapter 3.2.5.1. --- Cell Viability Assay --- p.41 / Chapter 3.2.5.2. --- Alkaline Phosphatase (ALP) Enzyme Activity --- p.42 / Chapter 3.2.5.3. --- Cell Morphology and Alkaline Phosphatase Cytochemistry --- p.42 / Chapter 3.2.6. --- Statistical Analysis --- p.43 / Chapter 3.3. --- Results --- p.43 / Chapter 3.3.1. --- Morphology --- p.43 / Chapter 3.3.2. --- Total Number of Viable Cells --- p.44 / Chapter 3.3.3. --- ALP Activity Absorbance --- p.44 / Chapter 3.3.4. --- ALP staining --- p.45 / Chapter 3.3.5. --- Qualitative Analysis --- p.45 / Chapter 3.3.6. --- Quantitative Analysis --- p.46 / Chapter 3.4. --- Discussion --- p.46 / Chapter 3.4.1. --- LIPUS have No Enhancing Effect on Proliferation of MSCs in Basal Medium Nor Osteogenic Medium --- p.47 / Chapter 3.4.2. --- LIPUS Stimulate Proliferation of MSCs in Early Period --- p.49 / Chapter 3.4.3. --- LIPUS Further Enhanced Osteogenesis of MSCs in Osteogenic Medium --- p.49 / Chapter 3.4.4. --- 10 mins LIPUS treatment for 7 days can positively enhance osteogenic differentiation --- p.50 / Chapter 3.4.5. --- Optimum Conditions of LIPUS was Cell Type Dependent --- p.51 / Chapter 3.4.6. --- LIPUS Promoted Osteogenesis in MSCs through Accelerated Mineralization --- p.52 / Chapter Chapter 4 --- Enhancement of Posterior Spinal Fusion The Effect of Tissue-Engineered MSC and Calcium Phosphate Ceramic composite treated with LIPUS in Vivo --- p.68 / Chapter 4.1. --- Introduction --- p.69 / Chapter 4.1.1. --- TCP Biomaterials --- p.70 / Chapter 4.2. --- Materials and Methods --- p.71 / Chapter 4.2.1. --- Materials and Reagents --- p.71 / Chapter 4.2.2. --- Preparation of MSC Derived Osteogenic Cells-tricalcium Phosphate Ceramics Composite --- p.73 / Chapter 4.2.3. --- Posterior Spinal Fusion Surgery --- p.74 / Chapter 4.2.4. --- In vivo LIPUS treatment --- p.75 / Chapter 4.2.5. --- Assessment of Fusion Mass --- p.76 / Chapter 4.2.6. --- Histology --- p.77 / Chapter 4.2.7. --- Statistical Analysis --- p.79 / Chapter 4.3. --- Results --- p.79 / Chapter 4.3.1. --- Fusion by Manual Palpation --- p.79 / Chapter 4.3.2. --- pQCT Analysis --- p.80 / Chapter 4.3.3. --- Histological Analysis --- p.81 / Chapter 4.4. --- Discussion --- p.85 / Chapter 4.4.1. --- Summary of the Findings from Different Assessment Methods --- p.85 / Chapter 4.4.2. --- Addition of MSCs to TCP ceramic in Spinal Fusion --- p.87 / Chapter 4.4.3. --- The Needs of Differentiated MSC in Spinal Fusion --- p.89 / Chapter 4.4.4. --- bFGF Masked the Effect of OS in MSC --- p.91 / Chapter 4.4.5. --- LIPUS Enhanced Bone Formation --- p.95 / Chapter 4.4.6. --- LIPUS Enhanced Bone Formation through Mineralization --- p.96 / Chapter 4.4.7. --- LIPUS Enhanced Spinal Fusion through Bone Remodeling-induced Fusion Mass --- p.97 / Chapter 4.4.8. --- LIPUS Enhanced Bone Formation through Endochondral Ossification --- p.99 / Chapter Chapter 5 --- In Vivo Monitoring of Spinal Fusion in Animal Model with High-resolution Peripheral