Дисертації з теми "(1,3;1,4)-beta-glucan"

An enzymatic assay designed for measurement of $\beta$-glucan in barley was modified to allow measurement of total $\beta$-glucan content in oats by manipulating the grinding and incubation protocol. Using the modified enzymatic assay, the range of genetic and environmental variation of $\beta$-glucan content in Canadian domestic and breeder's lines of oats was assessed using several cultivars grown in 5 locations in eastern Canada in 3 growing seasons. Analysis of variance indicated that the predominant source of variation was genetic. A second assay using Flow Injection Analysis (FIA) to measure $\beta$-glucan was also evaluated. Although a high correlation was observed for the results of the two methods (r = 0.90), the results obtained using FIA tended to be somewhat lower than those obtained using the modified enzymatic assay: the enzymatic assay was judged to be more accurate for estimation of total $\beta$-glucan in oats. Nevertheless, because of its greater speed and simplicity, FIA would be a valuable screening tool for routine-applications. Using the enzymatic assay, $\beta$-glucan content was also measured in 18 primitive species of Avena to evaluate possible sources of germplasm for expanding the range of $\beta$-glucan content currently available in domestic cultivars. A comparison of $\beta$-glucan content with protein content, oil content and thousand kernel weight in domestic oats showed that these quality parameters are independent of $\beta$-glucan concentration in oats. Scanning microspectrofluorometry was used to map $\beta$-glucan distribution in single kernels of oats: differences were observed within single kernels, and also among kernels from different cultivars of oats. Microscopic examination suggests that the different distribution patterns are due to differences in cell wall thickness adjacent to the germ and around the periphery of the kernel, and also to differences in cell size and shape in the central endosperm. A high $\beta$-glucan (Marion) and a low $\beta$-glucan oat cultivar (OA516-2) were selected for isolation and preliminary characterization of the endosperm cell walls, which are the major source of $\beta$-glucan in the oat kernel. It was concluded that the differences in $\beta$-glucan content that were observed in whole groats were not due to differences in the composition of isolated endosperm cell walls, but to variation in cell size and cell wall thickness in different areas of the groats. (Abstract shortened by UMI.)

This research was conducted to determine the potential of hulless winter barley (Hordeum vulgare L.) as an improved feed crop in the mid-Atlantic region. Winter barley is an excellent crop in rotation with soybean (Glycine max L.); however, production of winter barley during the past few years has decreased mainly due to low market prices, even though the mid-Atlantic region is a feed grain deficient area. Therefore, value added traits need to be developed in order for barley production to continue in the region. In the first part of this study, the objectives were to: (i) evaluate the agronomic performance and potential of six experimental hulless winter barley lines compared with two commercial hulled cultivars; (ii) determine and compare fiber, b-glucan, protein, and fat concentrations, and true metabolizable energy, corrected for nitrogen (TMEn) among these genotypes; and (iii) evaluate the genetic potential of winter hulless barley accessions from the world collection for use as parents in hulless breeding programs. Six hulless lines all derived from the cross VA75-42-45/SC793556//CI2457 were acquired from Clemson University in South Carolina. The six lines were evaluated for yield, test weight, heading date, plant height, and lodging. These hulless lines along with two hulled cultivars were planted in replicated yield plots in four states with a total of eight locations, and were managed according to standard recommended practices. Grain from each of the hulless lines and hulled checks, along with that of Trical 498 triticale (X Triticosecale) and Jackson wheat (Triticum aestivum L.) were analyzed for fiber, b-glucan, fat, protein, and ash concentration, and TMEn value. Eight hundred and seven winter or facultative habit hulless barley lines were obtained from the USDA-ARS National Small Grains Collection in Aberdeen, ID. These lines were screened for reaction type to races 8 and 30 of barley leaf rust (Puccinia hordei) and to a composite population of powdery mildew (Blumeria graminis f. sp. hordei). These accessions also were planted in observation rows to evaluate heading date, plant height, lodging, and seed threshability. The hulless lines yielded 23% less, but had 13% higher test weights than the hulled check cultivars. There was no difference between hulled and hulless barley in heading date and plant height. Hulless lines had a higher protein and lower fiber concentration than hulled barley. They also had higher b-glucan and fat concentrations than triticale or wheat. TMEn was similar between hulled and hulless barley, triticale, and wheat. Approximately 100 hulless barley lines from the world collection were selected for potential use as parents among 800 accessions tested, based on evaluations of lodging, plant height, threshability, and seed color. In the second part of the study the objectives were to determine the effects of (i) hulled and hulless barley, and (ii) b-glucanase on the performance of broilers fed different diets from 21 to 42 days of age. Diets comprised of 30% hulless or hulled barley, and a standard corn (Zea mays)/soybean meal diet with and without b-glucanase enzyme were evaluated to determine the effects of barley on gut viscosity, carcass weight, gain, percent shell, and feed efficiency in 21 to 42 day old broilers. In the first year, diets comprised of hulless lines SC890573 and SC860972, and the hulled cultivar Callao were compared to a standard check diet. In the second year SC860972 was replaced with SC880248 due to the inability to secure a sufficient amount of seed. Each year one hulled and two hulless barley diets were compared to a standard diet. Each diet was fed with and without enzyme, for a total of eight diets. Broilers 21 days of age were fed the diets until day 42 when they were processed. There was a significant decrease (P<0.05) in gut viscosity of birds fed diets with enzyme compared to birds fed diets without enzyme; however, gut viscosity did not affect weight gain or percent shell. Barley substituted at the 30% level did not have a significant effect on broiler performance, nor did the addition of enzyme. Absence of enzyme effect was attributed to bird age, since older birds are able to hydrolyze b-glucan more effectively than juveniles. The potential of hulless barley as an improved feed source for the poultry and swine industry is great for the mid-Atlantic region. Increases in grain yield are currently being realized through focused breeding efforts, and hulless lines exhibit positive nutritional components that combine favorable attributes of both wheat and hulled barley. Barley substituted at the 30% level in the diets of broilers did not cause any detrimental effects. Addition of hulless barley may potentially lead to a reduction in cost per pound of gain of broilers, and provide an alternative crop for mid-Atlantic region grain producers and feeders.
Ph. D.

Doctor of Philosophy
Department of Biochemistry
Ramaswamy Krishnamoorthi
Insect [beta]-1,3-glucan recognition protein ([beta]GRP), a soluble receptor in the hemolymph, binds to the surfaces of bacteria and fungi and activates serine protease cascades that promote destruction of pathogens by means of melanization or expression of antimicrobial peptides. Delineation of mechanistic details of these processes may help develop strategies to control insect-borne diseases and economic losses. Multi-dimensional nuclear magnetic resonance (NMR) techniques were employed to solve the solution structure of the Indian meal moth (Plodia interpunctella) [beta]GRP N-terminal domain (N-[beta]GRP), which is sufficient to activate the prophenoloxidase (proPO) pathway resulting in melanin formation. This is the first determined three-dimensional structure of N-[beta]GRP, which adopts an immunoglobulin fold. Addition of laminarin, a [beta]-1,3 and [beta]-1,6 link-containing glucose polysaccharide (∼6 kDa) that activates the proPO pathway, to N-[beta]GRP results in the loss of NMR cross-peaks from the backbone [subscript]1[subscript]5N-[subscript]1H groups of the protein, suggesting the formation of a large complex. Analytical ultracentrifugation (AUC) studies of formation of the N-[beta]GRP:laminarin complex show that ligand binding induces self-association of the protein-carbohydrate complex into a macro structure, likely containing six protein and three laminarin molecules (∼102 kDa). The macro complex is quite stable, as it does not undergo dissociation upon dilution to submicromolar concentrations. The structural model thus derived from this study for the N-[beta]GRP:laminarin complex in solution differs from the one in which a single N-[beta]GRP molecule has been proposed to bind to a triple-helical form of laminarin on the basis of a X-ray crystal structure of the N-[beta]GRP:laminarihexaose complex. AUC studies and phenoloxidase activation measurements made with designed mutants of N-[beta]GRP indicate that electrostatic interactions between the ligand-bound protein molecules contribute to the stability of the N-[beta]GRP:laminarin complex and that a decreased stability results in a reduction of proPO activation. These novel findings suggest that ligand-induced self-association of the [beta]GRP:[beta]-1,3-glucan complex may form a platform on a microbial surface for recruitment of downstream proteases, as a means of amplification of the pathogen recognition signal. In the case of the homolog of GNBPA2 from Anopheles gambiae, the malaria-causing Plasmodium carrier, multiligand specificity was characterized, suggesting a functional diversity of the immunoglobulin domain structure.

Pre-harvest glyphosate is applied to cereal grains to remove weeds. However, it has been claimed that oat compositions are affected by pre-harvest glyphosate. Research was conducted to evaluate differences in properties of β-glucan in the treated versus untreated oat groats. Two oat cultivars (Rockford and Souris) were grown at Minot and Prosper, North Dakota in 2015, and glyphosate was sprayed during the soft dough stage, physiological maturity stage, or not applied. β-Glucan viscosity was not significantly (p > 0:05) affected by treatment at soft dough or physiological maturity stages. Use of glyphosate at the soft dough stage significantly (p < 0:05) reduced the percentages of β-glucan content and solubility versus untreated samples. Treatment at soft dough and physiological maturity stages significantly (p < 0:05) increased β-glucan molecular weights compared to untreated controls. Therefore, glyphosate can be applied at the physiologically mature stage of grain development because β glucan properties from the groats were not negatively affected.

The goal of this research was the development of a method to extract pure (1,3/1,6)-β-glucans from Saccharomyces cerevisiae. These β-glucans are of pharmaceutical importance because an animal’s immune system can recognize glucan molecules, and these molecules can act as immunomodulators, essentially turning on the immune system. The problem in the past has been that previously published methods produce β-glucans with low side chain lengths and few branching occurrences. This issue was tackled by a multivariable approach that reduced extraction steps, initial sample size, and concentrations of reagents used. This method has been shown to produce greater yields of β-glucans while maintaining high purity. Analyses such as 1H-NMR and GC-MS have been used to confirm the content of the extracted glucans. Ideally, this research will generate interest for further β-glucan studies and ultimately be utilized pharmacologically with immunocompromised individuals.

