Les interactions entre protéines et polysaccharides dépendent des conditions environnementales et de leurs propriétés intrinsèques. La cosolubilité, la complexation et l’incompatibilité en sont le résultat. La complexation et l’incompatibilité ont démontré une amélioration des propriétés fonctionnelles de systèmes mixtes comparativement aux biopolymères pris individuellement. L’incompatibilité étant souvent la règle, cette recherche a pour but d’approfondir les connaissances sur les propriétés fonctionnelles des systèmes mixtes protéines-polysaccharides en conditions de compatibilité ou d’interactions associatives et de caractériser les nouvelles fonctionnalités qui en découlent. Un premier système isolat de protéines de lactosérum-xanthane a été étudié pour ses aptitudes à la gélification en conditions de compatibilité induites suite à une variation de pH et du ratio protéines-polysaccharides. Suivant l’application d’un traitement thermique, la solution est passée de compatible à incompatible. Les gels démontraient une augmentation du module élastique (G’) due à l’incompatibilité telle qu’observée par microscopie confocale à balayage laser. L’ajout de NaCl a augmenté cette incompatibilité et l’a rendue excessive au-delà d’une concentration critique entraînant une chute du G’. La compatibilité a ensuite été étudiée sur un système isolat de protéines de lactosérum-pectine. Suite à une variation de pH, de la concentration en biopolymères et du ratio protéines-polysaccharides, des conditions de compatibilité ont été confirmées par les mesures d’absorbance et du nombre de charges. Cette compatibilité a mené à une diminution de la viscosité en solution diluée due à la formation de complexes solubles alors qu’en solution concentrée, la complexation l’a plutôt augmentée. Un système modèle de yogourt ferme a finalement été étudié suite à l’incorporation de isolat de protéines de lactosérum et de pectine préalablement complexés et stabilisés. Les concentrations en protéines et en solides totaux ont été maintenues constantes. Les mélanges laitiers ont été acidifiés au glucono-delta-lactone. Les résultats démontrent que l’incorporation de complexes à différentes concentrations entrave la formation d’un réseau protéique homogène provoquant une diminution de la fermeté du gel et une augmentation de la synérèse. Les observations microscopiques appuient ces résultats. Les solutions mixtes permettent de développer de nouvelles propriétés fonctionnelles. Cependant, une meilleure connaissance de ces mélanges est nécessaire pour en arriver à des propriétés fonctionnelles variées et précises dans les formulations alimentaires. / Protein polysaccharide interactions depend on both environmental conditions and intrinsic properties. Results are co-solubility, complexation and incompatibility. Complexation and incompatibility have demonstrated improvement of functional properties of mixed systems compared to those of the individual components. Incompatibility being the rule, the aim of this study is to widen knowledge on functional properties of mixed protein-polysaccharide systems in presence of compatibility or associative interactions and to characterise the new emerging functional properties. A first mixed system of whey protein isolate-xanthan has been studied for its gelling abilities following pH and protein-polysaccharide ratio variations. Following application of a thermal treatment, the solution passed from compatible to incompatible. Gelation was demonstrated by an increase in elastic modulus (G’) due to incompatibility as observed by confocal laser scanning microscopy. Addition of NaCl increased this incompatibility and made it excessive at a certain critical concentration leading to loss in G’. Compatibility has then been studied on a whey protein isolate-pectin system. Varying pH, biopolymer total concentration and protein-polysaccharide ratio allowed soluble complex formation which was confirmed with the measurement of absorbance and the number of charge. This compatibility led to a decrease in viscosity in diluted solution due to soluble complex formation while in concentrated solution, complexation rather increased it. A model system of firm yogurt was finally studied following incorporation of whey protein isolate and pectin first complexed and stabilised. Protein and total solid concentrations were kept constant. Milky solutions were acidified with glucono-delta-lactone. Results demonstrate that incorporation of complexes at different concentrations hampers the formation of a homogenous protein network provoking a decrease in gel stiffness and an increase in syneresis. Microscopic observations supported these conclusions. Mixed solutions prepared by carefully condunting complex formation allow the design of new functional properties. However, a better knowledge of these mixes is necessary in order to achieve varied and precise functional properties in food formulation.

