Made available in DSpace on 2015-04-14T13:35:25Z (GMT). No. of bitstreams: 1 437015.pdf: 2405577 bytes, checksum: 64fe8d7f54de71f570c0f7a29d9c6838 (MD5) Previous issue date: 2012-01-16 / Cellular therapy represent a new frontier for the treatment of several diseases, including diseases related to central nervous system. Human umbilical cord blood is an attractive source of stem cells; however, it has a heterogeneous population with a small amount of mesenchymal stem cells. Currently, cell reprogramming induced by different methodologies can confer pluripotency to differentiated adult cells. The objective of the study was to evaluate the reprogramming potential of fibroblasts and neural differentiation after co-culture with umbilical cord blood mononuclear cells. This study was approved by the Ethics Committee of PUCRS (CEP 11/05504). Cells were obtained from four human umbilical cord blood. Cell suspensions were fractionated at gradient of density 1077. The mononuclear cells of each sample were cultured for 7 days and later, the culture was infected with 105 cells of mouse fibroblast cell line NIH 3T3 and co-cultured for 6 days. To evaluate the pluripotency it was performed RNA extraction and later, amplification using primers specific for pluripotency genes. The differentiation was also confirmed by the adipogenic and osteogenic differentiation. Neural differentiation of the reprogrammed cells was obtained by the method described by Song et. al (2008) and evaluated by immunofluorescence. Population growth was quantified by hemocytometer. All co-cultured cells showed adipogenic and osteogenic differentiation capacity. After co-cultivation cells began transcribing the pluripotency gene KLF4. Statistically significant differences in the parameters area, diameter, optical density and fractal dimension were observed by confocal microscopy in reprogrammed and neural differentiated cells. The population of reprogrammed cells doubled every three days until the addition of the last medium of neural differentiation, when cultures became quiescent until the end of the neural differentiation. The contact in form of co-cultivation of fibroblasts with umbilical cord blood mononuclear fraction for six days promoted the reprogramming of these cells to a indifferentiation stage, allowing the induction of neural differentiation later. / As terapias celulares representam uma nova fronteira para o tratamento de diversas doen?as, inclusive patologias relacionadas ao sistema nervoso central. O sangue de cord?o umbilical humano ? uma atrativa fonte de c?lulas-tronco, por?m, apresenta uma popula??o heterog?nea com pouca quantidade de c?lulas-tronco mesenquimais. Atualmente, a reprograma??o celular induzida atrav?s de metodologias distintas, pode conferir pluripot?ncia ?s c?lulas adultas diferenciadas. O objetivo deste trabalho foi avaliar o potencial de reprograma??o de fibroblastos e a diferencia??o neural ap?s co-cultivo com fra??o mononuclear de sangue de cord?o umbilical. Foram obtidas c?lulas da fra??o mononuclear de quatro cord?es umbilicais humanos de pacientes atendidas no Centro Obst?trico do Hospital S?o Lucas da PUCRS. As suspens?es celulares foram fracionadas em gradiente de densidade 1077. A fra??o mononuclear de cada amostra foi cultivada por sete dias e ap?s, contaminada com 105 c?lulas da linhagem de fibroblasto de camundongos NIH 3T3 e co-cultivadas por 6 dias. Para avalia??o da pluripot?ncia, foi realizada extra??o de RNA e posterior amplifica??o utilizando oligonulceot?deos espec?ficos para os genes de pluripot?ncia Oct3/4, Sox2 e KLF4. O fen?tipo mesenquimal foi confirmado pela diferencia??o adipog?nica e osteog?nica. A neurodiferencia??o das c?lulas reprogramadas seguiu o m?todo descrito por Song et. al (2008) e foi avaliada por imunofluoresc?ncia. O crescimento populacional foi quantificado em hemocit?metro. Todas as c?lulas co-cultivadas mostraram capacidade de diferencia??o adipog?nica e osteog?nica. Ap?s o co-cultivo, as c?lulas passaram a transcrever o gene de pluripot?ncia KLF4. Diferen?as estatisticamente significativas nos par?metros ?rea, di?metro, densidade ?ptica e dimens?o fractal foram observadas por microscopia confocal nas c?lulas reprogramadas e neurodiferenciadas. A popula??o de c?lulas reprogramadas duplicou a cada tr?s dias at? o momento da adi??o do ultimo meio de neurodiferencia??o, quando as culturas tornaram-se quiescentes at? o final do per?odo de neurodiferecia??o. O contato em forma de co-cultivo dos fibroblastos com a fra??o mononuclear de cord?o umbilical por seis dias promoveu a reprograma??o dessas c?lulas a um est?gio com certa plasticidade, permitindo a indu??o posterior de neurodiferencia??o.

Made available in DSpace on 2015-04-14T13:35:34Z (GMT). No. of bitstreams: 1 440859.pdf: 1769440 bytes, checksum: 1b3b990746636f5c1f4b9a971b320293 (MD5) Previous issue date: 2012-03-26 / Introduction: As a chronic disease which is highly prevalent, epilepsy has an important impact on the affected person's psychic/cognitive functioning and their family and social relationships. Among refractory epilepsies, epilepsy of the temporal lobe (ETL) is the most frequent in adults. Studies about lesions on the hippocampus and their effects found that patients with ETL presented verbal and visual memory deficits, depending on the affected hemisphere. Refractoriness to clinical treatment leads to the recommendation of surgery when it is seen that up to that point there have not been any other therapeutic alternatives available. In this context, there is a glimpse of a new treatment possibility for refractory epilepsies, using stem cells. These present a large capacity for proliferation and auto-renovation and the capacity to respond to external stimuli and give rise to different, more specialized cell lines. Objectives: To evaluate the effect of autologous stem cell transplants of bone marrow on the memory functions of patients with refractory epilepsy of the mesial temporal lobe. Patients and Methods: The selection of patients was done at the Outpatient Center for Clinical Epilepsy Research at PUCRS S?o Lucas Hospital [Ambulat?rio de Pesquisa Cl?nica em Epilepsia do Hospital S?o Lucas da PUCRS]. The 13 patients submitted themselves to therapy, and they were evaluated before and after the stem cell treatment (3 and 6 months after). The tests utilized for neuropsychological evaluation (pre and post-therapy) include the Wechsler Memory Scale revised (WMS-R), the Rey Auditory Verbal Learning Test (RAVLT) and the Taylor Complex Figure. Results and Conclusions: The results indicate a significant increase in the neuropsychological scores over time. In six out of seven utilized tests, the increase of average score obtained was significant. In only one test (visual reproduction I WMS R), the increase showed limitrofe statistical significance (between 5% and 10%). Given the favorable evolution of those patients analyzed, the stem cell transplant showed itself to be feasible, safe and with important perspectives for rehabilitation as well as psychosocial and professional reinsertion. / Introdu??o: A epilepsia, enquanto condi??o cr?nica de alta preval?ncia tem impacto importante no funcionamento ps?quico/cognitivo e nas rela??es familiares e sociais da pessoa acometida. Entre as epilepsias refrat?rias, a epilepsia do lobo temporal (ELT) ? a mais