No description available.

Glycosaminoglycans (GAGs) are linear polysaccharides expressed ubiquitously on animal cell surfaces and within extracellular matrices. GAGs usually occur as parts of proteoglycans and often accomplish their biological functions through their interactions with proteins. GAG oligosaccharides for this work were produced via enzymatic digest of heparin, followed by gel filtration and ion exchange chromatography. Two tetrasaccharide species obtained from this digest were characterised using 1H and 13C NMR spectroscopy. Complement factor H (fH) is a regulatory protein of the alternative pathway of the complement system, a major component of human innate immunity. Acting as a cofactor to factor I, fH inhibits C3b-initiated complement activation on host cells, protecting cells from auto immune attack. This study focused on the interaction of factor H with GAGs, which are thought to be among the markers allowing factor H to distinguish between self and non self surfaces. Binding studies of two heparin-binding sites in fH are presented. These include the C-terminal modules 19 and 20 (fH~19-20) and fH~7-8. FH~7, fH~7-8 and fH~19-20 were produced recombinantly in various isotope forms. The techniques used to study the protein-GAG interactions in this work encompass NMR spectroscopy, mass spectrometry, gel mobility shift assays (GMSA) and chemical cross linking. Several genetic studies suggest that a common polymorphism in the heparin-binding module fH~7, Y402H, plays a role in the development of age-related macular degeneration (AMD). The work presented here included preparation and backbone resonance assignment of a 13C, 15N- labelled sample of fH~ 7-8 via triple resonance NMR experiments. Further NMR experiments were employed to investigate the role of the lysine and arginine sidechains of fH~7 in GAG binding. These studies were combined with the preparation and characterisation of a covalently cross linked GAG-protein complex using NMR and mass spectrometry. A range of fH~19-20 mutations that are linked to a severe kidney disease, atypical haemolytic uraemic syndrome (aHUS), were characterised using GMSA. No correlation between the disease and the heparin binding properties of the aHUS mutants was observed. The mutant proteins were also characterised with respect to their ability to compete with full-length fH in a physiological complement assay. Simultaneous binding of WT fH~19-20 to GAGs and C3d, the relevant fragment of C3b, was assessed using NMR. NMR experiments were also conducted with NK1, which comprises the two N-terminal heparin-binding modules of hepatocyte growth factor/scatter factor (HGF/SF), and heparin as well as dermatan sulfate-derived GAGs. Relaxation studies on a human defensin, HBD2, were performed to assess the role of GAGs in HBD2 self-association.

Submitted in fulfilment for the degree of Doctor of Philosophy Department of Clinical Microbiology and Infectious Diseases School of Pathology Faculty of Health Sciences University of Witwatersrand 2017. / Background Vaccination of pregnant women with conserved group B Streptococcus (GBS) surface proteins has the potential to confer serotype independent protection against invasive GBS disease. Susceptibility to early onset (<7 days of age) GBS disease in infants is associated with maternal recto-vaginal colonisation, low circulating maternal antibodies and reduced trans-placental transfer of antibodies specific to GBS capsular polysaccharides (CPS). Additionally, invasive GBS disease beyond infancy has been reported in individuals with underlying conditions associated with immunosuppression including HIV infection. In this study we undertook retrospective analysis of serum IgG titres against select GBS surface proteins in relation to invasive GBS disease risk factors. Methods Multiplex Luminex immunoassay was used to measure serum IgG titres against GBS capsular polysaccharides of serotype Ia, Ib, III and V and surface proteins Fibrinogen binding surface Antigen (FbsA), GBS Immunogenic Bacterial Adhesin (BibA), Surface immunogenic protein (Sip), gbs0393, gbs1356, gbs1539, gbs0392; and lipoproteins gbs0233, gbs2106 and Foldase PsrA. Furthermore, in vitro expression of Sip, gbs2106, gbs0393 and gbs1356 proteins on the surface of clinical GBS isolates was assessed by flow cytometry using protein specific polyclonal antibodies generated in rabbits. Results Retrospective