Includes bibliographical references. / As part of an international collaborative programme concerned with the elucidation of the molecular structures of capsular polysaccharides (the K-antigen) produced by strains of the bacterial genus Klebsiella, the capsular material of serotype K71 has been investigated, and that of serotypes K36 and K64 re-examined, by novel enzymic and spectroscopic methods. The cultivation and employment of bacteriophages which are capable of cleaving (by specific glycanase action) the isolated, cognate bacterial polysaccharide in vitro has yielded highly significant oligosaccharides. These may represent the repeating unit in the polysaccharide or be derivatives resulting from conversion of uronic acid to the 4,5-unsaturated analogue where, as found for serotype K64, the mode of cleavage is β-elimination not hydrolysis. The oligosaccharides thus generated have proved to be far more amenable to chemical and spectroscopic studies than their parent polymers, thereby facilitating complete characterisation of the molecular structures of the original polysaccharides. Chemical methods applied to these oligosaccharides included specific degradations by periodate oxidation and acid-, alkali- or enzyme- catalysed hydrolysis, products being identified by methylation analysis (involving the extensive use of gas-liquid chromatography coupled to mass spectrometry) and spectroscopic studies (mass and n.m.r.). During the course of these investigations it became apparent that the structures of the intact oligosaccharides (containing six or seven sugar residues) could be determined almost entirely from spectroscopic analysis, chiefly by detailed two-dimensional n.m.r. studies involving the use of high field spectrometers and the application of homo- and heteronuclear shift correlated spectroscopy, the sequence of sugar units being confirmed by mass spectrometric analysis of the permethylated derivatives. Methylation analysis of the oligosaccharides derived from Klebsiella serotype K36 proved that the glucuronic acid residue is linked through 0-2, and not 0-4 as published by others; this finding was corroborated during characterisation of the monomeric oligosaccharide by mass- and n.m.r. spectroscopy. Bacteriophage φ64 was shown to cleave the cognate K64 exopolysaccharide by a β-elimination process; the resulting hex-4-enuronic acid, present as a terminal group in the derived oligosaccharide was fully characterised by hydrogenation and g.l.c.-m.s. of acetylated products, and by detailed n.m.r. studies including long-range heteronuclear experiments. Finally the structure of the heptasaccharide repeating unit of the Klebsiella K71 capsular polysaccharide was established by spectroscopic analysis of the oligosaccharides derived by bacteriophage φ71 cleavage of the polymer; features of the proposed structure were confirmed by chemical degradation studies performed on the native polysaccharide.

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Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

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Lai Kin Ming Connie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 121-141). / Abstracts in English and Chinese. / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Introduction on growth cycle of mushroom --- p.1 / Chapter 1.2 --- Literature review of mushroom biological activities --- p.3 / Chapter 1.2.1 --- Various bioactivities of mushroom --- p.3 / Chapter 1.2.2 --- Components responsible for various bioactivities of mushrooms --- p.3 / Chapter 1.3 --- Mushroom polysaccharides and polysaccharide-protein complexes --- p.5 / Chapter 1.3.1 --- Polysaccharides important for antitumor effects --- p.5 / Chapter 1.3.2 --- Polysaccharide-protein complexes important for antitumor effects --- p.7 / Chapter 1.4 --- Structure-function relationship of antitumor activities of polysaccharides --- p.8 / Chapter 1.4.1 --- Effect of molecular mass --- p.8 / Chapter 1.4.2 --- Effect of linkages --- p.9 / Chapter 1.4.3 --- Effect of degree of branching --- p.9 / Chapter 1.4.4 --- Effect of conformation --- p.10 / Chapter 1.5 --- Immunomodulatory effects of mushroom polysaccharides and polysaccharide-protein complexes --- p.11 / Chapter 1.5.1 --- Immunomodulatory effects of polysaccharides --- p.11 / Chapter 1.5.1.1 --- Bioactive polysaccharides in Lentinus edodes --- p.11 / Chapter 1.5.1.2 --- Bioactive polysaccharides in Ganoderma lucidum --- p.12 / Chapter 1.5.2 --- Immunomodulatory