Esponjas são sésseis e possuem um esqueleto constituído por elementos inorgânicos microscópicos (espículas), fibras de colágeno e/ou combinações destes dois componentes. Embora sem proteções físicas evidentes tais como conchas ou espinhos, são pouco predadas, sendo este fato atribuído principalmente a presença de metabólitos secundários ativos. Recentemente, um papel dos componentes esqueléticos como deterrente foi experimentalmente confirmado, mas sua importância relativa ainda é bastante debatida. Algumas estrelas-do-mar são consideradas como espongívoras, e a literatura existente geralmente coloca que a digestão é realizada externamente, sem ingestão do esqueleto da presa. Desta forma, neste trabalho foram verificados o comportamento alimentar da estrela-do-mar Echinaster brasiliensis em relação às esponjas, o destino dos componentes esqueléticos e seus efeitos na fisiologia do organismo. Observações de campo e experimentos em laboratório foram efetuados para determinar a estratégia de consumo utilizada pela estrela. O processo digestivo do material esquelético (espículas e/ou fibras) foi acompanhado até a eliminação. Foi verificado que E. brasiliensis se alimenta de diferentes espécies de esponjas, mas não consome apenas estes organismos, sendo mais generalista do que o suposto. As preferências no consumo das esponjas são determinados por fatores diversos, como a quantidade de matéria orgânica ou formato do organismo predado. A ingestão ocorre com a eversão do estômago, mas ao contrário do descrito para outras espécies, E. brasiliensis ingere componentes esqueléticos, sendo espículas encontradas inseridas no epitélio digestivo. Entretanto, essa ingestão é baixa e aparentemente não causa injúrias severas ao organismo, sendo as espículas descartadas no material fecal / Sponges are sessile and have a skeleton made of microscopic inorganic elements (spicules), collagen fibers and/or combinations of these two components. Although without clear physical defenses such as shells or spines, they are not preyed upon, and this is attributed mainly to the presence of active secondary metabolites. Recently, a role of skeletal components as deterrents has been experimentally confirmed, but its relative importance is still debated. Some starfishes are regarded as spongivorous, and the existing literature generally considers that the digestion is performed externally, without intake of the sponge skeleton. In this study the feeding behavior of the starfish Echinaster brasiliensis in relation to sponges, the fate of skeletal components and their effects on the starfish physiology were studied. Field observations and laboratory experiments were conducted to determine the consumption strategies used by the starfish. The digestive process of the skeletal material (spicules and/or fibers) was followed until the elimination. It was found that E. brasiliensis feeds on different species of sponges, but does not consume only these organisms, being more generalist than assumed. Preferences in the consumption of sponges were determined by several factors, such as the amount of organic matter or shape of the preyed organism. The intake occurs by the eversion of the stomach, but unlike what is described for other species E. brasiliensis ingests skeletal components, and spicules were found inserted in the digestive epithelium. However, this intake is small and apparently does not cause severe injuries to the starfishes and the spicules are discarded in fecal material

Orientador: Antonio Carlos de Laurentiz / Co-orientador: João Martins Pizauro Junior / Banca: Alan Peres Ferraz de Melo / Banca: Andréia Fróes Galuci Oliveira de Souza / Resumo: O objetivo deste trabalho foi avaliar a viabilidade da utilização do resíduo do óleo de soja (goma de soja) como um emulsificante na dieta de frangos de corte em fase inicial (1 a 21 dias). Foram realizados dois experimentos, uma para fonte lipídica de origem vegetal (óleo de soja) e outro para fonte lipídica de origem animal (gordura suína), sendo utilizados 600 pintos de corte macho da linhagem Cobb® 500, distribuídos em um esquema fatorial (3x4) sendo três níveis de goma (0%; 1,25%; 2,5%) e quatro níveis de fonte lipídica (0%; 1,3%; 2,6%; 3,9%), totalizando em 12 tratamentos com 5 repetições para cada um dos experimentos. Foram analisados parâmetros de desempenho, coeficientes de digestibilidade e atividades de