by Liu Chi. / "July, 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 270-283). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Thesis (PhD (Microbiology))--University of Stellenbosch, 2005. / Biomass is the sole foreseeable sustainable source of organic fuels, chemicals and materials. It is a rich and renewable energy source, which is abundant and readily available. Primary factors motivating the use of renewable enrgy sources include the growing concern over global climate change and the drastic depletion of non-renewable resources. Among various forms of biomass, cellulosic feedstocks have the greatest potential for energy production from. The biggest technological obstacle to large-scale utilisation of cellulosic feedstocks for the production of bioethanol as a cost-effective alternative to fossil fuels is the general absence of low-cost technology for overcoming the recalcitrance of cellulosic biomass. A promising strategy to overcome this impediment involves the production of cellulolytic enzymes, hydrolysis of biomass and fermentation of resulting sugars to ethanol in a single process step via a single microorganism or consortium. Such “consolidated bioprocessing” (CBP) offers very large cost reductions if microorganisms, such as the yeast Saccharomyces cerevisiae, can be developed that possess the required combination of efficient cellulose utilisation and high ethanol yields. Cellulose degradation in nature occurs in concert with a large group of bacteria and fungi. Cellulolytic microorganisms produce a battery of enzyme systems called cellulases. Most cellulases have a conserved tripartite structure with a large catalytic core domain linked by an O-glycosylated peptide to a cellulose-binding domain (CBD) that is required for the interaction with crystalline cellulose. The CBD plays a fundamental role in cellulose hydrolysis by mediating the binding of the cellulases to the substrate. This reduces the dilution effect of the enzyme at the substrate surface, possibly by helping to loosen individual cellulose chains from the cellulose surface prior to hydrolysis. Most information on the role of CBDs has been obtained from their removal, domain exchange, site-directed mutagenesis or the artificial addition of the CBD. It thus seems that the CBDs are interchangeable to a certain degree, but much more data are needed on different catalytic domain-CBD combinations to elucidate the exact functional role of the CBDs. In addition, the shortening, lengthening or deletion of the linker region between the CBD and the catalytic domain also affects the enzymatic activity of different cellulases. Enzymes such as the S. cerevisiae exoglucanases, namely EXG1 and SSG1, and the Saccharomycopsis fibuligera β-glucosidase (BGL1) do not exhibit the same architectural domain organisation as shown by most of the other fungal or bacterial cellulases. EXG1 and SSG1 display β-1,3-exoglucanase activities as their major activity and exhibit a significant β- 1,4-exoglucanase side activity on disaccharide substrates such as cellobiose, releasing a free glucose moiety. The BGL1 enzyme, on the other hand, displays β-1,4-exoglucanase activity on disaccharides. In this study, the domain engineering of EXG1, SSG1 and BGL1 was performed to link these enzymes to the CBD2 domain of the Trichoderma reesei CBHII cellobiohydrolase to investigate whether the CBD would be able to modulate these non-cellulolytic domains to function in cellulose hydrolysis. The engineered enzymes were constructed to display different modular organisations with the CBD, either at the N terminus or the C terminus, in single or double copy, with or without the synthetic linker peptide, to mimic the multi-domain organisation displayed by cellulases from other microorganisms. The organisation of the CBD in these recombinant enzymes resulted in enhanced substrate affinity, molecular flexibility and synergistic activity thereby improving their ability to act and hydrolyse cellulosic substrates, as characterised by adsorption, kinetics, thermostability and scanning electron microscopic (SEM) analysis. The chimeric enzyme of CBD2-BGL1 was also used as a reporter system for the development and efficient screening of mutagenised S. cerevisiae strains that overexpress CBD-associated enzymes such as T. reesei cellobiohydrolase (CBH2). A mutant strain WM91 was isolated showing up to 3-fold more cellobiohydrolase activity than that of the parent strain. The increase in the enzyme activity in the mutant strain was found to be associated with the increase in the mRNA expression levels. The CBH2 enzyme purified from the mutant strain did not show a significant difference in its characteristic properties in comparison to that of the parent strain. In summary, this research has paved the way for the improvement of the efficiency of the endogenous glucanases of S. cerevisiae, and the expression of heterologous cellulases in a hypersecreting mutant of S. cerevisiae. However, this work does not claim to advance the field closer to the goal of one-step cellulose processing in the sense of technological enablement; rather, its significance hinges on the fact that this study has resulted in progress towards laying the foundation for the possible development of efficient cellulolytic S. cerevisiae strains that could eventually be optimised for the one-step bioconversion of cellulosic materials to bioethanol.

Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Cellulose and hemicellulose represents the two most abundant groups of renewable polysaccharides known to man. Apart from their presence in plant material, they also contribute to a significant portion of inexpensive readily available material, such as wastes and bypro ducts from forestry / agricultural origin. The chemical composition of plant material varies, but the biomass content consists of approximately 75% carbohydrate polymers (cellulose and hemicellulose) and 25% lignin. The enzymes required for the degradation of cellulose and hemicellulose are collectively called cellulases and hemicellulases. These enzymes have a broad spectrum of industrial applications including the production of fuel ethanol through fermentations, reducing the amount of chlorine required for bleaching in the pulp and paper industry, increasing dough volume in the baking industry, improving digestion and nutritional value of animal feed, increasing clarification and enhancing the filterability of wine, beer and fruit juice, etc. Therefore, a large potential market exists for cellulases and hemicellulases provided their production is economical and the product, authentic. Aspergilli occur in a wide variety of habitats including soil, stored food and feed products and decaying vegetation. The advantages for using A. niger as host for heterologous enzyme production include good protein secretion, industrial fermentation technology dating as far back as 1919, being a non-pathogenic fungus with GRAS status, no special substrate or cultivation requirements, FDA approval of numerous enzymes (homologous and heterologous) produced, etc. In this study an Aspergillus expression vector was constructed using the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (gpdp) of A. niger and the glucoamylase terminator (glaAT) of Aspergillus awamori. The cDNA copies of the eg! and xyn2 genes of Trichoderma reesei, cbhl-4 of Phanerochaete chrysosporium, man! of Aspergillus aculeatus and xyn3 of Aspergillus kawachii were introduced into the expression vector, respectively. All the plasmids were co-transformed with plasmid p3SR2 to A. niger and transformants selected for stable plasmid integration into the genome of the host. The recombinant enzymes EgI, Xyn2, Cbhl-4, Man! and XynC were successfully expressed and secreted at activity levels of 2300, 8000, 500, 6000 and 900 nkatlml, respectively. The enzymes were produced as functional entities and were subsequently characterized. The EgI, Xyn2 and ManI were evaluated as feed additives for the possible use in the animal feed industry. Improved biomass gain was observed with in vivo studies on poultry. With the possible mass production of heterologous enzymes in mind, a simple medium had to be devised for their inexpensive production. Molasses medium (available from the South African sugar industry) was therefore evaluated and the cultivation conditions optimized for it's possible use as cultivation substrate for A. niger. The evaluation was done on the grounds of EgI and Xyn2 activity produced which was monitored over time. This study highlighted the possible use of A. niger for the heterologous production of enzymes, the use of industrial substrate for cultivation and paved the way for the high level expression of industrially important genes at low cost and a positive environmental impact. / AFRIKAANSE OPSOMMING: Sellulose en hemisellulose verteenwoordig die twee vollopste herwinbare polisakkariede bekend. Behalwe vir hul teenwoordigheid in plantmateriaal, dra hulle ook by tot 'n beduidende fraksie van goedkoop, maklik bekombare materiaal soos afval- en byprodukte van bosbou I landbou oorsprong. Soos te verwagte, varieër die chemiese samestelling van die plantmateriaal, maar die biomassa-inhoud bestaan uit naastenby 25% lignien en 75% koolhidraatpolimere (sellulose and hemicellulose). Die ensieme benodig vir die afbraak van sellulose en hemisellulose staan gesamentlik as sellulases en hemisellulases bekend. Hierdie ensieme het 'n breë spektrum van industriële toepassings insluitende die produksie van brandstofalkohol d.m.v. fermentasies, vermindering in die hoeveelheid chloor benodig vir die bleikproses in die pulp-en-papier industrie, toename in deegvolume in die bakkersindustrie, verbetering van verteerbaarheid en verhoging van voedingswaarde van dierevoer, toename in verheldering en verbeterde filtreerbaarheid van wyn, bier en vrugtesap, ens. Dus bestaan daar 'n groot potensiële mark vir sellulases en hemisellulases, mits hul produksie ekonomies en die produk outentiek is. Aspergilli kom in 'n wye verskeidenheid van omgewings voor, insluitende grond, gestoorde voedsel- en voerprodukte asook ontbindende plante materiaal. Die voordele vir die gebruik van A. niger as gasheer vir heteroloë ensiemproduksie sluit in 'n goeie proteïen produseerder, industriële fermentasietegnologie dateer sover terug as 1919, 'n nie-patogeniese fungus met GRAS-status, benodig geen spesiale substrate of kwekingskondisies nie, FDA goedkeuring vir 'n groot aantal ensieme (homoloog sowel as heteroloog) wat reeds geproduseer word, ens. In hierdie studie is 'n Aspergillus uitdrukkingsvektor gekonstrueer deur van die konstitutiewe gliseraldehied-3-fosfaat dehidrogenase promoter (gpdp) van A. niger en die glukoamilase termineerder (glaAT) van Aspergillus awamori gebruik te maak. Die cDNA kopiee van die die eg! en xyn2 van Trichoderma reesei, cbhl-4 van Phanerochaete chrysosporium, man! van Aspergillus aculeatus en die xynC van Aspergillus kawachii was onderskeidelik na die uitdrukkingsplasmied oorgedra. Alle plasmiede is gesamentlik met die p3 SR2 plasmied na A. niger getransformeer en vir stabiele integrasie in die gasheergenoom geselekteer. Die rekombinante ensieme Egl, Xyn2, Cbhl-4, Manl en Xyn3 is suksesvol uitgedruk en teen aktiviteitsvlakke van 2300, 8000, 500, 6000 en 900 nkat/ml, onderskeidelik uitgeskei. Die ensieme is as funksionele entiteite geproduseer en vervolgens gekaraktiriseer. Die Egl, Xyn2 en Manl is as voertoevoegings vir die moontlike gebruik in die dierevoerindustrie geëvalueer. Verbeterde biomassa toename is in die in vivo studie op pluimvee waargeneem. Met die moontlikheid van grootskaalse heteroloë ensiemproduksie in gedagte, moes 'n eenvoudige substraat vir hul goedkoop produksie gevind word. Molasse medium (verkrygbaar vanaf die Suid Afrikaanse suiker industrie) was derhalwe geëvalueer en die kwekingskondisies geoptimiseer vir die moontlike gebruik as kwekingssubstraat vir A. niger. Vir die evaluasie is die Egl en Xyn2 aktiwiteite onder verskillende toestande geproduseer en oor tyd gemonitor. Hierdie studie beklemtoon die moontlike gebruik van A. niger vir heteroloë produksie van ensieme, die gebruik van industriële substrate as kwekingsmedium en baan die weg vir ekonomiese, hoëvlakuitdrukking van industrieelbelangrike ensieme met 'n positiewe implikasie op die omgewing.

