Introdução: A técnica de criopreservação de tecido ovariano tem sido vista como tratamento promissor e se apresenta como a principal maneira de preservar a fertilidade em pacientes pré-púberes e em mulheres que necessitam de tratamento do câncer de imediato. Contudo, atualmente, ainda existem obstáculos em relação ao autotransplante de tecido ovariano criopreservado, devido a fatores como lesão isquêmica, assim como danos causados pelo processo durante o congelamento, bem como a escolha do melhor local para o enxerto. Objetivo: Verificar a possível restauração da função ovariana, analisando a histologia do ovário transplantado em ratas adultas estéreis, após transplante autólogo de tecido ovariano criopreservado em fase pré-púbere. Métodos: Foram utilizadas 45 ratas Wistar com 30 dias de idade, que foram divididas aleatoriamente em três grupos: Grupo Controle (n = 15), férteis normais; Sham (n = 15), submetidas à ooforectomia bilateral; Transplante (n = 15), submetidas à ooforectomia bilateral, seguida de transplante autólogo na região dorsal entre as escápulas. A partir do d35, foram realizadas observações quanto à maturidade sexual, através da análise da abertura vaginal e de esfregaços vaginais, para avaliação do ciclo estral. Após observação da fase do ciclo estral, os animais foram eutanasiados. E, amostras de tecidos foram coletadas e processadas para avaliação histológica dos implantes ovarianos; considerando: organização estrutural do tecido transplantado e adjacente, bem como o desenvolvimento folicular. Resultados: Quanto às avaliações de maturidade sexual, através das análises de abertura vaginal e da análise microscópica do material obtido dos esfregaços vaginais, foi possível observar que os animais do Grupo Controle, que eram férteis ciclaram normalmente. As ratas do Grupo Sham e Transplante não apresentaram ciclo regular, permanecendo em diestro. As avaliações histológicas das amostras de tecido de ovário pré-púbere, implantados em fêmeas adulto jovens, evidenciaram degeneração ovariana; uma vez que estes apresentaram fibrose e áreas de necrose, o que provavelmente impossibilitou o desenvolvimento folicular, nas ratas que receberam o transplante. Conclusão: A técnica de transplante de tecido ovariano em ratas é uma técnica relativamente simples de ser executada, e se mostrou eficaz na manutenção do massa corporal dos animais durante o período observado. Este achado sugere que houve produção hormonal, oriunda do ovário transplantado, fato este que encoraja as pesquisas neste sentido, a fim de se obter uma técnica que restaure a produção de folículos viáveis em pacientes estéreis. Apesar de ter apresentando indícios de falência do enxerto e isquemia no tecido transplantado, os resultados preliminares desta investigação precisam ser complementados com estudos adicionais, a fim de buscar as melhores condições para a obtenção de maior eficácia dos transplantes autólogos de tecido ovarianos criopreservados. / Introduction: Ovarian tissue cryopreservation is a promising treatment and it is presented as the main way to preserve fertility in prepubertal patients and women who need cancer treatment immediately. However still remain obstacles related to the ovarian tissue cryopreserved autograft due to ischemic injury, damage caused by the freezing process and selecting the best location for the graft. Objective: Investigate a possible restoration of the ovarian function by analyzing the histology of the ovary transplanted into sterile adult rats after autologous transplantation of ovarian tissue cryopreserved in prepubertal phase. Methods: 45 Wistar rats, 30 days old,which were randomly divided into three groups: control group (n = 15), normal fertile; Sham group (n = 15), underwent bilateral oophorectomy; Transplantation group (n = 15), underwent bilateral oophorectomy followed by autologous transplantation in the scapular area. From the d35, sexual maturity was observed by examining the vaginal opening and vaginal smears, for evaluation of the estrous cycle. After observing the phase of the estrous cycle, the animals were euthanized. The tissue samples were collected and processed for histological evaluation of ovarian implants; where structural organization of the transplanted tissue and adjacent as well as follicular development were analyzed. Results: Regarding sexual maturity evaluations, observed by vaginal opening analysis and microscopic analysis of material obtained from vaginal swabs, we could observe that the animals in the control group cycled normally. The rats of Sham and Transplant Group showed no regular cycle, staying in diestrus phase. The histological assessments of prepubertal ovarian tissue samples implanted in young adult females showed ovarian degeneration, since they had areas of necrosis and fibrosis, which probably impeded the follicular development in these rats. Conclusion: The ovarian tissue transplantation technique in rats is a relatively simple technique, and is effective in body mass maintenance of animals during the observed period. This finding suggests that there were hormone production originated from the transplanted ovaries, and this, encourages research in order to obtain a technique to restore the production of viable follicles in sterile patients. Despite presenting evidence of graft failure and ischemia in the transplanted tissue, the preliminary results of this investigation need to be supplemented with additional studies in order to get the best conditions for achieving greater effectiveness of autologous transplantation of cryopreserved ovarian tissue.

