A APECED é uma doença que apresenta autoimunidade e susceptibilidade a Candida albicans. Nosso grupo observou que a proteína AIRE participa da via da Dectina-1, importante contra a C. albicans. Neste projeto, investigamos como a ausência de AIRE influencia em eventos para a eliminação do patógeno via Dectina-1. Assim, avaliamos o burst oxidativo, expressão de moléculas do sistema NADPH oxidase e fagocitose por células de paciente com APECED e células THP-1 silenciadas para AIRE. Não houve diferença no burst oxidativo e na expressão dos componentes do sistema NADPH oxidase por estas células e as células silenciadas fagocitam menos que as células selvagens. Observamos que não há diferença na expressão flavocitocromo b558 e p40phox do paciente comparado ao controle. Em paralelo, mostramos que as células do paciente apresentaram um burst oxidativo e fagocitose diminuídos comparado ao controle. Estes resultados sugerem que há um defeito no reconhecimento via Dectina-1, gerando uma diminuição da fagocitose que pode dificultar sua eliminação. / The APECED is a syndrome with autoantibodies and Candida albicans susceptibility. Our group has noted that AIRE protein is required for dectin-1 signaling, important against C. albicans. In this project, we investigate how the absence of AIRE influences in events for elimination of pathogen via dectin-1. We evaluated reactive oxygen species production, expression of NADPH oxidase molecules and phagocytosis by APECED pacient cells or AIRE silent THP-1 cells. We didnt observe differences in oxidative burst and expression of NADPH oxidase components by these cells and silent cells phagocytize less than wild-type cells. We observed no difference in flavocytochrome b558 and p40phox expression in paciente cells and control. In parallel, we showed that pacient cells has a decrease in burst oxidative and phagocytosis compared to control. Our results suggest that there is a defect in pathogen recognition via dectin-1, resulting in decrease on phagocytosis that can hamper their elimination.

Pentoxifilina (PTF), um derivado da metilxantina, tem ação terapêutica como agente antifibrótico. In vitro, a PTF causa inibição na produção de colágeno, glicosaminoglicanos e fibronectina, bem como promove acentuada redução na proliferação dos próprios fibroblastos de quelóides. A PTF na concentração de 1.000 g/mL foi seletiva no controle da inibição da síntese protéica pelos fibroblastos. O objetivo deste estudo foi verificar o comportamento in vitro de fibroblastos oriundos de cicatrizes hipertróficas (HSHF) e de pele não-cicatricial (NHF) na presença e ausência de PTF (1.000 g/mL), quanto à: proliferação celular, produção de colágeno e capacidade dos fibroblastos gerarem contração em modelo experimental de matriz de colágeno. Para tanto, foram utilizados fibroblastos cultivados a partir de amostras de cicatrizes hipertróficas e pele não-cicatricial doadas, com finalidade de pesquisa, pelo banco de Tecidos do Instituto Central do Hospital das Clínicas da FMUSP. Culturas celulares expostas a PTF apresentaram diminuição na proliferação celular em HSHF (46,35%) e em NHF (37,73%) (p<0,0001). Na presença de PTF, foi observada seletividade de inibição na síntese de colágenos, havendo inibição mais expressiva de colágeno tipo III em HSHF e de colágeno tipo I em NHF (p<0,0001). O modelo experimental de matriz de colágeno povoada por fibroblastos de cicatriz hipertrófica apresentou contração menor (12%) na presença de PTF (p<0,0001), em relação à sua ausência / Fibroblasts are thought to be partially responsible for the persisting contractile forces that result in burn contractures. Using a monolayer and fibroblast populated collagen lattice (FPCL) three-dimensional (3D) model we subjected hypertrophic scar and non-cicatricial fibroblasts to the antifibrogenic agent pentoxifylline (PTF) 1000g/mL attempting to reduce proliferation, collagen type I and III synthesis and contraction in this 3D model. Fibroblasts were isolated from post burn hypertrophic scars (HSHF) and non-scarred skin (NHF). Cells were grown in monolayer or incorporated into FPCL\'s and exposed to PTF. In monolayer, cell number proliferation was reduced (46.35% in HSHF group and 37.73% in NHF group) p<0,0001. The PTF also demonstrated to be selective on collagen type I and III synthesis inhibition suggesting higher inhibition of collagen type III on HSHF group and more evident inhibition of type I on NHF group. FPCL\'s containing PTF had surface areas reduced in about 12% p<0,0001. PTF showed inhibition effects on cell proliferation and reduced contraction in both HSHF and NHF

