Autoimmune neutropenia (AIN) is characterized by antibody mediated peripheral destruction of neutrophils. Since there is no effective treatment, antibiotics have to be used frequently for recurrent infections. Five selected patients with serologically proven AIN were treated with r-metHuG- CSF at 5-8 μg/kg body weight (300-480 μg) daily: the dose and frequency of r-metHuG-CSF was reduced after neutrophil counts above 1.0 x 109/l were obtained. R-metHuG-CSF is effective in AIN and causes a sustained rise in ANC which can he maintained on a low dose administered twice or thrice weekly.

Granulocyte colony-stimulating factor (GCSF) is currently the most widely used cytokine for stem cell mobilization. There are few studies suggesting GCSF administration may induce activation of both coagulation and endothelial cells that could favor the developing of thrombotic events. We report a 58-year-old female with vasculitis and renal impairment. She was found to have an underlying monoclonal gammopathy of unknown significance (MGUS). The monoclonal protein was felt to play a role in her underlying renal disease and peripheral neuropathy. She was considered a candidate for peripheral blood stem cell transplantation to manage the monoclonal protein. During stem cell mobilization with GCSF, she developed worsening of anemia; thrombocytopenia and worsening of renal function. She was diagnosed with thrombotic microangiopathy (TMA) which was successfully treated with therapeutic plasma exchange and rituximab. It is possible that GCSF may have directly (activating endothelial cells) or indirectly (activation of underlying autoimmune disorder) contributed to TMA in this patient.

Une des stratégies pour limiter les neutropénies induites par la chimiothérapie est l’utilisation de granulocyte-colony stimulating factor (G-CSF). Nous avons développé, par une approche de population, un nouveau modèle pharmacocinétique/pharmacodynamique capable de décrire la cinétique des neutrophiles des patients traités au carboplatine, qu’ils aient ou non reçu du G-CSF. Les simulations réalisées à partir de ce modèle ont montré que le G-CSF n’était pas bénéfique chez tous les patients et que la formulation à action longue semblerait plus efficace que les autres formulations. Nous avons également établi des règles de décision permettant d’une part de prédire le risque de neutropénie sévère, et d’autre part d’identifier précocement les patients pour lesquels le G-CSF peut avoir un effet bénéfique. / Granulocyte colony-stimulating factor (G-CSF) is often used in cancer patients receiving cytotoxic drugs to prevent or reduce high grade neutropenia. We developed a new population pharmacokinetic/pharmacodynamic model to describe neutrophil time-course in carboplatin-treated patients, whether or not they received G-CSF. Model simulations showed that G-CSF was not as beneficial as expected in some patients and that the onceper- cycle formulation was more efficient than other formulations. Model-based decision rules were also built to anticipate prolonged high grade neutropenia and early identify patients for whom G-CSF was beneficial.

L’immunologie de la reproduction englobe les principes de l’immunologie générale et les aspects spécifiques de la reproduction et du développement. Les Colony Stimulating Factors (CSFs) sont une illustration de l'application médicale de ce domaine. Dans la famille des CSFs, le Granulocyte-Colony Stimulating Factor (G-CSF) apparaît aujourd'hui comme une thérapie innovante dans divers cas d'échec de la reproduction, bien que ses cibles et ses effets ne soient pas encore clairement établis. Dans ce travail, à travers une revue sur les CSFs dans la reproduction, une étude consacrée aux gènes cibles du G-CSF dans l'endomètre humain, et une étude consacrée aux effets de la supplémentation systémique en G-CSF sur l’implantation embryonnaire murine, nous avons essayé d'approcher certains mécanismes d'action possibles pour cette cytokine. Dans les modèles murins fertiles et pro-abortifs, la supplémentation systémique en G-CSF, ciblant spécifiquement l’endomètre préimplantatoire, modifie les taux d’implantation