O desequilíbio no turnover ósseo decorrente dos efeitos do alcoolismo crônico está relacionado à apoptose dos osteócitos, diminuição da espessura do osso medular e cortical e pelas alterações na atividade e diferenciação de osteoblastos. Devido à necessidade de tratamentos regenerativos e reconstrutivos associado ao alcoolismo, os xenoenxertos tem providenciado uma possibilidade terapêutica como material de preenchimento de defeitos ósseos. Em vista disso, este trabalho teve como objetivo analisar o comportamento do biomaterial Bonefill® em defeitos críticos realizados em calvária de ratos, comparar a interferência do alcoolismo experimental na neoformação óssea, além de avaliar a influência do tipo de dieta líquida sobre a massa corporal dos animais. Foram utilizados 40 ratos machos (Rattus norvegicus) da linhagem Wistar, com 60 dias de idade, separados aleatoriamente em Grupo Controle (GC) os animais receberam água como dieta líquida e Grupo Experimental (GE) os animais receberam etanol a 25%, cada grupo composto por 20 ratos. O GC foi sub-dividido em GAC (Grupo Água Coágulo) correspondente ao defeito direito na calota craniana do animal e GAB (Grupo Água Biomaterial), defeito do lado esquerdo do animal. O GE foi sub-dividido em GEC (Grupo Etanol Coágulo) correspondente ao defeito direito da calota craniana e GEB (Grupo Etanol Biomaterial) defeito do lado esquerdo do animal. Foi realizada uma osteotomia circular de 5 mm de diâmetro no osso parietal direito e esquerdo. Os defeitos GAC e GEC foram preenchidos com coágulo sanguíneo e os defeitos GAB e GEB com osso bovino cortical inorgânico. Cinco animais de cada grupo foram eutanasiados nos períodos de 10, 20, 40 e 60 dias pós-cirúrgico. Os resultados da análise histológica mostraram que no período de 10 dias, no GAB e GEB as partículas do enxerto ósseo estavam circundadas por tecido de granulação e células inflamatórias, apresentou uma pequena formação de osso imaturo principalmente nas margens do defeito. No período de 20 dias foi obsevado presença de vasos sanguíneos e no GAB e GEB algumas partículas parcialmente circundadas por tecido ósseo neoformado. Os períodos de 40 e 60 dias exibiram áreas de formação óssea nas margens e ao redor de algumas partículas. O osso neoformado encontrava-se em um estágio mais avançado de remodelação, porém sem preenchimento completo do defeito e sem sinais de reabsorção da superfície do biomaterial. Na análise histomofométrica o percentual de formação óssea entre o GAC versus GEC e GAB versus GEB, para cada período experimental, não apontou diferença estatisticamente significante. Com relação à massa corporal o GC, nos períodos de 20, 40 e 60 dias pós-cirurgia a massa corporal aumentou, 7,9%, 6,6% e 14,1%, respectivamente. No GE ocorreu uma perda de 9,2% da massa corporal após 10 dias da cirurgia, e aos 40 e 60 dias ocorreu ganho de massa corporal de respectivamente 5,9% e 6,4% nos animais. Conclui-se que o biomaterial Bonefill® não promoveu uma maior neoformação óssea em defeitos críticos; a dieta alcoólica não atingiu os seus efeitos nocivos na formação óssea, e a dieta líquida de etanol, quando comparada à de água, interferiu negativamente na massa corporal dos animais. / The imbalance in bone turnover resulting from the effects of chronic alcoholism is related to apoptosis of osteocytes, decrease of trabecular and cortical bone thickness and changes in activity and differentiation of osteoblasts. Due to the need for regenerative and reconstructive treatments associated with alcoholism, xenografts has provided a therapeutic possibility as a filling material of bone defects. Therefore, the aims of this study were to analyze the Bonefill® biomaterial behavior in critical defects created on the calvaria of rats; to compare the interference of experimental alcoholism in bone formation, as well as to evaluate the influence of 2 type of liquid diet on animal body weight. 40 male Wistar rats were used (Rattus norvegicus), with 60 days of age. The rats were randomly separated into Control Group (CG, nº = 20), which received water as liquid diet and Experimental Group (EG, nº = 20), these rats consumed ethanol 25%. CG was subdivided into WCG (Water Clot Group), corresponding to the right calvaria defect and WBG (Water Biomaterial Group), left side defect of the calvaria. EG was subdivided into ECG (Ethanol Clot Group), corresponding to the right calvaria defect and EBG (Ethanol Biomaterial Group), left side defect of the calvaria. A circle osteotomy of 5 mm in diameter was performed in the right and left parietal bone. The bone defects in WCG and ECG were filled with blood clot and the defects in WBG and EBG were filled with inorganic bovine bone. 5 animals from each group were euthanized at periods of 10, 20, 40 and 60 days after surgery. The histological analysis showed that at 10 days, WBG and EBG, the particles of bone graft were surrounded by granulation tissue and inflammatory cells, it also showed a small immature bone formation mainly at the margins of the defects. At 20 days it was observed blood vessels in WBG and EBG and some graft particles partially surrounded by new bone. At 40 and 60 days it was exhibited some areas of bone formation at the margins and around a few particles. The newly formed bone was in a more advanced stage of bone remodeling, however it did not showed neither complete filling nor signs of resorption of the biomaterial surface. Histomorphometric analysis on the percentage of bone formation between WCG versus ECG and WBG versus EBG, for each trial period, showed no statistical significant difference. Regarding the body mass CG, at 20, 40 and 60 days the body mass increased 7.9%, 6.6% and 14.1%, respectively. The EG had a loss of body weight of 9.2% after 10 days of surgery and at 40 and 60 days it showed a body mass gain of 5.9% and 6.4%, respectively. It can be concluded that Bonefill® did not promote an increase bone formation in critical defects; the alcohol diet did not achieve its adverse effects on bone formation, and the liquid diet containing ethanol, compared to the liquid water diet had a negative effect on animal body weight.

