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>

A mucosite oral é um dos efeitos adversos mais frequente e debilitantes em pacientes submetidos ao transplante de medula óssea (TMO). O Laser de Baixa Potência (LBP) tem sido estabelecido como importante ferramenta na prevenção de mucosite, durante o condicionamento com quimioterapia de altas doses no TMO. No entanto, protocolos que suportam tais intervenções variam e os fatores de riscos para mucosite em pacientes com diferentes tipos de neoplasias e condicionamentos mesmo recebendo a prevenção com o LBP são ainda desconhecidos. Este trabalho teve como objetivo avaliar a prevalência de mucosite, sua relação com os parâmetros clínicos e fatores preditivos em pacientes submetidos ao TMO e que receberam a prevenção com LBP. Foi realizada uma análise retrospectiva de 374 pacientes consecutivos que foram submetidos ao TMO no A.C. Camargo Cancer Center, entre o período de janeiro de 2006 a janeiro de 2013. Todos os pacientes receberam profilaxia para mucosite oral com LBP, utilizando protocolo único, desde o primeiro dia do condicionamento até o D+2 (2 dias após a infusão da medula óssea). Os pacientes continuaram a receber o LBP nos casos de mucosite oral >= grau 2 até a remissão completa das lesões. Os dados clínicos como neutropenia febril, dor em boca, uso de alimentação parenteral e o uso de morfina foram coletados diariamente através dos prontuários dos pacientes desde o primeiro dia de internação até a alta hospitalar. As variáveis clínicas como idade, peso e função renal foram coletadas no dia da internação para o condicionamento. Dos 374 pacientes selecionados para este estudo, 37 (9%) pacientes foram excluídos, totalizando assim, 337 pacientes. Destes, 43 (12,76%) não apresentaram mucosite, 166 (49,25%) manifestaram mucosite grau 1, 84 (24,93%) grau 2, 32 (9,50%) grau 3 e 12 (3,56%) grau 4. Os pacientes com mucosite grau ? 2, apresentaram uma média de 1,4 dias de dor em boca comparado com 9,2 dias nos pacientes com mucosite ? 3 (p<0,0001). Em relação ao tempo de hospitalização, nos pacientes com mucosite grau ? 2, a média foi de 27,16 dias comparado com 36,07 nos com mucosite grau ?3 (p<0,0001). Através do modelo de regressão logística, observou-se que a cada aumento em uma unidade de creatinina a chance de ocorrer mucosite grau ? 3 foi 4,3 vezes maior (RC= 4,37; 95% IC: 1,68 - 11,32; p=0,0024). Além disso, os pacientes submetidos ao transplante alogênico apresentaram cerca de 5,97 vezes mais chance de apresentar mucosite grau ? 3 comparado com os pacientes submetidos ao transplante autólogo (RC= 5,97; 95% IC: 3,02 - 11,99; p<0,001). O estudo concluiu que a incidência e intensidade da mucosite oral severa foi baixa nos pacientes submetidos ao TMO, provavelmente devido à profilaxia com o LBP. Além disso, quanto maior a severidade da mucosite oral, maior o tempo de dor em boca, uso de alimentação parenteral, uso de morfina e período de internação. O transplante alogênico e o aumento no nível sérico de creatinina foram considerados fatores de risco para ocorrência de mucosite oral severa. Novos estudos são necessários para definir protocolos específicos para o LBP nestes pacientes com maior risco para o desenvolvimento de mucosite severa. / Oral mucositis is one of the most common and debilitating adverse effects in patients undergoing bone marrow transplantation (BMT). The low-power laser (LBP) has been established as an important tool in the prevention of mucositis, during conditioning with high-dose chemotherapy in BMT. However, protocols that support such intervention vary and risk factors for mucositis in patients with different types of neoplasms and conditioning protocols are still unknown. This study aimed to assess the prevalence of mucositis, its relationship with clinical parameters and predictive factors in patients undergoing BMT and receiving prevention with LBP. A retrospective analysis was performed concerning 374 patients who were submitted consecutively to BMT at the AC Camargo Cancer Center, from