Introdução: O transplante de células-tronco hematopoiéticas (TCTH) tornou-se um procedimento terapêutico mundialmente aceito sobretudo pelo impacto positivo na sobrevida e na qualidade de vida dos pacientes com doenças onco-hematológicas. No entanto, a mortalidade ainda é alta e influenciada por fatores de natureza individual e terapêutica. Objetivo: Analisar a mortalidade relacionada ao transplante (MRT) nos pacientes submetidos ao TCTH e seus fatores associados. Método: Coorte prospectiva realizada com 60 pacientes internados na unidade de TCTH do Hospital de Clínicas da Universidade Estadual de Campinas. Os dados foram obtidos pela análise diária dos prontuários. A variável dependente foi a MRT e as variáveis independentes foram demográficas e de evolução clínicas, incluindo escore de risco pré-TCTH (EBMT) e o SAPS II. Na análise dos dados foram utilizados os testes Qui-quadrado, Exato de Fisher, o teste t de Student e Mann-Whitney. Na análise da MRT utilizou-se o método de kaplan-Meier e o Modelo de Cox. Considerou-se nível de significância igual a 5%. Resultados: A MRT foi de 15% aos cem dias do TCTH, de 18,9% no grupo de pacientes de TCTH alogênico e de 8,7% para os de TCTH autólogo. A infecção foi a principal causa de óbito. Na amostra, o tempo médio de sobrevida dos pacientes foi de 83,2 dias (DP 32,7). No grupo de pacientes não sobreviventes a maioria pertencia ao sexo masculino, com média de idade de 48,7 anos e diagnóstico principal de leucemia. Quanto à gravidade destes pacientes, o escore de risco pré-TCTH (EBMT) foi de 4,1 pontos e do SAPS II geral foi de 52,6 pontos, o que correspondeu a um risco médio de morte de de 38,4%. Os fatores associados à MRT, em cem dias, foram faixa etária (p=0,0306), presença de infecção (p=0,0216), número de infecções (p=0,0386), ocorrência de enxertia (p<0,0001), uso de ventilação mecânica (p<0,0001) e de drogas vasoativas (p<0,0001). O índice de gravidade SAPS II foi fator preditor para MRT (p=0,0001). Conclusão: O índice de gravidade SAPS II, preditor para MRT em cem dias, mostrou que o paciente submetido ao TCTH é grave e necessita de cuidado especializado e intensivo. / Hematopoietic stem cells transplantation (HSCT) has become a therapeutic procedure accepted worldwide, particularly because of its positive impact on survival and quality of life of patients with onco-hematological diseases. However, the mortality is still high and it is influenced by factors of individual and therapeutic kinds. Objective: To analyze the transplant-related mortality (TRM) on patients submitted to HSCT and its associated factors. Methodology: Prospective cohort study with 60 patients hospitalized in the HSTC unit of the Clinical Hospital of the State University of Campinas (Unicamp). Data was obtained by daily analysis of the medical records. The dependent variable was the TRM and the independent variables were demographic and clinical development, including pre-HSCT risk score (EBMT) and SAPS II. For data analysis were used the Chi-square, Fishers exact tests, Students t-test, Mann-Whitney. On TRM analysis were used Kaplan-Meier and Cox Model method. It was considered a significance level of 5%. Results: The TRM was 15% to a hundred days of HSCT, 18,9% to allogeneic HSCT patients and 8,7% to autologous HSCT. Infection was the main cause of death. In the sample, the median survival time of the patients was 83,2 days (DP 32,7). In the group of non-surviving patients the most were male, with an average age of 48,7 years and the main diagnosis was leukemia. Regarding to the severity of these patients, the pre-HSCT risk score (EBMT) was 4,1 points and general SAPS II was 52,6 points, which corresponds to an average death risk of 38,4%. The TRM associated factors on a hundred days were age (p=0,0306), presence of infection (p=0,0216), number of infections (p=0,0386), occurrence of grafting (p<0,0001), mechanical ventilation use (p<0,0001) and vasoactive drugs (p<0,0001). The severity rate SAPS II was a predictive factor for TRM (p=0,0001). Conclusion: The severity rate SAPS II was predictive for TRM on a hundred days and showed that the patient submitted to HSCT is severe and demands specialized and intensive care.

