This thesis describes the application of mass spectrometry (MS) to glycoprotein and oligosaccharide analysis. Glycosylated proteins are involved in cell-cell and cell-matrix recognition. Applications of trypsin and proteinase K to hydrolyze glycoproteins into glycopeptides that are compatible with MS and MS/MS analysis are investigated. For successful site-specific analysis of glycans, glycopeptides with short peptide (3-8 residues) are needed. Although trypsin is an important enzyme for protein identification, proteinase K is superior for site-specific glycan analysis due to its potential to hydrolyze every glycoprotein to short glycopeptides. The gas-phase dissociation pathways, kinetics and energetics of protonated oligosaccharides are described. The oligosaccharides dissociate via cleavage at the glycosidic linkages during thermal activation. Using double resonance experiments, it was established that oligosaccharides undergo sequential and parallel fragmentation reactions. Furthermore, dissociation of product ions to secondary ions was confirmed. Arrhenius activation parameters, Ea and A for protonated alpha- and beta-linked D-glucopyranose oligosaccharides are reported.

Heparan sulfates (HSs) are a class of anionic carbohydrate chains found at cell surfaces and in the extracellular matrix where they interact with a number of proteins. HS is characterized by extreme structural heterogeneity, and has been implicated in a number of biological phenomenon like embryogenesis, morphogen gradient formation and signalling of growth factors such as FGF, PDGF etc. Despite the characteristic structural heterogeneity, evidence from compositional studies show that the HS structure is expressed in a tightly regulated manner, implying a functional significance, which is most likely in the modulation of cell behaviour through HS-protein interactions. The lack of molecular tools has, however, hampered the understanding of HS structures with functional significance. This work therefore aims at characterizing the structural requirements on HS involved in the interaction with the anti-HS phage display antibodies HS4C3, AO4B08 and HS4E4 and a selected growth factor PDGF-BB. The characterization was done with the help of tailored oligosaccharide libraries generated from sources bearing structural resemblance to HS. The work has thus made available tools that preferentially recognize certain structural features on the HS chain and will aid in the further study of HS structure and its regulation. Evidence is also provided to support the notion that HS protein interactions can occur in multiple manners, utilizing any of the structural features on the HS chain.

La fibrose kystique (FK) est une maladie génétique causée par la mutation du gène codant pour la protéine CFTR (Cystic Fibrosis Transmembrane conductance Regulator). Celle-ci présente un défaut sur le canal à ions chlorure affectant ainsi entre autres la viscosité des muqueuses au niveau du système respiratoire. Cet environnement est alors propice aux colonisations bactériennes opportunistes sous la forme de biofilm. Burkholderia cepacia, bacille Gram-négatif mobile, multi-résistant aux antibiotiques et hautement transmissible, s'avère d'une extrême virulence pour les patients atteints de FK. Cette bactérie pathogène désigne en fait un ensemble de neuf souches rassemblées sous le nom de « complexe B. cepacia » (CBC). Au moins huit de ces neuf souches produisent un exopolysaccharide nommé Cepacian. Ceci est constitué d'un motif de répétition heptasaccharidique composé notamment de l'enchaînement α-D-Rhap-(1→4)-α-D-GlcpA. Le D-rhamnose (ou 6-deoxy-D-mannose) est un sucre rare et un composant de glycoconjugués des parois de bactéries pathogènes mais est absent chez l'homme. Ce dernier est donc un excellent candidat antigénique dans le cadre de la préparation d'un vaccin entièrement synthétique et spécifique. L'élaboration de celui-ci consiste en un activateur universel immunogénique peptidique (Tc-épitope) et/ou protéique (semi-synthétique) fonctionnalisé par une unité antigénique spécifique. Dans ce but, un tri-O-saccharide de constituant de LPS de B. cepacia et composé majoritairement de D-rhamnose, ainsi que des fragments du motif répétition de l'exopolysaccharide du CBC ont été synthétisés. Des méthodes de synthèses orthogonales ont été optimisées avec de hauts rendements sur les cinq types de sucres du CBC, spécifiquement le D-rhamnose, et l'acide glucuronique ainsi qu'une série de glycosylations. La synthèse linéaire en 18 étapes conduit à l'α-D-Rhap-(1→4 )-α-D-Galp-(1→3)-α-D-Rhap avec 11.3% de rendement global. La dernière étape sera la conjugaison de Tc-épitope par ce dernier afin d'obtenir un vaccin entièrement synthétique. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Vaccin synthétique, T-cell épitope, Burkholderia cepacia, Fibrose kystique, Oligosaccharide, D-rhamnose, Complexe B. cepacia.

