published_or_final_version / Biological Sciences / Master / Master of Philosophy

Les travaux décrits dans ce mémoire sont consacrés au développement de nouveaux dérivés amphiphiles de l'acide hyaluronique (HA) obtenus par greffage de chaînes alkyle hydrophobes le long de la chaîne macromoléculaire hydrophile. La stratégie de greffage présentée a été optimisée afin de pouvoir transposer la synthèse à l'échelle industrielle. Le comportement en milieu aqueux des polymères natifs et les propriétés viscoélastiques des systèmes associatifs ont été étudiés par des mesures rhéologiques en écoulement et en régime dynamique. Une modulation des propriétés physico-chimiques est obtenue par la variation de la longueur de la chaîne du HA, de l'hydrophobicité des greffons et/ou de l'introduction dans le système associatif des molécules hôtes (alpha et beta cyclodextrines) ou des tensioactifs. Des expériences de titration calorimétrique couplées à de la RMN bidimensionnelle (ROESY), à des mesures de viscosités et à la modélisation moléculaire ont permis, suite à une analyse détaillée, d'élucider les interactions entre des HA alkylés et des cyclodextrines. Au cours de ce travail, nous avons présenté différents types de produits industriels développés par des sociétés européennes et américaines en tant que viscosupplément, la viscosupplémentation étant l'application visée pour nos dérivés développés dans le cadre de cette thèse.

Submitted in partial fulfillment for the Degree of Master of Applied Sciences in Biotechnology, Durban University of Technology, Durban, South Africa, 2016. / Prebiotics are defined as non-digestible oligosaccharides (NDOs) or polysaccharides (NDPs), which promote the growth of beneficial lactic acid bacteria in the colon. Algae are rich in polysaccharides and can be exploited as prebiotics for functional food ingredients to improve human and animal health. Currently, inulin is the most widely used ingredient in the prebiotics market, which is produced from live plants and requires expensive production processing. There is a vast repository of marine life with algae as a major source of nutrients. Therefore, this study provides an alternative source for prebiotic production and examines marine and freshwater algae that promote the growth of two strains of Lactobacillus delbrueckii subs. (Lactobacillus lactis and Lactobacillus bulgaricus) and one strain of Bifidobacterium spp. (Bifidobacterium longum). Monosaccharides of the oligosaccharide fraction of marine and freshwater algal extracts were investigated with the use of thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) after acidic hydrolysis of cell matrix polysaccharides. A total of fifty-five marine and freshwater aqueous algal extracts were assessed for their effect on the growth of L. lactis, B. longum and L. bulgaricus over a 96 hour period. Relative to the negative control, 34.5% algal extracts showed improved growth on one or more probiotic bacteria. The optimum time for maximum bacterial growth was noted at 48 h for all the tested aqueous algal extracts. Five marine and freshwater algal cultures (Spirulina platensis, Chlorococcum spp., Dunaliella salina, Scenedesmus magnus, Chlorella spp. and algal extract no. 48) from various aquatic environments in Kwa-Zulu Natal showed the best growth dynamics and demonstrated the greatest potential as sources of biomass for prebiotic production. These algal extracts were able to significantly increase the growth of at least one of the three probiotic bacteria (p < 0.05). Aqueous algal extract from S. platensis was regarded as the best algal source for prebiotics as it demonstrated a greater stimulatory effect on the growth of all three probiotic bacteria (L. lactis, B. longum and L. bulgaricus) compared to tested aqueous algal extracts and the inulin used as a positive control. The results obtained by HPLC for characterization confirmed TLC data, as xylose and galactose were detected by both chromatograms. These data indicated that xylose and galactose were present in aqueous algal extracts from S. magnus and S. platensis and galactose in aqueous algal extract no. 48. Xylose was most abundant in aqueous algal extracts from S. platensis (3mg/ml) and S. magnus (2.3mg/ml). In conclusion aqueous algal extracts from S. platensis, Chlorococcum, D. salina, S. magnus, Chlorella and algal extract no. 48 are potential sources for prebiotic production. Spirulina platensis extract was regarded as the best algal source. Xyose and galactose characterized by HPLC in algal extracts make up oligosaccharides that function as prebiotic compounds for stimulation of probiotic bacteria. There is a great scope for successful production of prebiotics from algal sources in South Africa. / M

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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

The simplification of the historical pieces of work samples' treatment is described in the dissertation. The recent methods have been inappropriate therefore, modification of the treatment was necessary. The dammar and copal resin, important restoration materials, were used in the experiment. HPLC-MS, HPLC-ELSD, GC-FID, GC-MS and pyrolysis GC- MS were applied. The concentration of fatty acids in the historical sculpture was determined by GC-MS, and it was compared to the content of fatty acids inside an egg. For identification of monosaccharide in the gum, GC-MS (hexamethyldisilazan, trimethylchlorsilan, and pyridine) is the suitable method. GC-MS determines the same composition of dammar resin as stated in the cited sources. The simple process for the samples' treatment was designed.