enzimas pancreáticas. No experimento onde se utilizou óleo de soja como fonte lipídica foi possível verificar melhorias de desempenho a partir dos 14 dias, e também melhora do coeficiente de digestibilidade do extrato etéreo e aumento nos níveis de energia metabolizável aparente. Quando utilizada a gordura suína como fonte lipídica não foram obtidas diferenças de desempenho relacionadas a inclusão da goma, mas foi possível observar efeitos diretos da interação da utilização da goma com a gordura nos níveis de energia metabolizável aparente / Abstract: The objective of this study was to evaluate the feasibility of using soybean oil residue (soy gum) as an emulsifier in broiler diet at an early stage (1-21 days). Two experiments were conducted, one for lipid source of vegetable (soybean oil) and one for fat source of animal origin (pork fat) and used 600 male broiler chicks of the lineage Cobb® 500, distributed in a factorial scheme (3x4) with three levels of gum (0%, 1.25%, 2.5%) and four lipid source levels (0%, 1.3%, 2.6%, 3.9%), totalizing 12 treatments with 5 replicates for each of the experiments. Performance parameters, digestibility coefficients and activities of pancreatic enzymes were analyzed. In the experiment which we used soy oil as a lipid source was possible to see performance improvements from 14 days, and also improves the digestibility coefficient of ether extract and increase the apparent metabolizable energy levels. When used in pork fat as lipid source, performance differences were not obtained related to the inclusion of soy gum, but we observed direct effects of the use of gum interaction with the fat in the apparent metabolizable energy levels / Mestre

Esponjas são sésseis e possuem um esqueleto constituído por elementos inorgânicos microscópicos (espículas), fibras de colágeno e/ou combinações destes dois componentes. Embora sem proteções físicas evidentes tais como conchas ou espinhos, são pouco predadas, sendo este fato atribuído principalmente a presença de metabólitos secundários ativos. Recentemente, um papel dos componentes esqueléticos como deterrente foi experimentalmente confirmado, mas sua importância relativa ainda é bastante debatida. Algumas estrelas-do-mar são consideradas como espongívoras, e a literatura existente geralmente coloca que a digestão é realizada externamente, sem ingestão do esqueleto da presa. Desta forma, neste trabalho foram verificados o comportamento alimentar da estrela-do-mar Echinaster brasiliensis em relação às esponjas, o destino dos componentes esqueléticos e seus efeitos na fisiologia do organismo. Observações de campo e experimentos em laboratório foram efetuados para determinar a estratégia de consumo utilizada pela estrela. O processo digestivo do material esquelético (espículas e/ou fibras) foi acompanhado até a eliminação. Foi verificado que E. brasiliensis se alimenta de diferentes espécies de esponjas, mas não consome apenas estes organismos, sendo mais generalista do que o suposto. As preferências no consumo das esponjas são determinados por fatores diversos, como a quantidade de matéria orgânica ou formato do organismo predado. A ingestão ocorre com a eversão do estômago, mas ao contrário do descrito para outras espécies, E. brasiliensis ingere componentes esqueléticos, sendo espículas encontradas inseridas no epitélio digestivo. Entretanto, essa ingestão é baixa e aparentemente não causa injúrias severas ao organismo, sendo as espículas descartadas no material fecal / Sponges are sessile and have a skeleton made of microscopic inorganic elements (spicules), collagen fibers and/or combinations of these two components. Although without clear physical defenses such as shells or spines, they are not preyed upon, and this is attributed mainly to the presence of active secondary metabolites. Recently, a role of skeletal components as deterrents has been experimentally confirmed, but its relative importance is still debated. Some starfishes are regarded as spongivorous, and the existing literature generally considers that the digestion is performed externally, without intake of the sponge skeleton. In this study the feeding behavior of the starfish Echinaster brasiliensis in relation to sponges, the fate of skeletal components and their effects on the starfish physiology were studied. Field observations and laboratory experiments were conducted to determine