Les films multicouches de polyélectrolytes sont des auto-assemblages de polymères chargés formant des films dont l'épaisseur peut être variée de quelques nm à quelques µm. Un nombre croissant de travaux concerne la compréhension de leur mécanisme d'auto-assemblage et leur utilité pour modifier les propriétés physico-chimiques, topographiques ou mécaniques de surface de (bio)matériaux. Dans cette thèse, nous avons étudié les propriétés de films à base de poly(L-lysine) et de polysaccharides connus pour leur rôle physiologique, notamment le hyaluronane, la chondroïtine sulfate et l'héparine. Les compositions internes de ces films mono-constituants ou à base de mélanges de polyélectrolytes ont été sondées. L'influence de la chimie des polyélectrolytes sur la formation des films, en particulier l'importance des groupements sulfates, a été mise en évidence. Leur potentiel comme vecteur de délivrance d'un facteur de croissance, la BMP-2, a été évalué. De fortes quantités de BMP-2 ont pu être chargées dans les films à base de hyaluronane. Nous avons pu contrôler les quantités insérées en faisant varier la composition chimique des films, leur épaisseur ou la concentration en BMP-2 de la solution de chargement. Puis nous avons mis en évidence une différenciation contrôlée de façon dose-dépendante de cellules C2C12 pluripotentes sur les films bioactifs : différenciation myogénique (en absence de facteur) ou ostéoblastique. De plus, nous montrons qu'un contact des cellules avec le film bioactif est nécessaire pour induire leur différenciation. La protéine est donc présentée par « la phase solide », ce qui constitue un mode de présentation du facteur proche des conditions physiologiques. Des résultats préliminaires obtenus en recouvrant des biomatériaux orthopédiques par les films bioactifs laissent penser que ces films offrent des perspectives intéressantes dans le domaine de la régénération osseuse in vivo. / Polyelectrolyte multilayer films are self-assembled architectures forming nm to µm thick films. During the last decade, they have emerged as an efficient way of modifying materials surface properties such as chemistry, physico-chemical properties, topography as well as mechanical properties. Thanks to the technology's versatility and ease of use, polyelectrolyte multilayer films are now recognized as a new tool for modifying biomaterial surfaces and mediating cell behaviours and implant bio-integration. In this thesis, we studied the properties of poly(L-lysine) and polysaccharide-based multilayer films and focused on their physical-chemical properties as well as on their internal composition. In particular, we studies the influence of their chemistry (presence of carboxylic or sulfate groups) on film formation and characteristics. Three polysaccharides with increasing sulfate group content were chosen for this purpose: hyaluronan, chondroitin sulfate and heparin. The capacity of these films to act as a drug delivery vehicle for BMP-2 (a growth factor able to induce osteo-differentiation) was then assessed. High BMP-2 amounts were successfully loaded and retained in the films in a controlled manner. The loaded amounts could be modulated by varying the film's chemistry, film thickness or BMP-2 concentration in the loading solution. We showed that it is possible to control the extent of C2C12 cell differentiation in osteoblasts when cultured on the bioactive films. Importantly, when no BMP-2 is loaded in the films, the cells differentiated in to myotubes, their most common differentiation pathway. Cells needed a direct contact with the bioactive films to respond to BMP-2, suggesting that BMP-2 is mainly presented to the cells from the solid phase. Preliminary in vivo tests on film-coated orthopaedic biomaterials are encouraging. They showed that these films are interesting candidates for surface modification of orthopaedic biomaterials and may foster bone regeneration.