analyses of serum antibody titres against GBS surface proteins and CPS were measured in children between 4-7 years of age who were either HIVinfected (n=68) or HIV-uninfected (n=77). Lower geometric meant titres (GMT, U/mL) against Sip and gbs2106 were detected in HIV-infected children (77.03 and 53.10) compared to HIV-uninfected children (196.41 and 139.11, p<0.001) respectively. Similar results were observed for antibodies against CPS, with HIV-infected children having lower IgG GMC for serotype Ib (p=0.012) and V (p=0.005). Protein specific antibody titres were measured in pregnant women at 20-25 and ≥37 weeks of gestation age who were either non-colonised or colonised with GBS in the rectal and/or vaginal tract. Acquisition of GBS colonisation in the vagina was associated with higher antibody titres compared to colonisation in the rectum for proteins Sip (p=0.049), Foldase (p=0.0094), gbs0233 (p=0.0039), gbs0393 (p=0.027), gbs1539 (p=0.0004) and gbs1356 (p=0.039). The likelihood of acquiring GBS colonisation during pregnancy was lower in women having median IgG titres against gbs0233 ≥200 U/mL (adjusted OR=0.47 [95% CI: 0.25-0.89], p=0.021) and gbs1539 ≥85 U/mL (adjusted OR=0.44 [95% CI: 0.24-0.82], p=0.01). The influence of maternal HIV infection on trans-placental transfer of antibodies against GBS surface proteins was evaluated by comparing IgG titres between 83 HIV-infected and 81 HIV-uninfected mother-newborn dyads. Maternal HIV infection was associated with reduced trans-placental transfer of antibodies, demonstrated by the difference in cord-maternal antibody ratios between HIV-infected and HIV-uninfected mothernewborn pairs for Sip (25.8%, p<0.001), Foldase (30.4%, p<0.001), gba0392 (36.5%, p=0.006), gbs0393 (32.9%, p<0.001), gbs1539 (39.2%, p<0.008), gbs2106 (35.7%, p<0.001) and BibA (19.4%, p=0.004). A case control study was used to evaluate the association of protein specific IgG titres and invasive disease in neonates and young infants born to GBS colonised mothers, including 116 with healthy infants and 66 with invasive GBS disease at <90 days of age. Lower IgG GMT were detected in neonates who developed early-onset disease compared to control infants for Sip (65.48 vs 145.43, p<0.001), Foldase (50.45 vs 109.87, p=0.005), gbs0393 (106.42 vs 221.81, p=0.006) and gbs1356 (45.34 vs 94.52, p=0.01). Similarly, young infants with late-onset disease had significantly lower IgG GMT (U/mL) compared to healthy controls for Sip (43.72 vs 79.69, p=0.04) and gbs2106 (30.46 vs 65.35, p=0.003). Infants born to women with Sip specific antibody titres ≥150 U/mL antibodies titres against Sip were 66% less likely to develop invasive disease. Of the 82 GBS isolates collected from mothers who deliverd term babies (39 with invasive GBS disease and 43 healthy controls) that were assessed for in vitro expression of specific proteins, Sip, gbs2106 and gbs0393 were expressed in 70.7% 93.9% and 87.8% GBS isolates respectively. The expression of gbs2106 on the surface of GBS was more frequent in strains of women with infants who developed invasive disease compared to those with healthy infants (100% vs 88.4%, p=0.028). The distribution of Sip and gbs0393 expression between GBS isolates from women of infants with invasive GBS disease and healthy controls was similar, 71.8% vs 69.8% (p=0.84) and 89.7% vs 86.0% (p=0.61) respectively. Among women carrying GBS strains expressing gbs2106, having antibody titres ≥100 U/mL was associated with 90% lower likelihood for delivering infants that develop invasive GBS disease (OR=0.11 [95% CI: 0.02-0.54], p=0.002). Conclusion Vulnerability to invasive GBS disease in HIV-infected immunocompromised individuals is possibly due to reduced antibody levels against CPS, Sip and gbs2106. Also, susceptibility to invasive GBS disease in infants may be exacerbated by maternal HIV-infection, which was associated with reduced transplacental transfer of antibody against GBS surface proteins. Higher maternal antibodies titres against Sip and gbs2106 proteins were associated with reduced odds of their infants developing invasive GBS disease. These data support consideration of Sip and gbs2106 proteins as possible vaccine candidates in pregnant women to protect their infants against invasive GBS disease. Furthermore, reduced GBS colonisation during pregnancy can be achieved by maternal immunisation of gbs0233 and gbs1539, which may subsequently result in lower rates of GBS transmission to newborns and thereby decreasing their risk for invasive GBS disease. / MT2017