RESUMO BALDISSERA, J.L.C. Avaliação das diferentes metodologias de realização do ensaio clonogênico e validação do método de criopreservação e ressuspensão do sangue de cordão umbilical e placentário criopreservado. 2015, 78 f. Dissertação. Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, 2015. O sangue de cordão umbilical e placentário (SCUP) tem sido utilizado como fonte de célulastronco hematopoéticas (CTHs) para transplante. A qualidade desse produto pode ser afetada durante as várias etapas do seu processamento. Neste estudo, foi avaliada a melhor metodologia de preparo da amostra para a realização do ensaio clonogênico (pura, diluída ou lavada) e validado o método de criopreservação e de ressuspensão das bolsas de SCUP. Foi avaliada também a funcionalidade da enzima aldeído desidrogenase (ALDH) como método para determinar a função das CTHs do SCUP, em 15 unidades criopreservadas pelo Laboratório de Criobiologia e Terapia Celular do Centro de Hematologia e Hemoterapia de Santa Catarina (HEMOSC). As unidades foram descongeladas em quatro etapas. O conteúdo dos segmentos e da bolsa foi coletado e ressuspenso com solução de albumina 5%, ACD 5% e solução fisiológica. A suspensão celular obtida foi utilizada para realização do ensaio clonogênico, avaliação da viabilidade celular, quantificação das células nucleadas (CN), CD34+ e das ALDH br . Os parâmetros tempo, custo e o resultado do ensaio clonogênico, utilizados para avaliar a metodologia, indicaram que a suspensão celular diluída é o melhor método a ser utilizado para a realização do ensaio clonogênico. A quantificação das CN e das células CD34+ totais pré-criopreservação e pós-criopreservação/descongelamento foi 8,3 (±1,9) x 10 8 e 8,2 (±2,0) x 10 8 (p = 0,3388) e 3,3 (±2,7) x 10 6 e 3,2 (±2,1) x10 6 (p = 0,4455), respectivamente. A quantificação das CN e das células CD34+ viáveis pré-criopreservação e pós-criopreservação/descongelamento foi 8,1 (±1,9) e 6,3 (±1,7) x 10 8 (p < 0,0001) e 3,27 (±2,0) x 10 6 e 2,8 (±1,8) x 10 6 (p = 0,0063), respectivamente. A porcentagem de células nucleadas e CD34+ viáveis no segmento proximal e na bolsa de 20 mL foi, respectivamente, 66,3 (±11,8) e 75 (35-93); 76,5 (±11,6) e 89 (75-100). No ensaio clonogênico foi observado crescimento médio de 31,8 (±7,6) unidades formadoras de colônias granulócito-macrófago (CFU-GM) x 10 5 CN plaqueadas obtidas da bolsa pós-criopreservação/descongelamento. Não foi encontrada correlação entre as células ALDH br /CD45 + viáveis e a quantificação das CFUGM ou das células CD34+ viáveis da bolsa pós-criopreservação/descongelamento. O coeficiente de correlação entre as células nucleadas e as células ALDEFLUOR bright da bolsa e do segmento pós-criopreservação/descongelamento foi (r) = 0,9399 com p < 0,0001 e (r) = 0,5478 com p = 0,0426, respectivamente. Foi encontrada correlação entre quantificação das células CD34+ e das CFU-GM da bolsa e do segmento pós-criopreservação/descongelamento. Esses dados indicam que o método utilizado para a realização da criopreservação e o descongelamento das unidades de SCUP encontra-se validado, e que o segmento pode ser utilizado como uma ferramenta de controle de qualidade para a seleção da unidade de SCUP para transplante. Palavras-chave: Validação. Sangue de cordão umbilical e placentário. Aldefluor. Célulastronco hematopoéticas. / ABSTRACT BALDISSERA, J.L.C. Evaluation of different methods of performing clonogenic assay and validation of the method of cryopreservation and resuspension of cryopreserved umbilical cord and placental blood. 2015. 78 f. Master Dissertation. Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, 2015. Umbilical cord and placental blood (UCPB) has been used as a source of hematopoietic stem cells (HSCs) for transplant. The quality of this product may be affected during the various stages of processing it. In this study, the author reviewed the best preparation methodology for performing the clonogenic assay (pure, diluted or washed) and validated the method of cryopreservation and resuspension of UCPB bags. The author also evaluated the functionality of the aldehyde dehydrogenase enzyme (ALDH) as a method to determine the function of umbilical cord and placental blood HSCs, in 15 cryopreserved units by the Laboratory of Cryobiology and Cell Therapy of the Center for Hematology and Hemotherapy of Santa Catarina (HEMOSC). The units were thawed in four steps. The content of the segments and the bag was collected and resuspended in a 5% albumin solution, 5% acid ci trate dextrose and saline solution. The cell suspension obtained was used to conduct the clonogenic assay, the assessment of cell viability, the quantification of nucleated cells (NC), CD34 + and ALDH br . The