La réparation osseuse est assurée par le recrutement constant de cellules souches/progéniteurs ostéo-compétents de nature hématopoïétique (CSH/PH), et mésenchymateuse (CSM). Une approche prometteuse pour le traitement des défauts osseux graves consisterait à favoriser le recrutement et la mobilisation des CSH/PH et des CSM à partir de la moelle osseuse vers le site de lésion. Plusieurs facteurs environnementaux sont connus pour moduler la prolifération, la mobilisation et la différenciation des progéniteurs ostéocompétents, dont l’hypoxie et l’hypodynamie (absence de contraintes mécaniques). Le but de ce travail de thèse a été d’investiguer in vivo l’impact de l’hypoxie respiratoire et de l’absence de contraintes mécaniques, appliquées séparément ou ensemble sur i) la mobilisation des progéniteurs ostéocompétents et sur ii) la réparation d’un défaut osseux cavitaire fémoral chez la souris. Sur un modèle murin dépourvu de défaut osseux, nos données montrent que l’hypoxie respiratoire est un agent mobilisateur des progéniteurs ostéocompétents, et qu’elle pourrait donc potentiellement exercer des effets bénéfiques sur la réparation osseuse. Toutefois, les effets de l’hypoxie sont modulés selon le statut hypodynamique ou non de l’animal. L’absence de contraintes mécaniques limite la mobilisation des progéniteurs érythrocytaires et mésenchymateux initiée par l’hypoxie, suggérant un effet potentiellement délétère de l’hypodynamie en condition hypoxique dans le contexte de la réparation osseuse. Chez les souris opérées, nous confirmons que l’hypoxie respiratoire déclenchée lors des phases de remodelage améliore la réparation du défaut osseux cavitaire. Une mobilisation des progéniteurs mésenchymateux et hématopoïétiques du fémur contra-latéral vers le fémur opéré est noté, mais le mode d’action de l’hypoxie passerait plutôt par une accélération du mécanisme de réparation dans la zone lésée. De façon intéressante, nous montrons que l’hypodynamie ne diminue pas le bénéfice apporté par l’hypoxie respiratoire à la réparation osseuse. En conclusion, ce travail de thèse identifie l’hypoxie respiratoire comme un candidat thérapeutique pertinent pour l’amélioration de la réparation osseuse. Bien que la perte des contraintes mécaniques module la biologie des cellules ostéoprogénitrices en absence de lésion, l’hypodynamie ne semble pas influencer la consolidation osseuse dans le cadre d’une amélioration de la réparation par un épisode hypoxique tardif. / Many environmental factors are known to influence bone cell fate, including proliferation, mobilization and differentiation of osteoprogenitor cells deriving from both hematopoietic and mesenchymal lineages. Among these factors, hypoxia and unloading (lack of mechanical loading / hypodynamia) are of particular interest. This study aims at investigating the impact of short-term hypobaric hypoxia and hindlimb unloading applied alone or in combination i) on the mobilization of osteocompetent progenitor cells on mice and ii) on the healing in a mouse model of surgical metaphyseal bone defect.In mice free of bone defect, our data indicate that respiratory hypobaric hypoxia acts as a mobilizing stimulus for osteoprogenitor cells. However, the effects of hypoxia in the bone marrow depend on whether mice are subjected to hindlimb unloading or not: hypodynamia tends to restrain the mobilization of both mesenchymal and erythroid progenitors under hypoxia. This suggests a potential detrimental influence of hypodynamia in the course of bone healing in hypoxic condition.In mice with surgery, we showed that hypobaric hypoxia during the remodelling process strongly enhances bone healing. A mobilization of both mesenchymal and hematopoietic progenitors is detected from the contralateral femur to the operated femur. In the lesion area, an acceleration of the repair process is evidenced. Interestingly, hindlimb unloading does not exert any negative influence on bone repair in our animal model. In conclusion, this study identifies delayed hypobaric hypoxia as a potent candidate to enhance bone healing. Even if unloading exerts significant effects on the biology of osteoprogenitor cells on mice free of bone defect, its influence is not detrimental for bone repair.