effects of polysaccharide-protein complexes --- p.12 / Chapter 1.5.2.1 --- Bioactive polysaccharide-protein complexes in Trametes versicolor --- p.13 / Chapter 1.5.3 --- Immunotherapeutic effects of mushroom polysaccharides --- p.14 / Chapter 1.6 --- Cell cycle and apoptosis --- p.14 / Chapter 1.6.1 --- Introduction of cell cycle --- p.14 / Chapter 1.6.2 --- Cell cycle regulation --- p.15 / Chapter 1.6.3 --- Antitumor effects through apoptotic gene regulation --- p.17 / Chapter 1.7 --- Mushroom sclerotium with antitumor activity --- p.20 / Chapter 1.7.1 --- Literature review on Pleurotus tuber-regium --- p.20 / Chapter 1.7.2 --- Literature review on Poria cocos --- p.22 / Chapter 1.7.3 --- Literature review on Polyporus rhinocerus --- p.23 / Chapter 1.8 --- Objectives --- p.23 / Chapter Chapter 2. --- Materials and Methods --- p.25 / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Mushroom sclerotia --- p.25 / Chapter 2.1.2 --- Animal Model --- p.25 / Chapter 2.1.3 --- Cell lines --- p.27 / Chapter 2.2 --- Methods --- p.28 / Chapter 2.2.1 --- Extraction Scheme for mushroom sclerotia --- p.28 / Chapter 2.2.1.1 --- Hot water extraction only --- p.28 / Chapter 2.2.1.2 --- Sequential extraction scheme --- p.28 / Chapter 2.2.2 --- Measurement of monosaccharide profile --- p.31 / Chapter 2.2.2.1 --- Acid Depolymerisation --- p.31 / Chapter 2.2.2.2 --- Neutral Sugar Derivatization --- p.31 / Chapter 2.2.2.3 --- Gas Chromatography (GC) --- p.32 / Chapter 2.2.3 --- High Pressure Liquid Chromatography (HPLC) --- p.33 / Chapter 2.2.3.1 --- Size exclusion chromatography --- p.33 / Chapter 2.2.3.2 --- Anion exchange chromatography --- p.34 / Chapter 2.2.4 --- Linkage analysis by methylation --- p.34 / Chapter 2.2.4.1 --- Preparation of partially methylated polysaccharides --- p.34 / Chapter 2.2.4.2 --- Preparation of partially methylated alditol acetates (PMAAs) --- p.37 / Chapter 2.2.4.3 --- Gas chromatography-Mass spectrometry (GC-MS) analysis --- p.37 / Chapter 2.2.5 --- Determination of total sugar by phenol-sulphuric acid Method --- p.38 / Chapter 2.2.6 --- Determination of acidic sugars by measurement of uronic acid content --- p.39 / Chapter 2.2.7 --- Determination of protein content by Lowry-Folin method --- p.40 / Chapter 2.2.8 --- Chemical modification by carboxymethylation --- p.41 / Chapter 2.2.9 --- In vitro antitumor assay --- p.41 / Chapter 2.2.9.1 --- Trypan blue exclusion assay --- p.42 / Chapter 2.2.9.2 --- MTT Assay --- p.42 / Chapter 2.2.10 --- Cell cycle analysis by Flow Cytometry --- p.43 / Chapter 2.2.11 --- In vivo antitumor and immunomodulatory assay --- p.44 / Chapter 2.2.11.1 --- Measurement on tumor growth --- p.44 / Chapter 2.2.11.2 --- Blood sampling for immunostimulatory effects --- p.45 / Chapter 2.2.12 --- Mouse Cytokine Array --- p.45 / Chapter 2.2.13 --- Quantification of Mouse IL-13 by ELISA --- p.46 / Chapter 2.2.14 --- Enumeration of peritoneal cells --- p.47 / Chapter 2.2.15 --- Enumeration of splenocytes --- p.49 / Chapter 2.2.16 --- Statistical methods --- p.50 / Chapter Chapter 3. --- Results and Discussion --- p.51 / Chapter 3.1 --- Yield of crude mushroom sclerotial extracts --- p.51 / Chapter 3.2 --- Chemical composition of crude mushroom sclerotial extracts --- p.57 / Chapter 3.2.1 --- Total carbohydrate content --- p.57 / Chapter 3.2.2 --- Uronic acid content --- p.58 / Chapter 3.2.3 --- Soluble protein content --- p.58 / Chapter 3.3 --- Monosaccharide profiles of mushroom sclerotial extracts by GC --- p.60 / Chapter 3.4 --- Chromatographic analyses of mushroom sclerotial extracts --- p.65 / Chapter 3.4.1 --- Molecular weight profile by size exclusion chromatography (SEC) --- p.65 / Chapter 3.4.2 --- Charge distribution by ion exchange chromatography (IEC) --- p.73 / Chapter 3.5 --- Antitumor effects of mushroom sclerotial extracts from hot water extraction alone --- p.73 / Chapter 3.5.1 --- In vitro antiproliferative study by HL-60 --- p.73 / Chapter 3.5.2 --- In vitro antiproliferative study by MCF-7 --- p.74 / Chapter 3.5.3 --- In vivo antitumor study by BALB/c mice --- p.75 / Chapter 3.6 --- Antitumor effects of extracts from sequential extraction scheme --- p.76 / Chapter 3.6.1 --- In vitro antiproliferative study by HL-60 --- p.76 / Chapter 3.6.2 --- In vitro antiproliferative study by MCF-7 --- p.78 / Chapter 3.6.3 --- In vivo antitumor study by BALB/c mice --- p.80 / Chapter 3.7 --- Comparison of in vitro and in vivo activities of mushroom sclerotial extracts --- p.82 / Chapter 