embryonnaire. Dans l’endomètre humain, certaines dérégulations préimplantatoires de gènes cibles du G-CSF ont également été observées chez les patients infertiles. L'influence du G-CSF sur ces gènes cibles a été également illustrée dans un modèle ex-vivo de culture endométriale. Ces cibles dont l’expression est influencée par le G-CSF sont décrites comme des molécules clés dans le processus implantatoire, intervenant sur l’adhésion embryonnaire, la migration cellulaire, le remodelage des tissus et l'angiogenèse locale. Ces données suggèrent des possibilités de diagnostic préventif et pré-conceptionnel de certains échecs de reproduction, considérés jusqu’à maintenant comme idiopathiques, et de thérapies innovantes orientées, afin d’optimiser la réceptivité du biosenseur endométrial afin de permettre une implantation embryonnaire harmonieuse et une grossesse évolutive. / Reproductive Immunology involves general immunology principles and special aspects of reproduction and development. Colony Stimulating Factors (CSFs) are an illustration of the medical application of this domain. In the CSF family, Granulocyte-Colony Stimulating Factor (G-CSF) appears today as a promising therapy in various cases of reproductive failure although its targets and effects are not clearly established. In this work, through a review on CSFs in reproduction, a study dedicated to human endometrial targets of G-CSF, and a study dedicated to systemic G-CSF supplementation effects on murine embryo implantation, we tried to approach some possible mechanisms of action of this cytokine. In the considered non-abortive and abortion-prone murine models, the timed systemic G-CSF supplementation, targeting specifically the pre implantation endometrium, influenced the embryo implantation process. Some pre conceptual human endometrial dysregulations of G-CSF target genes were also observed in infertile patients. The endometrial influence of G-CSF on these target genes was also illustrated in an ex-vivo model. These molecules under G-CSF influence are described as critically involved in embryo implantation process, by influencing embryo adhesion, cell migration, tissue remodelling and angiogenesis. These data suggest possible pre-conceptual preventive diagnosis of such reproductive failures and future orientated therapies to optimise the endometrial biosensor and the further embryo implantation and ongoing pregnancy.

In under a century, melioidosis, the infection due to Burkholderia pseudomallei, has emerged from Whitmore’s series of glanders-like infections amongst the morphia addicts in Burma to a major cause of mortality in northeastern Thailand and northern Australia. Also endemic in other parts of south-east Asia, melioidosis may have varied presentations ranging from severe, overwhelming infection to chronic, low grade disease. Observational evidence had suggested that granulocyte colony stimulating factor (G-CSF), a naturally occurring substance produced by the body in response to infection, may have been useful in reducing the high mortality associated with the more severe forms of this infection. Other observations linked the occurrence of this disease to various environmental factors, such as contamination of drinking water and the annual rainfall. This thesis explores and attempts to quantify these associations. There are three parts to this thesis. In the first part, I reviewed the epidemiology and management of patients with melioidosis. The use of G-CSF and meropenem was associated with a fall in mortality, although other factors may have at least partially contributed to this effect. In the second part, I progressed towards a clinical trial of G-CSF. There was no other evidence supporting the use of G-CSF in severe sepsis and ethical issues precluded a trial in Darwin. There was not evidence from laboratory models of G-CSF action in melioidosis to support the use of G-CSF in patients, although there remained some doubt regarding the applicability of such models to human disease. I examined clinical methods to identify patients at high risk of death from melioidosis. A simple scoring system based on clinical and laboratory parameters was developed and externally validated. However, clinical definitions of severe sepsis appeared to be better predictors of mortality. A clinical trial based on clinical definitions was commenced in Thailand. In the final part, I explored the question of whether different strains or B. pseudomallei or different environmental conditions caused different patterns of infection. There was no evidence that strain types of this bacterium determine the pattern or severity of disease, but weather conditions appeared to influence the distribution of disease in northern Australia.