皮膚是人體最大的器官，具有多種功能，其中最重要的功能之一就是作為身體內部和外界環境之間的的保護屏障。完整地修復這一保護屏障是創傷癒合和組織再生領域的一個重要內容。本論文探討了人類臍帶被覆上皮細胞 (cord lining epithelial cells, CLECs)作為一種幹細胞來源，可用于表皮重建的潛能. / 本論文的第二章對CLECs的體外分離和增殖進行了詳細地描述。這一類細胞具有較長的染色體端粒，較高的增殖潛能和傳代能力。同時，它們表達上皮幹細胞和多能性幹細胞的標誌性表面抗原。它們還具有多種分化潛能，包括成脂、成骨和成軟骨。然而當皮下異種移植後，它們並不會形成畸胎瘤。 / 本論文的第三章對CLECs的免疫特性進行了評估。結果顯示CLECs不但具有低免疫原性，還具有免疫調節功能。它們表達典型性的一型主要組織相容性複合體(MHC class I)，即人白細胞ABC抗原(HLA-ABC)，但不表達典型性的二型主要組織相容性複合體(MHC class II)，即人白細胞DR抗原(HLA-DR)。它們同時還表達非典型性的MHC class I, 包括人白細胞G抗原和人白細胞E 抗原(HLA-G和HLA-E), 但不表達共激分子(CD40, CD80和CD86)。此外，體外檢測還發現它們表達適度的促炎/抗炎細胞因子和大量的生長因子. / 本論文的第四章對CLECs在表皮重建應用中的潛能進行了考察。結果顯示無論在體外器官培養還是異種移植動物模型中，CLECs都能形成分層的上皮結構，與用表皮細胞構建的分層上皮結構相類似。而且在CLECs構建的皮膚替代物中證實了有表皮分化標誌性抗原的表達。 / 結論：本論文證明了CLECs具有幹細胞樣特性但無致瘤性，具有低免疫原性和表皮分化的可塑性。研究結果支持CLECs在創傷癒合和皮膚再生領域的臨床應用可行性. / The skin is the largest organ in the body and has multiple functions. One of the most important functions is to serve as a protective barrier between the internal and external environments of the body. Restoration of the integrity of this protective barrier is an essential aspect of wound healing and tissue regeneration. In this thesis, the potential of human umbilical cord lining epithelial cells (CLECs) as a source of stem cells with appropriate differentiation capacity for epidermal reconstitution has been explored. / The isolation and propagation of CLECs from human umbilical cord lining epithelium were described in Chapter II. The cells presented a long telomere length and had high proliferative potential and passaging capability. They were also shown to display both epithelial and pluripotent stem cell markers. They were capable of multipotent differentiation, including adipogenesis, osteogenesis and chondrogenesis. However, they didn’t form teratoma after subcutaneous xenotransplantation until 12 weeks. / The immune properties of CLECs in vitro were assessed in Chapter III. The cells were shown to have low immunogenicity but high immunosuppressive function. They expressed classical major histocompatibility complex (MHC) class I antigens (HLA-ABC), but not MHC class II antigen (HLA-DR). They also expressed non-classical MHC class I antigens (HLA-G and HLA-E), but lacked the expression of the co-stimulatory molecules (CD40, CD80 and CD86). Moreover, they expressed moderate pro/anti-inflammatory cytokines and multiple growth factors both in cell supernatants and cell lysates. / The potential of CLECs for epidermal reconstitution was investigated in Chapter IV. In both organotypic culture and xenotransplantation model, CLECs were capable of generating a stratified epithelial structure, which is similar to that constructed by using keratinocytes. Furthermore, the expression of epidermal differentiation markers was verified in CLEC-constructed skin substitutes. / In conclusion, the stem cell-like properties of CLECs have been demonstrated in the present study. In addition to the lack of tumorigenicity, CLECs also have low immunogenicity and significant plasticity in epidermal differentiation. The findings support the potential clinical application of CLECs in wound healing and skin regeneration. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cai, Yijun. / "October 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 114-129). / Abstract also in Chinese. / Abstrac --- p.i / Table of Contents --- p.v / Abbreviations --- p.vii / List of Figures --- p.viii / List of Tables --- p.x / Chapter Chapter I --- Introduction --- p.1 / Skin --- p.3 / Wound healing --- p.6 / Wound regeneration and repair --- p.6 / Recent history of wound treatment --- p.9 / Skin substitutes --- p.11 / Stem cells for wound treatment --- p.14 / Stem cells overview --- p.15 / Adult stem cells --- p.16 / Fetal stem cells --- p.18 / Amniotic membrane derived stem cells --- p.19 / Umbilical cord stem cells --- p.22 / Hypothesis and Specific aims --- p.24 / Chapter Chapter II --- The Isolation and Characterization of the Stem Cell-like Properties of Human Umbilical Cord Lining Epithelial Cells --- p.28 / Introduction --- p.28 / Materials and methods --- p.30 / Results --- p.47 / Discussion --- p.62 / Conclusion --- p.67 / Chapter Chapter III --- The assessment of the Immune Properties of Human Umbilical Cord Lining Epithelial Cells --- p.69 / Introduction --- p.69 / Materials and methods --- p.72 / Results --- p.75 / Discussion --- p.83 / Conclusion --- p.88 / Chapter Chapter IV --- The Investigation of the Potential of Human Umbilical Cord Lining Epithelial Cells for the Epidermal Reconstitution --- p.89 / Introduction --- p.89 / Materials and methods --- p.91 / Results --- p.94 / Discussion --- p.101 / Conclusion --- p.104 / Chapter Chapter V --- Summary and Future Plan --- p.105 / Summary --- p.105 / Future plan --- p.108 / Acknowledgements --- p.113 / References --- p.114 / Appendix --- p.130

