Orientador: Aulus Cavalieri Carciofi / Resumo: Derivados da parede celular de levedura têm sido estudados por seu efeito prebiótico. Recentemente, preparações purificadas e mais solúveis foram desenvolvidas, com vista a aumentar seu efeito fisiológico. Este estudo teve por objetivo avaliar os efeitos da inclusão de dois diferentes extratos da parede celular de leveduras, um convencional (PCL) e outro com maior porcentagem de mananoligossacarídeos solúveis (PCLs) sobre a digestibilidade, produtos de fermentação nas fezes e alguns parâmetros imunológicos de cães adultos hígidos. Foi empregada uma única formulação, desdobrada em três tratamentos: CON – controle, sem adição de parede celular de levedura; PCL – adição de 0,3% de parede celular de levedura convencional; PCLs - adição de 0,3% de parede celular de levedura com elevada solubilidade de mananoligossacarídeos (MOS). Foram utilizados 24 cães beagle adultos, com oito repetições por ração, em delineamento em blocos casualizados. Cada bloco teve duração 30 dias, sendo avaliados no início e no final do período as concentrações séricas de fator de necrose tumoral alfa e interleucinas 6 e 10, produção ex vivo de intermediários reativos do oxigênio e nitrogênio por células mononucleares e polimorfonucleares de sangue periférico, capacidade fagocítica de monócitos e neutrófilos e concentração de IgA nas fezes. Adicionalmente, foi avaliada a digestiblidade aparente dos nutrientes, produção e qualidade das fezes, ácidos graxos de cadeia curta e ramificada, lactato, amônia, pH e... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Derivates of yeast cell wall have been studied by its prebiotic effect. Recently, more purified and soluble preparations were developed, attempting to increase their biological actions. This study evaluated the inclusion of two yeast cell wall preparations, one conventional (YCW), and another with higher solubility of the mannan oligosaccharide fraction (YCWs), on their effects on nutrient digestibility, fermentation products on feces, and some immunological parameters of dogs. A single food formulation was used, unfolded on the following treatments: CON – control, without yeast cell wall addition; YCW – addition of 0.3% of a conventional yeas cell wall extract; YCWs – addition of 0.3% of a yeast cell wall extraction with elevated mannan oligosaccharides solubility. Twenty-four adult beagle dogs were used, eight dogs per food, distributed on a randomized block design. Blocks lasted 30 days, and tumor necrosis factor alpha and interleukins 6 and 10, ex vivo production of hydrogen peroxide and nitric oxide by peripheral neutrophils and monocytes, phagocytic index, and fecal IgA were evaluated at the beginning and ending of each period. Additionally, nutrient digestibility, feces production and quality, and fermentation products, pH, and biogenic amines were quantified on feces. Results were evaluated by variance analysis and compared by Tukey test (P<0.05). For the immunological parameters, the basal values were used as a covariate. The inclusion of YCWs reduced fat digestibili... (Complete abstract click electronic access below) / Mestre