January 2006 to January 2013. All patients received prophylaxis for oral mucositis with LBP using only protocol from the first day of conditioning until D + 2 (2 days after the infusion of the bone marrow). Patients continued to receive the LBP in the case of oral mucositis grade >= 2 until the complete remission of the lesions. Clinical data, such as febrile neutropenia, mouth pain, parenteral nutrition usage and the use of morphine were collected daily through the medical records of patients from the first day of admission until discharge. The clinical variables such as age, weight and renal function data were collected on the day of admission for conditioning. Of the 374 patients selected for this study, 37 (9%) were excluded, totaling 337 patients. Of these, 43 (12.76%) had no mucositis, 166 (49.25%) expressed mucositis grade 1, 84 (24.93%) grade 2, 32 (9.50%) grade 3 and 12 (3.56 %) grade 4. Patients with mucositis grade <= 2, showed an average of 1.4 days of pain in the mouth compared with 9.2 days in patients with mucositis >= 3 (p <0.0001). Regarding the length of hospital stay in patients with mucositis grade <= 2, the average was 27.16 days compared to 36.07 in with mucositis grade >= 3 (p <0.0001). The logistic regression model showed that each increase of one unit of creatinine drives the chance of mucositis grade >= 3 4.3 times higher (OR = 4.37; 95% CI: 1.68 to 11.32 ; p = 0.0024). In addition, patients undergoing allogeneic transplantation showed about 5.97 times more likely to have grade >= 3 mucositis compared with patients undergoing autologous transplantation (OR = 5.97; 95% CI: 3.02 to 11.99 p <0.001). The study found that the incidence and intensity of severe oral mucositis was low in patients undergoing BMT, probably due to prophylaxis with LBP. In addition, the greater the severity of oral mucositis, the longer the pain in mouth, parenteral feeding use, morphine and time of hospitalization. Allogeneic transplantation and increase in serum creatinine level were considered risk factors for the occurrence of severe oral mucositis. Further studies are needed to define specific protocols for LBP in these patients at higher risk for developing severe mucositis.

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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.

A mucosite oral, também chamada recentemente de mucosite do trato gastrointestinal, continua sendo um importante efeito colateral que pode comprometer o resultado do transplante de células tronco hematopoéticas. Ela pode ocorrer em 100% dos pacientes submetidos ao transplante alogênico de células-tronco e a maior incidência neste pode ser atribuída à administração de metotrexate. A ocorrência de mucosite ulcerativa está relacionada ao aumento dos custos hospitalares, a redução da sobrevida em 100 dias e infecção sistêmica aumentando o risco de sepse. A última década foi muito importante para a compreensão da mucosite oral, incluindo a predisposição genética dos indivíduos e alterações nas enzimas responsáveis a metabolização de quimioterápicos. Recentemente, o polimorfismo C677T no gene metilenotetrahidrofolato redutase (MTHFR) têm ganhado enfoque na relação com a incidência da mucosite. Esta enzima metaboliza o metotrexate e a ela é atribuída maior ou menor atividade levando a modificações na metabolização do fármaco. Poucos trabalhos prospectivos e caso-controle são encontrados na literatura corrente com relação ao polimorfismo C677T e a incidência da mucosite. O objetivo deste estudo foi uma análise prospectiva caso-controle da relação do polimorfismo MTHFR C677T com a incidência da mucosite. Além disso, a influência da condição de saúde bucal (presença de placa dental e inflamação gengival) com a incidência de mucosite oral foi analisada. Foram inseridos 97 pacientes divididos em 2 grupos: 35 pacientes submetidos ao transplante alogênico e 62 pacientes submetidos ao transplante autólogo. A mediana de idade foi de 41,5 anos. O regime de condicionamento consistiu de busulfano e