Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Food Technology in the Faculty of Applied Sciences at the Cape Peninsula University of Technology / The aim of this study was to evaluate bambara groundnut [BGN] non-starch polysaccharides [NSP] subject to the incorporation into model and food systems with a view to establish their functional and physicochemical properties. BGN insoluble dietary fibre [BGNIF] and soluble dietary fibre [BGNSF] were successfully extracted from four varieties (black-eye: BLE, red: RED, brown: BRN and brown-eye: BRE). Physicochemical properties evaluated revealed the high bulk density of all BGNIF and BGNSF varieties, which could contribute to cost-effective packaging. The microstructures of BGNIFs were irregular in shape with different sizes. The colour parameters (lightness, redness, yellowness, chroma and hue angle) differed significantly [p ≤ 0.05] across all BGNIF and BGNSF varieties; and indicated a yellowish-red colour for BGNIFs and a light yellow colour for BGNSFs. Negligible amounts of condensed tannins [CT] were found in BGNIFs (0.014 – 0.160 mg.g-1). Higher amounts polyphenols [PP] were present in BGNSFs (45.42 – 55.90 mg.g-1 gallic acid equivalents [GAE]) compared to the amount PP in BGNIFs (6.14 – 15.56 mg.g-1 GAE). Major sugars identified were arabinose/galactose, xylose and mannose in BGNIFs, and xylose and mannose in BGNSFs. The functional properties evaluated revealed high swelling capacity of BGNIFs (6.37 – 7.72 ml.g-1) and no significant [p > 0.05] difference in water retention capacity. Fat absorption capacity ranged from 1.38 – 1.52 g oil.g-1 dry weight for BGNIFs and 4.04 – 4.55 g oil.g-1 dry weight for BGNSFs. Variability in BGNIF (91.2%) and BGNSF (79.4%) physicochemical and functional properties could both be explained by two principal components (BGNIF component 1: PP, redness, yield; and component 2: xylose, yellowness and chroma; BGNSF component 1: yellowness, chroma, mannose content; and component 2: redness, fat absorption and fructose content). Following an IV optimal mixture design, an optimum white bread formulation was obtained using 59.5% water, 4.3% yeast and 8.5% BGNIF. Bread enriched with the four BGNIF varieties (BLE, RED, BRN and BRE) were tested for several physicochemical properties. Significant [p ≤ 0.05] differences existed between the control and BGNIF enriched loaves for crumb grain characteristics (including pore area distribution, feret angle, circularity, roundness and aspect ratio). Specific loaf volume of BGNIF enriched loaves ranged from 3.33 – 3.85 ml.g-1 and were significantly [p ≤ 0.05] lower compared to the control bread (4.16 ml.g-1). Favourable texture characteristics obtained with the BGNIF enriched breads were lower hardness, chewiness and gumminess compared to the control loaf. Crust and crumb colour parameters (lightness, redness, yellowness, chroma and hue angle) were significantly [p ≤ 0.05] different across all loaves. BRE BGNIF bread (3.43 ± 0.20) had the significantly [p ≤ 0.05] lowest crumb colour difference compared to the control bread; whilst BRN (1.72 ± 0.42) and BRE (2.44 ± 0.78) loaves had the lowest significant [p ≤ 0.05] crust colour difference compared to the control. Favourable chemical properties were the high total dietary fibre [TDF] (7.14 – 8.33%) content of all BGNIF enriched loaves compared to the control loaf (4.96%). Significant [p ≤ 0.05] differences were also observed for some loaves for moisture content, condensed tannins and polyphenol content. Variability in bread physicochemical properties was differentiated by three components (component 1: bread textural properties; component 2: specific loaf volume and bread lightness; component 3: crumb colour parameters) which accounted for a cumulative variation of 92.8%. All bread loaves were also sensorially acceptable as rated moderately like to like very much (>3 rating on a 5-point hedonic scale) by consumers for all parameters (appearance, crust and crumb colour, aroma, taste, texture and overall acceptability) evaluated. Furthermore, brown BGNSF was tested for stabilising effects in an orange beverage emulsion. BGNSF and orange oil were varied at two levels each based on a 22 augmented factorial design and the effects determined on the equilibrium backscattering [BS] flux as emulsion stability indicator. The BS profiles which resulted from the Turbiscan stability analysis revealed flocculation at low rates as the major destabilisation mechanism. The optimal formulation producing a stable emulsion was identified as low oil (6%) and high BGNSF (30%) concentrations. The objective of this study was therefore achieved and showed that positive physicochemical and functional properties are associated with BGNIF and BGNSF from black-eye, red, brown and brown-eye varieties. Furthermore, the incorporation of BGN fibres in white bread and a beverage emulsion was shown to contribute positive technological properties in these systems.