Foram analisados 106 pacientes portadores de Linfoma de Hodgkin (LH) com recidiva da doença ou refratários ao tratamento inicial que foram submetidos ao transplante autólogo de células tronco hematopoiéticas (TCTH), na ordem consecutiva de sua realização, entre o mês de abril de 1993 a dezembro de 2007 em um único Centro Brasileiro: o Serviço de Transplante de Medula Óssea da FMUSP. O grupo teve a mediana etária de 28 anos, 55 pacientes (51,9%) eram do sexo masculino e houve predomínio da raça branca (87,6%). A mediana de seguimento clínico foi de 56,4 meses. Todos pacientes foram submetidos no pré TCTH a protocolos de tratamento convencionais, sendo que o uso dos protocolos MOPP, ABVD e sua associação foram utilizados em 97 pacientes (91,5%). Os pacientes foram classificados, de acordo com a resposta ao tratamento utilizado antes do TCTH, sendo 38,1% considerados refratários e 61,9% responsivos. Dos responsivos, 54 pacientes estavam em segunda remissão completa (85%). Tratamento com quimioterapia em alta dose pré TCTH foi feito em 45 (42,4%) dos pacientes (salvamento). A mobilização das células tronco hematopoiéticas foi realizada com ciclofosfamida 120 mg/kg dividida em dois dias seguido de fator estimulador de colônias de granulócitos (G-CSF) na dose de 6 a 17 mcg/kg em 83 (78%) pacientes. Em 25 pacientes (22%) houve falha de mobilização e a coleta foi realizada por múltiplas punções da medula óssea em centro cirúrgico. O valor mediano de células CD 34 coletadas foi de 2,6 x 106 células CD34/Kg de peso do paciente. Os condicionamentos mais utilizados foram BEAM e CVB, e não se encontrou diferença na sobrevida em relação ao regime empregado (p=0,17). A mediana de enxertia das células transplantadas foi de 12 dias. A sobrevida global após o TCTH pelo método de Kaplan-Meier foi, respectivamente, de 86% e 70% aos 5 e 10 anos. Não influenciaram a sobrevida na análise univariada o sexo, o estadio da doença e a presença de massa tumoral extensa. O principal fator preditivo de melhor sobrevida foi a presença de resposta a quimioterapia pré TCTH (p=0,0095) e hemoglobina maior que 10g/dL ao diagnóstico (p=0,0229). A mortalidade relacionada ao procedimento até o centésimo dia após o TCTH foi de 3,74%, e a principal causa de mortalidade tardia após TCTH foi a recidiva da doença / The study enrolled 106 patients with classic Hodgkin disease (HD) refractory or relapsed after initial treatment who underwent to autologous hematopoietic stem cell transplantation (HSCT) between April 1993 and December 2007. Median age was 28 years and 55 (51,9%) patients were male. Ninety three (87,6%) of patients were white. All patients underwent to conventional chemotherapy protocols prior HSCT. The use of MOPP, ABVD protocols and their associations were used in 97 (91,5%) of the patients. Disease classification was done according to the response to initial treatment and comprised 38,1% refractory and 61,9% responsive patients. In the group of responsive, 54 (85%) patients were in second complete remission. High dose chemotherapy prior HSCT was done as salvage in 45 (42,4%) patients. Stem cell mobilization was done after cyclophosphamide 120mg/kg divided in two days. Granulocyte-colony stimulating factor (G-CSF) 617 mcg/kg was given after cyclophosphamide in 83 (78%) patients. Twenty five (22%) patients failed the mobilization and stem cell harvest was done by bone marrow aspirations. The median number of CD34 collected was 2.6 x 106/L. Preparative regimen mostly used comprised BEAM and CVB and no differences was observed in overall survival (p=0.17). Median time to engraftment was 12 days. Median time of follow-up was 56.4 months. The overall survival (OS) was calculated by the Kaplan-Meier method and was 86% and 70% at 5 and 10 years, respectively. In the univariate analysis, response to initial treatment (p=0.009) and hemoglobin greater than 10g/dL at the time of diagnosis (p=0.02) were factors that influenced better OS. The gender, stage of disease and presence of bulky disease were not significant regarding OS in the univariate analysis. Treatment-related mortality (TRM) in 100-days was 3.74%. The major cause of late mortality was relapse of the disease