Mutações no gene Hfe causam a forma mais comum da hemocromatose hereditária, doença caracterizada por acúmulo progressivo de ferro nos tecidos parenquimatosos. Um estudo prévio conduzido em nosso laboratório (Am J Cardiol 88(4):388-91, 2001) encontrou associação entre mutação do gene Hfe e cardiomiopatia isquêmica, sugerindo que o acúmulo de ferro no tecido cardíaco pode ser um fator que potencializa as agressões ao sistema cardiovascular. O objetivo do presente trabalho foi testar a hipótese de que o ferro aumenta a susceptibilidade ao risco cardiovascular. A análise de dados de 318 pacientes seguidos durante 10 anos indicou que variantes genéticas do Hfe estão associadas com maior mortalidade em pacientes com insuficiência cardíaca de diferentes etiologias. Em seguida, verificou-se o acúmulo de ferro no coração, aorta e fígado ao longo de 1, 3, 6 e 12 meses em camundongos FVB. Para mimetizar os efeitos deletérios do ferro no ser humano, validamos proteínas envolvidas no metabolismo do ferro em camundongos e tratamos os animais com 10 mg diárias de ferro dextrano durante 4 semanas. Os resultados sem a sobrecarga de ferro já apontaram acúmulo de ferro significativo no coração e no fígado ao longo de 12 meses de vida, consistente com a ideia de aumento progressivo de risco cardiovascular associado ao envelhecimento. A sobrecarga de ferro foi associada com maior mortalidade e deterioração da função cardíaca. Os camundongos tratados com ferro apresentaram diminuição da fração de ejeção, redução da espessura do septo, maior remodelamento cardíaco e aumento do volume nuclear dos cardiomiócitos. Para entender as modulações gênicas causadas pelo ferro no coração, foi medida a expressão dos transcritos primários de mRNA relativo para os genes Hfe e para a hepcidina, encontrando-se ambos os genes significativamente menos expressos nos animais tratados com ferro em comparação ao grupo que só recebeu salina. Por fim, com o intuito de estudar em condições mais controladas o comportamento cardíaco frente à sobrecarga de ferro, foram comparados dois protocolos de extração primária de cardiomiócitos ventriculares de ratos neonatos para testes farmacológicos com ferro in vitro. O enriquecimento de cardiomiócitos in vitro se estabeleceu por dois métodos: separação por gradiente de percoll (Per) e por uma pré-seleção nomeada pre plating (PP). As células cardíacas foram mantidas por 8 dias em cultura e avaliações do metabolismo, produção de espécies reativas de oxigênio e contratilidade foram medidas. Ambos os métodos foram eficientes para a obtenção de células cardíacas, entretanto, as células extraídas por protocolo PP apresentaram metabolismo aumentado, com maior consumo de glicose e produção de lactato. Por diferentes parâmetros testados o protocolo PP apresentou maior estresse oxidativo, porém sem modular a quantidade de glutationas reduzidas e oxidadas. Notadamente, o protocolo PP apresentou maior atividade contrátil com aumento dos batimentos e maior influxo intracelular de cálcio. Células cardíacas extraídas pelo método PP foram tratadas com citrato de amônia férrico com doses de 50 ?g/mL e 100 ?g/mL e, após 24 horas, foi possível observar aumento significativo de apoptose. Desta forma, os modelos celulares em questão apresentam-se como importantes ferramentas para a identificação de mecanismos moleculares e celulares associados aos efeitos deletérios causados pelo ferro. Em conjunto, os resultados do presente trabalho apoiam a hipótese de que o acúmulo de ferro no tecido cardíaco aumenta a susceptibilidade cardiovascular. Trabalhos futuros permitirão melhor compreensão dos mecanismos envolvidos no acúmulo de ferro no coração ao longo do envelhecimento em pacientes com insuficiência cardíaca / Mutations in Hfe gene lead to the most common form of hereditary hemochromatosis, an autosomal recessive disease associated with iron accumulation in parenchymal tissues. In a previous study conducted in our laboratory (Am J Cardiol 88(4):388-91, 2001), genetic variation in the Hfe gene was associated with ischemic cardiomyopathy, suggesting that higher cardiac concentrations of iron aggravates injuries on the cardiovascular system. The aim of the present study was to test the hypothesis that iron increases susceptibility to cardiovascular risk. Analysis of data from 318 patients with 10-year follow-up showed that genetic variation in the Hfe gene was associated with higher mortality among patients with heart failure due to cardiomyopathy of different etiologies. Next, we demonstrated iron accumulation in heart, aorta, and liver in mice (FVB background) aged 1, 3, 6, and 12 months. To mimic the deleterious effect of iron observed in humans, we validated proteins playing a major role in iron metabolism and treated mice with 10 mg of iron-dextran daily for 4 weeks. Results showed that even without iron overload there is significant iron accumulation in the heart and liver with time, at 12 months of age, consistent with the idea that there is a progressive age-related increase in cardiovascular risk. Iron overload was associated with higher mortality in mice and impairment of cardiac function; in response to iron treatment ejection fraction and septum thickness were reduced, while cardiac remodeling and myocyte nuclear volume were increased. To understand the underlying mechanisms associated with iron-mediated modulation of genes in the heart, we assessed Hfe and hepcidin mRNA expression and found that these genes were significantly less expressed in iron-treated