Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 185-219).

Medicamentos biológicos são obtidos a partir de fluidos biológicos ou tecidos de origem animal por procedimentos biotecnológicos e, a partir do vencimento das suas patentes, surge a possibilidade da produção de suas cópias, os chamados biossimilares. Este tema, além de polêmico, por ainda apresentar divergências de entendimento da classe científica, também engloba 4 das 5 classes terapêuticas de medicamentos mais vendidas, e apresenta evolução crescente no mercado farmacêutico. Com o aumento da demanda, cresce o interesse na produção de medicamentos biológicos de alta qualidade, com a mesma eficácia, porém a preços mais baixos. Dessa forma, é possível entender a responsabilidade das regulamentações, principalmente no que diz respeito aos biossimilares, a fim de que eles respeitem os requisitos mínimos necessários para serem comparáveis ao seu medicamento biológico novo. Assim, este trabalho teve como objetivo avaliar questões técnico-regulatórias e os requisitos de qualidade para registro de medicamentos biológicos e biossimilares humanos frente a diferentes Autoridades Sanitárias mundiais. A análise foi baseada em três moléculas biológicas, sendo a clássica heparina e moléculas novas, filgrastim e infliximabe. Foi constatado que na teoria, a legislação brasileira é baseada em regulamentos internacionais, especialmente da Federal and Drug Administration (FDA) e European Medicines Agency (EMA), e que na prática, o Brasil tem se mostrado mais conservador na extrapolação de indicação e na aprovação dos biossimilares. Ainda, foi possível notar que independente do país, as Farmacopeias ainda necessitam de aprimoramento com relação a este tema, pois em sua maioria, não existe padronização dos parâmetros e testes a serem realizados. Pesquisa demonstrou que o conhecimento sobre biossimilares ainda não está consolidado entre profissionais médicos e que, portanto, há necessidade de programas para esclarecimentos, com a finalidade de estimular seu uso, quando possível e com custos mais interessantes. / Biological drugs are obtained from biological fluids or animals tissues by biotechnological procedures and, from the expiration of their patents, the possibility of producing their \"copies\", the so-called biosimilars, arises. In addition to being a controversial subject, as it still presents divergences of understanding by the scientific class, it also encompasses 4 of the 5 therapeutic classes of best-selling drugs, and it presents an increasing evolution in the pharmaceutical market. As demand increases, interest in the production of high-quality biological drugs with the same effectiveness, but at lower prices, also increases. In this way, it is possible to understand the responsibility of regulations, especially with regard to biosimilars, so that they comply with the minimum requirements needed to be comparable to their reference biological medicine. Thus, the objective of this project was to evaluate technical and regulatory topics, as well as quality requirements for the registration of human biological and biosimilar medicines under the perspective of different Health Authorities around the world. The analysis was based on three biological molecules, being the classic heparin and new molecules, filgrastim and infliximab. It was found that in theory, Brazilian regulation is based on international regulations, especially the Federal and Drug Administration (FDA) and the European Medicines Agency (EMA), and that in practice, Brazil has been more conservative in the extrapolation of indication and approval of biosimilars. Also, it was possible to note that, regardless the country, Pharmacopoeias still need to be improved for this topic, since in general, there is no standardization of the parameters and tests to be performed. Research showed that the knowledge about biosimilars is not yet consolidated among doctors and that, therefore, there is a need for clarification programs, with the purpose of stimulating their use, when possible and at lower costs.

Background: Although the growth-factor G-CSF is widely used to prevent granulotoxic side effects of cytotoxic chemotherapies, its optimal use is still unknown since treatment outcome depends on many parameters such as dosing and timing of chemotherapies, pharmaceutical derivative of G-CSF used and individual risk factors. We showed in the past that a pharmacokinetic and dynamic model of G-CSF and human granulopoiesis can be used to predict the performance of yet untested G-CSF schedules. However, only a single chemotherapy was considered so far. In the present paper, we propose a comprehensive model of chemotherapy toxicity and combine it with our cell kinetic model of granulopoiesis. Major assumptions are: proportionality of cell numbers and cell loss, delayed action of chemotherapy, drug, drugdose and cell stage specific toxicities, no interaction of drugs and higher toxicity of drugs at the first time of application. Correspondingly, chemotherapies can be characterized by a set of toxicity parameters which can be estimated by fitting the predictions of our model to clinical time series data of patients under therapy. Data were either extracted from the literature or were received from cooperating clinical study groups. Results: Model assumptions proved to be feasible in explaining granulotoxicity of 10 different chemotherapeutic drugs or drug-combinations applied in 33 different schedules with and without G-CSF. Risk groups of granulotoxicity were traced back to differences in toxicity parameters. Conclusion: We established a comprehensive model of combined G-CSF and chemotherapy action in humans which allows us to predict and compare the outcome of alternative G-CSF schedules. We aim to apply the model in different clinical contexts to optimize and individualize G-CSF treatment.