published_or_final_version / abstract / Biological Sciences / Doctoral / Doctor of Philosophy

La présente étude concerne la synthèse de matériaux à base de polysaccharides d’origine naturelle. Dans un premier temps, nous nous sommes intéressés à la synthèse en milieu homogène d’esters gras de cellulose, de nature de chaîne grasse et de degré de substitution variables. Les analyses chimiques, thermiques, mécaniques et structurales de ces produits en fonction de la longueur des chaînes grasses greffées (de C8 à C18) ont démontré que l’allongement du substituant greffé influençait les propriétés étudiées. De plus, un modèle structural a été proposé pour les chaînes cellulosiques estérifiées, dans lequel ces chaînes semblent s’organiser en couches avec les substituants gras greffés totalement déployés perpendiculairement au squelette cellulosique et non enchevêtrés entre eux.Dans une seconde partie, la valorisation d’un film cellulosique a été envisagée dans le domaine médical. Le choix s’est porté sur un film de laurate de cellulose fortement substitué. Des tests biologiques de cytocompatibilité et de cytotoxicité ont été réalisés sur ce matériau à partir de différents types de cellules. Les résultats des tests in-vitro obtenus concluent à la non-toxicité de ce film, ce qui rend prometteuse son application en tant que biomatériau.Enfin, la synthèse de dérivés polysaccharidiques a été réalisée dans le but de remplacer les dérivés pétrochimiques, utilisés comme superplastifiants dans la formulation du béton. Trois polysaccharides ont été testés, avec ou sans modification chimique: la cellulose et l’amidon. Les premiers résultats indiquent que les dérivés amidonnés sulfonés semblent être de bons candidats pour la substitution de tels additifs pétrochimiques. / This study deals with the synthesis of a natural-polysaccharide-based-material. First of all, we focused on the synthesis of fatty cellulose esters in a homogeneous media, with different fatty chains and various degrees of substitution. Chemical, thermal, mechanical and structural analyses of these products revealed the influence of the grafted fatty chain length (from C8 to C18) on these properties. Moreover, a structural model was purposed for the acylated cellulosic chains, in which these chains seem to be organized in a layered type structure, and where the fatty substituents are fully extended perpendicular to the cellulosic skeleton, and without interdigitated.Then, one of these cellulosic films was studied in order to be used in a medical application. A cellulose laurate film with high degree of substitution was chosen. Biological tests such as cytocompatibility and cytotoxicity were performed on this material from different type of cells. The results of the in-vitro tests conclude to the non-toxicity of this bio-based film. These results are promising for their application of as biomaterial.Finally, the synthesis of polysaccharide derivatives was performed in order to find alternatives to petrochemical derivatives compounds, used as superplasticizers in concrete formulation. Three native or chemically-modified polysaccharides were tested: cellulose and starch. First results indicate that starch sulfonate derivatives seem to be promising candidates to the substitution of such petrochemicals additives.

University of Macau / Institute of Chinese Medical Sciences

Em muitas espécies vegetais alguns órgãos desempenham mais do que uma função em certos estágios da vida. Raízes, caules ou folhas começam a acumular substâncias de reserva e, dependendo da sua origem, podem ser transformados em órgão de reserva, como tubérculos, bulbos, rizóforos e raízes tuberosas. Entre os compostos de reversa, os carboidratos são responsáveis por diversas funções, tais como fonte de energia, proteção contra a seca e temperaturas extremas. Os campos rupestres são caracterizados por um clima mesotérmico, com três a cinco meses de seca, correspondendo ao inverno, e seis a oito meses de chuvas, que corresponde ao verão. Os solos são rasos, salinos e com afloramentos rochosos. A flora possui um alto grau de endemismo, sugerindo a existência de estratégias adaptativas metabólicas para sobreviver aos estresses ambientais. O objetivo do presente trabalho foi realizar uma triagem dos carboidratos não estruturais em diferentes órgãos de espécies herbáceas predominantes destas regiões e a análise anatômica do sistema subterrâneo de quatro espécies de Asteraceae para visualização e localização dos cristais de inulina. Foram coletadas 26 espécies em três regiões distintas: 14 na Serra do Cipó e, cinco na Serra de Itacambira (estado de Minas Gerais), e sete na Serra Dourada (estado de Góias), representantes das famílias Amaranthaceae, Orchidaceae, Eriocaulaceae, Velloziaceae, Apiaceae, Apocynaceae e Asteraceae, sendo a última a mais representativa em números de espécies. Carboidratos solúveis foram quantificados colorimetricamente