Le tissu osseux est caractérisé par sa matrice minéralisée qui est soumise à des activités de formation et de résorption assurant son renouvellement et son remaniement tout au long de la vie. En cas de lésions, l’os est capable de se réparer naturellement de façon à rétablir son intégrité et ses propriétés physiques. Cependant, certaines pathologies ou interventions chirurgicales peuvent aboutir à des pertes massives de substance osseuse et le processus naturel d’autoréparation est alors insuffisant. En première intention, la greffe osseuse est envisagée (autogreffe et allogreffe), néanmoins, du fait d’une disponibilité réduite et des risques de rejet et de transmission d’agents infectieux, cette technique n’est pas réalisable dans toutes les situations cliniques. Le chirurgien peut alors avoir recours à des biomatériaux ostéoconducteurs mais ceux-ci ne sont utilisables que dans le cas de comblement de défauts de petite taille car ils sont simplement un support passif à la néoformation osseuse. Ces limites pourraient être dépassées grâce au concept d’ingénierie tissulaire, en concevant des biomatériaux innovants ayant un fort pouvoir ostéogène conféré notamment par des facteurs de croissance ou des cellules ostéoprogénitrices. Dans notre travail, nous avons cherché à mettre au point un nouveau produit d’ingénierie tissulaire permettant la réparation de défauts osseux. La stratégie envisagée repose sur l’association d’un support tridimensionnel et de cellules souches adultes dérivées du tissu adipeux humain (ASC). L’originalité du système provient de la matrice tridimensionnelle, qui est un hydrogel thermosensible composé de monomère synthétique Glycosyl-Nucléoside-Fluoré (GNF) de faible poids moléculaire. Dans le domaine de la régénération osseuse, les hydrogels cellularisés sont généralement utilisés comme matrice associée à des molécules ostéogéniques (BMP2, Béta-Glycérophosphate) ou à des ions (Calcium : Ca2+, Phosphate : PO42-) pour permettre la differenciation ostéoblastique des cellules encapsulées dans le gel. Cependant, dans notre travail, nous n’avons pas fait appel à ces facteurs ostéogéniques. Notre étude a révélé que l’hydrogel de GNF possède les critères essentiels pour être utilisé en clinique : la non-toxicité, la biocompatibilité, la biodégradabilité, l’injectabilité et la biointégration. Des injections de complexe gel/ASC réalisées en site ectopique chez l’animal ont démontré que le gel se forme in situ en moins de 20 minutes et que les cellules encapsulées ont survécu pendant plusieurs mois. In situ, les ASC se sont différerenciées en ostéoblastes matures, exprimant la phosphatase alcaline et l’ostéocalcine et synthétisant une matrice extracellulaire riche en phosphate de calcium. Ces travaux ont donc permis de développer un produit d’ingénierie tissulaire innovant, associant un support tridimensionnel, l’hydrogel de GNF, à une composante cellulaire, les ASC. Cette matrice cellularisée apparaît prometteuse comme système injectable pour des applications cliniques de régénération osseuse. / Bone tissue is characterized by its mineralized matrix which is subject to formation and resorption activities ensuring its renewal and remodeling throughout the life. In case of damage, the bone can repair itself naturally to restore its integrity and its physical properties. Nevertheless, some pathologies or surgical procedures can lead to massive loss of bone and the natural process of self-repair is insufficient. First line, the bone graft is considered (autograft and allograft), however, due to reduced availability and risks of rejection and transmission of infectious agents, this technique is not feasible in all clinical situations. The surgeon can then make use of osteoconductive biomaterials but these are only usable in the case of filling of small defects because they are simply passive scaffold for bone formation. These limits may be exceeded through the concept of tissue enginee- ring, designing innovative biomaterials with high osteogenic power conferred by particular growth factors or osteoprogenitor cells. In our work we seek to develop a new product of tissue engineering to repair bone defects. The proposed strategy is based on the combination of a three-dimensional scaffold and adult stem cells derived from human adipose tissue (ASC). The originality of this system comes from the three-dimensional matrix, which is a thermosensitive hydrogel composed of synthetic monomeric Glycosyl-Nucleoside-Fluorinated (GNF) low molecular weight. In the field of bone regeneration, hydrogels are generally used as cellularized matrix molecules associated with osteogenic (BMP2, Beta-Glycerophosphate) or ions (Calcium : Ca2+, Phosphate : PO42-) to allow osteoblast differentiation of cells encapsulated in the gel. However, in our work, we have not used these osteogenic factors. Our study revealed that the hydrogel of GNF has the essential criteria to be used in clinical practice : non-toxicity, biocompatibility, biodegradability, injectability and biointegration. Injections of gel/ASC complex performed in animal ectopic site have showed that the gel is formed in situ within 20 minutes and encapsulated cells survived and proliferated for several months. In situ, ASC were differentiated into mature osteoblasts expressing alkaline phosphatase and osteocalcin and synthesizing an extracellular matrix rich in calcium phosphate. So, this work has allowed the development of an innovative product for tissue engineering, combining a three-dimensional scaffold, the GNF based hydrogel, a cellular component, the ASC. This cellularized matrix appears promising as injection system for clinical applications of bone regeneration.

Un régime alimentaire déséquilibré et/ou la présence de composés contaminantsexogènes peuvent modifier la signalisation endocrine et l’homéostasie des lipides et induirel’obésité. Les travaux réalisés dans le cadre de cette thèse ont permis, dans un premier temps,de développer une méthode simple et rapide, dénommée "zebrafish obesogenic (ZO) test",pour identifier in vivo, par utilisation de la larve de poisson zèbre, des facteurs qui peuventaugmenter ou diminuer la taille de l’adipocyte blanc et ainsi moduler le niveau de l’adiposité(Tingaud-Sequeira, Ouadah, Babin, J. Lipid Res. 52, 1765-1772, 2011). Ce test permetd’identifier des composés et des mélanges de molécules obésogènes et anti-obésogènes etfournit des informations pertinentes pour l'évaluation des risques liés leur présence maiségalement pour élucider les mécanismes impliqués. Les travaux ont, dans un second temps,permis d’apporter des réponses quant aux modalités d’action d’un obésogène puissant, lechlorure de tributylétain, contaminant retrouvé très largement dans notre environnement.Cette molécule agit sur l’adipocyte blanc à une concentration de l’ordre du nano molaire viales récepteurs nucléaires RXR et LXR, et non pas via les isoformes PPARgamma/delta(Ouadah et Babin, manuscrit en préparation). / An unbalanced diet and / or the presence of exogenous compounds contaminants mayalter endocrine signaling and lipid homeostasis and induce obesity. The work done in thisthesis have, at first, developed a simple and rapid method, called "zebrafish obesogenic (ZO)test" to identify in vivo by using the zebrafish larva, the factors that may increase or decreasethe size of the white adipocyte and therefore modulate the level of adiposity (Tingaud-Sequeira, Ouadah, Babin, J. Lipid Res. 52, 1765-1772, 2011). This test helps to identifycompounds and mixtures of obesogenic and anti-obesogenic molecules and providesinformation relevant to the risk assessment of their presence but also to elucidate themechanisms involved. Work in a second time allowed to answer as to how the action oftributyltin chloride, a powerful obesogenic contaminant found widely in the environment.This molecule acts in vivo on white adipocytes in a concentration of the order of nano molarvia nuclear receptors LXR and RXR, and not via the PPARgamma isoforms / delta (Ouadahand Babin, manuscript in preparation).

