by Wang He-Xiang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 161-179). / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / LIST OF CONTENTS --- p.v / LIST OF TABLES --- p.xi / LIST OF FIGURES --- p.xii / LIST OF ABBREVIATIONS --- p.xvi / Chapter CHAPTER 1. --- General Introduction --- p.1 / Chapter CHAPTER 2. --- Literature Review --- p.5 / Chapter 2.1. --- Lectins --- p.5 / Chapter 2.1.1. --- Aspects of lectins --- p.5 / Chapter 2.1.2. --- Isolation and purification of lectins --- p.8 / Chapter 2.1.3. --- Characteristics of lectins --- p.9 / Chapter 2.1.4. --- Effects of lectins on biological activities --- p.10 / Chapter 2.1.4.1. --- The role of lectins in plant defence --- p.11 / Chapter 2.1.4.2. --- The specificity of some legume lectins --- p.13 / Chapter 2.1.4.3. --- Some properties of animal lectins --- p.14 / Chapter 2.1.4.4. --- Hypotensive activity of the lectins --- p.18 / Chapter 2.1.4.5. --- Lectins in immunology --- p.20 / Chapter 2.2. --- Mushroom Lectins and Polysaccharides --- p.24 / Chapter 2.2.1. --- General aspects of mushroom lectins and polysaccharides --- p.24 / Chapter 2.2.2. --- Mushroom lectins --- p.25 / Chapter 2.2.2.1. --- Hericium erinaceum lectin --- p.26 / Chapter 2.2.2.2. --- Lactarius deterrimus lectin --- p.26 / Chapter 2.2.2.3. --- Laetiporus sulfureus lectin --- p.27 / Chapter 2.2.2.4. --- Grifola frondosa lectin --- p.28 / Chapter 2.2.2.5. --- Volvariella volvacea lectin --- p.28 / Chapter 2.2.2.6. --- Flammulina veltipes lectin --- p.29 / Chapter 2.2.2.7. --- Ischnoderma resinosum agglutinin --- p.31 / Chapter 2.2.2.8. --- Lectins from Agaricus spp --- p.31 / Chapter 2.2.3. --- Mushroom polysaccharides --- p.34 / Chapter 2.2.3.1. --- Lentinan --- p.35 / Chapter 2.2.3.2. --- "PSK (trade name, Krestin)" --- p.35 / Chapter 2.2.3.3. --- PSP (Polysaccharopeptide) --- p.37 / Chapter 2.2.3.4. --- PSPC (polysaccharide-peptide complex) --- p.38 / Chapter CHAPTER 3. --- Isolation and Characterization of Two Distinct Lectins from the Cultured Mycelium of the Edible Mushroom Tricholoma mongolicum --- p.44 / Chapter 3.1. --- Introduction --- p.44 / Chapter 3.2. --- Materials and Methods --- p.45 / Chapter 3.2.1. --- Strain and culture condition --- p.45 / Chapter 3.2.2. --- Extraction --- p.46 / Chapter 3.2.3. --- Purification --- p.46 / Chapter 3.2.4. --- Hemagglutination activity --- p.47 / Chapter 3.2.5. --- Test of hemagglutination inhibition by various carbohydrates --- p.47 / Chapter 3.2.6. --- MW estimation by gel filtration and SDS- PAGE --- p.48 / Chapter 3.2.7. --- Glycoprotein staining with PAS reagent --- p.49 / Chapter 3.2.8. --- Carbohydrate content --- p.49 / Chapter 3.2.9. --- Thermal stability --- p.49 / Chapter 3.2.10. --- pH stability --- p.49 / Chapter 3.2.11. --- Effect of cations --- p.50 / Chapter 3.2.12. --- Amino acid analysis --- p.50 / Chapter 3.2.13. --- Antiproliferative activity of lectins --- p.50 / Chapter 3.2.14. --- Statistics --- p.51 / Chapter 3.3. --- Results --- p.51 / Chapter 3.3.1. --- Extraction and purification --- p.51 / Chapter 3.3.2. --- General characteristics of lectins --- p.52 / Chapter 3.3.3. --- Antiproliferative activity of lectins --- p.54 / Chapter 3.4. --- Discussion --- p.55 / Chapter 3.5. --- Summary --- p.58 / Chapter CHAPTER 4. --- The Immunomodulatory and Antitumor Activities of Lectins from the Mushroom Tricholoma mongolicum --- p.79 / Chapter 4.1. --- Introduction --- p.79 / Chapter 4.2. --- Materials and Methods --- p.81 / Chapter 4.2.1. --- Lectins --- p.81 / Chapter 4.2.2. --- Animals --- p.81 / Chapter 4.2.3. --- Assay for antitumor activity --- p.81 / Chapter 4.2.4. --- Assessment of tumor growth and host survival after lectin treatment --- p.82 / Chapter 4.2.5. --- Mitogenic activity of lectins --- p.82 / Chapter 4.2.6. --- Production of nitrite ions in response to lectin treatment --- p.83 / Chapter 4.2.7. --- Preparation of concanavalin A-stimulated lymphokines --- p.84 / Chapter 4.2.8. --- Assay for macrophage activating factor --- p.85 / Chapter 4.2.9. --- Production of tumor necrosis factor (TNF) --- p.86 / Chapter 4.2.10. --- Bioassay for tumor necrosis factor --- p.86 / Chapter 4.2.11. --- Statistics --- p.87 / Chapter 4.3. --- Results --- p.87 / Chapter 4.3.1. --- Antitumor activity --- p.87 / Chapter 4.3.2. --- Assessment of tumor growth and host survival --- p.87 / Chapter 4.3.3. --- Mitogenic activity --- p.88 / Chapter 4.3.4. --- Production of nitrite ions --- p.89 / Chapter 4.3.5. --- Production of macrophage activating factor --- p.89 / Chapter 4.3.6. --- Tumor necrosis factor assay --- p.90 / Chapter 4.4. --- Discussion --- p.90 / Chapter 4.5. --- Summary --- p.94 / Chapter CHAPTER 5. --- Hypotensive and Vasorelaxing Activities of a Lectin (TML-1) from the Edible Mushroom Tricholoma mongolicum --- p.109 / Chapter 5.1. --- Introduction --- p.109 / Chapter 5.2. --- Materials and