e analisados cromatograficamente por CCD e HPAEC/PAD. Amido foi quantificado por método enzimático e cristais de inulina foram visualizados sob luz polarizada. Frutanos foram detectados nos órgão subterrâneos de reserva de todas as espécies de Asteraceae e Amaranthaceae. A maior concentração de frutose total foi encontrada em Gomphrena marginata (Amaranthaceae), compreendendo 30% da massa seca de seus órgãos subterrâneos. Lessingianthus psilophyllus e Richterago polymorpha (Asteraceae) também contêm altas porcentagens de açúcares solúveis (34% e 33%, respectivamente), dos quais 26 e 27% correspondem aos frutanos. Todas as Asteraceae apresentaram frutanos da série homóloga da inulina com alto grau de polimerização. Gomphrena agrestis e Gomphrena marginata (Amaranthaceae) apresentaram frutanos da série dos levanos. Vellozia mínima e Barbacenia plantaginea (Velloziaceae) apresentaram os oligossacarídeos da série da rafinose. De todas as espécies estudadas, somente Habenaria caldensis, Oncidium hidrophylum (Orchidaceae), Mandevilla tenuifolia (Apocynaceae) and Klotzschia brasiliensis (Apiaceae) acumulam amido como principal polissacarídeo de reserva em seus órgãos subterrâneos, enquantoem Leiothrix curvifolia (Eriocaulaceae) o amido foi detectado nos caules. Cristais de inulina foram visualizados n as quatro Asteraceae analisadas e se e localizam principalmente no cilindro vascular. Foi observada também, a ocorrência de estruturas secretoras em Chresta curumbensis e Strophopappus glomeratus. Este trabalho fornece informações úteis para expandir o conhecimento de estratégias fisiológicas das plantas para sobreviverem a condições ambientais adversas, como ocorre nos campos rupestres, e contribuir para estabelecer estratégias de conservação para a biodiversidade tropical. / In many plant species some organs perform more than one function at certain stages of the life cycle. Roots, stems or leaves begin to accumulate reserve substances and depending on the origin may be transformed into storage organs like tubers, bulbs, rhizophores and tuberous roots. Among other storage compounds, carbohydrates are assigned several functions such as source of energy and protection against drought and extreme temperatures. Rocky fields are characterized by mesothermal climate, with three to five months of dry season in winter, and seven to eight months of humidity in summer. The soils are shallow, sandy and with rocky outcrops. The flora has a high degree of endemism suggesting the existence of metabolic adaptive strategies to overcome environmental stresses. The aim of this work was to carry out a screening of reserve compounds accumulated in different organs of predominant herbaceous species, and to analyze the localization of inulin crystals in the underground system in four Asteraceae species.Twenty-six species of the following families, Amaranthaceae, Orchidaceae, Eriocaulaceae, Velloziaceae, Apiaceae, Apocynaceae and Asteraceae were collected in three regions: 14 at \"Serra do Cipó\" and five at \"Serra de Itacambira\" (state of Minas Gerais), and seven at \"Serra Dourada\" (state of Goiás). The Asteraceae was the most significant in species number. Soluble carbohydrates were quantified colorimetrically and analyzed chromatographically by TLC and HPAEC/PAD, and starch was quantified by enzymatic assay. Inulin crystals were visualized under polarized light. Fructans were detected in underground reserve organs of all the Asteraceae and Amaranthaceae species. The highest concentration of total fructose was found in Gomphrena marginata (Amaranthaceae) comprising 30% of the underground organ dry mass. Lessingianthus psilophyllus and Richterago polymorpha (Asteraceae) also contained high percentages of soluble carbohydrates on a dry mass basis (34% and 33%, respectively), from which 26% and 27% corresponded to fructans. All the Asteraceae analyzed presented the inulin homologous series with a high degree of polymerization while Gomphrena agrestis and G. marginata (Amaranthaceae) presented the levan series. Vellozia minina and Barbacenia plantaginea presented the raffinose family oligosaccharides. Of all the analyzed species, only Habenaria caldensis, Oncidium hidrophylum (Orchidaceae), Mandevilla tenuifolia (Apocynaceae) and Klotzschia brasiliensis (Apiaceae) accumulate starch as the main reserve carbohydrate in the underground organs while in Leiothrix curvifolia (Eriocaulaceae) starch is accumulated in stems. Inulin crystals were visualized mainly in the vascular cilynder. in the four Asteraceae analyzed. Secretory structures were identified in Strophopappus glomerathus and Chresta curumbensis This work provides information to enhance the knowledge on physiological strategies used by plants to survive adverse environmental conditions such as those predominating in rocky fields, and may contribute for the establishment of conservation strategies of tropical biodiversity.

