Choi, Man Wing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 126-139). / Abstracts in English and Chinese. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Antigen presenting cells (APC) in Immune systems --- p.1 / Chapter 1.1.1 --- Dendritic cells --- p.2 / Chapter 1.1.1.1 --- Differentiation of dendritic cells in mice --- p.2 / Chapter 1.1.1.2 --- Maturation of dendritic cells --- p.3 / Chapter 1.1.1.3 --- Stimulation and polarization of T cells stimulated by dendritic cells --- p.6 / Chapter 1.1.2 --- Monocyte and macrophage --- p.7 / Chapter 1.1.2.1 --- Differentiation of monocyte and macrophage in humans --- p.7 / Chapter 1.1.2.2 --- Changes involved in differentiation of monocytes into macrophages --- p.9 / Chapter 1.2 --- "Isolation, structure and activity of mushroom polysaccharides" --- p.13 / Chapter 1.2.1 --- Sources of mushroom polysaccharides --- p.13 / Chapter 1.2.2 --- Extraction methods --- p.14 / Chapter 1.2.3 --- Structure-Activity Relationship (SAR) of mushroom polysaccharides --- p.15 / Chapter 1.2.4 --- Previous studies on immunomodulatory effects of mushroom sclerotial polysaccharides --- p.18 / Chapter 1.3 --- Recognition of β-glucan by specific receptors --- p.20 / Chapter 1.3.1 --- Complement Receptor 3 (CR3) --- p.22 / Chapter 1.3.1.1 --- Introduction of CR3 --- p.22 / Chapter 1.3.1.2 --- Expressions of CR3 to recognize fungi --- p.22 / Chapter 1.3.2 --- Dectin-1 --- p.24 / Chapter 1.3.2.1 --- Introduction of Dectin-1 --- p.24 / Chapter 1.3.2.2 --- Structure of Full-length Dectin-1 --- p.26 / Chapter 1.3.2.2.1 --- Isoforms of Dectin-1 in Mice --- p.28 / Chapter 1.3.2.2.2 --- Isoforms of Dectin-1 in Humans --- p.28 / Chapter 1.3.2.3 --- Immune responses triggered by of Dectin-1 --- p.29 / Chapter 1.3.3 --- Toll-like 2 receptor (TLR2) --- p.31 / Chapter 1.3.3.1 --- Introduction of TLR2 --- p.31 / Chapter 1.3.3.2 --- Structure of TLR2 --- p.33 / Chapter 1.3.3.3 --- Immune responses triggered by TLR2 --- p.34 / Chapter 1.4 --- Research Objectives --- p.35 / Chapter Chapter 2 --- Materials and Methods --- p.38 / Chapter 2.1 --- Materials --- p.38 / Chapter 2.1.1 --- Mushroom sclerotia --- p.38 / Chapter 2.1.1.1 --- Polysaccharide extraction from mushroom sclerotia --- p.38 / Chapter 2.1.2 --- Antibodies and reagents --- p.41 / Chapter 2.1.3 --- Human acute leukocyte monocytic cell line and culture medium --- p.42 / Chapter 2.1.4 --- Preparation of murine bone marrow-derived immature dendritic primary cells (immature BMDCs) --- p.43 / Chapter 2.2 --- Methods --- p.45 / Chapter 2.2.1 --- Chemical Analysis --- p.45 / Chapter 2.2.1.1 --- Measurement of monosaccharide profile --- p.45 / Chapter 2.2.1.1.1 --- Acid depolymerisation --- p.45 / Chapter 2.2.1.1.2 --- Neutral sugar derivatization --- p.45 / Chapter 2.2.1.1.3 --- Gas chromatography (GC) --- p.46 / Chapter 2.2.1.2 --- Determination of total sugar by phenol-sulfuric acid method --- p.47 / Chapter 2.2.1.3 --- Determination of protein content by Lowry-Folin Method --- p.48 / Chapter 2.2.1.4 --- Size exclusion chromatography by high pressure liquid chromatography (HPLC) --- p.49 / Chapter 2.2.1.5 --- Endotoxin detection --- p.50 / Chapter 2.2.2 --- Measurement of Bioactivities --- p.51 / Chapter 2.2.2.1 --- Trypan blue exclusion assay --- p.51 / Chapter 2.2.2.2 --- MTT cell proliferation assay --- p.51 / Chapter 2.2.2.3 --- BrdU cell proliferation assay --- p.53 / Chapter 2.2.2.4 --- Expression of cell surface markers --- p.54 / Chapter 2.2.2.5 --- Phagocytosis / Endocytosis of FITC-labeled dextrans --- p.55 / Chapter 2.2.2.6 --- Nitric oxide production assay --- p.55 / Chapter 2.2.2.7 --- Reactive oxygen species production --- p.57 / Chapter 2.2.2.8 --- Determination of cytokine profile using cytokine antibody array --- p.58 / Chapter 2.2.2.9 --- Cell cycle analysis --- p.59 / Chapter 2.2.2.10 --- Expression of surface receptors --- p.60 / Chapter 2.2.2.11 --- Statistical analysis --- p.61 / Chapter Chapter 3 --- Results and Discussion --- p.61 / Chapter 3.1 --- Chemical characteristics of sclerotial polysaccharides --- p.61 / Chapter 3.1.1. --- The yield of sclerotial polysaccharides --- p.61 / Chapter 3.1.2 --- Total carbohydrate content of sclerotial polysaccharides --- p.65 / Chapter 3.1.3 --- Protein content of sclerotial polysaccharides --- p.66 / Chapter 3.1.4 --- Monosaccharide profiles of sclerotial polysaccharides from PR by gas chromatography (GC) --- p.66 / Chapter 3.1.5 --- Molecular weight of sclerotial polysaccharides from PR by size exclusion chromatography (SEC) --- p.69 / Chapter 3.1.6 --- Endotoxin test --- p.73 / Chapter 3.2 --- Immune responses for human monocytic cell line THP-1 --- p.74 / Chapter 3.2.1 --- MTT cell viability assay --- p.74 / Chapter 3.2.2 --- BrdU cell proliferation assay --- p.75 / Chapter 3.2.3 --- Change in cell morphology of THP-1 --- p.79 / Chapter 3.2.4 --- Phenotypic maturation of THP-1 --- p.81 / Chapter 3.2.5 --- Up-regulated phagocytic ability of THP-1 --- p.84 / Chapter 3.2.6 --- Increased nitrite production in THP-1 --- p.86 / Chapter 3.2.7 --- Production of reactive oxygen species --- p.88 / Chapter 3.2.8 --- Human cytokines profile array --- p.90 / Chapter 3.2.9 --- Cell cycle analysis --- p.93 / Chapter 3.2.10 --- Surface receptors expression --- p.95 / Chapter 3.2.11 --- Summary --- p.98 / Chapter 3.3 --- Immune responses for murine immature BMDCs --- p.102 / Chapter 3.3.1 --- Inhibition effects on murine immature BMDCs --- p.102 / Chapter 3.3.2 --- Change in cell morphology of murine immature BMDCs --- p.103 / Chapter 3.3.3 --- Phenotypic maturation of murine immature BMDCs --- p.105 / Chapter 3.3.4 --- Down-regulation of endocytosis in murine immature BMDCs --- p.106 / Chapter 3.3.5 --- Increased nitrite production --- p.109 / Chapter 3.3.6 --- Decreased expression of CD 11c in PRW-treated immature BMDCs --- p.109 / Chapter 3.3.7 --- Cytokine profile detection --- p.112 / Chapter 3.3.8 --- Surface receptors expression --- p.116 / Chapter 3.3.9 --- Summary --- p.119 / Chapter Chapter 4 --- Conclusion and future works --- p.123 / Appendix --- p.125 / References --- p.126