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2013-03-22T14:03:31Z No. of bitstreams: 1 Tulio_Queto.pdf: 43727261 bytes, checksum: 24d32a6a536f42194f28e8e2b42a6fe1 (MD5) / Made available in DSpace on 2013-03-22T14:03:31Z (GMT). No. of bitstreams: 1 Tulio_Queto.pdf: 43727261 bytes, checksum: 24d32a6a536f42194f28e8e2b42a6fe1 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / A provocação por via respiratória com ovalbumina (OVA) em camundongos sensibilizados promove, na medula óssea (MO), eosinopoiese, resposta exacerbada à Interleucina(IL)-5, e mudanças no padrão de resposta em cultura à eotaxina, à IL- 13 e aos antiinflamatórios não esteroidais (NSAIDs). Em cultura de MO, a Prostaglandina (PG) E2 induz apoptose de eosinófilos, por via dependente de NO sintase indutível (iNOS) e ligante de CD95 (CD95L), enquanto a dexametasona (DEXA) promove eosinopoiese, gerando eosinófilos agregados e morfológicamente imaturos e protege contra da apoptose induzida por PGE2, provavelmente por mecanismos que regulem a expressão ou ativação de integrinas. No presente trabalho avaliamos se: a) in vitro, os efeitos dos NSAIDs, da IL-13 e da eotaxina dependem da produção de cisteinil-leucotrienos (CisLT) e da sinalização via receptor 1 de CisLT (CysLT1R); b) in vitro, a DEXA regula expressão de VLA-4, que promoveria a agregação e imaturidade dos eosinófilos, e se PGE2 contrapõe a ação da DEXA; c) in vivo, G-CSF e dietilcarbamazina (DEC) promoveriam eosinopenia medular e sistemica e se inibiriam a inflamação pulmonar alérgica. Resultados: a) NSAIDs, eotaxina e IL-13 potencializam a eosinopoiese via produção de CisLT e sinalização via CysLT1R. Os NSAIDs ainda protegem os eosinófilos da apoptose induzida por PGE2 exógena. b) A interação farmacológica entre PGE2 e DEXA modificam a ação de ambas, de forma estreitamente relacionada sobre a expressão de VLA-4 e, em condições específicas, esses agentes sinergizam gerando quantidades aumentadas de eosinófilos maduros. c) A DEC, inibidor da síntese de LTs, que na filariose experimental possivelmente atua via iNOS, inibe a eosinopoiese e os efeitos da provocação sobre o pulmão e a MO, através da via iNOS-CD95L. O Fator Estimulante de Colônias de Granulócitos (G-CSF), estimulante da neutropoiese, igualmente inibiu a inflamação pulmonar alérgica através da inibição da eosinopoiese. Este trabalho é parte do projeto intitulado "Eosinofilia na Asma Experimental", licenciado pela Comissão de Ética no Uso de Animais (CEUA) da Fiocruz, sob nos L010/04 e L002/09. / Our laboratory has previously shown that airway allergen challenge in ovalbumin-sensitized mice rapidly induces an increase in bone-marrow (BM) eosinophil production (eosinopoiesis), along with an increased response to Interleukin(IL)-5 in BM culture, changes in the ex vivo responses to cytokines and immunomodulators, including nonsteroidal anti-inflammatory drugs (NSAIDs) and cysteinyl-leukotrienes (CysLT), and colonization of the lungs by eosinophil progenitors. Early in the course of IL-5-induced eosinophil differentiation in BM culture, Prostaglandin E2 (PGE2) induces apoptosis, through a pathway dependent on the inducible isoform of NO synthase (iNOS) and the ligand for CD95 (CD95L). NSAIDs enhance eosinopoiesis and protect eosinophils in this critical period from exogenous PGE2, through a novel mechanism of action at the celular level. In this study, we show that indomethacin and aspirin act through endogenously synthesized CysLT, by establishing the essential roles of 5-lipoxygenase, LTC4 synthase and CysLT1R