Three experiments were conducted to evaluate the impacts of removing antibiotic growth promoters (AGP) on broiler performance and yield and to evaluate alternative products as potential replacements. In experiment one, approximately 552,000 broilers were reared in four solid-wall, tunnel ventilated houses that were divided into two paired-house facilities, each assigned one of two dietary treatments. The treated group received basal diets containing salinomycin (SAL), roxarsone (ROX) and AGP, while the control group received the same diets without ROX and AGP. Removal of ROX and AGP had no affect on average body weight and feed efficiency, while livability was significantly affected negatively by the removal of ROX and AGP. Tender, wing, drum and percentage of total white meat showed significant improvements in yield during the study, while all other parts were not affected by removal of ROX and AGP. In experiment two, an investigation was conducted to evaluate the effects on performance from feeding Bacillus subtilis spores (Gallipro®, Chr Hansen A/S, Denmark), as a direct-fed microbial additive, to commercial broiler chickens. Birds were divided among two paired-house facilities. The treatment group received basal diets supplemented with B. subtilis spores, while the control group was fed the same basal diets containing an AGP. Feed conversion ratio was significantly lower for the treatment group, while average body weight, coccidiosis lesion scores, and footpad scores were not affected by the treatments. In experiment three, 6,000 broiler chickens were equally divided among four treatment groups and reared to 49 d to determine the effectiveness mannan oligosaccharides (MOS, Bio-Mos®, Alltech, Nicholasville, Kentucky, USA) as an alternative for an AGP program and MOS plus Natustat™ (NAT, Alltech, Nicholasville, Kentucky, USA) as an alternative to an enteric health program (AGP+anticoccidial drug). Average body weight for the control (CON) and antibiotic (ANT) groups was significantly different from the MOS+NAT group, but not the MOS group. Carcass front half, carcass hind half, frame and skin yields were improved for all treatments when compared to the MOS+NAT group. Conversely, percent total white meat yield was improved with the inclusion of MOS when compared to the ANT group. The findings of this research suggest that the removal of AGP from the diets of commercial broiler chickens does not affect the performance and yield of the birds over a one year production period. Furthermore, B. subtilis spores and mannan oligosaccharides provide acceptable alternatives to an AGP program.

Extraction of fructo-oligosaccharide syrups from grated jerusalem artichoke (JA) tubers was studied by water at 20-60&deg / C by determining the yield, degree of polymerization (DP), product profile (DP of up to 6) and prebiotic effect using Lactobacillus plantarum on samples harvested between October and April, stored for 0-20 days. The optimum solvent to solid ratio was 4, the duration of shaking water bath extraction was 40 min and yield based on JA were 12-17%. Temperature was found to improve yield and functionality, and citric acid, at 26 mM, improved the color and darkness by 70 and 80%, respectively. Short-time (1 min) microwaving prior to extraction increased the yield by about 20%, decreased the amount of sugars with DP 1 and 2 and increased the amounts of oligosaccharides (OS) with DP 3-6, although the prebiotic effect increased only slightly / while the color and darkness of the syrup were tripled. Ultrasound-assisted-extraction (USE) gave best performance at 3 min duration / decreased the amounts of sugars with DP 1-2, increased the amounts of OS with DP 3-6, with 18% decrease in the yield. The better functionality of USE syrups were also indicated by 2.5 times higher growth rate of L.plantarum. The application of USE at 60&deg / C compared to 20&deg / C almost tripled the amounts of functional sugars. In order to obtain the largest proportion of monosaccharide units as functional sugars, 10 day storage at 4&deg / C after harvest was indicated. Ultrasonication did not affect the color but the darkness was doubled. The density and viscosity of all the syrups were practically the same.

Phosphorylated molecules play vastly important roles in the environment and biological systems. The main focus of this work sought to expand the current collection of synthetic phosphate receptors to include the binding of oligosaccharide phosphates. To this end, the synthesis of a boronic acid - appended porphyrin whose selectivity could be tuned through nucleic acid selection was pursued through a number of synthetic routes. Though difficulties were encountered with synthetic reactivities and product solubility, these efforts culminated in the development of a bis-boronic acid-substituted porphyrin soluble in DMSO/water mixtures that displays fluorescence quenching upon the addition of specific saccharide derivatives. In efforts to ease the synthetic burden, the creation of a DNA-based self-assembled receptor system was also investigated. Further, this work included the synthesis of phosphorylated derivatives of gemcitabine, a nucleoside chemotherapeutic drug, with the goal of developing targeted delivery systems for the improved treatment of cancer. Progress in each of these areas is discussed. / text

No description available.

The objective of these studies was to evaluate the effect of supplying mannan oligosaccharides (MOS) to pig diets on both weanling pig and sow performances. Nursery pigs challenged with LPS to stimulate an immune challenge had increased body temperature and respiratory rate as well as elevated serum cortisol and TNF-α concentration, MOS-supplemented pigs had a lower rectal temperature and respiratory rate which implied that MOS improves some aspect of the immune function of piglets. MOS supplementation in sow diets during late gestation and lactation had no effect on litter size, but the piglets from MOS-fed sows were heavier at birth (P = 0.04), at weaning (P = 0.03), and during the entire nursery period (P < 0.01). Moreover, milk fat and protein levels as well as the Ig concentrations in milk from MOS-fed sows were numerically higher (2 – 12%; P > 0.10) than control sows. Overall, MOS supplementation in the nursery diet may have limited effects on the growth performance, but may have some beneficial influence on pigs under immune challenge. Furthermore, including MOS to the sow diet during late gestation and lactation can potentially improve piglet body weight as well as growth during the suckling and nursery periods.