Lau Tung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 142-157). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Abreviations --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Pancreas and its functions --- p.1 / Chapter 1.1.1 --- Structure of pancreas --- p.1 / Chapter 1.1.2 --- Exocrine function --- p.4 / Chapter 1.1.3 --- Endocrine function --- p.7 / Chapter 1.1.3.1 --- Pancreatic islet and islet cells --- p.7 / Chapter 1.1.3.2 --- Regulation of insulin secretion --- p.10 / Chapter 1.1.3.3 --- Mechanism for glucose-stimulated insulin release --- p.14 / Chapter 1.1.3.4 --- Bi-phase response of insulin secretion --- p.16 / Chapter 1.2 --- Pancreatic Renin-Angiotensin System --- p.19 / Chapter 1.2.1 --- Circulating RAS and local RAS --- p.19 / Chapter 1.2.2 --- RAS inhibitors --- p.25 / Chapter 1.2.2.1 --- Angiotensin converting enzyme inhibitor --- p.25 / Chapter 1.2.2.2 --- Non-specific Ang II receptor blocker --- p.28 / Chapter 1.2.2.3 --- Specific AT1 receptor antagonist --- p.29 / Chapter 1.2.2.4 --- Specific AT2 receptor antagonist --- p.30 / Chapter 1.2.3 --- RAS and Pancreas --- p.30 / Chapter 1.2.3.1 --- Expression and localization of pancreatic RAS --- p.30 / Chapter 1.2.3.2 --- Regulation of pancreatic RAS and its clinical relevance --- p.32 / Chapter 1.3 --- Islet Transplantation and RAS --- p.34 / Chapter 1.3.1 --- Whole pancreas and islet transplantation --- p.34 / Chapter 1.3.2 --- Problems encountered in islet transplantation --- p.36 / Chapter 1.3.3 --- Potential role of RAS in islet transplantation --- p.38 / Chapter 1.4 --- Diabetes Mellitus and RAS --- p.40 / Chapter 1.4.1 --- Diabetes Mellitus --- p.40 / Chapter 1.4.2 --- Type 1 diabetes and its animal model --- p.42 / Chapter 1.4.3 --- Type 2 diabetes and its animal model --- p.44 / Chapter 1.4.4 --- RAS blockade in diabetes patients --- p.46 / Chapter 1.4.5 --- Potential role of RAS in Diabetes Mellitus --- p.47 / Chapter 1.5 --- Aims of Study --- p.49 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Experimental animals and mouse models --- p.50 / Chapter 2.1.1 --- Experimental animals for islet isolation and transplantation --- p.50 / Chapter 2.1.2 --- Mouse model for type 2 diabetes --- p.51 / Chapter 2.2 --- Islet isolation and transplantation --- p.52 / Chapter 2.2.1 --- Enzymatic islet isolation --- p.52 / Chapter 2.2.2 --- Islet transplantation --- p.53 / Chapter 2.3 --- Biological assay on islet functions --- p.53 / Chapter 2.3.1 --- Measurement of islet insulin release --- p.53 / Chapter 2.3.2 --- Measurement of islet glucose oxidation rate --- p.56 / Chapter 2.3.3 --- Measurement of islet (pro)insulin biosynthesis --- p.59 / Chapter 2.3.4 --- Measurement of islet total protein synthesis --- p.60 / Chapter 2.4 --- Chronic losartan treatment --- p.62 / Chapter 2.5 --- Perfusion experiment of transplanted islet graft --- p.62 / Chapter 2.6 --- Insulin content of the islet graft --- p.63 / Chapter 2.7 --- Islet graft (pro)insulin and total protein biosynthesis --- p.64 / Chapter 2.8 --- Real-time RT-PCR Analysis --- p.64 / Chapter 2.8.1 --- Design of primers and probes --- p.67 / Chapter 2.8.2 --- Use of internal control --- p.69 / Chapter 2.8.3 --- RT-PCR reaction --- p.69 / Chapter 2.8.4 --- Calculation using the comparative CT method --- p.70 / Chapter 2.9 --- Western Blot Analysis --- p.71 / Chapter 2.10 --- Immunocytochemistry --- p.72 / Chapter 2.11 --- Statistical data analysis --- p.73 / Chapter Chapter 3 --- Results / Chapter 3 .1 --- Effect of Angiotensin II and Losartan on islet insulin release --- p.74 / Chapter 3.1.1 --- Insulin release from normal islets --- p.74 / Chapter 3.2 --- "Effect of Angiotensin II and Losartan on islet glucose oxidation rate, (pro)insulin and total protein biosynthesis" --- p.77 / Chapter 3.2.1 --- Glucose oxidation rate of isolated normal islets --- p.77 / Chapter 3.2.2 --- (pro)insulin and total protein biosynthesis of isolated normal islets --- p.77 / Chapter 3.3 --- Regulation of RAS components in islet transplantation --- p.81 / Chapter 3.3.1 --- Expression of RAS components in endogenous islets and transplanted islets --- p.81 / Chapter 3.3.2 --- Localization of AT1-receptor in endogenous islets --- p.87 / Chapter 3.3.3 --- Expression of AT1-receptor protein in endogenous and transplanted islets --- p.89 / Chapter 3.3.4 --- Relative abundance of RAS components in kidney and liver --- p.91 / Chapter 3.3.5 --- Insulin release from perfused transplanted islet graft --- p.93 / Chapter 3.3.5 --- (pro)insulin and total protein biosynthesis of transplanted islet graft --- p.96 / Chapter 3.4 --- Effect of Angiotensin II and losartan on diabetic islets --- p.99 / Chapter 