receptors, as well as the cytoprotective effect of CysLT against exogenous PGE2. The similarity between the effects of NSAIDs and those of eotaxin and IL-13 prompted us to reevaluate the contribution of endogenous CysLT to the effects of these cytokines. We confirmed that eotaxin and IL-13 act through this mechanism, and expand therefore the list of agents that, through CysLT, enhance eosinopoiesis, protecting immature eosinophils from apoptosis induced through the iNOS-CD95L pathway. Dexamethasone promotes BM eosinopoiesis, generating aggregated, cytologically immature eosinophils, which are nevertheless protected from PGE2- induced apoptosis. We examined therefore the possibility that dexamethasone upregulates integrin expression/activation, thereby maintaining an immature celular phenotype in cultured eosinophils, while PGE2 would have opposite effects on both integrin function and cytological maturation. We show that the proapoptotic effects of PGE2 are profoundly modified by its pharmacological interaction with dexamethasone, paralleling the effects of both drugs on integrins, and leading to a synergic generation of increased numbers of mature eosinophils, in very specific experimental conditions. Diethylcarbamazine (DEC) is an antihelminthic drug that blocks leukotriene synthesis, and possibly acts in experimental filarial infection through iNOS. We have examined, for the first time, the effects of DEC in a model of allergic pulmonary inflammation, showing that DEC is very effective in preventing the impact of airway allergen challenge on BM and lungs, through the in vivo operation of the iNOS-CD95L pathway. Granulocyte Colony-stimulating Factor (G-CSF), which stimulates neutropoiesis, mobilizes CD34+ hemopoietic progenitors from BM, and exerts complex immunoregulatory effects, was shown in our study to have a strong impact in a murine model of allergic pulmonary inflammation. Like DEC, G-CSF suppressed BM eosinopoiesis, although through a different mechanism, since DEC suppressed neutrophilia in the lungs with no effect on BM neutrophilia/neutropoiesis, while G-CSF promoted neutropoiesis and induced blood neutrophilia, even though it suppressed eosinopoiesis. This work was part of the Research Project “Eosinophilia in Experimental Asthma”, licensed by the Committee on the Ethical Use of Laboratory Animals (CEUA) at FIOCRUZ, under numbers L010/04 and L002/09.

No description available.

Introdução: Várias são as propostas descritas para tratar farmacologicamente a lesão traumática da medula espinal. A metilprednisolona já foi padronizada para uso clínico. O fator estimulador de colônia de granulócitos (G-CSF) tem sido promissor em estudos experimentais e clínicos. Não há pesquisas quanto ao efeito da associação dos dois fármacos. Objetivo: Avaliar o efeito do tratamento com o fator estimulador de colônia de granulócitos associado a metilprednisolona na lesão medular aguda experimental em ratos. Material e métodos: Foram avaliados 40 ratos Wistar submetidos a lesão medular moderada com o NYU-Impactor. Os animais foram divididos em quatro grupos de 10 ratos. O Grupo Controle não recebeu tratamento; o Grupo G-CSF, foi tratado com G-CSF no momento da lesão e diariamente ao longo dos cinco dias subsequentes; o Grupo Metilprednisolona, com metilprednisolona durante 24 horas; e o Grupo G-CSF/Metilprednisolona, com metilprednisolona durante 24 horas e G-CSF no momento da lesão e ao longo de cinco dias. Os animais foram mantidos vivos durante 42 dias; a avaliação funcional foi realizada com a aplicação da escala funcional de Basso, Beattie e Bresnahan (BBB) nos dias 2, 7, 14, 21, 28, 35 e 42 subsequentes à lesão. A avaliação dos potenciais evocados motores foi realizada no dia 42 e a avaliação histológica da lesão da região da medula espinal lesada, realizada logo após a eutanásia ocorrida no dia 42. Resultados e conclusões: A associação de metilprednisolona e G-CSF no tratamento do traumatismo medular contuso experimental em ratos promoveu melhora neurológica avaliada pela escala BBB superior à melhora promovida pela metilprednisolona e G-CSF quando utilizadas isoladamente. A associação teve também