Coccidiosis is a costly parasitic disease to the poultry industry with multiple prevention methods being explored to control its impact. One approach under development is the use of -glucans, which are carbohydrates from cell walls of various plant species. The first study evaluated the feeding effects of algae- derived -glucans on performance and responses of broilers during a coccidiosis challenge. Cobb 500 broilers (n=1280) were fed a control diet, control supplemented with 150 g/MT Algamune (BG), 100 g/MT Algamune ZPC (BGZn), or 0.01% Salinomycin (Sal). On d 15, challenged birds received mixed Eimeria inoculum. Measurements were taken on d 7, 14, 21, and 28, and lesion scores assessed on d 21. The challenge resulted in reduced BW, and higher feed conversion ratio (FCR) was observed in the challenged birds with Sal and BGZn. Escherichia coli (E. coli) is normally commensal to the gastrointestinal tract, but certain serotypes cause disease in domestic poultry. A subsequent study was conducted to evaluate the feeding effects of algae-derived glucan (1,3 -glucan) on performance of broiler chickens during an E. coli challenge. Cobb 500 broilers (n=900) were fed a control diet, control + 25 mg/kg of -glucan, or control + 100 mg/kg of -glucan. On d 0, litter was sprayed with E. coli inoculum. Measurements were taken on d 7, 14, 21, and 28. -glucan supplementation increased BW gain andlowered FCR. The results from these studies offer some insight to the effects of -glucans on poultry and their potential to offset negative effects caused by infectious challenges.
Master of Science

O objetivo deste trabalho foi avaliar os efeitos da inclusão de leveduras, na forma de parede celular e hidrolisada, na dieta de poedeiras sobre o desempenho produtivo, qualidade dos ovos e viabilidade econômica da suplementação destes aditivos. Dois experimentos foram realizados, para cada um deles foram alojadas 256 poedeiras distribuídas em delineamento inteiramente casualizado em 8 repetições com 8 aves. Foram avaliados os níveis de 0, 225, 450 e 900 g/t de parede celular de levedura na dieta das aves durante o período de 21 a 67 semanas de idade. Os resultados demonstraram um aumento no consumo de ração, produção e massa de ovos, além de uma maior altura do albúmen e unidade Haugh. A espessura da casca e a cor da gema também foram influenciadas pelos tratamentos. A análise da viabilidade econômica demonstrou que mesmo despendendo mais com a alimentação houve um aumento da margem bruta média, devido ao aumento da produção de ovos. Portanto, o uso da parede celular de levedura apresentou efeitos benéficos sobre o desempenho produtivo das poedeiras, como uma melhora na qualidade interna e externa dos ovos, aliado a uma maior rentabilidade da atividade. Para a levedura hidrolisada foram avaliados os níveis de 0, 1, 2 e 4 kg/t. Dois períodos foram analisados, 21 a 40 e 48 a 67 semanas de idade. A adição de levedura hidrolisada na dieta de poedeiras não apresentou melhoria das características analisadas no primeiro período, porém os benefícios surgiram a partir do segundo período. O uso da levedura além de gerar uma menor quantidade de ovos não comerciais, promoveu o aumento do consumo de ração, produção, massa e peso dos ovos, melhoria na qualidade dos ovos em relação a unidade Haugh, altura do albúmen, espessura e resistência da casca; e ainda melhorou a conversão alimentar por massa e dúzia de ovos. A suplementação de levedura hidrolisada para poedeiras proporcionou incremento da margem bruta total média em todos os níveis testados, demonstrando-se viável economicamente. A inclusão de levedura hidrolisada na dieta de poedeiras de 48 a 67 semanas de idade exerceu efeito maximizador do desempenho produtivo e qualidade de ovos, sendo economicamente viável seu uso.
The purpose of this study was to evaluate the effects of including yeast in the form of cell wall and hydrolyzed, in the diet of laying hens on production performance, egg quality and economic viability of these feed additives. Two experiments were conducted, for each of them 256 laying hens were housed and distributed in a completely randomized design with 8 repetitions of 8 birds. The level of 0, 225, 450 and 900 g/t of yeast cell wall were evaluated in the diet of birds from 21 to 67 weeks of age. The results showed an increase in feed intake, egg production and mass as well as a higher albumen height and Haugh unit. The shell thickness and yolk color were also affected by treatments. The economic viability analysis demonstrated that even spending more on feed an increase in the average gross margin due to increased egg production was observed. Therefore, the cell wall yeast showed a beneficial effect on production performance of laying hens, represented by an improvement of internal and external egg quality, combined with higher profitability. Hydrolyzed yeast was evaluated at the levels of 0, 1, 2 and 4 kg/t. Two periods were analyzed, 21-40 and 48-67 weeks of age. The addition of hydrolyzed yeast in the diet of laying hens did not show improvement of the analyzed characteristics in the first period, however the benefits occurred in the second period. The use of yeast although generate a minor amount of non-commercial eggs, promoted an increase in feed consumption, production, egg mass and weight, improving quality of eggs in relation to Haugh unit, albumen height, shell thickness and resistance; and also improves the feed conversion by eggs mass and dozen. The supplementation of hydrolyzed yeast for laying hens provided increase overall average gross margin on all tested levels, demonstrating to be economically viable. The inclusion of hydrolyzed yeast in the diet of laying hens from 48-67 weeks of age maximized production performance and egg quality, being its use economically viable.

Resumo: Em peixes, o 'beta'- glucano apresenta uma potente função imunoestimulante sendo cada vez maior a sua utilização como suplemento alimentar, aumentando significantemente a resistência à exposição infecciosa. Este prebiótico tem função de prevenir a colonização de patógenos por intensificar a ativação de macrófagos, proporcionando benefícios ao trato gastrointestinal e resultando em melhor desempenho e resistência a doenças. Este estudo teve a finalidade de avaliar os efeitos do 'beta' - glucano adicionado às rações peletizadas e extrusadas sobre o desempenho produtivo, indicadores de estresse, perfil hematológico e sobrevivência do pacu. Este experimento foi conduzido no Laboratório de peixes ornamentais, do Centro de Aqüicultura da UNESP (CAUNESP), em Jaboticabal, São Paulo. Foram utilizados 640 juvenis de pacu, com 24,7 ± 2,0 g, distribuídos em 32 aquários de vidro (130L). Os parâmetros físico - químicos da água foram mensurados quinzenalmente. Os peixes foram alimentados duas vezes ao dia, uma pela manhão e outra ao fim do dia. Os experimentos apresentaram um delineamento experimental inteiramente casualizado em esquema fatorial 2 x 4, utilizando níveis de inclusão do 'beta'- glucano de: 0 (controle), 0,1%, 0,2% e 0,3% por kg/ ração. Os níveis de'beta'-glucano avaliados neste estudo, não proporcionaram ganhos significativos no desempenho produtivo de juvenis de pacu, porém o tratamento 0,3% apresentou melhores resultados no GP, PF e TCE. A administração do 'beta'- glucano na dieta, durante todo período experimental, provocou alterações nos parâmetros hematológicos e indicadores de estresse do pacu. Os peixes alimentados com o 'beta'-glucano apresentaram maior resistência à infecção da bactéria A. hidrophila. Sendo assim, o tratamento 0,1% apresenta um custo/kg inferior e garante eficácia na saúde de juvenis de pacu.
Abstract: In fish, glucano has shown a potent immunostimulant function. The use of glucano is increasing significantly the resistance to diseases after infectious exposition. This prebiotic may be prevent the bacterial colonization, and activated macrophages, been beneficial to the digestive tract, resulting in better performance and disease resistance. This study will evaluate the glucano effects added in palletized and extruded diets of fish, analyzing fish perfOrmance, stress indicators, hematological profile and survival of pacu. This study was driven in Laboratory of ornamental fish, on Centro de Aqüicultura of UNESP (CAUNESP), in Jaboticabal, São Paulo. Were used 640 pacu juveniles, with 24,7 ± 2,0 g, distributed in 32 aquarium (130 L). The physical and chemical water parameters were measured every two weeks. Fish were fed twice a day, in the morning and another at the end of the day. In this trial were used 640 pacu juveniles (24.7 ± 2.0 g) distribuided in 32 aquariums (20 fish/aquarium). Throughout the experimental period, water remained at 26.5 oC and the others limnological parameters (dissolved oxygen, pH, alkalinity, ammonia and conductivity) stayed within normal values for the specie. The experimental trial design was entirely casualized in factorial scheme 2 x 4, evaluating two proceeding of diets (extruded and pelletized) and four 'beta' - glucan levels in diets: 0 (control), 0.1%, 0.2% and 0.3% with four repetitions. In this study,'beta' - glucan levels do not provide significant gains on pacu juveniles performance, but treatment with 0,3% - glucan showed better results of weight gain, weight final and specific growth rate. The administration of glucan in the diet, caused changes in hematological parameters and stress indicators in pacu. The fishes fed with glucan showed greater resistance to infection with A. hidrophila. Thus, treatment with 0,1% of glucan presented a lower cost/kg and shows efficiency in health of pacu juveniles.
Orientador: João Batista Kochenborger Fernandes
Coorientador: Elisabeth Criscuolo Urbinati
Banca: Sérgio Fonseca Zaiden
Banca: Fabiana Pilarski
Mestre

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Em peixes, o 'beta'- glucano apresenta uma potente função imunoestimulante sendo cada vez maior a sua utilização como suplemento alimentar, aumentando significantemente a resistência à exposição infecciosa. Este prebiótico tem função de prevenir a colonização de patógenos por intensificar a ativação de macrófagos, proporcionando benefícios ao trato gastrointestinal e resultando em melhor desempenho e resistência a doenças. Este estudo teve a finalidade de avaliar os efeitos do 'beta' - glucano adicionado às rações peletizadas e extrusadas sobre o desempenho produtivo, indicadores de estresse, perfil hematológico e sobrevivência do pacu. Este experimento foi conduzido no Laboratório de peixes ornamentais, do Centro de Aqüicultura da UNESP (CAUNESP), em Jaboticabal, São Paulo. Foram utilizados 640 juvenis de pacu, com 24,7 ± 2,0 g, distribuídos em 32 aquários de vidro (130L). Os parâmetros físico – químicos da água foram mensurados quinzenalmente. Os peixes foram alimentados duas vezes ao dia, uma pela manhão e outra ao fim do dia. Os experimentos apresentaram um delineamento experimental inteiramente casualizado em esquema fatorial 2 x 4, utilizando níveis de inclusão do 'beta'– glucano de: 0 (controle), 0,1%, 0,2% e 0,3% por kg/ ração. Os níveis de'beta'-glucano avaliados neste estudo, não proporcionaram ganhos significativos no desempenho produtivo de juvenis de pacu, porém o tratamento 0,3% apresentou melhores resultados no GP, PF e TCE. A administração do 'beta'- glucano na dieta, durante todo período experimental, provocou alterações nos parâmetros hematológicos e indicadores de estresse do pacu. Os peixes alimentados com o 'beta'-glucano apresentaram maior resistência à infecção da bactéria A. hidrophila. Sendo assim, o tratamento 0,1% apresenta um custo/kg inferior e garante eficácia na saúde de juvenis de pacu.
In fish, glucano has shown a potent immunostimulant function. The use of glucano is increasing significantly the resistance to diseases after infectious exposition. This prebiotic may be prevent the bacterial colonization, and activated macrophages, been beneficial to the digestive tract, resulting in better performance and disease resistance. This study will evaluate the glucano effects added in palletized and extruded diets of fish, analyzing fish perfOrmance, stress indicators, hematological profile and survival of pacu. This study was driven in Laboratory of ornamental fish, on Centro de Aqüicultura of UNESP (CAUNESP), in Jaboticabal, São Paulo. Were used 640 pacu juveniles, with 24,7 ± 2,0 g, distributed in 32 aquarium (130 L). The physical and chemical water parameters were measured every two weeks. Fish were fed twice a day, in the morning and another at the end of the day. In this trial were used 640 pacu juveniles (24.7 ± 2.0 g) distribuided in 32 aquariums (20 fish/aquarium). Throughout the experimental period, water remained at 26.5 oC and the others limnological parameters (dissolved oxygen, pH, alkalinity, ammonia and conductivity) stayed within normal values for the specie. The experimental trial design was entirely casualized in factorial scheme 2 x 4, evaluating two proceeding of diets (extruded and pelletized) and four 'beta' - glucan levels in diets: 0 (control), 0.1%, 0.2% and 0.3% with four repetitions. In this study,'beta' – glucan levels do not provide significant gains on pacu juveniles performance, but treatment with 0,3% – glucan showed better results of weight gain, weight final and specific growth rate. The administration of glucan in the diet, caused changes in hematological parameters and stress indicators in pacu. The fishes fed with glucan showed greater resistance to infection with A. hidrophila. Thus, treatment with 0,1% of glucan presented a lower cost/kg and shows efficiency in health of pacu juveniles.