3.4.1 --- Expression of RAS components in diabetic pancreas --- p.99 / Chapter 3.4.2 --- Localization of AT1 receptors in diabetic pancreas --- p.105 / Chapter 3.4.3 --- Insulin release from islets of type 2 diabetic mice --- p.107 / Chapter 3.4.4 --- (pro)insulin and total protein biosynthesis of islets from type 2 diabetic mice --- p.112 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Effect of angiotensin II and losartan on islet insulin release --- p.116 / Chapter 4.2 --- Existence of local RAS in pancreatic islets --- p.119 / Chapter 4.3 --- Regulation of islet RAS components in transplanted islets --- p.122 / Chapter 4.4 --- Clinical relevance of islet RAS in transplantation --- p.125 / Chapter 4.5 --- Regulation of islet RAS by type 2 diabetes --- p.126 / Chapter 4.6 --- Clinical relevance of islet RAS in type 2 diabetes --- p.134 / Chapter 4.7 --- Conclusion --- p.140 / Chapter 4.8 --- Further studies --- p.141 / Chapter Chapter 5 --- Bibliography --- p.142

La transplantation hépatique (TH) est une intervention à risque hémorragique, au cours de laquelle toutes les étapes de la coagulation (hémostase primaire, hémostase secondaire, fibrinolyse) sont perturbées. Nous avons d’abord montré que les facteurs de risque de saignement et transfusion étaient difficiles à identifier et n’étaient pas cliniquement très pertinents. Puis nous avons montré que la thromboélastométrie (ROTEM®) pouvait diagnostiquer la thrombopénie et l’hypofibrinogénémie au cours de la TH, même si l’utilisation d’un algorithme basé sur le ROTEM® ne diminuait pas le saignement ni la transfusion par rapport à un algorithme basé sur les résultats du laboratoire. De plus le ROTEM® manquait de sensibilité pour détecter l’hyperfibrinolyse. La thrombine est l’enzyme-clé de la cascade de la coagulation. Le Calibrated Automated Thrombogram (CAT®) est la méthode de référence de génération de thrombine. Nous avons cherché des moyens rapides d’évaluation de la génération de thrombine, utilisable en routine et sur échantillons individuels. Le Thrombodynamics-4D® (TD4D) permettait à la fois l’étude de la formation et de la propagation du caillot de fibrine dans le temps et l’espace et de la génération de thrombine. Une hyperfibrinolyse survient au cours de 20 à 66% des TH. Elle majore le saignement et la transfusion. Le diagnostic rapide de l’hyperfibrinolyse permettrait un traitement rapide et ciblé par antifibrinolytique. Le Lysis Timer était plus sensible que le ROTEM® pour détecter les hyperfibrinolyses. Le TD4D permettait également de visualiser la lyse du caillot. L’utilisation en routine de ces nouveaux appareils nécessite la validation des résultats sur plasma frais, après accélération des étapes pré-analytiques (centrifugation rapide) et des études cliniques pour les positionner au sein d’algorithmes transfusionnels. / Liver transplantation (LT) is a bleeding procedure, in which all the haemostatic steps (primary haemostasis, secondary haemostasis, fibrinolysis) are impaired. We have first shown the predictive factors of bleeding and transfusion were difficult to determine and were of poor clinical relevance. Then, we showed that thromboelastometry (ROTEM®) could detect thrombocytopenia and hypofibrinogenemia during LT. However, the utilisation of an algorithm based on ROTEM® results did not led to less bleeding and transfusion when compared to an algorithm based on laboratory results. Moreover, ROTEM® lacked sensitivity to detect hyperfibrinolysis. Thrombin is the key-enzyme of coagulation cascade. The Calibrated Automated Thrombogram (CAT®) is the reference test for thrombin generation. We searched for rapid tools to evaluate thrombin generation, usable in routine and on individual plasma samples. The Thrombodynamics-4D® (TD4D) enabled in the same time study of fibrin clot formation and propagation in time and space and thrombin generation. Hyperfibrinolysis is encountered in 20 to 66% of LT procedures. It is associated with more bleeding and transfusion. Rapid diagnosis of hyperfibrinolysis would allow a quick and target treatment with antifibrinolytic drugs. Lysis Timer was more sensitive than ROTEM® to detect hyperfibrinolysis. TD4D also visualized clot lysis. Routine utilization of these new devices now requires validation of its results in fresh plasma, after acceleration of pre-analytic steps (rapid centrifugation) and clinical studies to find their place in transfusion algorithms.