Orientador: Eliana Gertrudes de Macedo Lemos / Coorientador: Tereza Cristina Luque Castellane / Banca: Maria de Lourdes Corradi Custodio da Silva / Banca: Luciana Maria Saran / Banca: Tsai Siu Mui / Banca: Jackson Antonio Marcondes de Souza / Resumo: As estruturas e alguns compostos produzidos pelos microrganismos apresentam elevada quantidade de cargas como, por exemplo, parede celular e biopolímeros. Os exopolissacarídeos (EPSs) de bactérias pertencentes à ordem Rhizobiales apresentam biopolímeros que têm sido muito estudados. Os EPSs são moléculas que apresentam grupos polares, esta característica é capaz de reduzir a tensão interfacial de mistura água/óleo e óleo/água. A formação de emulsões é um fator importante para a biodegradação de óleos e hidrocarbonetos. As cargas presentes na parede celular das células bacterianas são de natureza predominantemente aniônica. Estas estruturas são capazes de interagir fortemente com cátions metálicos e desempenham um papel importante no sequestro ou imobilização de íons no ambiente. A aplicação dos isolados de rizóbios são interessantes ferramentas biotecnológicas no processo de biorremediação, por estes microrganismos não apresentarem toxicidade à saúde e ao meio ambiente. Dos isolados de rizóbios pertencentes ao Laboratório de Bioquímica de Microrganismos e Plantas, da FCAV/UNESP, foram selecionados quatro, que demonstraram elevada produção de goma 'in vitro'. Este trabalho teve como objetivo investigar o potencial biotecnológico, de isolados de Rhizobium spp. (SEMIA 4064 e JAB6) e dois isolados de Mesorhizobium spp. (LMG 6125 e LMG 11892), como bioemulsificantes e biossorventes. Para a identificação dos isolados foi sequenciada a região 16S rRNA (1500pb). Foram estudados a produção de EPS e aplicação desses biopolímeros como bioemulsificantes de óleos e hidrocarbonetos. Investigando a Reologia e a ... / Abstract: The structures and some compounds produced by microorganisms have a high amount of charges such as, for example, cell wall and biopolymers. The exopolysaccharides (EPSs) of bacteria belonging to the order Rhizobiales have biopolymers that have been widely studied. The EPSs are molecules that have polar groups, this feature they are can to reduce the interfacial tension of water/oil and oil/water. The formation of emulsions is important factor for the biodegradation of oils and hydrocarbons. The charges present in the cell wall on the bacterial cells are predominantly of anionic in nature. These structures are capable of interacting strongly with metal ions and play an important role in sequestering or immobilization of ions in the environment. The application of the populations of rhizobia are interesting biotechnological tools in bioremediation process, these organisms do not present toxicity to health and the environment. Rhizobial isolates belonging to the Laboratory of Biochemistry of Plants and Microorganisms, FCAV / UNESP, four were chosen that demonstrated high gum production in vitro. This work aimed to investigate the potential of biotechnology of the isolates of Rhizobium spp. (SEMIA 4064 and JAB6) and two strains of Mesorhizobium spp. (LMG 6125 and LMG 11892), as biosorbents and bioemulsifiers. For the identification of the isolates were done sequencing of the gene 16S rRNA (1500pb). The production and application of these EPSs were studied as bioemulsifiers of oils and hydrocarbons. Investigating the monosaccharides composition and rheology of the EPSs. These isolates were explored for sensitivity of live cells and the process of removal of dead cells by copper ions (Cu2+) and chromium (Cr6+). The sequencing showed that the isolates LMG 6125 and LMG 11892 were not consistent with the genus and species previously described / Doutor