Thesis (M.S.)--University of Cincinnati, 2007.
Advisors: Tiina Reponen PhD, Sergey Grinshpun PhD, Linda Levin PhD. Title from electronic thesis title page (viewed June 3, 2009). Includes abstract. Keywords: (1-3)Beta-D-Glucan; pollen; fungal spores; variation. Includes bibliographical references.

In the UK, dietary fibre intake is below the recommended level of 30 g/day. The manipulation of behavioural change is challenging, hence finding alternative ways to improve diet is important. The development of functional foods such as bread with added functional ingredients such as β-glucan and black tea may be more feasible and acceptable than changing to a new eating pattern. β-Glucan and black tea are often eaten separately, however there may be a food-matrix interaction between starch, protein (gluten), tea (poly)phenols and β-glucan when added together in a bread. We hypothesise that β-glucan and black tea will be digested slowly and display a blunted postprandial glycaemia. Some undigested residues will reach the colon, where it will be metabolised to short chain fatty acids (SCFA). SCFA, particularly propionate, have the potential to increase satiety by stimulating G protein receptors, however the effects on food intake need to be tested. This project described: i) development of a functional bread containing black tea, BT; β- glucan, βG; β-Glucan and black tea, βGBT) and compare it to normal white bread (WB) (study 1); ii) determination of bread palatability, perceived satiety and subsequent energy intake following ingestion (study 2); iii) determination of postprandial glucose and insulin responses, and appetite hormones (CCK, PYY and GLP-1) among healthy volunteers (study 3 – in vivo study). In study 1, the breads were developed and tested for starch functionality, antioxidant potential and in vitro fermentability mimicking human colonic fermentation. βG and βGBT breads reduced early (10-min) in vitro starch hydrolysis and this could be due to action of β-glucan that ‘protected’ some of the starch granules (microscopic study) against amylolysis. Digestion with α-amylase increased antioxidant potential and total (poly)phenols content of BT and βGBT breads compared with WB. In vitro propionate concentration did not increase significantly when fermented with β-glucan. High inter- individual variation was observed for individual SCFA production. The addition of black tea had no apparent effect on SCFA production. Study 2 is a randomised, crossover study design conducted in healthy volunteers. Breads were given as breakfast and perceived satiety (perceived fullness, hunger, satiety, desire to eat and prospective food intake) was measured postprandially for 3 h. Ad libitum lunch was given after 3 h and energy intake estimated. BT bread was the most acceptable among all breads. βG and βGBT breads showed adverse taste, texture and palatability but showed similar overall acceptability as WB and BT breads. Female subjects showed lower preference for taste, texture and palatability of βG and βGBT compared with WB. βG and βGBT had positive effects on perceived satiety as follows: 1) decreased hunger; 2) increased fullness; and 3) decreased desire to eat. However, eating βG and βGBT at breakfast did not reduce energy intake at lunch compared with WB. Study 3 was similar to study 2. Only βG bread showed significantly lower glucose TAUC0-180 min compared with BT and βGBT but has no apparent effect on insulin response. No significant changes were observed for CCK and GLP-1 responses for all breads. However, βG and βGBT showed lower PYY TAUC0-180 min compared with BT. In vitro starch hydrolysis did not correlate with in vivo postprandial glycaemic responses. In conclusion, these studies suggest that breads with β-glucan and/or black tea have positive effects on perceived satiety in vivo and show good overall acceptability. However, there is no clear evidence that they affect appetite regulation. Breads containing 7 g β- glucan per 50 g of available carbohydrate reduced in vivo glucose response without altering insulin responses. There was no additional effect of adding black tea together with β-glucan to bread on the in vivo postprandial glycaemic response. It is too early to generalise the results from in vitro batch fermentation and starch hydrolysis and this needs to be considered when planning future dietary interventions looking at both in vitro and in vivo studies. Overall this study concluded that adding soluble dietary fibre to bread is feasible in controlling glycaemic responses and may help increase daily dietary fibre intake.

En los últimos 10 años, diferentes inmunoestimulantes han sido probados en más de 18 especies de peces, incluyendo carpa, turbot, salmón del atlántico, dorada, trucha, entre otros. Los compuestos que han sido estudiados son muy variados e incluyen componentes bacterianos, polisacáridos, extractos de plantas, algas y animales, factores nutricionales, e incluso hormonas y citoquinas. Sin embargo, a pesar del gran interés que han generado estos estudios, los inmunoestimulantes dietarios comercialmente disponibles contienen principalmente sólo β-glucanos. La mayoría de los estudios están basados en la respuesta celular de los peces, como actividad fagocítica, especies reactivas de oxígeno (ROS), y mediciones en sangres, como contenido de IgM en suero. La gran mayoría de estos estudios han mostrado resultados positivos, pero se sabe muy poco sobre los mecanismos moleculares que hay detrás de la respuesta a la administración de inmunoestimulantes a través de la dieta. Por lo anteriormente mencionado, el objetivo principal de este trabajo es evaluar a respuesta transcriptómica de las branquias de peces alimentados con dietas inmunoestimulantes, utilizando dos acercamientos, el molecular y celular. Experimentalmente, 360 doradas (Sparus aurata) sanas, de un peso promedio de 38±7.3 g fueron separados en 27 tanques y alimentados con dos dietas inmunoestimulantes manufacturadas por Skretting (Dieta A y Dieta B) y una dieta control (Dieta C, Skretting). Cada dieta fue administrada los peces a una razón del 3% de su masa corporal dos veces al día, con un periodo de pre-aclimatación de 14 días. Muestras de agallas fueron tomadas a 2, 7, 14 y 28 días de administración de la dieta. Todas las muestras fueron divididas en dos para realizar un análisis de microarray (Microarray específico para dorada 44K, diseñado por Agilent) y para un análisis in situ. Los datos de microarray fueron analizados con dos métodos, con un análisis referente al control y un análisis en loop. Los resultados del microarray mostraron la expresión diferencial de genes relacionados a procesos inmunológicos, como inflamación, activación y respuesta de células T, apoptosis, entre otros, sin embargo la intensidad y magnitud de la respuesta no fue muy alta. Los resultados del análisis in situ mostró que algunos de estos transcritos se localizaron en un tipo celular ubicado en la lamela secundaria de las agallas de dorada. Estas células, podrían ser células clorhídricas o linfocitos T, pero más estudios hacen falta para poder confirmar estas hipótesis.
Over the past 10 years, different immunostimulants have been tested in more than 18 fish species including: Carp, Yellow croaker, Turbot, Atlantic salmon and Seabream, amongst others. The compounds tested are varied including bacterial components, polysaccharides, animal, plant and algae extract, nutritional factors, and even hormones and cytokines and some synthetics such as Levamisole. However even although a lot of interest and studies have been carried out, commercially available immunostimulant diets mainly contain β-glucans. The majority of the studies reported are based upon cellular response assays such as phagocyte activity and ROS and simple blood measurements such as total serum IgM content. All studies have shown positive results, but little is known about the underlying molecular response to dietary administration of immunostimulants. In order to evaluate the transcriptomic response in gills we analyzed and evaluated gene expression profiles associated with exposure to immunostimulant diets over time, using both a molecular and cellular approach. Experimentally, 360 healthy Gilthead Seabream (Sparus aurata) of average body weight of 38±7.3 g were separated in 27 tanks and fed with two Skretting immunostimulant diets (Diet A and Diet B) and a control diet (Diet C). Each diet were fed at a feeding rate of 3% of body weight twice daily for 28 days with a period of 14 days of pre-acclimation. Gills samples were taken at 2, 7, 14 and 28 days post diet. All samples were divided for microarray analysis (specific Sparus aurata 44K microarray, Agilent custom design) and in situ hybridization (ISH) analysis. A diet dependent and a loop analysis were carried out, with control diet as a reference point. Microarray results shown a differential expression of genes associated to immunological processes such as inflammation, T and B cell response amongst others but the intensity and magnitude of the modulation of these responses was not high. ISH analysis showed localization of immunological transcripts in a specific cellular type in the primary lamellae of gilthead seabream gills.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Biorigin Sa
Vários derivados da parede celular da levedura Saccharomyces cerevisiae conhecidamente agem sobre a imunidade, no entanto a ação, especialmente da fração beta-glucano, foi pouco demonstrada em cães. Para o estudo dos possíveis efeitos sobre a imunidade na espécie canina foram empregadas quatro dietas isonutrientes, contendo uma fonte de parede celular de levedura (PCL), duas fontes de beta-glucano (BG1 e BG2) e uma dieta controle (CT). Foram utilizados 24 cães da raça beagle, adultos, divididos em quatro grupos de seis animais. As dietas foram fornecidas por um período total de 126 dias e as avaliações incluíram hemograma e avaliações bioquímicas, dosagem de anticorpos anti-Leptospira, imunofenotipagem de linfócitos sanguíneos, avaliação da concentração de IgA em fezes, teste de hipersensibilidade cutânea tardia e dosagens de citocinas em sobrenadante de cultura celular. Os animais foram submetidos a desafio antigênico com vacina contra leptospirose no dia 42. Os dados foram avaliados pelo procedimento GLM do SAS, sendo as médias comparadas pelo teste de Tukey (p≤0,1). Nos exames bioquímicos houve discreta variação entre os tratamentos e ao longo dos dias. No hemograma notou-se aumento dos linfócitos para BG2. A dosagem de anticorpos anti-Leptospira, mostrou baixos títulos, não havendo boa resposta à vacinação. A imunofenotipagem revelou um aumento dos linfócitos T totais, T helper, T citotóxicos e linfócitos B no grupo BG2 e de linfócitos T citotóxicos e linfócitos B para o grupo PCL. Apesar da variação da concentração de IgA fecal ao longo dos dias, os tratamentos não influenciaram tais parâmetros. O teste de hipersensibilidade cutânea tardia mostrou um aumento na resposta à inoculação da vacina, para os grupos PCL e BG2. Na dosagem de citocinas em sobrenadante de cultura celular, apenas foi observada diferença na quantificação de TNF-α,...
Some products from the cell wall of the yeast Saccharomyces cerevisiae are known to act on the immunity, however this action, especially of the beta-glucan fraction, has never been demonstrated in dogs. To study these effects on the immunity of dogs four isonutrient diets were made, containing one source of yeast cell wall (YCW), two sources of beta-glucan (BG1 and BG2) and a control diet (CT). 24 adult beagle dogs were used, divided in four groups of six animals. Diets were given for a 126 days period. Evaluations included complete blood count and biochemistry profile, quantification of antibodies against Leptospira, immunophenotyping of blood lymphocytes, IgA concentration in feces, delayed-type hypersensitivity test and quantification of cytokines in cell culture supernatant. Animals were exposed to antigen challenge, by the vaccine against leptospirosis on day 42. Data were analyzed by the GLM procedure of the SAS software and the means were compared by the Tukey test (p<0,01). Biochemistry profile showed slight differences among the groups. A increase in lymphocyte count was observed for BG2 treatment. Quantification of antibodies against Leptospira showed low titles, with poor response to vaccination. Immunophenotyping revealed an increase during the time in total T cells, helper and cytotoxic T cells, and B lymphocytes for BG2 and of cytotoxic T cells and B lymphocytes for YCW group. Despite the variation in fecal IgA concentration during the time, treatments did not influence these parameters. Delayed-type hypersensitivity test showed an increased response to the vaccine inoculation, for YCW and BG2 groups. In the quantification of cytokines in cell culture supernatant the only difference observed was in TNF-α concentration, being BG2 higher than CT. We concluded that both yeast cell wall and beta-glucan fraction act on dogs` immunity