parameters of time, cost and the result of the clonogenic assay, used to evaluate the methodology, indicated that the diluted cell suspension is the best method to be used when performing a clonogenic assay. The quantification of the nucleated cells (NC) and the total CD34+ cells pre-cryopreservation and post-cryopreservation/thawing was 8,3 (±1,9) x 10 8 and 8,2 (±2,0) x 10 8 (p = 0,3388) and 3,3 (±2,7) x 10 6 and 3,2 (±2,1) x10 6 (p = 0,4455), respectively. The quantification of the NC and CD34+ viable cells pre-cryopreservation and post-cryopreservation/thawing was 8,1 (±1,9) and 6,3 (±1,7) x 10 8 (p < 0,0001) and 3,27 (±2,0) x 10 6 and 2,8 (±1,8) x 10 6 (p = 0,0063), respectively. The percentage of viable nucleated cells and CD34+ viable cells in the proximal segment and in the 20mL bag was 66,3 (±11,8) and 75 (35-93); 76,5 (±11,6) and 89 (75-100), respectively. In the clonogenic assay an average growth of 31,8 (± 7,6) colony-forming granulocyte-macrophage units (CFUGM) x 10 5 NC plated, obtained from the post-cryopreservation/thawing bag was observed. No correlation between the ALDH br /CD45 + viable cells and the quantification of CFU-GM or CD34+ viable cells obtained from the bag post cryopreservation was found. The coefficient of correlation between nucleated cells and ALDEFLUOR bright cells from the bag and segment after cryopreservation were (r) = 0, 9399 with p < 0, 0001 and (r) = 0, 5478 with p = 0,0426, respectively. A correlation between quantification of CD34+ cells and CFU-GM bag and segment cells after cryopreservation/thawing was found. This data indicates that the method used to perform the cryopreservation and thawing of the UCPB unit has been validated, and that the segment can be used as a tool for quality control when making the selection of UCPB for transplant. Keywords: Validation. Umbilical cord and placental blood. Aldefluor. Hematopoietic stem cells.

Introdução: O transplante de células-tronco hematopoiéticas (TCTH) tornou-se um procedimento terapêutico mundialmente aceito sobretudo pelo impacto positivo na sobrevida e na qualidade de vida dos pacientes com doenças onco-hematológicas. No entanto, a mortalidade ainda é alta e influenciada por fatores de natureza individual e terapêutica. Objetivo: Analisar a mortalidade relacionada ao transplante (MRT) nos pacientes submetidos ao TCTH e seus fatores associados. Método: Coorte prospectiva realizada com 60 pacientes internados na unidade de TCTH do Hospital de Clínicas da Universidade Estadual de Campinas. Os dados foram obtidos pela análise diária dos prontuários. A variável dependente foi a MRT e as variáveis independentes foram demográficas e de evolução clínicas, incluindo escore de risco pré-TCTH (EBMT) e o SAPS II. Na análise dos dados foram utilizados os testes Qui-quadrado, Exato de Fisher, o teste t de Student e Mann-Whitney. Na análise da MRT utilizou-se o método de kaplan-Meier e o Modelo de Cox. Considerou-se nível de significância igual a 5%. Resultados: A MRT foi de 15% aos cem dias do TCTH, de 18,9% no grupo de pacientes de TCTH alogênico e de 8,7% para os de TCTH autólogo. A infecção foi a principal causa de óbito. Na amostra, o tempo médio de sobrevida dos pacientes foi de 83,2 dias (DP 32,7). No grupo de pacientes não sobreviventes a maioria pertencia ao sexo masculino, com média de idade de 48,7 anos e diagnóstico principal de leucemia. Quanto à gravidade destes pacientes, o escore de risco pré-TCTH (EBMT) foi de 4,1 pontos e do SAPS II geral foi de 52,6 pontos, o que correspondeu a um risco médio de morte de de 38,4%. Os fatores associados à MRT, em cem dias, foram faixa etária (p=0,0306), presença de infecção (p=0,0216), número de infecções (p=0,0386), ocorrência de enxertia (p<0,0001), uso de ventilação mecânica (p<0,0001) e de drogas vasoativas (p<0,0001). O índice de gravidade SAPS II foi fator preditor para MRT (p=0,0001). Conclusão: O índice de gravidade SAPS II, preditor para MRT em cem dias, mostrou que o paciente submetido ao TCTH é grave e necessita de cuidado especializado e intensivo. / Hematopoietic stem cells transplantation (HSCT) has become a therapeutic procedure accepted worldwide, particularly because of its positive impact on survival and quality of life of patients with onco-hematological diseases. However, the mortality is still high and it is influenced by factors of individual and therapeutic kinds. Objective: To analyze the transplant-related mortality (TRM) on patients submitted to HSCT and its associated factors. Methodology: Prospective cohort study with 60 patients hospitalized in the HSTC unit of the Clinical Hospital of the State University of Campinas (Unicamp). Data was obtained by daily analysis of the medical records. The