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Com base na necessidade de obter novas formulaÃÃes mais eficientes e diante das propriedades apresentadas pelas molÃculas oriundas de algas marinhas, acredita-se que estas possam ser eficazes no processo de cicatrizaÃÃo. A lectina da alga marinha vermelha Pterocladiella capillacea (PcL) e os polissacarÃdeos da alga vermelha Solieria filiformis (SfP) inicialmente foram analisados em ensaio de toxicidade. PcL foi aplicada no ensaio do edema de pata seguido da dosagem de mieloperoxidase (MPO). Avaliou-se o efeito da lectina da alga Pterocladiella capillacea (PcL) e os polissacarÃdeos das algas Solieria filiformis (SfP) na cicatrizaÃÃo de feridas induzidas em ratos. Ambas as molÃculas foram submetidas a ensaios microbiolÃgicos e analisadas quanto ao efeito no processo de cicatrizaÃÃo em feridas limpas induzidas no dorso de ratos. SfP foi utilizado como um possÃvel veÃculo para a administraÃÃo de PcL e comparado ao Carbopol 940 (C). Os gÃis (0,9%) foram submetidos a anÃlise reolÃgica e entÃo aplicados nas lesÃes durante um perÃodo de tratamento de 10 (dez) dias, utilizando kollagenase como controle. O processo de cicatrizaÃÃo foi avaliado quanto ao tamanho das feridas, dosagem de MPO e anÃlise histolÃgica. PcL e SfP nÃo demonstraram toxicidade quanto aos parÃmetros de peso corpÃreo, ÃrgÃos e dosagens bioquÃmicas. Entretanto a anÃlise histolÃgica mostrou pequenas alteraÃÃes no fÃgado e rim. PcL (1, 3 e 9 mg/kg, i.v.) reduziu o edema induzido por carragenana e quando administrada com seu inibidor mucina nÃo foi possÃvel verificar a reduÃÃo do edema o qual foi confirmado pela dosagem de MPO. As duas molÃculas foram aplicadas em ensaios microbiolÃgicos e nÃo inibiram o crescimento de nenhum micro-organismo testado, os quais tambÃm nÃo foram capazes de utilizar SfP como fonte de carbono. A anÃlise reolÃgica mostrou que os SfP utilizados na formulaÃÃo dos gÃis (PcL+SfP e SfP) apresentaram a caracterÃstica de um pseudoplÃstico. A anÃlise macroscÃpica das feridas mostrou uma reduÃÃo da Ãrea da lesÃo nos animais tratados com PcL+SfP e PcL+C (53,5 e 60%, respectivamente) no sexto dia de administraÃÃo. Na anÃlise histolÃgica, nÃo foi observado infiltrado inflamatÃrio acentuado nos tecidos obtidos atà o 4 dia da administraÃÃo dos gÃis (PcL+SfP e PcL+C) e Kollagenase (controle positivo). No 6 dia, os animais nÃo tratados e os tratados apenas com SfP mostraram infiltrado inflamatÃrio. A dosagem de MPO demonstrou reduÃÃo no processo inflamatÃrio nas amostras contendo PcL, cujo resultado corrobora com a anÃlise histolÃgica. Em conclusÃo, PcL auxiliou no reparo de feridas, sugerindo seu uso futuro como uma possÃvel ferramenta para o tratamento de lesÃes. O papel biolÃgico e farmacolÃgico das lectinas e polissacarÃdeos de algas marinhas faz parte de uma Ãrea de estudos ainda pouco explorada, onde muito conhecimento deverà ser investido visto que estas biomolÃculas podem ser promissoras para a indÃstria farmacÃutica. / Based on the need for new formulations that are more efficient and on the properties provided by molecules derived from seaweed, it is believed that these can be effective in healing process. The lectin from the red seaweed Pterocladiella capillacea (PcL) and the polysaccharides of red algae Solieria filiformis (SfP) were initially analyzed in toxicity testing. PcL was applied to the paw edema test followed by measurement of myeloperoxidase (MPO). We evaluated the effect of the seaweed Pterocladiella capillacea lectin (PcL) and algal polysaccharides Solieria filiformis (SfP) in healing wounds in rats induced. Both molecules were submitted to microbiological tests and assayed for the effect on wound healing in wounds clean induced on the back of rats. SfP was used as a possible vehicle for the administration of PcL and compared to Carbopol 940 (C). The gels (0.9%) were analyzed rheological and then applied to the lesions during a treatment period of 10 days, using kollagenase  as control. The healing process was evaluated on the size of the wounds, levels of MPO and histological analysis. The molecule SfP and PcL is not toxic for the parameters of body weight, organ and biochemical measurements. However, the histological analysis showed minor changes in liver and kidney. PcL (1, 3 and 9 mg / kg, i.v) reduced the edema induced by carrageenan and its inhibitor when administered with mucin was not possible to check the reduction of edema which was confirmed by measurement of MPO. The two molecules were used in microbiological