O transplante autólogo de células-tronco hematopoéticas (TACTH) tem se mostrado mais eficaz como tratamento das formas graves da esclerose sistêmica (ES) do que a imunossupressão convencional (IS), porém os mecanismos imunológicos envolvidos com a resposta terapêutica não estão completamente elucidados. Células mononucleares do sangue periférico e soro/plasma foram coletados de 31 pacientes com ES antes e semestralmente, até 36 meses pós-transplante, e de 16 pacientes com ES não-transplantados tratados com IS. A função tímica foi avaliada por RT-qPCR dos valores de b- e signal joint (sj)-T-cell receptor excision circles (TREC), sendo a taxa de proliferação intratímica (n) calculada pela fórmula: n=LOG(sjTREC/bTREC)/LOG2. A história replicativa das células B e a função medular foram quantificadas pelos valores de coding-joint (Cj) e sj-kappa-deleting recombination excision circles (sjKREC) e a taxa de proliferação das células B no sangue periférico (N) foi calculada pela fórmula: N=LOG(Cj/sjKREC)/LOG2. O comprimento telomérico foi avaliado por RT-qPCR e estimado pela razão T/S (Telomere repeat copy number/Single-copy gene copy number). As células recém-emigradas do timo (RET) CD3+CD4+CD31+CD45RA+, T reguladoras (Tregs) CD4+CD25hiFoxP3+(GITR+/CTLA-4+), naïve CD19+CD27-IgD+, Bm2 CD19+CD38lowIgD+, B reguladoras (Bregs) CD19+CD24hiCD38hi, senescentes CD8+CD28- CD57+ e exaustas PD1+ foram quantificadas por citometria de fluxo. O TCR foi sequenciado por sequenciamento de nova geração e o perfil de citocinas séricas inflamatórias e pró- fibróticas foi avaliado por CBA-Flex e ELISA. Observamos que os valores de sjTREC e bTREC diminuíram aos 6 meses pós-TACTH, retornando a valores basais aos 12 meses, correlacionando com o número de RET e promovendo maior diversidade do TCR. Não houve mudança na taxa de divisão de timócitos. A contagem de Tregs aumentou aos 12 meses pósTACTH, correlacionando com valores de sjTREC e apresentando maior expressão de GITR e CTLA-4. A partir dos 12 meses, até o final do acompanhamento, os valores de sjKREC aumentaram, enquanto que os de Cj permaneceram estáveis, correlacionando com aumento da contagem de células B naïve e Bm2, e resultando em uma menor taxa de divisão de células B. Houve aumento de Bregs de 6 meses a um ano após o TACTH, cujos níveis correlacionaramse com aqueles de sjKREC, e apresentando maior produção de IL-10 mediante estímulo com CPG±CD40L do que antes do transplante. O comprimento telomérico diminuiu aos 6 meses pós-TACTH e correlacionou-se com níveis elevados de células senescentes que expressavam FoxP3, aliado a um aumento de expressão de PD1 pelas células T e redução dos níveis séricos de IL-6, IL-1b e proteína C reativa. Seis pacientes recaíram após o transplante, apresentando menor expressão de FoxP3, GITR e CTLA-4 pelas Tregs, diminuição da contagem de Breg e da diversidade do TCR. Adicionalmente, a remissão clínica foi associada a maior expressão de PD1 por células T e B e baixos níveis séricos de TGF-b, IL-6, IL-1b, IL-17A, MIP-1a, GCSF e IL-12. Portanto, o aumento de células T e B reguladoras geradas de novo pós-TACTH, associado à renovação do repertório de células T, alta expressão de PD1 e baixos níveis séricos de mediadores inflamatórios e prófibróticos, estão relacionadas com a resposta clínica dos pacientes com ES ao transplante. / Autologous hematopoietic stem cell transplantation (AHSCT) is more effective for patients with severe systemic sclerosis (SSc) than conventional immunosuppression (IS). However, the immunological mechanisms associated with the therapeutic efficacy of AHSCT are not fully elucidated. Peripheral blood mononuclear cells and serum/plasma were collected from 31 SSc patients before and semiannually, until 36 months post-transplant, and from 16 nontransplanted SSc patients treated with IS. Thymic function was measured by RT-qPCR quantification of ?- and signal joint (sj)-T-cell receptor excision circles (sjTREC) and intrathymic T-cell division (n) was calculated by the formula: n=LOG(sjTREC/?TREC)/LOG2. Bcell replication history and bone marrow function were assessed by coding-joint (Cj) and sjkappa-deleting recombination excision circles (sjKREC). B-cell divisions in the peripheral blood (N) were calculated by the formula: N=LOG(Cj/sjKREC)/LOG2. CD3+CD4+CD31+CD45RA+ recent thymic emigrants (RTE), CD4+CD25hiFoxP3+ (GITR+/CTLA-4+) regulatory T-cells (Tregs), CD19+CD27-IgD+ naïve B-cells, CD19+CD38lowIgD+ Bm2 B-cells, CD19+CD24hiCD38hi regulatory B-cells (Bregs), CD8+CD28-CD57+ senescent cells and PD1+ exhausted cells were quantified by FACS (fluorescence-activated cell sorting). The T-cell receptor (TCR) was sequenced by New Generation Sequencing and the profile of inflammatory and pro-fibrotic serum cytokines was evaluated by CBA-Flex (cytometric bead-array) and ELISA (enzyme-linked immunosorbent assay). sjTREC and ?TREC values decreased at 6 months post-AHSCT, returning to pretransplant values at 12 months, correlating with RTE counts and associated with higher diversity of the TCR. There was no change in thymocyte division rates. At 12 months postAHSCT, Treg counts increased and correlated with sjTREC values, presenting increased expression of GITR and CTLA-4 when compared to pre-transplant levels. From 12 months until the end of follow-up, sjKREC values increased, while those of Cj remained stable, correlating with increased counts of naïve and Bm2 B-cells, resulting in reduced rate of B-cell division. There was an increase of Breg frequency from 6-months until one-year after AHSCT, correlating with sjKREC values and presenting higher IL-10 production after stimulation with CPG±CD40L than before transplantation. Telomere length decreased at 6 months post-transplant and correlated with elevated levels of FoxP3-expressing senescent cells, together with increased expression of PD1 by T-cells and reduced serum IL-6, IL-1b and C-reactive protein levels. Six patients relapsed after transplantation, presenting lower expression of FoxP3, GITR, CTLA-4 by Tregs, decreased Breg counts and reduced TCR diversity. In addition, clinical remission was associated with increased PD1 expression by T and B cells and low serum levels of TGF-?, IL-6, IL-1, IL-17A, MIP-1, G-CSF and IL-12. Therefore, newly-generated regulatory T and B cells after AHSCT, associated with T-cell repertoire renewal, high PD1 expression and low serum levels of inflammatory and profibrotic mediators associate with clinical outcomes of SSc patients after AHSCT.