Cette thèse visait à caractériser la composition en polysaccharides des fibres de bois de tension chez le peuplier et établir de possibles corrélations avec les mécanismes de création de la tension. A cette fin, nous avons étudié le bois de jeunes peupliers qui ont poussé sous conditions contrôlées. Nous avons pu déterminer la composition en polysaccharides des couches G isolées ainsi que la structure de ces polysaccharides. L’évolution de cette composition au long de la différentiation du bois de tension et du bois opposé a été évaluée à l’aide de 178 anticorps dirigés contre les principaux polysaccharides pariétales. La fonction de deux protéines à arabinogalactanes avec domaine fascicline-like a été également étudiée à l’aide des anticorps produits à cet effet. La génétique inverse a été également utilisée pour compléter l’étude de la fonction d’une de ces protéines dans la formation du bois de tension. Les résultats obtenus ont mis en évidence une évolution de la composition de la couche G au cours de la différenciation. Des différences entre la composition de la couche S2 des fibres du bois de tension et du bois opposé ont aussi été détectées. Une grande quantité de pectines de type RG-I a été décelé dans la couche G, molécules qui pourraient participer à la formation d’un gel. Le gonflement de ce gel serait responsable de la mise en tension des microfibrilles de cellulose. Cependant, nos travaux ne montrent aucune évidence pour la présence de xyloglucanes dans la couche G. Des indices en faveur de l’implication des FLAs dans la construction de la couche G ont été également trouvés dans cette étude. Ce travail de thèse ouvre des perspectives pour l’identification du déterminisme moléculaire à l’origine de la création de la tension dans le bois de tension. / This work aimed at characterizing the composition in polysaccharides of poplar tension wood fibres and a possible correlation with mechanisms for creating tension. Firstly, we isolated G-layers from tension wood formed in young poplars which grows in a greenhouse under controlled conditions and determine its polysaccharides composition and the structure of these polysaccharides. Therefore, we investigate the polysaccharide distribution during tension and opposite wood fibres differentiation by using 178 antibodies raised against the major wall polysaccharides. The distribution and function of two fasciclin-like arabinogalactan proteins were studied using two antibodies produced to this end and the function of one of the proteins were studied by reverse genetics. This work show an evolution in the polysaccharides composition of G-layer through its differentiation and also differences concerning S2 layer composition between tension and opposite wood fibres. No evidences for the presence of xyloglucans in the G-layer. However, this work shows the presence of a high quantity of RG-I type pectin which may be implicated in a gel-like structure formation which swelling could be responsible for tension creation in cellulose microfibrils. Our results suggest an implication of fasciclin-like arabinogalactan proteins in G-layer construction. Further, this work opens up new perspectives towards identification of molecular basis of tension creation in tension wood.

