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京都大学 / 新制・課程博士 / 博士(医学) / 甲第24502号 / 医博第4944号 / 新制||医||1064(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 朝長 啓造, 教授 伊達 洋至 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Submitted by FERNANDA HERRERA DA COSTA (ferherreracosta@gmail.com) on 2018-05-24T14:02:59Z No. of bitstreams: 1 dissertação final.pdf: 14796170 bytes, checksum: b56cc2f9884a2482b189d839e1ebdcca (MD5) / Approved for entry into archive by Silvana Alvarez null (silvana@ict.unesp.br) on 2018-06-06T19:33:04Z (GMT) No. of bitstreams: 1 godoi_fhc_me_sjc.pdf: 14796170 bytes, checksum: b56cc2f9884a2482b189d839e1ebdcca (MD5) / Made available in DSpace on 2018-06-06T19:33:04Z (GMT). No. of bitstreams: 1 godoi_fhc_me_sjc.pdf: 14796170 bytes, checksum: b56cc2f9884a2482b189d839e1ebdcca (MD5) Previous issue date: 2018-03-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A sobrevida de pacientes submetidos a tratamentos para câncer de cabeça e pescoço está aumentando e a busca por reabilitação é necessária para promover qualidade de vida. O objetivo deste estudo é avaliar o perfil da expressão gênica de transcritos relacionados na osteogênese e osteoclastogênese em uma cultura primária de células mesenquimais de fêmures de ratos submetidos a radioterapia e colocação de implantes de titânio. Setenta e dois ratos receberam implantes de titânio em ambos fêmures. Os animais foram divididos em quatro grupos: 1) Grupo controle (C): cirurgia de colocação dos implantes; 2) Implante + irradiação imediata (IrI): irurgia de implantes e seguido de irradiação imediata; 3) Implante + irradiação tardia (IrT): cirurgia de implantes e irradiação após 4 semanas; 4) Irradiação prévia + implantes (IrP): irradiação e após 4 semanas cirurgia de implantes. A dose de irradiação foi de 30 Gy fracionadas em duas sessões. As eutanásias nos períodos de 3, 14 e 49 dias após o tratamento. A células cultivadas sofreram diferenciação em osteoblastos. A expressão gênica dos genes Fosfatase alcalina (Alp), Colágeno 1 (Col-1), Integrina !1 (Itg ! ), Osteocalcina (Bglap), Osteopontina (Osp), Osteonectina (Osn), Sialoproteína Óssea (Bsp), Fator de crescimento transformador (Tgf- ! ), Osterix (Osx),Fator relacionado ao Runt (Runx2), Fator estimulador de colônias de macrófagos (M-csf), Interleucina -6 (Il-6) Apolipoproteína E (Apo-E), Prostaglandina E2 (PgE2), foram avaliados por qRT-PCR e os resultados validados por ELISA. A expressão mais alta de Alp foi encontrada no grupo IrP (p=0.0001) e foi subexpressa nos grupos IrI e IrT (p<0.0001 e p=0.0041 respectivamente). Resultados similares foram encontrados nos transcritos de Itg !, On, Bsp, Osx e Runx2. mRNA do Tgf- ! foi hiperexpresso em todos os grupos principalmente aos 49 dias. Depois de 49 dias, os níveis de proteína da Bglap e Il-6 foram correlacionados com a expressão do mRNA. A radioterapia imediata altera a atividade de diferenciaçãO das células mesenquimais dos fêmures de ratos. / Prognosis of patient submitted to head and neck cancer treatment has increased and the oral rehabilitation becomes necessary to improve their life quality. The aim of this study was to evaluate the gene expression profile of transcripts related to osteogenesis and osteoclastogenesis in primary culture of mesenchymal cells from rat femurs submitted to radiotherapy and installation of pure titanium implant. Seventy two rats received titanium implants in both femurs. The animals were divided in four groups: 1- Control (C) implant surgery; 2- Implant + immediate irradiation after 24 hours (IrI); 3- Implant + late irradiation after 4 weeks (IrL); 4- Implant + Previous irradiation 4 weeks before surgery (IrP) irradiation. The irradiation dose was 30Gy fractioned in two. The animals were euthanized in day 3, 14 and 49 after surgery. The mesenchymal cells from femurs were extracted and cultivated. The differentiation into osteoblastic cells was verified by calcification nodules formation. The gene expression of Alkaline Phosphatase (Alp), Collagen 1 (Col 1), Integrin β1 (ItgB1), Osteocalcin (Bglap), Osteopontin (Osp), Osteonectin (Osn), Bone Sialoprotein (Bsp), Transforming Growth Factor β-type (Tgf-β), Osterix (Osx), Runt-related transcription factor 2 (Runx2), Macrophage Colony- Stimulating Factor (M–csf), Interleukin-6 (Il-6), Apolipoprotein E (ApoE) and Prostaglandin E2 (PgE2) were evaluated by qRT-PCR and the results were validated by ELISA test. Higher mRNA of Alp expression was found in IrP group (p=0.0001) and it was downregulated in IrI and IrT groups (p<0.0001 and p=0.0041, respectively). Similar results were found for transcript levels of ItgB1, Osn, Bsp, Osx and Runx2. mRNA of Tgf-β was overexpressed in all groups mainly in 49 days. After 49 days, Osn and Bsp transcripts were downregulated in the 3 groups evaluated. The Bglap and IL-6 protein level was correlated to their mRNA expression. The radiotherapy alters immediately the differentiation and activity of mesenchymal cells from rat femurs; however these cells seem to recover becoming suitable for receiving implants. / FAPESP: 2016/20103-7