efeito sinérgico que resultou em melhora nos parâmetros histológicos no local da lesão. Não houve diferença entre os grupos quanto à avaliação neurofisiológica / Introduction: There are several proposals to pharmacologically treat traumatic spinal cord injury. Methylprednisolone has already been standardized for clinical use. Granulocyte colony stimulating factor (G-CSF) has been promising in experimental and clinical studies. There is no research on the effect of the association of the two drugs. Objective: to evaluate the effect of combined treatment of the granulocyte colony-stimulating factor (G-CSF) associated with methylprednisolone in experimental acute spinal cord injury in rats. Material and methods: Forty male Wistar rats were submitted to a moderate spinal cord injury with the NYU-Impactor. The animals were divided into four groups of ten rats each. The Control Group was not treated; the G-CSF Group was treated with G-CSF at the time of injury and daily over the next five days; the Methylprednisolone Group was treated with methylprednisolone for 24 hours; the G-CSF/methylprednisolone Group, was treated with methylprednisolone for 24 hours and G-CSF at the time of injury and daily over the next five days. The animals were kept alive for 42 days; Functional evaluation was performed using the Basso, Beattie and Bresnahan (BBB) score on days 2, 7, 14, 21, 28, 35 and 42 following the spinal cord injury. Evaluation of motor evoked potentials was held and histological examination of the lesion of the spinal cord was done immediately after euthanasia on day 42. Results and conclusions: The combination of methylprednisolone and G-CSF in the treatment of experimental spinal cord injury in rats promoted neurological improvement as assessed by BBB scale with greater improvement than with methylprednisolone or G-CSF when used alone. The combination of treatment had also a synergistic effect resulting in improvement in histological parameters at the injury site. There was no difference between groups regarding neurophysiological evaluation

Mobilização inadequada de células progenitoras hematopoéticas (CPH) tem sido observada em 10 - 30% dos pacientes submetidos a transplante de medula óssea (TMO) autogênico para tratamento de doenças onco-hematológicas. Os fatores relacionados com má resposta à mobilização ainda não estão totalmente estabelecidos. Apresentamos uma análise retrospectiva de pacientes submetidos à TMO autogênico com o objetivo de identificar variáveis associadas com resposta ruim ao regime de mobilização utilizado. Casuística e Métodos: Fizeram parte desta análise 307 pacientes com diferentes diagnósticos, tratados com TMO autogênico em uma única Instituição, no período de Abril de 2001 a Abril de 2007. Todos os pacientes incluídos no estudo foram submetidos a um único regime de mobilização baseado na administração de ciclofosfamida (dose total de 60-120 mg/kg de peso IV) e fator estimulador de colônias de granulócitos (G-CSF) (dose diária de 6 - 17 ug/(kg de peso)/dia SC). O sucesso na resposta ao regime de mobilização foi definido quando um número maior ou igual a 2,0x10 (6) células CD34 + /(kg de peso) foi coletado do sangue periférico com até três procedimentos de leucaférese. Resultados: Dos pacientes analisados, 260 apresentaram sucesso na mobilização (84,7%). Nestes pacientes, um número mediano de 3,67 (2,0 - 46,0) células CD34+ /(kg de peso) foi coletado por paciente com um número mediano de 1 (1-3) procedimento de leucaférese. O insucesso na mobilização foi observado em 47 pacientes (15,3%): 24 (7,8%) que foram submetidos à coleta de CPH de sangue periférico, porém não coletaram número maior ou igual 2,0x10 (6) células CD34+/(kg de peso) com pelo menos três procedimentos de leucaférese; e, 23 (7,5%) foram submetidos à coleta de CPH por punção da medula óssea, por não terem atingido número mínimo de 10 células CD34+/mm3 no sangue periférico para realização de leucaférese. De acordo com análise univariada, os fatores associados com o insucesso foram: diagnóstico (P < 0,0001), tempo de doença (P < 0,0001), número prévio de ciclos de quimioterapia (P = 0,0001), exposição