assays and not inhibit growth of any microorganism tested and unable to use SfP as carbon source. The rheological analysis showed that the SfP used in the formulation of the gels (PcL+SfP and SfP) had the characteristic of a pseudoplastic. Macroscopic analysis of wounds showed a reduction in lesion area in the animals treated with PCL, PCL+SfP, PCL+C (53.5 and 60% respectively) on the sixth day of administration. In histological analysis, there was no severe inflammatory infiltrate in the tissues obtained until 4th day of administration of the gels (PcL and PcL+SfP, PcL+C) and Kollagenase (positive control). On day 6, the untreated animals and those treated only with SfP showed inflammatory infiltrate. The measurement of MPO showed a reduction in the inflammatory process in the samples containing PcL, whose results corroborate the histological analysis. In conclusion, PcL aid in wound repair, suggesting its use as a possible future tool for the treatment of lesions. The biological and pharmacological role of lectins and polysaccharides of seaweed is part of a study area little explored, where a lot of knowledge should be invested since these biomolecules can be promising for the pharmaceutical industry.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Polysaccharide nanoparticles obtained from Anadenanthera macrocarpa tree exudates (angico gum, AG) and chitosan (CH) were produced by polyelectrolyte complex formation as well as by graft polymerization of acrylic acid onto a AG backbone. The polyelectrolyte complexes (PECs) were obtained by using AG and its carboxymethylated derivatives with degrees of substitution 0.20 and 0.63 as polianions. Reaction parameters investigated were polymer concentration, molar mass, order of solution addition, charge ratio (n+/n-) and AG degree of substitution of carboxymethylated samples. Data revealed that nanoparticles formed by adding AG to CH (AGCH) are in the range of 17.4 to 41.9 nm. Increasing polyelectrolyte concentration led to a decrease in particle size. Larger sizes were observed for charge ratios (n+/n-) > 1. Regarding the molar mass effect, the higher the chitosan molar mass, the larger are the particle sizes. Atomic Force Spectroscopy revealed that for samples with charge ratio n+/n- = 1, particle sizes were 10 and 14 nm, for high (CHa) and low (CHb) chitosan molar mass, respectively. These figures are lower than those analyzed by light scattering measurements (25.1 and 28.2 nm respectively for AGCHa at n+/n- = 1). Nanoparticles prepared using carboxymethylated AG (CMA) have their sizes strongly dependent on charge ratio; for those samples obtained from CMA with degree of substitution (DS) 0.20 and CHa, size distribution is similar to that of AGCHa samples. For samples synthesized with 0.63 DS, size distribution is dependent on charge ratio: for n+/n-< 0.8 similar distribution is observed as those of AGCHa and CMCHa; for n+/n- > 0.8 larger particles (140 - 636 nm) are produced. CMACHa with 0.63 DS, n+/n- = 4, was used as a matrix for chloroquine encapsulation resulting in a efficiency of encapsulation of 43 %. In vitro release was investigated at pH 5.8 and 7.4, a slower release profile being observed at pH 5.8, whereby only 20 % of drug was released in 22 days. Faster release was observed at pH 7.4, where 84 % of drug was released in 6 h, completion being achieved only after 10 days. Data obtained seem to point out that this system can be used for oral drug administration. AG nanoparticles were also produced by acrylic acid radical polymerization on AG backbone, using cerium ammonium nitrate (CAN) as initiator and methylene bisacrylamide as crosslinking agent. It was investigated the effects of glycosidic unit/acrylic acid (GU/AA) and AA/CAN molar ratios on particle size. AG/AA nanoparticles were found to have sizes in the range 6.4 â 19.4 nm, according to GU/AA ratio and an average reaction yield of 56.6 %. Decreasing initiator concentration resulted in low particle size and high polidispersity. By inserting AA onto AG backbone, smaller particle size was obtained than homopolymer one (417 nm). No pH response was found for AG/AA