by Wong Chun-kwok. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 233-246). / ABSTRACT --- p.I / ACKNOWLEDGEMENTS --- p.V / ABBREVIATIONS --- p.VI / PUBLICATIONS --- p.IX / CHAPTER / Chapter 1. --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- EFFECTOR CELLS MEDIATING ANTI一TUMOR IMMUNITY --- p.3 / Chapter 1.1.1 --- CYTOTOXIC T LYMPHOCYTES --- p.4 / Chapter 1.1.2 --- MACROPHAGES --- p.4 / Chapter 1.1.3 --- NATURAL KILLER CELLS --- p.5 / Chapter 1.1.4 --- LYMPHOKINE ACTIVATED KILLER CELLS --- p.7 / Chapter 1.1.5 --- TUMOR-INFILTRATING LYMPHOCYTES --- p.8 / Chapter 1.2 --- BIOLOGICAL RESPONSE MODIFIERS : THE NEW IMMUNOTHERAPY --- p.9 / Chapter 1. 3 --- CYTOKINES AS BIOLOGICAL RESPONSE MODIFIERS IN CANCER THERAPY --- p.12 / Chapter 1.3.1 --- INTERFERONS --- p.12 / Chapter 1.3.2 --- TUMOR NECROSIS FACTOR-ALPHA --- p.13 / Chapter 1.3.3 --- INTERLEUKIN-1 --- p.15 / Chapter 1.3.4 --- INTERLEUKIN-2 --- p.16 / Chapter 1.3.5 --- GRANULOCYTES /MACROPHAGES COLONY-STIMULATING FACTORS --- p.16 / Chapter 1.3.6 --- EPIDERMAL GROWTH FACTOR --- p.17 / Chapter 1.3.7 --- TRANSFORMING GROWTH FACTOR-BETA --- p.17 / Chapter 1.4 --- BIOACTIVE POLYSACCHARIDES FROM CHINESE MEDICINAL HERBS ACT AS BIOLOGICAL RESPONSE MODIFIERS --- p.18 / Chapter 2. --- AIM AND SCOPE OF INVESTIGATION --- p.27 / Chapter 3. --- MATERIALS AND METHODS --- p.30 / Chapter 3.1 --- MATERIALS --- p.30 / Chapter 3.2 --- METHODS --- p.39 / Chapter (I) --- "EXTRACTION, FRACTIONATION AND CHARACTERIZATION OF PSEUDOSTELLARIA HETEROPHYLLA" / Chapter 3.2.1 --- Hot water extraction and stepwise alcohol precipitation --- p.39 / Chapter 3.2.2 --- "Determination of carbohydrate, protein, uronic acid contents" --- p.41 / Chapter 3.2.3 --- Gel filtration --- p.41 / Chapter 3.2.4 --- Anion-exchange chromatography --- p.41 / Chapter 3.2.5 --- Paper chromatography --- p.42 / Chapter 3.2.6 --- Gas liquid chromatography --- p.43 / Chapter 3.2.7 --- Determination of molecular weight by high performance liquid chromatography --- p.44 / Chapter 3.2.8 --- SDS-polyacrylamide gel electrophoresis --- p.44 / Chapter 3.2.9 --- Determination of the bio´ؤtoxicity of samples --- p.46 / Chapter 3.2.10 --- Treatment of samples with sodium periodate or acetic acid --- p.46 / Chapter (II) --- ASSAYS OF IMMUNOMODULATORY ACTIVITIES OF PSEUDOSTELLARIA HETEROPHYLLA ON LYMPHOCYTES / Chapter 3.2.11 --- Isolation and preparation of cells --- p.48 / Chapter 3.2.12 --- In vitro lymphocyte transformation assay --- p.50 / Chapter 3.2.13 --- Mixed lymphocyte culture --- p.50 / Chapter 3.2.14 --- Depleting mouse T cells by anti-Thy-1.2 antibody plus complement treatment --- p.51 / Chapter 3.2.15 --- Depleting mouse B cells by anti-mouse B cell antibody plus complement treatment --- p.51 / Chapter 3.2.16 --- Haemolytic plaque assay --- p.52 / Chapter 3.2.17 --- Delayed-type hypersensitivity --- p.53 / Chapter 3.2.18 --- Immunofluorescent assay for interleukin-2 receptor expression --- p.54 / Chapter 3.2.19 --- Assay of murine interleukin-2 --- p.55 / Chapter (III) --- ASSAYS OF IMMUNOMODULATORY ACTIVITIES OF PSEUDOSTELLARIA HETEROPHYLLA ON MACROPHAGES / Chapter 3.2.20 --- Assay of murine interleukin-1 --- p.55 / Chapter 3.2.21 --- In vivo migration of macrophages --- p.56 / Chapter 3.2.22. --- Assay of phagocytic activity of peritoneal macrophages --- p.56 / Chapter 3.2.23 --- Northern blotting of mRNA of β-actin gene extracted from peritoneal exudate cells --- p.57 / Chapter (IV) --- ASSAYS OF ANTI-TUMOR ACTIVITIES OF PSEUDOSTELLARIA HETEROPHYLLA / Chapter 3.2.24 --- Assay of anti-tumor activity in vitro --- p.62 / Chapter 3.2.25 --- Assay of anti-tumor activity in vivo --- p.63 / Chapter 3.2.26 --- Priming effect of different fractions for the induction of TNF-α in mice --- p.63 / Chapter 3 .2.27 --- In vitro stimulation of TNF-α release from resting peritoneal macrophages --- p.64 / Chapter 3.2.28 --- Effects of P. heterophylla polysaccharides on TNF-α and IFN-gamma production as well as EAT growth in vivo --- p.64 / Chapter 3.2.29 --- Macrophage-mediated cytostatic activity --- p.65 / Chapter 3 2.30 --- Assay of lymphokine-activated killer cell activity --- p.66 / Chapter 3 2.31 --- Assay of natural killer cell activity --- p.67 / Chapter 3.2.32 --- Assay of tumor-infiltrating lymphocytes --- p.68 / Chapter (V) --- ASSAYS FOR THE EFFECTS OF PSEUDOSTELLARIA HETEROPHYLLA ON THE PROLIFERATION AND DIFFERENTIATION OF MURINE BONE MARROW CELLS AND MYELOID LEUKAEMIC Ml CELLS / Chapter 3.2.33 --- Assay of proliferation of murine bone marrow cells --- p.69 / Chapter 3.2.34 --- Assay of differentiation of murine bone marrow