Les cytokines, produites lors de l’activation du système immunitaire, ont la capacité d’agir au niveau du système nerveux central et d’induire diverses altérations comportementales. Lorsque l’activation du système de l’immunité innée devient chronique, ces altérations comportementales peuvent évoluer en véritables symptômes neuropsychiatriques. La physiopathologie des symptômes neuropsychiatriques qui se développent dans un contexte d’inflammation chronique à bas bruit, c’est-à-dire caractérisé par une activation chronique des processus immunitaires mais à un niveau relativement faible, est peu connue et reste à déterminer. L’implication de l’inflammation chronique à bas bruit dans les symptômes de fatigue et les altérations cognitives constitue l’élément d’étude principal de ce travail de thèse. Les troubles métaboliques, tels que l’obésité et le diabète de type 2, sont de bons modèles pour une telle étude. Ces deux pathologies sont en effet caractérisées par une inflammation chronique à bas bruit qui proviendrait, au moins en partie, du tissu adipeux. De plus, la fatigue et les altérations cognitives sont fréquentes chez les patients souffrant de troubles métaboliques. Compte tenu du rôle connu de l’inflammation dans la physiopathologie de ces altérations comportementales, leur développement dans des contextes de troubles métaboliques pourrait également être lié à l’activation chronique à bas bruit de processus inflammatoires. Différents objectifs ont été définis pour tester cette hypothèse : 1) caractériser et spécifier les symptômes de fatigue et les altérations cognitives chez des patients diabétiques ou obèses ; 2) évaluer la relation entre inflammation systémique et état inflammatoire du tissu adipeux ; 3) étudier l’association de l’inflammation chronique à bas bruit avec les symptômes de fatigue et les altérations cognitives des patients souffrant de troubles métaboliques. Nos résultats indiquent que la fatigue, en particulier la fatigue générale et physique, représente une caractéristique fondamentale des troubles métaboliques. Des perturbations cognitives, se traduisant par un ralentissement psychomoteur dans un test de temps de réaction ainsi qu’une altération de performance dans une tâche de planification spatiale, ont également été décelées chez les patients diabétiques de type 2, particulièrement ceux sous insulinothérapie, et chez les patients obèses. Des altérations mineures étaient également mesurées dans une tâche d’empan spatial rétrograde chez les patients obèses. En ce qui concerne les données biologiques, nos résultats indiquent diverses associations entre l’inflammation systémique et l’expression des marqueurs inflammatoires (cytokines inflammatoires, dont le MCP1, et marqueurs des cellules T) dans le tissu adipeux viscéral des patients obèses. De façon intéressante, l’inflammation systémique à bas bruit était associée aux dimensions de fatigue (générale, mentale, réduction des activités et de la motivation) et aux altérations de performance dans les tests ciblant les fonctions exécutives. Dans l’ensemble, ces résultats supportent l’hypothèse de l’implication des macrophages et des lymphocytes T du tissu adipeux dans l’état inflammatoire systémique associé à l’obésité. Il suggère en outre que l’inflammation systémique à bas bruit pourrait participer au développement de la fatigue et des altérations cognitives chez les patients souffrant de troubles métaboliques. Ce travail de thèse offre une caractérisation précise des symptômes de fatigue et des altérations cognitives associées aux troubles métaboliques. En outre, ce travail apporte d’importantes informations sur les relations de l’inflammation chronique à bas bruit avec ces symptômes, et permet d’affiner les hypothèses relatives à l’implication de processus inflammatoires dans la physiopathologie de ces altérations. / Cytokines produced during the activation of the immune system have the ability to act within the central nervous system and to induce a large number of behavioral alterations. When the activation of immune system becomes chronic and unregulated, these behavioral alterations may lead to the development of neuropsychiatric symptoms. The pathophysiology of neuropsychiatric symptoms that develop in conditions of chronic low-grade inflammation context (i.e., characterized by a chronic but low activation of inflammatory processes), remains unknown. The main aim of this thesis was to investigate the involvement of low-grade inflammation in the development of fatigue symptoms and cognitive alterations in patients with metabolic disorders including obesity and type 2 diabetes. These conditions are characterized by a chronic low-grade inflammatory state, manifesting by higher blood concentrations of inflammatory factors. This inflammatory state would originate, at least partially, from the adipose tissue. Moreover, fatigue symptoms and cognitive alterations are common in metabolic disorders. Given the role of inflammation in the physiopathology of these symptoms, their development could also rely on chronic low-grade inflammatory processes. Several objectives were defined to test this hypothesis: 1) to characterize fatigue symptoms and cognitive alterations in obese and diabetic patients; 2) to assess the relationship of systemic inflammation with the inflammatory state of the adipose tissue; and 3) to investigate the association of low-grade inflammation with fatigue symptoms and cognitive alterations in patients with metabolic disorders. Fatigue symptoms and cognitive function were respectively assessed using the multidimensional fatigue inventory (MFI) and the neuropsychological tests automated battery CANTAB in diabetic patients (type 1 and type 2) and in obese patients before and after bariatric surgery. A control group was included for each model (obesity and type 2 diabetes). Circulating concentrations of inflammatory markers, as well as expression of inflammatory markers in the visceral adipose tissue of obese patients, were measured. Our results indicate that fatigue symptoms, especially in the dimensions of general and physical fatigue, represent fundamental characteristics of patients suffering from metabolic disorders. In addition, cognitive alterations (psychomotor slowing and alterations in spatial planning performance) were measured in type 2 diabetic patients, more particularly those under insulin treatment, and in obese patients. Slight alterations in the test of backward spatial span were measured in obese patients. With respect to biological data, our results indicate significant relationships between systemic inflammation and inflammatory markers (inflammatory cytokines, including MCP1, and T-cell markers) in the visceral adipose tissue of obese patients. Interestingly, chronic low-grade inflammation was associated with fatigue symptoms (general fatigue, mental fatigue, reduced activity and motivation) and performance alterations in tests assessing executive functions. Altogether, these data support the hypothesis of the involvement of the adipose macrophages and T lymphocytes in the systemic inflammatory state associated with obesity. Moreover, these results suggest that systemic low-grade inflammation associated with metabolic disorders may contribute to the physiopathology of fatigue and cognitive alterations in these conditions. In conclusion, these studies provide a precise characterization of fatigue symptoms and cognitive alterations associated with metabolic disorders, such as obesity or type 2 diabetes. In addition, this thesis work gives interesting information about the relationships of chronic low-grade inflammation and fatigue and cognitive symptoms, and refines hypotheses regarding the involvement of inflammatory processes in the physiopathology of these symptoms in patients with diabetes or obesity.