A esclerose múltipla (EM) é uma doença autoimune desmielinizante progressiva imunomediada por linfócitos T auto-reativos, que provocam uma cascata imunológica, amplificando a inflamação local. No Diabetes mellitus tipo 1 (DM1), existem linfócitos T auto reativos destroem as células beta do pâncreas, causando deficiência na produção de insulina. O desenvolvimento de terapêuticas específicas fica limitado pela etiologia indefinida destas doenças, apesar de avanços na terapêutica antiinflamatória e imunossupressora. Uma alternativa de tratamento atual para tais doenças é o transplante autólogo de células tronco hematopoéticas (TACTH). O presente estudo, observacional do tipo transversal, com a coleta de dados de caráter retrospectivo, tem como objetivo identificar as reações adversas manifestadas pelos pacientes diabéticos ou de esclerose múltipla, submetidos ao TACTH no período de 2004 a dezembro de 2010. Para a coleta de dados elaborou-se dois instrumentos que foram submetidos à validação aparente e de conteúdo por três juízes. A amostra final do estudo foi constituída pela obtenção dos dados de 72 prontuários, sendo 23 de pacientes diabéticos e 49 de pacientes com EM. Em relação aos pacientes diabéticos 16 pertenciam ao sexo masculino e a idade média foi 18,26 anos. Todos possuíam positividade para o anticorpo anti-carboxilase do ácido glutâmico (antiGAD65). Quanto aos pacientes com EM, trinta e três pertenciam ao sexo feminino e idade média foi de 37,2 anos. O subtipo da doença mais frequente foi o surto-remissivo em 21 (42,9%) pacientes. A escala expandida do estado de incapacidade (EDSS) variou entre 3,0 e 6,5. Em relação às reações adversas manifestadas pelos pacientes diabéticos foram mais frequentes os calafrios, febre, cefaléia, náusea e vômito e nos pacientes com esclerose múltipla foram retenção hídrica e cefaléia. As principais intervenções de enfermagem identificadas para os pacientes diabéticos e com EM foram monitorização dos sinais vitais, coleta de hemocultura, otimização da administração de medicamentos antieméticos, controle da infusão da globulina antitimocitária, orientações sobre alimentação e para reduzir o risco de queda. Os pacientes com DM1 apresentam reações mais agudas e necessitam de monitorização contínua. Já os pacientes com EM são mais dependentes dos cuidados de enfermagem, exigindo maior tempo de cuidados prestados pelo profissional. Embora o DM1 e a EM sejam doenças distintas, percebe-se que na prática clínica, exigem do enfermeiro uma excelência no cuidado, quer pelas particularidades do tratamento realizado ou pelas singularidades de cada uma delas. / Multiple sclerosis (MS) is a progressive demyelinating autoimmune disease, immune- mediated by auto-reactive T lymphocytes, which provoke an immunological cascade, enhancing the local inflammation. In type 1 diabetes mellitus (DM1), self-reactive T lymphocytes exist that destroy ? cells in the pancreas, causing insulin production deficiency. The development of specific therapeutics is limited by these diseases\' undefined etiology, despite advances in anti-inflammatory and immunosuppressive therapy. A current treatment alternative for these diseases is autologous hematopoietic stem cell transplantations (AHSCT). The aim of this observational and cross-sectional study with retrospective data collection is to identify the adverse reactions manifested by diabetic or MS patients who were submitted to AHSCT between 2004 and December 2010. For data collection, two instruments were elaborated, submitted to face and content validation with the help of three experts. The final study sample comprised data from 72 patient files, 23 from diabetic and 49 from MS patients. As for the diabetic patients, 16 were male and the mean age was 18.26 years. All were positive for the anti-glutamic acid decarboxylase (antiGAD65) antibody. Concerning MS patients, 33 were female and the mean age was 37.2 years. The most frequent disease subtype was relapsing-remitting in 21 (42.9%) patients. The expanded disability status scale (EDSS) score ranged between 3.0 and 6.5. As for the adverse reactions the diabetic patients manifested, shivers, fever, migraine, nausea and vomiting were the most frequent, while fluid retention and migraine were the most frequent among multiple sclerosis patients. The main nursing interventions identified for the diabetic and MS patients were vital sign monitoring, blood culture collection, optimization of anti-emetic drug administration, control of anti- thymocyte globulin infusion, dietary orientations and advice to reduce the risk of falls. DM1 patients present more acute reactions and need continuous monitoring. MS patients are more dependent on nursing care, demanding lower professional care time. Although DM1 and MS are distinct conditions, in clinical practice, they demand excellent care from nurses, whether due to the particularities of the treatment or the singularities of each disease.