animals compared with the saline solution group. Lastly, to study cardiac behavior in the face of iron overload under well-controlled conditions we compared two protocols for primary extraction of neonatal rat cardiomyocytes for in vitro pharmacological tests: Percoll (Per) and pre plating (PP) extraction methods. Cardiac cells were used after 8 days and we measured metabolism, ROS production, and contractility. Both methods were effective in obtaining a high yield of cardiomyocytes. Nevertheless, cells extracted using PP protocol presented higher metabolic rate, as suggested by increased lactate production and glycolysis rate. In the PP protocol there was an increased oxidative stress, notwithstanding without modulating the amount of oxidized and reduced glutathione peroxidase. Notably, we found an increased contractile activity for pre-platting-prepared cells, with increased beating rate and higher calcium influx. Cardiac cells extracted by PP exposed to ferric ammonium citrate with doses of 50?g/mL and 100?g/mL, after 24 hours, displayed significant increased apoptosis. The cell models examined can be considered important tools for the identification of cell and molecular mechanisms associated with the harmful effects caused by iron. Taken together, the results of the present study support the hypothesis that cardiac tissue iron accumulation increases cardiovascular susceptibility. Further studies will help to unravel the mechanisms involved in cardiac iron accumulation throughout the aging process in patients with heart failure

O mastocitoma é um dos mais freqüentes neoplasmas que acometem a pele do cão, e novas modalidades terapêuticas vêm sendo buscadas visando seu controle. O presente trabalho descreve o estabelecimento e caracterização do modelo xenotransplantável de mastocitoma canino para testes de substâncias com potencial antineoplásico in vitro e in vivo. O animal doador da amostra tumoral apresentava um mastocitoma grau 3. O mastocitoma canino foi estabelecido com sucesso nos camundongos atímicos BALB/c nu/nu. O tumor apresenta as mesmas características histológicas do fragmento tumoral daquele do animal doador ao longo das passagens. Em média, 7 dias a 3 semanas são necessários para o aparecimento do nódulo palpável e cerca de 60 dias para o volume tumoral alcançar mais de 500 mm3. O cultivo celular das células de mastocitoma canino se mantém por aproximadamente 1 mês, às vezes mais, mas o tempo de cultivo é limitado. Há perda progressiva das características iniciais de cultivo como perda de granulação, aumento de adesão e diminuição de células em suspensão. A seguir, foram realizados testes in vitro e in vivo neste modelo com as substâncias de origem natural epigalocatequina-3-galato (EGCG, principal composto do chá-verde), tricostatina A (TSA, produto metabólico da bactéria Streptomyces sp) e resíduo butanólico da Pfaffia paniculata (RBPP) (Ginseng brasileiro). Nas doses utilizadas, a EGCG mostrou efeito estimulatório, não tendo sido testada in vivo. A TSA e o RBPP apresentaram efeitos inibitórios sobre as células de mastocitoma canino in vitro com diminuição da viabilidade celular detectada com o corante vital Azul de Tripan e diminuição do crescimento celular avaliada com o ensaio do MTT. A avaliação morfológica das células pela coloração Laranja de Acridina/Brometo de Etídio mostrou vários debris celulares e células apoptóticas no tratamento com TSA, e alterações morfológicas como vacuolização nas células tratadas com RBPP, incluindo células apoptóticas. A avaliação do ciclo celular avaliada por citometria de fluxo mostrou aumento das células em fase sub-G1 nas células tratadas com TSA, e diminuição nas fases G1 e G2, e aumento nas fases sub-G1 e S no tratamento com RBPP. As substâncias foram então testadas no modelo xenotransplantável de mastocitoma canino. Apesar do marcante efeito inibitório da TSA nos ensaios in vitro, o mesmo não aconteceu in vivo com as doses investigadas, não demonstrando diferença em relação ao controle. Já o RBPP mostrou efeito inibitório com a dosagem de 1,5 mg/animal com tratamento intratumoral no modelo in vivo. Estes dados mostram que o modelo estabelecido é estável e viável e a importância da complementação com os ensaios in vivo para a confirmação dos efeitos observados in vitro. / The mast cell tumor is one of the most common neoplasms that involve the skin of the dog, and new treatment modalities have been searched for its control. This paper describes the establishment and characterization of a xenotransplantable canine mast cell tumor model for testing substances with anticancer potential in vitro and in vivo. The tumor sample was original from a donor animal that showed a grade 3 mast cell tumor. The canine mast cell tumor was successfully established in athymic mice BALB/c nu/nu. The tumor has same histological characteristics retained during their passages in culture. On average, 7 days to 3 weeks are needed for the appearance of a palpable mass and about 60 days for tumor volume reaching more than 500 mm3. Cell cultivation of