O desenvolvimento gradual e recente de uma epidemia mundial de obesidade alavancou sobremaneira o estudo dessa condição e de suas comorbidades metabólicas. No âmbito fisiopatológico, múltiplos estudos demonstraram a expressão aumentada de mediadores inflamatórios no tecido adiposo de animais e humanos obesos, o acúmulo local de macrófagos, e um papel central da inflamação no desequilíbrio da homeostase metabólica local e sistêmica na obesidade. A definição de um papel ativo dos macrófagos, e portanto da imunidade inata, na rede inflamatória do tecido adiposo, evocou a hipótese de que, similarmente a outras condições inflamatórias crônicas como a aterosclerose, a obesidade também contaria com a importante participação de elementos da imunidade adaptativa, como as células T e suas citocinas, em sua fisiopatologia. Com base nessas considerações, os objetivos principais desse estudo foram: 1) avaliar a presença das células T e o papel do interferon-gama (IFNy), clássica citocina T-helper 1 (ou Th1), na inflamação do tecido adiposo; e 2) estudar mecanismos de acúmulo das células T no tecido adiposo na obesidade, particularmente a participação do receptor CXCR3 nesse processo. Experimentos de citometria de fluxo mostraram que o tecido adiposo visceral de camundongos C57BL/6 obesos após consumo de dieta rica em gorduras apresentou maior número de macrófagos e também de células T, CD4+ e CD8+, em comparação a controles que receberam dieta pobre em gorduras. A expressão de I-Ab, marcador do complexo de histocompatibilidade principal classe II (MHC II) murino, também foi maior no tecido adiposo dos animais obesos, sugerindo a presença local da atividade de apresentação de antígeno com consequente ativação das células T. Quando estimuladas in vitro, células T derivadas do tecido adiposo de camundongos obesos produziram mais IFNy do que aquelas isoladas de controles, novamente sugerindo a ativação dessas células em um contexto de obesidade. Na análise das possíveis funções do IFNy no tecido adiposo, a estimulação da linhagem de células 3T3-L1 diferenciadas em adipócitos com IFNy recombinante resultou na produção aumentada de quimiocinas de macrófagos, como a proteína quimiotática de monócito (MCP-1), e de quimiocinas de células T, como a proteína 10 induzida por IFNy (IP-10) e monocina induzida por IFNy (MIG). A estimulação de adipócitos com o sobrenadante de células Th1 cultivadas in vitro, com abundante concentração de IFNy, também levou à produção aumentada de IP-10. Em análise mais ampla, através de microarray, dos possíveis efeitos do IFNy na expressão gênica de adipócitos, o tratamento dessas células com 100 U/ml de IFNy resultou na expressão aumentada de diversas quimiocinas e seus receptores em comparação ao grupo tratado com placebo. Similarmente à estimulação de células isoladas com IFNy, a incubação de tecido adiposo ex vivo de camundongos com essa citocina também resultou em secreção aumentada de IP-10, MIG e fator de necrose tumoral alfa (TNFy). A investigação do papel do IFNy na inflamação do tecido adiposo in vivo envolveu camundongos com deficiência de IFNy e controles, ambos os grupos submetidos a dieta rica em gorduras (obesos) ou pobre em gorduras (não obesos). Camundongos obesos deficientes em IFNy apresentaram expressão reduzida de mRNA de genes inflamatórios como TNFalfa e MCP-1 no tecido adiposo; acúmulo local reduzido de macrófagos; e melhor tolerância à glicose em comparação aos controles sob mesma dieta. Animais com deficiência de apolipoproteína E (ApoE) e também do receptor de IFNy também apresentaram em seu tecido adiposo a expressão reduzida de mRNA de genes inflamatórios, particularmente relacionados às células T, como IP-10, MIG, e o receptor CXCR3, em comparação aos controles com deficiência única de ApoE. Resultados in vitro e in vivo sugerem conjuntamente um importante papel do IFNy, e portanto, das células T e da imunidade adaptativa, na rede inflamatória do tecido adiposo na obesidade, com consequente impacto metabólico sistêmico. A presença de células T ativadas no tecido adiposo e seu acúmulo diferencial na obesidade motivaram também a pesquisa de potenciais mecanismos quimiotáticos reguladores desse processo. CXCR3, receptor das quimiocinas de células T, IP-10, MIG e quimiocina alfa de células T IFNy-induzida (I-TAC), é expresso preferencialmente em células T ativadas, e detém papel central na migração dessas células em outras condições inflamatórias crônicas, como a aterosclerose. Em camundongos com deficiência de CXCR3 e que receberam dieta rica em gorduras por 8 ou 16 semanas, o tecido adiposo apresentou significativamente menos células T, incluindo as células CD4+ e CD8+, em comparação a controles submetidos a mesma dieta. Os números similares de células T e outras populações de leucócitos no baço e sangue periférico dos animais deficientes em CXCR3 e controles fortalecem o conceito de um efeito do CXCR3 sobre o acúmulo de células T no tecido adiposo, independentemente do número de células circulantes e periféricas. Os camundongos deficientes em CXCR3 apresentaram também maior tolerância à glicose e expressão reduzida de mRNA de mediadores inflamatórios em seu tecido adiposo em comparação aos controles após 8 semanas de dieta rica em gorduras. No entanto, a diferença na tolerância à glicose entre os dois grupos tornou-se não significativa após 16 semanas de dieta gordurosa, coincidindo com redução substancial na expressão de mRNA de mediadores anti-inflamatórios (como interleucina-10 [IL-10] e Arginase 1), e número reduzido de células T regulatórias no tecido adiposo de camundongo s deficientes em CXCR3 em relação a controles. Esses resultados sugerem que o CXCR3 é capaz de regular o acúmulo de células T de diferentes subtipos, com perfil proinflamatório ou anti-inflamatório. Em conclusão, nossos resultados revelam um importante papel da citocina Th1 IFNy na rede inflamatória do tecido adiposo na obesidade em camundongos, sugerindo a participação fundamental das células T e portanto, da imunidade adaptativa nesse cenário. Além disso, o receptor CXCR3 contribui significativamente para o acúmulo das células T, incluindo as células T regulatórias, no tecido adiposo desses animais / The gradual and recent development of a worldwide epidemic of obesity greatly leveraged the study of this condition and its metabolic comorbidities. In the pathophysiologic context, multiple studies have demonstrated increased expression of inflammatory mediators in adipose tissue of obese animals and humans, the local macrophage accumulation, and a