All these results advance our understanding on how stimulatory agents can be used to increase the production of useful metabolites by submerged fermentation of mushroom mycelium and indicate its potential biotechnological applications. / By use of one- and two-dimensional gels in proteomic analysis, some functional mycelial proteins that were differentially expressed by the addition of Tween 80 were identified. The up-regulation of heat shock proteins might help to maintain cellular viability under environmental stress. A down-regulation of YALI0E34793p and an up-regulation of ATP citrate lyase isoform 2 might suppress the activity of TCA cycle and subsequently stimulated the EPS production. Up-regulation of fatty acid synthase alpha subunit FasA might promote the synthesis of long-chain fatty acids and their incorporation into the mycelial cell membranes. Up-regulation of mitogen-activated protein kinase might facilitate the signal transduction in these processes. / The BPS, a highly branched glucomannan produced by the addition of Tween 80 in the fermentation broth of PTR mycelium had similar carbohydrate and protein content, monosaccharide composition and glycosidic linkages except by having a significantly lower molecular weight when compared to those of the control. Both BPS, with and without addition of Tween 80, could significantly inhibit (p < 0.05) the in vitro growth of a chronic myelogenous leukemia cells K562 in a dose dependent manner, with an estimated IC50 value of 43.7 and 47.6 microg/mL, respectively. / The effects of different kinds of stimulatory agents including fatty acids, surfactants and organic solvents were compared. The optimum results were achieved when 3.0 g/L Tween 80 was added to the fermentation broth on the 5th day of the fermentation, to give a maximum increase of 51.3 and 41.8% (p < 0.05) in the yield of mycelial biomass and BPS production, respectively. / The underlying mechanisms by which Tween 80 could increase the mycelial growth and EPS production in PTR were investigated by three novel approaches including changes in the nutrient uptake by mycelium, the morphology of mycelial pellets, and the fatty acid composition in the mycelial cell membrane. Firstly, the addition of Tween 80 significantly increased the glucose consumption rate by the mycelium, implying that the efficiency of nutrient uptake from the fermentation broth was enhanced. Secondly the addition of Tween 80 could extend the growth period of the mycelium possibly by maintaining the intact structure of the mycelial pellets and preventing its disintegration caused by shear stress in the fermentation system. Thirdly, the addition of Tween 80 could increase the incorporation of oleic acid which was a constituent of Tween 80 itself into the mycelial cell membrane of PTR, altering its fatty acid composition and increase the cell membrane permeability. The first two results explained the enhancement in the mycelial growth and EPS production while the last one was related to the extracellular transport of EPS to the fermentation broth. / This study aimed at comparing the effectiveness in the use of stimulatory agents with different chemical structures for enhancing the production of mycelial biomass and exopolysaccharide (BPS) by submerged fermentation of an edible mushroom Pleurotus tuber-regium (PTR). The chemical characteristics and antitumor activity of the BPS produced with and without the addition of the most effective stimulatory agent (Tween 80 which is a permitted food additive) were also compared. The underlying mechanisms by which Tween 80 could exert its effect on the mushroom mycelium were investigated by using chemical methods and microscopic techniques as well as proteomic analysis. / Zhang, Bobo. / Adviser: Chi Keung Peter Cheung. / Source: Dissertation Abstracts International, Volume: 73-08, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 132-156). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