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Manejos inerentes da piscicultura intensiva, como o transporte, desencadeiam resposta de estresse nos animais, podendo causar perdas na produtividade. Como alternativa, o β-glucano, um polissacarídeo derivado da parede celular de cereais, bactérias e fungos, vem sendo muito utilizado na aquicultura pelo seu efeito imunoestimulante. Há evidências de que o β-glucano minimiza os efeitos negativos do estresse por atuar no sistema imune, porém é pouco investigado o seu efeito direto sobre a resposta clássica de estresse. Neste contexto, o presente estudo avaliou, em juvenis de pacu, o uso oral de 0.1% de duas gerações de β-glucano, de diferentes graus de pureza e processamentos, na resposta de estresse e no sistema imune inato, após transporte e inoculação com Aeromonas hydrophila. Avaliamos a concentração de cortisol e glicose plasmáticos como indicadores da resposta de estresse, a atividade respiratória de leucócitos, a atividade hemolítica do sistema complemento, a atividade de lisozima e contagem total e diferencial de leucócitos, como indicadores do sistema imune inato, e o hematócrito, número de eritrócitos, concentração de hemoglobina e volume corpuscular médio de eritrócitos como indicadores hematológicos. As duas gerações de β-glucano utilizadas modularam os níveis de cortisol circulantes, mantendo os níveis elevados até 24 horas após o transporte, sem alteração após o desafio bacteriano. O β-glucano aumentou a atividade hemolítica do sistema complemento e de lisozima após o manejo e após a inoculação bacteriana, e manteve a população de leucócitos circulantes após recuperação de leucopenia, evidenciando o efeito imunoestimulante. A análise geral dos resultados deste estudo sugere o fortalecimento da resposta imune de juvenis de pacu alimentados por 15 dias antes de manejo estressante, com ração contendo 0.1% β-glucano derivado da parede celular de levedura (Sacaromyces cerevisiae).
Inherent handling in intensive fish farming, such as transport, trigger stress response in animals, and may cause losses in productivity. As an alternative, the β-glucan, a polysaccharide derived from the cell walls of cereals, bacteria and fungi, has been widely used in aquaculture due their immunostimulatory effect. The β-glucan minimizes the negative effects of stress by acting on the immune system, but it is little investigated its direct effect on the classical stress response. In this context, the present study evaluated in pacu, the oral administration of 0.1% of two generations of β-glucan, with different degrees of purity and processing methods, on the stress and innate immune system responses, after transport and inoculation with Aeromonas hydrophila. We evaluated the plasma concentration of cortisol and glucose, as stress response indicators, the respiratory activity of leukocytes, the hemolytic activity of the complement system, lysozyme activity and total and differential counts of leukocytes as innate immune system indicators, and hematocrit, number of erythrocytes, hemoglobin concentration, and mean corpuscular volume of erythrocytes as hematological indicators. The two generations of β-glucan modulated the circulating levels of cortisol, keeping the high levels 24 hours after transportation, without changes after the bacterial challenge. The β-glucan increased the hemolytic activity of the complement system and lysozyme after handling and after bacterial inoculation, and kept the population of circulating leukocytes after recovery of leukopenia, demonstrating the immunostimulatory effect. The general results of this study suggest the strengthening of the immune response of pacu juveniles fed for 15 with feed containing 0,1% β-glucan derived from yeast cell wall (Sacaromyces cerevisiae) days before stressful handling.
CNPq: 138990/2014-0

Orientadores: Marcia Regina Braga, Marcia Maria Camargo de Morais
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Fruto-oligossacarídeos (FOS) são frutanos de baixo peso molecular produzidos por microorganismos. O interesse em FOS vem aumentando uma vez que eles são considerados ingredientes funcionais benéficos à saúde humana. Com o objetivo de analisar como a produção de FOS e a composição da parede celular de fungos filamentosos é afetada pela fonte de carbono, os fungos Fusarium solani (URM 3338) e Neocosmospora vasinfecta (URM 3329) foram cultivados em meios contendo cinco fontes de carbono diferentes (sacarose, inulina, glucose, frutose ou glucose mais frutose, todos a 1%) e coletas foram realizadas aos 5, 10 e 15 dias de crescimento. A partir do meio de cultivo filtrado foram analisados o pH, teores de açúcar total, açúcares redutores e proteínas, a presença de FOS e atividades enzimáticas invertásica e inulinásica. A partir do micélio, a biomassa foi quantificada e a parede celular foi isolada e sua composição em açúcares neutros, ácidos urônicos e quitina analisada. Foi avaliada também a expressão relativa de genes de síntese de parede celular b-1,3-glucano sintase e quitina sintases. Os dois fungos utilizaram todas as fontes de carbono crescendo nas diferentes condições. Atividade de hidrólise foi detectada no meio contendo sacarose ou inulina para o fungo F. solani, gerando glucose, frutose e fruto-oligossacarideos como produtos havendo utilização dos monossacarídeos. O micélio deste fungo apresentou alterações visíveis no crescimento em meio sólido apenas no meio com frutose, mas foi observada igual quantidade de quitina da parede celular deste fungo quando crescido por cinco dias em sacarose e inulina, mas em menor quantidade com relação aos demais meios. As análises de expressão relativa de genes mostraram indução do gene da b-1,3-glucano sintase e repressão do gene quitina sintase 5 em sacarose e inulina com relação a condição frutose. Estes dados sugerem que a alteração na composição da parede celular do F. solani pode ter relação com a secreção de enzimas nos meios sacarose e inulina. Para N. vasinfecta, quando crescido em sacarose foi observada atividade de transfrutosilação, com a liberação de glucose e síntese de 1-cestose (FOS) no meio. Transfrutosilação também foi observada no meio que teve inulina como fonte de carbono. O micélio deste fungo apresentou alterações visíveis em meio sólido nas condições frutose e inulina, sendo mais hialino do que nas demais condições. A quantidade de quitina na parede celular deste fungo crescido por cinco dias foi maior nas condições frutose e inulina com relação às demais. As análises de expressão relativa de genes mostraram indução dos genes de quitina sintase 4 e 5 nestas duas condições em relação à sacarose. A partir dos resultados, pode-se concluir que as fontes de carbono oferecidas foram utilizadas pelos fungos, que as mesmas afetaram a composição de açúcares da parede celular e a expressão de genes de síntese de componentes da parede e que estes fungos são promissores para a produção de FOS, pois possuem enzimas que hidrolisam a inulina, além de enzimas que sintetizam oligossacarídeos a partir de sacarose por transfrutosilação
Abstract: Fructooligosaccharides (FOS) are low molecular weight fructans produced by microbes and plants. Interest in FOS has been increasing since they are considered as functional food ingredients with benefical effects in human nutrition. With the aim of examining how the production of FOS and the composition of the cell wall of filamentous fungi are affected by the carbon source, Fusarium solani (URM 3338) and Neocosmospora vasinfecta (URM 3329) were cultured in media containing five different carbon sources (sucrose, inulin, glucose, fructose or glucose plus fructose) and samples were taken at 5, 10 and 15 days of growth. From the filtered culture medium, pH, total carbohydrates, reducing sugars and proteins, the presence of FOS and inulinase and invertase activities were analyzed. Mycelium biomass was measured and the cell wall was isolated and its composition in neutral sugars, uronic acids and chitin analyzed. The expression of b-1,3-glucan synthase and chitin synthase genes was also evaluated. Both fungi utilized all the carbon sources for growing. In sucrose- and inulin-containing media, hydrolytic activity was detected in F. solani generating glucose, fructose and FOS as products. When grown on solid culture media, visible changes were observed in mycelium of this fungus only in fructose, but the amount of chitin in the cell wall was higher in the sucrose and inulin-containing media when compared to other carbon sources. The expression b-1,3-glucan synthase gene was induced and chitin synthase 5 gene repressed on sucrose and inulin media. N. vasinfecta showed transfructosilation activity when was grown in sucrose, with release of glucose and synthesis of 1-kestose (FOS) in the culture medium. Transfructosilation was also observed in the inulin-containing medium. The mycelium showed visible changes when the fungus was cultured in solid medium with fructose or inulin as carbon sources. The amount of chitin in the cell wall of this fungus when grown for five days in inulin or fructose was higher in comparison to other carbon sources. The analysis of gene expression showed induction of chitin synthase 4 and 5 genes in these two conditions in relation to sucrose. From the results it can be concluded that the carbon sources affected growth, enzymic activity, composition of the cell wall and gene expression in F. solani and N. vasinfecta, and that these fungi are promising organisms for FOS production since they secrete enzymes that hydrolyze inulin or synthesize oligosaccharides from sucrose by transfructosylation
Doutorado
Biologia Celular
Doutora em Biologia Celular e Estrutural