central role of inflammation in the imbalance of local or systemic metabolic homeostasis in obesity. The concept of an active role of macrophages and thus of innate immunity in the inflammatory network of adipose tissue, suggested the hypothesis that, similar to other chronic inflammatory conditions such as atherosclerosis, obesity also count on the participation of important elements of adaptive immunity such as T cells and their cytokines in its pathophysiology. Based on these considerations, the main objectives of this study were: 1) to evaluate the presence of T cells and the role of interferon-gamma (IFNy), classic T-helper 1 (Th1) cytokine, in adipose tissue inflammation, and 2) to study mechanisms of T cell accumulation in adipose tissue in the context of obesity, particularly the involvement of CXCR3 receptor in this process. Flow cytometry experiments showed that the visceral fat tissue of C57BL/6 obese mice fed a high fat diet showed a greater number of macrophages and also T cells, including CD4+ and CD8+ cells, compared to controls fed a low-fat diet. The expression of I-Ab, murine marker of class II major histocompatibility complex (MHC II), was also higher in adipose tissue of obese animals, suggesting the presence of local antigen presentation and consequent T cell activation. When stimulated in vitro, T cells derived from adipose tissue of obese mice produced more IFNy than those isolated from controls, again suggesting the activation of these cells in the context of obesity. In the analysis of possible functions of IFNy in adipose tissue, stimulation of 3T3 -L1 cells differentiated into adipocytes with recombinant IFNy resulted in enhanced production of macrophage chemokines, such as monocyte chemotactic protein-1 (MCP-1) and T-cell chemokines, such as interferon gamma-induced protein 10 (IP-10) and monokine induced by gamma interferon (MIG). The stimulation of adipocytes with the supernatant of in vitro cultured Th1 cells, with abundant levels of IFNy, has also led to increased IP-10 production. In a broader analysis, by microarray, of the possible effects of IFNy on adipocyte gene expression, treatment of these cells with 100 U/ml of IFNy resulted in increased expression of chemokines and their receptors in comparison to the placebo group. Similarly to the stimulation of isolated cells with IFNy, incubation of ex vivo adipose tissue with this cytokine also resulted in increased IP-10, MIG and tumor necrosis factor alpha (TNFalpha) secretion

INTRODUÇÃO: As células-tronco hematopoiéticas (CTH) do sangue do cordão umbilical (SCU) têm sido utilizadas com sucesso para o tratamento de doenças malignas e não malignas. No entanto, algumas unidades de SCU podem apresentar baixa quantidade de células nucleadas totais (CNT). Algumas abordagens têm sido sugeridas para evitar problemas em relação à baixa concentração de CTH no transplante, como a administração de duas unidades de SCU para o paciente e a expansão ex vivo de CTH. OBJETIVO: Avaliar as taxas de proliferação celular na expansão ex vivo do SCU em sistema de cocultura com células-tronco mesenquimais (CTM) obtidos a partir de diferentes fontes com alta e baixa confluência e adicionando-se ou não coquetel de citocinas no meio de cultura. MÉTODOS: Este estudo foi aprovado pelo Comitê de Ética de Pesquisa (CAPPesq) do Hospital das Clínicas da Faculdade de Medicina da USP. A coleta do SCU (n =10) foi realizada após o nascimento do bebê e expulsão da placenta. O processamento foi realizado utilizando o método de redução de volume, o qual consiste em depleção de eritrócitos. As amostras de CTM provenientes do endotélio vascular do cordão umbilical foram obtidas de doadores diferentes (n=3) e o tecido adiposo (n=3) do inventário do LIM-31. A expansão das CNT e das células com expressão de marcadores CD133+/CD34+ foram observados depois de sete dias de cultura. Além disso, o ensaio para análise de unidades de formadoras de colônias (UFC) foi realizado em todas as amostras antes e depois da expansão do SCU. Para a expansão em sistema de cocultura foi separado dois grupos para ambas as fontes de CTM (Grupo I - cocultura com adição de coquetel de citocinas vs. Grupo II - cocultura sem citocinas). RESULTADOS: Após sete dias, no grupo I com cocultura confluente, a taxa de proliferação de CNT foi duas vezes maior ao comparar com cocultura subconfluente (35 vs. 16 vezes). No mesmo grupo também foi possível evidenciar elevada taxa de proliferação de células CD133+/CD34+. O índice de proliferação das UFC no grupo I aumentou até oito vezes. A cocultura subconfluente tanto do endotélio vascular do cordão umbilical como do tecido adiposo apresentou menor rendimento em comparação as CTM confluentes. A expansão das células na presença de citocinas apresentou maior proliferação celular ao comparar às coculturas sem adição de citocinas. CONCLUSÃO: Este estudo mostrou que para alto rendimento de células do SCU, o sistema de cocultura requer adição de coquetel de citocinas e CTM confluente independentemente da fonte utilizada / INTRODUCTION: Umbilical cord blood (UCB) hematopoietic stem cells have been successfully used for the treatment of both malignant and non-malignant diseases. Nevertheless, some UCB units could have low total nucleated cells (TNC) dose. Several approaches have been suggested to avoid inadequacy problems of hematopoietic stem cells (HSC) number for transplantation, such as administration of two UCB units to the patient and HSC ex vivo expansion. OBJECTIVE: Evaluate UCB ex vivo expansion proliferative rates in a high and low mesenchymal stem cells (MSC) confluence feeder layer obtained from different MSC sources and by adding or not cytokines cocktail into the medium. METHODS: This study was approved by the Research Ethic Committee (CAPPESQ) of Hospital das Clínicas da Faculdade de Medicina da USP. The collection of UCB (n=10) was made after delivery of the infant and the expulsion of placenta. Processing was performed using volume reduction method which consists in red blood depletion. MSC samples from umbilical cord endothelium were obtained from three different donors and adipose tissue (n=3) obtained from LIM31\'s pattern inventory. The total nucleated cell (TNC), expression of hematopoietic surface markers such as CD133+/CD34+ were observed after seven days of culture. Beyond that, colony forming unit assay (CFU) was performed before and after UCB expansion. The expansion by coculture method was observed in two groups (Group I - coculture with cytokines cocktail added vs. Group II- coculture without cytokines cocktail) for both MSCs sources. RESULTS: After seven days, analysis of confluent coculture showed that TNC proliferation rate ware almost 2 times higher than in subconfluent coculture (35 vs. 16-fold) in Group I and also revealed higher proliferative rate in CD133+/CD34+ cells considering. CFU showed similar increase after seven days of culture in comparison of day 0 (up to 8-fold). Subconfluent coculture for both umbilical cord endothelium and adipose tissue showed lower yield compared with those with high MSC confluence. The expansion in the presence of cytokines showed higher cell proliferation compared to the cocultures without addition of cytokines. CONCLUSION: This study showed that coculture system may require the addition of cytokines cocktail in the media and confluent MSC regardless of source for high yield of UCB cells