prévia a agentes alquilantes (P = 0.0003) e a mitoxantrone (P = 0,0006), contagem de plaquetas pré-mobilização <150.000/mm3 (P = 0,0006) e intervalo entre o início da mobilização e o pico de células CD 34+ no sangue periférico (P < 0,0001). Idade, sexo, atividade da doença e envolvimento medular ao início da mobilização, tratamento prévio com radioterapia e exposição a análogos da platina não mostraram correlação significativa na resposta à mobilização. Após análise multivariada, as variáveis que permaneceram associadas com insucesso na mobilização foram: diagnóstico (P = 0,0232), número prévio de ciclos da quimioterapia (P = 0,0167), tratamento prévio com mitoxantrone (P = 0,0285) e contagem de plaquetas pré-mobilização < 150.000/mm3 (P = 0,0423). Conclusão: A carga cumulativa de quimioterapia administrada, exposição prévia à mitoxantrone, contagem de plaquetas pré-mobilização e diagnóstico foram os fatores independentes relacionados com a falha na resposta à mobilização. Os achados obtidos podem auxiliar no reconhecimento de pacientes de risco para resposta ruim à mobilização e permitir um planejamento alternativo ou mais agressivo no regime de mobilização para este grupo de pacientes. / Inadequate stem cells mobilization is seen in 10-30% of patients undergoing autotransplantation for hematologic malignancies. Factors affecting peripheral blood progenitor cell (PBSC) mobilization have not been clearly established. We retrospectively reviewed the data of patients treated by autologous bone marrow transplantation (BMT) with the aim to identify factors associated with poor PBSC mobilization. Design and Methods: We evaluated 307 patients with different diagnoses, submitted to autologous BMT between April 2001 and April 2007. PBSC were collected following mobilization with cyclophosphamide (60-120 mg/kg of weight IV) and granulocyte-colony stimulating factor (G-CSF) (dose of 6-17 ug/kg of weight/day SC). Success in mobilization was defined when > ou = a 2,0x10(6) CD34+ cells/(kg weight) could be collected from the peripheral blood with a maximum of three leukapheresis procedures. Clinical and laboratory parameters at the time of mobilization were analyzed for correlations with the number of CD34+ cells collected. Results: Two hundred and sixty patients (84.7%) presented success in mobilization. In this group, a median of 3.67 (2.0-46.0) CD34+ cells/(kg weight) was collected per patient in a median of 1(1-3) leukapheresis procedure. Poor response to mobilization was observed in 47 patients (15.3%): 24 (7.8%) were submitted to PBSC collection but didn\'t collected at least 2.0 x 106 CD34+ cells/(kg weight) with three leukapheresis procedures and 23 (7.5%) didn\'t reach an absolute number count of 10 CD34+ cells/mm3 in the peripheral blood to start collection by leukapheresis. In univariate analysis poorer PBSC mobilization was associated with diagnosis (Pp < 0.0001), time interval from the diagnosis to mobilization (P < 0.0001), number of cycles of previous chemotherapy (P = 0.0001), previous treatment with alkylating agents (P = 0.0003) and mitoxantrone (P = 0.0006), platelet count <150.000/mm3 before mobilization (P = 0.0006) and interval between mobilization and peak of CD34+ cells in peripheral blood (P < 0.0001). No significant correlation was found with age, gender, disease status, marrow involvement at mobilization, prior radiation therapy and exposition to platin analogues. In the stepwise regression model, diagnosis (P = 0.0232), number of cycles of previous chemotherapy (P = 0.0167), previous treatment with mitoxantrone (P = 0.0285) and platelet count <150.000/mm3 before mobilization (P = 0.0423) were found to be independent negative predictive factors for CD34+ cells mobilization. Conclusion: Cumulative load of chemotherapy, exposition to Mitoxantrone, platelet count just prior to mobilization and diagnosis were independent factors related to poor progenitor cells mobilization. These results could help in the previously recognition of patients at risk for poor or no response to mobilization and allow to plan an alternative or more aggressive regimen for this group of patients.