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Tang Kwan-Yee. / Thesis submitted in: September 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 86-94). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.i / LIST OF FIGURES --- p.v / LIST OF TABLES --- p.viii / ABSTRACT --- p.ix / ACKNOWLEDGEMENT --- p.xiii / DECLARATION --- p.xiv / ABBREVIATIONS --- p.xv / Chapter CHAPTER ONE --- INTRODUCTION / Chapter 1.1. --- Structure and conformation of Curdlan --- p.1 / Chapter 1.2. --- Important of Curdlan --- p.3 / Chapter 1.3. --- Conventional methods on molecular weight and molecular weight distribution on polysaccharide --- p.4 / Chapter 1.3.1. --- Laser Light Scattering --- p.4 / Chapter 1.3.2. --- Size Exclusion Chromatography --- p.5 / Chapter 1.3.3. --- Viscometry --- p.6 / Chapter 1.4. --- Polysaccharide analysis using MALDI-TOF-MS --- p.7 / Chapter 1.5. --- Matrix-Assisted Laser Desorption／Ionization Mass Spectrometry --- p.9 / Chapter 1.6. --- Outline of project --- p.13 / Chapter CHAPTER TWO --- INSTRUMENTATION AND EXPERIMENTAL / Chapter 2.1. --- Instrumentation --- p.14 / Chapter 2.1.1. --- Vacuum system --- p.14 / Chapter 2.1.2. --- Laser-based Ion Source --- p.14 / Chapter 2.1.3. --- Time-of-flight Mass Analyzer --- p.18 / Chapter 2.1.4. --- Detector and Data System --- p.22 / Chapter 2.2. --- Experimental --- p.22 / Chapter 2.2.1. --- Extraction of water-soluble and water-insoluble Curdlan --- p.22 / Chapter 2.2.2. --- Fractionation of water-soluble Curdlan samples --- p.23 / Chapter 2.2.3. --- Fractionation of water-insoluble Curdlan samples --- p.23 / Chapter 2.2.4. --- Phenol-sulfuric acid test --- p.24 / Chapter 2.2.5. --- Sample preparation in MS --- p.24 / Chapter 2.2.6. --- Calibration of MALDI-TOFMS --- p.24 / Chapter 2.3. --- Data Analysis --- p.25 / Chapter CHAPTER THREE --- OPTIMIZATION OF MALDI EXPERIMENTAL CONDITIONS FOR ANALYSIS OF CURDLAN / Chapter 3.1. --- Introduction --- p.27 / Chapter 3.2. --- Experimental --- p.28 / Chapter 3.2.1. --- Sample preparation method for analysis of water-soluble Curdlan --- p.29 / Chapter 3.2.2. --- Sample preparation method for analysis of water- insoluble Curdlan --- p.29 / Chapter 3.2.2.1 --- Aqueous DHB matrix --- p.30 / Chapter 3.2.2.2 --- Aqueous DHB/NH4F matrix --- p.30 / Chapter 3.2.2.3 --- Non-aqueous DHB matrix (in DMSO) --- p.30 / Chapter 3.2.2.4 --- Non-aqueous DHB/3AQ matrix (in DMSO) --- p.30 / Chapter 3.2.2.5 --- Aqueous DHB/3AQ matrix --- p.31 / Chapter 3.2.3. --- Fractionation of the water-soluble and water-insoluble Curdlan --- p.31 / Chapter 3.3. --- Results and Discussion --- p.32 / Chapter 3.3.1. --- Development of matrix system for analysis of water- insoluble Curdlan --- p.35 / Chapter 3.3.2. --- MALDI analysis of water-soluble and water-insoluble Curdlans --- p.39 / Chapter 3.3.3 --- MALDI analysis of fractionated water-soluble and water- insoluble Curdlans --- p.42 / Chapter 3.4. --- Conclusion --- p.48 / Chapter CHAPTER FOUR --- "EVALUATION OF MW AND MWD OF DMSO- SOLUBLE CURDLAN BY MALDI-TOF-MS, UV- VIS, REFRACTOMETRIC AND GC-MS ANALYSIS" / Chapter 4.1. --- Introduction --- p.51 / Chapter 4.2. --- Experimental --- p.54 / Chapter 4.2.1. --- Gel permeation chromatography of water-insoluble Curdlan --- p.54 / Chapter 4.2.2. --- Matrix-assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry --- p.55 / Chapter 4.2.3. --- Ultraviolet Absorption Spectrometry (UV-VIS) --- p.55 / Chapter 4.2.4 --- Refractometry Analysis (RI) --- p.56 / Chapter 4.2.5 --- Gas Chromatography Mass Spectrometry (GC-MS) --- p.56 / Chapter 4.3. --- Results --- p.57 / Chapter 4.3.1 --- MALDI analysis --- p.57 / Chapter 4.3.2 --- Ultra-violet Absorption Spectrometry (UV-VIS) --- p.61 / Chapter 4.3.3 --- Refractometry Analysis (RI) --- p.62 / Chapter 4.3.4 --- Gas chromatography Mass Spectrometry (GC-MS) --- p.66 / Chapter 4.4. --- Discussion --- p.79 / Chapter 4.5 --- Conclusion --- p.83 / Chapter CHAPTER FIVE --- CONCLUDING REMARKS --- p.84 / REFERENCES --- p.86 / APPENDIX / Appendix A Chemical Structure of Curdlan --- p.95 / Appendix B Structure of Universal Matrices Used in MALDI-TOF- MS --- p.96 / "Appendix C Structure of Aniline Blue fluorochrome Sodium 4,4- [carbonyldis-(benzene-4,l-diyI)bis(imino)]bisbenzene- sulphonate" --- p.97 / Appendix D Structure of common internal standard for GCMS analysis --- p.97 / Appendix E Structure of Trim ethylsilylated reagent and Trimethylsilylated glucose --- p.98 / Appendix F The table of raw data for graph plotting in RI analysis. --- p.99 / Appendix G The table of raw data used for graph plotting in UV-VIS analysis. --- p.100 / Appendix H The table of raw data used for graph plotting in GC-MS analysis. --- p.101