melfalano ou regime BEAM - becenum, etoposide, citarabina e melfalano (para a Doença de Hodgkin e Linfoma não Hodgkin). A profilaxia da doença do enxerto contra o hospedeiro foi feita com ciclosporina e metotrexate, no transplante alogênico. Não foi feito resgate com ácido folínico durante a administração de metotrexato. Os resultados mostraram que o polimorfismo C677T não foi significativo no grupo de estudo em comparação com o grupo controle na previsão de incidência e severidade da mucosite oral. No entanto, a incidência e gravidade da mucosite oral foram influenciadas pela condição de saúde bucal. Em conclusão, o polimorfismo C677T da MTHFR não foi relacionado ao oral mucosite, mas o estado de saúde oral foi um fator importante no desenvolvimento da mucosite. Estes resultados reforçam a importância de um dentista na equipe multiprofissional de assistência a estes pacientes. / Oral mucositis remains an important side-effect and life-threatening complication of hematopoietic stem cell transplantation. It can occurs in 100% of patients underwenting allogeneic stem cell transplantation. The differences in incidence between allogeneic and autologous transplantation may be due to methotrexate administration in the first. Ulcerative mucositis is related to increase hospitalar costs, reduced 100-days survival and systemic infections leading to sepsis risk. The last decade was very important to the understanding of oral mucositis, including genetics changes in enzymes responsible to drug metabolization, as the C677T polymorphism in the methylenetethrahidrofolate reductase gene (MTHFR). A prospective evaluation of oral mucositis in relation to the C677T MTHFR polymorphism was done. Also, the influence of oral health condition (presence of dental plaque and gingival inflammation) with the incidence of oral mucositis was analyzed. A cohort of 97 patients (35 allogeneic-study group and 62 autologous-control group) with median age of 41.5 years was evaluated. Conditioning regimen comprised busulfan and melphalan or becenum based conditioning regimen (BEAM becenum, etoposide, cytarabin and melphalan. GVHD prophylaxis comprised cyclosporine A plus short course of methotrexate in allogeneic transplantation. No rescue with folinic acid was done in the methotrexate administration. Results showed that C677T polymorphism was not significant in the study group compared with control group in predicting incidence and severity of oral mucositis. However, the incidence and severity of oral mucositis was influenced by oral health condition. In conclusion, C677T MTHFR polymorphism was not related to oral mucositis, but oral health status was an important factor in developing mucositis. These findings reinforce the importance of a dentist in the multiprofessional team to assist these patients.

O Transplante de Células-Tronco Hematopoéticas (TCTH) trata-se da substituição das Células-Tronco Hematopoéticas (CTH) doentes de um indivíduo por CTH normais com o objetivo de normalizar a hematopoese do receptor. O cateter venoso central (CVC) tornou-se indispensável para viabilizar tal terapêutica pela segurança na infusão das CTH e dos quimioterápicos. Para o TCTH são utilizados cateteres do tipo Hickman ou duplo lúmen, ambos de longa permanência. O curativo adesivo utilizado no sítio de inserção do cateter é essencial para a fixação do cateter, proteção contra agentes externos e prevenção de infecção. Há forte evidência clínica no uso do filme transparente gel de clorexidina (CHX) como uma alternativa no controle de infecções relacionadas ao cateter. A adesividade deste curativo confere-lhe uma possível permanência de até sete dias aderido à pele, o que evita repetidas aplicações e remoções do curativo, diminui a frequência na manipulação do cateter, a ocorrência de infecção e irritação cutânea local. Observa-se que a adesividade do curativo nem sempre permite a permanência por sete dias, sendo necessárias trocas não planejadas, podendo causar lesões à integridade cutânea. Neste estudo, objetivou-se