cells --- p.70 / Chapter 3.2.35 --- Assay of differentiation of Ml cells --- p.71 / Chapter 3.2.36 --- Induction of GM-CSF from bone marrow cells and Ml cells --- p.71 / Chapter (VI) --- ASSAYS OF THE IMMUNORESTORATIVE PROPERTIES OF PSEUDOSTELLARIA HETEROPHYLLA / Chapter 3.2.37 --- Immunorestoration in tumor-bearing mice --- p.72 / Chapter 3.2.38 --- Immunorestoration in aged mice --- p.72 / Chapter 3.2.39 --- Immunorestoration in cyclophosphamide- treated mice --- p.73 / Chapter 3.2.40 --- Statistical analysis --- p.73 / Chapter 4. --- "EXTRACTION, FRACTIONATION AND CHARACTERIZATION OF MITOGENIC FRACTIONS FROM PSEUDOSTELLARIA HETEROPHYLLA" / INTRODUCTION --- p.74 / RESULTS --- p.76 / Chapter 4.1 --- Extraction and fractionation of Pseudostellaria heterophylla --- p.76 / Chapter 4.2 --- Gel filtration and anion-exchange chromatography --- p.76 / Chapter 4.3 --- Characterization of bioactive fractions from Pseudostellaria heterophylla --- p.79 / Chapter 4.4 --- Mitogenic activity of fraction PH-I on murine lymphocytes in vitro --- p.96 / Chapter 4.5 --- Mitogenic effect of PH-I on murine lymphocytes in vivo --- p.102 / Chapter 4.6 --- Effect of PH-I on polyclonal B cell activation --- p.102 / Chapter 4.7 --- Adjuvant effect of PH-I on antibody response to SRBC in vivo --- p.106 / Chapter 4.8 --- Evidences to support the mitogenic activity of PH-I is due to its polysaccharide rather than due to the contamination by LPS --- p.106 / Chapter 4.9 --- The effects of PH-I on IL-2 production and IL-2 receptor expression on murine lymphocytes in vitro --- p.110 / Chapter 4.10 --- The mitogenic activity of the purified fractions on murine lymphocytes in vitro --- p.110 / Chapter 4.11 --- Adjuvant effect of PH-I Ab on antibody response to SRBC in vivo --- p.116 / Chapter 4.12 --- Mitogenic effect of PH-I C on murine lymphocytes in vivo --- p.116 / Chapter 4.13 --- Evidences to support the mitogenic activity of PH-I Ab is due to its polysaccharide rather than due to the contamination by LPS --- p.122 / DISCUSSION --- p.122 / Chapter 5. --- IMMUNOMODULATING AND ANTI-TUMOR ACTIVITIES OF ALCOHOL- INSOLUBLE FRACTION (PH-I) FROM THE HOT WATER EXTRACT OF PSEUDOSTELLARIA HETEROPHYLLA / INTRODUCTION --- p.133 / RESULTS --- p.135 / Chapter 5.1 --- Effect of PH-I on cytokine production --- p.135 / Chapter 5.2 --- In vivo activation of macrophages by PH-I --- p.135 / Chapter 5.3 --- Effect of PH-I on the activation of β-actin gene transcription in peritoneal macrophages --- p.142 / Chapter 5.4 --- Effect of PH-I on the in vitro growth of various tumor cell lines --- p.142 / Chapter 5.5 --- Immunorestoration of PH-I on the mitogenic response in EAT-bearing mice --- p.147 / DISCUSSION --- p.147 / Chapter 6. --- IMMUNOMODULATING AND ANTI-TUMOR ACTIVITIES OF PURIFIED FRACTIONS SEPARATED FROM PSEUDOSTELLARIA HETEROPHYLLA / INTRODUCTION --- p.154 / RESULTS --- p.157 / Chapter 6.1 --- In vitro anti-tumor activities of P. heterophylla --- p.157 / Chapter 6.2 --- In vivo anti-tumor activities of P. heterophylla --- p.165 / Chapter 6.3 --- Effect of P. heterophylla fractions on induction of delayed-type hypersensitivity --- p.165 / Chapter 6.4 --- Effect of PH-I fraction on the cytotoxic alloreactive T lymphocytes in vitro --- p.165 / Chapter 6.5 --- Effect of P. heterophylla on the production of TNF-α and IFN-gamma --- p.170 / Chapter 6.6 --- Effect of P. heterophylla on the activation of macrophages --- p.176 / Chapter 6.7 --- "Effect of P. heterophylla on the activation of NK, LAK and TIL" --- p.181 / Chapter 6.8 --- The effect of combined treatment of EAT-bearing mice with P. heterophylla amd Mur-TNF-α on the growth of EAT cells in vivo --- p.181 / Chapter 6 9 --- Immunorestorative activities of P. heterophylla in aged mice and cyclophosphamide-treated mice --- p.187 / DISCUSSION --- p.187 / Chapter 7. --- EFFECTS OF PSEUDOSTELLARIA HETEROPHYLLA ON PROLIFERATION AND DIFFERENTIATION OF MURINE BONE MARROW CELLS AND MYELOID LEUKAEMIC Ml CELLS / INTRODUCTION --- p.200 / RESULTS --- p.202 / Chapter 7.1 --- Effect of P. het erophyl1a on the proliferation and differentiation of murine bone marrow cells --- p.202 / Chapter 7 .2 --- Effects of P. heterophyl la on the proliferation and differentiation of murine myeloid leukaemia Ml cells --- p.205 / Chapter 7 .3 --- Effects of P. heterophylla on GM-CSF production by bone marow cells and myeloid leukaemia Ml cells --- p.214 / DISCUSSION --- p.218 / Chapter 8. --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.223 / BIBLIOGRAPHY --- p.233