freq?ente em adultos. Estudos sobre les?es no hipocampo e seus efeitos, constataram que pacientes com ELT apresentam d?ficits de mem?ria verbal e visual, dependendo do hemisf?rio acometido. A refratariedade ao tratamento cl?nico, leva ? indica??o cir?rgica, visto que at? o momento, n?o est?o dispon?veis outras alternativas terap?uticas. Neste contexto, se vislumbra uma nova possibilidade de tratamento para as epilepsias refrat?rias, utilizando-se c?lulas-tronco (CT). Estas apresentam grande capacidade de prolifera??o e auto-renova??o e capacidade de responder a est?mulos externos dando origem a diferentes linhagens celulares mais especializadas. Objetivos: Avaliar o efeito do transplante aut?logo de c?lulas-tronco da medula ?ssea (CTMO) nas fun??es de mem?ria de pacientes com epilepsia refrat?ria do lobo temporal mesial. Pacientes e M?todos: A sele??o dos pacientes foi realizada no Ambulat?rio de Pesquisa Cl?nica em Epilepsia do Hospital S?o Lucas da PUCRS. Os 13 pacientes que se submeteram ? terapia, foram avaliados antes e ap?s o tratamento com c?lulas-tronco (3 meses e 6 meses depois). Os testes utilizados para avalia??o neuropsicol?gica: Escala de Mem?ria Wechsler-revisada (WMS-R), Teste de Aprendizado de Mem?ria Verbal de Rey (RAVLT) e Figura Complexa de Taylor. Resultados e Conclus?es: Os resultados indicam um aumento significativo nos escores neuropsicol?gicos ao longo do tempo. Em seis dos sete testes utilizados, o acr?scimo no escore m?dio obtido foi significativo e, em apenas um teste (WMS R visual imediato), o acr?scimo apresentou signific?ncia estat?stica lim?trofe (entre 5% e 10%). Dada a evolu??o favor?vel dos pacientes analisados, o transplante de CTMO mostrou-se fact?vel, seguro e com perspectivas importantes para a reabilita??o e reinser??o psicossocial e profissional.

Made available in DSpace on 2015-04-14T13:35:39Z (GMT). No. of bitstreams: 1 447914.pdf: 1273209 bytes, checksum: bc6f4a88f17be9566f98a33586d6f9a5 (MD5) Previous issue date: 2013-03-22 / Epilepsy affects 1% of the world population and 30% of these patients are refractory to available medication. Stem cells host hope in the treatment of epilepsy. Given their ability to proliferate, differentiate and production of factors which may activate endogenous mechanisms to restore the injured brain. Knowing that the administration of bone marrow mononuclear cells (BMMC) in animals have therapeutic potential in an experimental model of epilepsy, the aim of this study is to investigate the mechanisms by which administered cells exert their beneficial. In order to better understand the mechanisms of action of transplanted cells, a comparative study was done to detect the expression of trophic factors as brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), transforming growth factor beta (TGF-R) and vascular endothelial growth factor (VEGF) in hippocampi of each experimental groups by ELISA. Experimental model of epilepsy was induced by pilocarpine injection (320 mg/kg; ip). Seizures were scored by Racine s scale. The duration of SE was controlled with diazepan (10mg/kg; ip; 90 minutes after SE onset). Twenty-two days after SE, rats were randomly assigned into groups: Control, Pilo, Pilo+BMMC evaluated in periods 3, 7 and 14 days after transplant. BMMC groups received cell transplantation (obtained from EGFP C57BL/6 mice) via tail vein (1x107 cells, 100L). While control animals received saline instead of pilocarpine. Pilocarpine-treated animals were monitored for the presence of spontaneous seizures for 22 days (7 days prior to cell transplant). Our results showed that there was a change in the protein expression of BDNF, GDNF, NGF, TGF-R and VEGF in the hippocampus of epileptic animals treated with BMMC compared to untreated epileptic animals and control, with variations in the expression of each factor at different times after transplantation. The expression of BDNF, GDNF, NGF and VEGF was high and TGF-R1 reduced after transplantation of BMMC compared to untreated epileptic animals. However, there was no difference in the expression of these factors in untreated epileptic animals compared to control animals, except TGF-R1, which proved to be high in the group of untreated epileptic animals compared to control animals. The results of this study provide additional data on the potential benefit of BMMC as well as provide insight into the mechanism by which BMMC promote functional recovery in epileptic rats. / A epilepsia atinge cerca de 1% da popula??o mundial, sendo que aproximadamente 30% desses pacientes n?o respondem ao tratamento medicamentoso. Por sua vez, as c?lulas-tronco t?m sido consideradas uma esperan?a de tratamento da epilepsia, visto que t?m grande capacidade de prolifera??o, diferencia??o e produ??o de fatores, podendo ativar mecanismos de restaura??o end?gena no c?rebro lesado. Sabendo-se que a administra??o de c?lulas mononucleares da medula ?ssea (CMMO) apresenta potencial terap?utico em um modelo experimental de epilepsia, o objetivo deste estudo ? investigar se o transplante das CMMO em ratos com epilepsia cr?nica modula a express?o de fatores tr?ficos. Com o intuito de melhor compreender os mecanismos de a??o das c?lulas transplantas foi realizada a detec??o da express?o de fatores tr?ficos como o fator neurotr?fico derivado do c?rebro (BDNF), fator neurotr?fico derivado da glia (GDNF), fator de crescimento neural (NGF), fator de crescimento transformador R1 (TGF-R1) e fator de crescimento endotelial vascular (VEGF) em diferentes per?odos ap?s transplante das CMMO nos hipocampos dos grupos experimentais atrav?s da t?cnica de ELISA. A pilocarpina (PILO) foi administrada nos animais (320 mg/kg i.p.,) para indu??o do modelo de epilepsia cr?nica. As crises comportamentais foram classificadas de acordo com a escala de Racine e a dura??o do SE foi controlada com diazepam (10 mg/kg, i.p., 90 minutos). Ap?s 22 dias, o total dos animais foi dividido em grupos: Controle, Pilo e Pilo+CMMO avaliados nos tempos de 3, 7 e 14 dias ap?s o transplante. Os grupos CMMO receberam transplante de c?lulas da camada mononuclear da medula ?ssea, obtidas de camundongos EGFP C57BL/6, via veia caudal (1x107 c?lulas, 100L). Os animais controle receberam solu??o salina nas mesmas condi??es do grupo transplantado. Os animais tratados com pilocarpina foram v?deo-monitorados durante sete dias pr?-transplante para observa??o de crises espont?neas recorrentes (CERs). Nossos resultados mostraram que houve altera??o da express?o proteica de BDNF, GDNF, NGF, TGF-R1 e VEGF nos hipocampos dos animais epil?pticos tratados com as CMMO em rela??o aos animais epil?pticos n?o tratados e controle, havendo varia??es da express?o de cada fator em diferentes tempos ap?s o transplante. A express?o dos fatores BDNF, GDNF, NGF e VEGF mostrou-se elevada e do TGF-R1 reduzida ap?s o transplante das CMMO em rela??o aos animais epil?pticos n?o tratados. Por?m, n?o houve diferen?a na express?o destes fatores nos animais epil?pticos n?o tratados em rela??o aos animais controle, exceto o TGF-R1, o qual se mostrou elevado no grupo de animais epil?pticos n?o tratados em rela??o aos animais controle. Os resultados deste estudo fornecem dados adicionais sobre o benef?cio do potencial das CMMO, bem como fornecer uma vis?o sobre o mecanismo pelo qual as CMMO favorecem a recupera??o funcional em ratos epil?pticos.