avaliar a adesividade e a irritação cutânea do filme transparente gel de CHX aplicado no sítio de inserção do CVC por meio de um estudo transversal, prospectivo, realizado em um hospital público de ensino, no período de novembro de 2013 a junho de 2014, com crianças e adultos de ambos os sexos com doenças autoimunes ou onco-hematológicas submetidos ao TCTH. A amostra desta pesquisa foi constituída por 25 pacientes. Onze (44%) apresentaram irritação cutânea caracterizada por perda de pele de extensão <=0,5cm, perda de pele de extensão > 0,5cm, placa eritematosa e vesículas. Destes, seis tiveram necessidade de suspensão do uso do curativo. A pele fragilizada devido ao uso de agentes quimioterápicos pode aderir fortemente ao curativo adesivo, sendo desprendida com o mesmo quando este é removido. A perda da integridade cutânea com remoção da camada protetora da pele facilita a entrada de microrganismos aumentando a susceptibilidade a infecções em pacientes imunossuprimidos. Em 55,6% das avaliações foi observado desprendimento do curativo e a região da fenda foi onde mais ocorreu desprendimento (43,4%). Esta é uma região da margem do curativo, assim denominada por possuir uma abertura no adesivo onde são posicionadas as vias do cateter. Infere-se que devido à mobilidade do paciente e manuseio das vias do cateter, com o passar dos dias, essa região da borda do curativo apresenta maior desprendimento. Em 8,4% das observações havia bolha de ar no gel de clorexidina envolvendo o sítio de inserção do cateter. A bolha de ar no gel de clorexidina significa que este não está em contato com a pele do paciente o que é preocupante quando envolve o sítio de inserção do cateter, pois a CHX não está exercendo sua função antisséptica neste local, onde há possibilidade de migração extra- lúmen de microrganismos à corrente sanguínea / Hematopoietic stem cell transplantation (HSCT) involves the replacement of sick hematopoietic stem cells (HSC) from an individual with normal HSC, with the aim to restore the recipient\'s hematopoiesis. Central venous catheters (CVC) have become indispensable to make this therapy feasible due to the safety of infusion of the HSC and the chemotherapeutic agents. In HSCT, long-term Hickman or long-term double lumen catheters are used. The adhesive dressing used in the site of insertion of the catheter is essential for the catheter fixation, protection against external agents and prevention of infection. There is strong clinical evidence on the use of transparent film dressing with chlorhexidine gel (CHX) as an alternative in the control of catheter-related infections. The adhesiveness of this dressing allows it to be used up to seven days once it adheres to the skin, which avoids repeated dressing applications and removals, and reduces the frequency of catheter handling, the occurrence of infection, and local skin rash. However, as observed, the dressing adhesiveness does not always allow it to be used for seven days, and unplanned replacements may be needed, which can cause lesions to the skin integrity. The objective of this study was to evaluate the adhesiveness and skin rash related to the transparent film dressing with CHX gel applied at the site of insertion of the CVC by means of a cross-sectional, prospective study developed at a public teaching hospital, between November 2013 and June 2014, with children and adults of both genders with autoimmune or onco-hematological diseases who were submitted to HSCT. The sample comprised 25 patients. Eleven (44%) presented skin rash characterized by skin loss to an extension of <=0.5 cm, skin loss to an extension of > 0.5 cm, erythematous plaque and vesicles. Of these, six had to interrupt the use of the dressing. Frail skin due to the use of chemotherapeutic agents can adhere strongly to the adhesive dressing, coming off with the dressing at its removal. The loss of skin integrity with the removal of the skin\'s protective layer facilitates the entrance of microorganisms, increasing susceptibility to infections in