by Lee Juon Kiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 139-152). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Acknowledgements --- p.iii / Abstract --- p.v / Table of contents --- p.ix / List of figures --- p.xv / List of tables --- p.xvii / List of graphs --- p.xviii / List of abbreviations --- p.xix / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter Chapter 2: --- Literature Review --- p.4 / Chapter 2.1 --- Human granulocyte colony-stimulating factor (hG-CSF) --- p.4 / Chapter 2.1.1 --- Physiological roles --- p.4 / Chapter 2.1.2 --- Molecular properties --- p.8 / Chapter 2.1.3 --- Biochemical properties --- p.9 / Chapter 2.1.4 --- Comparison to G-CSF of other specie --- p.10 / Chapter 2.1.5 --- Clinical application --- p.11 / Chapter 2.1.6 --- Economic value --- p.13 / Chapter 2.2 --- Expression systems producing recombinant hG-CSF --- p.15 / Chapter 2.2.1 --- Bacteria --- p.15 / Chapter 2.2.2 --- Yeasts --- p.17 / Chapter 2.2.3 --- Animal cell lines --- p.18 / Chapter 2.2.4 --- Transgenic animals --- p.19 / Chapter 2.2.5 --- Transgenic plants --- p.20 / Chapter 2.3 --- Plant as bioreactors --- p.21 / Chapter 2.3.1 --- Characteristics of using plant as bioreactors --- p.22 / Chapter 2.3.2 --- Transgenic plants producing hematopoietic growth factors --- p.24 / Chapter 2.3.2.1 --- Granulocyte-macrophage colony-stimulating factor (GM-CSF) --- p.24 / Chapter 2.3.2.2 --- Erythropoietin (Epo) --- p.26 / Chapter 2.3.3 --- Arabidopsis and tobacco as model plants --- p.27 / Chapter 2.3.3.1 --- Arabidopsis --- p.28 / Chapter 2.3.3.2 --- Tobacco --- p.28 / Chapter 2.3.4 --- Phaseolin and its regulatory sequences --- p.29 / Chapter 2.4 --- Plant transformation methods --- p.31 / Chapter 2.4.1 --- Agrobacterium-mediated transformation --- p.31 / Chapter 2.4.1.1 --- Tissue culture methods --- p.31 / Chapter 2.4.1.2 --- Non-tissue culture (In planta) methods --- p.32 / Chapter 2.4.2 --- Direct DNA uptake transformation --- p.33 / Chapter 2.4.2.1 --- Chemical methods --- p.33 / Chapter 2.4.2.2 --- Electrical methods --- p.34 / Chapter 2.4.2.3 --- Physical methods --- p.34 / Chapter Chapter 3: --- Materials and Methods --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Chemicals --- p.37 / Chapter 3.3 --- Bacterial strains --- p.37 / Chapter 3.4 --- Chimeric gene construction --- p.37 / Chapter 3.4.1 --- Cloning of pTZ/Phas/His/EK/hG-CSF --- p.41 / Chapter 3.4.2 --- Cloning of pBK/Phas/SP/His/EK/hG-CSF --- p.44 / Chapter 3.4.3 --- Cloning of pBK/Phas/SP/hG-CSF --- p.47 / Chapter 3.4.4 --- Confirmation of sequence fidelity of chimeric genes --- p.50 / Chapter 3.4.5 --- Cloning of chimeric genes into Agrobacterium binary vector --- p.51 / Chapter 3.5 --- Expression in Arabidopsis --- p.52 / Chapter 3.5.1 --- Agrobacterium GV3101/pMP90 transformation --- p.52 / Chapter 3.5.2 --- Arabidopsis transformation --- p.53 / Chapter 3.5.2.1 --- Plant materials --- p.53 / Chapter 3.5.2.2 --- Vacuum infiltration --- p.54 / Chapter 3.5.3 --- Screening of successful R1 transformants --- p.55 / Chapter 3.5.4 --- Screening of hemizygous and homozygous transgenic Arabidopsis --- p.56 / Chapter 3.5.5 --- GUS assay --- p.57 / Chapter 3.5.6 --- Genomic DNA extraction --- p.57 / Chapter 3.5.7 --- Southern blot analysis --- p.58 / Chapter 3.5.8 --- Total RNA extraction from developing siliques --- p.59 / Chapter 3.5.9 --- Northern blot analysis --- p.60 / Chapter 3.5.10 --- Protein extraction and Tricine SDS-PAGE --- p.61 / Chapter 3.5.11 --- Western blot analysis --- p.62 / Chapter 3.5.12 --- Functional