Methods --- p.111 / Chapter 5.2.1. --- Animals --- p.111 / Chapter 5.2.2. --- In vivo blood pressure measurement in rats --- p.112 / Chapter 5.2.3. --- Study employing blockade of autonomic ganglion transmission --- p.113 / Chapter 5.2.4. --- Study employing alpha-adrenergic blockade --- p.113 / Chapter 5.2.5. --- Study employing beta-adrenergic blockade --- p.114 / Chapter 5.2.6. --- Study employing cholinergic blockade --- p.114 / Chapter 5.2.7. --- Study employing histaminergic blockade --- p.114 / Chapter 5.2.8. --- Study employing inhibitor of the renin- angiotensin system --- p.115 / Chapter 5.2.9. --- Preparation of right atrium for in vitro studies --- p.115 / Chapter 5.2.10. --- Preparation of aorta for in vitro studies --- p.116 / Chapter 5.2.11. --- Adenosine receptor binding assays --- p.116 / Chapter 5.2.12. --- Effect of methylene blue on the hypotensive activity of TML-1 --- p.118 / Chapter 5.2.13. --- Statistics --- p.118 / Chapter 5.3. --- Results --- p.118 / Chapter 5.3.1. --- Blood pressure changes in vivo --- p.118 / Chapter 5.3.2. --- Pharmacological studies using receptor antagonists --- p.119 / Chapter 5.3.3. --- Adenosine receptor binding assay --- p.119 / Chapter 5.3.4. --- Effects on the right atrium in vitro --- p.120 / Chapter 5.3.5. --- Effect of TML-1 on vascular relaxation --- p.120 / Chapter 5.3.6. --- Effect of methylene blue on the hypotensive activity of TML-1 --- p.120 / Chapter 5.4. --- Discussion --- p.120 / Chapter 5.5. --- Summary --- p.123 / Chapter CHAPTER 6. --- A Polysaccharide-Peptide Complex with Immunoenhancing and Antitumor Activities from Cultured Mycelia of the Mushroom Tricholoma mongolicum --- p.134 / Chapter 6.1. --- Introduction --- p.134 / Chapter 6.2. --- Materials and Methods --- p.135 / Chapter 6.2.1. --- Extraction --- p.135 / Chapter 6.2.2. --- Purification --- p.135 / Chapter 6.2.3. --- PSP for purpose of comparison --- p.136 / Chapter 6.2.4. --- Polysaccharide and protein contents --- p.136 / Chapter 6.2.5. --- MW determination of F1 using gel filtration --- p.136 / Chapter 6.2.6. --- Animals --- p.136 / Chapter 6.2.7. --- Antiproliferative activity assay --- p.137 / Chapter 6.2.8. --- Mitogenic activity --- p.137 / Chapter 6.2.9. --- Production of nitrite ions --- p.138 / Chapter 6.2.10. --- Macrophage activating factor assay --- p.138 / Chapter 6.2.11. --- Antitumor activity assay --- p.139 / Chapter 6.2.12. --- Statistics --- p.139 / Chapter 6.3. --- Results --- p.140 / Chapter 6.3.1. --- Purification of polysaccharide-peptide complex --- p.140 / Chapter 6.3.2. --- Antiproliferative activity --- p.140 / Chapter 6.3.3. --- Mitogenic activity in vitro --- p.140 / Chapter 6.3.4. --- Molecular weight of Fl --- p.141 / Chapter 6.3.5. --- Mitogenic activity in vivo --- p.141 / Chapter 6.3.6. --- Production of nitrite ions --- p.141 / Chapter 6.3.7. --- Production of macrophage activating factor --- p.141 / Chapter 6.3.8. --- Antitumor activity in vivo --- p.142 / Chapter 6.4. --- Discussion --- p.142 / Chapter 6.5. --- Summary --- p.144 / GENERAL DISCUSSION --- p.155 / CONCLUSIONS --- p.158 / REFERENCES

by Tang Yan Chi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 156-168). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABBREVIATIONS --- p.II / ABSTRACT --- p.V / ABSTRACT (CHINESE) --- p.VII / TABLE OF CONTENT --- p.1 / GENERAL INTRODUCTION --- p.4 / Chapter 1.1. --- Immunotherapy in Cancer --- p.4 / Chapter 1.1.2. --- Cytokines used in Immunotherapy in Cancer --- p.5 / Chapter 1.1.2.1. --- Interferons (IFNs) --- p.6 / Chapter 1.1.2.2. --- interleukins (ILs) --- p.11 / Chapter 1.1.2.3. --- Tumor Necrosis Factor-Alpha (TNF-α) --- p.14 / Chapter 1.2. --- Immunomodulatory and Anti-tumor Activities of Plant Polysaccharides --- p.17 / Chapter 1.3. --- Previous Studies on the Capsular Polysaccharides (CPS) from Klebsiella pneumoniae --- p.21 / Chapter 1.4. --- Activation of TNF-α Production from Macrophages by Bacterial Polysaccharides --- p.26 / Chapter 1.5. --- Chemotherapy of Cancer --- p.29 / Chapter 1.5.1. --- Types of Chemotherapeutic Drugs --- p.29 / Chapter 1.5.1.1. --- Alkylating Agents --- p.29 / Chapter 1.5.1.2. --- Plant Alkaloids --- p.31 / Chapter 1.5.1.3. --- Anti-metabolites --- p.32 / Chapter 1. 