Opportunistic infection is always a potentially life threatening complication in pediatric oncology patients and hematopoietic stem cell transplant recipients. With the advances in various disease treatment protocols, the overall and event-free survivals of this high risk population improve significantly. In this thesis, the author reported a number of original studies to discuss different strategies in prevention of this serious complication. Firstly, the author demonstrates that pediatric oncology patients are still vulnerable to various vaccine-preventable infectious diseases up to 18 months after stopping chemotherapy. For those vaccine-preventable infectious diseases, pediatric oncology patients can mount a significant and persistent immune response to common inactivated vaccine (namely diphtheria-tetanus-pertussis vaccine). For non-vaccine preventable infectious diseases, regular monitoring of plasma viral load and strategic use of antiviral agents as pre-emptive or prophylactic agent is an effective approach to prevent infection. In hematopoietic stem cell transplant setting, adoptive transfer of acquired immunity from donor to recipient and incorporation of this parameter in donor selection process can be considered. The findings of the studies can be applied to clinical setting. The future direction of our studies includes the immune responses of other common vaccines namely pneumococcal vaccine and pandemic influenza vaccine in high risk population. The role of transfer of donor's varicella zoster immunity in prevention of herpes zoster infection in transplant recipient can be further explored. With the advances in supportive care of our vulnerable patients, the survival rate is expected to be further improved in the future. / by Frankie Wai Tsoi, Cheng. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (M.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 193-208). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.

Allogeneic haematopoietic stem cell transplantation (HSCT) has been used successfully to treat children and adults with high-risk or relapsed hematopoietic malignancies, marrow failure syndromes, and hereditary immunodeficiency disorders. When initially developed, allogeneic HSCT was conceived as a method of rescuing patients from the toxic side effects of dose-intensive chemoradiotherapy. Due to transplant-related toxicities, the application of myeloablative allogeneic HSCT has been limited to younger patients without organ dysfunctions. Since the early 1990s many groups of investigators have explored strategies using less intensive preparative regimens that would allow engraftment of hematopoietic progenitor cells from either identical or non-identical donors. These reduced-intensity conditioning (RIC) regimens result in less tissue damage, less inflammatory cytokine secretion, and possibly lower rates of graft-versus-host disease (GVHD) and non-relapse mortality (NRM). Such non-myeloablative approach, or "mini-transplant", has been suggested to benefit older patients as well as in conditions in which traditional myeloablative conditioning regimens are associated with high rates of non-relapse mortality. / Allogeneic HSCT is the only curative therapy for many patients with myeloid malignancies or myelodysplastic syndrome (MDS). The development of reduced-intensity preparative regimens may allow the extension of this form of treatment to older and patients with coexisting medical illness. On the other hand, relapse after transplantation remains the most important cause of treatment failure in patients with refractory acute myeloid leukemia (AML) or MDS, and is associated with poor survival. Evaluation of prognostic factors may help to improve the results of myeloablative and RIC allogeneic HSCT in this group of patients. Furthermore, the impact of comorbidities on outcomes of RIC allogeneic HSCT in this group of patients with refractory AML or MDS needs to be defined. / The application of embryonic and adult stem cells in regenerative and reparative therapies of non-hematopoietic diseases is emerging rapidly. Human umbilical cord blood (UCB) is a rich source of hematopoietic stem cells and mesenchymal progenitor cells. Although clinical experience to date with UCB has focused on hematological application, early preclinical studies support the hypothesis that multipotential stem cells derived from UCB exhibit functional characteristics similar to that observed in adult marrow-derived stem cells in mediating vascular and organ regenerative capabilities. However, the application of these preclinical findings in clinical setting needs to be further studied. Mini-transplant of human UCB may be an effective approach to repair organ damage in patients with non-hematological diseases. / Wong Siu Ming Raymond. / Adviser: Joseph J.Y. Sung. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (M.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 187-223). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.