Orientador: Janete Apparecida Desidério / Banca: Sonia Marli Zingaretti / Banca: Liliane Marcia Mertz Henning / Resumo: Com o atual cenário de mudanças climáticas, observa-se a tendência a eventos de seca mais longos e recorrentes, desta forma a obtenção de plantas mais tolerantes à seca figura como um dos principais investimentos dentro da ciência e tecnologia nacional. Os oligossacarídeos da família das rafinoses (RFOs) desempenham múltiplas funções nas plantas e sabe-se que estes são acumulados nos tecidos vegetais em situações de déficit hídrico, garantindo a estabilidade das membranas celulares, consequentemente mantendo as funções vitais da planta. Por sua vez, a enzima galactinol sintase (GolS, EC 2.4.1.123), catalisa o primeiro passo na biossíntese dos oligossacarídeos dos RFOs desempenhando um importante papel regulador na partição do carbono entre sacarose e RFOs. Desta forma, o objetivo do presente trabalho foi introduzir em soja, via Agrobacterium tumefaciens, a construção gênica 35S:AtGolS2 e caracterizar molecularmente e fisiologicamente os eventos obtidos sob déficit hídrico. Para o processo de transformação, a cultivar convencional de soja BRS 184 foi utilizada e os eventos obtidos foram caracterizados quanto ao número de cópias através da técnica de qPCR. Para a análise da expressão gênica constitutiva o RNA total dos eventos, em condições bem irrigadas, foi extraído e a expressão determinada via RT-qPCR. A taxa de segregação foi calculada através do teste do X2 (p≤ 0.05). Com base nos resultados obtidos, dois eventos (2Ia1 e 2Ia4) foram selecionados para serem analisados quanto a respostas moleculares e fisiológicas sob déficit hídrico induzido em condições de casa de vegetação. Os resultados mostraram que nas plantas do evento 2Ia4 o maior acumulo de água associado a menor área foliar na condição controle levou a manutenção das trocas gasosas causado pela redução na transpiração foliar, maior acúmulo de água no substrato e acúmulo de transcritos de rafinose e galactinol nos tecidos / Abstract: With the current scenario of climate change, there is a tendency to longer and recurrent drought events, thus obtaining more drought tolerant plants figure as a major investment in the national science and technology. Raffinose family oligosaccharides (RFOs) plays multiple functions in plants and it is known that these are accumulated in plant tissues in water deficit situations, guaranteeing the stability of cell membranes, thus maintaining the vital functions of the plant. In turn, galactinol synthase (GolS, EC 2.4.1.123) catalyzes the first step in the biosynthesis of RFOs plays an important regulatory role in carbon partitioning between sucrose and orphans. Thus, our objective was to introduce gene construction 35S:AtGolS2 via Agrobacterium tumefaciens in soybean plants and characterize molecularly and physiologically events obtained under water deficit. In this context, the conventional soybean BRS 184 was used in the transformation process and the soybean events were molecularly characterized in regard to the transgene copy number by qPCR technique. For the analysis of constitutive gene expression total RNA of events, well-watered conditions, was extracted and the expression determined by RT-qPCR. The segregation rate was calculated using the X2 test (p ≤ 0.05). Based on our results, two events (2Ia1 and 2Ia4) were selected to be analyzed for physiological responses under drought simulated under greenhouse conditions. The results showed that the plants 2Ia4 event the largest accumulation of water associated with lower leaf area in the control condition led to maintenance of gas exchange caused by the reduction in leaf transpiration, increased water accumulation in the substrate and accumulation of raffinose and galactinol transcripts in tissues. Thus, the increased levels of these carbohydrates would have made these act as osmoprotectors, enabling the recommendation of 2Ia4 plants breeding programs aimed at tolerance to drought / Mestre