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-09-04T17:11:04Z No. of bitstreams: 1 Ticiana Xavier Ferreira Avaliação do potencial terapeutico do ....pdf: 16365064 bytes, checksum: 9af2a14308bdaa00d28e7b6a6711fc5b (MD5) / Made available in DSpace on 2012-09-04T17:11:04Z (GMT). No. of bitstreams: 1 Ticiana Xavier Ferreira Avaliação do potencial terapeutico do ....pdf: 16365064 bytes, checksum: 9af2a14308bdaa00d28e7b6a6711fc5b (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A doença de Chagas, causada pelo protozoário Trypanosoma cruzi, é uma das mais importantes causas de insuficiência cardíaca na América. A terapia celular vem sendo investigada como uma possível opção terapêutica para pacientes com doenças cardiovasculares. Neste estudo foram avaliados os efeitos da terapia com célulastronco mesenquimais em um modelo experimental de cardiomiopatia chagásica crônica. Camundongos C57BL/6 foram infectados com 1000 tripomastigotas da cepa Colombiana de T. cruzi e, após seis meses de infecção, foram tratados com célulastronco mesenquimais derivadas de tecido adiposo humano (CTTAs) ou com meio DMEM (controle). Antes e após 1 e 2 meses de tratamento, os animais chagásicos e controles normais foram submetidos à avaliação cardíaca, incluindo eletrocardiograma, ecocardiograma e teste ergoespirométrico. O grupo tratado recebeu duas injeções intraperitoneais de CTTAs (1x106 células / dose), com um mês de intervalo entre as duas doses. Todos os animais foram sacrificados sob anestesia após 2 meses de tratamento, para análise histopatológica do coração. Não foi observada melhora de arritmias e da função cardiovascular no grupo tratado com CTTAs, porém secções de corações de camundongos deste grupo apresentaram uma redução significativa do número de células inflamatórias (p< 0,0001 ) e da área de fibrose (p< 0,01) em comparação com animais chagásicos tratados com DMEM. A dosagem de 22 citocinas séricas dois meses após o tratamento mostrou um aumento da maioria destas citocinas em animais chagásicos crônicos quando comparados aos controles não-infectados, sendo algumas destas moduladas após a terapia celular. Deste modo, conclui-se que as CTTAs foram capazes de reduzir inflamação e fibrose no coração de camundongos cronicamente infectados por T. cruzi, porém não teve efeitos na função cardíaca dois meses após o transplante. / Chagas disease, caused by the protozoan Trypanosoma cruzi, is one of the most important causes of heart failure in America. Cell therapy has been investigated as a possible therapeutic option for patients with cardiovascular diseases. This study evaluated the effects of therapy with mesenchymal stem cells in an experimental model of chronic Chagas cardiomyopathy. C57BL/6 mice were infected with 1000 trypomastigotes of the Colombian strain of T. cruzi and, after six months of infection, were treated with mesenchymal stem cells derived from human adipose tissue (ADSCs) or DMEM (control). Before and after 1 and 2 months of treatment, chagasic mice and normal controls underwent cardiac evaluation including electrocardiogram, echocardiography and cardiopulmonary exercise test. The treated group received two intraperitoneal injections of ADSCs (1x106 cells / dose), with one month interval. All animals were sacrificed under anesthesia after two months of treatment for histopathology analysis of the heart. No improvement was observed for arrhythmias and cardiovascular function in mice treated with ADSCs, but sections of hearts in this group had a significantly reduced number of inflammatory cells (p<0.0001) and area of fibrosis (p<0.01), compared to animals treated with DMEM. The measurement of 22 serum cytokines two months after treatment showed an increase of most of these cytokines in chronic chagasic animals when compared to uninfected controls, and some of which modulated after cell therapy. Thus, we conclude that the ADSCs were able to reduce inflammation and fibrosis in the hearts of mice chronically infected with T. cruzi, but had no effect on cardiac function two months after transplantation.