Wong Ka-kei. / Thesis submitted in: December 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 123-139). / Abstracts in English and Chinese. / THESIS COMMITTEE --- p.i / ACKNOWNLEDGEMENT --- p.ii / ABSTRACT (ENGLISH VERSION) --- p.iii / ABSTRACT (CHINESE VERSION) --- p.v / LIST OF TABLES --- p.ix / ABBREVIATIONS --- p.xiii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Edible mushrooms --- p.1 / Chapter 1.1.1 --- Classification and terminology --- p.1 / Chapter 1.1.2 --- Mode of nutrition --- p.3 / Chapter 1.1.3 --- World consumption --- p.3 / Chapter 1.1.4 --- Nutritional values of edible mushroom --- p.6 / Chapter 1.1.5 --- Medicinal values of mushrooms --- p.7 / Chapter 1.2 --- Mushroom mycelium --- p.11 / Chapter 1.2.1 --- Uses and applications --- p.11 / Chapter 1.2.2 --- Submerged fermentation (SmF) --- p.12 / Chapter 1.2.3 --- Factors affecting the growth of mycelium in submerged fermentation --- p.14 / Chapter 1.2.3.1 --- Nutritional requirements - Carbon sources --- p.14 / Chapter 1.2.3.2 --- Nutritional requirements ´ؤ Nitrogen sources --- p.16 / Chapter 1.2.3.3 --- Nutritional requirements ´ؤ Minerals --- p.16 / Chapter 1.2.3.4 --- Environmental factors ´ؤ Temperature --- p.17 / Chapter 1.2.3.5 --- Environmental factors - Aeration --- p.17 / Chapter 1.2.3.6 --- Environmental factors - Agitation --- p.18 / Chapter 1.2.4 --- Optimization of growth of mycelium and production of EPS --- p.18 / Chapter 1.3 --- Mushroom polysaccharides --- p.21 / Chapter 1.3.1 --- Biologically active mushroom polysaccharides --- p.21 / Chapter 1.3.2 --- Chemical structures of mushroom polysaccharides --- p.21 / Chapter 1.3.2.1 --- β-glucans --- p.23 / Chapter 1.3.2.2 --- α-glucans --- p.25 / Chapter 1.3.2.3 --- Mannans --- p.26 / Chapter 1.3.2.4 --- Protein-bound polysaccharides --- p.26 / Chapter 1.3.2.5 --- Other heteroglycans --- p.28 / Chapter 1.4 --- Mushrooms under investigation --- p.28 / Chapter 1.4.1 --- Pleurotus tuber-regium (Fr.) Sing. (PTR) --- p.28 / Chapter 1.4.2 --- Agrocybe cylindracea (AC) --- p.30 / Chapter 1.4.3 --- Grifola frondosa (GF) --- p.31 / Chapter 1.5 --- Objectives and experimental design --- p.32 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.35 / Chapter 2.1 --- Source of mushroom mycelium --- p.35 / Chapter 2.2 --- Effect of different carbon sources on submerged fermentation --- p.37 / Chapter 2.2.1 --- Production of mycelium by submerged fermentation using 250 mL and 1L shake-flasks --- p.37 / Chapter 2.2.2 --- Scale-up production of mycelium of PTR using fermentor --- p.39 / Chapter 2.2.3 --- Concentration of dissolved oxygen in 250 mL and 1L shake-flasks. --- p.39 / Chapter 2.3 --- Isolation and fractionation of mushroom polysaccharides --- p.40 / Chapter 2.3.1 --- Isolation of exo-polysaccharides (EPS) from culture medium by ethanol precipitation --- p.40 / Chapter 2.3.2 --- Isolation of EPS from culture medium by ultra-filtration --- p.40 / Chapter 2.3.3 --- Hot water extraction of PTR mycelium --- p.41 / Chapter 2.3.4 --- Fractionation of HWE by fractional ethanol precipitation --- p.41 / Chapter 2.4 --- Chemical composition of HWE and EPS --- p.42 / Chapter 2.4.1 --- Phenol-sulphuric acid method --- p.42 / Chapter 2.4.2 --- Modified Lowry method --- p.43 / Chapter 2.4.3 --- Monosaccharide composition analysis of HWE and EPS --- p.43 / Chapter 2.4.3.1 --- Acid depolymerization --- p.43 / Chapter 2.4.3.2 --- Neutral sugar derivatization --- p.44 / Chapter 2.4.3.3 --- Determination of neutral sugar composition by gas chromatography (GC) --- p.45 / Chapter 2.4.3.4 --- Uronic acid content --- p.46 / Chapter 2.5 --- Structural studies of HWE and EPS --- p.47 / Chapter 2.5.1 --- High Pressure Liquid Chromatography (HPLC) --- p.47 / Chapter 2.5.2 --- Methylation study and gas chromatography- mass spectrometry (GC-MS) --- p.48 / Chapter 2.5.2.1 --- Preparation of dry dimethyl sulfoxide (DMSO) --- p.48 / Chapter 2.5.2.2 --- Preparation of methylsulfinyl methyl sodium (CH3SOCH2-Na+) --- p.48 / Chapter 2.5.2.3 --- Methylation --- p.49 / Chapter 2.5.2.4 --- Extraction of methylated polysaccharide --- p.49 / Chapter 2.5.2.5 --- Acid depolymerization and preparation of aditol acetate derivatives --- p.50 / Chapter 2.5.2.6 --- Determination of partially methylated alditol acetates (PMAAs) by gas chromatography-mass spectrometry (GC-MS) --- p.50 / Chapter CHAPTER 3 --- RESULTS AND DISCUSSION --- p.51 / Chapter 3.1 --- "Production of mycelium and EPS of PTR, AC and GF by submerged fermentation in 250 mL shake-flask with liquid medium containing different carbon sources" --- p.51 / Chapter 3.1.1 --- "Mycelial biomass production of PTR, AC and GF" --- p.51 / Chapter 3.1.2 --- "Production of EPS of PTR, AC and GF" --- p.57 / Chapter 3.1.3 --- "Characterization of EPS of PTR, AC and GF" --- p.62 / Chapter 3.1.3.1 --- Carbohydrate and protein content --- p.62 / Chapter 3.1.3.2 --- Monosaccharide composition --- p.67 / Chapter 3.1.4 --- Summary --- p.72 / Chapter 3.2 --- "Production of mycelium, EPS of PTR by submerged fermentation in 1L shake-flask and 8L fermentor with liquid medium containing different carbon sources" --- p.75 / Chapter 3.2.1 --- Mycelial production of PTR --- p.75 / Chapter 3.2.2 --- EPS Production of PTR --- p.80 / Chapter 3.2.3 --- Chemical characteristics of EPS of PTR --- p.83 / Chapter 3.2.3.1 --- Carbohydrate and protein content --- p.83 / Chapter 3.2.3.2 --- Monosaccharide composition --- p.85 / Chapter 3.2.4 --- Structural characteristics of EPS of PTR --- p.87 / Chapter 3.2.4.1 --- Molecular weight of EPS of PTR by HPLC --- p.87 / Chapter 3.2.4.2 --- Glycosyl linkages of EPS of PTR by GC-MS of PMAA --- p.90 / Chapter 3.2.5 --- Summary --- p.93 / Chapter 3.3 --- Hot water extraction of mycelium of PTR from the scale-up submerged fermentation in 1L shake-flask and 8L fermentor with liquid medium containing different carbon sources --- p.95 / Chapter 3.3.1 --- Yield of hot water extract (HWE) of mycelium of PTR --- p.95 / Chapter 3.3.2 --- Chemical characteristics of HWE of PTR --- p.101 / Chapter 3.3.2.1 --- Carbohydrate and protein content --- p.101 / Chapter 3.3.2.2 --- Monosaccharide composition --- p.104 / Chapter 3.3.3 --- Structural characteristics of HWE of PTR --- p.112 / Chapter 3.3.3.1 --- Molecular weight of HWE of PTR by HPLC --- p.112 / Chapter 3.3.3.2 --- Glycosyl linkages of HWE of PTR by GC-MS ofPMAA --- p.116 / Chapter 3.3.4 --- Summary --- p.119 / Chapter CHAPTER 4 --- CONCLUSIONS AND FUTURE WORKS --- p.120 / Chapter 4.1 --- Conclusions --- p.120 / Chapter 4.2 --- Future works --- p.121 / REFERENCES --- p.123

Le glycogène et l’amidon sont les formes de polysaccharides de réserve les plus répandues. Ils sont tous deux constitués de résidus de glucose liés en α-1,4, et branchés en α-1,6. Bien qu’ils se distinguent fortement par leurs propriétés physico-chimiques, l’amidon a évolué à partir d’un métabolisme du glycogène préexistant. Il est apparu après l’endosymbiose primaire du plaste qui a eu lieu il y a plus d’un milliard d’années entre une cellule eucaryote et une cyanobactérie. Il a été proposé que l’endosymbiose primaire du plaste ait impliqué la manipulation du métabolisme des polysaccharides de réserve par une bactérie intracellulaire obligatoire pathogène qui appartient à l’ordre des Chlamydiales. Afin de comprendre l'histoire évolutive des gènes du métabolisme du glycogène, et enquêter sur l’implication d’une bactérie de l’ordre des Chlamydiales dans l’endosymbiose plastidiale, nous nous sommes intéressés à l’évolution des polysaccharides chez les cyanobactéries et les Chlamydiales. Nous avons donc disséqué le fonctionnement de ce métabolisme chez Cyanobacterium sp. CLg1, une cyanobactérie de l’ordre des Chroococcales qui accumule simultanément de l’amidon et du glycogène. D’autres part, nous avons étudié l’évolution du métabolisme des polysaccharides de réserve chez les Chlamydiales. La plupart des enzymes du métabolisme du glycogène des Chlamydiales ont été caractérisées. Les résultats de la caractérisation renforcent notre théorie de ménage à trois et l’implication des Chlamydiales dans l’établissement de l’endosymbiose primaire du plaste. L’impact du métabolisme du glycogène des Chlamydiales sur l’évolution de celui des plantes et des animaux est discuté. / Glycogen and starch are the most commonly found forms of storage polysaccharides. They both consist solely of glucose residues linked and branched respectively through