Les extraits polysaccharidiques totaux (EPS) produits par LOCA-1 et LOCA-2, deux souches de levures isolées de la peau de raisin en Champagne viticole, ont été isolés en vue de leur caractérisation tant biochimique que fonctionnelle. La caractérisation structurale par GC démontre que ces EPS sont des hétéropolymères complexes de haut poids moléculaires (~2.106 g/mol) et sont composés de monomères de mannose, glucose, xylose et d’acide glucuronique, et deux types de substituants mis en évidence par analyse MALDI (sulfate et phosphate). L’élucidation de l’enchaînement structural des résidus osidiques au sein de ces EPS, sur la base des analyses GC/MS et RMN, a mis en évidence la présence d’une ossature principale linéaire constituée d’unités α-(1→3)-D-mannopyranosyles. Toutefois, des différences sont notables entre ces deux souches du même genre. Pour la souche LOCA-1, de courtes chaines latérales de β-(1,2)-xyloses (2-5 résidus) se ramifient à la chaine principale sur ses positions C-2 et/ou C-6. A l’opposé, la chaine mannosidique principale, plus longue chez la souche LOCA-2 (˃40 unités), est substituée en ses positions 2 et/ou 4 par des antennes de xylomannanes. Les caractéristiques structurales de ces EPS n’avaient jamais été observées auparavant chez d’autres microorganismes. La seconde partie de ce travail de thèse est dédiée aux propriétés fonctionnelles (pouvoirs moussant, gélifiant, viscosifiant) de ces EPS. Des propriétés viscosifiantes et moussantes tout à fait exceptionnelles ont été observées à l’issue d’une analyse comparative avec des biopolymères industriels commercialisés.Les propriétés intrinsèques des polymères naturels produits par ces souches indigènes de baies de raisin, en font des candidats potentiels pour une exploitation dans divers domaines d’applications biotechnologiques, et notamment oenologique. / The total polysaccharide extracts (EPS) produced by LOCA-1 and LOCA-2, two yeast strains collected from grape skin in Champagne, were isolated for both their biochemical and functional characterization. Their structural characterization by GC analysis show that EPS were complex heteropolymers with high molecular weight (~2.106 g/mol), and were composed of mannose, glucose, xylose and glucuronic acid as monosaccharide constituents, and 2 types of substituents (sulphate and phosphate) evidenced by MALDI analysis. Elucidation of the structural enchainment of these EPS carbohydrates based on GC-MS and NMR analyses revealed in both studied cases, a linear main backbone built up of α-(1→3)-D-mannopyranosyl residues. However differences have been noted between these two strains. For LOCA-1 EPS, some short side chains of β-(1→2)-xyloses (2-5 residues) are branched to the main linear backbone on its C-2 and/or C-6 positions. In contrast, the LOCA-2 main backbone that is more extended (˃40 units) than the former, is substituted on its C-2 and/or C-4 positions by xylomannan antennas. This is the first report of these yeasts’ polysaccharides with such structural characteristics. The second part of this thesis is devoted to the functional properties (foaming, gelling, and viscosifying abilities) of the EPS. Very exceptional viscosifying and foaming properties were observed after a comparative analysis with some marketed industrial biopolymers.The intrinsic properties of these natural polymers produced by these grape berries indigenous yeast strains, make them potential candidates for operating in various fields of biotechnology applications, especially enology.