3.8 --- Dose-response relationship of hot water extract from PR on cancer cell lines --- p.85 / Chapter 3.8.1 --- In vitro dose-response antiproliferation of PR-W and PR-HWE on HL-60 --- p.85 / Chapter 3.8.2 --- In vitro dose-response antiproliferation of PR-W on K562 and S180 --- p.88 / Chapter 3.8.3 --- In vivo dose-response relationship of PR-W on S180 --- p.91 / Chapter 3.9 --- Flow cytometric analysis of PR-W on cancer cell lines --- p.92 / Chapter 3.9.1 --- Antiproliferative effect of PR-W on HL-60 --- p.92 / Chapter 3.9.2 --- Antiproliferative effect of PR-W on K562 --- p.95 / Chapter 3.9.3 --- Proposed mechanisms of cell cycle arrest by PR-W --- p.98 / Chapter 3.10 --- Host-mediated antitumor mechanism of PR-W --- p.100 / Chapter 3.10.1 --- Mouse cytokine array --- p.100 / Chapter 3.10.2 --- Quantification of IL-13 by ELISA --- p.105 / Chapter 3.10.3 --- Immunostimulatory effects of PR-W on mice --- p.109 / Chapter 3.11 --- Correlation between antitumor activity and structure of mushroom sclerotial extract from hot water extraction alone --- p.114 / Chapter Chapter 4. --- Conclusions and Future works --- p.118 / List of References --- p.121 / Related Publications --- p.142

Athymic nude mice were employed as the in vivo model to study the detailed mechanism of how the three sclerotial polysaccharides act to inhibit the growth of human xenografted tumors in vivo. Using immunohistochemical staining, it was found that the presence of F4/80 + macrophages was related to the reduction of tumor size of the HL-60 xenograft. mRNA extracted from the spleens were reverse-transcribed to cDNA and detected by real-time PCR so that a variety of genes related to the toll-like receptors being up-regulated or down-regulated due to the injection of mushroom sclerotial polysaccharides were determined. Combining the results from dectin-1 regulation, it was concluded that both hot water-soluble sclerotial polysaccharides, PTRW and PRW, having a structure of polysaccharide-protein complexes were responsible for activating and thus binding to CR3 or toll-like receptors while PRSon with structure of pure beta-glucan was responsible for activating the expression of dectin-1 receptor, which led to the subsequent activation of host immune system in immunopotentiation and antitumor activities. / In the future, further investigation of the detailed structure of mushroom sclerotial polysaccharides is required to explain the immunomodulatory mechanism so that the effective dosage for immunomodulation as well as antitumor effects can be determined. Furthermore, phage display can be applied to find out any novel glucan receptors specific to the mushroom sclerotial polysaccharides. / In vitro antitumor study indicated that PTRW had a significant (p<0.05) inhibitory effect (>40%) on the human monocytic leukemic cells (THP-1) in addition to HL-60 and K562 cells. In vitro immunomodulatory study showed that both PRW and PRSon had significant proliferative effects (p<0.05) on human normal spleen monocyte/macrophage cell, MD. Moreover, PRSon was shown to have a significant increase (p<0.05) in the growth of human natural killer cells, NK-92M1; however, PTRW showed a significant inhibition (p<0.05) on this cell line. / Mushroom sclerotia have a rich source of polysaccharides when compared with fruit bodies. It was previously reported that the polysaccharides from novel mushroom sclerotia, namely, Pleurotus tuber-regium and Polyporus rhinocerus, had potent in vitro and in vivo antitumor effects. In this project, hot water-soluble sclerotial polysaccharides of Pleurotus tuber-regium (PTRW), hot water-soluble and sonication-assisted cold alkali-soluble sclerotial polysaccharides of Polyporus rhinocerus (PRW and PRSon, respectively) were chosen for investigation of their in vivo and in vitro immunomodulatory effects. / Polysaccharides have long been proposed to exert their antitumor and thus immunomodulating functions through glucan receptors and among the four being discovered, Dectin-1 has drawn most attention recently. In the in vivo study, PRSon showed an increase in the expression of Dectin-1 on mice spleen MNCs while PTRW showed an increase in the expression of the previously widely-reported complement receptor (CR3). There was also an increase of Dectin-1 expression on PEC in the mice injected with PRSon. In the in vitro study, the three mushroom sclerotial polysaccharides were incubated with NK-92M1, MD and THP-1 cells. There was a significant increase (p<0.05) of Dectin-1 expression on NK-92MI cells incubated with PTRW. On the other hand, PTRW caused a significant decrease ( p<0.05) of Dectin-1 expression while PRSon showed a significant increase (p<0.05) on THP-1 cells. The cytokine profile of extra-cellular media indicated that the inhibition of THP-1 cells by PTRW should be related to the innate immunity. In the in vitro study, human primary immune cells, CD56+ NK cells were used to incubate with sclerotial polysaccharides and there was a significant stimulation (p<0.05) of their growth when compared with the control. / The in vivo immunomodulatory study was carried out by injecting the abovementioned sclerotial polysaccharides intraperitoneal to 7-8 weeks old healthy male BALB/c mice. The spleens excised from groups injected with PTRW and PRW were found to have significant increase of weight ( p<0.001). Flow cytometric analysis revealed that the NK cell population in spleen mononuclear cells (MNCs) of mice injected with PRW and PRSon was increased when compared with the control. In addition, ail three sclerotial polysaccharides showed a large increase of T helper cell population as well as CD4+/CD8+ ratio in spleen MNCs. On the other hand, the macrophage population in peritoneal exudates cells (PEC) was found to be increased in the groups of mice injected with PTRW and PRW. / Lai, Kin Ming Connie. / Adviser: Cheung Chi Keung. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3412. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 120-137). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Les biofilms ont longtemps été décrits comme des organisations évolutives de microorganismes, attachés à une surface et englués dans une matrice contenant, entre autre, des polysaccharides. En partant de ce constat BioFilm Control a souhaité cribler des microorganismes pour la production d’exopolysaccharides, en utilisant le BioFilm Ring Test® (BRT). Le principe repose sur la coincubation de microorganismes avec des particules magnétiques en microplaque. Les particules sont plus ou moins attirées par un aimant en fonction du stade d’organisation du biofilm. En se formant, il piège, dans sa matrice visqueuse, les particules qui perdent leur mobilité. Celle-ci est révélée par une aimantation qui provoque l’apparition d’un spot (pas de biofilm) ou non (biofilm). Une analyse d’images quantifie ce processus et permet de le standardiser. La démarche a consisté dans un premier temps à vérifier le comportement de microorganismes modèles producteurs de polysaccharides (bactéries et microalgues) avec le BRT. L’étude a été étendue au criblage d’une banque de lactobacilles. Les résultats inattendus ont orienté l’étude vers l’analyse du rôle exact des polysaccharides et plus généralement de l’implication des macromolécules dans la structuration du biofilm. Pour cela, la dégradation séquentielle de chaque famille macromoléculaire a été réalisée via des enzymes dépolymérisantes sur les biofilms de Leuconostoc mesenteroides et Bacillus sp. Au regard des résultats obtenus, l’utilisation du BRT a été étendue à la caractérisation qualitative et quantitative d’enzymes de dégradation de polysaccharides. / Biofilms were described for a long time as evolutionary structures elaborated by microorganisms, fixed on a surface and maintained in a polysaccharidic matrix. From this assessment, BioFilm Control chose to screen microorganisms for their capacity to produce exopolysaccharides (EPS), using theBioFilm Ring Test® (BRT). The principle is the co-incubation of magnetic particles with microbial culture on microplates. The mobility of particles depends on the stage of biofilm formation. During this formation, particles are trapped in the matrix and loose their mobility. Revelation is induced by magnet which causes a spot in the absence of biofilm. The pictures analysis quantifies this phenomenon and standardizes different results. This approach was realised, at first step, by the test of EPS-producing bacteria or microalgae with the BRT. The study was extended to the screening of a lactobacilli collection. Unexpected results guided the research toward the understanding of the role of macromolecules in biofilm structuring. To study their implication, sequential enzymatic degradation has been achieved for each macromolecular family of Leuconostoc mesenteroïdes and Bacillus sp. biofilms. Using the results, BRT was then appreciated as a suitable method to detect and quantify polysaccharide degrading enzymes.