Quantitative Computed Tomography-A New Pilot Study --- p.122 / Chapter 5.1. --- Introduction --- p.123 / Chapter 5.2. --- Materials and Methods --- p.124 / Chapter 5.2.1. --- Animal Groupings --- p.124 / Chapter 5.2.2. --- Preparation of MSC Derived Osteogenic Cells-tricalcium Phosphate Ceramics Composite --- p.124 / Chapter 5.2.3. --- Posterior Spinal Fusion Operation Procedures --- p.125 / Chapter 5.2.4. --- LIPUS treatment --- p.125 / Chapter 5.2.5. --- High-resolution Peripheral Quantitative Computed Tomography …- --- p.125 / Chapter 5.2.6. --- Analysis with HR-pQCT --- p.126 / Chapter 5.3. --- Result --- p.128 / Chapter 5.3.1. --- Qualitative Observations from HR-pQCT Images --- p.128 / Chapter 5.3.2. --- Quantitative Analysis --- p.129 / Chapter 5.4. --- Discussion --- p.130 / Chapter Chapter 6 --- "Overall Summary, Discussion and Conclusion" --- p.140 / Chapter 6.1. --- Overall Summary and Discussion --- p.141 / Chapter 6.2. --- Limitations and Further Studies --- p.145 / Chapter 6.3. --- Conclusions --- p.147 / Chapter 6.4. --- Summary Flowchart of the whole thesis --- p.148 / References --- p.153

Smad7作為轉化生長因數-β信號通路中的負性調節因子為人所知，異常的Smad7表達通常會引發癌症及組織纖維化等疾病。而目前對於其在骨重建及其相關疾病中的作用尚未有研究。本研究利用Smad7部分敲除小鼠來探索Smad7在骨重建，骨質疏鬆以及間充質幹細胞分化等方面的作用。 / 本研究所用的Smad7部分敲除小鼠模型來源於已有報導過的Smad7ΔE1(KO)小鼠。該小鼠體內Smad7基因組外顯子I的翻譯區被替換，導致部分蛋白失及其功能破壞。研究結果表明，KO小鼠在6、12、24周齡時股骨遠端幹骺端均有不同程度下降的骨小梁數目、厚度，骨礦化率，骨密度，骨體積分數，及其上升的骨小梁間隙和破骨細胞表面。骨髓來源間充質幹細胞的多向分化實驗表明，KO組呈現出抑制性的成骨能力，表現為鈣結節形成減少，鹼性磷酸酶活性下降，早晚期成骨標記基因表達下降。該組亦表現出促進性的成脂能力，有較多及較早的脂滴形成，成脂標記基因表達上升。而對於骨髓來源巨噬細胞的體外破骨誘導實驗表明，KO組有更多且更大的破骨細胞形成，較大的骨吸收面積，以及上升的破骨標記基因表達。卵巢切除小鼠模型的研究表明，術後4、8、16周，KO组的股骨遠端幹骺端对比野生组有更大程度下降的骨形态学参数，以及明顯升高的破骨細胞融合標記蛋白的表達。體外實驗表明KO组有更多且更大的破骨細胞形成，以及更大面積的骨吸收。積雪草酸曾被證實在肝纖維化模型中誘導Smad7 基因的表達，也在本實驗中用以研究對骨質疏鬆疾病的作用。卵巢切除動物模型連續給藥8周後，骨質疏鬆的現象有明顯逆轉，表現為升高的骨形态学参数，及下降的股骨內破骨細胞融合標記蛋白的表達。 / 總結，本研究證實了Smad7在骨骼發育重建及骨疾病的病理機理等方面的研究提供了突破性的見解。部分敲除Smad7可以導致抑制性的成骨能力，促進性的破骨能力，以及損傷性的骨重建，亦會加速骨質疏鬆的進程，并可作為全新的藥物治療靶點，提示Smad7 本身對於骨重建及骨代謝的保護性作用，為代謝性骨疾病的研究及其臨床藥物開發提供了更廣泛的前景。 / Smad7 has been well documented as a negative regulator of TGF-β signaling, and its altered expression often leads to human diseases such as cancer and fibrosis. However, the role of Smad7 in regulating bone remodeling and related diseases remains unclear. We performed both in vivo and in vitro experiments as well as disease model and drug therapy studies using both wild-type (WT) and Smad7ΔE1 (KO) mice to investigate the functional role of Smad7 in bone remodeling, osteoporosis, and MSCs differentiation. / The Smad7ΔE1 mice were generated by replacing part of the exon1 of Smad7 gene as reported, which resulted in truncated protein and partial loss of Smad7 function. Mice were genotyped by PCR. The μ-CT, histological assays and bone histomorphometric assays in metaphysic region of the femurs showed lower trabecular number (TbN), trabecular thickness (TbTh), mineral apposition rate (MAR), higher trabecular separation (TbSp) and Osteoclast Surface (Oc.S/BS & Oc.N/BS) in