canine mast cell tumor is maintained for approximately one month, sometimes more, but the cultivation time is limited. There is progressive loss of the initial features of culture such as loss of granulation, increased adhesion and decreased cell suspensions. The following tests were performed in vitro and in vivo in this model, by using the naturally occurring substance epigallocatechin-3-gallate (EGCG, the major compound of green tea), trichostatin A (TSA, metabolic product of the fungus Streptomyces sp) and butanolic residue of the Pfaffia paniculata (RBPP) (Brazilian ginseng). At the used doses, EGCG showed stimulatory effect in vitro, but has not been tested in vivo. The TSA and RBPP showed inhibitory effects on the canine mastocytoma cells in vitro with a decrease in cell viability detected with the vital dye Trypan blue and a decrease in cell growth assessed by the MTT assay. The morphological evaluation of cells stained by Acridine Orange/Ethidium Bromide showed several cellular debris and apoptotic cells in the treatment with TSA, and morphological changes such as vacuolization in cells treated with RBPP, including apoptotic cells. The evaluation of the cell cycle measured by flow cytometry showed an increase of cells in sub-G1 phase in cells treated with TSA, and a decrease in both G1 and G2 phases and increased sub-G1 and S in the treatment RBPP. The substances were then tested in the xenotransplantable model of canine mast cell tumor. Despite the remarkable inhibitory effect of TSA in vitro, it did not happen in vivo with the doses studied, showing no difference compared to control. RBPP already had an inhibitory effect with the dosage of 1.5 mg/animal treatment with intratumoral in vivo model. These data show that the established model of murine xenotransplantable mastocytoma is stable and viable and has importance as a complement in vivo of the tests with antineoplastic drugs performed in vitro.

Tout épithélium glandulaire sécrétoire est constitué d'une unité structurale et fonctionnelle commune, l'acinus. C'est une architecture sphérique pluricellulaire parfaitement différentiée et polarisée qui, reconstruite en culture 3D, mime l'organisation réelle du tissu. Etudier les déterminants environnementaux et génétiques qui gouvernent la transformation d'un acinus en sphéroïde s'apparentant à une tumeur est l'un des enjeux majeurs des modèles in vitro. Un des défis actuels est d'adapter ces modèles in vitro à des conditions de culture 3D qui soient compatibles avec la réalisation de cribles génétiques en 3D, basés par exemple sur l'ARN interférence (RNAi). Cependant, les formats standards de culture 3D et les méthodes analytiques ne sont pas compatibles aux cribles haut-débit. Ils ne permettent pas de contrôler la taille et la distribution des acini, sont dépendants d'immuno-marquages et les acquisitions sont longues. Par ailleurs, la microscopie confocale et vidéomicroscopie offrent un champ d'observation restreint qui ne permet pas d'observer un grand nombre de structures 3D en même temps, pour permettre une analyse statistique. Ainsi, dans le but i) de développer des modèles cellulaires appropriés en 3D, ii) d'adresser des questions fondamentales relatives au cancer de la prostate et iii) de réaliser des cribles RNAi dans un contexte plus pertinent que la culture 2D, j'ai développé des outils innovants au format microsystèmes adaptés à l'analyse haut-débit d'un grand nombre d'objets 3D. En optimisant les conditions de culture cellulaire 3D sur le modèle de la lignée cellulaire RWPE1, j'ai pu récapituler les étapes de formation des acini prostatiques et montrer que la formation du lumen est indépendante de la polarité et est gouvernée par deux mécanismes, « hollowing » et cavitation. / In all secretory epithelia from glandular tissues, there is a common structural and functional unit, the acinus. It is a well polarized and organized pluricellular structure that is spontaneously reconstructed in 3D culture, therefore closely mimics the real structure we find in vivo. For my purpose, acini are used as models for tumor initiation and cancer development. One of the objectives of Biomics laboratory is to identify the genetic and microenvironmental determinants of prostate acini morphogenesis and polarity. The strategy is based on High-Throughput (HT) RNA interference (RNAi)-based screening. To meet this objective, my project was to develop appropriate 3D cell models which closely mimic the cyst-like and duct-like structure of prostate. By optimizing conventional 3D culture in Matrigel, I could recapitulate prostate acini morphogenesis and showed that lumen formation is independent to the polarity, which appears later. However, the conventional 3D cell culture formats and analytical tools are not suited for HT Screening (HTS). They lack control over acini size, are label-dependant and therefore time-consuming and labor intensive. Also, classical microscopy offers a very limited field of view and hence does not allow observing a large amount of 3D structures for statistical analysis.