immunosuppressed patients. In 55.6% of the evaluations, there was dressing detachment, and it was greater in the opening region (43.4%), which is a region in the dressing border with a break in the adhesive where the catheter is placed. Given the mobility of the patient and the handling of the catheter, over the days, this region of the dressing border presents greater detachment. In 8.4% of the observations there were air bubbles in the chlorhexidine gel involving the site of insertion of the catheter. Air bubbles in the chlorhexidine gel means that it is not in contact with the patient\'s skin, which is a cause of concern when the site of insertion of a catheter is involved, since CHX is not exerting its antiseptic function in this location, which can lead to extra-lumen migration of microorganisms to the bloodstream

O transplante autólogo de células-tronco hematopoéticas é indicado no tratamento de doenças autoimunes graves e refratárias ou que comprometem a qualidade de vida do paciente. Tal tratamento possui sólidas bases experimentais e clínicas e para alguns já apresenta superioridade ao tratamento convencional. No entanto, é um procedimento considerado agressivo, de alto custo financeiro, que pode acarretar eventos adversos, complicações, fatores de tensão física e psíquica para o paciente e sua família. Essa complexidade exige uma assistência de enfermagem especializada, capaz de assistir o paciente em cada uma das fases do tratamento, a fim de identificar os riscos, as intercorrências e propondo intervenções adequadas. Além disso, é fundamental que o paciente adquira conhecimentos e habilidades para se adaptar às condições impostas pela terapêutica, assim reunirá recursos de autonomia para realizar seu autocuidado. Frente ao exposto, as tecnologias podem ajudar e facilitar a aprendizagem, pois abordam o conteúdo por meio de várias formas e formatos. Nesse contexto, a enfermagem depara-se com o desafio de integrar o uso da Internet® no cuidado prestado ao paciente. A utilização dessa rede enfoca, principalmente, a disponibilização de informação de saúde ou estabelecimento de contato virtual para providenciar informação acessada de forma rápida e adequada à demanda da pessoa. Desta forma, o objetivo deste estudo foi desenvolver um ambiente virtual de aprendizagem com orientações sobre transplante autólogo de células-tronco hematopoéticas para doenças autoimunes. Trata-se de um estudo metodológico, com o objetivo de desenvolver o ambiente virtual de aprendizagem, website, com orientações sobre transplante autólogo de células-tronco hematopoéticas para doenças autoimunes. O website foi construído conforme o modelo de design instrucional, o qual percorreu as seguintes etapas: análise, design, desenvolvimento e implementação. Este foi desenvolvido em plataforma web, na linguagem de marcação Hypertext Markup Language, utilizando-se o programa WebAcappella, Responsive Website Creator 5 e disponibilizado no endereço eletrônico: http://www.transplantardai.com.br. O conteúdo do website foi estruturado nos seguintes tópicos: História, Transplante, Doenças Autoimunes, Links Interessantes, Orientações, Fala Equipe e Dúvidas Frequentes. Os ícones e menus foram criados de modo que o conteúdo atraia o usuário, sem cansar ou distraí-lo, a fim de otimizar os recursos disponíveis no ambiente e facilitar o acesso à busca dessas informações. Assim sendo, o presente estudo desenvolveu um ambiente virtual de aprendizagem que pode ser uma ferramenta utilizada para orientar, cuidar e interagir / Autologous hematopoietic stem cell transplantation is indicated for the treatment of severe or refractory autoimmune diseases, or those which compromise patients\' quality of life. This treatment has solid experimental and clinical foundations, and in some cases, it outranks conventional treatments. However, it is considered an aggressive and high-cost procedure that may lead to adverse events, complications, and factors of physical and psychic tension to patients and their families. This complexity