dependent variable was the TRM and the independent variables were demographic and clinical development, including pre-HSCT risk score (EBMT) and SAPS II. For data analysis were used the Chi-square, Fishers exact tests, Students t-test, Mann-Whitney. On TRM analysis were used Kaplan-Meier and Cox Model method. It was considered a significance level of 5%. Results: The TRM was 15% to a hundred days of HSCT, 18,9% to allogeneic HSCT patients and 8,7% to autologous HSCT. Infection was the main cause of death. In the sample, the median survival time of the patients was 83,2 days (DP 32,7). In the group of non-surviving patients the most were male, with an average age of 48,7 years and the main diagnosis was leukemia. Regarding to the severity of these patients, the pre-HSCT risk score (EBMT) was 4,1 points and general SAPS II was 52,6 points, which corresponds to an average death risk of 38,4%. The TRM associated factors on a hundred days were age (p=0,0306), presence of infection (p=0,0216), number of infections (p=0,0386), occurrence of grafting (p<0,0001), mechanical ventilation use (p<0,0001) and vasoactive drugs (p<0,0001). The severity rate SAPS II was a predictive factor for TRM (p=0,0001). Conclusion: The severity rate SAPS II was predictive for TRM on a hundred days and showed that the patient submitted to HSCT is severe and demands specialized and intensive care.

O saco vitelino é o único anexo embrionário presente em todas as espécies dos embriões vertebrados, répteis, aves e mamíferos. Em mamíferos domésticos o saco vitelino é inicialmente grande, pois nestas espécies ele é transitório. Após a implantação, surge no mesênquima lateral à notocorda agrupamentos de células, denominados ilhotas sanguíneas, que representam os progenitores dos sistemas vascular e hematopoético: os hemangioblastos. Os hemangioblastos centrais das ilhas sanguíneas formam as primeiras células-tronco hematopoéticas, enquanto os hemangioblastos periféricos se diferenciam em angioblastos, os precursores dos vasos sanguíneos. O desenvolvimento inicial da atividade hematopoética no saco vitelino conduz a hipótese de que esse tecido é o local primário de desenvolvimento hematopoético e que as células-tronco derivadas dele semeiam os outros sítios intraembriônicos. Foi possível observar nas análises microscópicas que realmente existe uma relação entre ambas linhagens. Nas análises de expressão gênica, alguns genes expressos pelo hemangioblasto apresentaram alta expressão nas análises D+0 e outros genes também específicos do hemangioblasto, porém em estágios secundários de diferenciação como os encontrados na região aórtica, a nível de endotélio hemogênico apresentaram altos níveis de expressão após 3 dias em cultivo. Concluímos portanto, que o saco vitelino por ser o local primário de formação das células sanguíneas e endoteliais nos estágios iniciais da embriogênese, por serem primitivas e, portanto não expressarem marcadores de células maduras na sua superfície, tornam estas células uma importante fonte de células-tronco relevante para a Terapia Celular para hemofilia e muitas outras doenças humanas. / The yolk sac is the single attachment embryo present in all species of vertebrate embryos, reptiles, birds and mammals. In domestic mammals the yolk sac is initially large, since these species it is transient. After implantation, appears in the lateral mesenchyme to the notochord cell clusters, called \"blood islands\" that represent the progenitors of vascular and hematopoietic systems: the hemangioblasts. The central islands hemangioblasts form the first blood hematopoietic stem cells, while peripheral hemangioblasts, the angioblastic differentiate into the precursors of blood vessels. The initial development of the yolk sac hematopoietic activity leads to the hypothesis that this tissue is the primary site of development and that hematopoietic stem cells derived from them sow other intraembryos sites. It was observed in the microscopic analysis that there is indeed a relationship between the two lineages. In the analysis of gene expression, some genes expressed by hemangioblasts showed high expression in D+0 and other specific genes also hemangioblasts, but in secondary stages of differentiation as found in the aortic region, the level of hemogenic endothelium showed high levels of expression after 3 days in culture. We therefore conclude that the yolk sac to be the primary site of formation of blood and endothelial cells in the early stages of embryogenesis, for its cells be primitive and therefore do not express markers of mature cells on the surface, these cells become an important source of cells relevant to stem cell therapy for hemophilia and many other human diseases.