nanoparticles. Bovine serum albumin (BSA) was doped in AG/AA nanoparticles, with an encapsulation efficiency of 50 % resulting in a low release profile, where 69 % of BSA being released in 30 days. / NanopartÃculas do polissacarÃdeo extraÃdo de exsudatos de Ãrvores da Anadenanthera macrocarpa (goma do angico, GA) foram produzidas utilizando como rotas de sÃnteses a complexaÃÃo polieletrolÃtica (CEP) com quitosana (QT) e a copolimerizaÃÃo por enxertia de Ãcido acrÃlico (AA). Na rota de complexaÃÃo polieletrolÃtica a goma do angico (GA) e seus derivados carboximetilados (CMA) com grau de substituiÃÃo (GS) de 0,20 e 0,63 foram utilizados como poliÃnions. Foram investigados alguns parÃmetros que influenciam o tamanho da nanopartÃcula tais como: concentraÃÃo dos polissacarÃdeos, ordem de adiÃÃo das soluÃÃes polieletrolÃticas, massa molar da quitosana, razÃo de carga dos polieletrÃlitos e grau de substituiÃÃo da goma do angico carboximetilada. NanopartÃculas formadas pela adiÃÃo de GA a quitosana de alta massa molar (QTa) (GAQTA) possuem valores de tamanho variando de 17,4 a 41,9 nm. O aumento da concentraÃÃo dos polieletrÃlitos ocasionou uma diminuiÃÃo nos tamanhos de partÃculas. Maiores valores de tamanho foram observados para nanopartÃculas com razÃo molar de cargas (n+/n-) > 1. Quanto maior a massa molar da quitosana maior o tamanho das nanopartÃculas. A variaÃÃo do tamanho com ordem de adiÃÃo dos polieletrÃlitos depende de outros fatores como razÃo molar de cargas e massa molar da quitosana. A anÃlise por microscopia de forÃa atÃmica das nanopartÃculas de GAQTa e GAQTb na razÃo de carga n+/n- = 1 apresentam partÃculas ovaladas com diÃmetros de 10 a 14 nm respectivamente. Estes valores sÃo inferiores ao obtidos por espalhamento de luz (25,1 e 28,2 nm respectivamente para GAQTa e GAQTb na razÃo de carga n+/n- = 1). O aumento da densidade de carga pela reaÃÃo de carboximetilaÃÃo afeta significantemente o tamanho das partÃculas. NanopartÃcula formadas com CMA GS 0,20 e quitosana de alta massa molar (QTA) apresenta valores similares ao observado para nanopartÃculas de GAQTa (14 a 30 nm). Quando o GS à aumentado para 0,63 as nanopartÃculas exibem comportamento diferente dependendo da razÃo de carga (n+/n-) na qual a nanopartÃculas foi sintetizada. Para valores de n+/n- < 0,8 os tamanhos de partÃculas sÃo similares aos observados para GAQTa e CMA0,20QTa, entretanto para n+/n- > 0,8 partÃculas maiores sÃo produzidas (~ 140 a 636 nm). O estudo de incorporaÃÃo e ensaio in vitro de liberaÃÃo de cloroquina foi realizado utilizando como matriz nanopartÃculas de CMA0,63QTa na razÃo n+/n- igual a 4 (431 nm) devido a esta apresentar maior eficiÃncia de encapsulamento do fÃrmaco. A nanopartÃcula incorporou 43 % de cloroquina. Em pH 5,8 a liberaÃÃo de cloroquina foi lenta, liberando apenas 20 % em 22 dias. Em pH 7,4 foi observado que a liberaÃÃo à mais rÃpida do que em pH 5,8 tendo liberado 84 % em 6 h, contudo a liberaÃÃo sà atinge o equilÃbrio em 10 dias. Analisando o tamanho de partÃcula e o comportamento da liberaÃÃo, observa-se que este sistema à promissor para administraÃÃo via oral deste fÃrmaco. A segunda rota de sÃntese foi via copolimerizaÃÃo radicalar por enxertia de Ãcido acrÃlico. A reaÃÃo foi realizada utilizando nitrato de amÃnio cÃrico (CAN) como iniciador e bis-acrilamida (MBA) como agente reticulante. Foram avaliados os efeitos da razÃo molar de unidade glicosÃdica/Ãcido acrÃlico (UG/AA) e AA/CAN no tamanho de partÃcula. As nanopartÃculas de goma do angico e Ãcido acrÃlico tiveram tamanhos variando de 6,4 a 19,4 nm dependendo da razÃo UG/AA e um rendimento mÃdio de 56,6 %.Diminuindo a quantidade de iniciador observa-se que o diÃmetro diminui, entretanto que a polidispersividade aumenta. NanopartÃculas sà com poli(Ãcido acrÃlico) apresentam tamanho superior aos valores observados em presenÃa de goma do angico (417 nm). Apesar da presenÃa de Ãcido acrÃlico as nanopartÃculas de GA/AA o tamanho da nanopartÃcula nÃo à sensÃvel ao pH do meio. A incorporaÃÃo de BSA em nanopartÃculas de GA/AA foi de 50 % sendo a liberaÃÃo da proteÃna bem lenta, tendo liberado apenas 69 % da BSA em 30 dias.