O transplante de células-tronco hematopoéticas é uma modalidade de tratamento baseada na infusão de células-tronco hematopoéticas. Para tanto, o paciente necessita de um cateter venoso central de longa permanência. Espera-se que este cateter permaneça in situ durante o tratamento, porém uma complicação frequente que leva a sua retirada é a ocorrência da infecção relacionada ao cateter. Neste contexto, a técnica de bloqueio/lock terapia pode ser usada na prevenção e controle da infecção relacionada ao cateter intravascular e consiste em manter o cateter sem uso durante um período de tempo com sua via preenchida por algum tipo de solução. No entanto, é uma técnica pouco estudada e com escassos resultados de sua efetividade. O presente estudo teve o objetivo de identificar a forma de utilização da técnica de bloqueio/lock terapia pela equipe de enfermagem nos centros de transplante de células-tronco hematopoéticas brasileiros cadastrados na Sociedade Brasileira de Transplante de Medula Óssea. Trata-se de um estudo descritivo de corte transversal. As variáveis foram analisadas por meio de estatística descritiva. A casuística compôs de 46 centros de transplante de células-tronco hematopoéticas, na qual foi enviado um questionário on-line composto de oito questões de múltipla escolha realizado no período de Junho a Novembro de 2014. A população respondente foi de vinte e dois centros (47,8%), com predomínio de dezoito (82%) centros da região sudeste. Observou-se que dezesseis (73%) dos centros conhecem a técnica de bloqueio e, desses, quatorze (87%) utilizam a mesma. Tal técnica é utilizada há mais de cinco anos por seis (43%) dos centros. A maioria dos centros respondentes utilizam a técnica para prevenção da infecção relacionada ao cateter venoso central e apenas cinco (36%) para o tratamento. Em relação à redução da taxa de infecção relacionada ao cateter, oito (57%) responderam que há redução, no entanto, na avaliação da eficácia na redução da infecção relacionada ao cateter, seis (43%) dos enfermeiros declararam-se parcialmente favorável ao uso da técnica de bloqueio. Independente do tipo de antibiótico utilizado seis (43%) utilizam antibiótico mais heparina e seis (43%) usam apenas vancomicina. Os resultados oferecem aos profissionais dos centros de transplantes de células-tronco hematopoéticas um panorama geral sobre o uso da técnica de bloqueio/lock terapia para os serviços, a fim de que conheçam uma estratégia que pode evitar a retirada precoce do cateter venoso central / Hematopoietic stem cell transplantation is a modality of treatment based on the infusion of hematopoietic stem cells, and for such procedure, the patient needs an indwelling long-term central venous catheter. This catheter is expected to remain in situ during treatment, however a frequent complication that leads to its removal is the occurrence of catheter-related infection. The therapy lock technique consists in keeping the catheter unused for some time with its route filled in by some type of solution, and it may be used to prevent and control intravascular catheter-related infection. Nonetheless, this technique is poorly studied and has few results as for its effectiveness. The aim of the present study was to identify the way the therapy lock technique has been used by the nursing staff of Brazilian hematopoietic stem cell transplantation centers registered in the Brazilian Society of Bone Marrow Transplantation. A descriptive cross-sectional study was conducted, and variables were analyzed by means of descriptive statistics. The sample of cases comprised forty six hematopoietic stem cell transplantation centers, which received an on-line questionnaire with eight multiple-choice questions, between June and November 2014. The respondent population was twenty two centers (47.8%), with prevalence of eighteen (82%) centers in the southeast region. The staff of sixteen (73%) centers knew the lock technique and, of these, the staff of fourteen (87%) used it. This technique has been used for over five years in six (43%) centers. Most of the centers used the technique to prevent catheter-related infection and only five (36%) used it for treatment. Regarding catheter-related infection rates, eight (57%) stated they were reduced, however, in the assessment of efficacy in the reduction of catheter- related infection, six (43%) nurses claimed to be partially favorable to the use of the lock technique. Regardless of the type of antibiotic used, six (43%) used antibiotic and heparin, and six (43%) used only vancomycin. Results provide professionals from the hematopoietic stem cell transplantation centers with a general panorama on the use of the therapy lock technique for the services, so they can learn a strategy that can prevent the early removal of the central venous catheter

Made available in DSpace on 2014-06-11T19:22:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-19Bitstream added on 2014-06-13T19:07:44Z : No. of bitstreams: 1 oliveira_lhb_me_arafcf.pdf: 553720 bytes, checksum: b0f8f04b07802f8bf467e43259c87fba (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / A maior expressão de alvos transcricionais e componentes da via NFkB é uma característica distintiva das células-tronco hematopoéticas (CTH) CD34+ de sangue de cordão umbilical (SCU) comparadas às CTH CD34+ de medula óssea (MO) e pode estar relacionada com o estado mais primitivo das CTH dos neonatos. No entanto, as células CD34+ são um grupo heterogêneo de célulastronco (CT) e progenitoras em diferentes estágios de maturação e diferenças na composição celular entre MO e SCU poderiam contribuir para os resultados mencionados. Estudos recentes têm identificado o marcador de superfície CD133, como um marcador de CT mais primitivas, expresso em uma subpopulação de células CD34bright, com um sugestivo potencial de hemangioblasto. Com o objetivo de caracterizar a composição celular de MO e SCU e identificar mecanismos moleculares envolvidos com a maior primitividade das células CD133+, propusemos avaliar o perfis imunofenotípico (quanto à expressão de CD34 e CD133) por citometria de fluxo e de expressão gênica de células CD34+ e células CD133+ selecionadas imunomagneticamente, de ambas as fontes, pelas técnicas de microarray e PCR em tempo real. Nossos resultados revelaram que enquanto a maioria das células CD133+ são CD34+, independente da fonte, as células CD34+ de SCU possuem uma porcentagem significativamente maior de células CD133+ do que às células CD34+ de MO. A análise de clusterização revelou que as células CD133+ de MO se agrupam com as células de SCU (CD34+ e CD133+), enquanto as CD34+ de MO aparecem como um grupo distinto. A comparação dos perfis de expressão gênica entre as células CD133+ e as células CD34+, revelou a hiper-expressão... / A higher expression of transcription targets and components of the NF-kB pathway is a distinctive feature of umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSC) when compared to bone marrow (BM) CD34+ HSC and this could be related to the more primitive state of the newborn’s HSC. However, CD34+ cells represent a heterogeneous group of cells composed by stem and progenitors cells in different developmental stages, and differences in cellular composition between both sources could contribute for these finding. The surface marker CD133 has been identified