Lau, Wai Hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 141-153). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

A mucosite bucal (MB) é uma complicação comum no tratamento do câncer e o desenvolvimento de intervenções efetivas para sua prevenção e tratamento são vistos como prioridade nos cuidados de suporte ao paciente oncológico. O objetivo do presente estudo foi investigar fatores de risco relacionados à incidência de mucosite bucal em pacientes submetidos a transplante de células progenitoras hematopoiéticas (TCPH) e em pacientes oncológicos pediátricos. Foram realizados dois estudos: o primeiro analisando a relação entre a incidência de mucosite bucal e o estado de saúde bucal, neutropenia, leucopenia e níveis de IL-1β em pacientes submetidos ao TCPH; e, o segundo, avaliando a incidência de mucosite bucal em pacientes oncopediátricos submetidos a diferentes protocolos quimioterápicos e sua relação com toxicidade hematológica, hepática e renal. Estudo 1: Foram avaliados 54 pacientes submetidos ao TCPH coletados dados demográficos e de à história médica foram coletados. Todos os pacientes foram avaliados quanto a saúde bucal através da análise do índice de placa (IP), índice gengival (IG), número de dentes cariados, perdidos e obturados (CPOD) e exame da mucosa bucal. Todos os pacientes receberam tratamento dentário e orientações de higiene bucal prévio bem como, fotobiomodulação (FBM) com laser de diodo InGaAlP como protocolo preventivo para mucosite bucal. Os pacientes foram avaliados diariamente desde o condicionamento ate o final do transplante. Avaliações de mucosite bucal, níveis de neutrófilos e leucócitos e análise de IL-1β foram realizados nos períodos de condicionamento, D+3 e D+8. Os pacientes que apresentaram gengivite severa anterior ao condicionamento para o transplante e que apresentaram neutropenia grave e leucopenia mostraram associação com o desenvolvimento OM. Os pacientes com mucosite bucal apresentaram níveis mais baixos de IL-1β. Estudo 2: Foram acompanhados 172 ciclos de quimioterapia realizados em 40 pacientes pediátricos. Dados de toxicidade hematológica (níveis de plaquetas, leucócitos, neutrófilos e hemoglobina), hepática (níveis de bilirrubina, TGO, TGP) e renal (creatinina e uréia) nos períodos D1, D5, D10 e D15 foram coletados. Avaliação do grau de mucosite bucal foi realizado diariamente a partir de D1 até D15. Os pacientes que desenvolveram mucosite receberam FBM 3 vezes por semana como tratamento. Os resultados mostraram que a mucosite bucal em pacientes oncológicos pediátricos tem relação com o tipo de protocolo quimioterápico utilizado, com a diminuição nos níveis de plaquetas, leucócitos e hemoglobina bem como, com o aumento dos níveis de bilirrubina. Os níveis de plaquetas e de bilirrubina podem ser considerados como fatores de risco para predizer o desenvolvimento de mucosite bucal. Conclui-se que ambos os trabalhos vieram a contribuir para a elucidação de fatores envolvidos no desenvolvimiento de mucosite bucal em pacientes submetidos ao TCPH e em pacientes oncopediátricos. / Oral mucositis (OM) is a common complication in cancer treatment. The development of effective interventions for prevention and treatment are seen as priority in supportive care cancer patients. The aim of this study was to investigate risk factors related to the incidence of oral mucositis in patients undergoing to hematopoietic stem cells transplantation (HSCT) and in pediatric oncology patients. Two studies were performed: the first analyzing the relationship between oral mucositis incidence with oral health status, neutropenia, leukopenia, and IL-1β levels in patients undergoing to HPCT; and the second, evaluating the incidence of oral mucositis in pediatric oncological patients undergoing to different chemotherapy protocols and their relationship with toxicity haematological, of liver and of kidney. Study 1: A total of 54 patients undergoing to HSCT were collected demographic data and medical history. All patients were evaluated for the oral health through plaque index (PI), gingival index (GI), number of decayed, missing and filled (DMF) and oral mucosa examination. All patients received prior dental and oral hygiene as well as photobiomodulation (PBM) InGaAlP diode laser as a preventive protocol for oral mucositis. Patients were evaluated daily from the conditioning until the end of transplantation. Reviews of oral mucositis, neutrophil ans leukocytes levels and IL- 1β analysis were performed in periods of conditioning, D+3 and D+8. Patients with previous severe gingivitis to conditioning for transplantation and who had severe neutropenia and leukopenia showed association with OM development. The oral mucositis patients had lower levels of IL-1β. Study 2: Wewre analyzed a total of 172 cycles of chemotherapy conducted in 40 oncological pediatric patients. Haematological toxicity data (levels of platelets, leukocytes, neutrophils, and hemoglobin), liver (bilirubin, GOT, GPT) and renal (creatinine and urea) in the periods D1, D5, D10 and D15 were collected. oral mucositis grade evaluation was performed daily from D1 to D15. Patients who developed oral mucositis received three times a week PBM as treatment. The results showed that oral mucositis in pediatric oncology patients is related to the type of chemotherapy protocol used with the decrease in the levels of platelets, leucocytes and hemoglobin as well as with the increase of the bilirubin level. The levels of platelets and bilirubin may be considered as risk factors to predict the development of oral mucositis. We conclude that both studies contributed to the elucidation of factors involved in the development of oral mucositis in patients undergoing HSCT and pediatric oncology patients.