5.1.4. --- Antitumor Antibiotics --- p.32 / Chapter 1.5.2. --- Chemotherapeutic Drugs in this Project --- p.34 / Chapter 1.5.2.1. --- actinomycin D (ACT D) --- p.34 / Chapter 1.5.2.2. --- Cyclophosphamide (CY) --- p.35 / Chapter 1.5.2.3. --- Doxorubicin (Dox) --- p.37 / Chapter CHAPTER TWO --- AIM AND SCOPE OF THIS DISSERTATION --- p.40 / Chapter CHAPTER THREE --- MATERIALS AND METHODS --- p.42 / Chapter 3.1 --- MATERIALS --- p.42 / Chapter 3.1.1. --- Animals --- p.42 / Chapter 3.1.2. --- Klebsiella pneumoniae Serotype 1 and Serotype 24 --- p.42 / Chapter 3.1.3. --- Cell lines --- p.43 / Chapter 3.1.4. --- "Buffers, culture media and chemicals" --- p.43 / "POLYCLONAL RABBIT ANTI-ACTIVE ERK1/2, ANTI-ACTIVE JNK 1/2 AND ANTI-ACTIVE P38WERE FROM PROMEGA. POLYCLONAL RABBIT ANTI-TOTAL JNK1, ANTI-TOTAL P38WERE FROM SANTA CRUZ. MONOCLONAL ANTI-MOUSE TOTAL ERK1 AND POLYCLONAL ANTI-RABBIT TOTAL ERK2 WERE PURCHASED FROM ZYMED" --- p.50 / Chapter 3.2. --- METHODS --- p.51 / Chapter 3.2.1. --- "Extraction, Purification and Characterization of K1 and K24 Capsular Polysaccharides (CPS) from Klebsiella pneumoniae" --- p.51 / Chapter 3.2.1.1. --- Extraction and Purification of K1 and K24 Klebsiella Capsular Polysaccharides (CPS) --- p.51 / Chapter 3.2.1.2. --- Gel filtration of K1 and K24 Klebsiella Capsular Polysaccharides --- p.52 / Chapter 3.2.1.3. --- Characterization of K1 and K24 Capsular Polysaccharides --- p.52 / Chapter 3.2.3. --- Assay for Macrophage Activating Activities of Klebsiella Capsular Polysaccharides --- p.55 / Chapter 3.2.3.1. --- Tumour Necrosis Factor-α (TNF-α) Production by Peritoneal Macrophages --- p.55 / Chapter 3.2.3.2. --- Effect of Capsular Polysaccharides on the activities of MAPK family in murine macrophages --- p.56 / Chapter 3.2.4. --- Assay for Anti-Tumor Activities of Klebsiella Capsular Polysaccharides --- p.63 / Chapter 3.2.4.1. --- Assay of in vivo Suppression of EAT growth by K1 and K24 Capsular Polysaccharides --- p.63 / Chapter 3.2.4.2. --- Assay of the Effect of CPS on the Survival of EAT-Bearing Mice --- p.64 / Chapter 3.2.5. --- "Assay for Anti-Tumor Activities of Combined Treatment of Klebsiella Capsular Polysaccharides and Chemotherapeutic Drugs (Actinomycin D (Act D), Cyclophosphamide (CY) and Doxorubicin (Dox)" --- p.65 / Chapter 3.2.5.1. --- "Assay of the Cytotoxic Effect of Chemotherapeutic Drugs： Actinomycin D (Act D), Cyclophosphamide (CY) and Doxorubicin (Dox), on wehi-164 cell line" --- p.65 / Chapter 3.2.5.2. --- "Assay of the Combined Treatment of the CPS and Chemotherapeutic Drugs (Act D, CY and Dox) on WEHI-164 cell line in vitro" --- p.66 / Chapter 3.2.5.3. --- Assay for Anti-Tumor Effect of Chemotherapeutic Drugs (Act D，CY and Dox) on EAT-bearing Mice in vivo --- p.67 / Chapter 3.2.5.4. --- "Assay of the Combined Treatment of the CPS and Chemotherapeutic Drugs (Act D, CY and Dox) on EAT-bearing Mice in vivo" --- p.68 / Chapter 3.2.5.5. --- "Assay of the Combined Treatment of the CPS and Chemotherapeutic Drugs (Act D, CY and Dox) on the survival of eat-bearing mice in vivo" --- p.72 / Chapter CHAPTER FOUR --- "EXTRACTION, PURIFICATION & CHARACTERIZATION OF KLEBSIELLA K1 & K24 CAPSULAR POLYSACCHARIDES" --- p.76 / Chapter 4.1. --- Extraction and Purification of K1 and K24 Capsular Polysaccharides from Klebsiella pneumoniae Serotype 1 and Serotype 24 --- p.76 / Chapter 4.2. --- Gel Filtration Chromatography of K1 and K24 Capsular Polysaccharides --- p.77 / Chapter 4.3. --- Characterization of K1 and K24 Capsular Polysaccharides --- p.81 / Chapter CHAPTER FIVE --- EFFECT OF THE K1 & K24 CAPSULAR POLYSACCHARIDES ON THE MACROPHAGE ACTIVITIES --- p.83 / Chapter 5.1. --- Effect ofK1 and K24 Capsular Polysaccharides on the in vitro induction of TNF- α Production of Murine Peritoneal Macrophages --- p.83 / Chapter 5.2. --- Effect of Capsular Polysaccharides on the activities of MAPK family in murine macrophages --- p.87 / Chapter CHAPTER SIX --- ANTI-TUMOR ACTIVITIES ON MURINE TUMOR CELL LINES OF KLEBSIELLA K1 &K24 CAPSULAR POLYSACCHARIDES --- p.94 / Chapter 6.1. --- Effect of K1 and K24 Capsular Polysaccharides on the In vivo Anti-tumor Activities on EAT-bearing Mice --- p.94 / Chapter 6.2. --- In vivo Anti-tumor Effect on the Survival of EAT-Bearing Mice by Treatment of K1 and K24 CPS --- p.97 / Chapter CHAPTER SEVEN --- "ANTI-TUMOR ACTIVITIES OF COMBINED TREATMENT OF KLEBSIELLA K1 &K24 CAPSULAR POLYSACCHARIDES AND CHEMOTHERAPEUTIC DRUGS: ACTINOMYCIN D, CYCLOPHOSPHAMIDE AND DOXORUBICIN" --- p.100 / Chapter 7.1. --- "Cytotoxic Effect Chemotherapeutic Drugs： Actinomycin D (Act D), Cyclophosphamide (CY) and Doxorubicin (Dox), on WEHI-164 cell line" --- p.100 / Chapter 7.2. --- "Cytotoxic Effect of the Combined Treatment of the CPS and the Chemotherapeutic Drugs (Act D, CY and Dox), on WEHI-164 cell line" --- p.104 / Chapter 7.3. --- "Anti-Tumor Effect of Chemotherapeutic Drugs (Act D, CY and Dox) on EAT-bearing Mice in vivo" --- p.111 / Chapter 7.4. --- "Combined Treatment of the CPS and Chemotherapeutic Drugs (Act D, CY and Dox) on the EAT Growth in vivo" --- p.115 / Chapter 7.5. --- "Combined Treatment of the CPS and Chemotherapeutic Drugs (Act D, CY and Dox) on the Survival of EAT-bearing Mice in vivo" --- p.122 / Chapter CHAPTER EIGHT --- GENERAL DISCUSSION --- p.135 / Chapter 8.1. --- "Extraction, Purification and Characterization of K1 and K24 Capsular Polysaccharides from Klebsiella pneumoniae Serotype 1 and Serotype 24" --- p.135 / Chapter 8.2. --- Effect of K1 and K24 Capsular Polysaccharides on the Macrophage Activation --- p.140 / Chapter 8.3. --- Anti-tumor Activities on Murine Tumor Cell LInes of Klebsiella K1 and K24 Capsular Polysaccharides --- p.145 / Chapter 8.4. --- "Anti-tumor Activities of Combined Treatment of K1 and K24 Capsular Polysaccharides and Chemotherapeutic Drugs： Actinomycin D, Cyclophosphamide and Doxorubicin" --- p.148 / Chapter CHAPTER NINE --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.152 / BIBLIOGRAPHY --- p.156