the consumption strategies used by the starfish. The digestive process of the skeletal material (spicules and/or fibers) was followed until the elimination. It was found that E. brasiliensis feeds on different species of sponges, but does not consume only these organisms, being more generalist than assumed. Preferences in the consumption of sponges were determined by several factors, such as the amount of organic matter or shape of the preyed organism. The intake occurs by the eversion of the stomach, but unlike what is described for other species E. brasiliensis ingests skeletal components, and spicules were found inserted in the digestive epithelium. However, this intake is small and apparently does not cause severe injuries to the starfishes and the spicules are discarded in fecal material

Chick embryos derive nutrients from the yolk during incubation and transition to intestinal absorption posthatch. Nutrient uptake is mediated by digestive enzymes and membrane bound transporter proteins. The objective of this study was to determine expression profiles of nutrient transporters and digestive enzymes during incubation in the yolk sac membrane (YSM) and small intestine of Leghorn and Cobb chickens derived from 22–30 wk (young) and 45–50 wk (old) breeder flocks. Genes examined included peptide transporter PepT1, amino acid transporters EAAT3, CAT1, and B0AT, monosaccharide transporters SGLT1 and GLUT5, and digestive enzymes APN and SI. Expression of these genes was measured in YSM at embryonic day (e) 11, 13, 15, 17, 19, 20, and 21 and small intestine at e15, e17, e19, e20, and e21. Absolute quantification real-time PCR assessed gene expression. PepT1, APN, and B0AT expression in YSM peaked between e15 and e17 and then decreased until e21, while expression increased over time in the small intestine. SGLT1 and EAAT3 expression increased over time in the small intestine and YSM. There was minimal gene expression of SI in the YSM, while the small intestine had high expression. GLUT5 and CAT1 expression decreased in the YSM, while peaking at e19 then decreasing in the small intestine. Breed and flock age affected expression levels in some genes. These results demonstrate that these genes show tissue- and development-specific patterns of expression and that the YSM expresses many digestive enzymes and nutrient transporters associated with the small intestine. / Master of Science

Thesis (D. Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2006 xii, 274 leaves / In Sub-Saharan Africa there is an urgent need to sustain and improve the quality of its food resources. Poverty eradication features high on the agenda of a number of world health organisations, while the number of underweight children in Africa continues to increase (Pellet, 1996). Providing nutritionally enhanced foods to the poor will help towards achieving this objective. Protein-energy malnutrition has been identified as one of the most important problems facing Africa, with maize as the staple diet (Nkama et al., 1995). However, a combination of several factors limits availability and the nutritional quality of maize. During starvation, energy and protein intakes decrease by 20-30%, with most of the children in Africa having an average protein intake of only 20 g per day (Igbedioh, 1996). Energy availability also affects protein utilization because of interrelationships of protein and energy metabolism (Elwyn, 1993). The diets of inhabitants in developing regions depend mainly on cereals (maize) for both protein and dietary energy which lacks indispensable amino acids, minerals, vitamins and carbohydrates. In light of these growing concerns an attempt was made to devise a scientific strategy to combat the nutritional shortfalls of maize meal. A multidisciplinary and concerted approach was followed within this project aimed at designing an improved thermostable amylase and applying the enzyme to nutritionally enhance maize meal. It was envisaged that the manipulation of maize meal, by the application of enzyme technology will improve the nutritional status of this staple food. The consequences is that an alternate solution for the eradication of an ailing, poverty stricken and malnourished African population is achievable. It is possible that the boundaries defining the limits of life will extend to even greater extremes through the application of novel technologies.