Made available in DSpace on 2015-04-14T13:35:45Z (GMT). No. of bitstreams: 1 450246.pdf: 1514408 bytes, checksum: 6ceddb0f6677e9485ec44236c23b7b0b (MD5) Previous issue date: 2013-03-25 / The spinal cord injury (SCI) is a condition that dramatically affects the quality of life of affected patients, has a high incidence and causes a high cost to the government and society. Existing treatments for SCI are only palliative nature, not being able to reverse the neurological damage caused by trauma. As a result, it is necessary to investigate new therapies that seek more effective solutions for these cases. The studies with bone marrow mononuclear cells (BMMC) have shown encouraging results, but still not definitive for clinical application, so the expansion of preclinical studies is essential. In this study we aimed to compare treatment outcomes between groups with 3 and 5 applications BMMC applications, analyzing motor function, and inflammation at the lesion site after treatment with BMMC by subarachnoid through via lumbar puncture. The experimental groups were divided into two groups, with different times of transplantation. A group of 3 applications of BMMC, the first application 48 hours, 9 days and 16 days after SCI and a second group with 5 BMMC applications, application in the first 48 hours, 9, 16, 23 and 30 days after by spinal cord injury. The BMMC of male wistar rats was applied via subarachnoid. The entire group received vehicle (saline). The animals were evaluated for motor function through the BBB scale, for the presence of BMMC in the lesion by PCR for the presence of Y chromossome of the cells, and the inflammatory process at the site of injury, analyzing IL-1β and TNF-α, by immunohistochemistry. Our results show an improvement in motor function in the groups treated with BMMC transplants. The immunohistochemical evaluation revealed no difference in the expression of inflammatory cytokines Il-1β and TNF-α at the site of injury in the groups treated with BMMC. The PCR analysis for the presence of chromossome of the cells was negative at the 37th day of the last application of BMMCs / O trauma-raquimedular (TRM) ? uma patologia que afeta drasticamente a qualidade de vida dos pacientes acometidos, apresenta alta incid?ncia e ocasiona um alto custo para o governo e a sociedade. Os tratamentos existentes para o TRM s?o apenas de cunho paliativo, n?o sendo capazes de reverter o dano neurol?gico ocasionado pelo trauma. Em fun??o disso, ? necess?rio investigar novas terapias que busquem solu??es mais efetivas para esses pacientes. Os estudos com c?lulas-tronco de medula ?ssea (CMMO) t?m demonstrado resultados animadores, mas ainda n?o definitivos para aplica??o cl?nica; assim, a amplia??o dos estudos pr?-cl?nicos ? indispens?vel. Neste estudo tivemos o objetivo de comparar os resultados do tratamento de CMMO pela via subaracnoidea (VS) atrav?s de pun??o lombar (PL), entre grupos com 3 aplica??es e 5 aplica??es de CMMO, analisando a fun??o motora e o processo inflamat?rio no local da les?o. Nossos grupos experimentais de estudo foram divididos em 2 grupos pela via de administra??o subaracn?idea, com tempos de transplante diferentes. Um grupo com 3 aplica?oes de CMMO, sendo a primeira aplica??o em 48h, 9 dias e 16 dias ap?s a les?o medular (LM) e um segundo grupo com 5 aplica??es de CMMO, primeira aplica??o em 48 h, 9, 16, 23 e 30 dias ap?s a Les?o Medular (LM) pela VS e cada grupo com seu controle de ve?culo, solu??o salina (SS). Os animais doadores eram machos e os receptores eram f?meas. As ratas foram avaliadas quanto ? fun??o motora, atrav?s da escala de Basso, Beattie and Bresnahan (BBB), quanto ? presen?a de CMMO na les?o, por meio da Rea??o em Cadeia da Polimerase (PCR) para a detec??o do cromossomo Y dos animais doadores, e quanto ao processo inflamat?rio no local da les?o, analisando a interleucina 1 beta (IL-1β) e o fator de necrose tumoral alfa (TNF-α), atrav?s de imuno-histoquimica. Nossos resultados demonstraram uma melhora da fun??o motora nos grupos tratados com transplantes de CMMO, sendo mais r?pida nos animais que receberam 7 cinco aplica??es. A avalia??o imuno-histoqu?mica revelou que n?o houve diferen?a na express?o das citocinas inflamat?rias IL-1β e TNF-α no local da les?o nos grupos tratados com CMMO. A an?lise de PCR n?o demonstrou c?lulas no local da les?o, quando analisadas no 37? dia

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2016-06-22T19:22:17Z No. of bitstreams: 1 TES_DANIEL_RODRIGO_MARINOWIC_COMPLETO.pdf: 16202491 bytes, checksum: 5bb7aaf0de3a700a163c7437ecf2adef (MD5) / Made available in DSpace on 2016-06-22T19:22:17Z (GMT). No. of bitstreams: 1 TES_DANIEL_RODRIGO_MARINOWIC_COMPLETO.pdf: 16202491 bytes, checksum: 5bb7aaf0de3a700a163c7437ecf2adef (MD5) Previous issue date: 2016-03-03 / Changes in the cerebral cortex development are presented as a group of distinct defects with a not well-defined pathogenesis. The focal cortical dysplasia (FCD) is one of the most frequent form of cortical development malformation, that encompasses multiple types of changes both in the cortical architecture and cytological abnormalities. It is an underlying pathology of a significant proportion of partial epilepsy refractory to drug treatment. Especially the limited number of cases and the lack of suitable experimental models rarely document the mechanisms involved with the genesis of FCD. The scarse predictive preclinical models that can be used to study the pathophysiology and to translate the therapeutic discovery from animal models to human use, strengthens the need to study the brain development from cells originated from patients that harbor these central nervous systems diseases. The generation of iPSC cells and the differentiation into specific cells and tissues will provide important testing and an unique ability to study the development and progress of CNS diseases. The objective of this study was to establish a