Ce travail de thèse se situe à l’interface de deux domaines : la culture cellulaire en trois dimensions et l’imagerie sans lentille.Fournissant un protocole de culture cellulaire plus réaliste sur le plan physiologique, le passage des cultures monocouches (2D) à des cultures tridimensionnelles (3D) - via l’utilisation de gels extracellulaires dans lesquels les cellules peuvent se développer dans les trois dimensions - permet de faire de grandes avancées dans de nombreux domaines en biologie tels que l’organogénèse, l’oncologie et la médecine régénérative. Ces nouveaux objets à étudier crée un besoin en matière d’imagerie 3D.De son côté, l’imagerie sans lentille 2D fournit un moyen robuste, peu cher, sans marquage et non toxique, d’étudier les cultures cellulaires en deux dimensions sur de grandes échelles et sur de longues périodes. Ce type de microscopie enregistre l’image des interférences produites par l’échantillon biologique traversé par une lumière cohérente. Connaissant la physique de la propagation de la lumière, ces hologrammes sont rétro-propagés numériquement pour reconstruire l’objet recherché. L’algorithme de reconstruction remplace les lentilles absentes dans le rôle de la formation de l’image.Le but de cette thèse est de montrer la possibilité d’adapter cette technologie sans lentille à l’imagerie des cultures cellulaires en 3D. De nouveaux prototypes de microscopes sans lentille sont conçus en parallèle du développement d’algorithmes de reconstructions tomographiques dédiés.Concernant les prototypes, plusieurs solutions sont testées pour converger vers un schéma alliant deux conditions. La première est le choix de la simplicité d’utilisation avec une culture cellulaire en boîte de Petri standard et ne nécessitant aucune préparation spécifique ou aucun changement de contenant. Cette condition entraînant de fortes contraintes géométriques sur l’architecture, la deuxième est de trouver la meilleure couverture angulaire possible des angles d’éclairage. Enfin, une version adaptée aux conditions en incubateur est développée et testée avec succès.Concernant les algorithmes, quatre types de solutions sont proposés, basées sur le théorème de diffraction de Fourier classiquement utilisé en tomographie diffractive optique. Toutes cherchent à corriger deux problèmes inhérents au microscope sans lentille : l’absence de l’information de phase, le capteur n’étant sensible qu’à l’intensité de l’onde reçue, et la couverture angulaire limitée. Le premier algorithme se limite à remplacer la phase inconnue par celle d’une onde incidente plane. Rapide, cette méthode est néanmoins source de nombreux artefacts. La deuxième solution, en approximant l’objet 3D inconnu par un plan moyen, utilise les outils de la microscopie sans lentille 2D pour retrouver cette phase manquante via une approche inverse. La troisième solution consiste à implémenter une approche inverse régularisée sur l’objet 3D à reconstruire. C’est la méthode la plus efficace pour compenser les deux problèmes mentionnés, mais elle est très lente. La quatrième et dernière solution est basée sur un algorithme de type Gerchberg-Saxton modifié avec une étape de régularisation sur l’objet.Toutes ces méthodes sont comparées et testées avec succès sur des simulations numériques et des données expérimentales. Des comparaisons avec des acquisitions au microscope classique montrent la validité des reconstructions en matière de tailles et de formes des objets reconstruits ainsi que la précision de leur positionnement tridimensionnel. Elles permettent de reconstruire des volumes de plusieurs dizaines de millimètres cubes de cultures cellulaires 3D, inaccessibles en microscopie standard.Par ailleurs, les données spatio-temporelles obtenues avec succès en incubateur montrent aussi la pertinence de ce type d’imagerie en mettant en évidence des interactions dynamiques sur de grandes échelles des cellules entres elles ainsi qu’avec leur environnement tridimensionnel. / This PhD work is at the interface of two fields: 3D cell culture and lens-free imaging.Providing a more realistic cell culture protocol on the physiological level, switching from single-layer (2D) cultures to three-dimensional (3D) cultures - via the use of extracellular gel in which cells can grow in three dimensions - is at the origin of several breakthroughs in several fields such as developmental biology, oncology and regenerative medicine. The study of these new 3D structures creates a need in terms of 3D imaging.On another side, 2D lens-free imaging provides a robust, inexpensive, non-labeling and non-toxic tool to study cell cultures in two dimensions over large scales and over long periods of time. This type of microscopy records the interferences produced by a coherent light scattered by the biological sample. Knowing the physics of the light propagation, these holograms are retro-propagated numerically to reconstruct the unknown object. The reconstruction algorithm replaces the absent lenses in the role of image formation.The aim of this PhD is to show the possibility of adapting this lens-free technology for imaging 3D cell culture. New lens-free microscopes are designed and built along with the development of dedicated tomographic reconstruction algorithms.Concerning the prototypes, several solutions are tested to finally converge to a scheme combining two conditions. The first requirement is the choice of simplicity of use with a cell culture in standard Petri dish and requiring no specific preparation or change of container. The second condition is to find the best possible angular coverage of lighting angles in regards of the geometric constraint imposed by the first requirement. Finally, an incubator-proof version is successfully built and tested.Regarding the algorithms, four major types of solutions are implemented, all based on the Fourier diffraction theorem, conventionally used in optical diffractive tomography. All methods aim to correct two inherent problems of a lens-free microscope: the absence of phase information, the sensor being sensitive only to the intensity of the incident wave, and the limited angular coverage. The first algorithm simply replaces the unknown phase with that of an incident plane wave. However, this method is fast but it is the source of many artifacts. The second solution tries to estimate the missing phase by approximating the unknown object by an average plane and uses the tools of the 2D lens-free microscopy to recover the missing phase in an inverse problem approach. The third solution consists in implementing a regularized inverse problem approach on the 3D object to reconstruct. This is the most effective method to deal with the two problems mentioned above but it is very slow. The fourth and last solution is based on a modified Gerchberg-Saxton algorithm with a regularization step on the object.All these methods are compared and tested successfully on numerical simulations and experimental data. Comparisons with conventional microscope acquisitions show the validity of the reconstructions in terms of shape and positioning of the retrieved objects as well as the accuracy of their three-dimensional positioning. Biological samples are reconstructed with volumes of several tens of cubic millimeters, inaccessible in standard microscopy.Moreover, 3D time-lapse data successfully obtained in incubators show the relevance of this type of imaging by highlighting large-scale interactions between cells or between cells and their three-dimensional environment.