Esta tese apresenta o estudo inédito da obtenção e caracterização de filmes finos de xiloglucanas obtidas de duas fontes diferentes, Hymenaea courbaril (HXG) e Tamarindus indica (TXG) sobre lâminas de Si/SiO2 e lâminas modificadas com grupos amino, assim como a aplicação desses filmes na imobilização da lectina concanavalina A (Con A) por medidas de elipsometria e microscopia de força atômica (AFM). A diferença na estrutura fina de cada XG influenciou na espessura e morfologia de seus filmes finos. Os oligômeros XXXXG presentes apenas na HXG favorecem as interações com a superfície, assim como já observado para interação entre XG-celulose [Lima e Buckeridge, 2001]. A imobilização de Con A foi realizada sobre filmes de carboximetilcelulose (CMC) de HXG, TXG e poli(metacrilato de metila) (PMMA), lâminas de Si/SiO2 e partículas poliméricas decoradas com polissacarídeo PMMA/CMC. A adsorção de Con A foi mais pronunciada sobre os filmes de CMC. As camadas de Con A formadas sobre HXG e TXG apresentam potencial utilização no reconhecimento e detecção de carboidratos para desenvolvimento de diagnósticos para algumas doenças, por não comprometerem o sítio de ligação específica da lectina na adsorção. No caso das partículas PMMA/CMC cobertas com uma camada de Con A, a alta estabilidade coloidal do sistema confere às partículas a possibilidade de explorar a propriedade de aglutinação específica da lectina no reconhecimento de carboidratos. A enzima hexoquinase (HK) foi imobilizada sobre dois diferentes tipos de partículas poliméricas decoradas com polissacarídeo, PMMA/CMC e poliestireno/quitosana (PS/CH). Na primeira, HK perdeu drasticamente sua atividade catalítica, ao contrário do observado quando a mesma enzima foi imobilizada sobre PS/CH, onde a HK manteve aproximadamente 50% de sua atividade enzimática, podendo ser armazenada em temperatura e pressão ambientes por 1 mês e ser reutilizada até 3 vezes. A estrutura da camada de hidratação e a composição química de cada um dos polissacarídeos (CMC ou CH) na superfície das partículas são diferentes, provocando diferenças na conformação das moléculas de HK quando imobilizadas sobre cada tipo de partícula. Medidas de forças de adesão utilizando as partículas poliméricas coladas na extremidade de cantilevers de AFM (Colloidal Probe Technique) permitiram medir as espessuras dessas camadas hidratadas de polissacarídeos nas superfícies das partículas PMMA/CMC e PS/CH, da ordem de (20 &#177; 10) nm e (35 &#177; 11) nm, respectivamente. Essas medidas de força também evidenciaram o potencial dos dois sistemas estudados (PMMA/CMC - Con A e PS/CH - HK) como métodos qualitativos para a detecção dos açúcares manose, glicose e frutose. / This thesis presents the study on thin film formation and characterization of xyloglucans extracted from two different sources, Hymenaea courbaril (HXG) and Tamarindus indica (TXG) onto Si/SiO2 wafers or amino-terminated wafers, as well as the application of these films as substrates for the immobilization of the lectin concanavalin A (Con A) by means of ellipsometry and atomic force microscopy (AFM). The difference in the fine structure of each XG exerted influence on film thickness and morphology. The oligomers XXXXG present only in HXG favor the interactions with the surfaces, as already observed for the interaction between XG-cellulose [Lima and Buckeridge, 2001]. Con A was immobilized onto films of carboxymethylcellulose (CMC), HXG, TXG and poly(methyl methacrylate) (PMMA), Si/SiO2 wafers and on polysaccharide decorated polymeric particles PMMA/CMC. The adsorption of Con A was more pronounced on CMC films. Con A layers formed on HXG and TXG present potential application in the recognition and detection of carbohydrates for diagnostic development, once the specific sugar binding site is not involved in the adsorption process. In the case of polymeric particles PMMA/CMC covered with a Con A layer, the high colloidal stability of the system confers to particles the possibility