Ce travail de thèse décrit la préparation et l'étude des propriétés d'auto-assemblage de nouveaux amphiphiles fonctionnalisés par des sucres. Des glycosides propargyliques du lactose et de la N-acétyl-glucosamine ont été conjugués par chimie click (cycloaddition de Huisgen catalysée par des sels de cuivre) à des dérivés de poly(ethyleneglycol) dont une des extrémités a au préalable été modifiée par une fonction azide et l'autre par un bloc hydrophobe de type polyphénylène ou bien aliphatique. Après une caractérisation par résonance magnétique nucléaire et spectrométrie de masse, les propriétés d'auto-assemblage de ces amphiphiles ont été étudiées par diffusion dynamique de la lumière (DLS), diffraction des rayons-X aux petits angles (SAXS) et microscopie électronique. Il a été montré qu'en phase aqueuse, les systèmes amphiphiles dérivés du PEG 900 s'auto-assemblent pour former de micelles de taille extrêmement régulière dont le diamètre moyen est de l'ordre de 10 nm. La présence et la biodisponibilité des sucres à la surface de ces nanoparticules ont également pu être démontrées par diffusion dynamique de la lumière avec les lectines PNA et WGA. Les interactions spécifiques observées entre les lectines et micelles associées aux propriétés d'encapsulation de ce type de nanoparticules permettent d'imaginer de futures applications pour la délivrance de médicaments ou encore l'imagerie médicale. / This thesis reports the preparation and characterization of new rod-coil amphiphiles functionalized with oligo- and polysaccharides through Huisgen 1,3-dipolar cycloaddition reactions between species functionalized by an azide group on one side and an terminal alkyne on the other catalyzed by copper. The amphiphiles were synthesized and characterized based on different hydrophobic parts conjugated with the polymer poly(ethylene oxide) PEO with a hydrophilic spacer arm and the oligo- and polyssaccharides 2-propargyl-2-acetamido-2-deoxy-β-D-glucopyranose (GlcNAc) and propargyl β-D-galactopyranosyl-(14)-β-D-glucopyranose (Lac). The amphiphiles synthesized were characterized in terms of their chemical structure and composition through nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, mass spectroscopy (MALDI-TOF-MS and ESI-MS) and high resolution mass spectroscopy (HRMS). After the dissolution in water, the amphiphiles self-associate in highly regular micelles with an average diameter of 2RH ~ 10 nm. Dynamic light scattering (DLS), transmission electron microscopy (TEM) and small angle x-ray scattering (SAXS) were used in order to investigate the structure and dynamics of these saccharide amphiphiles. The presence of carbohydrate epitopes on the surface of the micelles and their bioavailability for the segmentation of lectin were also demonstrated by DLS. Specific interactions of GlcNAc and Lac residues with lectins from wheat germ agglutinin (WGA) and peanut agglutinin (PNA), respectively, reveal the potential applications of such amphiphilic derivatives of carbohydrates as vectorizing systems, both simple and specific to a drug delivery site.

No description available.

This thesis reports the use of capillary electrophoresis (CE) for the analysis of carrageenans, anionic polysaccharides extracted from red seaweeds and widely used in the food industry for their gelling and thickening properties. The three main types, kappa, iota and lambda, differ in the number of sulfate groups and the presence or absence of a 3,6-anhydro bridge in the disaccharide residue repeat unit. CE separates analytes according to their charge to frictional coefficient ratios, therefore it is suitable to separate these biopolymers. In order to detect polysaccharides in CE, our approach consisted in derivatising the reducing ends of the saccharides by reductive arnination with a fluorophore, l-arninopyrene-3,6,8- trisulfonate (APTS). This allowed sensitive detection by laser induced fluorescence. Method development gave optimal conditions for separation using a polyvinyl alcohol coated capillary and a 25 mM ammonium acetate, pH 8.0 background electrolyte. The effects of changes of both instrumental parameters (temperature, injection mode, field strength) and, the composition of the BGE (concentration and pH) are reported, and explained in terms of the physical chemistry of the BGE and the biopolymers. The conditions of the derivatisation reaction were studied in order to minimise degradation due in particular to acid catalysis and to reduction of the reacting sites occurring in competition with derivatisation. Characterisation of the derivatised carrageenans by SEC-MALLS- RI was performed and showed that the extent of degradation occurring during the labelling reaction was a maximum of 40 % for kappa and 20 % for iota and lambda. The presence of the label APTS in excess and its reaction with the reagents during the labelling reaction produces peaks interfering with those from the carrageenan. A sample clean-up was therefore required before injection onto CEo A comparison was made of a range of clean-up procedures (centrifugation, dialysis, preparative SEC) to remove side products of the reaction and salts and to concentrate the carrageenans. Various seaweed extracts were analysed, including standards of carrageenans not available commercially. This study revealed that carrageenans are complex structures, and often occurring as hybrids between sUb-types. CE has the ability to characterise these hybrids, unlike spectroscopic methods which detect individual residues. When using actual food products, preliminary steps such as defatting and dialysis were found to be necessary to allow satisfactory detection of carrageenans. Finally the strategy for sample purification, derivatisation, clean-up and separation was successfully applied to additive mixtures used as raw materials in the food industry and to finished products (jelly, dairy products). CE has proved to be a fast and sensitive method to identify and provide semi-quantitative information on carrageenans present in such mixtures.