the KO mice at 6, 12, to 24 weeks old; as well as lower bone mineral density (BMD) and bone volume fraction (BV/TV) at 24 weeks old in the KO mice. The in vitro BM-MSCs multi-lineage differentiation studies showed the suppressed osteogenic potential in the KO group with fewer mineralized nodules, lower ALP activity and expression of Col1A1, Runx2 and OCN; while the adipogenic potential was elevated with more lipid droplets formation and higher expression of Adipsin and C/EBPα. The osteoclastogenic potential of KO mice BMMs was also elevated, showing higher osteoclasts activity and larger resorptive areas, as well as elevated expression of TRAP and CTR. Both in vivo and in vitro studies of the osteoporotic models showed that the KO mice had lower BMD, TbTh, and higher TbSp compared to the WT mice at 4, 8, 16 weeks after OVX, similar results of lower BV/TV and TbN were observed at 4 weeks after OVX in the KO mice. The RANKL-induced osteoclastogenesis potential was elevated compared to WT mice, with more and bigger osteoclasts, larger resorptive areas, as well as elevated expression of TRAP and CTR. The osteoclastic cell fusion was also enhanced. Treatment of Asiatic acid (one traditional Chinese medicine that has been proved to induce the expression of Smad7 as reported) in the OVX mice reversed the osteoporotic process with increase BMD, BV/TV, TbN, TbTh, and decreased TbSp compared to the untreated group. The osteoclastic cell fusion was suppressed after AA treatment. / Partial loss of Smad7 function leads to impaired bone remodeling in vivo, reduced osteogenesis and enhanced osteoclastogenesis in vitro, and also accelerates the osteoporotic development and osteoclastic cell fusion. Asiatic acid may be a novel potential drug for prevention of osteoporosis. Our findings provide new evidences for a better understanding of the biological functions of Smad7 in bone remodeling and its therapeutic potential for metabolic bone diseases. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Nan. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 131-153). / Abstracts also in Chinese.

Imatinib mesylate has been associated with the changes in bone turnover. We report a case of the development of tissue calcification in a patient on long-term therapy with this drug. A 48-year-old male patient with gastrointestinal stromal tumor and liver metastasis complained of abdominal pain. His treatment included hepatic artery chemoembolization and partial hepatectomy in addition to chronic imatinib mesylate for 4 years. On physical examination, he had a peritoneal mass just beneath the laparotomy incision scar that, after resection, was found to be dystrophic bone formation. Based on the previous studies suggesting bone changes due to chronic therapy with imatinib mesylate, we believe that the patient's new bone formation was causally related to the use of this drug. To our knowledge, there are no similar reported cases in the literature.