The glycosylation pattern of a chimeric heavy chain antibody (EG2) produced from CHO cells was affected by the glucose concentration (0-25 mM) of cultures established at high density (>106 cells/ml) over 24 h. The resulting proportion of non-glycosylated Mab was directly correlated to the exposure time of cells to media depleted of glucose. Deprivation of glucose for the full 24 h resulted in a 52% non-glycosylated Mab fraction. Analysis of steady state levels of intracellular lipid-linked oligosaccharides (LLOs) showed that under glucose limitation there was a reduction in the amount of full length LLO (Glc3Man9GlcNac2), with a concomitant increase in the smaller mannosyl-glycans (Man2-5GlcNAc2). Glycan microheterogeneity was quantified by galactosylation and sialylation indices (GI and SI), which showed a direct correlation to the cell specific glucose uptake. These findings are important in relation to the low substrate that may occur in fed-batch cultures for Mab production.

Orientador: Maria Cristina Thomaz / Banca: Jane Maria Bertocco Ezequiel / Banca: Rafael Neme / Resumo: Foram conduzidos três experimentos, com o objetivo de avaliar os efeitos da adição de diferentes níveis de prebiótico na alimentação de leitões, sobre o desempenho, desenvolvimento da mucosa intestinal, parâmetros fisiológicos, digestibilidade e metabolismo de suínos. O prebiótico utilizado foi o mananoligossacarídeo MOS. No 1º experimento, foram avaliados o desempenho e a incidência de diarréia em 30 leitões fêmeas, dos 22 aos 63 dias de idade, com peso inicial de 6,28 l 1,40 kg. A utilização de MOS nas rações de leitões recém - desmamados piorou o ganho de peso dos animais dos 22 aos 63 dias de idade, porém não teve efeito sobre a incidência de diarréia. No 2º experimento foram avaliadas a morfometria do intestino delgado e mensurados os pesos do fígado e do pâncreas, assim como, o pH dos conteúdos do estômago, intestino delgado e ceco. Foram utilizados 15 leitões fêmeas dos 22 aos 63 dias de idade, com peso inicial de 5,74 l 0,93 kg. Em ambos os experimentos, os tratamentos foram: Controle positivo CP (Dieta basal + antibiótico); Controle negativo - CN (Dieta basal); CN + 0,25% de Prebiótico; CN + 0,50% de Prebiótico; CN + 0,75% de Prebiótico, e utilizou-se o delineamento em blocos ao acaso para controlar diferenças inicias de peso. Não foram observadas diferenças em nenhum dos parâmetros avaliados. No 3º experimento foram determinadas as digestibilidades dos nutrientes e da energia e o coeficiente de metabolizabilidade da energia e a energia metabolizável, das rações controle e com 0,5% de prebiótico. Utilizou-se 16 suínos machos castrados, com peso médio de 24,8 l 2,79 kg, e os blocos controlaram a repetição no tempo...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Three experiments were set to evaluate the effects of adding different levels of mannanoligossacharides (MOS, prebiotic) to the diet of piglets on their performance, development of intestinal mucosa, physiological parameters, digestibility and metabolism.. Corn and soybean meal were the major feed components. The first experiment evaluated the development and occurrence of diarrhea in 30 female piglets at 22 and 63 days of age, with an initial weight of 6.28 l 1.40 kg. Prebiotic additions to the diets worsen the growth performance of animals in Phase 3, but did not influence on the diarrhea incidence. The second experiment evaluated the morphometry of the small bowel, liver and pancreas weight, and the pH of stomach, small bowel and cecum contents. Fifteen female piglets were used at 22 and 63 days of age, with an initial weight of 5.74 l 0.93 kg. Both experiments were arranged in randomized blocks design to control initial weight difference, with the following treatments: positive control PC (base diet + antibiotic); negative control - NC (base diet); NC + 0.25% of prebiotic; NC + 0.50% of prebiotic; NC + 0.75% of prebiotic. No significant difference was observed in any of the studied parameters. In the third experiment, nutrient and energy digestibility were determined in feeds containing prebiotic (0.5%) or not (control). Sixteen castrated male pigs weighing 24.8 l 2.79 kg were used, and randomized blocks design was used so blocks controlled replications during time. Nutrient digestibility and feed energy were determined through the total feces collection method, using ferric oxide as fecal marker. The digestibility coefficient of the mineral matter and the digestible mineral matter were higher (P<0.05) in the control feed than in the feed containing prebiotic...(Complete abstract, click electronic address below) / Mestre