Photobacterium profundum SS9 is a γ-proteobacterium which grows optimally at 15°C and 28 MPa (a psychrophilic piezophile) and can grow over a range of temperatures (2-20oC) and pressures (0.1-90 MPa). Previous research had demonstrated that P. profundum SS9 adapts its membrane proteins and phospholipids in response to growth conditions. In this study, methodology was developed for growing P. profundum SS9 under cold temperatures and high pressures in both liquid and solid cultures. The effect of changing growth conditions on cell envelope polysaccharides was then investigated. The lipopolysaccharide (LPS) profile of a rifampicin resistant P. profundum SS9 derivative, SS9R, was shown to change at 0.1 MPa with respect to temperature and at 15°C with respect to pressure. Compositional analysis showed that the LPS was almost entirely composed of glucose. This provides evidence that, under these conditions, the major polysaccharide produced by P. profundum SS9 is a glucan. Two putative polysaccharide mutants, FL26 & FL9, were previously isolated from a screen for cold-sensitive mutants of P. profundum SS9R. Both mutants displayed an increased sensitivity to cold temperatures on solid medium and were unaffected in their growth at high pressure. FL26 was found to exhibit an LPS alteration similar to previously published O-antigen ligase mutants, providing evidence that this mutant is likely to lack O-antigen ligase. Interestingly, FL26 was also shown to have a reduced ability to form biofilms and had increased swimming motility. This suggests that there are a number of changes which occur in FL26 in the absence of O-antigen. FL9 was found to have an altered LPS and capsular polysaccharide (CPS), similar to an E. coli wzc mutant. In E. coli, Wzc is involved in the polymerisation and transport of CPS, disruption of which can also lead to LPS alterations. The LPS and CPS alterations may lead to the cold-sensitivity phenotype, either individually or in combination. In conclusion, alterations in the cell envelope polysaccharides were shown to affect cold temperature sensitivity on solid agar. Cold-sensitivity is most likely directly related to the LPS alterations and stability of the membrane under cold temperatures. Exopolysaccharides (EPS) have previously been shown to affect desiccation and freezethaw resistance, making it is possible that the CPS plays a similar role in this case.

Orientador: Maria Célia Bertolini / Banca: Cleslei Fernando Zanelli / Banca: Ana Paula Ulian de Araújo / Banca: Maria Teresa Marques Novo Mansur / Banca: Marcos Roberto de Mattos Fontes / Resumo: O fungo Neurospora crassa tem sido amplamente usado como um organismo modelo para os aspectos fundamentais da biologia dos eucariotos. Neste trabalho, foi investigado o papel funcional de uma ciclina de N. crassa (PCL-1), codificada pela ORF NCU08772 e ortóloga a Pcl10 de Saccharomyces cerevisiae. Na levedura, a proteína Pcl10, em conjunto com a proteína quinase dependente de ciclina Pho85, fosforila a enzima glicogênio sintase, a enzima regulatória da síntese de glicogênio. A fosforilação resulta na inativação da enzima e, portanto, em diminuição do acúmulo de glicogênio. A linhagem pcl-1 de N. crassa apresentou um atraso na germinação dos conídios e um retardo na progressão do ciclo celular quando comparado com a linhagem selvagem, sugerindo que esta ciclina pode regular o desenvolvimento e a divisão celular. Além disto, a linhagem nocauteada acumulou níveis mais elevados de glicogênio que a linhagem selvagem indicando o papel na regulação do metabolismo deste carboidrato. A fosforilação da enzima glicogênio sintase de N. crassa (GSN) foi analisada na linhagem nocaute através de análises de atividade enzimática, e os resultados mostraram que a GSN apresentou baixo índice de fosforilação, portanto alta atividade durante o crescimento, um resultado que pode explicar o alto acúmulo de glicogênio observado. Este resultado foi confirmado por análise de 2D-PAGE seguida por western blot, utilizando anticorpos anti-GSN. GSN apresentou na linhagem mutante isoformas m... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