by Wong Chun-kwok. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 233-246). / ABSTRACT --- p.I / ACKNOWLEDGEMENTS --- p.V / ABBREVIATIONS --- p.VI / PUBLICATIONS --- p.IX / CHAPTER / Chapter 1. --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- EFFECTOR CELLS MEDIATING ANTI一TUMOR IMMUNITY --- p.3 / Chapter 1.1.1 --- CYTOTOXIC T LYMPHOCYTES --- p.4 / Chapter 1.1.2 --- MACROPHAGES --- p.4 / Chapter 1.1.3 --- NATURAL KILLER CELLS --- p.5 / Chapter 1.1.4 --- LYMPHOKINE ACTIVATED KILLER CELLS --- p.7 / Chapter 1.1.5 --- TUMOR-INFILTRATING LYMPHOCYTES --- p.8 / Chapter 1.2 --- BIOLOGICAL RESPONSE MODIFIERS : THE NEW IMMUNOTHERAPY --- p.9 / Chapter 1. 3 --- CYTOKINES AS BIOLOGICAL RESPONSE MODIFIERS IN CANCER THERAPY --- p.12 / Chapter 1.3.1 --- INTERFERONS --- p.12 / Chapter 1.3.2 --- TUMOR NECROSIS FACTOR-ALPHA --- p.13 / Chapter 1.3.3 --- INTERLEUKIN-1 --- p.15 / Chapter 1.3.4 --- INTERLEUKIN-2 --- p.16 / Chapter 1.3.5 --- GRANULOCYTES /MACROPHAGES COLONY-STIMULATING FACTORS --- p.16 / Chapter 1.3.6 --- EPIDERMAL GROWTH FACTOR --- p.17 / Chapter 1.3.7 --- TRANSFORMING GROWTH FACTOR-BETA --- p.17 / Chapter 1.4 --- BIOACTIVE POLYSACCHARIDES FROM CHINESE MEDICINAL HERBS ACT AS BIOLOGICAL RESPONSE MODIFIERS --- p.18 / Chapter 2. --- AIM AND SCOPE OF INVESTIGATION --- p.27 / Chapter 3. --- MATERIALS AND METHODS --- p.30 / Chapter 3.1 --- MATERIALS --- p.30 / Chapter 3.2 --- METHODS --- p.39 / Chapter (I) --- "EXTRACTION, FRACTIONATION AND CHARACTERIZATION OF PSEUDOSTELLARIA HETEROPHYLLA" / Chapter 3.2.1 --- Hot water extraction and stepwise alcohol precipitation --- p.39 / Chapter 3.2.2 --- "Determination of carbohydrate, protein, uronic acid contents" --- p.41 / Chapter 3.2.3 --- Gel filtration --- p.41 / Chapter 3.2.4 --- Anion-exchange chromatography --- p.41 / Chapter 3.2.5 --- Paper chromatography --- p.42 / Chapter 3.2.6 --- Gas liquid chromatography --- p.43 / Chapter 3.2.7 --- Determination of molecular weight by high performance liquid chromatography --- p.44 / Chapter 3.2.8 --- SDS-polyacrylamide gel electrophoresis --- p.44 / Chapter 3.2.9 --- Determination of the bio´ؤtoxicity of samples --- p.46 / Chapter 3.2.10 --- Treatment of samples with sodium periodate or acetic acid --- p.46 / Chapter (II) --- ASSAYS OF IMMUNOMODULATORY ACTIVITIES OF PSEUDOSTELLARIA HETEROPHYLLA ON LYMPHOCYTES / Chapter 3.2.11 --- Isolation and preparation of cells --- p.48 / Chapter 3.2.12 --- In vitro lymphocyte transformation assay --- p.50 / Chapter 3.2.13 --- Mixed lymphocyte culture --- p.50 / Chapter 3.2.14 --- Depleting mouse T cells by anti-Thy-1.2 antibody plus complement treatment --- p.51 / Chapter 3.2.15 --- Depleting mouse B cells by anti-mouse B cell antibody plus complement treatment --- p.51 / Chapter 3.2.16 --- Haemolytic plaque assay --- p.52 / Chapter 3.2.17 --- Delayed-type hypersensitivity --- p.53 / Chapter 3.2.18 --- Immunofluorescent assay for interleukin-2 receptor expression --- p.54 / Chapter 3.2.19 --- Assay of murine interleukin-2 --- p.55 / Chapter (III) --- ASSAYS OF IMMUNOMODULATORY ACTIVITIES OF PSEUDOSTELLARIA HETEROPHYLLA ON MACROPHAGES / Chapter 3.2.20 --- Assay of murine interleukin-1 --- p.55 / Chapter 3.2.21 --- In vivo migration of macrophages --- p.56 / Chapter 3.2.22. --- Assay of phagocytic activity of peritoneal macrophages --- p.56 / Chapter 3.2.23 --- Northern blotting of mRNA of β-actin gene extracted from peritoneal exudate cells --- p.57 / Chapter (IV) --- ASSAYS OF ANTI-TUMOR ACTIVITIES OF PSEUDOSTELLARIA HETEROPHYLLA / Chapter 3.2.24 --- Assay of anti-tumor activity in vitro --- p.62 / Chapter 3.2.25 --- Assay of anti-tumor activity in vivo --- p.63 / Chapter 3.2.26 --- Priming effect of different fractions for the induction of TNF-α in mice --- p.63 / Chapter 3 .2.27 --- In vitro stimulation of TNF-α release from resting peritoneal macrophages --- p.64 / Chapter 3.2.28 --- Effects of P. heterophylla polysaccharides on TNF-α and IFN-gamma production as well as EAT growth in vivo --- p.64 / Chapter 3.2.29 --- Macrophage-mediated cytostatic activity --- p.65 / Chapter 3 2.30 --- Assay of lymphokine-activated killer cell activity --- p.66 / Chapter 3 2.31 --- Assay of natural killer cell activity --- p.67 / Chapter 3.2.32 --- Assay of tumor-infiltrating lymphocytes --- p.68 / Chapter (V) --- ASSAYS FOR THE EFFECTS OF PSEUDOSTELLARIA HETEROPHYLLA ON THE PROLIFERATION AND DIFFERENTIATION OF MURINE BONE MARROW CELLS AND MYELOID LEUKAEMIC Ml CELLS / Chapter 3.2.33 --- Assay of proliferation of murine bone marrow cells --- p.69 / Chapter 3.2.34 --- Assay of differentiation of murine bone marrow cells --- p.70 / Chapter 3.2.35 --- Assay of differentiation of Ml cells --- p.71 / Chapter 3.2.36 --- Induction of GM-CSF from bone marrow cells and Ml cells --- p.71 / Chapter (VI) --- ASSAYS OF THE IMMUNORESTORATIVE PROPERTIES OF PSEUDOSTELLARIA HETEROPHYLLA / Chapter 3.2.37 --- Immunorestoration in tumor-bearing mice --- p.72 / Chapter 3.2.38 --- Immunorestoration in aged mice --- p.72 / Chapter 3.2.39 --- Immunorestoration in cyclophosphamide- treated mice --- p.73 / Chapter 3.2.40 --- Statistical analysis --- p.73 / Chapter 4. --- "EXTRACTION, FRACTIONATION AND CHARACTERIZATION OF MITOGENIC FRACTIONS FROM PSEUDOSTELLARIA HETEROPHYLLA" / INTRODUCTION --- p.74 / RESULTS --- p.76 / Chapter 4.1 --- Extraction and fractionation of Pseudostellaria heterophylla --- p.76 / Chapter 4.2 --- Gel filtration and anion-exchange chromatography --- p.76 / Chapter 4.3 --- Characterization of bioactive fractions from Pseudostellaria heterophylla --- p.79 / Chapter 4.4 --- Mitogenic activity of fraction PH-I on murine lymphocytes in vitro --- p.96 / Chapter 4.5 --- Mitogenic effect of PH-I on murine lymphocytes in vivo --- p.102 / Chapter 4.6 --- Effect of PH-I on polyclonal B cell activation --- p.102 / Chapter 4.7 --- Adjuvant effect of PH-I on antibody response to SRBC in vivo --- p.106 / Chapter 4.8 --- Evidences to support the mitogenic activity of PH-I is due to its polysaccharide rather than due to the contamination by LPS --- p.106 / Chapter 4.9 --- The effects of PH-I on IL-2 production and IL-2 receptor expression on murine lymphocytes in vitro --- p.110 / Chapter 4.10 --- The mitogenic activity of the purified fractions on murine lymphocytes in vitro --- p.110 / Chapter 4.11 --- Adjuvant effect of PH-I Ab on antibody response to SRBC in vivo --- p.116 / Chapter 4.12 --- Mitogenic effect of PH-I C on murine lymphocytes in vivo --- p.116 / Chapter 4.13 --- Evidences to support the mitogenic activity of PH-I Ab is due to its polysaccharide rather than due to the contamination by LPS --- p.122 / DISCUSSION --- p.122 / Chapter 5. --- IMMUNOMODULATING AND ANTI-TUMOR ACTIVITIES OF ALCOHOL- INSOLUBLE FRACTION (PH-I) FROM THE HOT WATER EXTRACT OF PSEUDOSTELLARIA HETEROPHYLLA / INTRODUCTION --- p.133 / RESULTS --- p.135 / Chapter 5.1 --- Effect of PH-I on cytokine production --- p.135 / Chapter 5.2 --- In vivo activation of macrophages by PH-I --- p.135 / Chapter 5.3 --- Effect of PH-I on the activation of β-actin gene transcription in peritoneal macrophages --- p.142 / Chapter 5.4 --- Effect of PH-I on the in vitro growth of various tumor cell lines --- p.142 / Chapter 5.5 --- Immunorestoration of PH-I on the mitogenic response in EAT-bearing mice --- p.147 / DISCUSSION --- p.147 / Chapter 6. --- IMMUNOMODULATING AND ANTI-TUMOR ACTIVITIES OF PURIFIED FRACTIONS SEPARATED FROM PSEUDOSTELLARIA HETEROPHYLLA / INTRODUCTION --- p.154 / RESULTS --- p.157 / Chapter 6.1 --- In vitro anti-tumor activities of P. heterophylla --- p.157 / Chapter 6.2 --- In vivo anti-tumor activities of P. heterophylla --- p.165 / Chapter 6.3 --- Effect of P. heterophylla fractions on induction of delayed-type hypersensitivity --- p.165 / Chapter 6.4 --- Effect of PH-I fraction on the cytotoxic alloreactive T lymphocytes in vitro --- p.165 / Chapter 6.5 --- Effect of P. heterophylla on the production of TNF-α and IFN-gamma --- p.170 / Chapter 6.6 --- Effect of P. heterophylla on the activation of macrophages --- p.176 / Chapter 6.7 --- "Effect of P. heterophylla on the activation of NK, LAK and TIL" --- p.181 / Chapter 6.8 --- The effect of combined treatment of EAT-bearing mice with P. heterophylla amd Mur-TNF-α on the growth of EAT cells in vivo --- p.181 / Chapter 6 9 --- Immunorestorative activities of P. heterophylla in aged mice and cyclophosphamide-treated mice --- p.187 / DISCUSSION --- p.187 / Chapter 7. --- EFFECTS OF PSEUDOSTELLARIA HETEROPHYLLA ON PROLIFERATION AND DIFFERENTIATION OF MURINE BONE MARROW CELLS AND MYELOID LEUKAEMIC Ml CELLS / INTRODUCTION --- p.200 / RESULTS --- p.202 / Chapter 7.1 --- Effect of P. het erophyl1a on the proliferation and differentiation of murine bone marrow cells --- p.202 / Chapter 7 .2 --- Effects of P. heterophyl la on the proliferation and differentiation of murine myeloid leukaemia Ml cells --- p.205 / Chapter 7 .3 --- Effects of P. heterophylla on GM-CSF production by bone marow cells and myeloid leukaemia Ml cells --- p.214 / DISCUSSION --- p.218 / Chapter 8. --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.223 / BIBLIOGRAPHY --- p.233