The hematopoietic stem cell (HSC) is characterized by its ability to self-renew and produce all mature blood cells throughout the life of an organism. This is tightly regulated to maintain a balance between survival, proliferation, and differentiation. The HSCs are located in specialized niches in the bone marrow thought to be low in oxygen, which is suggested to be involved in the regulation of HSC maintenance, proliferation, and migration. However, the importance of hypoxia in the stem cell niche and the molecular mechanisms involved remain fairly undefined. Another important regulator of human HSCs maintenance is the tyrosine kinase receptor FLT3, which triggers survival of HSCs and progenitor cells. Mutations in FLT3 cause constitutively active signaling. This leads to uncontrolled survival and proliferation, which can result in development of acute myeloid leukemia (AML). One of the purposes with this thesis is to investigate how survival, proliferation and self-renewal in normal HSCs are affected by hypoxia. To study this, we used both in vitro and in vivo models with isolated Lineage-Sca-1+Kit+ (LSK) and CD34-Flt3-LSK cells from mouse bone marrow. We found that hypoxia maintained an immature phenotype. In addition, hypoxia decreased proliferation and induced cell cycle arrest, which is the signature of HSCs with long term multipotential capacity. A dormant state of HSCs is suggested to be critical for protecting and preventing depletion of the stem cell pool. Furthermore, we observed that hypoxia rescues HSCs from oxidative stress-induced cell death, implicating that hypoxia is important in the bone marrow niche to limit reactive oxidative species (ROS) production and give life-long protection of HSCs. Another focus in this thesis is to investigate downstream pathways involved in tyrosine kinase inhibitor-induced cell death of primary AML cells and cell lines expressing mutated FLT3. Our results demonstrate an important role of the PI3K/AKT pathway to mediate survival signals from FLT3. We found FoxO3a and its target gene Bim to be key players of apoptosis in cells carrying oncogenic FLT3 after treatment with tyrosine kinase inhibitors. In conclusion, this thesis highlights hypoxic-mediated regulation of normal HSCs maintenance and critical effectors of apoptosis in leukemic cells expressing mutated FLT3. / <p>On the day of the defence date the title of article II was "Hypoxia, via hypoxia-inducible factor (HIF)-1, mediates low cell cycle activity and preserves the engraftment potential of mouse hematopoietic stem cells" and one of the authors is no longer included in the article.</p>

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No. of bitstreams: 1 Dissertação - Nadja Pinto Garcia.pdf: 2876829 bytes, checksum: 836b1ac1a926c0202497b6aaef613039 (MD5) Previous issue date: 2011-11-24 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The mesenchymal stem cells (MSCs) have regenerative potential by its plasticity and ability to modulate the immune response with immunossupressive effects and secretion of a broadspectrum cytokines. The G-CSF is a potent cell growth factor and has the ability to mobilize SCs into peripheral blood. The aim of this study was to evaluated the influence of different doses of G-CSF on the kinetics at HSC and MSCs mobilization into peripheral blood and the G-CSF effect on the cytokine profile produced by these cells in vitro. We used six groups of 12 female Swiss mice, control group and five doses groups 1, 2, 3, 4 and 5 of G-CSF. The HSCs and MSCs mobilized were identified using cell markers performed by flow cytometry. The peripheral blood (PB) and bone marrow (BM) samples were used of each group to obtain the HSCs and MSCs, which were cultivated in vitro. Cytokines were measured in supernatants of PB and BM cultures by Cytometric Bead Array (CBA). The MSCs mobilized peak occurred with 4 doses of G-CSF in BM and 5 doses in PB. The HSC peaked occurred with 2 doses of G-CSF in BM and 4 doses in PB. There was a greater mobilization of MSCs than HSC, but that reason MSC/HSC was even greater in BM. The BM Cultures doses 3, 4 and 5 of G-CSF showed fibroblastoid adherent cells while in the PB cultures it was in cultures doses 2 and 3 doses of G-CSF. There was an increased production of IL-6, TNF-α in early cultivation of the SCs both BM and PB. The IFN-γ was increased in the initial phase, but peaked at the end of cultivation. The IL-2, IL-4, IL-17A and IL-10 cytokines had similar behavior reaching peak concentration in the late stage of cultivation. In the analysis of high frequency of cytokine producers for each dose of G-CSF in vivo, we observed same behavior cytokine in BM and PB cultures. Most of the cytokines produced in both BM and PB cultures of five doses showed significant differences about lower doses of G-CSF. This study suggested a possible influence of G-CSF to mobilize cells, as is known, but also in the production of several inflammatory and