cellular model of focal cortical dysplasia through the generation of induced pluripotent stem cells (iPSC) from fibroblasts derived from affected patients. Human fibroblasts were obtained from skin biopsies from two patients and cultured up to the fifth passage. Immunofluorescence analysis of AKT/mTOR signaling pathways was performed in the dysplastic brain tissue. iPSC cells were generated from fibroblasts through transfection with a viral vector containing the genes OCT4, KLF4, SOX2, and C-MYC and characterized by immunohistochemistry. Cell migration co-cultured with fibroblast and iPSC was investigated. Morphological and molecular characterization of neurodifferentiated iPSC were performed by immunohistochemistry and PI3K/ATK/mTOR signaling pathway analysis. Both patients were diagnosed with FCD type IIb. The AKT and mTOR phosphorylated and non-phosphorylated was higher in brain sections from patient 01. iPSC clones from patients and controls were generated from skin fibroblasts. Both iPSC from patients and controls showed polarization and morphology similar to nerve cells. In the cell migration assay, fibroblasts derived from FCD migrate with greater intensity within 24 hours (p<0,0001) and 48 hours (p<0,001), and iPSC cells did not present difference in cell migration. During neurodifferentiation, iPSC cells from patients with FCD showed lower values of 4EBP-1, ?-catenin, CIAP-1, CIAP-2 and PI3K, and higher values of MCL 1 gene expression. Changes in cell migration in adult tissue, uncontrolled cell proliferation, cell adhesion protein deficiency, and alterations in the expression of genes responsible for apoptosis and PI3K pathway that are implicated with an complete and well successful CNS formation. Alterations in some of these were detected in cells and iPSC from patients with FCD and may be related to the ethiopathology of the disease. / As altera??es do desenvolvimento do c?rtex cerebral apresentam-se como um grupo de malforma??es com uma distin??o e patog?nse ainda n?o bem definidas. A displasia cortical focal (DCF) ? uma das formas mais frequentes de malforma??es do desenvolvimento cortical, sendo a patologia subjacente a uma parcela significativa de epilepsias parciais refrat?rias ao tratamento medicamentoso. A displasia cortical focal engloba m?ltiplos tipos de altera??es tanto na arquitetura cortical quanto em anormalidades citol?gicas. Palmini e colaboradores classificaram as displasias corticais focais de acordo com observa??es na subst?ncia branca e na arquitetura da camada cortical. Os mecanismos envolvidos na g?nese da DCF s?o pouco investigados, principalmente pelo n?mero limitado de casos e a falta de modelos experimentais adequados e suas causas provavelmente est?o relacionadas a muta??es som?ticas. A falta de modelos pr?-cl?nicos preditivos que possam ser utilizados no estudo da fisiopatologia e o hist?rico enfraquecido quando se trata em traduzir a descoberta terap?utica de modelos animais para o uso humano, fortalece a necessidade de estudar o desenvolvimento cerebral a partir de c?lulas originadas do pr?prio paciente. A gera??o de c?lulas iPSC e diferencia??o tecidual espec?fica de c?lulas de pacientes acometidos por doen?as neurol?gicas relevantes possui um valor inestim?vel para a realiza??o de testes al?m de fornecerem uma capacidade adicional e ?nica para estudar o desenvolvimento inicial e a progress?o das patologias associadas ao SNC. O objetivo do presente trabalho ? estabelecer um modelo celular de Displasia Cortical Focal atrav?s da gera??o de c?lulas-tronco pluripotentes induzidas (iPSC) a partir de fibroblastos de pacientes afetados. Fibroblastos humanos foram obtidos de bi?psias de pele de dois pacientes com DCF e foram cultivados at? a quinta passagem. Foi realizada an?lise imunopatol?gica e das vias de sinaliza??o AKT/mTOR no tecido cerebral displ?sico. C?lulas iPSC foram geradas a partir dos fibroblastos atrav?s da exposi??o a vetores virais contendo os genes OCT4, KLF4, SOX2, e C-MYC e caracterizadas por imunohistoqu?mica. Foi realizado ensaio de migra??o celular dos fibroblastos (24h, 48h e 72h) e das iPSC (3 dias e 7 dias). As c?lulas iPSC foram neurodiferenciadas e analisadas nos per?odos de 14 dias, 22 dias e 35 dias. Nesses per?odos, foram realizadas an?lises morfol?gicas, imunohistoqu?mica (polariza??o) e moleculares (genes relacionados a via PI3K/ATK/mTOR). Ambos os pacientes foram diagnosticados com DCF tipo IIb. O paciente 01 apresentou valores maiores que o paciente 02 em rela??o ? ?rea marcada das vias AKT e mTOR, tanto na via fosforilada como n?o fosforilada no tecido cerebral, com diferen?as estatisticamente significativas. Clones iPSC dos pacientes e controles foram gerados e caracterizados a partir dos fibroblastos de pele. Tanto as iPSC dos pacientes quanto do controle apresentaram morfologia e polariza??o semelhante a c?lulas nervosas ap?s protocolo de neurodiferencia??o. No ensaio de migra??o celular, fibroblastos com DCF migraram com mais intensidade em 24 horas (p<0,0001) e 48 horas (p<0,001). As c?lulas iPSC n?o apresentaram diferen?a no potencial de migra??o celular nos per?odos analisados. Durante o protocolo de neurodiferencia??o, as c?lulas iPSC dos pacientes com DCF apresentaram valores menores de express?o dos genes 4EBP-1, ?-Catenina, CIAP-1, CIAP-2 e PI3K, e valores mais elevados da express?o de MCL 1. Altera??es na migra??o celular no tecido adulto e em processos como de prolifera??o celular acentuada, defici?ncia de prote?na de ades?o celular, altera??o na express?o de genes respons?veis pelo controle de apoptose e altera??o na via PI3K respons?vel no sistema nervoso central pela sobreviv?ncia celular, controle de apoptose, migra??o neuronal, desenvolvimento morfol?gico dos neur?nios e forma??o das neurotransmiss?es, puderam ser evidenciadas nas c?lulas dos pacientes com DCF em rela??o aos pacientes controle e podem estar relacionadas com a forma??o do c?rebro com displasia.