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The growing attention to the health, the food industry and market look to provide foods with functional properties to the consumers. The soluble fibers, as beta-glucan, present this property and have been studied about yours physiologic and technologic effects. The beta-glucan produce a gel with high viscosity, and research with the objective of alter the viscosity and swelling power for facilitate your incorporation in the foods has been accomplished. However, these modifications can alter the physiologic effects. Besides, don t exist studies about effect of oxidative treatment on the beta-glucan properties. The objective of this study was evaluate the effect of oxidative treatment with hydrogen peroxide in different concentrations (0,3; 0,6; 0,9%) and two times of reaction, 30 and 60 minutes, on the beta-glucan from oat and, later, apply in cheese bread in 2, 3, 4% levels. the oxidative treatment increase the carbonyl and carboxyl groups, affected the swelling power and increase glucose release after chemic digestion. The oxidative treatment increase bile acid binding capacity, however, didn t alter the fat binding capacity. Has decrease in hardness, adhesiveness and gumminess, as well as viscosity of gel with the oxidative treatment. The oxidized beta-glucan decrease the expansion factor and increase the firmness of cheese bread, with exception of the treatment with native beta-glucan at 2% and oxidized with 0,9% of H2O2/30 min at 3%.
Em vista da crescente preocupação com a saúde, a indústria e o mercado de alimentos buscam proporcionar aos consumidores alimentos com propriedades funcionais. As fibras solúveis, como a beta-glicana, apresentam essa propriedade e estão sendo estudadas quanto a seus efeitos fisiológicos e tecnológicos. A betaglicana forma um gel de alta viscosidade e, para facilitar a sua incorporação aos alimentos, são realizadas pesquisas de modificação da beta-glicana, com o intuito de alterar as suas características como viscosidade e poder de intumescimento. No entanto, com essas modificações, os seus efeitos fisiológicos podem também ser alterados. Além disso, não há estudos sobre o efeito de tratamentos oxidativos sobre as propriedades da beta glicana. O objetivo deste estudo foi avaliar o efeito do tratamento oxidativo com peróxido de hidrogênio em diferentes concentrações (0,3; 0,6 e 0,9%) com dois tempos de reação, 30 e 60 minutos em beta-glicana extraída da aveia e, posteriormente, utilizá-la na formulação de pães de queijo nos níveis 2, 3 e 4%. O tratamento oxidativo promoveu aumento de grupos carbonila e carboxila, alterou o poder de intumescimento da beta-glicana e aumentou a liberação de glicose após digestão química. A capacidade de ligação com ácidos biliares aumentou com os tratamentos oxidativos, entretanto, não houve alteração da capacidade de ligação com gordura. Houve diminuição da dureza, adesividade e gomosidade, bem como da viscosidade do gel com os tratamentos oxidativos. A adição de beta-glicana oxidada promoveu diminuição do índice de expansão dos pães de queijo, e aumento da firmeza, com exceção dos tratamentos com betaglicana nativa a 2% e oxidada com 0,9% de H2O2/30 min a 3%.

In Southeast Asia, the family Pangasiidae is important for commercial fisheries and aquaculture. Pangasianodon hypophthalmus (striped catfish) is the most economically important species farmed in Vietnam, with a total export value of 1.7 billion USD in 2012. Intensive aquaculture can lead to problems with major outbreaks of disease and Edwardsiella ictaluri and Aeromonas hydrophila represent two important bacterial pathogens in P. hypophthalmus aquaculture. Immunostimulants have proven to be a very useful food additive for the aquaculture industry, since they can be easily fed to fish to enhance their immune response at times of stress and to improve resistance to disease. The immune system of pangasius catfish has not been fully described, despite the recent growth in aquaculture for this species, and little is known about the effects of immunostimulants on disease resistance. Understanding the immune response is very important in order to evaluate the health status of the fish and assist in control of disease (including prevention) so that production levels by the aquaculture industry can be sustained. The aims of this thesis were to develop and standardise methods to elucidate and measure immune responses in P. hypophthalmus and then to use these with relevant disease models (A. hydrophila and E. ictaluri) and immunomodulators (β-glucans from different sources and at different doses) to determine if bacterial diseases can be controlled, and which functional immune responses and immune genes could be correlated with disease resistance. As a variety of different species from family Pangasiidae are economically important for aquaculture, initial work focused on the characterisation of the immunoglobulin IgM molecule in these species, and anti-P. hypophthalmus IgM mAbs were tested to determine if they cross-reacted between different Pangasiidae species (Chapter 2). Although affinity purification of IgM from the different fish species resulted in a purer preparation ammonium sulphate precipitation (14% w/w), the latter proved faster and easier to perform. The heavy (H) and light (L) chains of IgM from P. hypophthalmus were estimated to be 70-72 kDa and 25-26 kDa, respectively, using SDS-PAGE (12.5%). The L chains of IgM in the other Asian fish species examined were similar in molecular weight to P. hypophthalmus, while the H chains varied (P. gigas and P. larnaudii 76kDa, P. sanitwongsei 69kDa, H. filamentus 73kDa, P. borcoti and H. wyckioides 75kDa, C. bactracus 74kDa, C. macrocephalus 73kDa and C. carpio 70kDa), as did the native IgM molecules. Sedimentation velocity ultracentrifugation was used to determine the molecular weight of the whole IgM molecule from P. hypophthalmus as an alternative to the more commonly used native gels that are run under non-denaturing conditions, although this technique proved more complex. Anti–P. hypophthalmus IgM monoclonal antibodies (mAbs) cross reacted with all of the Pangasiidae species and were successfully applied in an enzyme-linked immunosorbent assay (ELISA) using mAb 23 to measure serum antibody response of P. hypoophthalmus following experimental infection with A. hydrophila by interperitoneal (I.P.) injection in Chapter 3 and E. ictaluri by immersion in Chapter 4. As P. hypophthalmus is a relatively new aquaculture species, there are few reports evaluating its immune response to pathogens. Thus, functional assays were standardised to evaluate both innate and adaptive immune responses of this species and then these assays used to compare immune response following stimulation with live and killed A. hydrophila. (Chapter3). Four treatment groups of 40 fish per group (53.2 ± 14.8g.) consisting of an untreated control group, a group injected I.P. with adjuvant (Montanide ISA 760 VG) only, a group injected with heat-killed A. hydrophila (1 x109 cfu ml-1 mixed with adjuvant), and a group injected with a subclinical dose of live A. hydrophila 2.7 x105 cfu ml-1 were used in the study. Samples were collected 0, 1, 3, 7, 14 and 21 days post injection (d.p.i.) to assess the immune response of fish. The results indicated that challenge with live or/and dead bacteria stimulated the immune response in P. hypophthalmus significantly above control groups with respect to specific antibody titre, lysozyme activity, phagocytosis and plasma peroxidase at 7 or/and 14 d.p.i. Moreover, on 21 d.p.i. total IgM, specific antibody titre and lysozyme activity from both live and dead A. hydrophila challenge groups were significantly different to the control groups. Differential immune responses between live and dead bacterial challenges were also observed as only live A. hydrophila significantly stimulated WBC counts and plasma peroxidase at 3 d.p.i. with the greatest increase in WBC counts noted at 21 d.p.i. and in phagocytosis at 14 d.p.i. By 21 d.p.i. only the macrophages from fish challenged with dead A. hydrophila showed significantly stimulated respiratory burst activity. Immunostimulants are food additives used by the aquaculture industry to enhance the immune response, and β-glucan is now commonly used for this purpose in aquaculture. In Chapter 4 the effect of the prebiotic β-glucan on the immune response and disease resistance of P. hypophthalmus was evaluated. The fish (60.3 ± 11.7 g.) were fed with a basal diet (control) or diets supplemented with fungal derived β-glucan at concentrations of 0.05 %, 0.1 %, or 0.2 % g/kg for four weeks. Fish fed 0.1 % commercial yeast derived β-glucan were also included as a positive control group. Samples were collected from fish on Days 0, 1, 3, 7, 14, 21 and 28. The results showed that fish fed with the highest two levels of fungal derived β-glucan had enhanced immune responses compared to the control group, with respiratory burst activity on all days examined and lysozyme activity on 7 days post feeding (d.p.f.) being significantly elevated (P<0.05) in the group fed with 0.2 % fungal derived β-glucan, while plasma anti-protease activity on 21 d.p.f., natural antibody titre on 3 d.p.f. and complement activity 7 d.p.f. and 14 d.p.i. were significantly enhanced (P<0.05) in the group fed 0.1 % fungal derived β-glucan. The lowest dose of fungal derived β-glucan (0.05 %) appeared insufficient to effectively stimulate the fish’s immune response. WBC count, respiratory burst, lysozyme activity and complement were useful as an early indication of immunostimulation (1 to 7 days). Four weeks after feeding with the different diets, the fish were experimentally infected with E. ictaluri by immersion using 8 x104 cfu ml-1 for 1 h and mortalities were monitored for 14 days. There was a great deal of variation in the level of mortalities within the four replicate tanks for each dietary group. Although the in vivo challenge results showed no statistical differences between the groups fed on the different diets, the highest mortalities were observed in group fed with the control diet and the lowest mortalities were observed in the groups fed with commercial yeast derived β-glucan and 0.2 % fungal derived β glucan. Immune gene expression following stimulation with β-glucan and challenge with E. ictaluri was investigated in Chapter 5.