as a very primitive marker, expressed in a subpopulation of CD34bright, with a proposal hemangioblast potential. Thus, in attempt to better characterize the cellular composition of UCB and BM and to identify molecular mechanisms related to the more primitive characteristics of CD133+ cells, we proposed to evaluate the immunophenotypic profile (expression of CD34 and CD133) by flow-cytometry and the gene expression profiles of immunomagnetically selected CD34+ and CD133+ cells, from both sources, by microarray and Real time PCR. Our results highlighted that, while almost all CD133+ cells are CD34+ independently of the evaluated source, the UCB CD34+ cells showed a significantly higher proportion of CD133 expression, compared to BM CD34+ cells. After obtaining the expression profiles from distinct HSC pooled samples generated by microarrays, cluster analysis showed that BM CD133+ cells preferentially grouped with UCB cells (CD34+ and CD133+) instead of BM CD34+ cells, which appeared as a very distinct profile The comparison between CD133+ and CD34+ samples revealed the over-expression of 47 transcriptional factors (TF) in CD133+ cells, many of them well-known and related... (Complete abstract click electronic access below)

Submitted by Fabíola Silva (fabiola.silva@famerp.br) on 2017-02-14T12:02:30Z No. of bitstreams: 1 lidianemiottobarretta_dissert.pdf: 1992056 bytes, checksum: ac02b0ad50d5f0c3528b7470603f6213 (MD5) / Made available in DSpace on 2017-02-14T12:02:30Z (GMT). No. of bitstreams: 1 lidianemiottobarretta_dissert.pdf: 1992056 bytes, checksum: ac02b0ad50d5f0c3528b7470603f6213 (MD5) Previous issue date: 2014-10-31 / Introduction: Hematopoietic Stem Cell Transplantation is a long, complex, and high-cost process, which demands an appropriate hospital infrastructure and a qualified multidisciplinary team. The major cause of morbidity and mortality in transplanted patients is infection. The insertion of a semi-implantable, long-term central venous indwelling catheter poses a risk of complications, such as obstruction, fracture or breakage, migration, infection, and death. Objectives: The aims of the present study were to characterize the patients undergoing hematopoietic stem cell transplantation and to identify complications related to the central venous catheter and to describe the associated complications between the variables (sex, age, diagnosis, kind of transplantation, type of catheter, site of catheter insertion, and the length of time with the catheter. Methods: This was a retrospective, quantitative study conducted at a teaching hospital in inland of São Paulo State. Transplanted patients’ names were retrieved from the medical records and from the nursing forms. Files were reviewed retrospectively. The study involved the analysis of 188 patients’ files from 2007 to 2011. Association between variables was compared using the Chi-square test and the univariate analysis. Continuous variables were compared using Student’s t-test. Independent variables were analyzed using the Mann-Whitney test, Anova and the Kruskal-Wallis test. P = 0.05 was considered statistically significant. We used the correspondence analysis and multivariate technique to analyze the categorical variables. Results: Most patients were married man with age ranging from 40 to 60 years. They worked in general services and came from the cities of São Paulo and Mato Grosso States. They were patients from the Unified Health System (SUS) diagnosed with Hodgkin's lymphoma, multiple myeloma and non-Hodgkin lymphoma undergoing autologous transplantation. The major complications were constipation, diarrhea, urinary tract infection, respiratory infection and graft versus host disease. However, most patients survived the transplant and were discharged. The Hickman catheter was the most used catheter, with a mean length of time with the catheter of 47.6 days. The fever/bacteremia complication was more incident in young males with non-Hodgkin lymphoma and autologous transplantation, who remained with the catheter for a long period in the subclavian vein. Conclusion: Characterizing the patients’ profile, identifying the diagnosis, complications of treatment, the current situation of the post transplanted patients, provide grants to nursing professionals in search of a safer practice with actions related to prevention. Complications of catheter and its relation to age, gender, medical diagnosis, type of transplant, catheter model, mean residence time and insertion site should be analyzed considering the conditioning protocol, which directly affects the occurrence of complications and injury Related catheter. / Introdução: O transplante de células tronco hematopoéticas é um processo longo, complexo, de alto custo, que exige infraestrutura hospitalar apropriada e equipe multiprofissional qualificada. As maiores causas de morbidade e mortalidade de pacientes transplantados são as infecções. Faz parte do transplante a inserção de um cateter venoso central semi-implantável de longa permanência, com risco de complicações como obstrução, fratura ou ruptura, migração, infecção e morte. Objetivos: Caracterizar os pacientes submetidos ao transplante de células tronco hematopoéticas; identificar as intercorrências associadas ao cateter venoso central e descrever a relação de correspondência entre as complicações e as variáveis: sexo, idade, diagnóstico médico, tipo de transplante, modelo de cateter, local de inserção e tempo de permanência com o cateter. Método: Estudo retrospectivo, quantitativo, realizado em um hospital de ensino do interior do estado de São Paulo, por meio do prontuário e planilhas de enfermagem de 188 pacientes transplantados entre 2007 e 2011. Foram realizados testes estatísticos de associação (qui-quadrado), de comparação univariada, teste T, para amostras independentes, Mann-Whitney, ANOVA e Kruskal-Wallis, todos ao nível de significância 0,05 e análise de correspondência e técnica multivariada para as variáveis categóricas. Resultados: A maioria dos pacientes era do sexo masculino, idade 40 a 60 anos, casada, trabalhavam na área de serviços gerais, procedentes de cidades do estado de São Paulo e Mato Grosso, atendidos pelo SUS, com diagnóstico de linfoma de Hodgkin, mieloma múltiplo e linfoma não Hodgkin, submetidos ao transplante autólogo. As complicações foram: constipação, diarreia, infecção urinária, infecção respiratória e doença do enxerto contra o hospedeiro, entretanto, a maioria sobreviveu ao transplante e teve alta hospitalar. O Hickman foi o cateter mais utilizado, com permanência média de 47,6 dias. A complicação febre/bacteremia mais incidente em jovens, sexo masculino, com Linfoma não Hodgkin e transplante autólogo, que permaneceram com o cateter por longo período, em veia subclávia. Conclusão: Caracterizar o perfil, identificar o diagnóstico, as complicações decorrentes do tratamento, a situação atual do paciente pós transplante, oferece subsídios aos profissionais de enfermagem na busca de uma prática mais segura. As complicações do cateter e suas relações com idade, sexo, diagnóstico médico, modalidade de transplante, modelo de cateter, tempo médio de permanência e local de inserção, devem ser analisadas considerando o protocolo de condicionamento, que interfere diretamente na ocorrência e agravo das complicações relacionadas ao cateter.