Choi Pui-yu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 113-123). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese Version) --- p.iii / Acknowledgements --- p.v / List of Abbreviations --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xii / List of Figures --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature Review / Chapter 2.1 --- Mushroom Polysaccharides with Antitumor Activities --- p.7 / Chapter 2.1.1 --- Antitumor β-Glucans --- p.7 / Chapter 2.1.2 --- Antitumor Heteroglycans --- p.9 / Chapter 2.1.3 --- Antitumor Polysaccharide-Protein Complexes --- p.12 / Chapter 2.2 --- Antitumor Activities and Structural Characteristics of Mushroom Polysaccharides --- p.15 / Chapter 2.3 --- Antitumor Effects of Mushroom Polysaccharides In Vitro --- p.19 / Chapter 2.4 --- Antitumor Effects of Mushroom Polysaccharides In Vivo --- p.21 / Chapter 2.5 --- Immunomodulatory Activities --- p.24 / Chapter 2.6 --- Activation of Cytokines by Mushroom Polysaccharides --- p.28 / Chapter 2.7 --- Induction of Nitric Oxide Synthase by Mushroom Polysaccharides --- p.32 / Chapter 2.8 --- Tremella aurantialba --- p.34 / Chapter Chapter 3 --- Materials and Methods --- p.35 / Chapter 3.1 --- Extraction --- p.35 / Chapter 3.1.1 --- Extraction of Crude Tremella aurantialba Polysaccharide --- p.35 / Chapter 3.1.2 --- Fractionation --- p.38 / Chapter 3.1.3 --- Polysaccharide and Protein Content Determination --- p.38 / Chapter 3.1.3.1 --- Phenol-Sulfuric Assay --- p.39 / Chapter 3.1.3.2 --- Lowry-Folin Method --- p.39 / Chapter 3.1.4 --- Gas Chromatography (GC) --- p.40 / Chapter 3.1.5 --- Modified Carbazole Assay --- p.41 / Chapter 3.1.6 --- High Performance Liquid Chromatography (HPLC) --- p.42 / Chapter 3.2 --- In Vitro Studies --- p.43 / Chapter 3.2.1 --- Maintenance of Cell Lines --- p.43 / Chapter 3.2.2 --- Effect on Cancer Cell Lines --- p.43 / Chapter 3.2.2.1 --- Trypan Blue Exclusion Methods --- p.44 / Chapter 3.2.2.2 --- MTT Assay --- p.44 / Chapter 3.2.3 --- Effect on Normal Cell Line --- p.45 / Chapter 3.2.4 --- Coulter Counter --- p.46 / Chapter 3.3 --- In Vivo Studies --- p.47 / Chapter 3.3.1 --- Animals --- p.47 / Chapter 3.3.2 --- Maintenance of Sarcoma 180 Cell Line --- p.47 / Chapter 3.3.3 --- Effect of TAP Fractions on Sarcoma 18 Solid Tumor --- p.48 / Chapter 3.3.3.1 --- Injection of TAP Fractions 24 h After Transplantation --- p.48 / Chapter 3.3.3.2 --- Injection of TAP Fractions 72 h After Transplantation --- p.49 / Chapter 3.4 --- Effect of TAP Fractions on Modulating mRNA Expression of Cytokines and Nitric Oxide Synthase --- p.51 / Chapter 3.4.1 --- Treatment of Mice --- p.51 / Chapter 3.4.2 --- Isolation of Splenocytes and Peritoneal Exduate Cells --- p.51 / Chapter 3.4.3 --- Extraction of Total mRNA from Splenocyte and Peritoneal Exduate Cells --- p.52 / Chapter 3.4.4 --- Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.53 / Chapter 3.4.4.1 --- Reverse Transcription --- p.53 / Chapter 3.4.4.2 --- Polymerase Chain Reaction --- p.56 / Chapter 3.4.5 --- DNA Sequencing --- p.57 / Chapter 3.5 --- Statistical Analysis --- p.58 / Chapter Chapter 4 --- Results --- p.59 / Chapter 4.1 --- Isolation and Characterization of TAP Fractions --- p.59 / Chapter 4.1.1 --- Percentage Yield of TAP Fractions --- p.59 / Chapter 4.1.2 --- Polysaccharide and Protein Content of TAP Fractions --- p.59 / Chapter 4.1.3 --- Relative Monosaccharide Contents in TAP Fractions --- p.60 / Chapter 4.1.4 --- Results of HPLC --- p.60 / Chapter 4.2 --- Effects of TAP Fractions In Vitro --- p.69 / Chapter 4.2.1 --- Effects of TAP Fractions on Suspension Cancer Cell Lines --- p.69 / Chapter 4.2.2 --- Effects of TAP Fractions on Adhesion Cancer Cell Lines --- p.69 / Chapter 4.2.3 --- Effects of TAP Fractions on Normal Cell Line --- p.70 / Chapter 4.2.4 --- Effect of TAP 2 on HL-60 Cell Line as Evaluated by Coulter Counter --- p.70 / Chapter 4.3 --- Antitumor Effect of TAP Fractions In Vivo --- p.78 / Chapter 4.4 --- Effect of TAP Fractions on Modulating mRNA Expressions of Cytokines and Nitric Oxide Sythase (NOS) --- p.83 / Chapter 4.4.1 --- Results of RT-PCR --- p.83 / Chapter 4.4.2 --- Sequencing --- p.84 / Chapter Chapter 5 --- Discussion --- p.91 / Chapter 5.1 --- Characterization of TAP Fractions --- p.91 / Chapter 5.2 --- Antitumor Effects of TAP Fractions In Vitro --- p.96 / Chapter 5.3 --- Furhter Study --- p.109 / Chapter Chapter 6 --- Conclusion --- p.111 / References --- p.113