Class of 2012 Abstract / Specific Aims: Autologous stem cell transplant (ASCT) is the current gold standard following induction therapy to improve survival of multiple myeloma (MM). Lenalidomide (LEN) is used for treatment of MM before ASCT, but exposure may impair autologous peripheral blood stem cell (PBSC) mobilization. Chemomobilization with cyclophosphamide (CTX) has not been evaluated in this setting. CTX + filgrastim was investigated to determine if LEN-associated mobilization impairment can be abrogated. Methods: 36 pts (group A=12 pts who received ≥2 cycles of LEN and group B=24 pts without LEN) were analyzed retrospectively. Baseline characteristics were matched (p>0.05 for all variables). All pts received CTX (median group B, 1.5g/m2; median group A, 3gm/m2(p=0.18)) and filgrastim 10μg/kg/day. Primary outcomes include number of CD34+ cells collected and number of leukapheresis sessions. Secondary outcomes include failure to collect CD34+ cells and total CD34+ cells collected after second leukapheresis. Main Results: Total median number of CD34+ cells collected in group B=9.15x106/kg CD34+ cells and group A=7.43x106/kg CD34+ cells (p=0.159). Median number of apheresis sessions in group B=2 and group A=3 (p=0.42). Two of 12 pts with antecedent LEN usage failed to collect while no patient without previous LEN exposure failed to collect (p=0.105). Total number of CD34+ cells collected after 2 apheresis sessions for group B=8.13x106/kg CD34+ cells and group A=3.34x106/kg CD34+ cells (p=0.06). Conclusions: Chemomobilization with CTX + filgrastim yields robust PBSC collections irrespective of antecedent lenalidomide. There was a trend towards lesser PBSC collection in LEN-treated pts.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 157-188).

Background: Although the growth-factor G-CSF is widely used to prevent granulotoxic side effects of cytotoxic chemotherapies, its optimal use is still unknown since treatment outcome depends on many parameters such as dosing and timing of chemotherapies, pharmaceutical derivative of G-CSF used and individual risk factors. We showed in the past that a pharmacokinetic and dynamic model of G-CSF and human granulopoiesis can be used to predict the performance of yet untested G-CSF schedules. However, only a single chemotherapy was considered so far. In the present paper, we propose a comprehensive model of chemotherapy toxicity and combine it with our cell kinetic model of granulopoiesis. Major assumptions are: proportionality of cell numbers and cell loss, delayed action of chemotherapy, drug, drugdose and cell stage specific toxicities, no interaction of drugs and higher toxicity of drugs at the first time of application. Correspondingly, chemotherapies can be characterized by a set of toxicity parameters which can be estimated by fitting the predictions of our model to clinical time series data of patients under therapy. Data were either extracted from the literature or were received from cooperating clinical study groups. Results: Model assumptions proved to be feasible in explaining granulotoxicity of 10 different chemotherapeutic drugs or drug-combinations applied in 33 different schedules with and without G-CSF. Risk groups of granulotoxicity were traced back to differences in toxicity parameters. Conclusion: We established a comprehensive model of combined G-CSF and chemotherapy action in humans which allows us to predict and compare the outcome of alternative G-CSF schedules. We aim to apply the model in different clinical contexts to optimize and individualize G-CSF treatment.