Orientador: Marco Antonio Bottino / Banca: Laís Regiane da Silva Concilio / Banca: Alexandre Luiz Souto Borges / Resumo: O objetivo deste estudo foi avaliar a topografia e a formação de biofilme na superfície de cerâmicas feldspáticas obtidas através de duas técnicas de confecção e dois tratamentos de superfície, assim como avaliar a viabilidade do crescimento de fibroblastos gengivais humanos (FMM-1) sobre estes materiais. Foram confeccionados 52 blocos de cada tipo de cerâmica feldspática: VM9 obtida através da técnica da estratificação e cerâmica Vita Blocs Mark II (VMII) para o sistema CAD/CAM (ambas, Vita Zahnfabrik). As superfícies dos blocos foram padronizadas em politriz nas dimensões de 4,5 x 4,5 x 1,5 mm e os blocos foram divididos em dois tratamentos de finalização de superfície: polimento com borrachas Ceramisté + pasta de polimento e aplicação de glaze spray + sinterização. Os parâmetros de rugosidade Ra e Rsm foram mensurados através de um rugosímetro de contato. As amostras foram esterilizadas e, em seguida contaminadas (n=10) para formação de biofilme heterotípico inicial de S. mutans, S. sanguinis e C. albicans, cuja aderência foi quantificada por contagem de unidades formadoras de colônias (UFC/mL). O teste MTT foi empregado para avaliação da viabilidade celular dos materiais ao crescimento de fibroblastos gengivais humanos (FMM-1) em 24 h e 7 dias (n=12). Foram realizadas análises qualitativas da superfície dos espécimes através de microscopia eletrônica de varredura (MEV) e perfilometria óptica 3D. A energia livre de superfície (ELS) foi calculada a partir de análises de gon... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract:The Objective of this study was to evaluate the topography and surface biofilm formation of feldspathic ceramics obtained through two techniques of preparation and two surface treatments, as well as to evaluate the viability of human gingival fibroblasts (FMM-1) growth on these materials. A total of 52 blocks of each type of feldspathic ceramic were made: VM9 obtained by the stratification technique and Vita Blocs Mark II (VMII) for the CAD/CAM system (both, Vita Zahnfabrik). The blocks' surfaces were standardized in a polishing machine to the dimensions of 4.5 x 4.5 x 1.5 mm and blocks were divided into two surface finishing treatments: polishing with Ceramisté rubbers + polishing with paste; and glaze application + sintering. The Ra and RSm roughness parameters were measured through a contact rugosimeter. Samples were sterilized and then contaminated (n = 10) for initial heterotypic biofilm formation of S. mutans, S. sanguinis and C. albicans, whose adherence was quantified by counting colony forming units (CFU/mL). The MTT test was used to evaluate the cellular viability of the materials to the growth of human gingival fibroblasts (FMM-1) in 24 h and 7 days (n=12). Qualitative analyzes of the specimens' surface were performed using a scanning electron microscopy (SEM) and 3D optical profilometry. Surface free energy (SFE) was calculated from goniometry analyzes of polar and apolar liquids in 10 samples of 15 x 15 x 1.5 mm. The results of Ra, RSm and ELS were subjected to 2-way ANOVA (Material x surface treatment) followed by Tukey's test (both, α=95%), and UFC (material x surface treatment x microorganisms) and MTT (material x surfacetreatment x time) data were evaluated by 3-way ANOVA and Tukey's test (α=95%). SEM and profilometry images were described. The polished ceramics presented lower roughness (Ra p=0.015; RSm p=0.049) ... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