by Li Ho Kin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 213-245). / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.iii / ABSTRACT --- p.viii / TABLE OF CONTENTS --- p.xi / Chapter CHAPTER 1: --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Effector Cells Mediating Anti-Tumour Immunity --- p.5 / Chapter 1.2.1 --- Cytotoxic T Lymphocytes --- p.6 / Chapter 1.2.2 --- Natural Killer Cells --- p.7 / Chapter 1.2.3 --- Lymphokine-Activated Killer Cells --- p.9 / Chapter 1.2.4 --- Macrophages --- p.10 / Chapter 1.2.5 --- Tumour-Infiltrating Lymphocytes --- p.12 / Chapter 1.3 --- Current Modalities for Cancer Treatment --- p.13 / Chapter 1.3.1 --- Surgery --- p.13 / Chapter 1.3.2 --- Radiation Therapy --- p.14 / Chapter 1.3.3 --- Chemotherapy --- p.14 / Chapter 1.3.4 --- Biologic Therapy --- p.15 / Chapter 1.4 --- Biological Response Modifiers as Cancer Therapeutics --- p.17 / Chapter 1.4.1 --- Monoclonal Antibodies --- p.20 / Chapter 1.4.2 --- Differentiating Agents --- p.21 / Chapter 1.4.3 --- Cytokines --- p.24 / Chapter 1.4.4 --- Bioactive Plant Polysaccharides --- p.30 / Chapter 1.5 --- Bacterial Polysaccharides as Potential Immunomodulators and Anti-Tumour Agents --- p.36 / Chapter 1.5.1 --- General Properties of Bacterial Polysaccharides --- p.36 / Chapter 1.5.2 --- Structure and Function of Bacterial Polysaccharides --- p.37 / Chapter 1.5.3 --- Immunomodulatory and Anti-Tumour Activities of Bacterial Polysaccharides --- p.39 / Chapter 1.5.4 --- General Properties of Klebsiella K7 Capsular Antigen --- p.41 / Chapter 1.6 --- Aims and Scopes of the Investigation --- p.44 / Chapter CHAPTER 2: --- MATERIALS AND METHODS --- p.46 / Chapter 2.1 --- Materials --- p.47 / Chapter 2.1.1 --- Animals --- p.47 / Chapter 2.1.2 --- Klebsiella pneumoniae Serotype7 --- p.47 / Chapter 2.1.3 --- Agar Medium for Cultivation of Klebsiella pneumoniae Serotype7 --- p.47 / Chapter 2.1.4 --- Cell Lines --- p.47 / Chapter 2.1.5 --- "Buffers, Culture Medium and Other Reagents" --- p.49 / Chapter 2.1.6 --- Antibodies --- p.54 / Chapter 2.1.7 --- Radioisotopes --- p.55 / Chapter 2.1.8 --- Recombinant Cytokines --- p.55 / Chapter 2.1.9 --- Oligonucleotide Primers and Internal Probes --- p.56 / Chapter 2.1.10 --- Reagents and Solutions for Gene Expression Analysis --- p.59 / Chapter 2.2 --- Methods --- p.64 / Chapter 2.2.1 --- "Extraction, Purification and Characterization of Klebsiella K7 Capsular Antigen" --- p.54 / Chapter 2.2.1.1 --- Extraction and Purification of K7 Capsular Antigen --- p.64 / Chapter 2.2.1.2 --- Gel Filtration of K7 Capsular Antigen --- p.65 / Chapter 2.2.1.3 --- Characterization of K7 Capsular Antigen --- p.67 / Chapter 2.2.1.4 --- Determination of the Bio-Toxicity of K7 Capsular Antigen --- p.67 / Chapter 2.2.1.5 --- In Vitro Cytotoxicity of K7 Capsular Antigen on Splenocytes --- p.53 / Chapter 2.2.2 --- Assay for the Hematopoietic and Mitogenic Activities of Klebsiella K7 Capsular Antigen --- p.69 / Chapter 2.2.2.1 --- Isolation and Preparation of Cells --- p.69 / Chapter 2.2.2.2 --- In Vitro Lymphocyte Transformation Assay --- p.70 / Chapter 2.2.2.3 --- In Vitro Assay of Thymocyte Proliferation --- p.70 / Chapter 2.2.2.4 --- Depleting Mouse T Cells by Anti-Thy-1.2 Antibody Plus Complement Treatment --- p.71 / Chapter 2.2.2.5 --- Depleting Mouse B Cells by Anti-mouse B Cell Antibody Plus Complement Treatment --- p.71 / Chapter 2.2.2.6 --- Depleting Macrophages from Spleen Cell Suspension --- p.71 / Chapter 2.2.2.7 --- Flow Cytometric Analysis of Different Cell Populations from Splenocytes --- p.72 / Chapter 2.2.2.8 --- In Vitro Assay of rmIL-3-Stimulated Proliferation of Murine Bone Marrow Cells --- p.73 / Chapter 2.2.2.9 --- In Vitro Cytotoxicity of K7 Capsular Antigen on Bone Marrow Cells --- p.73 / Chapter 2.2.2.10 --- Colony Assay of Murine Bone Marrow Cells --- p.73 / Chapter 2.2.2.11 --- Assay of Differentiation of Murine Bone Marrow Cells --- p.74 / Chapter 2.2.3 --- Assay for Macrophage Activating Activities of Klebsiella K7 Capsular Antigen --- p.75 / Chapter 2.2.3.1 --- Preparation of Murine Peritoneal Exudate Cells (PEC) --- p.75 / Chapter 2.2.3.2 --- Assay of Phagocytic Activity of Peritoneal Macrophages --- p.75 / Chapter 2.2.3.3 --- In Vitro Macrophage-Mediated Cytostatic Activity --- p.76 / Chapter 2.2.3.4 --- Nitric Oxide (NO) Production of Peritoneal Macrophages --- p.76 / Chapter 2.2.3.5 --- In Vivo Migration of Macrophages --- p.77 / Chapter 2.2.3.6 --- Tumour Necrosis Factor (TNF) Production by Peritoneal Macrophages --- p.77 / Chapter 2.2.3.7 --- TNF Bioassay --- p.78 / Chapter 2.2.3.8 --- Gene Expression Analysis in Peritoneal Macrophages --- p.78 / Chapter 2.2.3.8.1 --- Preparation of Cell Lysate --- p.78 / Chapter 2.2.3.8.2 --- RNA Isolation --- p.79 / Chapter 2.2.3.8.3 --- Reverse Transcription of RNA --- p.80 / Chapter 2.2.3.8.4 --- Polymerase Chain Reaction (PCR) --- p.80 / Chapter 2.2.3.8.5 --- Agarose Gel Electrophoresis --- p.82 / Chapter 2.2.3.8.6 --- 3' End Labelling of Oligonucleotide Probes --- p.82 / Chapter 2.2.3.8.7 --- Dot Blot Hybridization --- p.83 / Chapter 2.2.3.8.8 --- DIG Chemiluminescent Detection --- p.83 / Chapter 2.2.4 --- Assay for Anti-Tumour Activities of Klebsiella K7 Capsular Antigen --- p.85 / Chapter 2.2.4.1 --- Assay of Tumour Cell Proliferation --- p.85 / Chapter 2.2.4.2 --- Assay of Anti-Tumour Activity In Vivo --- p.85 / Chapter 2.2.4.3 --- Assay of Lymphokine-Activated Killer Cell Activity --- p.85 / Chapter 2.2.4.4 --- Assay of Natural Killer Cell Activity --- p.87 / Chapter 2.2.4.5 --- Assay for Differentiation-Associated Characteristics of Myeloid Leukemia Cells --- p.88 / Chapter 2.2.4.5.1 --- Determination of the Viability of Myeloid Leukemia JCS cells --- p.88 / Chapter 2.2.4.5.2 --- Assessment of Cell Morphology --- p.88 / Chapter 2.2.4.5.3 --- Surface Antigen Immunophenotyping --- p.88 / Chapter 2.2.4.5.4 --- Assay of Non-specific Esterase Activity --- p.89 / Chapter 2.2.4.5.5 --- Assay of Phagocytosis --- p.90 / Chapter 2.2.5 --- Statistical Analysis --- p.90 / Chapter CHAPTER 3: --- "EXTRACTION, PURIFICATION & CHARACTERIZATION OF KLEBSIELLA K7 CAPSULAR ANTIGEN" --- p.91 / Chapter 3.1 --- Introduction --- p.92 / Chapter 3.2 --- Results --- p.94 / Chapter 3.2.1 --- Extraction and Purification of K7 Capsular Antigen from Klebsiella pneumoniae Serotype7 --- p.94 / Chapter 3.2.2 --- Gel Filtration Chromatography of K7 Capsular Antigen --- p.94 / Chapter 3.2.3 --- Characterization of K7 Capsular Antigen --- p.97 / Chapter 3.2.4 --- Determination of Bio-toxicity and Cellular Toxicity of K7 Capsular Antigen --- p.97 / Chapter 3.3 --- Discussion --- p.101 / Chapter CHAPTER 4: --- HEMATOPOIETIC AND MITOGENIC ACTIVITIES OF KLEBSIELLA K7 CAPSULAR ANTIGEN --- p.103 / Chapter 4.1 --- Introduction --- p.104 / Chapter 4.2 --- Results --- p.107 / Chapter 4.2.1 --- Effect of K7 Capsular Antigen on the In Vitro Proliferation of Murine Bone Marrow Cells --- p.107 / Chapter 4.2.2 --- Effect of K7 Capsular Antigen on rmIL-3-Stimulated Proliferation of Murine Bone Marrow Cells --- p.107 / Chapter 4.2.3 --- Effect of K7 Capsular Antigen on the In Vitro Differentiation of Murine Bone Marrow Cells --- p.112 / Chapter 4.2.4 --- Effect of K7 Capsular Antigen on the In Vitro Murine Bone Marrow Colony Formation --- p.112 / Chapter 4.2.5 --- Effect of K7 Capsular Antigen on the Viability of Splenocytes In Vitro --- p.117 / Chapter 4.2.6 --- In Vitro Mitogenic Effect of K7 Capsular Antigen on Splenocytes and Thymocytes --- p.117 / Chapter 4.2.7 --- Characterization of the Lymphocyte Population(s) Responding to K7 Capsular Antigen --- p.121 / Chapter 4.2.8 --- Effect of Polymyxin B Sulphate on the Mitogenic Activity of K7Capsular Antigen --- p.125 / Chapter 4.2.9 --- In Vivo Mitogenic Activity of K7 Capsular Antigen --- p.125 / Chapter 4.2.10 --- Flow Cytometric Analysis of Splenocytes from K7 Capsular Antigen-Treated Mice --- p.128 / Chapter 4.2.11 --- In Vitro Co-mitogenic Activity of K7 Capsular Antigen --- p.128 / Chapter 4.3 --- Discussion --- p.132 / Chapter CHAPTER 5: --- ACTIVATION OF MACROPHAGES BY KLEBSIELLA K7CAPSULAR ANTIGEN --- p.136 / Chapter 5.1 --- Introduction --- p.137 / Chapter 5.2 --- Results --- p.140 / Chapter 5.2.1 --- Effect of K7 Capsular Antigen on the Phagocytic Activity of Macrophages In Vitro --- p.140 / Chapter 5.2.2 --- Effect of K7 Capsular Antigen on the In Vivo Migration of Macrophages --- p.140 / Chapter 5.2.3 --- Effect of K7 Capsular Antigen on the In Vitro Cytostatic Activity of Picolinic Acid (PLA)-Activated Macrophages --- p.144 / Chapter 5.2.4 --- Effect of K7 Capsular Antigen on Nitric Oxide (NO) Production by Macrophages In Vitro --- p.144 / Chapter 5.2.5 --- Effect of K7 Capsular Antigen on the In Vitro Production of Tumour Necrosis Factor (TNF) by Macrophages --- p.148 / Chapter 5.2.6 --- Effect of K7 Capsular Antigen on the In Vitro Induction of Gene Expression in Macrophages --- p.148 / Chapter 5.3 --- Discussion --- p.166 / Chapter CHAPTER 6: --- ANTI-TUMOUR ACTIVITIES OF KLEBSIELLA K7 CAPSULAR ANTIGEN --- p.171 / Chapter 6.1 --- Introduction --- p.172 / Chapter 6.2 --- Results --- p.174 / Chapter 6.2.1 --- Effect of K7 Capsular Antigen In Vitro Growth of Various Tumour Cell Lines --- p.174 / Chapter 6.2.2 --- Effect of K7 Capsular Antigen on the In Vitro Growth of EAT Cells --- p.174 / Chapter 6.2.3 --- Effect of K7 Capsular Antigen on the Activation of LAK cells --- p.185 / Chapter 6.2.4 --- Effect of K7 Capsular Antigen on the Activation of NK Cells --- p.185 / Chapter 6.2.5 --- Effect of K7 Capsular Antigen on the Induction of Monocytic Differentiation of the Murine Myeloid Leukemia JCS Cells --- p.185 / Chapter 6.2.5.1 --- Effect of K7 Capsular Antigen on the Induction of Morphological Changes of JCS Cells --- p.188 / Chapter 6.2.5.2 --- Effect of K7 Capsular Antigen on the Expression of Macrophage Differentiation Antigen on Leukemia JCS Cells --- p.192 / Chapter 6.2.5.3 --- Effect of K7 Capsular Antigen on the Induction of Non-specific Esterase Activity in Leukemia JCS Cells --- p.192 / Chapter 6.2.5.4 --- Effect of K7 Capsular Antigen on the Stimulation of Phagocytic Activity in Leukemia JCS Cells --- p.192 / Chapter 6.3 --- Discussion --- p.198 / Chapter CHAPTER 7: --- CONCLUSIONS & FUTURE PERSPECTIVES --- p.202 / REFERENCES --- p.213