Made available in DSpace on 2014-12-17T14:05:23Z (GMT). No. of bitstreams: 1 JoanaCMT_TESE.pdf: 5399741 bytes, checksum: 9af0d84203a9976314d4eb5bba7e4067 (MD5) Previous issue date: 2013-03-25 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Human mesenchymal stem cells (MSC) are powerful sources for cell therapy in regenerative medicine. The long time cultivation can result in replicative senescence or can be related to the emergence of chromosomal alterations responsible for the acquisition of tumorigenesis features in vitro. In this study, for the first time, the expression profile of MSC with a paracentric chromosomal inversion (MSC/inv) was compared to normal karyotype (MSC/n) in early and late passages. Furthermore, we compared the transcriptome of each MSC in early passages with late passages. MSC used in this study were obtained from the umbilical vein of three donors, two MSC/n and one MSC/inv. After their cryopreservation, they have been expanded in vitro until reached senescence. Total RNA was extracted using the RNeasy mini kit (Qiagen) and marked with the GeneChip ? 3 IVT Express Kit (Affymetrix Inc.). Subsequently, the fragmented aRNA was hybridized on the microarranjo Affymetrix Human Genome U133 Plus 2.0 arrays (Affymetrix Inc.). The statistical analysis of differential gene expression was performed between groups MSC by the Partek Genomic Suite software, version 6.4 (Partek Inc.). Was considered statistically significant differences in expression to p-value Bonferroni correction &#706;.01. Only signals with fold change &#707; 3.0 were included in the list of differentially expressed. Differences in gene expression data obtained from microarrays were confirmed by Real Time RT-PCR. For the interpretation of biological expression data were used: IPA (Ingenuity Systems) for analysis enrichment functions, the STRING 9.0 for construction of network interactions; Cytoscape 2.8 to the network visualization and analysis bottlenecks with the aid of the GraphPad Prism 5.0 software. BiNGO Cytoscape pluggin was used to access overrepresentation of Gene Ontology categories in Biological Networks. The comparison between senescent and young at each group of MSC has shown that there is a difference in the expression parttern, being higher in the senescent MSC/inv group. The results also showed difference in expression profiles between the MSC/inv versus MSC/n, being greater when they are senescent. New networks were identified for genes related to the response of two of MSC over cultivation time. Were also identified genes that can coordinate functional categories over represented at networks, such as CXCL12, SFRP1, xvi EGF, SPP1, MMP1 e THBS1. The biological interpretation of these data suggests that the population of MSC/inv has different constitutional characteristics, related to their potential for differentiation, proliferation and response to stimuli, responsible for a distinct process of replicative senescence in MSC/inv compared to MSC/n. The genes identified in this study are candidates for biomarkers of cellular senescence in MSC, but their functional relevance in this process should be evaluated in additional in vitro and/or in vivo assays / C?lulas-tronco mesenquimais humanas (CTMH) s?o muito ?teis na terapia celular. O longo per?odo de cultivo pode resultar em senesc?ncia replicativa ou estar relacionado com o aparecimento de altera??es cromoss?micas respons?veis pela aquisi??o de um car?ter tumorig?nico in vitro . Neste estudo, foi comparado o transcriptoma de CTMH jovens e senescentes obtidas de diferentes doadores. Al?m disso, pela primeira vez, o perfil de express?o de CTMH com uma invers?o cromoss?mica parac?ntrica (CTMH/inv) foi comparado ao de CTMH que possuem cari?tipo normal (CTMH/n) em passagens jovens e senescentes de cultivo in vitro . As CTMH utilizadas neste estudo foram isoladas da veia do cord?o umbilical de tr?s dadores, dois CTMH/n e de um CTMH/inv. Ap?s a criopreserva??o, elas foram expandidas in vitro at? alcan?arem a senesc?ncia. O RNA total foi extra?do utilizando o RNeasy mini kit (Qiagen), marcado, purificado e fragmentado com o ? 3 GeneChip IVT expresso Kit (Affymetrix, Inc.). Subsequentemente, o RNA fragmentado foi hibridado no microarranjo Affymetrix Human Genome U133 Plus 2.0 (Affymetrix, Inc.). A an?lise estat?stica da express?o diferencial foi realizada usando o Partek Suite Software Genomic, vers?o 6.4 (Partek, Inc.). Foram consideradas estatisticamente significativas as diferen?as na express?o com valor de P &#706;0.01 corrigido com Bonferroni. Apenas os sinais com fold change &#707;3.0 foram inclu?dos na lista de diferencialmente expressos. Diferen?as na express?o g?nica observadas no estudo dos microarranjos foram confirmadas por resultados de RT-PCR em tempo real. Para a interpreta??o biol?gica dos dados foram utilizados: IPA (Ingenuity Systems) para an?lise de enriquecimento de fun??es; STRING 9,0 para a constru??o de redes de intera??es; Cytoscape 2,8 para a visualiza??o das redes e an?lises de gargalos com o aux?lio do software GraphPad Prism 5.0. O pluggin BiNGO do Cytoscape foi utilizado para avaliar a representa??o de categorias funcionais no Gene Ontology nas redes biol?gicas. A compara??o entre senescentes e jovens em cada grupo de CTMH mostrou que h? uma diferen?a no perfil de express?o, sendo maior nas senescentes do grupo CTMH/inv. Os resultados tamb?m mostraram que h? diferen?a nos perfis de express?o entre as CTMH/inv e CTMH/n, sendo maior a diferen?a quando as c?lulas est?o senescentes. Novas xiv redes foram identificadas para genes relacionados com a resposta ao longo do tempo de cultivo nos dois grupos de CTMH. Foram identificados genes que podem coordenar fun??es importantes mais enriquecidas nas redes, como por exemplo, CXCL12, SFRP1, EGF, SPP1, MMP1 e THBS1. A interpreta??o biol?gica destes dados sugere que a popula??o de c?lulas CTMH/inv tem diferentes caracter?sticas constitucionais, relacionadas com o seu potencial de prolifera??o, diferencia??o e resposta a est?mulos, respons?veis por um processo de senesc?ncia replicativa em CTMH/inv distinto das CTMH/n. Os genes identificados neste estudo s?o candidatos a marcadores da senesc?ncia celular em CTMH, mas a sua relev?ncia funcional neste processo deve ser testada em experi?ncias adicionais in vitro e/ou in vivo