La niche des cellules souches hématopoïétiques représente un compartiment complexe et radiosensible. Sa protection est nécessaire pour la restauration de l'hématopoïèse faisant suite à la myélosuppression due à l'exposition aux rayonnements ionisants. Nous avons dans un premier temps étudié l'effet des RI sur les progéniteurs endothéliaux et mésenchymateux de la niche par une étude de radiosensiblilité et une étude d'évaluation de la mort cellulaire. Nous avons proposé par la suite une stratégie innovante de thérapie génique basée sur la sécrétion locale et à court terme du morphogène Sonic hedgehog visant à favoriser la réparation de niche vasculaire et de stimuler les cellules souches hématopoïétiques et les cellules progénitrices résiduelles. Nous avons étudié la réponse hématopoïétique des singes irradiés à 8-Gy gamma après une seule injection intra-osseuse de cellules souches mésenchymateuses xénogéniques, multipotentes et d'origine adipocytaire transfectées avec un plasmide pIRES2-eGFP codant la protéine Shh. La durée de thrombocytopénie et celle de neutropénie ont été significativement réduites chez les animaux greffés et les clonogènes sont normalisés à partir du 42e jour. Les aires sous la courbe des numération des plaquettes et des neutrophiles entre 0 et 30 jours ont été significativement plus élevée chez les animaux traités que chez les témoins. La greffe d'explants de MatrigelTM colonisés ou non avec des ASC chez des souris immunodéprimées a démontré une activité pro-angiogénique notable des ASC transfectées avec le plasmide Shh . Le suivi à long terme (180 à 300 jours) a confirmé une reconstitution durable dans les quatre singes greffés. Globalement cette étude suggère que la greffe de cellules souches multipotentes Shh-peut représenter une nouvelle stratégie pour la prise en charge des dommages radio-induits de la niche. / The hematopoietic stem cell niche represents a complex radiosensitive compartment whose protection is required for recovery from radiation-induced myelosuppression. We initially studied RI effects on endothelial and mesenchymal progenitors by an evaluating radiosensitivity and cell death. Then, we have proposed a new gene therapy strategy based on local and short term secretion of Sonic hedgehog morphogene to favour vascular niche repair and to stimulate residual hematopoietic stem and progenitor cells. We investigated the hematopoietic response of 8-Gy gamma irradiated monkeys to a single intra-osseous injection of xenogeneic multipotent mesenchymal stem cells transduced with a Shh pIRES2 plasmid. Thrombocytopenia and neutropenia duration were significantly reduced in grafted animals and clonogenics normalized from day 42. Areas under the curve of PLTs and ANCs between day 0 and day 30 were significantly higher in treated animals than in controls. Grafting MatrigelTM colonized or not with ASC in immunocompromized mice demonstrated a notable pro-angiogenic activity for Shh-ASC. Long term follow up (180-300 days) confirmed a durable recovery in the four grafted monkeys. Globally this study suggests that grafting Shh-multipotent stem cells may represent a new strategy to cure radiation-induced niche damage.