O tecido adiposo marrom (TAM), caracterizado pela presença da proteína termogênica UCP-1, é conhecido há muitas décadas como um tecido termogênico em mamíferos, porém sua significância clínica em humanos era considerada pequena, com exceção de neonatos, até que o desenvolvimento e uso de métodos de PET-FDG terem demonstrado que humanos adultos também possuem TAM ativo, especialmente após exposição ao frio. Essa descoberta levou a um enorme aumento nas pesquisas sobre o assunto, já que sua ativação, levando a um aumento do gasto energético, poderia, pelo menos na teoria, ser uma possível arma no tratamento da obesidade e diabetes tipo 2 e sua redução ou ausência ser uma causa de ganho de peso. Muitos compostos vêm sendo estudados como possíveis recrutadores e ativdadores desse tecido. A melatonina é um deles, embora nenhum estudo tenha sido feito em humanos. A melatonina, um hormônio pineal sintetizado à noite com um papel crítico na sincronização do ritmo circadiano, é estudado há várias décadas como um regulador chave do metabolismo energético em diversas espécies animais. Ratos pinealectomizados ganham peso e tem distúrbios metabólicos durante sua vida, e a suplementação noturna de melatonina, reverte estas alterações, sem redução da ingesta alimentar. Devido a isso, uma hipótese é que o papel central da melatonina no metabolismo energético inclui sua função no gasto energético, possivelmente relacionado à ativação do TAM. Muitos modelos experimentais, a maioria em animais hibernantes, demonstraram o papel da melatonina no recrutamento do TAM. Nesse estudo, o objetivo é determinar se a suplementação de melatonina para indivíduos e animais de experimentação (ratos Wistar) deficientes de melatonina aumenta sua ativação. Foi encontrado que, em ratos Wistar, animais pinelaectomizados possuem uma capacidade termogênica do TAM reduzida após exposição ao frio comparado com a temperatura ambiente, e a suplementação de melatonina normaliza essa capacidade termogênica. Esse dado sugere um papel da melatonina na resposta máxima de ativação do TAM após um desafio ao frio agudo. Também foi observado um aumento de expressão de UCP-1 (RNA) em animais repostos com melatonina, tanto em controles como em pinealectomizados, e animais pinealectomizados não repostos apresentam uma expressão de UCP-1 menor que um grupo controle. Em humanos, a suplementação de melatonina aumenta o volume e atividade do TAM em quatro indivíduos pinealectomizados (por tumores pineais) com baixo nível de melatonina no basal, analisado por tomografia de emissão de prótons acoplada a ressonância magnética (PET-RM). Embora a análise do TAM em ambos os protocolos tenha sido distinta, seus resultados apontam para a mesma regulação positiva do TAM pela melatonina. A termografia infravermelha (TIV) foi também realizada em humanos, com aumento de atividade de TAM após exposição ao frio, poréma correlação entre as respostas com a TIV e o PET-RM foi moderada e não significativa. Diferenças entre o protocolo frio e limitação da TIV em indivíduos mais obesos podem ter contribuído para esses resultados. Uma relação positiva da suplementação de melatonina nos lípides (principalmente colesterol e triglicérides) também foi encontrada, porém sem impacto na gordura hepática / Brown adipose tissue (BAT), characterized by the presence of the thermogenic protein UCP-1 have long been known as a thermogenic tissue in mammals, however its significance in humans was considered minor, with the exception of newborns, until FDG-PET exams demonstrated that human adults still have active BAT, especially after cold exposure. This prompted to an incredible increase in research on the field, since its activation, leading to increased energy expenditure could, at least theoretically, be a possible tool for the treatment of obesity and type 2 diabetes and its reduction or absence be a cause of weight gain. Many compounds aiming to recruit and activate BAT have been studied. Melatonin has been one of them, although no study has been performed in humans. Melatonin, a pineal hormone synthetized at night with a critical role in the synchronization of circadian rhythms, has long been studied as a key regulator of energy metabolism in many animal species. Pinealectomized rats gain weight and have metabolic disturbances during life, and the circadian supplementation of melatonin, at night, reverts these alterations, without decrease in energy intake. Due to that, it is hypothesized that a main role of melatonin in energy metabolism includes its action on energy expenditure, possibly related to activation of BAT. Many experimental models, mainly in hibernating animals, have shown a role of melatonin on BAT recruitment. In the present study, we ought to determine if the supplementation of melatonin for melatonin deficient subjects and experimental animals (Wistar rats) increases BAT activation. We found, in Wistar rats, that pinealectomized animals have a reduced BAT thermogenic capacity after acute cold exposure compared with ambient temperature, and melatonin supplementation in this animals leads to normalization of BAT thermogenic capacity. This data suggests a role of melatonin in improving the maximal response of BAT after an acute challenge. We also found that melatonin supplementation increases UCP-1 RNA expression both in control and pinealectomized rats, and pinealectomized rats without supplementation have a reduced UCP-1 expression compared with controls. In humans, we found that melatonin supplementation increased BAT volume and activity in four pinealectomized (due to pineal tumors) individuals with low melatonin at baseline, analyzed by Positron Emission Tomography associated with magnetic resonance (PET-MR). Although the analysis of BAT in both studies was different, their results point to the same positive regulation of BAT by melatonin. We also performed infrared termography (IRT) in humans, but the results were not conclusive since although we also found an increase in BAT activity measured in Watts, the correlation between the methods was moderate. The difference may be due to different protocols of cold exposure between methods, probably inadequate in IRT, as well as maybe to a limitation of IRT in more obese individuals. We also found that melatonin supplementation in melatonin deficient humans may have a positive impact on blood lipid concentrations, (mainly total cholesterol and triglycerides) but, at least for the time studied, does not appear to have an impact on liver fat