by Ho Cheong Yip. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 152-164). / ACKNOWLEDGMENTS --- p.I / ABBREVIATIONS --- p.II / ABSTRACT --- p.VI / CHAPTER / Chapter 1 --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Immunomodulators of biological origin --- p.1 / Chapter 1.2 --- Effector cells involved in anti-tumor immunity --- p.3 / Chapter 1.2.1 --- Cytotoxic T lymphocytes --- p.3 / Chapter 1.2.2 --- Macrophages --- p.4 / Chapter 1.2.3 --- Natural killer cells --- p.5 / Chapter 1.2.4 --- Lymphokine-activated killer cells --- p.7 / Chapter 1.2.5 --- Tumor-infiltrating lymphocytes --- p.9 / Chapter 1.3 --- Cytokines involved in immunomodulation and anti-tumor immunity --- p.11 / Chapter 1.3.1 --- Interleukin-1 --- p.11 / Chapter 1.3.2 --- Interleukin-2 --- p.13 / Chapter 1.3.3 --- Interleukin-3 --- p.14 / Chapter 1.3.4 --- Tumor necrosis factor-alpha --- p.15 / Chapter 1.3.5 --- Interferons --- p.18 / Chapter 1.4 --- Carbohydrates used as potential immunopotentiating agents --- p.19 / Chapter 1.5 --- General properties of K1 capsular antigen --- p.20 / Chapter 2 --- AIM AND SCOPE OF THIS DISSERTATION --- p.22 / Chapter 3 --- MATERIALS AND METHODS --- p.24 / Chapter 3.1 --- Materials --- p.24 / Chapter 3.2 --- Methods --- p.33 / Chapter (I) --- "Extraction, purification and characterization of K1 capsular antigen from Klebsiella pneumoniae" / Chapter 3.2.1 --- Extraction and purification of Kl capsular antigen --- p.33 / Chapter 3.2.2 --- Gel filtration of K1 capsular antigen --- p.35 / Chapter 3.2.3 --- Characterization of K1 capsular antigen --- p.35 / Chapter 3.2.3.1 --- Determination of carbohydrate and protein contents --- p.35 / Chapter 3.2.3.2 --- Determination of uronic acid content --- p.35 / Chapter 3.2.4 --- Determination of bio-toxicity of K1 capsular antigen --- p.35 / Chapter 3.2.5 --- Treatment of K1 capsular antigen with sodium periodate --- p.36 / Chapter 3.2.6 --- Treatment of K1 capsular antigen with sodium hydroxide followed by acetic acid neutralization --- p.36 / Chapter (II) --- Isolation and preparation of cells / Chapter 3.2.7 --- Murine splenocytes --- p.37 / Chapter 3.2.8 --- Removal of red blood cells and dead cells using Ficoll-paque gradient method --- p.37 / Chapter 3.2.9 --- Murine thymocytes --- p.37 / Chapter 3.2.10 --- Murine peritoneal exudate cells (PEC) --- p.38 / Chapter 3.2.11 --- Bone marrow cells --- p.38 / Chapter (III) --- Assays of immunomodulatory activities of K1 capsular antigen on lymphocytes / Chapter 3.2.12 --- In vitro mitogenic assay --- p.39 / Chapter 3.2.13 --- In vivo mitogenic assay --- p.39 / Chapter 3.2.14 --- "In vitro co-mitogenic assay of K1 capsular antigen with Polymyxin B sulfate, LPS or Con A" --- p.40 / Chapter 3.2.15 --- In vitro mitogenic effect of K1 capsular antigen on lymphocyte sub-populations --- p.40 / Chapter 3.2.16 --- Assay of murine interleukin-2 --- p.41 / Chapter 3.2.17 --- Assay of murine interleukin-1 --- p.41 / Chapter (IV) --- Assays of immunomodulatory activities of K1 capsular antigen on macrophages and bone marrow cells / Chapter 3.2.18 --- In vivo migration of macrophages --- p.42 / Chapter 3.2.19 --- Assay of interleukin-1 produced from murine macrophages --- p.42 / Chapter 3.2.20 --- Assay of nitric oxide produced from murine macrophages --- p.43 / Chapter 3.2.21 --- Assay of proliferation of murine bone marrow cells --- p.44 / Chapter 3.2.22 --- Assay of differentiation of murine bone marrow cells --- p.44 / Chapter (V) --- Assays of anti-tumor activities of K1 capsular antigen / Chapter 3.2.23 --- In vitro cytostatic effect of K1 capsular antigen on murine tumor cell lines --- p.45 / Chapter 3.2.24 --- In vivo anti-tumor activities of K1 capsular antigen --- p.45 / Chapter 3.2.25 --- In vitro stimulation of TNF-α-like factor release from thioglycollate-elicited murine peritoneal macrophages by K1 capsular antigen --- p.46 / Chapter 3.2.26 --- Effects of K1 capsular antigen on TNF-α production in normal mice in vivo --- p.47 / Chapter 3.2.27 --- Effects of K1 capsular antigen