Les plaquettes sanguines ont comme rôle principal d’arrêter les saignements. Elles sont produites dans la moelle osseuse par des mégacaryocytes (MK) qui proviennent de la différenciation des cellules souches hématopoïétiques (CSH). L’objectif de ma thèse a été d’identifier les éléments cellulaires du microenvironnement contrôlant la mégacaryopoïèse. Mon travail a permis d’identifier une population particulière de progéniteurs hépatiques du foie foetal capable de promouvoir in vitro les étapes précoces de la mégacaryopoïèse à partir de CSH humaines et murines (Brouard et al., 2017). Le rôle des cellules endothéliales (EC) dans les étapes tardives de maturation a été étudié après purification à partir de moelle humaine dans des expériences de co-culture avec des MK prédifférenciés. Mes résultats montrent que ces EC ont la propriété unique, par comparaison avec des EC d’autres tissus, de promouvoir la maturation des MK. Une analyse transcriptionnelle différentielle a permis d’identifier des effecteurs possibles ouvrant des pistes pour mieux comprendre les mécanismes de la mégacaryopoïèse et pour améliorer la production des plaquettes en culture. / The main role of platelets is to stop bleeding. They are produced in the bone marrow by megakaryocytes (MK) that are produced by the differentiation of hematopoietic stem cells (HSC). The objective of my thesis was to identify the cellular elements of the microenvironment controlling megakaryopoiesis. My work has identified a particular population of hepatic progenitors from the fetal liver capable of promoting in vitro the early stages of megakaryopoiesis from human and murine HSC (Brouard et al., 2017). The role of endothelial cells (EC), purified from human bone marrow, in late maturation stages was studied in co-culture experiments with predifferentiated MK. My results show that these EC have the unique property in comparison with EC from other tissues, of promoting the maturation of MK. A differential transcriptional analysis identified possible effectors that could lead to a better understanding of the mechanisms of megakaryopoiesis and improve platelet production in culture.

Since the first successful derivation of human embryonic stem cells (hESC), rapid progress has been attained in the development of strategies in differentiation of these cells into various neural lineages, with the fundamental objective of using these cells for replacement and repair of damaged neuronal circuits in the central nervous system (CNS). Of particular interest are midbrain dopaminergic (mDA) neurons, which play a central role in regulation of voluntary movement. Degeneration or loss of function of mDA neurons in the nigrostriatal pathway is associated with Parkinson disease (PD). Stromal-Derived Inducing Activity (SDIA) is recognized as one of the most efficient methods in restricting ESC differentiation to a dopaminergic lineage, and refers to the property of mouse stromal cell lines such as PA6 or MS5 to cause ESC to differentiate to DA neurons. Although this strategy has been extensively used to generate mDA neurons from hESC, the biochemical nature of SDIA is yet unknown.  In the present study mDA neurons were generated from the BG01V2 hESC line by SDIA. To examine whether SDIA exerts its effect directly on hESC and is responsible for early dopaminergic induction, neural progenitor cells (NPC) were enyzmatically isolated from the co-cultures and allowed to differentiate in feeder-free conditions. The isolated cells were committed to a mesencephalic neural lineage, and were capable of maintaining their phenotype and developing into postmitotic mDA neurons in feeder-free conditions. The mDA neurons showed neuronal excitability and dopamine transporter function. The in vitro proliferation and differentiation of the NPC was also investigated by a BrDU incorporation assay. Next, the maintenance of cellular memory and capacity for proliferation of the mesencephalic NPC was assessed. The NPC could be expanded in vitro by five-fold as neurospheres for up to two weeks while retaining their DA differentiation potential, but did not retain a stable phenotype over extended periods of time. Preliminary transplantation experiments of neurospheres in striatal lesioned animals indicated, however, that these cells could survive and conserve their phenotype in vivo. To gain additional insight into the biochemical role of SDIA in early dopaminergic induction of hESC, the separate contributions of cell surface activity and secreted factors were examined. The data revealed that the PA6 cell surface activity promoted cell survival and was mainly responsible for enhanced neurogenesis of hESC, whereas secreted factors provided DA lineage-specific instructions. In order to identify the soluble factors responsible for the DA phenotype-inducing component of SDIA, the gene expression profile of PA6 cells was compared to that of cell lines lacking the DA-inducing property. A number of soluble factors known to be associated with CNS development that were highly expressed in PA6 cells were identified as potential DA differentiation-inducing candidates. These differentially-expressed genes included stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). When these factors, termed SPIE, were applied to the hESC, they induced dopaminergic neuronal differentiation of hESC line, BG01V2 and other karyotypically normal hESC lines in vitro. Thus, it appears that SPIE comprises the DA phenotype-inducing property of SDIA. This may provide a simple and direct means of differentiating hESC to form DA neurons in a single step, without a requirement for co-culture, animal cell lines, or animal products. / QC 20100916