Made available in DSpace on 2014-12-17T14:05:20Z (GMT). No. of bitstreams: 1 DeborahAC_TESE.pdf: 2567069 bytes, checksum: 17be3c292d241e19b0a9d1323613899f (MD5) Previous issue date: 2012-04-13 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Human multipotent mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, have become an important and attractive therapeutic tool since they are easily isolated and cultured, have in vitro expansion potential, substantial plasticity and secrete bioactive molecules that exert trophic effects. The human umbilical cord as a cell source for cell therapy will help to avoid several ethical, political, religious and technical issues. One of the main issues with SC lines from different sources, mainly those of embryonic origin, is the possibility of chromosomal alterations and genomic instability during in vitro expansion. Cells isolated from one umbilical cord exhibited a rare balanced paracentric inversion, likely a cytogenetic constitutional alteration, karyotype: 46,XY,inv(3)(p13p25~26). Important genes related to cancer predisposition and others involved in DNA repair are located in 3p25~26. Titanium is an excellent biomaterial for bone-implant integration; however, the use can result in the generation of particulate debris that can accumulate in the tissues adjacent to the prosthesis, in the local bone marrow, in the lymph nodes, liver and spleen. Subsequently may elicit important biological responses that aren?t well studied. In this work, we have studied the genetic stability of MSC isolated from the umbilical cord vein during in vitro expansion, after the cryopreservation, and under different concentrations and time of exposition to titanium microparticles. Cells were isolated, in vitro expanded, demonstrated capacity for osteogenic, adipogenic and chondrogenic differentiation and were evaluated using flow cytometry, so they met the minimum requirements for characterization as MSCs. The cells were expanded under different concentrations and time of exposition to titanium microparticles. The genetic stability of MSCs was assessed by cytogenetic analysis, fluorescence in situ hybridization (FISH) and analysis of micronucleus and other nuclear alterations (CBMN). The cells were able to internalize the titanium microparticles, but MSCs preserve their morphology, differentiation capacity and surface marker expression profiles. Furthermore, there was an increase in the genomic instability after long time of in vitro expansion, and this instability was greater when cells were exposed to high doses of titanium microparticles that induced oxidative stress. It is necessary always assess the risks/ benefits of using titanium in tissue therapy involving MSCs, considering the biosafety of the use of bone regeneration using titanium and MSCs. Even without using titanium, it is important that the therapeutic use of such cells is based on analyzes that ensure quality, security and cellular stability, with the standardization of quality control programs appropriate. In conclusion, it is suggested that cytogenetic analysis, FISH analysis and the micronucleus and other nuclear alterations are carried out in CTMH before implanting in a patient / C?lulas mesenquimais estromais multipotentes, tamb?m conhecidas como c?lulas-tronco mesenquimais humanas (CTMH), s?o c?lulas multipotentes utilizadas em v?rias pesquisas de terapia celular, pois apresentam a capacidade de se diferenciar em m?ltiplas e diferentes linhagens, t?m grande capacidade de autorrenova??o e de expans?o in vitro, excelentes propriedades imunossupressoras e s?o capazes de secretar mol?culas bioativas que exercem efeitos tr?ficos. O cord?o umbilical ? uma fonte de CTMH cuja extra??o n?o necessita de um procedimento invasivo, al?m de n?o envolver controv?rsias ?ticas, pol?ticas e religiosas. Um dos problemas que envolvem linhagens de CTMH de diferentes fontes ? a possibilidade de ocorr?ncia de altera??es cromoss?micas e instabilidade gen?tica, que podem aparecer durante a expans?o in vitro. Al?m disso, as CTMH de um dos cord?es apresentaram uma altera??o cromoss?mica constitucional: invers?o parac?ntrica no bra?o curto do cromossomo 3, cari?tipo: 46,XY,inv(3)(p13p25~26). Em 3p25-26, est?o localizados v?rios genes de grande import?ncia biol?gica, como genes envolvidos com o reparo de DNA e outros respons?veis pelo desenvolvimento de tumores. O tit?nio ? um dos materiais mais utilizado para fabrica??o de implantes ortop?dicos e dent?rios, e ? considerado um excelente biomaterial, entretanto, as part?culas derivadas de pr?teses acumulam-se nos tecidos periprost?ticos e na medula ?ssea local, disseminam-se para linfonodos, f?gado e ba?o. As implica??es biol?gicas em longo prazo da dissemina??o sist?mica de part?culas de metais e seus efeitos cito e genot?xicos n?o est?o bem caracterizados. Neste trabalho investigamos a estabilidade gen?tica de CTMH isoladas da veia do cord?o umbilical durante a expans?o in vitro, ap?s a criopreserva??o, e em diferentes condi??es de cultivo, na presen?a e na aus?ncia de tit?nio, antes e ap?s o aparecimento de c?lulas senescentes no cultivo. As c?lulas foram isoladas, expandidas, diferenciadas em osteoblastos, adip?citos e condroblastos e analisadas com citometria de fluxo para comprovar que s?o c?lulas-tronco mesenquimais. As CTMH foram tratadas com diferentes doses/ tempo de exposi??o ? micropart?culas de tit?nio. A avalia??o da estabilidade gen?tica das CTMH foi realizada atrav?s da an?lise do cari?tipo, de hibrida??o in situ por fluoresc?ncia (FISH) e da an?lise do micron?cleo e outras altera??es nucleares (CBMN). Ficou estabelecido que as CTMH foram capazes de internalizar as micropart?culas de tit?nio, mas as c?lulas mant?m sua capacidade de prolifera??o, diferencia??o e preservam os mesmos marcadores de membrana. Al?m disso, demonstrou-se que existe um aumento na instabilidade gen?tica com o decorrer do tempo de expans?o in vitro, e esta instabilidade foi maior na presen?a de grande concentra??o de micropart?culas de tit?nio que induzem estresse oxidativo. ? necess?rio sempre avaliar os riscos/ benef?cios da utiliza??o do tit?nio na terapia tecidual envolvendo CTMH, considerando a biosseguran?a da utiliza??o da regenera??o ?ssea guiada que utiliza CTMH e tit?nio. Mesmo n?o se utilizando o tit?nio, ? importante que o uso terap?utico de tais c?lulas seja baseado em an?lises que garantam a qualidade, seguran?a e estabilidade celular, com a padroniza??o de programas de controle de qualidade adequados. Como conclus?o, sugere-se que a an?lise citogen?tica, FISH e a an?lise do micron?cleo e outras altera??es nucleares sejam realizadas nas CTMH antes de implantar num paciente, sejam elas cultivadas por longo tempo ou n?o

Made available in DSpace on 2015-04-14T13:35:16Z (GMT). No. of bitstreams: 1 432976.pdf: 13950952 bytes, checksum: 85bef78387928925cf69df3245a8d8b9 (MD5) Previous issue date: 2011-03-30 / Os defeitos ?sseos podem ser considerados cr?ticos ou n?o, de acordo com sua capacidade de reparo normal pelo organismo evitando sequelas ?sseas. O tempo decorrido para ocorrer esse reparo acabar? trazendo alguma sequela tempor?ria para o paciente. Esse estudo teve como objetivo avaliar e comparar a influ?ncia de c?lulas mesenquimais indiferenciadas, cultivadas sob membranas de col?geno, no reparo de defeitos ?sseos em f?mur de ratos. Dos 14 animais utilizados na pesquisa, dois foram doadores de medula ?ssea e doze compuseram a amostra. Os animais foram submetidos a quatro danos teciduais no f?mur direito: Grupo 1, com c?lulas mesenquimais indiferenciadas dilu?das em soro fisiol?gico e recobertas por membrana de col?geno; Grupo 2, com c?lulas mesenquimais indiferenciadas dilu?das em gel de hidroxipropilmetilcelulose (HPMC); Grupo 3, com c?lulas mesenquimais indiferenciadas dilu?das em gel de HPMC e recobertas com membrana de col?geno; e Grupo 4, controle: reparo espont?neo. Quatro animais foram eutanasiados nos per?odos de 7, 18 e 30 dias p?s-operat?rios e avaliados por meio de histologia com hematoxilina e eosina em micorscopia de luz e demicroscopia eletr?nica de varredura. Todos os grupos estudados tiveram reparoda cavidade ?ssea formada, muito embora somente o grupo controle tenha apresentado forma??o de cortical ?ssea na sua por??o externa em 30 dias. O presente estudo sugere que o uso de c?lulas mesenquimais indiferenciadas n?o contribui para acelerar o processo de reparo ?sseo em defeitos n?o cr?ticos. O uso desses biomateriais pode inclusive atrasar o processo de reparo ?sseo, uma vez que o organismo ter? que reabsorver esse biomaterial, ao mesmo tempo que dever? estar formando novo osso