by Li Chi-kong. / "September 2002." / Thesis (M.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 223-251). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

La transplantation d’îlots pancréatiques est une des thérapies proposées aux diabétiques de type 1. Cependant, la perte d’un nombre considérable d’îlots durant le processus est un frein à l’expansion de la thérapie (en culture : anoïkis et hypoxie ; lors de la greffe : réactions inflammatoires intenses déclenchées lors de l’infusion intra-portale). L’objectif de ce travail était de valider un biomatériau permettant d’optimiser la transplantation d’îlots. Nous avons pu démontrer l’efficacité du plasma réticulé sur la survie des îlots en culture via une diminution drastique de l’anoïkis. Nous avons également mis au point une technique de greffe intra-tissulaire à l’aide d’HPMC combinée à du plasma, permettant de reverser le diabète de manière semblable à la greffe hépatique. Ces travaux ont donc permis de valider l’importance d’un support de culture adapté, mais aussi du site receveur de la greffe et placé l’omentum comme site de choix pour ce type de greffe. / Pancreatic islet transplantation is one of the therapies proposed to type 1 diabetic patients. However, the considerable loss of islets during the process is an obstacle to the expansion of this therapy (culture: anoïkis and hypoxia; at graft time: intense inflammatory reactions triggered by intra-portal islet infusion). The objective of this work was to validate a biomaterial that can optimize islet transplantation. We were able to demonstrate the effectiveness of cross-linked plasma on the islet survival in culture through a drastic reduction of anoikis. We also design a new intra-tissular grafting technique using HPMC combined with plasma, which reversed diabetes in a manner similar to the liver. This work allowed validating the importance of an adapted culture support, but also the one of the recipient site. Also, this work placed the omentum as an excellent recipient site for this kind of transplant.

Kwok, Po Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 124-130). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.i / Abstract --- p.ii / 中文摘要 --- p.v / Acknowledgements --- p.vii / List of Figures --- p.viii / List of Tables --- p.x / Table of Abbreviations --- p.xii / Contents --- p.xix / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- The Biology of Human Umbilical Cord Perivascular Cells (HUCPVs) and Their Potential Applications in Tissue Regeneration / Chapter 2.1 --- INTRODUCTION --- p.5 / Chapter 2.1.1 --- Stem cells --- p.5 / Chapter 2.1.2.1 --- Embryonic stem cells --- p.6 / Chapter 2.1.2.2 --- iPS cells --- p.7 / Chapter 2.1.2.3 --- Somatic stem cells --- p.8 / Chapter 2.1.3 --- Mesenchymal stem cells --- p.9 / Chapter 2.1.4 --- Pericytes --- p.11 / Chapter 2.1.5 --- CD146 positive MSCs --- p.12 / Chapter 2.1.6 --- Human umbilical cord perivascular cells (HUCPVs) --- p.13 / Chapter 2.1.7 --- The biology of stem cell microenvironment (niche) --- p.14 / Chapter 2.1.8 --- Current applications of HUCPVs --- p.17 / Chapter 2.1.9 --- Regenerative medicine --- p.17 / Chapter 2.1.10 --- Applications of stem cells in bone regeneration --- p.19 / Chapter 2.2 --- MATERIALS AND METHODS --- p.22 / Chapter 2.2.1 --- Cell culture --- p.22 / Chapter 2.2.2 --- Preparation of Human Umbilical Cord Perivascular (HUCPV) cells --- p.22 / Chapter 2.2.2.1 --- Isolation of Human Umbilical Cord Perivascular (HUCPV) cells from human umbilical cord --- p.22 / Chapter 2.2.2.2 --- Purification of HUCPV cells --- p.23 / Chapter 2.2.3 --- Immunocytochemsitry --- p.24 / Chapter 2.2.4 --- Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) --- p.25 / Chapter 2.2.4.1 --- Isolation of total cellular RNA --- p.25 / Chapter 2.2.4.2 --- Complementary DNA (cDNA) synthesis --- p.26 / Chapter 2.2.4.3 --- Polymerase chain reaction (PCR) --- p.26 / Chapter 2.2.5 --- Quantitative real-time reverse transcriptionpolymerase chain reaction (qRT-PCR) --- p.30 / Chapter 2.2.6 --- In vitro differentiation assays --- p.33 / Chapter 2.2.6.1 --- Osteogenic differentiation --- p.33 / Chapter 2.2.6.2 --- Adipogenic differentiation --- p.33 / Chapter 2.2.6.3 --- Chondrogenic differentiation --- p.34 / Chapter 2.2.6.4 --- In vitro chondrogenic differentiation on