L’obtention de substrats „cyto-favorables”, aptes à soutenir la régénération tissulaire, impose l’utilisation de biomatériaux qui portent des domaines de reconnaissance cellulaire, comme par exemple les scléroprotéines et certains polysaccharides. La membrane des cellules spécifiques aux tissus conjonctifs dispose de mécanismes qui facilitent l’ancrage aux substrats solides ou à l’état de gel où se retrouvent des macromolécules ou des fibrilles de (atelo) collagène, associées ou non à l’acide hyaluronique. On peut générer de tels substrats par des techniques de rassemblement moléculaire spontané ordonné (tout comme dans le cas de la restructuration du collagène quasi-natif pour former des fibrilles), ou induite physico-chimiquement ensuite stabilisé morphologiquement (tout comme dans le cas de la préparation des hydrogels mixtes, atelocollagène–hyaluronate de sodium, diversement réticulés ensuite transformés en cryo- ou vitri-gels). Dans le cadre de la thèse, nous étudions les moyens d’obtention et de purification des précurseurs bio-macromoléculaires nécessaires, par la suite, à l’obtention de substrats „cyto-favorables”, ainsi que leurs modalités de génération et de caractérisation. Les méthodes de restructuration auxquelles on en appelle sont de nature physico-chimique (la co-précipitation contrôlée dans des mélanges binaires et ternaires d’atelocollagène et d’hyaluronate de sodium), ou chimique (la réticulation par des ponts moléculaires à longueur minimale). On a étudié les possibilités de mélanger de l’atelocollagène (aK) avec deux types de polysaccharides, le hyaluronate de sodium (NaHyal) et le gellane. On a établi des formulations et les procédures optimales pour obtenir des hydrogels avec des caractéristiques rhéologiques contrôlables, et avec la réactivité et la morphologie capables de permettre la fixation et la prolifération des fibroblastes. On constate que les hydrogels et cryogels obtenus à partir des mélanges 5:1 aK:NaHyal réticulés avec du 1,4-butanediol diglycidyl éther ont des propriétés rhéologiques qui permettent leurs manipulation dans les conditions des techniques de culture cellulaire. Ils ne présentent pas de cytotoxicité et ils assurent la viabilité cellulaire dans les milieux de culture standards. La morphologie des cryogels obtenus montre une macro-porosité qui dépend de la formulation des mélanges et peu la technique d'obtention. La présence de gellane dans les mélanges conduit à une séparation de phases, même à faible concentration, soulignant la diversité des caractéristiques de substrats. / Obtaining "cyto-favourable" substrates able to support tissue regeneration leads to use biomaterials holding cellular recognition domains, as scleroproteins and some polysaccharides as examples. Cellules membranes specific to conjunctive tissues have mechanisms making easier the anchoring to solid or gel substrates where macromolecules or fibrils of (aceto)collagen, associated or not to hyaluronic acid, are found. Such substrates may be generated using spontaneous molecular gathering (as in native collagen restructuration to fibrils), or physico-chemically induced (as the preparation of mixed hydrogels then transformed in cryo- or vitri-gels). In this thesis, were studied the obtaining and purification of bio-macromolecular presursors necessary to obtaining "cyto-favourable" substrates, and the procedures to generate and characterize them. Used restructuration methods are of physico-chemical nature (controlled co-precipitation in binary and ternary mixtures of acetocollagen and sodium hyaluronate) or chemical one (crosslinking).The mixture of acetocolagen (aK) with two polysaccharides, sodium hyaluronate (NaHyal) and gellan were investigated. Formulations and optimal conditions were established to obtain hydrogels with controlled rheological characteristics, and reactivity and morphology able to allow fibroplast fixation and proliferation. Hydrogels and cryogels prepared from 5:1 aK:NaHyal crosslinked with 1,4-butanediol diglycidyl ether were defined as the best materials we have prepared. They do not show any cytotoxicity and they ensure the cellular viability within standard cellule culture media. The cryogel morphology shows macro-porosity depending on the formulation but a few on the obtaining process. The presence of gellan in the mixtures leads to a phase separation, even at low concentration.