A hipercolesterolemia familiar (HF) é uma doença causada por um grupo de alterações genéticas que resultam em altas concentrações de colesterol no sangue e aumento na prevalência de aterosclerose subclínica e risco de eventos coronarianos precoces. Apesar da importância do colesterol como fator causal da aterosclerose na HF, o curso desta última é variável e influenciado por outros fatores de risco. A gordura pericárdica é um compartimento da gordura visceral e está associada à presença de aterosclerose coronária subclínica em populações sem HF. Este estudo avaliou a associação da gordura pericárdica com a presença e extensão da aterosclerose coronária subclínica em pacientes com HF. Noventa e sete pacientes com HF diagnosticada por critérios clínicos, confirmada geneticamente em 67% dos casos, foram submetidos a angiotomografia de coronárias e determinação do escore de cálcio (CAC). Foram analisadas a presença de placas, de estenose luminal > 50%, de CAC > 0 e do percentil de CAC > 75. Para se quantificar a extensão e gravidade da aterosclerose coronária subclínica foram avaliados de forma contínua a CAC, o Segment-Involvement Score (SIS) e o Segment-Stenosis Score (SSS). O volume de gordura pericárdica foi aferido por método semiautomático e dividido em dois compartimentos: gordura epicárdica (localizada dentre do saco pericárdico) e mediastinal (localizada fora do pericárdio). Para avaliar a associação dos volumes de gordura pericárdica com a aterosclerose subclínica foram ajustados modelos de regressão logística e linear. Os pacientes tinham idade média de 45 (±13) anos, com predomínio do sexo feminino. Foi encontrada presença de placas coronarianas e de CAC em 47,4% e 45,4% dos pacientes, respectivamente. Idade, colesterol total, LDL-C, HDL-C, apolipoproteína A-I, apolipoproteína B, presença de xantomas de tendão de Aquiles e clearance de creatinina foram associados a aterosclerose coronária subclínica na análise univariada. Os volumes de gordura pericárdica se associaram à presença de síndrome metabólica, hipertensão arterial, idade, IMC, a circunferência abdominal, o colesterol não-HDL, triglicerídeos e glicemia de jejum. Na análise multivariada em modelos ajustados para idade, sexo, tabagismo, HDL-C, LDL-C, circunferência abdominal, síndrome metabólica e uso prévio de estatinas, a gordura epicárdica foi associada independentemente com o percentil de CAC > 75 e foi diretamente proporcional a intensidade da CAC, SSS e SIS. Em conclusão, a gordura epicárdica associou-se independentemente à maior extensão e gravidade de aterosclerose coronária subclínica em pacientes com HF / Familial hypercholesterolemia (FH) is a disease caused by a group of genetic mutations resulting in high blood cholesterol and elevated prevalence of subclinical atherosclerosis and early coronary events. Although high cholesterol is the driving cause of atherosclerosis in FH, the course of the latter is variable and is affected by other risk factors. Pericardial fat (PF) is a visceral fat compartment that is associated to the presence of subclinical atherosclerosis in non-FH populations. The present study sought to determine the association of PF with the presence and extent of subclinical coronary atherosclerosis in FH subjects. Ninety-seven patients with clinical diagnosis of FH, genetically confirmed in 67%, were submitted to coronary tomography angiography and coronary artery calcium (CAC) quantification. The presence of plaques, luminal stenosis > 50%, CAC > 0, CAC percentile above 75 were evaluated. In order to evaluate the extent and severity of subclinical atherosclerosis, the CAC scores, Segment-Involvement Score (SIS) and Segment-Stenosis Score (SSS) were also measured. Pericardial fat volumes were measured by semi automated method and divided in two compartments: epicardial fat (located inside the pericardial sac) and mediastinal fat (located outside pericardial sac). Logistic regression and linear models tested the association of PF volumes with subclinical coronary atherosclerosis. Patients were predominantly female, with mean age of 45 (± 13) years. Coronary plaques and CAC were found respectively in 47.4% and 45.4% of patients. Age, total cholesterol, LDL-C, HDL-C, apolipoproteins A-I and B, the presence of Achilles xanthomas and creatinine clearance were associated with subclinical coronary atherosclerosis in univariate analysis. PF volumes were associated with the presence of metabolic syndrome, hypertension age, BMI, abdominal circumference non-HDL-cholesterol triglycerides and fasting glucose. On multivariate analysis in models adjusted for age, sex, smoking, HDL-C, LDL-C, abdominal circumference, metabolic syndrome and previous statin use epicardial fat was independently associated with CAC > 75th percentile, and was directly proportional to the intensity of CAC, SSS and SIS. In conclusion, epicardial fat was independently associated with a greater extension and severity of subclinical atherosclerosis in FH patients