Glucansucrases são enzimas que atuam em reações de síntese de polissacarídeos e oligossacarídeos. Para que esses biocatalisadores sejam aplicados em escala industrial, é desejável ótimas estabilidades térmica e operacional, o que pode ser alcançado com a imobilização de enzimas. Como alternativa aos suportes sólidos amplamente estudados, está a quitosana, polímero que não apresenta toxicidade e possui alta biocompatibilidade e alta afinidade com proteínas. Outra possibilidade promissora na imobilização de enzimas, é a síntese dos agregados enzimáticos entrecruzados (CLEAs), os quais apresentam alta atividade catalítica e alta estabilidade. Contudo, uma peculiaridade das glucansucrases quando produzidas em meio contendo sacarose é a camada de polímero que as envolve, e que bloqueia o acesso aos grupos reativos na superfície da proteína. No caso da expressão heteróloga das glucansucrases em Escherichia coli essa dificuldade pode ser contornada. Além disso, o uso da mutagênese sítio-dirigida pode proporcionar modificações de aminoácidos na superfície da enzima, tais como os resíduos Lys, Cys, His, com o intuito de que melhorias na imobilização sejam alcançadas. Sendo assim, na primeira etapa desse trabalho, uma extensa discussão é apresentada em relação às metodologias de imobilização de dextransucrase encontradas na literatura. A seguir, estudos referentes à imobilização da dextransucrase de Leuconostoc mesenteroides B-512 F em esferas de quitosana ativadas com glutaraldeído foram realizados. Esse imobilizado apresentou alta atividade catalítica (197 U/g) quando utilizada a carga de proteína de 400 mg/g de suporte. Além disso, observou-se que a imobilização covalente e os açúcares maltose e glicose promoveram proteção à enzima em temperaturas de 40 ºC e 50 ºC. Na etapa seguinte, a produção e a caracterização de CLEAs de dextransucrase de L. mesenteroides B-512 F foram investigados. Demonstrou-se que o tratamento com a dextranase foi essencial para a imobilização da glucansucrase e que o isopropanol foi o melhor agente precipitante. Os CLEAs apresentaram pH e temperatura ótimos de 3,0 e 60 ºC, respectivamente, enquanto que a dextransucrase imobilizada nas esferas de quitosana funcionalizada com glutaraldeído apresentaram os valores de 4,5 e 20 ºC. Ambas formas imobilizadas apresentaram boa estabilidade operacional na síntese de oligossacarídeos uma vez que após 10 ciclos, 40 % de atividade residual foi observada. Por fim, estão apresentados estudos sobre a modelagem das estruturas tridimensionais e a mutagênese sítio-dirigida das glucansucrases DSR-S vardel Δ4N and ASR C-APY del. Os modelos preditos demonstraram boa qualidade e a mutagênese sítio-dirigida não promoveu perdas significativas na atividade enzimática dos mutantes. Somente o mutante DSR_S326C mostrouse inativo. Os resultados obtidos sugerem que a imobilização da dextransucrase foi satisfatória e que cada técnica possibilita diferentes características ao imobilizado. Além disso, os imobilizados foram adequados para síntese de dextrana e oligossacarídeos. / Glucansucrases are enzymes that catalyze the synthesis of polysaccharides and oligosaccharides. In order to assure continuous processing and reuse of the biocatalyst in industrial applications, enzyme immobilization techniques are required to promote good thermal and operational stabilities. Among the several solid supports for enzyme immobilization, chitosan shows interesting properties because it is non-toxic, it is biocompatible, and it has high protein affinity. Other possibility is the production of cross-linked enzyme aggregates (CLEAs), which presents high catalytic activity and good stability. However, glucansucrases have a particularity when produced in sucrose medium, since a polymer layer surrounds the protein, blocking the access to reactive groups on the enzyme surface. To overcome this problem, it is possible to make the heterologous production of glucansucrases in Escherichia coli. Likewise, the site-directed mutagenesis may promote changes in the amino acids located on the surface to improve immobilization parameters. Therefore, this work aimed to discuss the several techniques applied for dextransucrase immobilization, and to design new immobilized biocatalysts. In a first step, it is presented a review about the distinct immobilization methodologies for dextransucrase. In a second study, an investigation about dextransucrase from Leuconostoc mesenteroides B-512 F immobilized on glutaraldehyde-activated chitosan particles was carried out. The novel immobilized biocatalyst showed 197 U/g (400 mg/g dried support) of catalytic activity. The covalent immobilization promoted protection against enzyme damages at 40 ºC and 50 ºC, whereas maltose and glucose acted as stabilizers. Furthermore, it was studied the production and characterization of CLEAs dextransucrase from L. mesenteroides B-512 F. It was demonstrated that dextranase treatment was crucial for immobilization. Isopropanol was chosen as the best precipitant agent. CLEAs presented optimal pH and temperature of 3.0 and 60 ºC, respectively, whereas it was found values of 4.5 e 20 ºC for dextransucrase immobilized on glutaraldehyde-activated chitosan particles. Both immobilized biocatalysts showed good operational stability in the oligosaccharides synthesis, exhibiting 40 % of residual activity after 10 cycles. Finally, the study concerning the homology modeling and site-directed mutagenesis of glucansucrases DSR-S vardel Δ4N and ASR C-APY del is presented. The predicted models showed good quality and it has been demonstrated that the site-directed mutagenesis did not promote significant losses in the variant enzyme activities. Only one mutant (DSR_S326C) had shown no dextransucrase activity. The results obtained in this work suggest that the immobilization of dextransucrase was satisfactory, also showing that each technique promotes different characteristics to the immobilized biocatalyst. Besides, these immobilized enzymes were feasible for the synthesis of dextran and oligosaccharides.