to explore the lectin property of specific agglutination with carbohydrates. The enzyme hexokinase (HK) was immobilized onto two different types of polysaccharide decorated polymeric particles, PMMA/CMC and polystyrene/chitosan (PS/CH). In the first one, HK drastically lost its catalytic activity. The same enzyme immobilized onto PS/CH kept approximately 50% of its enzymatic activity, even after storing under room temperature and pressure for 1 month. Moreover upon immobilizing HK could be reused up to 3 times. The structure of the hydration layer and the chemical composition due to the presence of each polysaccharide (CMC or CH) on the polymeric particle surface provided different environment, generating differences in the conformation of immobilized molecules of HK on each particle. Measurements of adhesion forces using the polymeric particles glued on the extremity of AFM cantilevers (Colloidal Probe Technique) allowed to measure the thicknesses of these hydrated polysaccharide layers on the surfaces of the PMMA/CMC and PS/CH particles as (20 &#177; 10) nm and (35 &#177; 11) nm, respectively. These force measurements had also evidenced the potential of the two studied systems (PMMA/CMC - Con A and PS/CH - HK) as qualitative methods for detection of the sugars mannose, glucose and fructose.

Os alimentos desempenham papel fundamental na nutrição e também na imunidade, pois podem conter substâncias que interagem direta ou indiretamente com o sistema imune, principalmente o de mucosas. Alguns polissacarídeos não-amido (PNAs) originados de plantas, algas e fungos comestíveis podem modular a função imune, contribuindo para a manutenção da saúde. A banana apresenta composição monossacarídica da parede celular similar à de plantas com efeitos imunomoduladores, permitindo formular a hipótese de que os PNAs dessa fruta também tenham essas propriedades. Assim, o objetivo foi extrair, purificar e caracterizar PNAs hidrossolúveis da polpa de banana das cultivares Nanicão e Thap Maeo e testá-los em cultivos de macrófagos RAW 267.4, células THP-1, macrófagos diferenciados por PMA THP-1 e células HL-60 in vitro avaliando a ação imunomoduladora através da dosagem de citocinas, NO e ensaios de fagocitose. As frações hidrossolúveis foram caracterizadas quanto ao seu conteúdo monossacarídico e ligações por hidrólise enzimática, identificando homogalacturonanos, mananos, arabinogalactanos, xilogalacturonanos e galactoglucomananos. As frações foram testadas para ausência de endotoxinas e viabilidade celular por MTT. Foram estabelecidas as doses não-tóxicas e ensaiadas as concentrações de 10, 50 e 200 &#181;g.ml-1 das frações polissacarídicas para os testes in vitro. Não houve toxicidade para macrófagos e monócitos, enquanto que para a linhagem de células pro-mielóciticas de leucemia humana HL-60, as frações se mostraram citotóxicas. Houve aumento de atividade fagocítica nas frações WSP, UFP e HTP quando comparadas ao controle negativo de células, assim como a produção de citocinas inflamatórias como IL-1&#946;, TNF-&#945;, IL-6, além da quimiocina IL-8 nas células oriundas de THP-1 e diminuição nos marcadores TLR-2 com aumento de CD14 nas células THP-1, além do aumento do tamanho celular promovido pelas frações polissacarídicas mostrando diferenciação para série granulocítica. Portanto, PNAs de ambas as cultivares de banana possuem potencial imunomodulador seja inflamatório, anti-inflamatório ou de diferenciação de células THP-1 imaturas in vitro. / Food plays a major role in nutrition as in immunity, as they may contain substances that interact directly or indirectly with the immune system, especially those located on mucous membranes. Some non-starch polysaccharides (NSPs) originate from plants, algae, and edible fungi can modulate immune function, contributing to the maintenance of health. The banana presents cell wall monosaccharide composition like the other plant polysaccharides with immunomodulatory effects allowing to formulate the hypothesis that