Tissue engineering strategies that combine porous biomaterial scaffolds with cells capable of osteogenesis or bioactive proteins have shown promise as effective bone graft substitutes. Attempts to culture bone tissue-engineering constructs thicker than 1mm in vitro often result in a shell of viable cells and mineralized matrix surrounding a necrotic core. To address this limitation, we developed a perfusion bioreactor system that improves mass transport throughout large cell-seeded constructs. Additionally, we established and validated 3-D computational methods to model flow and shear stresses within the microporosity of perfused constructs. Micro-CT scanning and analysis techniques were used to non-destructively monitor mineral development over time in culture. CFD modeling of axial perfusion through cylindrical scaffolds with a regular microarchitecture revealed a uniform flow field distributed throughout the scaffold. Perfusion resulted in a 140-fold increase in mineral deposition at the interior of 3 mm thick polymer scaffolds seeded with rat bone marrow stromal cells. The total detected mineral volume tripled as the construct length was increased from 3 to 9 mm. Increasing scaffold length to 9 mm did not affect the mineral volume fraction (MVF) within the full volume of each construct. Mineral volume, spatial distribution, density, particle size and particle number were then quantified on cell-seeded constructs in 5 different culture environments. The effect of time varying flow conditions was compared with continuous perfusion as well as two different control cell culture methods in an attempt to enhance mineralized matrix within the constructs. Intermittent elevated perfusion and dynamic culture in an orbital rocker plate produced the greatest amount of mineral within 9 mm long constructs compared to low continuous flow and high continuous flow cases. Together, these studies indicate that dynamic culture conditions enhance construct development with regards to cell viability, mineralized matrix deposition, growth rate, and distribution. Furthermore, these techniques provide a rational approach to selecting perfusion culture conditions that optimize the amount and distribution of mineralized matrix production. Finally, the established perfusion bioreactor, in combination with micro-CT analysis, provides a foundation for evaluating new scaffolds and cell types that may be useful for the development of effective bone graft substitutes.

Traumatic brain injury (TBI) results from a physical insult to the head and often results in temporary or permanent brain dysfunction. However, the cellular pathology remains poorly understood and there are currently no clinically effective treatments. The overall goal of this work was to develop and characterize a novel three-dimensional (3-D) in vitro paradigm of neural trauma integrating a robust 3-D neural co-culture system and a well-defined biomechanical input representative of clinical TBI. Specifically, a novel 3-D neuronal-astrocytic co-culture system was characterized, establishing parameters resulting in the growth and vitality of mature 3-D networks, potentially providing enhanced physiological relevance and providing an experimental platform for the mechanistic study of neurobiological phenomena. Furthermore, an electromechanical device was developed that is capable of subjecting 3-D cell-containing matrices to a defined mechanical insult, with a predicted strain manifestation at the cellular level. Following independent development and validation, these novel 3-D neural cell and mechanical trauma paradigms were used in combination to develop a mechanically-induced model of neural degeneration and reactive astrogliosis. This in vitro surrogate model of neural degeneration and reactive astrogliosis was then exploited to assess factors influencing neural stem cell (NSC) survival and integration upon delivery to this environment, revealing that specific factors in an injured environment were detrimental to NSC survival. This work has developed enabling technologies for the in vitro study of neurobiological phenomena and responses to injury, and may aid in elucidating the complex biochemical cascades that occur after a traumatic insult. Furthermore, the novel paradigm developed here may provide a powerful experimental framework for improving treatment strategies following neural trauma, and therefore serve as a valid pre-animal test-bed.