α-1,4 and α-1,6 glycosidic bonds. Although they considerably differ in their physicochemical properties, starch evolved in only a few steps in eukaryotes from the pre-existing eukaryotic glycogen metabolism. It appeared, after primary plastidial endosymbiosis which took place over one billion years ago between an ancestral cyanobacterium and a heterotrophic eukaryotic host. This endosymbiosis has been recently proposed in the “Ménage à Trois Hypothesis” (MATH) to have been triggered and facilitated through manipulation of glycogen metabolism by obligate intracellular bacteria pathogens, belonging to the order Chlamydiales. In order to understand the evolutionary history of glycogen metabolism genes, and to investigate the possible nature of the Chlamydiales involvement in primary plastid endosymbiosis, we probed the evolution of storage polysaccharide metabolism in both extant Chroococcales and Chlamydiales. So we dissected the functioning of the metabolism in Cyanobacterium sp. CLg1, a cyanobacterium of order Chroococcales that simultaneously accumulate starch and glycogen. On the other hand, we investigated the evolution of storage polysaccharide metabolism in Chlamydiales. Several recombinant enzymes of glycogen metabolism were thus characterized. Our characterization strengthens the MATH and the implication of the Chlamydiales in the establishement of primary plastidial endosymbiosis. The importance of our findings with respect to the evolution of glycogen metabolism in animals and plants is discussed.

L'amidon et le glycogène définissent les deux polysaccharides de réserve les plus répandus dans le monde vivant. L'apparition de l'amidon chez les eucaryotes coïncide avec un événement d'endosymbiose unique qui s'est déroulé il y a 1,6 milliards d'années entre une cyanobactérie et un eucaryote primitif. De cet événement sont apparues trois lignées photosynthétiques : les Chloroplastida (plantes terrestres et algues vertes), les Rhodophyceae (algues rouges et organismes qui en sont dérivés par endosymbiose secondaire) et les Glaucophyta. La voie de biosynthèse de l'amidon chloroplastique chez les Chloroplastida est relativement bien caractérisée. Pour celle des Rhodophyceae (amidon cytoplasmique) des études ont déjà été entreprises mais les informations obtenues sont encore incomplètes et non représentatives de la lignée en général. Enfin pour les Glaucophyta, qui ont divergé de façon plus précoce après l'évènement d'endosymbiose aucune information sur le métabolisme de son amidon (cytoplasmique) n'était disponible. Afin d'appréhender la voie de biosynthèse utilisée par ces organismes nous avons entrepris la caractérisation de la voie métabolique de l'amidon de Cyanophora paradoxa (Glaucophyta) ainsi que celle de Chondrus crispus (Rhodophyceae). Ce manuscrit de thèse présente essentiellement des informations sur le métabolisme de l'amidon floridéen chez Cyanophora paradoxa, l'étude entreprise chez Chondrus crispus étant encore à l'état préliminaire. / Starch and glycogen both define the most wide-spread storage polysaccharide in the world. The starch appearance in the eukaryotic lineage coincides with a single endosymbiotic event occurring 1,6 billion years ago between a cyanobacteria and a primitive eukaryotic hosto From this event appeared three photosynthetic lines: Chloroplastida (land plants and green algae), Rhodophyceae (red algae and organisms which are diverted from the Rhodophyceae by secondary endosymbiosis) and Glaucophyta. The plastid starch biosynthesis pathway in Chloroplastida is relatively weIl characterized. For the Rhodophyceae (cytosolic starch) studies have already been initiated but the information obtained is still incomplete and not representative of the lineage. Finally for the Glaucophyta, which diverged in a more premature way after the event of endosymbiosis no information about this (cytosolic) starch metabolism was available. To discover the biosynthesis pathway used by these organisms, we decided to perform the complete characterization of the starch biosynthetic pathway for two differents organisms: The Glaucophyta named Cyanophora paradoxa and the Rhodophyceae named Chondrus crispus. This manuscript of PhD mainly presents information on the Floridian starch metabolism of Cyanophora paradoxa. The starch characterisation of Chondrus crispus is currently at the preliminary stage and will require further development.