CARACTERISATION DE VACCINS A BASE DE GLUCIDESVariables influençant l'immunogénicité et propriétés physico-chimiques des vaccins glycoconjuguésDe nombreux aspects peuvent influer sur l'immunogénicité des vaccins conjugués et les principales variables étudiées jusqu'ici sont la taille du fragment saccharide et la nature des glycosides: taux de protéine dans le conjugué purifié, la stratégie de conjugaison, nature de l’espaceur et la protéine porteuse.La taille de la partie saccharidique et le ratio de cette partie / protéine a été étudiée dans différents travaux de Seppälä et Mäkelä,.Dans l'une des premières études sur l'effet de la taille sur l'immunogénicité de la protéine conjuguées à des dextrans, il a été montré que des dextrans de faible poids moléculaire conjugué à l’albumine sérique de poluet induit des réponses anti-dextran fortes chez la souris. L’augmentation de la taille du dextran, a abouti à une réduction de l’immunogénicité. Peeters et al. a montré qu’un tétramère synthétique de Hib unité capsulaire polysaccharide, conjuguée à un support protéique, induit chez des souris adultes, des niveaux d'anticorps primates non humains comparables à un conjugué Hib commercial. Ces niveaux sont plus élevées que ceux induits par un trimère répété, ce qui indique que pour le Hib un minimum de huit sucres est nécessaire pour une bonne réponse immunologique. Laferrière et al. a trouvé peu d'influence de la longueur de la chaîne glucidique sur l'immunogénicité des vaccins antipneumococciques conjugués chez la souris. Pozsgay et al. a étudié chez la souris, l'immunogénicité de l’oligosaccharides du LPS de Shigella dysenteriae de type 1 conjugué à l'albumine sérique humaine (HSA). Les auteurs ont constaté que les octa-, dodéca-, et des fragments de hexadécasaccharides induit des niveaux élevés d'anticorps IgG après trois injections. Ces niveaux sont supérieurs à ceux obtenus avec un conjugué tétrasaccharidique. L'influence du ratio glucides / protéine est différente pour les trois conjugués. Le conjugué octasaccharide-HSA avec la plus forte densité provoque une bonne réponse immunitaire, tandis que dans le cas des conjugués dodéca- et hexadécasaccharides, la densité médiane est optimal. Ces études suggèrent que la longueur de la chaîne d'oligosaccharides et le chargement de l’haptène peuvent être liés entre eux pour déterminer l'immunogénicité des vaccins glycoconjugués.L'espaceur est une molécule linéaire courte qui est généralement liée à la chaîne polysaccharidique et la protéine ou de fragments. Il ya des évidences dans la littérature qui suggèrent que les espaceurs rigides, contraints comme le maléimide cyclohexyle, provoquent une importante quantité d'anticorps indésirables, avec le risque de conduire à une réponse immunitaire éloignée de l'épitope ciblé sur la haptène. L'utilisation d'un alkyle souple type maleimido a été rapporté comme un moyen de surmonter l'immunogénicité observée précédemment. Un certain nombre de transporteurs protéiques ont été utilisés jusqu'ici dans l'évaluation préclinique et clinique de vaccins conjugués. Des protéines telles que les anatoxines diphtériques et tétaniques, qui dérivent des toxines respectives, après la décontamination chimique avec le formaldéhyde, ont été initialement choisies comme transporteur en raison de inocuité (tétanos et la vaccination contre la diphtérie). CRM 197, un mutant non toxique de la toxine 61 de la diphtérie a été largement utilisé comme support pour Hib. Un complexe protéique de la membrane externe de méningocoque du sérogroupe B a été utilisé par Merck comme support pour leur vaccin conjugué Hib. GSK dans leur vaccin antipneumococcique, conjugué multivalent, introduit la protéine D Hib liée à la plupart des polysaccharides inclus dans le vaccin. L'équipe de John Robbins fait un large usage de la forme recombinante non toxique de l’exo-toxine de Pseudomonas aeruginosa comme support contre Staphylococcus aureus de type 5 et 8 ainsi que pour Salmonella. / CHARACTERIZATION OF CARBOHYDRATE BASED VACCINES Variables influencing the immunogenicity and physicochemical properties of glycoconjugate vaccinesMany aspects can influence the immunogenicity of conjugate vaccines and the main variables investigated so far are the size of the saccharide moiety, the saccharide:protein ratio in the purified conjugate, the conjugation strategy, the nature of the spacer and the protein carrier. The size of the saccharide moiety and saccharide/protein ratio were investigated in different works such as Seppälä and Mäkelä in one of the first studies on the effect of size and chemistry on the immunogenicity of dextrans-protein conjugates found that dextrans of low molecular weight