La synthèse d'inhibiteurs puissants et selectifs de glycosidases, enzymes impliquées dans de nombreux processus biologiques, permet d'accéder à des agents thérapeutiques potentiels. L'objectif de ce travail était de synthétiser de nouveaux analogues du salacinol, un puissant inhibiteur naturel d'alpha - glucosidases possédant une stucture originale zwitterionique, et d'évaluer les propriétés inhibitrices des composes obtenus sur six glycosidases commerciales. Nous avons donc préparé plusieurs iminosucres, analogues de la désoxynojirimycine et de l'acide pipécolique, par une méthode originale basée sur l'utilisation d'isoxazolines. par couplage de ces iminosucres ou de différents thiosucres avec deux sulfares cycliques, nous avons synthétisé six nouveaux analogues azotés et neuf nouveaux analogues soufrés du salacinol. L'un de ces analogues soufrés s'est révélé être un très bon inhibiteur de la béta - glucosidase d'amandes.

Ce travail de thèse décrit la préparation et l'étude des propriétés d'auto-assemblage de nouveaux amphiphiles fonctionnalisés par des sucres. Des glycosides propargyliques du lactose et de la N-acétyl-glucosamine ont été conjugués par chimie click (cycloaddition de Huisgen catalysée par des sels de cuivre) à des dérivés de poly(ethyleneglycol) dont une des extrémités a au préalable été modifiée par une fonction azide et l'autre par un bloc hydrophobe de type polyphénylène ou bien aliphatique. Après une caractérisation par résonance magnétique nucléaire et spectrométrie de masse, les propriétés d'auto-assemblage de ces amphiphiles ont été étudiées par diffusion dynamique de la lumière (DLS), diffraction des rayons-X aux petits angles (SAXS) et microscopie électronique. Il a été montré qu'en phase aqueuse, les systèmes amphiphiles dérivés du PEG 900 s'auto-assemblent pour former de micelles de taille extrêmement régulière dont le diamètre moyen est de l'ordre de 10 nm. La présence et la biodisponibilité des sucres à la surface de ces nanoparticules ont également pu être démontrées par diffusion dynamique de la lumière avec les lectines PNA et WGA. Les interactions spécifiques observées entre les lectines et micelles associées aux propriétés d'encapsulation de ce type de nanoparticules permettent d'imaginer de futures applications pour la délivrance de médicaments ou encore l'imagerie médicale.

Vibrio vulnificus is an opportunistic human and animal pathogen that is predominantly found in estuarine waters. In aquatic ecosystems, it colonizes filter-feeders, such as oysters, and has been found to form biofilms on the surface of various marine organisms, including plankton, algae, fish, eels, and crustaceans. The bacterium can spontaneously develop a rugose phenotype, which is associated with the production of polysaccharide(s) that impart a raised, wrinkled appearance to cells, copious biofilm formation, and increased stress resistance. Biofilm and rugose colony development, along with pellicle and aggregate formation, are believed to be crucial for the environmental survival and persistence of V. vulnificus. As the biosynthesis of polysaccharide(s) is a key feature linking these physiological processes, the main objectives of this study were to identify polysaccharide loci contributing to survival phenotypes in V. vulnificus and to gain insight into the regulation of these loci. Two polysaccharide loci (brp and rbd) were found to contribute to biofilm formation. The brp locus is regulated by the second messenger c-di-GMP and by at least two transcriptional regulators BrpR and BrpT. Lesions in glycosyltransferases in the locus or in either of the regulators abated the inducing effects of c-di-GMP on biofilm formation. The rbd locus is regulated not by c-di-GMP, but instead by a response regulator (RbdG) belonging to the TCRS family, which is encoded within the locus. The biofilms associated with the expression of the brp and rbd polysaccharides were structurally unique and simultaneous expression of both loci dramatically enhanced pellicle formation. Each locus also provides unique survival characteristics; the development of rugosity and stress resistance could be attributed to brp expression whereas rbd expression augmented aggregate formation. The ability of V. vulnificus to differentially regulate expression of the brp and rbd polysaccharides may allow the bacterium to “fine tune” its biofilm lifestyle to maximally benefit from the characteristics associated with each locus.