on TNF-α production as well as EAT growth in vivo --- p.48 / Chapter 3.2.28 --- In vitro induction of macrophage-mediated cytostasis on tumor cells --- p.48 / Chapter 3.2.29 --- In vitro induction of macrophage-mediated cytolysis on tumor cells --- p.49 / Chapter 3.2.30 --- In vivo induction of alloreactive cytotoxic T cells --- p.49 / Chapter 3.2.31 --- In vitro induction of lymphokine-activated killer cell activity --- p.50 / Chapter 3.2.32 --- In vivo induction of lymphokine-activated killer cell activity --- p.51 / Chapter 3.2.33 --- In vivo induction of lymphokine-activated killer cell activity in tumor-bearing mice --- p.52 / Chapter 3.2.34 --- In vivo induction of lymphokine-activated killer cell activity in tumor-bearing mice with in vitro culture --- p.52 / Chapter 3.2.35 --- Phenotypic characterization of LAK cells by flow cytometry --- p.53 / Chapter 3.2.36 --- Winn-type tumor-inhibition of LAK cells --- p.54 / Chapter 3.2.37 --- Adoptive transfer of LAK cells to tumor-bearing mice --- p.54 / Chapter 3.2.38 --- In vivo stimulation of tumor-infiltrating lymphocytes --- p.55 / Chapter 3.2.39 --- Statistical analysis --- p.57 / Chapter 4 --- "EXTRACTION, PURIFICATION AND CHARACTERIZATION OF K1 CAPSULAR ANTIGEN FROM KLEBSIELLA PNEUMONIAE" / Introduction --- p.58 / Results --- p.59 / Chapter 4.1 --- Extraction and purification of Kl capsular antigen from Klebsiella pneumoniae --- p.59 / Chapter 4.2 --- Gel filtration of Kl capsular antigen --- p.59 / Chapter 4.3 --- Characterization of Kl capsular antigen --- p.62 / Chapter 4.4 --- Determination of toxicity (LC50) of Kl capsular antigen by brine shrimp assay --- p.62 / Discussion --- p.62 / Chapter 5 --- IMMUNOMODULATORY ACTIVITIES OF K1 CAPSULAR POLYSACCHARIDE ANTIGEN FROM KLEBSIELLA PNEUMONIAE / Introduction --- p.67 / Results --- p.69 / Chapter 5.1 --- Mitogenic activity of Kl capsular antigen on murine lymphocytes in vitro --- p.69 / Chapter 5.2 --- Mitogenic activity of K1 capsular antigen on murine lymphocytes in vivo --- p.69 / Chapter 5.3 --- Evidences to support the mitogenic activity of K1 capsular antigen is due to its polysaccharide structure rather than due to the contamination by LPS --- p.74 / Chapter 5.4 --- Effect of Kl capsular antigen on IL-2 production from murine lymphocytes in vitro --- p.79 / Chapter 5.5 --- Effect of K1 capsular antigen on IL-1 -like factor production from murine thymocytes in vitro --- p.79 / Chapter 5.6 --- Immunopotentiating activities of Kl capsular antigen on macrophages --- p.85 / Chapter 5.6.1 --- In vivo migration of macrophages in K1-treated mice --- p.85 / Chapter 5.6.2 --- Effect ofKl capsular antigen on the macrophage EL-1-like factor production in vitro --- p.85 / Chapter 5.6.3 --- Effect ofKl capsular antigen on the macrophage nitric oxide (NO) production in vitro --- p.85 / Chapter 5.7 --- Effects of Kl capsular antigen on the proliferation and differentiation of murine bone marrow cells --- p.89 / Discussion ´ب --- p.94 / Chapter 6 --- ANTI-TUMOR ACTIVITIES OF K1 CAPSULAR POLYSACCHARIDE ANTIGEN FROM KLEBSIELLA PNEUMONIAE / Introduction --- p.100 / Results --- p.102 / Chapter 6.1 --- In vitro cytostatic effects of Kl capsular antigen on murine tumor cell lines --- p.102 / Chapter 6.2 --- In vivo anti-tumor activities of Kl capsular antigen --- p.102 / Chapter 6.3 --- Effects of Kl capsular antigen on TNF-α production and on EAT growth in vivo --- p.110 / Chapter 6.4 --- In vitro induction of macrophage-mediated cytostatic effect on tumor cells by K1 capsular antigen --- p.110 / Chapter 6.5 --- In vitro induction of macrophage-mediated cytolytic effect on tumor cells by K1 capsular antigen --- p.115 / Chapter 6.6 --- Effect of Kl capsular antigen on the activation of alloreactive cytotoxic T cells --- p.115 / Chapter 6.7 --- Effect of Kl capsular antigen on the activation of lymphokine- activated killer (LAK) cells in vitro and in vivo --- p.115 / Chapter 6.8 --- Phenotypic characterization of LAK cells by flow cytometry --- p.123 / Chapter 6.9 --- Winn-type tumor inhibition assay of LAK cells --- p.126 / Chapter 6.10 --- Adoptive transfer of LAK cells to tumor-bearing mice --- p.132 / Chapter 6.11 --- Effect ofKl capsular antigen on the activation of tumor- infiltrating lymphocytes (TILs) in tumor-bearing mice --- p.132 / Discussion --- p.136 / Chapter 7 --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.146 / BIBLIOGRAPHY --- p.152