Poon Shuk-ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 113-125). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abstract (Chinese Version) --- p.iv / Table of Contents --- p.vi / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiii / Chapter Chapterl 1 --- ntroduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.5 / Chapter 2.1 --- Mushroom Polysaccharides From Basidiomycetes --- p.5 / Chapter 2.1.1 --- Antitumor and Immunomodulatory Activity --- p.6 / Chapter 2.1.2 --- Antiviral Activity --- p.9 / Chapter 2.1.3 --- Hypoglycermic Activity --- p.11 / Chapter 2.1.4 --- Free Radical Scavenging Activity --- p.11 / Chapter 2.2 --- Mushroom Dictyophora indusiata --- p.13 / Chapter 2.2.1 --- Nutritional Value --- p.13 / Chapter 2.2.2 --- Structural Characteristic of Dictyophora indusiata Polysaccharides --- p.14 / Chapter 2.2.3 --- Biological Activity --- p.16 / Chapter 2.3 --- In vivo Antitumor Study --- p.19 / Chapter 2.4 --- Induction of Cytokines Production in Immune System --- p.21 / Chapter 2.5 --- In vitro Antitumor Study --- p.25 / Chapter 2.6 --- Cell Cycle Regulation --- p.28 / Chapter Chapter 3 --- Materials & Methods --- p.34 / Chapter 3.1 --- Extraction --- p.34 / Chapter 3.1.1 --- Extraction of Dictyophora indusiata Polysaccharides --- p.34 / Chapter 3.1.2 --- Purification of Dictyophora indusiata Polysaccharides --- p.35 / Chapter 3.1.2.1 --- Preparation of DEAE-cellulose Ion Exchanger --- p.35 / Chapter 3.1.2.2 --- Fractionation --- p.35 / Chapter 3.2. --- Characterization of Dictyophora indusiata Polysaccharides --- p.39 / Chapter 3.2.1 --- Polysaccharide Content Determination --- p.39 / Chapter 3.2.2 --- Protein Content Determination --- p.39 / Chapter 3.2.3 --- Gas Chromatography (GC) --- p.40 / Chapter 3.2.4 --- Uronic Acid Content Determination --- p.42 / Chapter 3.2.5 --- High Performance Liquid Chromatography (HPLC) --- p.43 / Chapter 3.3 --- In vivo Studies --- p.44 / Chapter 3.3.1 --- Animals --- p.44 / Chapter 3.3.2 --- Maintenance of Sarcoma 180 Cell Line --- p.44 / Chapter 3.3.3 --- Effect of DI3 Fraction on Sarcoma 180 Solid Tumor --- p.45 / Chapter 3.3.4 --- Effect of DI3c Fraction on Tumor Necrosis Factor-Alpha (TNF-α) and Interleukin 2 (IL-2) Production --- p.47 / Chapter 3.3.4.1 --- Treatment of Mice --- p.47 / Chapter 3.3.4.2 --- Preparation of Mouse Serum --- p.47 / Chapter 3.3.4.3 --- Enzyme-linked Immunosorbent Assay (ELISA) for TNF-α Production --- p.48 / Chapter 3.3.4.4 --- Enzyme-linked Immunosorbent Assay (ELISA) for IL-2 Production --- p.49 / Chapter 3.4 --- In vitro Studies --- p.51 / Chapter 3.4.1 --- Maintenance of Cell Lines --- p.51 / Chapter 3.4.2 --- Effect on Cancer Cell Lines --- p.52 / Chapter 3.4.3 --- Cytotoxicity on Normal Cell Line --- p.52 / Chapter 3.4.4 --- Trypan Blue Exclusion Method --- p.53 / Chapter 3.4.5 --- MTT Assay --- p.54 / Chapter 3.4.6 --- BrdU Incorporation --- p.55 / Chapter 3.5 --- Statistical Analysis --- p.56 / Chapter Chapter 4 --- Results --- p.57 / Chapter 4.1 --- Extraction and Fractionation of Polysaccharides from Dictyophora indusiata --- p.57 / Chapter 4.1.1 --- Percentage Yield of Crude DI Polysaccharides --- p.57 / Chapter 4.1.2 --- Percentage Yield of DI3 Fractions --- p.57 / Chapter 4.2 --- Characterization of DI3 Fractions --- p.62 / Chapter 4.2.1 --- Polysaccharide and Protein Contents of DI3 Fractions --- p.62 / Chapter 4.2.2 --- Relative Monosaccharide and Uronic Acid Content in Different DI3 Fractions --- p.62 / Chapter 4.2.3 --- Estimated Molecular Weight of DI3 Fractions --- p.65 / Chapter 4.3 --- Antitumor Effect of Dictyophora indusiata Polysaccharides In vivo --- p.70 / Chapter 4.3.1 --- In vivo Antitumor Effect of Crude DI Polysaccharides --- p.70 / Chapter 4.3.2 --- In vivo Antitumor Effect of Various Fractions of DI3 --- p.70 / Chapter 4.3.3 --- Effect of DI3c on TNP-α and IL-2 Production in Mice --- p.78 / Chapter 4.4 --- In vitro Effects of DI3 Fractions on Cell Density and Viability on Normal and Cancer Cell Lines --- p.86 / Chapter 4.4.1 --- Effects of DI3 Fractions on Cell Density and Viability of Human Leukemic HL-60 and K-562 and Mouse Sarcoma 180 Cells --- p.86 / Chapter 4.4.2 --- Effects of DI3 Fractions on the Growth of Human Liver Cancer HepG2 and Normal Monkey Kidney Vero Cells --- p.86 / Chapter 4.4.3 --- Effect of DI3b Fraction on Proliferation of HL-60 Cells Determined by BrdU Incorporation --- p.94 / Chapter Chapter 5 --- Discussions --- p.96 / Chapter 5.1 --- Extraction and Characterization of DI3 Fractions --- p.96 / Chapter 5.2 --- Antitumor Effects of Dictyophora indusiata Polysaccharides --- p.101 / Chapter 5.3 --- Further Studies --- p.109 / Chapter Chapter 6 --- Conclusion --- p.111 / References --- p.113

La paroi végétale est une structure complexe composée principalement de polysaccharides (cellulose, hémicellulose et pectine), de lignine et de protéines. Elle est impliquée dans de nombreuses fonctions essentielles à la vie de la cellule végétale. De plus, les constituants de cette paroi, que sont les polysaccharides et la lignine, représentent la plus grande source de carbone renouvelable de la planète. Ceci en fait des cibles de choix notamment pour la production d'énergies « vertes ». Toutefois, l'utilisation des polysaccharides tels que les hémicelluloses constituant la paroi végétale reste, à l'heure actuelle, limitée du fait de la difficulté à les dégrader. Ces dernières années, un effort important a été mis en œuvre pour identifier et caractériser de nouvelles enzymes, telles que les glycosides hydrolases, permettant de dégrader efficacement la biomasse végétale. Dans le but de découvrir de nouvelles enzymes impliquées dans la dégradation de la biomasse végétale, des chercheurs de l'équipe « Catalyse et Ingénierie Moléculaire Enzymatiques » du LISBP ont décidé d'explorer le métagénome d'organismes connus pour dégrader la biomasse végétale. Deux espèces animales ont fait l'objet d'analyses : tout d'abord les termites qui sont considérés comme les champions de la dégradation de la biomasse végétales et souvent comparés à des bioréacteurs, et le ver de terre. Des banques métagénomiques de trois espèces différentes de termites ainsi qu'une banque métagénomique de ver de terre ont ainsi été créées. Dans ces travaux de thèse deux des banques métagénomiques de termites, celle de Nasutitermes corniger et celle de Termes hispaniolae, ont fait l'objet d'une étude afin de comparer le potentiel hémicellulolytique de ces deux espèces. Après sélection de nombreux clones positifs sur substrats chromogéniques de chacune des deux banques, séquençage puis annotation taxonomique et fonctionnelle, un grand nombre d'enzymes et principalement des glycosides hydrolases, a pu être identifié. Les résultats montrent que le métagénome de Nasutitermes corniger présente majoritairement des enzymes à activité endoglycosidase alors que le métagenome de Termes hispaniolae possède plutôt des enzymes à activité exoglycosidase. Toutes les activités trouvées dans chacune des espèces de termite sont en bonne corrélation avec l'alimentation du termite. De plus, nous avons observé que le microbiote intestinal des deux termites ne possèdent pas les mêmes embranchements bactériens majoritaires et nous avons pu voir que le microbiote de Termes hispaniolae est plus diversifié ce qui corrèle aussi avec l'alimentation des deux termites. D'autre part, dans la banque métagénomique du ver de terre, l'annotation fonctionnelle a révélé une enzyme intéressante. Il s'agit d'une enzyme annotée par B. Henrissat (responsable de la base de données CAZy) comme étant une glycoside hydrolase putative mais n'appartenant à aucune des 135 familles de glycosides hydrolases existantes. Cette enzyme putative, appelée GH* présente des similitudes avec les GH de la famille 5 sans pour autant