The use of polysaccharides as building blocks in the development of drugs and contrast agents delivery systems is rapidly growing. This can be attributed to the outstanding virtues of polysaccharides such as biocompatibility, biodegradability, upgradability, multiple reacting groups and low cost. The focus of this thesis was to develop and characterize novel starch based hydrogels and nanoparticles for delivery of drugs and imaging agents. To this end, two different systems were developed. The first system includes polymer and nanoparticles prepared by graft polymerization of polymethacrylic acid and polysorbate 80 onto starch. This starch based platform nanotechnology was developed using the design principles based on the pathophysiology of breast cancer, with applications in both medical imaging and breast cancer chemotherapy. The nanoparticles exhibited a high degree of doxorubicin loading as well as sustained pH dependent release of the drug. The drug loaded nanoparticles were significantly more effective against multidrug resistant human breast cancer cells compared to free doxorubicin. Systemic administration of the starch based nanoparticles co-loaded with doxorubicin and a near infrared fluorescent probe allowed for non-invasive real time monitoring of the nanoparticles biodistribution, tumor accumulation, and clearance. Systemic administration of the clinically relevant doses of the drug loaded particles to a mouse model of breast cancer significantly enhanced therapeutic efficacy while minimizing side effects compared to free doxorubicin. A novel, starch based magnetic resonance imaging (MRI) contrast agent with good in vitro and in vivo tolerability was formulated which exhibited superior signal enhancement in tumor and vasculature. The second system is a co-polymeric hydrogel of starch and xanthan gum with adjustable swelling and permeation properties. The hydrogels exhibited excellent film forming capability, and appeared to be particularly useful in controlled delivery applications of larger molecular size compounds. The starch based hydrogels, polymers and nanoparticles developed in this work have shown great potentials for controlled drug delivery and biomedical imaging applications.

Vibrio vulnificus is an opportunistic human and animal pathogen that is predominantly found in estuarine waters. In aquatic ecosystems, it colonizes filter-feeders, such as oysters, and has been found to form biofilms on the surface of various marine organisms, including plankton, algae, fish, eels, and crustaceans. The bacterium can spontaneously develop a rugose phenotype, which is associated with the production of polysaccharide(s) that impart a raised, wrinkled appearance to cells, copious biofilm formation, and increased stress resistance. Biofilm and rugose colony development, along with pellicle and aggregate formation, are believed to be crucial for the environmental survival and persistence of V. vulnificus. As the biosynthesis of polysaccharide(s) is a key feature linking these physiological processes, the main objectives of this study were to identify polysaccharide loci contributing to survival phenotypes in V. vulnificus and to gain insight into the regulation of these loci. Two polysaccharide loci (brp and rbd) were found to contribute to biofilm formation. The brp locus is regulated by the second messenger c-di-GMP and by at least two transcriptional regulators BrpR and BrpT. Lesions in glycosyltransferases in the locus or in either of the regulators abated the inducing effects of c-di-GMP on biofilm formation. The rbd locus is regulated not by c-di-GMP, but instead by a response regulator (RbdG) belonging to the TCRS family, which is encoded within the locus. The biofilms associated with the expression of the brp and rbd polysaccharides were structurally unique and simultaneous expression of both loci dramatically enhanced pellicle formation. Each locus also provides unique survival characteristics; the development of rugosity and stress resistance could be attributed to brp expression whereas rbd expression augmented aggregate formation. The ability of V. vulnificus to differentially regulate expression of the brp and rbd polysaccharides may allow the bacterium to “fine tune” its biofilm lifestyle to maximally benefit from the characteristics associated with each locus.

No description available.