anti-inflammatory cytokines and possibly stimulate and modulate the differentiation of MSCs. / As células-tronco mesenquimais (CTM) apresentam potencial regenerativo não somente pela sua plasticidade, mas também pela sua capacidade de modular a resposta imunológica com efeitos imunossupressores e secreção de um largo espectro citocinas. O G-CSF é um potente fator de crescimento celular e tem a capacidade de mobilizar as CTs para o sangue periférico permitindo fácil obtenção destas células. O objetivo deste estudo foi avaliar a influência de diferentes doses de G-CSF na cinética de mobilização das CTHs e CTMs para o sangue periférico e no perfil de citocinas produzidas in vitro por essas células. Foram utilizados 6 grupos com 12 camundongos fêmeas Swiss, grupo controle e 5 grupos de doses 1, 2, 3, 4 e 5 de G-CSF. As CTHs e CTMs mobilizadas foram identificadas por meio de marcadores celulares específicos por citometria de fluxo. As amostras de CTHs e CTMs, de sangue periférico (SP) e medula óssea (MO) foram cultivadas in vitro e as citocinas foram dosadas nos sobrenadantes destas culturas pela técnica de Cytometric Bead Array (CBA). O pico de CTMs mobilizadas ocorreu com 4 doses na MO e com 5 doses de G-CSF no SP. As CTHs atingiram o pico com 2 doses de G-CSF na MO e com 4 doses no SP. Houve uma maior mobilização de CTMs do que CTHs, porém essa razão CTM/CTH ainda foi maior na MO. As culturas de MO das doses 3, 4 e 5 de G-CSF apresentaram células aderentes fibroblastóides, enquanto que foram observadas nas culturas de sangue de 2 e 3 doses de G-CSF. Houve uma maior produção das citocinas IL-6, TNF-α na fase inicial do cultivo das CTs, tanto MO quanto SP. O IFN-γ apresentou-se elevado na fase inicial, porém atingiu um pico no final do cultivo. As citocinas IL-2, IL-4, IL-17A e IL-10 tiveram um comportamento semelhante atingindo pico de concentração na fase tardia do cultivo. Na análise da frequência de altos produtores de citocinas para cada dose administrada de G-CSF in vivo, observou-se um comportamento semellhante das citocinas das culturas de MO e SP. A maioria das citocinas produzidas nas culturas de 5 doses tanto de MO quanto de SP apresentaram diferença significativa com relação a doses inferiores de G-CSF. Esse estudo sugeriu uma possível influência do G-CSF não somente na mobilização, como já é conhecido, mas também na produção de várias citocinas inflamatórias e anti-inflamatótrias, podendo possivelmente atuar no estímulo da diferenciação e modulação das CTMs.

Abstract This thesis describes the design, fabrication and operation of a microfluidic device for the screening of biomolecule mixture surface mediated effects. The characterisation of a surface immobilisation strategy that will allow the robust attachment of candidate biomolecules on a substrate for use in cell culture applications. This is carried out in the form of a modified and optimised layer-by-layer surface immobilisation strategy and its subsequent thorough and robust characterisation. This was achieved by compiling and critically analysing large amounts of quartz crystal microbalance with dissipation (QCM-D) data and the model utilised to provide meaningful, physical data as an output. QCM-D data was combined with surface plasmon resonance (SPR) data to validate the assumptions used within the QCM-D model package. Further evidence demonstrating the presence of the multilayer, as described by QCM-D and SPR, is achieved using x-ray photoelectron spectroscopy (XPS). These results show that the multilayer surface is robustly attached to the substrate and consists of a large amount of water whilst being able to immobilise mixtures of four proteins. A custom protocol for fabricating these two layer devices was devised and is presented. Scale limitations have been overcome to provide mixing capabilities for large extracellular matrix molecules to be immobilised on the previously described, microfluidically generated surface immobilisation strategy. The optimisation and characterisation of the mixing within this microfluidic device, affected by the incorporated staggered herring bone mixer is also shown. Using dynamic force spectroscopy (DFS) along with a custom designed force curve data processing and analysis package, the spatial localisation of a mixture of four immobilised biomolecules was determined. The aim of this study was to compare the spatial localization of a mixture of four biomolecules created by; standard cell culture protocols (adsorbed from bulk onto tissue culture polystyrene) and a surface created via microfluidic deposition on top of a previously described surface immobilisation strategy. The design and robust application of this custom analysis package allows the definition of a “Barricade of Specificity” such that interactions between an antibody functionalised AFM tip and a surface composed of a mixture of proteins, to be categorised as either a “true” specific interaction, or a non-specific interaction. The