appartenir à cette famille du fait notamment de l'absence du résidu catalytique nucléophile conservé. Une étude structurale et fonctionnelle de GH* a donc été menée. Les expériences ont permis de prouver que GH* est une endo-xylanase ayant une préférence pour les arabinoxylanes et les xylooligosaccharides de degré de polymérisation d'au moins 5 ou 6. La structure tridimensionnelle de GH* à 1,6Å de résolution a été obtenue par cristallographie des rayons X par remplacement moléculaire à l'aide d'une GH5. Cette structure a permis de confirmer l'identité du résidu acide/base identifié par alignement de séquences et d'émettre une hypothèse sur l'identité du résidu nucléophile. Enfin des mutants de GH* pour ces deux résidus ont été obtenus et ont confirmé leur implication dans l'activité de l'enzyme. / Plant cell wall is a complex structure surrounding plant cells mainly composed by polysaccharides (cellulose, hemicellulose and pectin), lignin and proteins. The plant wall maintains and imposes the size and shape of cells. It is also important for exchanges between cells and extra cellular medium. The polysaccharides of this cell wall are the largest renewable carbon source on the earth, which makes them good targets to produce green energies. Because plant cell wall is difficult to degrade, its use for biofuels for is still limited. However, some organisms are able to efficiently degrade this biomass. Exploring the diversity of the living word to discover new effective biocatalysts has grown considerably last years, because of the emergence of metagenomics. In this context and to discover new enzymes involved in the degradation of plant biomass, the team « Catalyse et Ingénierie Moléculaire Enzymatiques » of LISBP decided to explore metagenome of organisms known to degrade plant biomass. Two animal families were chosen for metagenomics analysis, the termite and earthworm. Metagenomics banks of three different species of termite and one metagenomics bank of an earthworm were created. In this thesis project, two of the three metagenomics banks of termites, the one from Nasutitermes corniger and the other one from Termes hispaniolae, were studied to compare the hemicellulolytic potential of these two species. After selection of many positive clones on chromogenic substrates of both banks, sequencing, taxonomic and functional annotations, a large number of enzymes and mainly glycoside hydrolases, could be identified. The results obtained shown that the trends observed during functional screens were maintained. Indeed, it appears that Nasutitermes corniger has a majority of endoglycosidases while Termes hispaniolae has mainly exoglycosidases. Thereby, families of enzymes highlighted allowed correlating their hydrolytic activities with the diet of these species. Furthermore, we observed that the intestinal microbiota of each termite is different. Indeed, both termites do not have the same majority bacterial phyla and the microbiota of Termes hispaniolae is more diverse than the one of Nasutitermes corniger. On the other hand, functional annotation of the metagenomics bank of the earthworm revealed an enzyme annotated as a glycoside hydrolase no belonging to any of the 135 glycoside hydrolase existing families. This enzyme, named GH*, seems to be close to GH5 but does not shown the nucleophilic catalyst residue perfectly conserved in this glycoside hydrolase family. A functional and structural study of GH* was then done. We have shown that GH* is an endo-xylanase which prefers arabinoxylans and xylooligosaccharides having a polymerization degree greater than 5. In addition, we determined the crystal structure of GH* at 1.6Å resolution. This 3D structure has confirmed the presence of the acid/base residue identified by sequence alignment and allowed us to hypothesize about the identity of the nucleophilic residue. Finally, mutants of GH* for these two residues were obtained and confirmed their involvement in the activity of the enzyme. We were able to progress in the understanding of structure/function relationships of this protein.