conjugated to chicken serum albumin, induced strong anti-dextran responses in mice, while increasing the dextrans' size resulted in reduced immunogenicity.47 Peeters et al. showed that a synthetic tetramer of Hib capsular polysaccharide repeating unit, conjugated to a protein carrier, induced in adult mice and non-human primates antibody levels comparable to a commercial Hib conjugate and higher than those induced by a trimer, indicating that for Hib a minimum of eight sugars is needed for a proper immunological response.48 Laferriere et al. found little influence of the carbohydrate chain length on the immunogenicity of pneumococcal conjugate vaccines in mice.49 Pozsgay et al. studied the immunogenicity in mice of synthetic Shigella dysenteriae type 1 LPS oligosaccharides conjugated to human serum albumin (HSA). The authors found that octa-, dodeca-, and hexadecasaccharide fragments induced high levels of lipopolysaccharide binding IgG antibodies in mice after three injections and were superior to a tetrasaccharide conjugate. The influence of the carbohydrate/protein ratio was different for the three conjugates. The octasaccharide-HSA conjugate with the highest density evoked a good immune response, while in the case of dodeca- and hexadecasaccharide conjugates, the median density was optimal.50 These studies suggest that oligosaccharide chain length and hapten loading might be interconnected in determining the immunogenicity of glycoconjugate vaccines. The spacer is a short linear molecule that is generally linked to the polysaccharide chain or to the protein or to both moieties, depending on the chemistry, used to facilitate the coupling between the protein and sugar. There are evidences in the literature which suggest that rigid, constrained spacers like cyclohexyl maleimide, elicit a significant amount of undesirable antibodies, with the risk of driving the immune response away from the targeted epitope on the hapten.51 52 The use of a flexible alkyl type maleimido spacer has been reported as a way to overcome the previous observed immunogenicity of cyclic maleimide linkers.53 A number of protein carriers have been used so far in preclinical and clinical evaluation of conjugate vaccines. 54 55 56 57 58 59 60Proteins such as diphtheria and tetanus toxoids, which derive from the respective toxins after chemical detoxification with formaldehyde, were initially selected as carrier because of the safety track record accumulated with tetanus and diphtheria vaccination. CRM197, a non-toxic mutant of diphtheria toxin61 which instead does not need chemical detoxification, has been extensively used as carrier for licensed Hib, pneumococcal, meningococcal conjugate vaccines and for other vaccines being developed. An outer membrane protein complex of serogroup B meningococcus has been used by Merck as carrier for their Hib conjugate vaccine.62 GSK in their multivalent pneumococcal conjugate vaccine introduced the use of the Hib-related protein D as carrier for most of the polysaccharides included into the vaccine.63 64 The team of John Robbins made extensive use of the recombinant non toxic form of Pseudomonas aeruginosa exo-toxin as carrier for Staphylococcus aureus type 5 and 8 as well as for Salmonella

Des solutions d'acides carboxyliques contenant principalement de l'acide formique sont obtenues par oxydation photo-Fenton des polysaccharides. Une irradiation dans le visible de 60 W sous des conditions douces suffit à fournir des résultats reproductibles. Les produits d'oxydation issus d'amidon de pomme de terre et d'amidon de blé ont montré des capacités de complexation comparables aux solutions commerciales d'acide gluconique et glucuronique.Un système d'oxydation innovant, comprenant des sels d'halogénures et du DMSO, convertit quantitativement le HMF en DFF. Cette méthode permet aussi la transformation one-pot du fructose en DFF avec de bons rendements. Selon l'étude mécanistique, le bromo-HMF serait un intermédiaire réactionnel. / Solutions of carboxylic acids, containing mainly formic acid, are produced under photo-Fenton conditions. Visible irradiation with a 60 W spot is sufficient to provide reproducible results under mild conditions. The oxidation products of potato starch and wheat starch have shown Ca sequestering properties similar to those of gluconic and glucuronic acids.Using halide salts and DMSO, an innovating method has been elaborated for the selective oxidation of HMF to DFF with quantitative yields. The one-pot transformation of fructose to DFF occurs with fair yields. Based on the mechanistic study, Br-HMF would be the reaction intermediate.

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.