Background: Haematotoxicity of conventional chemotherapies often results in delays of treatment or reduction of chemotherapy dose. To ameliorate these side-effects, patients are routinely treated with blood transfusions or haematopoietic growth factors such as erythropoietin (EPO) or granulocyte colony-stimulating factor (G-CSF). For the latter ones, pharmaceutical derivatives are available, which differ in absorption kinetics, pharmacokinetic and -dynamic properties. Due to the complex interaction of cytotoxic effects of chemotherapy and the stimulating effects of different growth factor derivatives, optimal treatment is a non-trivial task. In the past, we developed mathematical models of thrombopoiesis, granulopoiesis and erythropoiesis under chemotherapy and growth-factor applications which can be used to perform clinically relevant predictions regarding the feasibility of chemotherapy schedules and cytopenia prophylaxis with haematopoietic growth factors. However, interactions of lineages and growth-factors were ignored so far. Results: To close this gap, we constructed a hybrid model of human granulopoiesis and erythropoiesis under conventional chemotherapy, G-CSF and EPO applications. This was achieved by combining our single lineage models of human erythropoiesis and granulopoiesis with a common stem cell model. G-CSF effects on erythropoiesis were also implemented. Pharmacodynamic models are based on ordinary differential equations describing proliferation and maturation of haematopoietic cells. The system is regulated by feedback loops partly mediated by endogenous and exogenous EPO and G-CSF. Chemotherapy is modelled by depletion of cells. Unknown model parameters were determined by fitting the model predictions to time series data of blood counts and cytokine profiles. Data were extracted from literature or received from cooperating clinical study groups. Our model explains dynamics of mature blood cells and cytokines after growth-factor applications in healthy volunteers. Moreover, we modelled 15 different chemotherapeutic drugs by estimating their bone marrow toxicity. Taking into account different growth-factor schedules, this adds up to 33 different chemotherapy regimens explained by the model. Conclusions: We conclude that we established a comprehensive biomathematical model to explain the dynamics of granulopoiesis and erythropoiesis under combined chemotherapy, G-CSF, and EPO applications. We demonstrate how it can be used to make predictions regarding haematotoxicity of yet untested chemotherapy and growth-factor schedules.

Thesis (Ph.D.). / Written for the Dept. of Mathematics and Statistics. Title from title page of PDF (viewed 2008/01/12). Includes bibliographical references.