requires specialized nursing care, capable of assisting patients in each phase of the treatment, so as to identify risks and propose appropriate interventions. In addition, patients must acquire knowledge and skills to adapt to the conditions imposed by the treatment, thus gathering autonomy resources to perform self-care. In the light of this, technologies can help and facilitate learning, as they approach content in several ways and formats. In this context, nursing faces the challenge of integrating the use of the Internet® in the care provided to patients. The use of this network focuses, mainly, on making health information available or establishing virtual contact to provide information that can be accessed fast and that is suitable to the person\'s demand. This was a methodological study, with the aim of developing a virtual learning environment and a website, with instructions on autologous hematopoietic stem cell transplantation for autoimmune diseases. The website was built according to an instructional design model, through the following steps: analysis, design, development, and implementation. It was developed on a web platform, using the Hypertext Markup Language and the programs WebAcappella and Responsive Website Creator 5, being available at http://www.transplantardai.com.br. The website content was structured in the following topics: History, Transplantation, Autoimmune diseases, Interesting links, Instructions, Talk to the team and Frequently asked questions. The icons and menus were created so that the content would be attractive to users, without making them tired or distracting them, in order to optimize the resources available in the environment and facilitate access to the search for information. Therefore, the present study developed a virtual learning environment that can be used for guidance, caring, and interaction

A mucosite oral é um dos efeitos adversos mais frequente e debilitantes em pacientes submetidos ao transplante de medula óssea (TMO). O Laser de Baixa Potência (LBP) tem sido estabelecido como importante ferramenta na prevenção de mucosite, durante o condicionamento com quimioterapia de altas doses no TMO. No entanto, protocolos que suportam tais intervenções variam e os fatores de riscos para mucosite em pacientes com diferentes tipos de neoplasias e condicionamentos mesmo recebendo a prevenção com o LBP são ainda desconhecidos. Este trabalho teve como objetivo avaliar a prevalência de mucosite, sua relação com os parâmetros clínicos e fatores preditivos em pacientes submetidos ao TMO e que receberam a prevenção com LBP. Foi realizada uma análise retrospectiva de 374 pacientes consecutivos que foram submetidos ao TMO no A.C. Camargo Cancer Center, entre o período de janeiro de 2006 a janeiro de 2013. Todos os pacientes receberam profilaxia para mucosite oral com LBP, utilizando protocolo único, desde o primeiro dia do condicionamento até o D+2 (2 dias após a infusão da medula óssea). Os pacientes continuaram a receber o LBP nos casos de mucosite oral >= grau 2 até a remissão completa das lesões. Os dados clínicos como neutropenia febril, dor em boca, uso de alimentação parenteral e o uso de morfina foram coletados diariamente através dos prontuários dos pacientes desde o primeiro dia de internação até a alta hospitalar. As variáveis clínicas como idade, peso e função renal foram coletadas no dia da internação para o condicionamento. Dos 374 pacientes selecionados para este estudo, 37 (9%) pacientes foram excluídos, totalizando assim, 337 pacientes. Destes, 43 (12,76%) não apresentaram mucosite, 166 (49,25%) manifestaram mucosite grau 1, 84 (24,93%) grau 2, 32 (9,50%) grau 3 e 12 (3,56%) grau 4. Os pacientes com mucosite grau ? 2, apresentaram uma média de 1,4 dias de dor em boca comparado com 9,2 dias nos pacientes com mucosite ? 3 (p<0,0001). Em relação ao tempo de hospitalização, nos pacientes com mucosite grau ? 2, a média foi de 27,16 dias comparado com 36,07 nos com mucosite grau ?3 (p<0,0001). Através do modelo de regressão logística, observou-se que a cada aumento em uma unidade de creatinina a chance de ocorrer mucosite grau ? 