The major components of wheat flour are keys to its functionality in processing and product quality. The major components, other than the lipids, are polymers: starch, protein, and non-starchy polysaccharides (NSP). In wheat NSP are primarily arabinoxylans (AX). These components are compartmentalized in the grain but are forced into close contact after the disruption caused by the milling process. These components further interact once water is added to the flour to create doughs and batters. It is these interactions and the water holding capacities of these polymeric components that are the unifying thread for most of this dissertation, other than the inclusion of nutritional traits in chapters 6 and 7. This dissertation consists of three independent studies, the last of which had two parts. Study one was "Effect of carbonate on co-extraction of arabinoxylans (AX) with glutenin macropolymer (GMP)". The aim of this study was to investigate if the level of AX in GMP increased under alkaline extraction conditions compared to extractions done in water. The amount of wet GMP obtained from alkaline extraction was greater than that from water extraction. Hard wheats had overall higher GMP wet weights than soft wheats. The level of AX in GMP extracted under alkaline conditions was greater than that in GMP extracted with water and the amount of increase was generally higher in soft wheats. Study two was "Optimization of water addition to noodle doughs". The aim of this study was to determine if a lubricated squeezing flow (LSF) technique could be useful in determination of optimum water addition to noodle doughs. Comparing the LSF method with alternative methods (Mixograph and sieving test), optimum water additions predicted by LSF for both salted and alkaline soft-wheat derived noodle doughs were equivalent or slightly higher than those predicted by the Mixograph and sieving test. For both salted and alkaline hard-wheat derived noodle doughs, optimum water additions predicted by the LSF method were substantial higher than those predicted by the Mixograph but equivalent or slightly higher than those predicted by the sieving test. Relaxation time was the most useful parameter in determining optimum water addition for the soft-wheat noodle doughs. The LSF method in its current form was found to be not adequate for all noodle types. Additional work with LSF parameters altered to improve sensitivity and with more of samples should be performed. Study three was "Determination of wheat quality and quantitative trait locus analysis". Part 1 was to measure a comprehensive set of quality phenotypes (including nutritional parameters) on a wheat population derived from the cross Tubbs [soft] x NSA98-0995 [hard]; (T x N). Part 2 was to identify if there were quantitative trait loci (QTL) associated with the traits determined in part 1. Considerable and transgressive segregation was observed for many of the studied traits. The transgressive segregation could useful, in that lines with superior soft-wheat quality can be identified that could be introduced quickly into the wheat breeding program from this elite x elite cross. Hardness index was significantly correlated with several important traits related to the solvent absorption capacity of the flour. Composite interval mapping detected a total of significant 28 QTLs on 10 wheat chromosomes for 15 end-use quality and nutrition traits in 2 harvest years. QTLs for total antioxidant activity (TAA) and total phenolic content (TPC) were identified for the first time. QTLs for TAA were on chromosomes 3B and 5BS, while the QTL for TPC was on chromosome 7AC. Hybridization between Tubbs and NSA surprisingly produced superior soft-wheat quality with potentially higher in nutritional values. The QTLs identified in this study could be useful in marker-assisted selection for future pre-selection of progeny from Tubbs or NSA. / Graduation date: 2012