Orientador: Aulus Cavalieri Carciofi
Banca: Helio Jose Montassier
Banca: Iracilda Zeppone Carlos
Resumo: Vários derivados da parede celular da levedura Saccharomyces cerevisiae conhecidamente agem sobre a imunidade, no entanto a ação, especialmente da fração beta-glucano, foi pouco demonstrada em cães. Para o estudo dos possíveis efeitos sobre a imunidade na espécie canina foram empregadas quatro dietas isonutrientes, contendo uma fonte de parede celular de levedura (PCL), duas fontes de beta-glucano (BG1 e BG2) e uma dieta controle (CT). Foram utilizados 24 cães da raça beagle, adultos, divididos em quatro grupos de seis animais. As dietas foram fornecidas por um período total de 126 dias e as avaliações incluíram hemograma e avaliações bioquímicas, dosagem de anticorpos anti-Leptospira, imunofenotipagem de linfócitos sanguíneos, avaliação da concentração de IgA em fezes, teste de hipersensibilidade cutânea tardia e dosagens de citocinas em sobrenadante de cultura celular. Os animais foram submetidos a desafio antigênico com vacina contra leptospirose no dia 42. Os dados foram avaliados pelo procedimento GLM do SAS, sendo as médias comparadas pelo teste de Tukey (p≤0,1). Nos exames bioquímicos houve discreta variação entre os tratamentos e ao longo dos dias. No hemograma notou-se aumento dos linfócitos para BG2. A dosagem de anticorpos anti-Leptospira, mostrou baixos títulos, não havendo boa resposta à vacinação. A imunofenotipagem revelou um aumento dos linfócitos T totais, T helper, T citotóxicos e linfócitos B no grupo BG2 e de linfócitos T citotóxicos e linfócitos B para o grupo PCL. Apesar da variação da concentração de IgA fecal ao longo dos dias, os tratamentos não influenciaram tais parâmetros. O teste de hipersensibilidade cutânea tardia mostrou um aumento na resposta à inoculação da vacina, para os grupos PCL e BG2. Na dosagem de citocinas em sobrenadante de cultura celular, apenas foi observada diferença na quantificação de TNF-α, ...(Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Some products from the cell wall of the yeast Saccharomyces cerevisiae are known to act on the immunity, however this action, especially of the beta-glucan fraction, has never been demonstrated in dogs. To study these effects on the immunity of dogs four isonutrient diets were made, containing one source of yeast cell wall (YCW), two sources of beta-glucan (BG1 and BG2) and a control diet (CT). 24 adult beagle dogs were used, divided in four groups of six animals. Diets were given for a 126 days period. Evaluations included complete blood count and biochemistry profile, quantification of antibodies against Leptospira, immunophenotyping of blood lymphocytes, IgA concentration in feces, delayed-type hypersensitivity test and quantification of cytokines in cell culture supernatant. Animals were exposed to antigen challenge, by the vaccine against leptospirosis on day 42. Data were analyzed by the GLM procedure of the SAS software and the means were compared by the Tukey test (p<0,01). Biochemistry profile showed slight differences among the groups. A increase in lymphocyte count was observed for BG2 treatment. Quantification of antibodies against Leptospira showed low titles, with poor response to vaccination. Immunophenotyping revealed an increase during the time in total T cells, helper and cytotoxic T cells, and B lymphocytes for BG2 and of cytotoxic T cells and B lymphocytes for YCW group. Despite the variation in fecal IgA concentration during the time, treatments did not influence these parameters. Delayed-type hypersensitivity test showed an increased response to the vaccine inoculation, for YCW and BG2 groups. In the quantification of cytokines in cell culture supernatant the only difference observed was in TNF-α concentration, being BG2 higher than CT. We concluded that both yeast cell wall and beta-glucan fraction act on dogs' immunity
Mestre

La constatació de la rellevància dels carbohidrats i glicoconjugats en processos de reconeixement cel·lular, regulació i senyalització, proliferació cel·lular i resposta immunitària, així com en funcions estructurals i energètiques reconegudes clàssicament, han portat a un interès creixent en aquestes biomolècules per part de la Glicobiologia i la Ciència dels Materials. Aquest fet ha comportat la necessitat de disposar d’eines complementàries o alternatives a la química convencional per a la síntesi d’aquestes biomolècules i abastir-ne així la demanda generada. Tot això ha propiciat el desenvolupament de noves metodologies sintètiques amb la incorporació d’etapes enzimàtiques. L’any 1998 es va desenvolupar la metodologia glicosintasa basada en un redisseny del centre actiu de glicosidases que actuen amb retenció de configuració per tal de suprimir-ne la seva activitat hidrolítica. L’ús d’aquests enzims modificats permeten la catàlisi eficient de la formació d’enllaços glicosídics quan s’empren donadors glicosídics activats amb la configuració anomèrica oposada a la del substrat de la reacció normal d’hidròlisi En la present Tesi s’aprofundeix en la metodologia glicosintasa tant a nivell mecanístic com aplicat. Es treballa amb dues glicosintases, el mutant E134A de la β-glucanasa de Bacillus lichenisformis i el mutant E383A de β-glucosidasa d’Streptomyces sp. amb les quals s’assoleixen les següents fites: S’estudia la funció del residu D136 en el centre actiu de la glicosintasa E134A de la β-glucanasa de Bacillus lichenisformis. L’anàlisi de dobles mutants, mostra que només el mutant E134A/D136E manté certa activitat glicosintasa, suggerint la seva funció com a residu assistent en el mecanisme enzimàtic. També s’obtenen les glicosintases més actives E134S i E134G de la -glucanasa de Bacillus licheniformis, on la substitució del E134 per Ser resulta en un enzim amb una activitat glicosintasa incrementada 5 vegades (en termes de kcat/KM) en comparació amb el mutant original E134A. Es demostra la utilitat de la glicosintasa E134A de Bacillus licheniformis per sintetitzar 1,3-1,4-β-glucans artificials per polimerització dels donadors disacàrid (Glcβ3GlcαF), trisacàrid (Glcβ4Glcβ3GlcαF) i tetrasacàrid (Glcβ4Glcβ4Glcβ3GlcαF). S’originen polisacàrids constituïts per unitats repetitives dels seus corresponents monòmers units per enllaços β-1,4. La morfologia del polisacàrid depèn de la unitat repetitiva que el forma de manera que per (β4Glcβ3Glc)n i (β4Glcβ4Glcβ4Glcβ3Glc)n s’obtenen esferulites mentre que per (β4Glcβ4Glcβ3Glc)n s’obté un precipitat amorf. Aquests polisacàrids constitueixen nous β-glucans amb estructures homogènies amb una proporció d’enllaços β-1,3 més elevada respecte els β-glucans existents a la natura. El grau de polimerització dels β-glucans obtinguts ve limitat per la solubilitat dels productes de reacció. S’observa que l’ús del mutant més actiu E134S permet estendre la polimerització i assolir polisacàrids d’alt pes molecular en funció de la concentració d’enzim. D’aquesta manera el grau de polimerització (DP) pot ser controlat per l’activitat enzimàtica. Per avaluar la inhibició per producte de l’activitat glicosintasa del mutant E134A se sintetitza l’octasacàrid (β4Glcβ4Glcβ4Glcβ3Glc)2, concloent que el producte de reacció actua com inhibidor competitiu malgrat no té un efecte significatiu en els rendiments preparatius dels productes de polimerització enzimàtica. D’altra banda s’estudia l’activitat glicosintasa del mutant E383A de la β-glucosidasa d’Streptomyces sp. S’observa que aquest presenta activitat hidrolítica i de transglicosidació, fet que queda palès amb la formació de productes secundaris en la reacció glicosintasa. Descartada que aquesta activitat hidrolítica provingui d’una contaminació d’enzim wt, es proposa un mecanisme d’hidròlisi i transglicosidació assistit per anió fluorur com a nucleòfil exogen per la formació d’aquests productes secundaris no esperats. Finalment s’immobilitza amb èxit la glicosintasa E383A de la β-glucosidasa d’Streptomyces sp. sobre reïna Chelating Sepharose FF. L’enzim immobilitzat no mostra millora de la seva estabilitat envers els dissolvents orgànics. No obstant, sí millora pel que fa a estabilitat d’emmagatzemament a 4ºC i pH 7 en comparació amb la seva forma lliure i permet la seva reutilització fins a 9 cicles mantenint el 90% de l’activitat inicial.
La constatación de la relevancia de los carbohidratos y glicoconjugados en procesos de reconocimiento celular, regulación y señalización, proliferación celular y respuesta inmunitaria, así como en las funciones estructurales y energéticas reconocidas de forma clásica, han llevado a un interés creciente en estas biomoléculas por parte de la Glicobiología y de la Ciencia de los Materiales. Este hecho ha comportado la necesidad de disponer de herramientas complementarias o alternativas a la química convencional para la síntesis de estas biomoléculas y poder abastecer así la demanda generada. Todo ello ha propiciado el desarrollo de nuevas metodologías sintéticas con la incorporación de etapas enzimáticas. En 1998 se desarrolló la metodología glicosintasa basada en un rediseño del centro activo de glicosidasas que actúan con retención de configuración con la finalidad de suprimir su actividad hidrolítica. El uso de estas enzimas modificadas permite la catálisis eficiente de la formación de enlaces glicosídicos cuando se utilizan dadores glicosídicos activados con la configuración anomérica opuesta a la del sustrato de la reacción normal de hidrólisis. En la presente Tesis se profundiza en la metodología glicosintasa tanto a nivel mecanístico como aplicado. Se trabaja con dos glicosintasas, el mutante E134A de la β-glucanasa de Bacillus licheniformis y el mutante E383A de la β-glucosidasa de Streptomyces sp., con las que se consiguen los siguientes hitos: Se estudia la función del residuo D136 en el centro activo de la glicosintasa E134A de la β-glucanasa de Bacillus licheniformis. El análisis con dobles mutantes, muestra que solo la variante E134A/D136D mantiene cierta actividad glicosintasa, sugiriendo su función como residuo asistente en el mecanismo enzimático. También se obtienen las glicosintasas más activas E134S y E134G de la β-glucanasa de Bacillus licheniformis, donde la sustitución del E134 por Ser da como resultado una variante con una actividad glicosintasa incrementada 5 veces (en términos de kcat/KM) en comparación con la variante original E134A. Se demuestra la utilidad de la glicosintasa E134A de Bacillus licheniformis para sintetizar 1,3-1,4-β-glucanos artificiales por polimerización de los dadores disacárido (Glcβ3GlcαF), trisacárido (Glcβ4Glcβ3GlcαF) y tetrasacárido (Glcβ4Glcβ4Glcβ3GlcαF). Se originan polisacáridos constituidos por unidades repetitivas de sus correspondientes monómeros unidos por enlaces β-1,4. La morfología del polisacárido depende de la unidad repetitiva que lo forma de manera que para el (β4Glcβ3Glc)n y el (β4Glcβ4Glcβ4Glcβ3Glc)n se obtienen esferulitas mientras que para el (β4Glcβ4Glcβ3Glc)n se obtiene un precipitado amorfo. Estos polisacáridos constituyen nuevos β-glucanos con estructuras homogéneas con una proporción de enlaces β-1,3 más elevada respecto a los β-glucanos existentes en la naturaleza. El grado de polimerización de los β-glucanos obtenidos viene limitado por la solubilidad de los productos de reacción. Se observa que el uso de la variante más activa E134S permite extender la polimeritzación y conseguir polisacáridos de alto peso molecular en función de la concentración de enzima. De esta manera el grado de polimerización (DP) puede controlarse mediante la actividad enzimática. Para evaluar la inhibición por producto de la actividad glicosintasa de la variante E134A se sintetiza el octasacárido (β4Glcβ4Glcβ4Glcβ3Glc)2, concluyendo que el producto de reacción actúa como inhibidor competitivo aunque no tiene un efecto significativo en los rendimientos preparativos de los productos de polimerización enzimática. Por otra parte, se estudia la actividad glicosintasa de la variante E383A de la β-glucosidasa de Streptomyces sp. Se observa que este mutante presenta actividad hidrolítica y de transglicosidación, hecho que queda patente con la formación de productos secundarios en la reacción glicosintasa. Una vez descartada que la actividad hidrolítica provenga de una contaminación por enzima wt, se propone un mecanismo de hidrólisis y transglicosidación asistido por anión fluoruro como nucleófilo exógeno para la formación de estos productos secundarios no esperados. Finalmente se inmoviliza con éxito la glicosintasa E383A de la β-glucosidasa de Streptomyces sp. sobre resina Chelating Sepharose FF. La enzima inmovilizada no muestra mejora en cuanto a estabilidad frente a disolventes orgánicos. No obstante, sí mejora en términos de estabilidad de almacenamiento a 4ºC y pH 7 en comparación con la forma libre y permite su reutilización hasta 9 ciclos manteniendo el 90% de la actividad inicial.
The verification of the relevance of carbohydrates and glycoconjugates in cellular recognition processes, regulation and signaling, cellular proliferation and immunologic response, as well as in structural and energetic functions, has led to an increasing interest in these biomolecules from Glicobiology and Material Science. This fact has turned into a necessity to endow this increasing demand with synthetic tools that are alternative or complementary to conventional chemistry. This has prompted the development of new synthetic methodologies with the inclusion of enzymatic steps. In 1998, Glycosynthase methodology was developed. Glycosynthases are mutated retaining glycosidases in which the catalytic nucleophile has been replaced by an inert residue, resulting in an enzyme hydrolytically inactive but able to catalyze the formation of glycosidic bonds with glycosyl fluoride donors having the opposite anomeric configuration than the normal substrate of the wild-type enzyme. This Thesis goes into detail about the Glycosynthase methodology both on a mechanistic and applied level. Two glycosynthases are used for this purpose, the E134A mutant of the -glucanase from Bacillus licheniformis and the E383A mutant of the -glucosidase from Streptomyces sp., with the following results: The function of D136 residue in the catalytic site of the E134A glycosynthase is studied. The analysis with double mutant shows that only the E134A/D136G mutant maintains some glycosynthase activity, suggesting that it acts as an assistant residue in the enzymatic mechanism. Additionally, the more active glycosynhases E134S and E134G are obtained, where the substitution of E134 for serine results in a five-fold reactivity increase (in terms ofkcat/KM) compared to the original E134A glycosynthase. The E134A glycosynthase derived frorm the Bacillus licheniformis -glucanase is able to synthesize artificial 1,3-1,4--glucans by polymerization of the following glycosyl fluoride donors: Glcβ3GlcαF, Glcβ4Glcβ3GlcαF and Glcβ4Glcβ4Glcβ3GlcαF. These polysaccharides are formed by repeating units of their corresponding monomers connected by 1,4 linkages. Polysaccharide morphology depends on the repeating unit, so that for the (β4Glcβ3Glc)n and the (β4Glcβ4Glcβ4Glcβ3Glc)n spherulites are observed whereas for the (β4Glcβ4Glcβ3Glc)n an amorphous precipitated is recovered. These polysaccharides are new artificial -glucans with regular structures presenting a 1,3 linkage content higher than observed in nature. The degree of polymerization of the new -glucans is limited by the solubility of the reaction products. The use of the more active E134S mutant allows reaching larger polymers and rendering high molecular mass polysaccharides as a function of enzyme concentration. In this way, the degree of polymerization (DP) can be modulated by enzyme activity. In order to evaluate the product inhibition of the glycosynthase reaction catalyzed by the E134A mutant, the octasaccharide substrate (β4Glcβ4Glcβ4Glcβ3Glc)2, is synthesized, concluding that the reaction product acts as a competitive inhibitor, even though there is not a significant effect on the preparative yields of the enzymatic polymerization products. On the other hand, the E383A glycosynthase derived from the Streptomyces sp. is studied. This mutant presents both hydrolytic and transglycosidation activity, thus the formation of secondary products on the glycosynthase reaction is observed. No contamination of wt enzyme is detected. An hydrolytic and transglycosidation mechanism concerning assisted by fluoride as an exogenous nucleophile is proposed as an explanation for the formation of unexpected secondary products. Finally, E383A β-glucosidasa from Streptomyces sp. is immobilized on Chelating Sepharose FF resin. Immobilization does not increase stability to organic solvents, but the storage stability at 4ºC and pH 7 is clearly enhanced. Additionally, the operation stability is improved, thus the immobilized enzyme maintains 90% of activity along 9 operational cycles.