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

by Ng Wing Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 156-174). / Abstracts in English and Chinese. / Acknowledgements --- p.iii / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.ix / List of Figures --- p.xiii / List of Tables --- p.xvi / List of Graphs --- p.xvii / List of Abbreviations --- p.xviii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.3 / Chapter 2.1 --- Human granulocyte colony-stimulating factor (hG-CSF) --- p.3 / Chapter 2.1.1 --- Historical background --- p.3 / Chapter 2.1.2 --- Physiological Roles --- p.5 / Chapter 2.1.3 --- Molecular properties --- p.8 / Chapter 2.1.4 --- Biochemical properties --- p.9 / Chapter 2.1.5 --- Comparison to G-CSF of other species --- p.11 / Chapter 2.1.6 --- Biological Activities --- p.12 / Chapter 2.1.7 --- Clinical Applications --- p.14 / Chapter 2.1.7.1 --- Clinical use in myelosuppressive chemotherapy and neutropenic fever --- p.14 / Chapter 2.1.7.2 --- Clinical use in bone marrow transplantation (BMT) and peripheral blood progenitor cell (PBPC) transplantation --- p.14 / Chapter 2.1.7.3 --- Clinical use in HIV infection --- p.16 / Chapter 2.1.7.4 --- Clinical use in diabetes mellitus --- p.17 / Chapter 2.1.7.5 --- Clinical use in severe chronic neutropenia --- p.18 / Chapter 2.1.7.6 --- Future prospects --- p.18 / Chapter 2.1.7.7 --- Dosages and adverse effects --- p.19 / Chapter 2.1.8 --- Economic value --- p.20 / Chapter 2.2 --- Plant as bioractor --- p.20 / Chapter 2.2.1 --- Medical molecular farming --- p.20 / Chapter 2.2.2 --- Commercial biopharmaceutical proteins --- p.25 / Chapter 2.2.3 --- Transgenic plants producing hematopoietic growth factors --- p.25 / Chapter 2.2.3.1 --- Granulocyte-macrophage colony-stimulating factor (GM-CSF) --- p.26 / Chapter 2.2.3.2 --- Interleukin-2 (IL-2) --- p.28 / Chapter 2.3 --- Rice as expression system --- p.29 / Chapter 2.3.1 --- Characteristics --- p.29 / Chapter 2.3.2 --- Advantages of using rice as bioreactor --- p.30 / Chapter 2.3.3 --- Previous studies --- p.31 / Chapter 2.3.4 --- Transformation method --- p.33 / Chapter 2.3.5 --- Super-binary vector --- p.34 / Chapter 2.4 --- Strategies for enhancing protein expression level --- p.36 / Chapter 2.4.1 --- Vacuolar targeting --- p.36 / Chapter 2.4.1.1 --- Protein targeting signals --- p.38 / Chapter 2.4.1.2 --- Binding protein of 80kDa (BP-80) --- p.39 / Chapter 2.4.1.3 --- a-Tonoplast intrinsic protein (α-TIP) --- p.39 / Chapter 2.4.1.4 --- Receptor homology region-transmembrane domain-Ring H2 motif (RMR) --- p.40 / Chapter 2.4.2 --- Fusion with glutelin in rice --- p.41 / Chapter 2.5 --- Hypotheses and aims of this study --- p.43 / Chapter Chapter 3 --- Materials and Methods --- p.45 / Chapter 3.1 --- Introduction --- p.45 / Chapter 3.2 --- Chemicals --- p.45 / Chapter 3.3 --- Bacterial strains --- p.46 / Chapter 3.4 --- Chimeric genes construction --- p.46 / Chapter 3.4.1 --- Protein targeting constructs --- p.51 / Chapter 3.4.2 --- Enterokinase site constructs --- p.60 / Chapter 3.4.3 --- Glutein signal peptide constructs --- p.65 / Chapter 3.4.4 --- Glutelin fusion constructs --- p.70 / Chapter 3.4.5 --- Sequence fidelity of chimeric genes --- p.77 / Chapter 3.4.6 --- Cloning of chimeric genes into rice super-binary vector --- p.77 / Chapter 3.5 --- Rice transformation --- p.79 / Chapter 3.5.1 --- Plant materials --- p.79 / Chapter 3.5.2 --- Agrobacterium transformation --- p.79 / Chapter 3.5.3 --- A grobacterium-mediated transformation of rice --- p.79 / Chapter 3.6 --- Transgenic expression --- p.81 / Chapter 3.6.1 --- Extraction of leaf genomic DNA --- p.81 / Chapter 3.6.2 --- Synthesis of DIG-labeled double-stranded DNA probe --- p.82 / Chapter 3.6.3 --- Southern blot analysis --- p.83 / Chapter 3.6.4 --- Extraction of total RNA from immature rice seeds --- p.84 / Chapter 3.6.5 --- Northern blot analysis --- p.85 / Chapter 3.6.6 --- Protein extraction --- p.86 / Chapter 3.6.7 --- Tricine SDS-PAGE --- p.86 / Chapter 3.6.8 --- Western blot analysis --- p.87 / Chapter 3.6.9 --- Enterokinase digestion of EK fusion proteins --- p.88 / Chapter 3.7 --- Confocal immunoflorescence studies of rhG-CSF in rice grain --- p.89 / Chapter 3.7.1 --- Preparation of sample sections --- p.89 / Chapter 3.7.2 --- Double-labeling of fluorescence probes --- p.89 / Chapter 3.7.3 --- Image collection --- p.90 / Chapter 3.8 --- Functional analysis of rhG-CSF --- p.91 / Chapter 3.8.1 --- Culture of NFS-60 cells --- p.91 / Chapter 3.8.2 --- MTT cell proliferation assay --- p.92 / Chapter 3.9 --- Bacterial expression of anti-hG-CSF --- p.93 / Chapter 3.9.1 --- pET expression in E. coli --- p.93 / Chapter 3.9.2 --- Purification of His-hG-CSF --- p.97 / Chapter 3.9.3 --- Immunization of rabbits --- p.97 / Chapter Chapter 4 --- Results --- p.99 / Chapter 4.1 --- Construction of chimeric genes for rice transformation --- p.99 / Chapter 4.2 --- "Rice transformation, selection and regeneration" --- p.103 / Chapter 4.3 --- Southern blot analysis --- p.105 / Chapter 4.4 --- Northern blot analysis --- p.109 / Chapter 4.5 --- Western blot analysis --- p.114 / Chapter 4.6 --- Enterokinase digestion of EK fusion proteins --- p.125 / Chapter 4.7 --- Confocal immunofluorescence studies of rhG-CSF in transgenic rice grain --- p.128 / Chapter 4.8 --- Functional analysis of rhG-CSF --- p.132 / Chapter 4.9 --- Bacterial expression of anti-hG-CSF --- p.135 / Chapter 4.9.1 --- Expression and purification of recombinant His-hG-CSF in E. coli --- p.135 / Chapter 4.9.2 --- Titer and specificity of the anti-serum --- p.137 / Chapter Chapter 5 --- Discussion --- p.139 / Chapter 5.1 --- Introduction --- p.139 / Chapter 5.2 --- Fusion of hG-CSF with protein sorting determinants --- p.141 / Chapter 5.3 --- Fusion of hG-CSF with rice glutelin --- p.145 / Chapter 5.4 --- Glutelin signal peptide --- p.146 / Chapter 5.5 --- O-glycosylation --- p.148 / Chapter 5.6 --- Enterokinase digestion --- p.148 / Chapter 5.7 --- Expression level of rhG-CSF --- p.149 / Chapter 5.8 --- Functional analysis of rhG-CSF --- p.151 / Chapter 5.9 --- Future perspectives --- p.151 / Chapter Chapter 6 --- Conclusion --- p.155 / References --- p.156