Submitted by (edna.saturno@ufrpe.br) on 2017-02-22T12:37:59Z No. of bitstreams: 1 Suely Santos Bezerra.pdf: 418523 bytes, checksum: 6cbf43e2cf1c398824dd661f7ea8c038 (MD5) / Made available in DSpace on 2017-02-22T12:37:59Z (GMT). No. of bitstreams: 1 Suely Santos Bezerra.pdf: 418523 bytes, checksum: 6cbf43e2cf1c398824dd661f7ea8c038 (MD5) Previous issue date: 2007-02-23 / It can be observed nowadays that the increase in the commercial shrimp produc-tion occurs concomitantly with an enhancement of the incidence of diseases. These last ones can cause innumerable damages to the shrimp farmers in the whole world. Amongst these diseases, it can be distinguished the Infectious Hypodermal and Hema-topoietic Necrosis (IHHN), with viral etiology, that can be characterized by not causing great mortality, but body deformities. In addition to this, it frequently leads shrimp to heterogenic growth. For viruses diagnosis histology is an efficient tool, allowing the verification of the health of these crustaceans. This study aimed the observation of the occurrence of IHHN characteristic histopatologic injuries in Litopenaeus vannamei, and its relation with parameters of culture of marine shrimp farms in the state of Per-nambuco. After multiple regression statistic analysis it could be concluded that the tem-perature of the water of the pound and its oxygen availability are narrowly related with the occurrence of those histopatologic injuries. / Ao mesmo tempo em que crescem os cultivos comerciais de camarão, observa-se o aumento da incidência de enfermidades que podem causar inúmeros prejuízos aos carcinicultores em todo o mundo. Dentre essas enfermidades, destaca-se a Infecção Hi-podermal e Necrose Hematopoiética (IHHN), de etiologia viral que, embora não cause grandes taxas de mortalidade, produz retardo no desenvolvimento e deformidades nos camarões. Para o diagnóstico dessa virose a histologia tem sido uma ferramenta bastante eficiente e que permite maior monitoramento da sanidade desses crustáceos. Desta for-ma, objetivou-se estudar a IHHN em Litopenaeus vannamei relacionando lesões histo-patológicas características e sua relação com variáveis de cultivo. Com base em regres-sões múltiplas conclui-se que existe uma alta relação entre as variáveis de cultivo tem-peratura e oxigênio e a ocorrência destas lesões.

Le Dimethyl Ampal Thiolester (DIMATE) est un inhibiteur des aldéhydes déshydrogénases (ALDHs) de type 1 et 3. L’intérêt croissant au cours de ces dernières années pour ces enzymes intra cytoplasmiques que sont les ALDHs, s’explique par leurs utilisations comme marqueurs pour distinguer les cellules souches, saines ou cancéreuses, au sein de différents tissus, comme le tissu hématopoïétique. Le traitement des Leucémies Aiguës Myéloïdes demeure une problématique clinique majeure. En effet, malgré un taux de rémission complète moyen d’environ 70% avec les traitements conventionnels, la survie moyenne des patients porteurs d’une LAM n’excède pas les 50% à 5 ans. A ce titre, le DIMATE, semble être un médicament d’avenir. Le DIMATE présente une toxicité majeure sur plusieurs lignées leucémiques humaines et sur des cellules souches leucémiques issues de patients. De manière remarquable, le DIMATE à ces doses anti-leucémiques présente une innocuité quasi-totale sur les cellules souches hématopoïétiques saines. In vivo, chez la souris, le DIMATE permet d’éradiquer spécifiquement les cellules leucémiques humaines xénogreffées et présente en monothérapie une efficacité similaire à l’association Cytarabine + Daunorubicine qui constitue le standard thérapeutique actuel. Ces résultats encourageants vont servir de support conceptuel à la mise en place prochaine d’essais cliniques. / The Dimethyl Ampal Thiolester (DIMATE) is a type 1 and 3 Aldehydes Dehydrogenases (ALDHs) inhibitor as an innovating treatment for AML. Interest in ALDH is due to its activity as a marker for identification of stem cell in different tissues. The different species of ALDHs control the levels of the endogenous apoptogenic aldehydes. Cancer cells protect themselves from the apoptogenic effect of these aldehydes by the ALDHs that oxidize them to their non-apoptogenic carboxylic acids. The vast majority of patients with AML achieve complete remission (CR) after standard induction chemotherapy. However, the majority subsequently relapses and dies of the disease. Therefore, AML remains a clinical challenge and new therapies are urgently needed. For this, DIMATE appears as a promising drug. In vitro, DIMATE is a powerful ALDH inhibitor and has a major cytotoxic activity on human AML cell lines. Moreover, DIMATE is highly active against leukemic population enriched in LSCs, but, unlike conventional chemotherapy, DIMATE is not toxic for healthy hematopoietic stem cells which retained after treatment their self-renewing and multi-lineage differentiation capacity. In immunodeficient mice, xenografted with human leukemic cells, DIMATE eradicates specifically human AML cells and spares healthy mouse hematologic cells. Moreover, DIMATE showed the same efficiency than the association Daunorubicin + Cyrabine, which is considered as the gold standard for AML induction. Results from our work open new therapeutic perspectives in AML and provide a conceptual support for initiation of a phase I-II clinical trials, but also innovating cellular therapy.