O envelhecimento traz modificações na composição corporal habitualmente não acompanhadas por mudança concomitante no índice de massa corporal (IMC). Assim, o IMC tem baixa acurácia para estimar risco de morte atribuído às mudanças de composição corporal em idosos. Entretanto, a maioria dos estudos de composição corporal nesta população utilizou medidas antropométricas ou métodos de alta acurácia mas de elevados custo e complexidade operacional (tomografia computadorizada e ressonância magnética). Atualmente, o melhor método na prática clínica para análise da composição corporal é a absorciometria por dupla emissão de raios-X (DXA). Porém, os poucos estudos que utilizaram DXA apresentam limitações, como análise não estratificada por sexo e avaliação global da gordura corporal, não separando gordura subcutânea e visceral. O objetivo do presente estudo foi avaliar a associação da composição corporal por DXA (incluindo o tecido adiposo visceral) e mortalidade geral (por todas as causas) e cardiovascular em uma população de idosos brasileiros da comunidade. Oitocentos e trinta e nove (839) indivíduos da comunidade (516 mulheres, 323 homens), com 65 anos ou mais, foram avaliados por questionário clínico, exames laboratoriais e composição corporal por DXA na visita inicial. A gordura corporal foi avaliada por índices de massa gorda total e pelo tecido adiposo visceral (VAT), sendo utilizado o scan de corpo total do DXA (HOLOGIC, QDR 4500, software APEX). Baixa massa muscular (BMM) foi definida como baixa massa muscular apendicular ajustada para gordura corporal, segundo método previamente descrito na literatura (NEWMAN, 2003). A mortalidade foi registrada durante o seguimento médio de aproximadamente 4 anos da avaliação inicial. Modelos de regressão logística, para homens e mulheres, foram utilizados para avaliar a associação entre composição corporal e mortalidade. Após 4,06 ± 1,07 anos de seguimento, houve 132 (15,7%) óbitos, sendo 57 (43,2%) por causas cardiovasculares. Em homens, após ajustes para múltiplas variáveis pertinentes, a presença de BMM (OR 11,36 IC95%: 2,21-58,37, p=0,004) e o VAT (OR 1,99 IC95%: 1,38-2,87, p < 0,001) aumentaram significativamente o risco de mortalidade geral enquanto a gordura corporal total foi associada com menor risco de morte (OR 0,48 IC95%: 0,33-0,71, p < 0,001). Resultados semelhantes foram encontrados para a mortalidade cardiovascular. Em mulheres, apenas a BMM foi preditor de mortalidade geral (OR 62,88 IC95%:22,59-175,0, p < 0,001) e mortalidade cardiovascular (OR 74,54 IC95%: 9,72-571,46, p < 0,001), não havendo associação entre massa gorda total ou visceral com mortalidade. Após os resultados encontrados, percebemos que os riscos associados às mudanças da composição corporal em idosos são diferentes de acordo com o sexo, e contrariam o que a literatura mostra para populações mais jovens em relação ao papel da gordura. Estes resultados sugerem que a composição corporal por DXA parece ser uma ferramenta promissora para avaliação da massa muscular, gordura corporal e risco de mortalidade em idosos, uma vez que consiste em metodologia precisa e de fácil aplicabilidade na prática clínica / Body composition changes resulting from ageing (decreased muscle mass and increased fat tissue) are frequently not accompanied by concomitant changes in body mass index (BMI). Thus, BMI has low accuracy to estimate death risk attributed to changes in body composition in older adults. Currently, the best method for body composition analysis in routine clinical practice is dual energy X-ray absorptiometry (DXA). However, the few studies on body composition by DXA and mortality risk in elderly have some limitations, such as analysis not compartmentalized (subcutaneous and visceral tissues) of body fat and appendicular muscle mass not adjusted for fat mass. Thus, we sought to investigate the association between body composition by DXA (including visceral adipose tissue [VAT]) and mortality in a longitudinal, prospective, population-based cohort of elderly subjects. Eight hundred and thirty nine (839) community-dwelling subjects (516 women, 323 men), 65 years or older, were assessed by questionnaire on clinical data, laboratory exams and body composition by DXA using Hologic QDR 4500A equipment. All analyses were performed at baseline. Both total fat and its compartments (eg. visceral adipose tissue [VAT]) were estimated. Low muscle mass (LMM) was defined as the presence of low appendicular muscle mass adjusted for fat. Mortality was recorded during 4 year-follow-up. Multivariate logistic regression models, for men and women, were used to compute odds ratios for all-cause and cardiovascular mortality. Over a mean follow-up of 4.06 ± 1.07 years, there were 132 (15.7%) deaths. In men, after adjustment for relevant variables, the presence of LMM (OR 11.36, 95% CI: 2.21-58.37, p=0.004) and VAT (OR 1.99 95%CI: 1.38-2.87, p < 0.001, for each 100g-increase) significantly increased all-cause mortality risk, while total body fat, measured by Fat Mass Index (FMI), was associated with decreased mortality risk (OR 0.48, 95% CI: 0.33-0.71, p < 0.001). Similar results were observed for cardiovascular mortality in men. In women, only the presence of LMM was a predictor of all-cause (OR 62.88, 95% CI: 22.59-175.0, p < 0.001) and cardiovascular death (OR 74.54, 95% CI: 9.72-571.46, p < 0.001). Both muscle mass and fat mass, including its compartments, impacts on all-cause and cardiovascular mortality risk in elderly. Moreover, their effects are different according to sex. Visceral fat and subcutaneous fat have opposite roles on mortality risk in elderly men, and this is distinct from what is observed in young adults. Thus, DXA seems to be a promising tool for evaluation risk of mortality in elderly, since it is easily applicable in clinical practice

L’obésité s’accompagne de pathologies comme le diabète de type 2 et les maladies cardiovasculaires, liées à des dérégulations métaboliques et endocriniennes du tissu adipeux blanc (TAB). Au cours du vieillissement, la perte de masse musculaire peut être associée à l’obésité et définit le concept d’obésité sarcopénique. Les traitements mis en œuvre pour contrecarrer ces pathologies n’ont qu’un succès très partiel. Il est donc opportun de développer des stratégies alternatives originales qui pourraient aboutir à des thérapeutiques ciblées. Notre équipe étudie les régulations métaboliques du TAB, source majeure de stockage de l’énergie de l’organisme. Les triglycérides stockés sont libérés à jeun grâce à la lipolyse qui libère les acides gras non-estérifiés (AGNE) et le glycérol dans le sang, comme source d’énergie des autres tissus. En plus de la β-oxydation des AGNE, leur ré-estérification partielle intervient pour limiter leur libération lors de la lipolyse. La glycéronéogenèse est nécessaire à la ré-estérification en situation de jeûne. Des études préalables ont montré que l'administration de citrulline (CIT) pendant trois mois à des rats vieillissants induit une diminution d’environ 40% de la masse viscérale du TAB. Cet acide aminé non protéique est un complément alimentaire donné au cours du vieillissement ou à des sportifs pour augmenter la masse musculaire. Nous avons étudié les effets de la CIT sur des cultures d’explants de TAB de rats. Dans la première partie de