by Li Chi Yeung. / Thesis submitted in: November 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 151-176). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Acknowledgement --- p.ii / Abstract (English Version) --- p.iii / Abstract (Chinese Version) --- p.v / Content Page --- p.vii / List of Tables --- p.xiii / List of Figures --- p.xv / Abbreviation --- p.xvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Diabetes Mellitus --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Economic Impact --- p.3 / Chapter 1.2 --- "Digestion, Absorption and Metabolism of Carbohydrates" --- p.4 / Chapter 1.2.1 --- Carbohydrate Digestion --- p.4 / Chapter 1.2.2 --- Carbohydrate Absorption --- p.6 / Chapter 1.2.3 --- Insulin Secretion --- p.6 / Chapter 1.3 --- Pathophysiology of Diabetes Mellitus --- p.7 / Chapter 1.3.1 --- Insulin-Dependent Diabetes Mellitus (lDDM) --- p.7 / Chapter 1.3.1.1 --- Genetics --- p.8 / Chapter 1.3.1.2 --- Autoimmunity --- p.9 / Chapter 1.3.2 --- Non-Insulin-Dependent Diabetes Mellitus (NlDDM) --- p.11 / Chapter 1.3.2.1 --- Insulin Resistance --- p.11 / Chapter 1.3.2.2 --- Impaired Insulin Secretion --- p.14 / Chapter 1.4 --- Management of Diabetes Mellitus --- p.15 / Chapter 1.4.1 --- Sulfonylureas --- p.15 / Chapter 1.4.2 --- Biguanides --- p.16 / Chapter 1.4.3 --- Problems Encountered in the Management of Diabetes --- p.16 / Chapter 1.4.4 --- Role of Dietary Fiber in the Management of Diabetes Mellitus --- p.18 / Chapter 1.4.4.1 --- Dietary Fiber and Gastric Emptying Time --- p.19 / Chapter 1.4.4.2 --- Dietary Fiber and Glucose Absorption in Small Intestine --- p.20 / Chapter 1.4.5 --- Other Natural Products used for Diabetes Treatment…… --- p.22 / Chapter 1.5 --- Mushrooms --- p.22 / Chapter 1.5.1 --- The Definition of Mushrooms --- p.23 / Chapter 1.5.2 --- Nutritional Values of Mushrooms --- p.24 / Chapter 1.5.3 --- Production of Mushrooms --- p.25 / Chapter 1.6 --- Medicinal (Antidiabetic) Properties of Mushrooms --- p.28 / Chapter 1.6.1 --- Ganoderma lucidum --- p.29 / Chapter 1.6.2 --- Tremella aurantia --- p.33 / Chapter 1.6.3 --- Auricularia auricula --- p.36 / Chapter 1.6.4 --- Grifola frondosa --- p.37 / Chapter 1.7 --- Medicinal Uses of Hericium erinaceus --- p.39 / Chapter 1.7.1 --- HeLa Cell Proliferation Inhibitors --- p.39 / Chapter 1.7.2 --- Induction of Growth of Nerve Cells --- p.42 / Chapter 1.7.3 --- Antitumour Activity --- p.42 / Chapter 1.7.4 --- Antidiabetic Effect --- p.43 / Chapter 1.8 --- Objectives --- p.45 / Chapter Chapter 2 --- Materials and Methods --- p.46 / Chapter 2.1 --- Extraction of Polysaccharides from the Fruiting Body of H. erinaceus --- p.46 / Chapter 2.1.1 --- Small-scale Extraction --- p.46 / Chapter 2.1.2 --- Large-scale Extraction --- p.47 / Chapter 2.2 --- Physico-Chemical Characterization of HE-polysaccharides --- p.52 / Chapter 2.2.1 --- Carbohydrate Content: Phenol-Sulfuric Acid Method --- p.52 / Chapter 2.2.2 --- Protein Content: Lowry Assay --- p.52 / Chapter 2.2.3 --- Uronic Acid Content --- p.53 / Chapter 2.2.4 --- Molecular Weight Determination by High Pressure Liquid Chromatography (HPLC) --- p.55 / Chapter 2.2.5 --- Determination of Monosaccharide Composition of Non-Starch Polysaccharides by Gas Chromatography (GC) --- p.56 / Chapter 2.2.5.1 --- Acid Depolymerisation --- p.56 / Chapter 2.2.5.2 --- Neutral Sugar Derivatisation --- p.56 / Chapter 2.2.5.3 --- Determination of Neutral Sugar Composition by Gas Chromatography (GC) --- p.57 / Chapter 2.2.6 --- Structural Study of Polysaccharides by Methylation --- p.59 / Chapter 2.2.6.1 --- Preparation of dry Dimethyl Sulfoxide (DMSO) --- p.59 / Chapter 2.2.6.2 --- Preparation of Methylsulfinyl Methyl Sodium (CH3SOCH2-Na+) from the dry DMSO and Sodium Hydride --- p.59 / Chapter 2.2.6.3 --- Methylation Procedure --- p.60 / Chapter 2.2.6.4 --- Preparation of Partially Methylated Alditol Acetates (PMAAs) --- p.61 / Chapter 2.2.6.5 --- Analysis of the PMAAs by GC --- p.62 / Chapter 2.2.7 --- The Measurement of Viscosity --- p.62 / Chapter 2.3 --- In vitro Hypoglycemic Tests of HE-Polysaccharides --- p.64 / Chapter 2.3.1 --- Glucose Dialysis Retardation Index (GDRl) --- p.64 / Chapter 2.3.1.1 --- Experimental Setup --- p.64 / Chapter 2.3.1.2 --- Measurement of Glucose in the Dialysate --- p.65 / Chapter 2.3.2 --- Inhibition of Amylolysis --- p.66 / Chapter 2.3.2.1 --- Experimental Setup --- p.66 / Chapter 2.3.2.2 --- Measurement of Maltose in the Dialysate --- p.66 / Chapter 2.4 --- In vivo Hypoglycemic Evaluation of HE-Polysaccharides --- p.67 / Chapter 2.4.1 --- Oral Glucose Tolerance Test (OGTT) --- p.67 / Chapter 2.4.2 --- Induction of Type l Diabetes in Normal BALB/c Mice --- p.69 / Chapter 2.4.2.1 --- lnduction Protocol --- p.69 / Chapter 2.4.2.2 --- Measurement of Plasma Glucose Level --- p.70 / Chapter 2.4.3 --- Hypoglycemic Test on Normal and Diabetic BALB/c Mice --- p.71 / Chapter 2.4.4 --- Measurement of Insulin Level by Enzyme-Linked Immunoadsorbent Assay (ELlSA) --- p.72 / Chapter 2.4.4.1 --- Plasma Samples used in ELlSA --- p.72 / Chapter 2.4.4.2 --- Assay Procedure --- p.73 / Chapter 2.5 --- Statistical Evaluation --- p.74 / Chapter Chapter 3 --- Results and Discussion --- p.75 / Chapter 3.1 --- Yield of Polysaccharides extracted from H. erinaceus --- p.75 / Chapter 3.2 --- Physico-chemical Properties of HE Polysaccharides --- p.79 / Chapter 3.2.1 --- "Carbohydrate, Protein and Uronic Acid Content" --- p.79 / Chapter 3.2.2 --- Monosaccharide Compositions --- p.83 / Chapter 3.2.3 --- Molecular Weight of the HE polysaccharides --- p.85 / Chapter 3.2.4 --- Structure of HE polysaccharides --- p.90 / Chapter 3.2.5 --- Conclusion for the Physico-chemical Properties of HE-Polysaccharides --- p.96 / Chapter 3.2.6 --- Viscosity of HE Polysaccharides --- p.99 / Chapter 3.3 --- In vitro Study of the Hypoglycemic Effect of HE-Polysaccharides --- p.101 / Chapter 3.3.1 --- Glucose Dialysis Retardation Index (GDRl) --- p.101 / Chapter 3.3.2 --- Inhibition of α-Amylase Activity --- p.105 / Chapter 3.4 --- In vivo Hypoglycemic Evaluation of HE-Polysaccharides --- p.109 / Chapter 3.4.1 --- In vivo Oral Glucose Tolerance Test (OGTT) in Normal Mice --- p.109 / Chapter 3.4.1.1 --- Oral Glucose Tolerance Test --- p.109 / Chapter 3.4.1.2 --- Effect of Change of Viscosity of HE