Thesis (D. Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2006 xii, 274 leaves / In Sub-Saharan Africa there is an urgent need to sustain and improve the quality of its food resources. Poverty eradication features high on the agenda of a number of world health organisations, while the number of underweight children in Africa continues to increase (Pellet, 1996). Providing nutritionally enhanced foods to the poor will help towards achieving this objective. Protein-energy malnutrition has been identified as one of the most important problems facing Africa, with maize as the staple diet (Nkama et al., 1995). However, a combination of several factors limits availability and the nutritional quality of maize. During starvation, energy and protein intakes decrease by 20-30%, with most of the children in Africa having an average protein intake of only 20 g per day (Igbedioh, 1996). Energy availability also affects protein utilization because of interrelationships of protein and energy metabolism (Elwyn, 1993). The diets of inhabitants in developing regions depend mainly on cereals (maize) for both protein and dietary energy which lacks indispensable amino acids, minerals, vitamins and carbohydrates. In light of these growing concerns an attempt was made to devise a scientific strategy to combat the nutritional shortfalls of maize meal. A multidisciplinary and concerted approach was followed within this project aimed at designing an improved thermostable amylase and applying the enzyme to nutritionally enhance maize meal. It was envisaged that the manipulation of maize meal, by the application of enzyme technology will improve the nutritional status of this staple food. The consequences is that an alternate solution for the eradication of an ailing, poverty stricken and malnourished African population is achievable. It is possible that the boundaries defining the limits of life will extend to even greater extremes through the application of novel technologies.

Dans le cadre de l'évaluation de la qualité des écosystèmes aquatiques, les réponses en lien avec l'énergie semblent particulièrement pertinentes pour appréhender l'état physiologique des êtres vivants et prévoir des effets populationnels. Dans ce contexte, l'objectif de ce travail a été de proposer un biomarqueur de l'assimilation de l'énergie chez un amphipode d'eau douce Gammarus fossarum. La caractérisation des activités enzymatiques digestives (amylase, cellulase et trypsine) et des réserves énergétiques (glycogène, lipides et protéines) soumises à l'influence de divers facteurs biotiques et abiotiques (sexe, stade reproducteur, température, conductivité et quantité de nourriture) a permis d'identifier l'amplitude des réponses étudiées afin d'améliorer l'interprétation des réponses obtenues en milieu naturel. Ces résultats ont également validé l'utilisation d'organismes mâles standardisés et nourris ad libitum dans le cadre d'approche active. D'autre part, la pertinence écologique des enzymes digestives en tant que biomarqueur a été évaluée en étudiant le lien existant entre une perturbation des réponses enzymatiques digestives au niveau sub-individuel et ses conséquences au niveau individuel sur le succès reproducteur des gammares. Par ailleurs, dans le cadre de deux campagnes de terrain, les mesures des activités enzymatiques digestives réalisées chez les organismes transplantés ont permis de discriminer les sites en fonction de leur qualité chimique et de proposer pour chaque marqueur une valeur référence et seuil traduisant un effet. Ainsi les activités enzymatiques digestives peuvent être utilisées comme outil pour établir un diagnostic de risque environnemental. / For the assessment of the aquatic ecosystems health, responses related to energy metabolism seem particularly relevant to understanding the physiological state of living organisms and provide population- effects. In this context, the objective of this work was to propose a biomarker of energy assimilation in a freshwater amphipod Gammarus fossarum. The characterization of digestive enzymes activities ( amylase , cellulase and trypsin) and energy reserves (glycogen , lipids and proteins) submited to the influence of various biotic and abiotic factors (sex, reproductive stage , temperature, conductivity and amount of food ) determine the range of responses studied to improve the interpretation of data obtained in the field. These results can also validate the use of standardized male fed ad libitum in caging approach. On the other hand, the ecological relevance of digestive enzymes was assess, the link between digestive enzyme impairments at sub -individual level and its consequences on the fitness of the gammarid population at individual level was established. Furthermore, in the context of two field campaigns, measures of digestive enzyme activities in organisms transplanted allowed to discriminate sites according to their chemical quality and to propose for each marker a benchmarck and a threshold value reflecting an effect. Thus, digestive enzyme activities can be used as diagnostic tool for environmental risk assessment.