Há evidências de que a ciclosporina, imunossupressor utilizado nos transplantes de células- tronco hematopoéticas, impregna nos cateteres de silicone quando os mesmos são utilizados para a sua infusão; podendo a coleta de amostras para dosagem sérica do medicamento por essa via resultar em níveis falsamente elevados. Apesar de já existirem dados comprovando a possibilidade de se coletar as amostras da via do cateter venoso central não utilizada para a infusão, há escassez de estudos e também controvérsias quanto ao melhor momento para realizar o procedimento, assim como divergências quanto ao volume de sangue ideal a ser descartado. Esse ensaio clínico controlado randomizado teve como objetivo verificar o efeito do tempo transcorrido entre a interrupção da infusão de ciclosporina e a coleta das amostras na dosagem sérica do medicamento, em relação à via utilizada para a coleta e ao volume de descarte. Os sujeitos foram aleatorizados em dois grupos. No grupo A, a coleta das amostras em acesso venoso periférico, via do cateter utilizada para a infusão da ciclosporina e via não utilizada para infusão foi realizada imediatamente após a interrupção da infusão do medicamento, sendo que na última a coleta foi realizada após descarte de 5 mL e de 10 mL de sangue. No grupo B os mesmos procedimentos foram realizados, porém cinco minutos depois da interrupção. Participaram 32 sujeitos adultos, a maioria do sexo masculino (68,75%), portadores de leucemia (59,37%), com doadores aparentados (84,37%) e histocompatibilidade total (90,62%). A coleta realizada previamente ao início da infusão da ciclosporina atestou ausência desta no sangue e impregnada nos cateteres. As demais foram realizadas nos 32 sujeitos depois de 24 horas do início da infusão; em 12 do grupo A e 16 do grupo B sete dias depois; e em nove do grupo A e 13 do B 14 dias após iniciado o uso do medicamento. O principal motivo que levou à interrupção da coleta foi a transição da ciclosporina para via oral (71,87%). Inicialmente, nas análises intra sujeitos, a diferença entre a dosagem sérica obtida na via do cateter utilizada para a infusão da ciclosporina e as outras vias foi significativa (p < 0,001), enquanto entre o acesso venoso periférico e via não utilizada para a infusão, independente do volume de descarte, não houve diferença (p > 0,05). Quando realizadas as comparações entre os grupos, não foram observadas diferenças (p > 0,05) quanto à influência do tempo transcorrido entre a interrupção da infusão e a coleta das amostras de sangue, independente da via de coleta utilizada e do volume descartado. Conclui-se que a via do cateter não utilizada para infusão da ciclosporina é segura para ser utilizada na coleta das amostras para dosagem sérica desse medicamento; e o procedimento pode ser realizado imediatamente após a interrupção da infusão, desde que empregada a técnica adequada com descarte de 5 mL de sangue. Assim evita-se que o sujeito, já fragilizado e submetido a um tratamento complexo, seja exposto a mais um procedimento doloroso e associado ao estresse que é punção venosa periférica / There is evidence that cyclosporine, an immunosuppressant used in hematopoietic stem cell transplantation, impregnate in silicone catheters when they are used for its infusion; and the sample collection for serum levels of medication through this line may result in falsely elevated levels. Although there are data demonstrating the possibility to collect samples through the line of the central venous catheter not used for the infusion, there are few studies and also controversies regarding the best time to perform the procedure, as well as disagreement about the optimal volume of blood to be discarded. This randomized controlled trial aimed to verify the effect of time elapsed between discontinuation of the infusion and serum sample collection in relation to the line used for collection and volume of discard. The subjects were randomized into two groups. In group A, samples collected from a peripheral venous access, through catheter line used for cyclosporine infusion and catheter line not used for cyclosporine infusion was performed immediately after discontinuation of the drug infusion, and the last collection was performed after discarding 5ml and 10ml of blood. In group B, the same procedures were done, but five minutes after the interruption. The participants were 32 adults, most males (68,75%), with leukemia (59,37%), with related donors (84,37%), and total histocompatibility (90,62%). The collection performed prior to the start of cyclosporine infusion attested absence of cyclosporine in blood and presence of it in catheters. The others were performed in 32 subjects after 24 hours prior the start of infusion; in 12 of group A and 16 of group B after seven days; and in 9 of group A and 13 of B fourteen days after starting the medication. The main reason that led to discontinuation of the collection was the switch of cyclosporine to oral administration (71,87%). Initially, in the intra-subject analysis, the difference between the serum levels obtained in the line used for cyclosporine infusion of and other lines was significant (p < 0,001), while there was no difference between the peripheral venous access and the line not used for infusion, independently of the volume of discard (p > 0,05). When performed comparisons between groups, no differences were observed (p > 0,05) in the influence of time elapsed between discontinuation of the infusion and collection of blood samples, regardless the line used for collection and volume of discard. It is concluded that the catheter line not used for infusion of cyclosporine seems to be safe for use in serum samples collection of this medication; and the procedure can be performed immediately after discontinuation of the infusion, since used the technique with adequate discard of 5ml of blood. This avoids that the subject, already weakened and subordinated to a complex treatment, be exposed to a painful and stressful procedure such as peripheral venipuncture