Currently, the production of palm oil leads to the destruction of rainforest. A more sustainable source of lipids could be obtained using abundant lignocellulosic waste (e.g. wheat straw) as a source of carbon in the form of polysaccharides. Some species of oleaginous yeast, grown on sugars, can be made to accumulate between 20-80% of their biomass as oil and so offer a promising alternative to terrestrial crops. In this thesis, the yeast Metschnikowia pulcherrima was selected for its resilience to contamination. Although not previously classified as oleaginous, a combination of low temperature and restricted nutrient availability prevented sporulation and consequently triggered levels of oil production in M. pulcherrima cultures of up to 47%. The potential of this yeast to produce lipids inexpensively on waste resources was investigated. This yeast was grown under non-sterile conditions at pilot scale with minimal temperature control. The possibility of growing M. pulcherrima on lignocellulose was studied on models and showed that it was tolerant to a range of sugars and inhibitors commonly found in hydrolysed lignocellulose. The yeast produced 6.04 g L-1 lipid when cultivated on a mixture of hexoses and pentoses. This was corroborated by demonstrating that the yeast could be cultured on oligomers and sugars produced by hydrolysing wheat straw. Evidence of cellulase production was observed, and this was utilised in a process involving mildly pretreated wheat straw, using a range of pretreatment processes and culture conditions to yield a maximum of 1.12 g L-1 lipid. The usefulness of the products of this cultivation in forming oil in water emulsions was evaluated, and some evidence of surface-active effects was found. Overall, M. pulcherrima was found to have phenotypes that would be highly beneficial in reducing the capital and running costs of a putative lipid production process.

Four phytopathogenic fungi were cultivated up to six days in media containing chitooligosaccharide mixtures differing in average DP and FA. The three different mixtures were named Q3 (which contained oligosaccharides ofDP2–DP10, withDP2–DP7 asmain components), Q2 (which contained oligosaccharides of DP2–DP12, with DP2–DP10 as main components) and Q1 (which derived from Q2 and contained oligomers of DP5–DP8 with hexamer and a heptamer as the main components). The novel aspect of this work is the description of the effect of mixtures of oligosaccharides with different and known composition on fungal growth rates. The growth rate of Alternaria alternata and Rhizopus stolonifer was initially inhibited by Q3 and Q2 at higher concentrations. Q1 had a growth stimulating effect on these two fungi. Growth of Botrytis cinerea was inhibited by Q3 and Q2, while Q1 had no effect on the growth of this fungus. Growth of Penicillium expansum was only slightly inhibited by higher concentrations of sample Q3, while Q2 and Q1 had no effect. The inhibition of growth rates or their resistance toward chitooligosaccharides correlated with the absence or presence of chitinolytic enzymes in the culture media, respectively.

The ability to produce monomolecular coatings with well-defined structural and functional properties is of key importance in biosensing, drug delivery, and many recently developed applications of nanotechnology. Organic chemistry has proven to be a powerful tool to achieve this in many research areas. Herein, we present the synthesis of three oligo(lactosides) glycosylated in a (1 → 3) manner, and which are further functionalized with amide-linked short alkanethiol spacers. The oligosaccharides (di-, tetra-, and hexasaccharide) originate from the inexpensive and readily available lactose disaccharide. These thiolated derivatives were immobilized onto gold surfaces, and the thus formed self-assembled monolayers (SAMs) on planar gold were characterized by wettability, ellipsometry and infrared reflection–absorption spectroscopy. Further, the ability of these SAMs to stabilize gold nanoparticles in saline solutions was also demonstrated, indicating that the oligosaccharides may be used as stabilizing agents in gold nanoparticle-based assays.

No description available.

Thesis (Ph. D.)--Institute of Paper Chemistry, 1966. / Bibliography: leaves 90-92.