Tripsinas são enzimas comuns à maioria dos insetos e de fundamental importância para a digestão inicial de proteínas. Desta forma, tornam-se alvos importantes para orientar a construção de plantas transgênicas resistentes a insetos. As tripsinas são serina endopeptidases que clivam cadeias protéicas na porção carboxílica de resíduos de aminoácidos básicos como lisina e arginina, sendo que a hidrólise da ligação peptídica formada por um resíduo de arginina é de duas a dez vezes mais eficiente que a hidrólise da ligação peptídica formada por lisina. A purificação das tripsinas de insetos de diferentes ordens e o estudo da especificidade de seus subsítios utilizando substratos de fluorescência apagada servem de base para a seleção de um método mais eficiente de inibição da sua atividade, assim como para desvendar as tendências evolutivas da especificidade destas enzimas. O trabalho dessa dissertação levou ao desenvolvimento de processos de purificação das tripsinas de Periplaneta americana, Tenebrio molitor, Musca domestica e Diatraea saccharalis. O estudo da especificidade dos subsítios S1, S2, S3 e S1\' das tripsinas demonstraram que, diferentemente das tripsinas dos outros insetos e das tripsinas de mamíferos, a tripsina de Diatraea saccharalis hidrolisa com maior eficiência substratos que apresentem lisina em P1, demonstrando uma diferença na especificidade primária desta enzima. Além disso, é possível verificar ao longo da evolução dos grupos de insetos estudados uma tendência a tornar os subsítios cada vez mais hidrofóbicos. / Trypsins are serine endopeptidases that hydrolyze peptide bonds at the carboxyl side of positively charged residues: arginine and lysine. Mammalian trypsin preferentially cleaves the peptide bond formed by arginine. Site directed mutagenesis has shown that trypsin specificity is related to residues present at the primary specificity site and to structural determinants like two surface loops. Differences in trypsins specificity may be the cause of some insects be resistant to serine endopeptidases plant inhibitors and to Bacillus thuringiensis toxins. Trypsins are usual enzymes in insects and are very important to protein digestion. There are few studies dealing with insect trypsin specificity. They generally consist in analyses of fragments formed by the action of the enzyme on peptide chains like insulin β chain. As these studies were semi-quantitative, insect trypsin specificity requires a better characterization. This dissertation describes the purification of trypsins from Periplaneta americana, Tenebrio molitor, Musca domestica and Diatraea saccharalis and the characterization of the specificity of the subsites S1, S2, S3 e S1\' by the use of quenched fluorescence peptide substrates. The results showed that trypsins from the mentioned insects have different specificities, including the primary specificity. Thus, Diatraea saccharalis trypsin cleaves at Lys more efficiently than at Arg, whereas the eontrary is true for the other insects. The data also showed that trypsin subsites tend to beeome more hydrophobic as the insects are more evolved.