3 foi 4,3 vezes maior (RC= 4,37; 95% IC: 1,68 - 11,32; p=0,0024). Além disso, os pacientes submetidos ao transplante alogênico apresentaram cerca de 5,97 vezes mais chance de apresentar mucosite grau ? 3 comparado com os pacientes submetidos ao transplante autólogo (RC= 5,97; 95% IC: 3,02 - 11,99; p<0,001). O estudo concluiu que a incidência e intensidade da mucosite oral severa foi baixa nos pacientes submetidos ao TMO, provavelmente devido à profilaxia com o LBP. Além disso, quanto maior a severidade da mucosite oral, maior o tempo de dor em boca, uso de alimentação parenteral, uso de morfina e período de internação. O transplante alogênico e o aumento no nível sérico de creatinina foram considerados fatores de risco para ocorrência de mucosite oral severa. Novos estudos são necessários para definir protocolos específicos para o LBP nestes pacientes com maior risco para o desenvolvimento de mucosite severa. / Oral mucositis is one of the most common and debilitating adverse effects in patients undergoing bone marrow transplantation (BMT). The low-power laser (LBP) has been established as an important tool in the prevention of mucositis, during conditioning with high-dose chemotherapy in BMT. However, protocols that support such intervention vary and risk factors for mucositis in patients with different types of neoplasms and conditioning protocols are still unknown. This study aimed to assess the prevalence of mucositis, its relationship with clinical parameters and predictive factors in patients undergoing BMT and receiving prevention with LBP. A retrospective analysis was performed concerning 374 patients who were submitted consecutively to BMT at the AC Camargo Cancer Center, from January 2006 to January 2013. All patients received prophylaxis for oral mucositis with LBP using only protocol from the first day of conditioning until D + 2 (2 days after the infusion of the bone marrow). Patients continued to receive the LBP in the case of oral mucositis grade >= 2 until the complete remission of the lesions. Clinical data, such as febrile neutropenia, mouth pain, parenteral nutrition usage and the use of morphine were collected daily through the medical records of patients from the first day of admission until discharge. The clinical variables such as age, weight and renal function data were collected on the day of admission for conditioning. Of the 374 patients selected for this study, 37 (9%) were excluded, totaling 337 patients. Of these, 43 (12.76%) had no mucositis, 166 (49.25%) expressed mucositis grade 1, 84 (24.93%) grade 2, 32 (9.50%) grade 3 and 12 (3.56 %) grade 4. Patients with mucositis grade <= 2, showed an average of 1.4 days of pain in the mouth compared with 9.2 days in patients with mucositis >= 3 (p <0.0001). Regarding the length of hospital stay in patients with mucositis grade <= 2, the average was 27.16 days compared to 36.07 in with mucositis grade >= 3 (p <0.0001). The logistic regression model showed that each increase of one unit of creatinine drives the chance of mucositis grade >= 3 4.3 times higher (OR = 4.37; 95% CI: 1.68 to 11.32 ; p = 0.0024). In addition, patients undergoing allogeneic transplantation showed about 5.97 times more likely to have grade >= 3 mucositis compared with patients undergoing autologous transplantation (OR = 5.97; 95% CI: 3.02 to 11.99 p <0.001). The study found that the incidence and intensity of severe oral mucositis was low in patients undergoing BMT, probably due to prophylaxis with LBP. In addition, the greater the severity of oral mucositis, the longer the pain in mouth, parenteral feeding use, morphine and time of hospitalization. Allogeneic transplantation and increase in serum creatinine level were considered risk factors for the occurrence of severe oral mucositis. Further studies are needed to define specific protocols for LBP in these patients at higher risk for developing severe mucositis.