Several innovative tandem mass spectrometric strategies have been developed for the structural determination and isomer differentiation of glycosides and oligosaccharides. Specifically, collisionally activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) are used in conjunction with derivatization methods designed to exploit variations in binding energies or attach chromophores. These include metal complexation incorporating modified neutral auxiliary ligands and covalent derivatization involving site-specific reactions. The elucidation of flavonoid isomers is accomplished by electrospray ionization tandem mass spectrometry (ESI-MS/MS) via formation and CAD of metal/flavonoid complexes containing an auxiliary ligand. Addition of a metal salt and a suitable neutral auxiliary ligand to flavonoids in solution results in the formation of [M(II) (flavonoid-H) ligand]⁺ complexes by ESI which, upon collisional activated dissociation, often result in more distinctive fragmentation patterns than observed for conventional protonated or deprotonated flavonoids. We compare and explore the use of alternative pyridyl ligands, with electron-releasing substituents including 4,7-diphenyl-1,10-phenanthroline. Using this technique, three groups of flavonoid glycoside isomers are differentiated, including glycosides of apigenin, quercetin and luteolin. A tunable ESI-MS/MS strategy for differentiation of flavone and flavanone diglycoside isomers based on metal complexation with auxiliary ligands that have electron-withdrawing substituents is reported. A series of auxiliary ligands with electronwithdrawing substituents was synthesized in order to tailor the relative metal binding affinities of the ligands and thus directly influence the stabilities, and consequently the dissociation pathways, of the complexes. Upon collisionally activated dissociation, the complexes yield fragmentation patterns in which the abundances of key diagnostic ions are enhanced, thus facilitating isomer differentiation. A strategy for increasing the efficiency of IRMPD in a quadrupole ion trap (QIT) based on another metal complexation strategy is described. Two IR-active ligands (IRALs) that have an IR-active phosphonate functional groups were synthesized. The IR-active groups were therefore incorporated into the analyte complexes via metal complexation. We demonstrate this new IRMPD approach for the structural characterization of flavonoids. The fragment ions obtained by IRMPD are similar to those obtained by CAD and allow facile isomer differentiation of flavonoids. Fourier transform infrared absorption attenuated total reflectance (FTIR-ATR) and energyvariable CAD experiments indicate that the high IRMPD efficiencies stem from the very large IR absorptivities of the IR-active ligands. A simplified method for determining the sequence and branching of oligosaccharides using IRMPD in a QIT is described. An IR-active boronic acid (IRABA) reagent was synthesized and subsequently used to derivatize the oligosaccharides prior to IRMPD analysis. The IRABA ligand is designed to both enhance the efficiency of the derivatization reaction and to facilitate the photon absorption process. The resulting IRMPD spectra display oligosaccharide fragments that are formed from primarily one type of diagnostic cleavage, thus making sequencing straightforward. The presence of sequential fragment ions, a phenomenon of IRMPD, permit the comprehensive sequencing of the oligosaccharides studied in a single stage of activation. The approach is demonstrated for two series of oligosaccharides, the lacto-Nfucopentaoses (LNFPs) and the lacto-N-difucohexaoses (LNDFHs).

Lipochitooligosaccharides (LCOs) are molecules secreted by rhizobia during the establishment of rhizobia-legume N2-fixing symbiosis. Some recent reports have shown that there are physiological effects on host and non-host rhizobia after LCO treatment. However, the cellular mechanisms underlying these observations are not known. Therefore, the study was aimed at assessing phenotypic changes by measuring photosynthesis, leaf area and dry weight on a non-host plant (tobacco). Our results did not show any significant physiological changes following LCO treatment. We also wanted to explore the molecular basis of changes in the plant cell by looking at gene and protein profiling following LCO treatment in a natural host plant (soybean) using real-time RT-PCR and SDS-PAGE. To do so, a reproducible stimulation method for soybean seed germination by LCO was successfully developed. The results obtained on soybean did not reveal significant differences in gene expression between water and LCO-treated seeds for the genes cdc2, WASI, ICL1 and 14-3-3 studied. In addition, LCO treatment did not change the protein profile compared to the water control in a significant way.