Polysaccharide in the Gl Tract of Mice --- p.114 / Chapter 3.4.2 --- Establishment of a Diabetic Murine Model --- p.120 / Chapter 3.4.3 --- Hypoglycemic Activity of HE-polysaccharides in Normal Mice --- p.123 / Chapter 3.4.4 --- Hypoglycemic Activity of HE-polysaccharides in Diabetic Mice --- p.126 / Chapter 3.4.5 --- Change of Plasma Insulin Level in the Hypoglycemic Test --- p.132 / Chapter 3.4.6 --- Comparison of Hypoglycemic Activity of HE-Polysaccharides in Normal and Diabetic mice --- p.139 / Chapter 3.4.6.1 --- Severity of Diabetic Conditions lnduced --- p.139 / Chapter 3.4.6.2 --- Change in Insulin Secretion --- p.140 / Chapter 3.4.6.3 --- Glucose Transporter --- p.140 / Chapter 3.5 --- Other Factors that Affect in vivo Hypoglycemic Activity of the HE-polysaccharides --- p.141 / Chapter 3.5.1 --- Route of Administration: Oral Feeding and Intraperitoneal Injection --- p.141 / Chapter 3.5.2 --- Molecular Mechanisms of Hypoglycemic Activity --- p.142 / Chapter 3.5.3 --- Glucose Toxicity --- p.144 / Chapter 3.5.3.1 --- Insulin Resistance --- p.144 / Chapter 3.5.3.2 --- Impaired Insulin Secretion --- p.145 / Chapter Chapter 4 --- Conclusions and Future Works --- p.147 / References --- p.151

by Liu Fang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 159-178). / ACKNOWLEGEMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.v / LIST OF TABLES --- p.ix / LIST OF FIGURES --- p.xi / ABBREVIATIONS --- p.xiv / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter Chapter 2. --- Literature Review --- p.7 / Chapter 2.1. --- Biologically active polysaccharides --- p.7 / Chapter 2.2. --- Antitumor activities of polysaccharides --- p.11 / Chapter 2.2.1. --- In vivo studies --- p.11 / Chapter 2.2.2. --- In vitro studies --- p.15 / Chapter 2.3. --- Antitumor mechanisms of polysaccharides --- p.17 / Chapter 2.4. --- Structure and antitumor activities of polysaccharides --- p.25 / Chapter 2.4.1. --- The effect of molecular mass --- p.26 / Chapter 2.4.2. --- The impact of branching configuration --- p.21 / Chapter 2.4.3. --- The relationship between antitumor activity and conformation --- p.28 / Chapter 2.4.4. --- Improvement of antitumor activity by chemical modification --- p.29 / Chapter 2.5. --- Other biological activities --- p.30 / Chapter 2.5.1. --- Antiviral activity --- p.30 / Chapter 2.5.2. --- Antimicrobial activity --- p.31 / Chapter 2.5.3. --- Free radical scavenging activity --- p.32 / Chapter 2.5.4. --- Hepatic protective effect --- p.32 / Chapter Chapter 3. --- Isolation and Characterization of a Polysaccharide-Protein Complex (PSPC) from Tricholoma lobayense --- p.34 / Chapter 3.1. --- Introduction --- p.34 / Chapter 3.2. --- Materials and methods --- p.36 / Chapter 3.2.1. --- Strain --- p.36 / Chapter 3.2.2. --- Culture conditions --- p.36 / Chapter 3.2.3. --- Extraction of T. lobayense --- p.39 / Chapter 3.2.4. --- Purification of polysaccharide-protein complex --- p.40 / Chapter 3.2.5. --- Molecular mass determination --- p.43 / Chapter 3.2.6. --- High performance liquid chromatography --- p.43 / Chapter 3.2.7. --- SDS-polyacrylamide gel electrophoresis --- p.44 / Chapter 3.2.8. --- Ultraviolet scanning --- p.44 / Chapter 3.2.9. --- Chemical analysis --- p.45 / Chapter 3.2.10. --- Experimental animals --- p.47 / Chapter 3.2.11. --- In vivo antitumor assay --- p.48 / Chapter 3.2.12. --- Safety tests --- p.49 / Chapter 3.2.13. --- Statistical analysis --- p.51 / Chapter 3.3. --- Results --- p.51 / Chapter 3.3.1. --- Extraction and purification --- p.51 / Chapter 3.3.2. --- Biochemical analysis --- p.52 / Chapter 3.3.3. --- Chemical analysis --- p.60 / Chapter 3.3.4. --- In vivo antitumor activity --- p.68 / Chapter 3.3.5. --- Safety evaluation --- p.68 / Chapter 3.4. --- Discussion --- p.75 / Chapter 3.5. --- Summary --- p.84 / Chapter Chapter 4. --- "Immunomodulating, Antitumor and other Biological Activities of Polysaccharide-Protein Complex (PSPC) from Tricholoma lobayense" --- p.85 / Chapter 4.1. --- Introduction --- p.85 / Chapter 4.2. --- Materials and methods --- p.87 / Chapter 4.2.1. --- Experimental animals --- p.87 / Chapter 4.2.2. --- Cultivation of tumor cells --- p.87 / Chapter 4.2.3. --- Preparation of peritoneal exudate cells and splenocytes --- p.87 / Chapter 4.2.4. --- Mitogenic response of T cells --- p.89 / Chapter 4.2.5. --- Responses of peritoneal exudate cells --- p.89 / Chapter 4.2.6. --- In vitro antitumor assay --- p.92 / Chapter 4.2.7. --- Transmission electron microscope --- p.93 / Chapter 4.2.8. --- Evaluation of other biological activities --- p.94 / Chapter 4.2.9. --- Statistical analysis --- p.99 / Chapter 4.3. --- Results --- p.99 / Chapter 4.3.1. --- Immunomodulating activity --- p.99 / Chapter 4.3.2. --- In vitro antitumor action --- p.107 / Chapter 4.3.3. --- Observation on tumor regression induced by PSPC --- p.107 / Chapter 4.3.4. --- Other biological actions --- p.112 / Chapter 4.4. --- Discussion --- p.121 / Chapter 4.4.1. --- Immunomodulating activity --- p.121 / Chapter 4.4.2. --- Antitumor activity --- p.125 / Chapter 4.4.3. --- Other biological activities --- p.127 / Chapter 4.5. --- Summary --- p.130 / Chapter Chapter 5. --- Induction of Gene Expression of Immunomodulating Cytokines by Polysaccharide-Protein Complex (PSPC) from Tricholoma lobayense --- p.132 / Chapter 5.1. --- Introduction --- p.132 / Chapter 5.2. --- Materials and methods --- p.135 / Chapter 5.2.1. --- Experimental animals --- p.135 / Chapter 5.2.2. --- Preparation of peritoneal exudate cells and splenocytes --- p.136 / Chapter 5.2.3. --- RNA extraction --- p.137 / Chapter 5.2.4. --- Reverse transcription- polymerase chain reaction --- p.137 / Chapter 5.2.5. --- Dot blot --- p.138 / Chapter 5.2.6. --- Hybridization --- p.141 / Chapter 5.3. --- Results --- p.142 / Chapter 5.3.1. --- mRNA phenotyping of cytokines and cytokine receptors in normal mice --- p.142 / Chapter 5.3.2. --- mRNA phenotyping of cytokines and cytokine receptors in tumor-bearing mice --- p.142 / Chapter 5.4. --- Discussion --- p.150 / Chapter 5.5. --- Summary --- p.153 / Chapter Chapter 6. --- General Discussion and Conclusion --- p.155 / REFERENCES --- p.159

Thesis (Academic dissertation)--University of Helsinki, 1998. / Includes bibliographical references. Also available on the World Wide Web.