Orientador: Lucia Maria Carareto Alves / Coorientador: Alessandro de Mello Varani / Banca: Hugo Miguel Preto de Morais Sarmento / Banca: Rodrigo Matheus Pereira / Banca: Everlon Cid Rigobelo / Banca: Silvana Pompéia do Val de Moraes / Resumo: O composto orgânico produzido a partir de carcaças, resíduos animais e vegetais é uma alternativa viável para a substituição total ou parcial dos fertilizantes minerais utilizados na atualidade. No processo de compostagem participam diferentes populações microbianas, e com isso o composto torna-se um sistema rico para utilização como fertilizante no solo, complementando assim as necessidades nutricionais e microbianas do meio ambiente. Entretanto, há poucos trabalhos envolvendo análise da diversidade bacteriana em solos sob uso de composto orgânico feito a partir de carcaças, e também pouco se conhece sobre o impacto ambiental do uso agrícola de composto orgânico na qualidade da água. Existe também a questão se há influência da água utilizada para irrigação na qualidade do solo. O objetivo central desse trabalho foi analisar a diversidade bacteriana e perfil funcional de um solo de horta e da água de um córrego utilizada para irrigação. E como objetivo secundário, através do isolamento de bactérias da água verificar o potencial biotecnológico de produção e uso de exopolissacarídeo como bioemulsificante de óleo e hidrocarbonetos. As amostras de solo e água utilizadas nesse trabalho foram coletadas na área do departamento rural da Fundação Parque Zoológico de São Paulo, em setembro de 2014. Todo material coletado foi transportado até o Laboratório de Bioquímica de Micro-organismos e de Plantas, onde realizou-se a extração de DNA total e sequenciamento através de tecnologia Ion ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The organic compost produced from carcasses, animal and vegetable waste is a viable alternative to full or partial replacement of mineral fertilizers used nowadays. In the composting process there are involved different microbial populations, and the compost becomes a rich system for use as a fertilizer in the soil, thereby supplementing the nutritional and microbial requirements of the medium. However, there are few studies involving the analysis of bacterial diversity in soil under use of organic compost made from carcasses, and also little is known about the environmental impact of agricultural use of organic compost in water quality. Furthermore, there is also the question of whether there is influence of the water used for irrigation on soil quality. Because of these questions, the central objective of this study was to analyze the bacterial diversity and functional profile of a soil from vegetable garden and freshwater used for irrigation from a local stream. As a secondary objective, we aimed to verify the production and biotechnology potential of a bacterial exopolysaccharide as oil and hydrocarbons bioemulsifier. Soil and freshwater samples used in this study were collected at rural department of the Zoo Foundation Park of São Paulo, in September 2014. All material collected was transported to the Laboratório de Bioquímica de Micro-organismos e de Plantas where we proceeded with the total DNA extraction experiments, sequencing through Ion Proton technology (Life Technologies), bacterial isolation, production and application of exopolysaccharide as bioemulsifier. By analyzing the metagenomic DNA it was observed that both freshwater and soil were plenty of bacterial communities normally found in agricultural areas under influence of organic amendments. Through the analysis of genes related to biogeochemical cycles, it was found abundance ... (Complete abstract click electronic access below) / Doutor

Ce travail a été consacré à l'étude expérimentale du procédé de foisonement en continu appliqué aux émulsions alimentaires. Son objectif était d'analyser l'effet combiné des technologies, des variables de formulations et des conditions opératoires du procédé. L'étude a porté sur trois familles d'émulsions alimentaires commerciales : les "acides" de type "fromages frais", les "salées" de type "sauce blanche" et les "sucrées" de type "topping de dessert lacté". Du point de vue technologique, trois foisonneurs continus ont été comparés : une colonne agitée à faible entrefer, un rotor/stator axial à dents et un échangeur à surface raclée. L'étude des paramètres opératoires inclut les effets d'échelle, de la vitesse d'agitation, du temps de séjour et de la pression d'opération. L'analyse de la puissance dissipée par les foisonneurs et l'effet de la substitution de la gélatine par les polysaccharides en qualité d'agent épaississant dans le topping ont également été abordés.

Graduation date: 1994

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