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-29T15:00:54Z No. of bitstreams: 1 TES_DANIEL_GONCALVES_BOECKEL_PARCIAL.pdf: 676511 bytes, checksum: cb9dde990f100982c7eac5a144973b47 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-29T15:01:09Z (GMT) No. of bitstreams: 1 TES_DANIEL_GONCALVES_BOECKEL_PARCIAL.pdf: 676511 bytes, checksum: cb9dde990f100982c7eac5a144973b47 (MD5) / Made available in DSpace on 2017-06-29T15:01:17Z (GMT). No. of bitstreams: 1 TES_DANIEL_GONCALVES_BOECKEL_PARCIAL.pdf: 676511 bytes, checksum: cb9dde990f100982c7eac5a144973b47 (MD5) Previous issue date: 2016-12-19 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The present research aims to characterize the mesenchymal stem cells of adipose origin (MSCAs) and to evaluate its use on the scaffold of hyaluronic acid (HA) as a cellular compound for bone tissue engineering. Firstly, cell characterization was performed through the collection of epididymal adipose tissue, isolation, culture and in vitro expansion. Through the morphological analysis of MSCAs culture it was possible to confirm elongated fusiform characteristic, centralized nucleus, extensions and adhesion to the plastic bottle. Expansion analysis also demonstrated a high cell proliferative index during the 26 days of in vitro culture. The Flow Cytometry test allowed the identification of the main surface markers that characterize the mesenchymal stem cells (CD29 and CD90) and negative for hematopoietic markers (CD31 and CD45). Moreover, MSCAs when induced to adipogenic and osteogenic media showed plasticity, since they were able to differentiate into adipocytes and osteoblasts respectively. The cell viability test was also performed in vitro, through the M.T.T (mitochondrial activity) of MSCAs on the HA scaffold. Concentrations of 100%, 75%, 50%, 25% and 15% were evaluated on the HA scaffold at times of 24, 48 and 72 hours. The results have shown cellular viability above 60% in almost all times and concentrations. Then, 50 critical bone defects were performed in the femoral region with 2 mm in diameter (one defect per femur) in 25 Lewis rats. The grafting treatments were divided as follows: I-negative control / only the defect (C); II-HA Scaffold; III- MSCAs; IV- MSCAs + HA and V- MSCAs previously osteoinduced + HA. After 23 days the rats were euthanized and had 5 femurs used for the microtomographic (?-CT) and histomorphometric analysis and 5 femurs used for the RT-PCR (Real-Time Polymerase Chain Reaction) analysis. The results for the ?-CT tests in the volume of osseous tissue parameter (VOT) and the percentage of osseous tissue (POT) did not present statistical differences among all groups. However, on the osseous contact surface (SCO) and osseous surface density (DSO) parameters, we had groups IV, III and V with higher indexes and differing statistically from the negative control groups I and group II. In histomorphometry we also had groups IV, V and III with greater area of regenerated bone tissue and differing with significance from groups I and II. The results were analyzed statistically by Analysis of Variance one way (ANOVA) and the level of significance was 5% (p <0.05). Regarding RT-PCR, a statistically significant difference was observed only when we evaluated osteonectin (ON) in which group II and V were more expressive in relation to groups III and IV. Regarding osteopontin (OP) and Type I collagen (Col1A), no differences were identified among the treated groups. The results were analyzed using Kruskal-Wallis non-parametric test and the significance level set was 5% (p <0.05). / A presente pesquisa tem por objetivo caracterizar as c?lulas-tronco mesenquimais de origem adiposa (CTMAs) e avaliar seu uso sobre a matriz de ?cido hialur?nico (AH) como composto celular para engenharia tecidual ?ssea. Primeiramente, foi realizada a caracteriza??o celular atrav?s da coleta do tecido adiposo epididimal, isolamento, cultivo e expans?o in vitro. Atrav?s da an?lise morfol?gica da cultura das CTMAs foi poss?vel confirmar caracter?stica fusiforme alongada, n?cleo centralizado, prolongamentos e ades?o ? garrafa pl?stica. A an?lise de expans?o tamb?m comprovou um alto ?ndice proliferativo celular, durante os 26 dias de cultura in vitro. J? o teste de citometria de fluxo permitiu a identifica??o dos principais marcadores de superf?cie que caracterizam as c?lulas-tronco mesenquimais (CD29 e CD90) e sendo negativo para marcadores hematopoi?ticos (CD31 e CD45). As CTMAS tamb?m quando induzidas aos meios adipog?nico e osteog?nico mostraram plasticidade, j? que foram capazes de diferenciarem-se em adip?citos e osteoblastos, respectivamente. Ainda in vitro, foi realizado o teste de viabilidade celular atrav?s do MTT (atividade mitocondrial), ap?s o contato das CTMAs sobre a matriz de AH. As concentra??es de 100%, 75%, 50%, 25% e 15% foram avaliadas sobre a matriz de AH nos tempos de 24, 48 e 72 horas. Os resultados comprovaram uma viabilidade celular acima de 60% em quase todos os tempos e concentra??es. Em seguida, foram realizados 50 defeitos ?sseos cr?ticos (DOC) na regi?o femoral com 2 mm de di?metro (um defeito por f?mur) em 25 ratos Lewis. Os tratamentos de enxertia realizados foram divididos da seguinte forma: I-controle negativo / apenas o defeito (C); II- matriz AH; III-CTMAs; IV-CTMAs + AH e V- CTMAs osteoinduzida + AH. Ap?s 23 dias os ratos sofreram eutan?sia e tiveram 5 f?mures utilizados para as an?lises microtomogr?ficas (?-CT) e histomorfom?trica e 5 f?mures utilizados para a an?lise por RT-PCR (Rea??o em cadeia da Polimerase em tempo Real). Os resultados para os testes por ?-CT no par?metro volume de tecido ?sseo (VTO) e porcentagem de tecido ?sseo (PTO) n?o apresentaram diferen?as estat?sticas entre todos os grupos. J?, nos par?metros superf?cie de contato (SCO) e densidade de superf?cie ?ssea (DSO) tivemos o grupo IV, III e V com maiores ?ndices e diferindo estatisticamente dos grupos controle negativo I e do grupo II. Na histomorfometria tamb?m tivemos os grupos IV, V e III com maiores ?reas de tecido ?sseo regenerado e diferindo com signific?ncia dos grupos I e II. Os resultados foram analisados estatisticamente pela An?lise de Vari?ncia de uma via (ANOVA) o n?vel de signific?ncia estabelecido foi de 5% (p < 0,05). Em rela??o ao RT-PCR, observou-se diferen?a estatisticamente significante apenas quando foi avaliada osteonectina (ON), sendo que o grupo II e V apresentaram maior express?o em rela??o aos grupos III e IV. Em rela??o ? osteopontina (OP) e ao col?geno Tipo I (Col1A) n?o foram identificadas diferen?as entres os grupos tratados. Os resultados foram analisados atrav?s do teste n?o param?trico de Kruskal-Wallis e o n?vel de signific?ncia estabelecido foi de 5% (p < 0,05).