ce travail, nous montrons que la CIT a un effet direct lipolytique et anti-glycéronéogénique sur les explants des rats qu’ils soient jeunes ou âgés. Cependant, la libération des AGNE du TAB des rats jeunes est limitée par une augmentation de la capacité oxydative du tissu. Avec l’âge, la masse du TAB augmente en parallèle à l’augmentation d’un état pro-inflammatoire. Afin de comprendre l’influence de ces deux paramètres indépendamment de l’âge, nous avons étudié dans la deuxième partie de ce travail, les effets de la CIT sur les explants de TAB de rats jeunes soumis à un régime contrôle (CD) ou hyperlipidique (HFD). Nous observons une augmentation, induite par la CIT, de la lipolyse et de la capacité ß-oxydative du TAB des rats quel que soit le régime, alors que la glycéronéogenèse est diminuée. Toutefois, les AGNE sont sélectivement libérés par le TAB de rats HFD, en relation avec une réduction drastique de leur ré-estérification. Le NO est un médiateur de ces effets. Dans une troisième partie, nous démontrons que la CIT agit directement sur le TAB de rats CD et HFD pour induire l'expression de la protéine découplante, UCP1, en lien avec le « brunissement » potentiel du TAB par cet acide aminé. Ces effets ne sont pas observés au sein du TAB des rats âgés. L’ensemble de nos résultats établit les bases pour de futures investigations visant à élucider les mécanismes par lesquels la CIT réduit la masse adipeuse et ouvre de nouvelles perspectives thérapeutiques pour lutter contre le surpoids et l’obésité sarcopénique. / Obesity is frequently associated with type 2 diabetes and cardiovascular diseases, related to metabolic and endocrine dysregulation of white adipose tissue (WAT). During aging, the loss of muscle mass may be associated with obesity and defines the concept of sarcopenic obesity. Treatments implemented to counteract these conditions showed a very partial success. It is therefore appropriate to develop original alternative strategies that could lead to targeted therapies. Our team studies the metabolic regulation of WAT, the major source of energy storage in the body. Non-esterified fatty acids (NEFA) and glycerol are released in the blood from stored triglycerides through lipolysis and used as a source of energy for other tissues. In addition to their β-oxidation, NEFA are re-esterified in part, a process that limits their release in the blood. Glyceroneogenesis is the pathway necessary to NEFA re-esterification in the fasting state. Previous studies showed that administration of citrulline (CIT) for three months to aging rats induced a decrease of approximately 40% of the visceral WAT mass. This non-protein amino acid is given as a dietary supplement during aging or sports to increase muscle mass. We studied the effects of CIT on explant cultures of rat WAT. In the first part of this work, we show that CIT exerts a direct lipolytic and anti-glyceroneogenic effect on explants from rats whether young or old. However, the release of NEFA from the explants of young rats is limited by an increase in the oxidative capacity of the tissue. During aging, WAT mass augments in parallel to the increase in a pro-inflammatory state. To understand the influence of these two parameters regardless of age, we studied in the second part of this work, the effects of CIT on WAT explants from young rats fed a control (CD) or high fat (HFD) diet. We show an CIT-induced increase in lipolysis and beta-oxidative capacity of WAT from rats whatever the diet, while glyceroneogenesis is reduced. However, NEFA are selectively released from WAT of HFD rats, in connection with a drastic reduction of their re-esterification. NO is a mediator of these effects. In the third part of this work, we show that CIT acts directly on WAT from CD and HFD rats to induce the expression of uncoupling protein, UCP1, in line with the potential "browning" of WAT by this amino acid. These effects were not observed in explants from old rats. Altogether our results establish the basis for future investigations aimed at elucidating the mechanisms by which CIT reduces body fat and open new therapeutic perspectives to fight overweight and sarcopenic obesity.

Os procedimentos para uso clínico de células-tronco derivadas de tecido adiposo (CT-TA) exigem grandes quantidades de células, por isso, em geral os protocolos envolvem a expansão e cultura celular in vitro. No entanto, as metodologias utilizadas rotineiramente para o cultivo de CT-TA envolvem a utilização de componentes xenobióticos, como a colagenase e o soro fetal bovino (SFB), que representam riscos potencias de reações imunológicas e transmissão de doenças infecciosas. Sendo assim, pretendeu-se no presente estudo analisar diferentes parâmetros metodológicos para isolamento e expansão de CT-TA, na ausência de componentes xenobióticos. Para tanto, as células-tronco foram isoladas por digestão enzimática ou dissociação mecânica e submetidas à expansão na presença de SFB ou lisado de plaquetas humano (LP). Os resultados mostraram que a metodologia de dissociação mecânica representa uma alternativa viável e eficiente para cultivo de CT-TA, e que o emprego de LP como suplemento para o meio de cultura aumentou de forma significativa a proliferação celular. Em função desses resultados, pode-se concluir que é possível a implementação de técnicas de isolamento e expansão de CT-TA, prescindindo-se de componentes xenobióticos. / The procedures for the clinical use of adipose-derived stem cells (ASC) require large amounts of cells, so in general protocols involve culture and cell expansion in vitro.However, the methods routinely used for the culture of ASC involves the use of xenobiotic components, such as collagenase and fetal bovine serum (FBS), that may representing potential risk of immunological reactions and the risk of transmission of infectious diseases. Thus, it was intended in this study to analyze different methodological parameters for the isolation and expansion of ASC in the absence of xenobiotic components. For this, stem cells were isolated by enzymatic digestion and mechanical dissociation and were submitted to expansion in the presence of FBS or human platelet lysate (PL). The results showed that the mechanical dissociation method represents an effective alternative to growing ASC, and that the use of PL as a supplement to the culture medium significantly increased cellular proliferation. In view of these results, we can conclude that it is possible to implement techniques for isolation and expansion of ASC, dispensing xenobiotic components.