Notre étude aborde deux points importants sur les interactions supramoléculaires dans les polymères : tout d'abord comment des polymères peuvent s'assembler sur des surfaces planes au moyen de d'interactions de type hôte/invité, puis sur les interactions entre polymères à l'échelle de la molécule unique. En particulier nous verrons comment ces interactions à courte portée influent sur l'adhésion des chaînes sur des surfaces chimiquement contrôlées. Notre choix s'est porté sur un polymère d'origine naturelle le chitosane fonctionnalisé respectivement par des B-cyclodextrines (hôte) et des adamantanes (invité) et dont les assemblages forment des gels. Dans une première partie nous montrerons la possibilité de créer des multicouches de polymère par la méthode Layer-by-Layer (LbL) à l'aide des interactions de type hôte/invité, assemblage toutefois limité par les interactions électrostatiques au sein de la structure. Dans une seconde partie nous étudions les interactions multivalentes hôte/invité entre les couches de polymères en mesurant la force d'interaction par AFM. Nous avons pu mettre en évidence les différentes contributions à la force d'interaction et montrer que les interactions hôte//invités dominent les interactions non spécifiques d'un ordre de grandeur / In this study we focused on two important points concerning supramolecular interactions in polymeric systems. First; how polymers self-assemble on planar surfaces through side-chain host-guest interactions. Second; how those polymers interact each other at the level of single chain and how the adhesion properties of polymers on the modified surfaces can be controlled with those short ranged specific interactions. For that purpose a natural polysaccharide, chitosan, was chosen as the polymeric backbone and was specifically modified with host (B-cyclodextrin) and guest (adamanatane) molecules. It is known that those modified polysaccharides interact each other through host-guest units and their supramolecular assemblies exhibiting a gel-like behavior in solution state. In the first part of the study we investigated the feasibility to use supramolecular interactions to construct functional polymer multilayers by using the Layer-by-Layer (LbL) self assembly approach. The driving force with the proposed system is host-guest interactions thus short ranged and sterically demanding as the structural fitting is necessary. Our results show that multiple host-guest interactions along the chitosan chain allow the self assembles of the modified chitosans on guest-attached surfaces. The number of layers is limited and possibly affected by the electrostatic charge of the chitosan backbone. In the second part of the study we used atomic force microscope (AFM) to probe the multivalent host-guest interactions between modified polymer layers by direct force measurement. By this technique, the main contributions to the interaction between modified chitosan layers could be identified. Adhesion properties of the modified chitosans have also been investigated. The work of adhesion is about an order of magnitude larger for those chitosan derivatives that can form host-guest complexes than for those where this is not possible.

Les fucosyltransférases sont les enzymes responsables du transfert d'un groupement fucose à partir du GDP-fucose sur des accepteurs variés (oligosaccharides, protéines,...). Chez l'homme, les rôles de ces glycosyltransférases sont importants dans un grand nombre de processus biologiques et pathologiques. De nombreuses fucosyltransférases existent chez les végétaux. Notamment FuT1, qui transfert un fucose en 1,2 sur un résidu galactose du xyloglucane : l'une des hémicelluloses majeures de la paroi des dicotylédones. Ce polysaccharide ramifié est très étudié en raison de ses applications actuelles et potentielles dans différents secteurs de l'industrie : textile, alimentation, pharmaceutique, etc. Les objectifs de ce doctorat ont été d'obtenir des informations biochimiques et structurales sur la fucosyltransférase AtFuT1 de la plante modèle Arabidopsis thaliana. Pour cela, une forme recombinante soluble de l'enzyme a été produite avec le système baculovirus /cellules d'insectes. De manière à obtenir suffisamment de protéines pour les études structurales, une méthode de culture en suspension des cellules a été mise en place au laboratoire. Un protocole de purification en deux étapes, impliquant une chromatographie par affinité puis par exclusion de taille, a permis d'obtenir à partir d'un litre de culture cellulaire entre un à deux mg de protéine pure et homogène. Ce résultat a permis de mieux comprendre le comportement de l'enzyme vis-à-vis de ses substrats (GDP-fucose et xyloglucane), d'obtenir des cristaux de la protéine et de résoudre la structure 3D d'AtFuT1 en complexe avec le GDP et un oligosaccharide dérivé du xyloglucane, à une résolution de 2,2 Å. La forme AtFut1 produite se comporte en solution et dans le cristal sous forme d'un dimère non covalent. La protéine adopte un repliement qui est un variant du type GT-B classique. Parallèlement à ces travaux, des tests d'activité glycosyltransférase ont été mis au point permettant le criblage de nombreuses conditions de réactions. L'ensemble des méthodes et techniques développées constitue une base utile, devant faciliter la caractérisation d'autres glycosyltransférases. / Fucosyltransferases are enzymes that transfer a fucose residue from GDP-fucose on varied acceptors (oligosaccharides, proteins). In Human, these glycosyltransferases are involved in many biological and pathological processes. Numerous fucosyltransferase exist in the plant kingdom. Among them, FuT1 transfers a fucose linked in 1,2 onto a galactose of xyloglucan: a major hemicellulose of dicots cell wall. This branched polysaccharide is intensively studied because of its current and potential industrial applications in textile, food, pharmaceuticals, etc. The main objective of this PhD program was to obtain biochemical and structural information on the fucosyltransferase AtFuT1 from the model plant Arabidopsis thaliana. A recombinant form of this protein has therefore been produced, using the baculovirus/insect cell system. In order to get sufficient amount of protein for structural studies, a suspension cell culture method has been set-up in the lab. A two-step purification protocol, involving affinity and size exclusion chromatography was established. The active, and highly pure recovered protein was used to determine the biochemical properties of the protein towards its substrates (GDP-fucose and xyloglucan), to get protein crystals and hence to solve its 3D structure in complex with GDP and a xyloglucan derived oligosaccharide (2.2 Å resolution). AtFuT1 behaves in solution and in crystallo as a non-covalent dimer. The protein adopts a variant of the classical GTB fold. In addition, novel glycosyltransferase assay have been designed allowing the screening of numerous reaction conditions. Methods and techniques that were developed during this study should be a useful base for the characterization of other glycosyltransferase.