Increased concerns on antibiotics used in aquaculture have promoted research toward alternative products. Immunostimulants have been approved to be good alternatives for antibiotics used in the culture of many species. A series of experiments were conducted under laboratory conditions to investigate the roles of immunostimulants, beta glucan (BG) and mannan oligosaccharides (MOS) in the culture of marron (Cherax tenuimanus), tropical rock lobster (Panulirus ornatus) and yabbies (Cherax destructor).BG showed survival, physiological and immunological improvements in marron through dietary supplementary levels of 0.1 to 0.2%. Dietary MOS at 0.2 to 0.4% inclusion level improved survival, health status and immune system of marron. Similar improvements of dietary MOS were observed in yabbies and tropical rock lobster. In addition, the growth of yabbies and tropical rock lobster increased with MOS supplemented diets. Dietary MOS also benefited marron, tropical rock lobster and yabbies by enhancing the biological functions of their digestive system. Marron and lobster fed MOS diets revealed a healthier gut condition with improvement in morphological structure and microbiota structure. MOS also changed digestive enzyme activities of yabbies. Further, dietary MOS lowered mortality of marron and tropical rock lobster when they were infected with bacteria. The resistance ability of marron to environmental stressors such as NH3 and air exposure during live transportation was also enhanced in marron fed MOS. In addition, there were no adverse effects of BG and MOS on the performances of above crustaceans.The current research implies that MOS and BG could be the used as alternatives to antibiotics in crustacean cultured under laboratory environment. In order to attain the optimum benefits of MOS and BG dietary supplementation in the commercial production of crustaceans, however, further research is suggested.

To understand and modify the secondary cell walls of plants the project group Enzyme Discovery in Hybrid Aspen for Fiber Engineering (EDEN) was founded composed of nine laboratories with funding from the European Commission. The main target of EDEN´s research is to genetically engineer fiber structure in order to produce transgenic trees with modified properties for the pulp and paper industries.
In this target framework, the Populus tremula x tremuloides xyloglucan endotransglycosylase (PttXET16A) was selected for in-depth study of its transglycosylase activity catalyzing cleavage and reconnection of xyloglucan molecules, which is proposed to be involved in secondary cell wall morphogenesis.
The creation of a family 16 carbohydrate active enzyme -glucanase/XET hybrids were attempted in order to design a chimeric enzyme with one or more of the following altered properties: specificity, activity, and or stability.
The two enzymes, Bacillus licheniformis 1,3-1,4--glucanase and Populus tremula x tremuloides xyloglucan endotransglycosylase, are members of the same enzymatic family and have highly homologous 3-dimensional structures. However, the enzymes exhibit different activities, one a hydrolase the other a transferase; different specificities, one accepts only linear glcosydic substrates while the other branched substrates; and different stabilities.
Hybrid enzyme construction represented an investigational challenge in order to understand what physical characteristics of both enzymes attribute to the specific pattern of activity and specificity observed.
Removal of the 1,3-1,4--glucanase major loop resulted in a folded protein which still maintained some β-glucan hydrolase activity. However, no xyloglucan endotransglycosylase-like activity or specificity was observed. Next, point mutations of the β-sheets forming the enzymatic binding site cleft were mutated to resemble PttXET16A residues. The final chimeric protein neither exhibited XET nor β-glucanase activities. Structural analysis by X-ray crystallography revealed a major unexpected structural rearrangement providing a clear insight for further enzyme engineering.
Amb la finalitat d'entendre i modificar la paret cel·lular secundària de les plantes, es va fundar el grup Enzyme Discovery in Hibrid Aspen for Fibern Engineering (EDEN) composat per nou laboratoris amb la finançament de la Comissió Europea. El principal objectiu de la recerca del grup EDEN és enginyar genèticament l'estructura de fibres per tal de produir arbres transgènics amb propietats modificades per les indústries de la polpa i el paper.
En el marc d'aquest projecte, es va seleccionar el Populus tremula x tremuloides xiloglucà endotransglicosilasa (PttXET16A) per estudiar en profunditat la seva activitat transglicosilasa catalitzant el trencament i la reconnexió de molècules de xiloglucà, el qual sembla estar involucrat en la morfogènesi de la paret cel·lular secundària.
D'aquesta manera, s'intentà crear una família 16 d'híbrids de l'enzim actiu amb carbohidrats -glucanasa/XET per tal de dissenyar un enzim quimèric amb una o més de les propietats següents alterades: especificitat, activitat i/o estabilitat.
Els dos enzims, Bacillus licheniformis 1,3-1,4--glucanasa i Populus tremula x tremuloides xiloglucà endotransglicosilasa, són membres de la mateixa família enzimàtica i tenen una gran homologia en les seves estructures en 3-dimensions. Tot i així, aquests enzims presenten diferents activitats, un presenta activitat hidrolasa i l'altre, transferasa; diferents especificitats, un accepta només substrats glicosílics lineals mentre l'altre, substrats ramificats; i diferents estabilitats.
La construcció d'un enzim híbrid representa un repte en la investigació amb la finalitat d'entendre quines característiques físiques dels dos enzims s'atribueixen al model específic de l'activitat i especificitat observada.
L'extracció del llaç més gran de l'1,3-1,4--glucanasa va resultar en l'obtenció d'una proteïna plegada que encara manté certa activitat hidrolasa del -glucà. Tot i això, no s'observà activitat o especificitat similar a la xiloglucà endotransglicosilasa. A partir d'aquí, es realitzaren mutacions puntuals a diferents punts de les fulles  que formen l'escletxa del lloc d'unió de l'enzim per assemblar-se als residus del PttXET16A. La proteïna quimèrica final tampoc presentava activitat XET ni -glucanasa. L'anàlisi de l'estructura per cristal·lografia de raigs X revelà una major reorganització estructural de l'esperada proveint el nou enzim d'un clar espai intern que obra moltes més portes a l'enginyeria de l'enzim.
Con la finalidad de entender y modificar la pared celular secundaria de las plantas, se fundó el grupo Enzyme Discovery in Hibrid Aspen for Fibern Engineering (EDEN) compuesto por nueve laboratorios con la financiación de la Comisión Europea. El principal objetivo de la búsqueda del grupo EDEN es ingeniar genéticamente la estructura de fibras para producir árboles transgénicos con propiedades modificadas para las industrias de la pulpa y el papel.
En el marco de este proyecto, se seleccionó el Populus tremula x tremuloides xiloglucán endotransglicosilasa (PttXET16A) para estudiar en profundidad su actividad transglicosilasa catalizando la rotura y la reconnexión de moléculas de xiloglucán, el cual parece estar involucrado en la morfogénesis de la pared celular secundaria. De esta forma, se intentó crear una familia 16 de híbridos de la enzima activa con carbohidratos -glucanasa/XET con la finalidad de diseñar una enzima quimérica con una o más de las propiedades siguientes alteradas: especificidad, actividad y/o estabilidad.
Las dos enzimas, Bacillus licheniformis 1,3-1,4--glucanasa y Populus tremula x tremuloides xiloglucà endotransglicosilasa, son miembros de la misma familia enzimática y tienen una gran homología en sus estructuras en 3-dimensiones. Aún así, estas enzimas presentan diferentes actividades, una tiene actividad hidrolasa y la otra, transferasa; diferentes especificidades, una acepta sólo sustratos glicosílicos lineales mientras la otra, sustratos ramificados; y diferentes estabilidades.
La construcción de una enzima híbrida representa un reto dentro de la investigación con la finalidad de entender qué características físicas de las dos enzimas se atribuyen al modelo específico de la actividad y especificidad observada. La extracción del lazo más grande de la 1,3-1,4--glucanasa resultó en la obtención de una proteína plegada que todavía mantiene cierta actividad hidrolasa del -glucán. Aún así, no se observó actividad o especificidad similar a la xiloglucán endotransglicosilasa. A partir de este punto, se realizaron mutaciones puntuales a diferentes puntos de las hojas  que forman la brecha del lugar de unión de la enzima por asemejarse a los residuos del PttXET16A. La proteína quimérica final tampoco presentaba actividad XET ni -glucanasa. El análisis de la estructura por cristalografía de rayos X reveló una mayor reorganización estructural de la esperada proveyendo la nueva enzima de un claro espacio interno que obre muchas más puertas a la ingeniería de la enzima.