Serina endopeptidases de insetos, principalmente tripsinas e quimotripsinas, estão envolvidas na digestão inicial de proteínas. Genes codificadores para estas enzimas estão organizados em famílias multigênicas tendo expressão diferencial de acordo com a dieta do inseto, estando envolvidos no desenvolvimento de resistência a diferentes metabólitos secundários vegetais. Para uma melhor compreensão desta interação, fez-se necessário o isolamento destas enzimas para insetos de diferentes ordens, bem como a caracterização de suas especificidades por duas abordagens: (a) caracterização cinética dos subsítios componentes do sítio de ligação de tripsinas e quimotripsinas, utilizando diferentes substratos, modificadores químicos e inibidores e (b) estudos estruturais por modelagem molecular, clonagem, expressão e cristalização destas enzimas de insetos. Além disso, estudos evolutivos por análise de distância possibilitaram uma caracterização inicial da interação insetoplanta. Estas determinações permitiram verificar que tripsinas de insetos apresentam diferenças de especificidade tanto dentre as diferentes ordens de insetos quanto em relação às tripsinas de vertebrados, sendo que as tripsinas da ordem Lepidóptera apresentam troca de especificidade primária hidrolisando preferencialmente substratos P1 Lys. Foram também observadas diferenças de hidrofobicidade para os subsítios caracterizados sendo que estes apresentam hidrofobicidades crescentes segundo o grau de complexidade dos insetos na sua escala evolutiva. A troca de especificidade e o aumento da hidrofobicidade podem permitir a hidrólise dos inibidores vegetais protéicos. A análise das sequências de tripsinas de insetos por Neighbor Joining (NJ) compõe uma árvore de distâncias topologicamente semelhante à árvore de relações filogenéticas determinadas por morfologia. A sobreposição de estruturas pré -determinadas de tripsina complexada a diferentes inibidores permite a identificação de posições de interação enzima-inibidor que justificam a classificação em grupos distintos de enzimas sensíveis ou resistentes a presença de inibidores na dieta de insetos. Da mesma forma: a caracterização da especificidade das quimotripsinas de insetos permitiu a separação de grupos distintos de quimotripsinas. Estes grupos são sustentados pela substituição do resíduo 59 em insetos polífagos que alimentam-se de plantas que contêm cetonas naturais reativas. Estas caracterizações demonstram a importância de um estudo detalhado da especificidade de serina endopeptidases possibilitando o desenho de moléculas apropriadas para inibição destas e desenvolvimento de estratégias de controle de insetos. / Insect serine endopeptidases, mairily trypsin and chymotrypsin are involved in initial protein digestion. Genes that encode these proteins are members of complex multigene families and are differentially expressed according to insects diet , thus being involved with resistance to plant metabolites. Purification of trypsins from different insect orders and chymotrypsins, as well as, characterization of their specificity are essential to a better understanding of this interaction. Characterization relied on two approaches: (a) kinetic characterization of the binding subsities of trypsins and chymotrypsins using different substrates, chemical modification and inhibition assays and (b) study of protein structure by molecular modelling and cloning, expression and crystallization of these enzymes. Besides that, evolutionary studies performed through distance analysis, permitted the investigation of plantinsect interaction. These characterizations showed that insect trypsins, in terms of specificity, are quite different from vertebrate trypsins and among insect orders. Lepidopterans trypsins have a distinct primary specificity, since they hydrolyses preferentially P1 Lys substrates, and present a crescent subsite hydrophobicity, which is directly correlated with the evolutionary scale. Both, the specificity exchange and the crescent hydrophobicity can allow the hydrolysis of vegetal proteic inhibitors. The analysis of trypsin sequences in Neighbor-Joining (NJ) algorithm yield a distance tree that is coherent with morphological phylogenetic relationships. The superposition of predicted structures of trypsins-inhibitors complexes permits to observe amino acid residues of interaction between enzyme-inhibitor, which support the distinction of different groups between sensitive and insensitive trypsins to the presence of inhibitors on insect diet. Similarly, characterization of insect chymotrypsins according to their specificity allowed us to classify these enzymes into different groups. These groups are supported by residue 59 replacements in polyphagous insects, which feed on plants bearing natural reactive ketones. These studies show the irnportance of a detailed study of serine endopeptidases, which may help in the development of better insect control strategies.

The anticancer activity of several platinum compounds is due to the formation of complexes with DNA. We hypothesize that the size and shape of the platinum compounds would impact interaction with proteins, and these interactions may be partly responsible for the anticancer activity. Chymotrypsin and subtilisin are serine proteases that have a histidine residue in the active site. We are investigating the inhibition of the digestive enzymes chymotrypsin and subtilisin by analogs of the anticancer drug cisplatin and trying to discern trends in the inhibition as the active site residues vary. In our research, we found that the enzyme subtilisin did not show any significant inhibition with different platinum compounds we used, while chymotrypsin showed inhibition only with the potassium tetrachloroplatinate and this inhibition is concentration dependent