Selon le modèle standard de l'hématopoïèse, la différenciation des cellules souches hématopoïétiques est un processus graduel de type arborescent. La première séparation a lieu au niveau de cellules multipotentes qui se scindent en progéniteurs lymphoïdes et myéloïdes communs. Bien que l'architecture de l'hématopoïèse humaine reste encore mal connue, de nombreux travaux suggèrent qu'elle ne suit pas le modèle standard. À ce jour encore, la question de l'existence d'un équivalent humain du CLP murin, n'a pas été tranchée. L'étude de l'hématopoïèse humaine soulève des problèmes méthodologiques. Ceci est lié au difficile accès au; prélèvements de moelle primaire et les études sur le sang placentaire ne reflètent pas complétement le développement médullaire. Dans ce travail, nous avons utilisé un modèle in vivo d'hématopoïèse foetale humaine chez la souris NSG par xénogreffe de progéniteurs du sang placentaire. La caractérisation faite sur les populations générées dans la moelle osseuse de souris a révélé que ce modèle reproduit l'hématopoïèse foetale humaine. Nous montrons que la lymphopoïèse foetale humaine présente une organisation originale caractérisée par une duplication des axes développementaux. Nos travaux mettent en évidence l'émergence indépendante de deux type de progéniteurs lymphoïdes à partir d'un intermédiaire multipotent: une population ancestrale CD127+générant principalement des lymphocytes B folliculaires, ainsi que des cellules ILC3 ; une population CD127- générant des lymphocytes T, des lymphocytes B de la zone marginale, et des cellules NK/ILC1. Ces résultats montrent que l'hématopoïèse humaine ne suit pas le modèle standard établi chez la souris. / The standard model of hematopoiesis proposes that hematopoietic differentiation is a stepwise bifurcation process. The first separation downstream of hematopoietic stem cells will segregate mutipotent progenitors into common lymphoid and myeloid progenitors. In human many evidences support the idea that human hematopoietic organization doesn't follow the classical model, but the question was not concluded and need for further investigation. Due to limited access to primary bone marrow samples and lack of appropriate in vivo model human studies face many difficulties. In this work, we used a xenogeneic model of human fetal hematopoiesis in immune-deficient mice to dissect the early stages of lymphoid development. This model relies on the injection of UCB CD34+ cells into NSG mice. Flow cytometry analysis and gene expression profiling of humanized mice BM populations revealed that this model faithfully reproduces human fetal hematopoiesis. Combining in vitro differentiation assays to molecular studies and genetic approaches, we show that fetal human lymphopoiesis displays a dual organization, split into an ancestral CD127+ CLP-like population devoid o myeloid potential that differentiate preferentially into follicular B cells and ILC3s, and into a previously undescribed CD127- population mainly dedicated to the generation of T, marginal zone B, NK, and ILC1s We also provide evidence that Early Lymphoid Progenitors emerge independently from multipotent developmental intermediates referred to as lympho-mono'dendritic progenitors. These results confirm that human hematopoiesis doesn't follow the standard model of hematopoietic differentiation established in the mouse.

The blood system consists of two main lineages: myeloid and lymphoid. The myeloid system consists of cells that are part of the innate immune response while the lymphoid system consist of cells that are part of humoral response. These responses protect our bodies from foreign pathogens. Thus, malignancies in these systems often cause complications and mortality. Scientists world wide have been researching alternatives to treat hematologic disorders and have explored induced pluripotent stem cells (iPSCs) and the conversion of one cell type to another. First, iPS cells were generated by overexpression of four transcription factors: Oct4, Sox2, Klf4 an cMyc. These cells closely resemble embryonic stem cells (ESCs) at the molecular and cellular level. However, the efficiency of cell conversion is less than 0.1%. In addition, many iPS colonies can arise from the same culture, but each has a different molecular signature and potential. Identifying the appropriate iPS cell lines to use for patient specific therapy is crucial. Here we demonstrate that our system is highly efficient in generating iPS cell lines, and cell lines with silent transgenes are most efficient in differentiating to different cell types . Second, we are interested in generating hematopoietic stem cells (HSCs) from fibroblasts directly, without going through the pluripotent state, to increase efficiency and to avoid complications associated with a stem cell intermediate. However, a robust hematopoietic reporter system remains elusive. There are multiple hematopoietic reporter candidates, but we demonstrate that the CD45 gene was the most promising. CD45 is expressed early during hematopoiesis on the surface of HSCs; and as HSCs differentiate CD45 levels increase. Furthermore, the CD45 reporter is only active in hematopoietic cells. We were able to confirm the utility of the CD45 reporter using an in vitro and an in vivo murine model. In conclusion, The goal of this research was to expand the knowledge of stem cell reprogramming, specifically the reprogramming of iPS cells. Furthermore, it is our desire that the CD45 reporter system will undergo further validation and find utility in clinical and cell therapy environments.

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.