(1→3)-β-D-Glucans are carbohydrate polymers that are present in the cell wall of various fungi and bacteria; they are pathogen associated molecular patterns that circulate during infection and modulate immunity. Our laboratory has previously established the pharmacokinetics of intravenously and orally administered glucans; the present studies investigated the renal excretion of (1→3)-β-D-glucans following intravenous and oral administration. Three fluorescently-labeled glucans were administered to adult male rats in the presence or absence of toxic challenge. Urine specimens were collected and analyzed by fluorescence spectroscopy, size-exclusion chromatography and GPC/MALLS. 71 ± 3% of fluorescence remained in the >5K MWCO fraction; this fraction showed a minor peak with a molecular mass (171 ± 11K) corresponding to injected glucan (~150K). Most excreted glucans were of lower molecular mass (13 ± 8.5K), indicating most (1→3)-β-D-glucans are excreted by the kidneys as smaller polysaccharides. The presence of urinary glucans may be an important indicator of fungal infection.

Beta-glukany jsou polysacharidy složeny z monomerů D-glukózy. V dnešní době se -glukany těší zvýšené pozornosti zejména kvůli imunomodulační aktivitě a využitelnosti ve farmaceutickém a potravinařském průmyslu. Saccharomyces cerevisiae je dodnes jediným kvasinkovým zdrojem požívaným v biotechnologické produkci. Avšak některé kvasinky z oddělení Basidiomycetes, které jsou schopny produkce lipidů a karotenoidů, mohou být využity rovněž jako alternativní zdroj -glukanů. Dizertační práce se zabývá možností a optimalizací produkce -glukanů a dalších mikrobiálních sacharidů u karotenogenních kvasinek a mikrořas. Testovány byli zástupci rodů Rhodotorula, Sporobolomyces, Cystofilobasidium a Dioshegia. Z nekarotenogenních kvasinek byly do screeningu zařazeny kvasinky rodu Metschnikowia, askomycetní kvasinky a z mikrořas zástupci zelených a červených řas. Experimentální část cílí rovněž na možnosti koprodukce dalších metabolitů, jako jsou lipidy, pigmenty a extracelulární polymery. První část experimentu se zabývá vlivem čtyř C/N poměrů (10:1, 40:1, 70:1 a 100:1) na produkci biomasy, -glukanů, karotenoidů a lipidů. Ze všech testovaných kmenů, S. cerevisiae CCY 21-4-102, C. infirmominiatum CCY 17-18-4, P. rhodozyma CCY 77-1-1 a R. kratochvilovae CCY 20-2-26 vykazovaly nejvyšší produkci -glukanů a byly proto vybrány k podrobnější optimalizaci, zejména osmotického stresu, teploty a zdroje dusíku v kultivačním médiu. Dodatečně, kmen R. kratochvilovae CCY 20-2-26 je schopný produkce extracelulárních glykolipidů a S. pararoseus CCY 19-9-6 extracelulárních polysacharidů. Následně bylo stanoveno množství -glukanů u dalších dvanácti kmenů S. cerevisiae a rovněž možnost produkce polysacharidů u mikrořas.

This project was carried out to explore the regulatory mechanism for (1,3;1,4)-β-glucan synthesis in barley (Hordeum vulgare L.) in both endosperm and leaf tissues. For the grain (1,3;1,4)-β-glucan content regulation study, transcriptional profiles of candidate genes involved in (1,3;1,4)-β-glucan synthesis and degradation, from the developing barley endosperm, were compared across parental lines that had been previously used for grain (1,3;1,4)-β-glucan QTL studies. Correspondence between differences in transcript levels of selected genes and the QTL detected in parental lines was analysed. In the high (1,3;1,4)-β-glucan parent of a mapping population with a grain (1,3;1,4)-β-glucan QTL near/at the location of the HvCslF6 gene, HvCSLF6 transcript levels increased sharply late in endosperm development. In contrast, HvCslF6 transcript levels did not differ between parents of a mapping population in which no grain (1,3;1,4)-β-glucan QTL had been mapped near/at the HvCslF6 gene. Co-transcription of a β-glucan exo-glucanase gene, HvExoIV gene with HvCslF9 early in endosperm development and with HvCslF6 late in endosperm development indicated that HvEXOIV could be involved in (1,3;1,4)-β-glucan synthesis. It has been reported that leaf (1,3;1,4)-β-glucan is degraded when plants are incubated in the dark for prolonged periods and is re-synthesized upon re-exposure to a normal day/night cycle. Thus, to investigate the regulation of leaf (1,3;1,4)-β-glucan, the transcript levels of (1,3;1,4)-β-glucan synthase genes and related genes were profiled during (1,3;1,4)-β-glucan mobilization upon dark incubation. Some of the genes that responded to prolonged dark incubation showed diurnal transcription patterns, even in continuous darkness. Among the (1,3;1,4)-β-glucan synthase candidate genes, only HvCslH1 was up-regulated upon dark incubation. Its transcripts quickly returned to control levels upon re-exposure to the normal day/night cycle. None of the (1,3;1,4)-β-glucan synthase genes were up-regulated upon re- exposure to normal day/night cycles. Consistent with what was observed for HvExoIV, HvCslF6 and HvCslF9 in developing endosperm, HvExoIV seemed to exhibit co-transcription gene with the HvCslH1.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2015

Cereals such as rice (Oryza sativa (Os)), barley (Hordeum vulgare (Hv)) and sorghum (Sorghum bicolor (Sb)) provide a considerable portion of our daily energy requirements. Their cell wall constituents, such as (1,3;1,4)-β-glucan, survive relatively intact through much of the upper human digestive system to reach the colon, where they are fermented by a range of commensal microorganisms. The products of this fermentation help reduce blood cholesterol levels and ameliorate diseases including coronary heart disease, type II diabetes and colorectal cancer. Efforts have therefore been directed toward understanding the regulation and mechanism of (1,3;1,4)-β-glucan biosynthesis to enhance the human health potential and industrial utility of cereal grain. Numerous reports suggest that the CELLULOSE SYNTHASE-LIKE F6 (CslF6) gene encodes the synthase responsible for producing the majority of (1,3;1,4)-β-glucan in cereals. These synthase genes contain species-specific polymorphisms that have been shown to influence the amount and structure of (1,3;1,4)-β-glucan produced when they are expressed heterologously in Nicotiana benthamiana and barley grain. Here, a chimeric approach exchanged sections of the barley (Hv) and sorghum (Sb) CSLF6 synthases to identify regions influencing (1,3;1,4)-β-glucan production and structure. Using this approach an 80 amino acid stretch, which contains the conserved TED and QxxRW motifs, was shown to be responsible for much of the difference in (1,3;1,4)-β-glucan production and structure between the barley and sorghum synthases. Of the six amino acid polymorphisms contained within this section, one affected polysaccharide structure whilst another dictated the amount of (1,3;1,4)-β-glucan. Co-expression in N. benthamiana was used to investigate CSLF6 modulation and complex formation. Results from a variety of chimeric, truncated and mutated constructs suggest that a highly variable section of unknown function, termed the class-specific region (CSR), and the NH2-terminal region of CSLF6 are separately able to mediate complex formation and increase (1,3;1,4)-β-glucan production. Expression of a construct missing the CSR indicated that the region was not structurally or functionally required for (1,3;1,4)-β-glucan synthesis in N. benthamiana. A PilZ domain responsible for cofactor binding and cellulose synthase activation in bacteria was also identified at the COOH-terminal end of the NH2-terminal region of CSLF6, and was shown to influence (1,3;1,4)-β-glucan production. Overall, the results presented here have furthered our understanding of the action of the CSLF6 isoform of the (1,3;1,4)-β-glucan synthase enzyme. This brings us closer to having the capacity to precisely control the synthase’s function, and allowing the prospect of manipulating cereal tissues to contain the optimal amount of (1,3;1,4)-β-glucan with a defined structure for specific human health and industrial applications.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2016.