gelfoam® --- p.34 / Chapter 2.2.7 --- Cytochemistry staining --- p.35 / Chapter 2.2.7.1 --- Alkaline Phosphatase staining --- p.35 / Chapter 2.2.7.2 --- Alizarin Red S staining --- p.35 / Chapter 2.2.7.3 --- Oil Red O staining --- p.36 / Chapter 2.2.7.4 --- Alcian Blue staining --- p.36 / Chapter 2.2.8 --- Scanning electron microscopy (SEM) --- p.37 / Chapter 2.2.9 --- Transmission electron microscopy (TEM) --- p.37 / Chapter 2.2.10 --- Paraffin tissue embedding --- p.38 / Chapter 2.2.10 --- Haematoxylin and Eosin staining --- p.38 / Chapter 2.3 --- RESULTS --- p.40 / Chapter 2.3.1 --- Isolation and purification of HUCPVs --- p.40 / Chapter 2.3.2 --- Osteogenic differentiation of HUCPVs under normoxia --- p.41 / Chapter 2.3.3 --- Osteogenic differentiation of HUCPVs under hypoxia --- p.42 / Chapter 2.3.4 --- Adipogenic differentiation of HUCPVs --- p.43 / Chapter 2.3.5 --- Chondrogenic differentiation of HUCPVs --- p.43 / Chapter 2.3.6 --- Chondrogenic differentiation of HUCPVs on gelfoam® --- p.44 / Chapter 2.4 --- DISCUSSION --- p.59 / Chapter Chapter 3 --- Calcium and Calcium-sensing Receptor (CaSR) in osteogenesis / Chapter 3.1 --- INTRODUCTION --- p.62 / Chapter 3.1.1 --- Metabolism of calcium --- p.62 / Chapter 3.1.2 --- Calcium-sensing receptor --- p.64 / Chapter 3.1.2.1 --- The molecular structure of calcium-sensing Receptor (CaSR) --- p.64 / Chapter 3.1.2.2 --- The expression pattern of calciumsensing receptor (CaSR) --- p.67 / Chapter 3.1.2.3 --- The physiological function of calcium-sensing receptor in different tissues or organs --- p.68 / Chapter 3.1.2.4 --- Regulatory role of calcium-sensing receptor in calcium sensing and homeostasis --- p.71 / Chapter 3.1.2.5 --- The role of calcium-sensing receptor in diseases --- p.72 / Chapter 3.1.2.6 --- Genetic animal models targeting calciumsensing receptor --- p.73 / Chapter 3.1.2.7 --- Calcium-sensing receptor in mesenchymal lineage Differentiation --- p.76 / Chapter 3.1.2.8 --- The role of calcium-sensing receptor in the skeleton --- p.76 / Chapter 3.1.3 --- Calcium-sensing receptor related pathway --- p.78 / Chapter 3.1.3.1 --- Cyclic AMP pathway --- p.78 / Chapter 3.1.3.2 --- Cyclic AMP response element-binding protein (CREB) --- p.80 / Chapter 3.2 --- MATERIALS AND METHODS --- p.83 / Chapter 3.2.1 --- Preparation of primary mouse osteoblasts (MOB) from long bone --- p.83 / Chapter 3.2.2 --- Preparation of primary mouse osteoblasts (CMOB) from calvaria --- p.84 / Chapter 3.2.3 --- Immunocytochemistry --- p.84 / Chapter 3.2.4 --- Osteogenic differentiation --- p.85 / Chapter 3.2.3 --- Quantitative real-time reverse transcriptionpolymerase chain reaction (qRT-PCR) --- p.85 / Chapter 3.2.4 --- Cell proliferation measurement by BrdU ELISA (colorimetric) assay --- p.85 / Chapter 3.2.5 --- Western blotting analysis --- p.86 / Chapter 3.2.5.1 --- Preparation of the protein lysate --- p.86 / Chapter 3.2.5.2 --- Protein quantitation --- p.86 / Chapter 3.2.5.3 --- SDS-PAGE --- p.87 / Chapter 3.2.5.4 --- Protein transfer --- p.87 / Chapter 3.2.5.5 --- Immunodetection --- p.88 / Chapter 3.2.6 --- cAMP EIA assay --- p.89 / Chapter 3.3 --- RESULTS --- p.91 / Chapter 3.3.1 --- "Expression of CD 146 and CaSR in HUCPVs, primary mouse long bone osteoblasts and MC3T3-E1 cell line" --- p.91 / Chapter 3.3.2 --- The effect of calcium treatment on the osteogenic differentiation potential of MC3T3-E1 cells under normoxia --- p.91 / Chapter 3.3.3 --- The effect of calcium treatment on the osteogenic differentiation potential of MC3T3-E1 cells under hypoxia --- p.92 / Chapter 3.3.4 --- The effect of calcium treatment on cell proliferation in primary mouse long bone osteoblasts --- p.93 / Chapter 3.3.5 --- The effect of calcium treatment on calcium-sensing receptor expression in primary mouse long bone osteoblasts --- p.94 / Chapter 3.3.6 --- The effect of calcium treatment on calcium-sensing receptor expression in HUCPVs --- p.95 / Chapter 3.3.7 --- The effect of calcium treatment on calcium-sensing receptor expression in primary mouse calvarian osteoblasts --- p.96 / Chapter 3.3.8 --- The effect of calcium treatment on cyclic AMP levels in primary mouse long bone osteoblasts --- p.97 / Chapter 3.4 --- DISCUSSION --- p.117 / Chapter Chapter 4 --- General Discussions --- p.121 / References --- p.124 / Appendices --- p.131