Sui-lam Wong. / Thesis (M.Phil.) -- Chinese University of Hong Kong. / Includes bibliographies.

Sun Baizhong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 75-79). / Abstracts in English and Chinese. / ABSTRACT --- p.i / ABSTRACT (IN CHINESE) --- p.ii / TABLE OF CONTENTS --- p.iii / LIST OF FIGURES --- p.v / LIST OF TABLES --- p.vii / ABBREVIATIONS --- p.vi / ACKNOWLEDGEMENT --- p.ix / DECLARATION --- p.x / Chapter CHAPTER ONE --- INTRODUCTION / Chapter 1.1 --- Polysaccharides --- p.1 / Chapter 1.2 --- Pharmacological importance of polysaccharides --- p.2 / Chapter 1.3 --- Polysaccharides from Ganoderma Lucidum --- p.3 / Chapter 1.4 --- Characterization of polysaccharides --- p.3 / Chapter 1.5 --- Matrix-assisted laser desorption / ionization --- p.5 / Chapter 1.6 --- Matrix-assisted laser desorption / ionization of polysaccharides --- p.8 / Chapter 1.7 --- Outline of the project --- p.10 / Chapter CHAPTER TWO --- INSTRUMENTATION AND EXPERIMENTAL / Chapter 2.1 --- Time-of-flight mass spectrometry --- p.12 / Chapter 2.1.1 --- Instrumentation --- p.15 / Chapter 2.1.1.1 --- Laser system --- p.16 / Chapter 2.1.1.2 --- Ion source --- p.16 / Chapter 2.1.1.3 --- Reflector --- p.19 / Chapter 2.1.1.4 --- Detector --- p.19 / Chapter 2.1.1.5 --- Data acquisition and manipulation --- p.20 / Chapter 2.2 --- Ultraviolet-visible spectrometer --- p.21 / Chapter CHAPTER THREE --- OPTIMIZATION OF MALDI CONDITIONS FOR POLYSACCHARIDE ANALYSIS USING DMSO AS SOLVENT / Chapter 3.1 --- Introduction --- p.22 / Chapter 3.2 --- Experimental --- p.25 / Chapter 3.2.1 --- Selection of matrix materials --- p.26 / Chapter 3.2.2 --- Selection of co-matrix materials --- p.26 / Chapter 3.2.3 --- Ratios of matrix-to-analyte --- p.26 / Chapter 3.2.4 --- Effect of sample drying temperature --- p.27 / Chapter 3.2.5 --- Sample loading methods --- p.27 / Chapter 3.3 --- Results and discussion --- p.27 / Chapter 3.3.1 --- Selection of matrix materials --- p.28 / Chapter 3.3.2 --- Selection of co-matrix materials --- p.30 / Chapter 3.3.3 --- Effect of matrix-to-co-matrix ratio --- p.32 / Chapter 3.3.4 --- Effect of sample drying temperature --- p.37 / Chapter 3.3.5 --- Effect of matrix-to-analyte ratio --- p.39 / Chapter 3.3.6 --- Evaluation of sample preparation protocols --- p.43 / Chapter 3.4 --- Conclusion --- p.46 / Chapter CHAPTER FOUR --- MOLECULAR AND MOLECULAR WEIGHT DISTRIBUTION OF WATER-INSOLUBLE POLYSACCHARIDES FROM GANODERMA LUCIDUM / Chapter 4.1 --- Introduction --- p.47 / Chapter 4.2 --- Experimental --- p.48 / Chapter 4.2.1 --- Extraction of water-insoluble polysaccharides from Ganoderma Lucidum --- p.48 / Chapter 4.2.2 --- Gel permeation chromatography of water-insoluble polysaccharides --- p.49 / Chapter 4.2.3 --- Phenol sulfuric acid assay --- p.51 / Chapter 4.2.4 --- MALDI-TOF analysis --- p.51 / Chapter 4.2.5 --- Bradford assay --- p.52 / Chapter 4.3 --- Results and discussion --- p.52 / Chapter 4.3.1 --- Extraction of water-insoluble polysaccharides from Ganoderma Lucidum --- p.52 / Chapter 4.3.2 --- Fractionation of water-insoluble polysaccharides using gel permeation chromatography (GPC) --- p.54 / Chapter 4.3.3 --- MALDI-TOF analysis of fractionated polysaccharides --- p.57 / Chapter 4.3.4 --- Carbohydrate content analysis of fractionated polysaccharides --- p.62 / Chapter 4.3.5 --- MW and MWD of water-insoluble polysaccharides extracted from Ganoderma Lucidum --- p.69 / Chapter 4.4 --- Conclusion --- p.71 / Chapter CHAPTER FIVE --- CONCLUDING REMARKS / Chapter 5.1 --- Conclusion --- p.73 / REFERENCES --- p.76 / APPENDIX --- p.81 / Appendix A Reaction scheme of carbohydrate with phenol in phenol-sulfuric acid assay --- p.81

Glycosaminoglycans (GAGs) are a group of structurally related, naturally occurring polysaccharides, found as the carbohydrate moieties of proteoglycans and sometimes as free polysaccharides. GAGs are expressed ubiquitously on animal cell surfaces and within extracellular matrices. All GAGs are sulfated to various degrees, except hyaluronan, which is always non-sulfated. In this project, GAGs and other sulfated carbohydrates were purified from different marine sources and an algae species living in soil, and their structures were characterised, mainly by NMR spectroscopy. Oversulfated dermatan sulfate (DS), fucosylated chondroitin sulfate (fCS) with interesting anti-metastatic properties, a sulfated fucosylated GlcNAc polymer, naturally-occurring cellulose sulfate and a polysaccharide with a repeating disaccharide unit of IdoA-Gal, were some of the more unusual carbohydrates identified. Common GAGs like hyaluronan, chondroitin sulfate A and C, DS and heparin/heparin sulfate-like polysaccharides were also purified from lumpsuckers and ragoworms. Different depolymerisation techniques were also investigated. The effects of Fentontype reaction and photochemical free radical depolymerisation on DS were studied. Elimination of the reducing end IdoA in the case of Fenton-type produced oligosaccharides was established. The oxidisation of reducing end GalNAc to Nacetylgalactosaminic acid, as a result of both depolymerisation techniques, was also identified. In addition, the effect of mild acid hydrolysis on fCS polysaccharides were studied and this technique was deemed unfitting for the depolymerisation of the molecule, as it causes defucosylation and partial desulfation. Finally, human beta-defensin 2 (HBD2) was expressed recombinantly, as a fusion protein with pE-Sumo, in an attempt to design a high yield expression system capable of producing correctly folded protein. The significance of NMR in the identification of the state of the protein and the need for an expression system capable of carrying out correctly the post-translational modifications was demonstrated.

No description available.

No description available.