analysis --- p.63 / Chapter 3.5.12.1 --- Culture ofNFS-60 cells --- p.64 / Chapter 3.5.12.2 --- MTT assay --- p.65 / Chapter 3.6 --- Expression in tobacco --- p.67 / Chapter 3.6.1 --- Agrobacterium LBA4404/pAL4404 transformation --- p.67 / Chapter 3.6.2 --- Tobacco transformation --- p.68 / Chapter 3.6.2.1 --- Plant materials --- p.68 / Chapter 3.6.2.2 --- Tobacco transformation using leaf-disc technique --- p.68 / Chapter 3.6.3 --- Regeneration of transgenic tobacco --- p.69 / Chapter 3.6.4 --- GUS assay --- p.70 / Chapter 3.6.5 --- Genomic DNA extraction --- p.70 / Chapter 3.6.6 --- Southern blot analysis --- p.70 / Chapter 3.6.7 --- Total RNA extraction from immature seeds --- p.70 / Chapter 3.6.8 --- Northern blot analysis --- p.71 / Chapter 3.6.9 --- Protein extraction and Tricine SDS-PAGE --- p.71 / Chapter 3.6.10 --- Western blot analysis --- p.71 / Chapter 3.6.11 --- Functional analysis --- p.71 / Chapter 3.6.11.1 --- Culture of NFS-60 cells --- p.72 / Chapter 3.6.11.2 --- MTT assay --- p.72 / Chapter Chapter 4: --- Results --- p.73 / Chapter 4.1 --- Chimeric gene construction --- p.73 / Chapter 4.1.1 --- Cloning of pTZ/Phas/His/EK/hG-CSF --- p.73 / Chapter 4.1.2 --- Cloning of pBK/Phas/SP/His/EK/hG-CSF --- p.75 / Chapter 4.1.3 --- Cloning of pBK/Phas/SP/hG-CSF --- p.77 / Chapter 4.1.4 --- Cloning of chimeric genes into Agrobacterium binary vector --- p.79 / Chapter 4.2 --- Expression in Arabidopsis --- p.81 / Chapter 4.2.1 --- Agrobacterium GV3101/pMP90 transformation --- p.81 / Chapter 4.2.2 --- Arabidopsis transformation and screening of R1 transformants --- p.83 / Chapter 4.2.3 --- Screening of hemizygous transgenic R1 Arabidopsis --- p.84 / Chapter 4.2.4 --- Screening of homozygous transgenic R2 Arabidopsis --- p.86 / Chapter 4.2.5 --- GUS assay --- p.88 / Chapter 4.2.6 --- Genomic DNA extraction --- p.89 / Chapter 4.2.7 --- Southern blot analysis --- p.91 / Chapter 4.2.8 --- Total RNA extraction from developing siliques --- p.93 / Chapter 4.2.9 --- Northern blot analysis --- p.94 / Chapter 4.2.10 --- Protein extraction and Tricine SDS-PAGE --- p.96 / Chapter 4.2.11 --- Western blot analysis --- p.99 / Chapter 4.2.12 --- Functional analysis --- p.103 / Chapter 4.3 --- Expression in tobacco --- p.108 / Chapter 4.3.1 --- Agrobacterium LBA4404/pAL4404 transformation --- p.108 / Chapter 4.3.2 --- Tobacco transformation and regeneration of transformants --- p.109 / Chapter 4.3.3 --- GUS assay --- p.111 / Chapter 4.3.4 --- Genomic DNA extraction --- p.112 / Chapter 4.3.5 --- Southern blot analysis --- p.114 / Chapter 4.3.6 --- Total RNA extraction from immature seeds --- p.116 / Chapter 4.3.7 --- Northern blot analysis --- p.116 / Chapter 4.3.8 --- Protein extraction and Tricine SDS-PAGE --- p.118 / Chapter 4.3.9 --- Western blot analysis --- p.120 / Chapter 4.3.10 --- Functional analysis --- p.123 / Chapter Chapter 5: --- Discussion --- p.126 / Chapter 5.1 --- Introduction --- p.126 / Chapter 5.2 --- Successful in producing biologically active rhG-CSF from transgenic plants --- p.128 / Chapter 5.2.1 --- Production level --- p.129 / Chapter 5.2.2 --- O-glycosylation --- p.130 / Chapter 5.2.3 --- Phaseolin signal peptide --- p.131 / Chapter 5.2.4 --- Functional analysis --- p.131 / Chapter 5.3 --- Comparison of the productivity of other expression systems producing rhG-CSF --- p.132 / Chapter 5.4 --- Comparison of the productivity of plants producing different human proteins --- p.135 / Chapter 5.5 --- Future perspectives --- p.137 / Chapter Chapter 6: --- Conclusion --- p.138 / References --- p.139