L'attention de la communauté scientifique, ainsi que le développement, pour les bioprocédés dédiés à la culture et à l'expansion de cellules souches mésenchymateuses (MSCs) pour la thérapie cellulaire et la médecine régénérative a considérablement grandi pendant ces dernières décennies. Une plus ample compréhension du lien entre la structure, la fonction et les propriétés des suspensions de cellules mésenchymateuses est devenue de première importance. Dans cette thèse, nous présentons tout d'abord les résultats d'une étude expérimentale portant sur l'écoulement de suspensions concentrées de cellules vivantes d'origine mésenchymateuse pour une grande gamme de concentration cellulaire. Nous caractérisons l'évolution de la viscosité relative en fonction de la contrainte de cisaillement appliquée pour des fractions volumiques cellulaires allant de 20 à 60%. Ces matériaux ont des empreintes rhéologiques compliquées mais très reproductibles, incluant des comportements de fluide à seuil, rhéofluidifiants ainsi que des fractures liées à la contrainte de cisaillement. Les propriétés rhéologiques de la suspension sont ensuite étudiées avec l'addition d'acide hyaluronique (HA), une biomolécule avec des séquences d'adhésion pour des récepteurs à la surface des cellules étudiées. Nous montrons que l'addition d'acide hyaluronique modifie substantiellement le comportement de la suspension et nous permet de contrôler les propriétés d'écoulement de la suspension à toutes les fractions volumiques. Cytométrie de flux et imagerie confocal à l'appui, nous montrons que l'effet observé est dû à un important changement dans la formation d'agrégats cellulaires dans la suspension, et donc dans l'envergure du réseau correspondant. La troisième partie de cette thèse porte sur l'ajout de polyéthylène glycol, une molécule qui n'est pas naturellement présente dans l'organisme mais fréquemment utilisée dans la formulation d'hydrogel. En utilisant trois types de PEG, l'influence de la charge des molécules est étudiée. Les résultats montrent que la charge est un paramètre important dans le contrôle des propriétés d'écoulement de suspensions cellulaires, car déterminant dans la formation et la compacité des agrégats. En considérant les agrégats comme des objets fractals, nous montrons qu'en prenant en compte les modifications de fractions volumiques avec le cisaillement, nous pouvons obtenir une courbe maitresse pour l'ensemble des conditions testées, et en extraire la force d'adhésion moyenne entre les cellules, au travers une population de plusieurs millions de cellules. Cette étude livre de nouveaux aspects sur la complexité des propriétés en écoulement de suspensions de cellules méchymateuses, adhérentes et concentrées, sur leur sensibilité à l'ajout de molécules, qu'elles soient naturellement présentes dans les tissues ou non, ainsi qu'une nouvelle méthode pour mesurer la force d'adhésion entre les cellules. / With the rapidly growing interest in the development of bioprocess systems to culture and expand mesenchymal stromal cells (MSCs) for cell therapy and regenerative medicine applications, greater understanding of the structure-function-property characteristics of mesenchymal cell suspensions is required. In this thesis, the results of a detailed experimental study into the flow behaviour of concentrated suspensions of living mesenchymal cells over a wide range of cell concentrations and in the presence of two macromolecules (hyaluronic acid and polyethylene glycol) often used in cellular therapy applications are presented. The change in the shear viscosity as a function of shear stress and shear rate for cell volume fractions varying from 20 to 60% are firstly presented, showing that these suspensions exhibit highly complex but reproducible rheological footprints, including yield stress, shear thinning and shear-induced fracture behaviours. The rheological properties of the suspension with the addition of hyaluronic acid (HA), a biomolecule with adhesion sequences for receptors on these types of cells, was then investigated. With the addition of HA, the rheology of these cell suspensions is significantly modified at all volume fractions. Using FACS and confocal imaging, we show that the observed effect of HA addition is due to it significantly modulating the formation of cellular aggregates in these suspensions, and thus the resultant volume spanning network. This understanding permits the rheology of concentrated mesenchymal cell suspensions to be tailored to suit particular processing scenarios. The third part of this project focused on the addition of polyethylene glycol, a molecule which is not naturally present in tissues but commonly utilised in hydrogels as injectable delivery vehicles for cells to sites of tissue damage. Using three different kinds of PEG, the influence of the charge of the molecules is investigated. The results show the charge is also a crucial parameter to tailor the flow behaviour of cell suspension when biomacromolecules are added, influencing the formation and the compactness of the cellular aggregates. Considering the aggregates as fractal structures, and by taking into account the changes in volume fractions with shear, a master curve for the range of conditions investigated was successfully achieved through the use of an analytical model. Critically, this model also permitted the estimation of the average adhesion force between cells, across a population of millions of cells. The outcomes of this study not only provide new insight into the complexity of the flow behaviours of concentrated, dynamically adhesive mesenchymal cell suspensions, and their sensitivity to associative biomolecule and synthetic molecule addition, but also a novel, rapid method by which to estimate adhesion forces between cells.