The nutritional value of distillers dried grains with solubles (DDGS) has not been assessed in swine. The nutritional value of corn and wheat DDGS, and possibilities to improve the nutritional value of wheat DDGS were for swine were investigated in two studies. <p>In study 1, two experiments were conducted to determine digestibility and digestible contents of energy, amino acids (AA) and P in corn and wheat DDGS and wheat grain, together with N and P excretion and growth performance in grower-finisher pigs. In experiment 1, 12 barrows (64.6 ± 6.4 kg) were fitted with ileal T-cannulae and had restricted access (2.6 x maintenance) to a wheat control diet or one of three diets with 40% corn, wheat+corn (4:1) or wheat DDGS. For energy, apparent total tract digestibility was highest for wheat (85%; P < 0.05) and did not differ among DDGS (77 to 79%; P > 0.10). Total tract digestible energy (DE) was highest for corn DDGS (4292 kcal kg-1 DM; P < 0.05) and tended to differ among wheat+corn and wheat DDGS and wheat (4038, 4019, and 3807, respectively; P = 0.06). For lysine, apparent ileal digestibility (AID) was highest for wheat (71%; P < 0.05) and did not differ among DDGS (59 to 63%; P > 0.10). The apparent ileal digestible lysine content was highest for corn DDGS (0.51% DM; P < 0.05), intermediate for wheat+corn and wheat DDGS (0.45 and 0.42), and lowest for wheat (0.37%). For P, total tract digestibility was lowest for wheat (15%; P < 0.05) and did not differ among DDGS samples (53 to 56%; P > 0.10). Total N excretion was highest for wheat+corn and wheat DDGS (55 and 58 g d-1; P < 0.05), intermediate for corn DDGS (44) and lowest for wheat (36). Total P excretion did not differ among DDGS (11 g d-1) and was lowest for wheat (8; P < 0.05). In experiment 2, 100 pigs (52.0 ± 3.3 kg) were fed a wheat-pea control diet or one of three 25%-DDGS (corn, wheat+corn or wheat) diets (3.375 Mcal DE kg-1; 2.50 g AID lysine Mcal-1 DE) for 5 wk. Overall, average daily feed intake (ADFI) and daily gain (ADG) were higher for wheat than DDGS (P < 0.05) but feed efficiency did not differ (P > 0.10). In summary, the nutritional value of wheat DDGS for swine is higher than wheat and lower than corn DDGS and feeding DDGS reduced growth performance, partly via a reduced ADFI, indicating that anti-nutritional factors in DDGS require further investigation.<p>In study 2, the effect of xylanase supplementation of wheat DDGS on nutrient digestibility and nutrient excretion was evaluated in grower-finisher pigs. Wheat-based diets with or without 40% wheat DDGS were tested with or without supplementary xylanase (4,000 U kg-1 feed) as a 2 x 2 factorial arrangement in a repeated Latin square design using eight barrows (29.4 ± 2.0 kg) fitted with ileal T-cannulae. Following a 6-day acclimation, faeces and urine were collected for 3 d, and ileal digesta for 2 d. The apparent ileal energy digestibility and DE content were not affected either by ingredient or xylanase (P > 0.05). The total tract energy digestibility and DE content were affected by ingredient (P > 0.05), but not by xylanase (P > 0.05). The total-tract energy digestibility was higher for wheat, but DE content was higher for wheat DDGS. The AID of arginine, isoleucine, leucine, phenylalanine, threonine, tryptophan and total AA were higher (P < 0.05), and of cysteine, histidine and lysine were similar (P > 0.05), and SID of phenylalanine was higher (P < 0.05), and of the other AA was similar (P > 0.10) for wheat DDGS compared to wheat. Supplementary xylanase improved AID and SID of most of the indispensable AA in wheat (P < 0.05), but not in wheat DDGS (P > 0.05). The apparent and standardized ileal AA contents were affected by ingredients (P < 0.05), but not by xylanase (P > 0.05). Digestible AA contents were higher for wheat DDGS than for wheat. The digestibility and digestible content of P were affected by ingredient and xylanse (P < 0.05). The P digestibility and digestible P contents were higher for wheat DDGS compared to wheat. Neither ingredient nor supplementary xylanase affected DM intake (P > 0.05). The DM excretion on daily basis and as a percentage of intake were affected by ingredient (P < 0.05), but not by xylanase (P > 0.05). Ingredients affected all N and P variables (P < 0.05), except percentage retained for both nutrients (P > 0.05). None of N variables (P > 0.05), but P intake and, retention on daily basis and as a percentage of intake were affected by xylanase (P < 0.05). The DM excretion and N and P intake, excretion and daily retention were higher for wheat DDGS compared to wheat. Lack of beneficial response to supplementary xylanase might be due to inappropriate enzyme level or insufficient substrate level of wheat DDGS. In addition, unidentified factors associated with fermentation and drying processes might constrain the nutritional value of wheat DDGS. Further studies are required to determine the proper xylanase inclusion level and/or to identify the factors associated with reduced nutrient digestibility of wheat DDGS.