this fruit NSPs also have these properties. The objective was to extract, purify and characterize water-soluble banana pulp NSPs of Nanicão and Thap Maeo cultivars and test them in vitro on cell lineages of RAW 267.4 macrophages, PMA differentiated THP-1 macrophages, THP-1 monocytes, and HL-60, evaluating their immunomodulatory action by the dosage of cytokines, NO and phagocytosis assays. Water-soluble fractions were characterized as to their content and monosaccharide linkages by enzymatic hydrolysis, identifying homogalacturonans, mannans, arabinogalactans, xylogalacturonan, and galactoglucomannans in its composition. Fractions were tested for the absence of endotoxins and cell viability by MTT. Non-toxic concentrations doses of 10, 50 and 200 &#181;g.ml-1 of the polysaccharide fractions were established for in vitro testing. The NSPs fractions WSP, UFP, and HTP tested promoted an increase in phagocytic activity in THP-1 PMA derived macrophages compared to negative control cells, as well as the production of inflammatory cytokines such as IL-1&#946;, TNF-&#945;, IL-6. An increase in the THP-1 CD14 cell receptor and decrease in CD33, CD123, CD11c and TLR-2 receptors were promoted by the banana polysaccharides. Besides, there was an increase in cell size showing cell differentiation towards granulocyte line and demonstrating that NSPs from both cultivars were immunomodulatory with a capacity of immature cell differentiation and proving that in vitro screening test is an efficient method to test other foodborne substances with immunomodulatory potential.

E. coli pertencentes ao sorogrupo O111 são incluem amostras com diferentes fatores de virulência que são responsáveis por gastroenterites por todo o mundo e surtos de diarreia com sangue e síndrome hemolítico-urêmica em países desenvolvidos. Resultados obtidos anteriormente mostraram que o LPS O111 é um bom candidato a antígeno para a formulação de uma vacina contra E. coli O111. Desta forma, o polissacarídeo O111 foi conjugado com proteínas arreadoras. Coelhos foram imunizados pela via subcutânea com o conjugado O111-citocromo c incorporado em sílica SBA-15 como adjuvante, os resultados mostraram que os anticorpos obtidos foram capazes de reconhecer e inibir a adesão de todas as categorias de E. coli pertencentes ao sorogrupo O111. O veículo carreador de antígeno, VaxcineTM , foi incorporado no conjugado O111-EtxB e camundongos foram imunizados pela via oral, o que resultou em resposta imune sistêmica e de mucosa contra o LPS O111. Os anticorpos obtidos foram capazes de reconhecer amostras de todas as categorias de E. coli O111. Os dados obtidos neste trabalho sugerem que a utilização do polissacarídeo O111 em uma formulação conjugada é capaz de prevenir surtos de diarreia causada por E. coli O111 independente do seu mecanismo de virulência. / The E. coli serogroup O111 include samples with different virulence factors that are responsible for enteritis throughout the world and for outbreaks of bloody diarrhea and hemolytic uremic syndrome in developed countries. Previous results obtained have shown that O111 LPS is a good candidate antigen for the development of a vaccine against E. coli O111. Therefore, the polysaccharide was conjugated with carrier proteins. Rabbits were immunized subcutaneously with the O111-cytochrome c conjugate incorporated in silica SBA-15 as an adjuvant, the results obtained show that the antibodies were able to recognize and inhibit the adhesion of all types of E. coli belonging to serogroup O111. The antigen delivery vehicle, VaxcineTM was incorporated into the O111-EtxB conjugate, and mice were immunized by gavage, resulting in systemic and mucosal immune response against O111 LPS. These antibodies were able to recognize all categories of E. coli O111. The results presented in this work indicate that an oral conjugated vaccine that targets the O111 antigen has the potencial to prevent diarrhea by O111 E. coli strains, regardless their mechanism of virulence.