Le travail présenté dans se manuscrit s'inscrit dans le domaine de la chimie et physico-chimie des polysaccharides. Une étude des propriétés physico-chimiques du xyloglucane en fonction de différentes conditions est d'abords réalisée, elle montre la difficulté d'obtenir des solutions stables à l'échelle moléculaire ainsi que la possibilité d'obtenir des nanoparticules à base de xyloglucanes. Le suivi de l'hydrolyse enzymatique du xyloglucane en solution par la diffusion de la lumière a mis en évidence l'agrégation des produits obtenus en solution. L'imagerie par la microscopie à force atomique a révélé l'importance de la surface dans le phénomène de dégradation. Enfin des dérivés de xyloglucane ont été obtenus par oxydation par le système TEMPO/NaOCl/NaBr, et des essais de préparation de conjugués xyloglucane-cytarabine ont été effectués. / The work presented in this manuscript is in the field of chemistry and physical chemistry of polysaccharides. A study of the physicochemical properties of xyloglucan under different conditions is carried around and showing the difficulty to obtaining stable solutions at the molecular level. The study also evidenced the possibility of obtaining nanoparticles based on xyloglucan. Monitoring of the enzymatic hydrolysis of xyloglucan in solution by light scattering indicated the aggregation of the degradation products. Imaging by atomic force microscopy revealed the importance of the surface in the degradation phenomenon. Finally xyloglucan derivatives were obtained by oxidation system TEMPO / NaOCl / NaBr, and test on the coupling of xyloglucan and cytarabine conjugates were made.

La reconstruction de lésions osseuses complexes reste un défi dans le domaine de la chirurgie orthopédique et maxillo-faciale. A ce jour, la technique de référence reste la greffe d’os autologue. Cependant cette technique présente de nombreuses limites (risque d’infection, morbidité au site de prélèvement). Dans ce contexte, l’ingénierie tissulaire peut apporter des solutions de reconstruction innovantes. En effet l’utilisation de matrices ostéoconductrices et ostéoinductrices permettrait de remplacer l’autogreffe. Le premier objectif de ce travail a été de mettre au point une matrice de polysaccharides, contenant des particules d’hydroxyapatite (HA) dopées avec du strontium (Sr), afin de stimuler à la fois la régénération osseuse, mais également l’angiogenèse. Les résultats obtenus in vitro et in vivo nous ont permis d’optimiser la formulation de cette matrice, en termes de quantités de particules d’hydroxyapatite dopées par différents taux de substitution en strontium, dispersées au sein de la matrice. La deuxième partie de ce travail a été consacrée à l’application de cette matrice à la technique de Masquelet afin de remplacer l’utilisation de l’autogreffe. Cette procédure chirurgicale en deux temps, basée sur la formation d’une membrane induite, est utilisée fréquemment en chirurgie orthopédique et maxillo-faciale. Le premier temps opératoire utilise un ciment chirurgical, le (poly(méthyl)méthalcrylate, PMMA) qui entraîne la formation d’une membrane induite vascularisée. Notre travail a été de remplacer ce ciment par du silicone et d’étudier l’influence de la radiothérapie sur la qualité et la fonction de la membrane ainsi formée. Les premiers essais d’évaluation de cette matrice ont été réalisés chez le rat après résection segmentaire du fémur, suivie d’une procédure de radiothérapie. Les perspectives de ce travail sont d’évaluer la performance de cette matrice dans une lésion mandibulaire de grand volume, après irradiation, chez le gros animal. / Reconstruction of large and complex bone defects remains a challenge for orthopaedic and maxillo-facial surgery. The gold standard strategy for bone reconstruction is the autologous bone graft. However, this approach still exhibits some limitations (infection risks, morbidity at the donor site). In this context, tissue engineering can provide innovative solutions for bone reconstruction. Indeed, the use of osteoconductives and osteoinductives matrices could replace autograft. Based on previous data obtained by our laboratory, the first objective of this work was to develop a composite matrix of polysaccharides containing hydroxyapatite (HA) particles doped with strontium (Sr), to stimulate both bone formation and angiogenesis. In vitro and in vivo results allow us to optimize the amount of HA particules and the ratio of Sr-substitution within the polysaccharide-based matrix. The second part of this work was to apply this biomaterial in the context of Masquelet approach. These two time procedure surgery, based on the formation of an induced membrane, is commonly used in orthopaedic and maxillo-facial surgery. The first chirurgical step uses a surgical cement (poly(methyl)methalcrylate, PMMA) to promote around it the formation of a vascularized membrane. Our work was to replace this cement by silicone and to study the influence of radiotherapy treatment on the quality and the function of this induced membrane. The first preclinical evaluation of this matrix has been performed on a rat femoral segmental bone defect, followed by a radiotherapy procedure. The perspectives of this work are to evaluate the performances of this matrix on irradiated segmental mandibular bone defect in large animal.