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2015-09-16T22:49:16Z No. of bitstreams: 1 474864 - Texto Completo.pdf: 4783570 bytes, checksum: 1da454a8aaea971c62e4fff0a6d841a2 (MD5) / Made available in DSpace on 2015-09-16T22:49:16Z (GMT). No. of bitstreams: 1 474864 - Texto Completo.pdf: 4783570 bytes, checksum: 1da454a8aaea971c62e4fff0a6d841a2 (MD5) Previous issue date: 2015-08-03 / Introduction : The cellular and physiological changes that accompany the aging process has pronounced effects in neurodegenerative diseases. Factors that produce neurodegeneration do so through different processes that culminate in a common signaling pathway cascade. Stem cells communicate via cellular signaling mechanisms, which is part of a complex system which governs and coordinates activities and cell functions. Understanding the mechanisms and forms of intercellular communication and their relationship in the activation of signaling cascades from the surface receptor to the cell nucleus during neurogenesis, will answer some pertinent questions to the mechanisms of action of stem cells in repair central nervous system. Objective : This study aimed to determine which RTKs being activated during neurogenesis of adult stem cells from human umbilical cord tissue. Methods : Adult stem cells were obtained from human umbilical cord tissue (n = 3) and subjected to specific inducers in neurodifferentiation means. The kit Human Phospho-RTK Array was used for evaluating the phosphorylation of the RTK and the expression determined by realtime PCR. Results : The mesenchymal stem cells obtained from umbilical cord tissue were confirmed by differentiation induction in adipocytes and osteocytes cells. The neurodifferentiation was confirmed by expression of genes that are expressed during neurogenesis in cells and neural cells, as well as immunocytochemistry technique markings which showed nuclear (DAPI) and cell processes (Fluoropan). During the neuroinduction processes and neurodifferentiation the phosphorylation levels of RTKs were variable, with no clear pattern. The Ryk receptor showed no significant changes in their levels of phosphorylation of the three samples studied and the different stages of differentiation, ie, mesenchymal, neuroinductions and neurodifferentiates cells. Conclusions : The tyrosine kinases are actively being phosphorylated during neuroinduction and neurogenesis, with varying standards between the different stages and between different samples, reflecting the variability of human adult stem cells. / Introdu??o : As altera??es celulares e fisiol?gicas que acompanham o processo de envelhecimento tem efeitos pronunciados nas doen?as neurodegenerativas. Os fatores que produzem neurodegenera??o o fazem atrav?s de diferentes processos, que culminam em uma via comum de cascata de sinaliza??o. As c?lulas-tronco se comunicam por mecanismos de sinaliza??o celular, que faz parte de um complexo sistema que governa e coordena as atividades e fun??es celulares. Compreender os mecanismos e as formas de comunica??o intercelular e a sua rela??o na ativa??o das cascatas de sinaliza??o, desde o receptor de superf?cie at? o n?cleo celular, durante a neurog?nese, permitir? responder algumas quest?es pertinentes aos mecanismos de a??o das c?lulas-tronco no reparo do sistema nervoso central. Objetivo : Este estudo teve como objetivo determinar quais os RTKs que est?o sendo ativados durante a neurog?nese de c?lulas-tronco adultas de tecido de cord?o umbilical humano. Metodologia : C?lulas-tronco adultas foram obtidas de tecido de cord?o umbilical humano (n=3) e submetidas a neurodiferencia??o em meios indutores espec?ficos. O kit Human Phospho-RTK Array foi utilizado para avalia??o da fosforila??o dos RTKs e a express?o determinada por PCR em tempo real. Resultados : As c?lulas-tronco mesenquimais obtidas do tecido do cord?o umbilical foram confirmadas atrav?s da indu??o de diferencia??o em c?lulas adip?citas e oste?citas. A neurodiferencia??o foi confirmada atrav?s da express?o de genes que s?o expressos nas c?lulas durante a neurog?nese e em c?lulas neurais, como tamb?m a t?cnica de imunocitoqu?mica que mostrou marca??es nucleares (DAPI) e prolongamentos celulares (Fluoropan). Durante os processos de neuroindu??o e neurodiferencia??o os n?veis de fosforila??o dos RTKs foram vari?veis, sem um padr?o definido. O receptor Ryk n?o apresentou altera??es significativas nos seus n?veis de fosforila??o entre as tr?s amostras estudadas e nas diferentes etapas de diferencia??o, isto ?, c?lulas-tronco mesenquimais, c?lulas neuroinduzidas e c?lulas neurodiferenciadas. Conclus?es : Os receptores tirosina quinases est?o sendo ativamente fosforilados durante a neuroindu??o e neurog?nese, com padr?es vari?veis entre as diferentes etapas e entre diferentes amostras, refletindo a variabilidade das c?lulas-tronco adultas humanas.