application of this Barricade of Specificity thus allows the spatial localisation of four immobilized biomolecules to be determined with a large degree of accuracy as a result of the large rage of non-specific interactions surveyed and the strict definition of a valid rupture force. The final chapter details the application of the microfluidic platform to enable high throughput screening of the effects of extracellular matrix (ECM) molecules, singly and in combination, with regards to the effect on the expression of cell surface markers on umbilical cord blood (UCB) derived CD34+ cells. Careful selection of candidate ECM molecules, cytokine and oxygen concentration has resulted in little difference in the effect on UCB derived CD34+ cells differentiation state after seven days in culture. The major effect has been the maturation towards lymphocyte and leukocyte precursors. However, of the four ECM molecules tested individually, in binary and in quaternary combinations, osteopontin (Opn) and laminin (Ln) demonstrated differences compared to other surfaces tested. In order to further assess the effect of these protein surfaces on the cell surface marker expression of UCB derived CD34+ cells, further tests are warranted for increased periods of time to enable greater discrimination in marker expression and thus increase our understanding of the fundamental biology of this rare and clinically useful cell source.

During embryonic development, generation of functional organs is dependent on proper interactions between different cell types. Elucidation of the mechanisms operating during organ formation might provide insights into the origin of many pathological disorders in the adult. Gene inactivation studies in mice have provided invaluable tool to study the function of genes critical for morphogenesis of distinct organs. A LIM-homeodomain transcription factor Lhx2 has previously been reported to play a role in fetal liver development and hematopoiesis, as its inactivation leads to lethal anemia due to underdeveloped liver. This thesis focuses on the function of Lhx2 in the development of these two organ systems. Reciprocal signaling between ventral foregut endoderm and mesenchyme of the septum transversum regulates the liver formation, expansion and differentiation. A fully formed liver is composed of endoderm-derived hepatocytes and cholangiocytes and a variety of mesenchyme-derived cell types, such as endothelial cells and hepatic stellate cells. In early stages of liver development Lhx2 is expressed in the liver-associated septum transversum mesenchyme, a part of which becomes integrated into the liver organ and develops into hepatic stellate cells. Functional Lhx2 expression in the hepatic mesenchyme is necessary for normal liver outgrowth and differentiation. Loss of Lhx2 from developing hepatic stellate cells leads to their activation and excessive deposition of collagen fibres, resulting in hepatic fibrosis and severely distorted liver architecture. Transfection of Lhx2 to human stellate cell line downregulates genes associated with stellate cell activation and fibrogenesis. Thus, Lhx2 is the first gene identified to negatively regulate events leading to hepatic fibrosis. Elucidation of the molecular mechanisms involved in this process might therefore be instrumental for the development of novel therapies useful in treatment of this disorder. Fetal liver is also a major site of hematopoiesis in the embryo and provides physiological conditions necessary for the efficient expansion of hematopoietic stem cells (HSCs). The hematopoietic defect observed in Lhx2-deficient embryos is cell-nonautonomous, indicating that Lhx2 might control secreted factors involved in the self-renewal of HSCs. This putative second role of Lhx2 has been investigated by analyzing the mechanism whereby Lhx2 expression generates in vitro self-renewing HSC-like cell lines. Interestingly, in agreement with the cell nonautonomous phenotype of the lethal anemia in Lhx2-/- embryos, the mechanism of self-renewal is dependent on Lhx2 expression and occurs via secreted factor(s). Identification of these factor(s) might potentially allow ex vivo expansion of HSCs for therapeutic purposes. The Lhx2-immortalized HSC-like cell lines share many basic features with HSCs and self-renew in vitro in presence of Steel factor (SF). SF/c-Kit signaling mediates a wide variety of biological activities in cells at many different levels in the hematopoietic hierarchy. We used the HSC-like cell lines as an in vitro model system to compare signal transduction pathways from c-Kit receptor in stem cells versus differentiated hematopoietic cells. HSCs require PI-3K dependent activation of Raf1-Mek-Erk cascade for their survival and self-renewal in response to SF, whereas activation of Erk is PI-3K independent in committed myeloid and mast cells. Thus, the mode of SF/c-Kit signaling is dependent on the differentiation status of the cells.