Hypoxia inducible factor (HIF)-1α is a heterodimerically-activated transcription factor central to the cellular response to hypoxic environments and is often upregulated in cancer. Binding of HIF-1α to the co-activator p300 is necessary for the hypoxia-induced transcription of many oncogenic proteins. The aim of this project was to develop novel small molecule inhibitors of the HIF-1α/p300 protein-protein interaction (PPI). Initial work focused on designing, validating and optimising two high-throughput competition binding assays to screen for inhibitors of the PPI (Chapter 2). Alongside these, zinc ejector assays for both p300 and KDM4A proteins were developed to probe the mechanism of action and selectivity. Analysis of hits from a natural product high-throughput screen (HTS) revealed two compound classes; benzoquinones and 2-substituted indandiones, which modulate the PPI. The potency of these series correlated with the reactivity of the core functional groups, which act as electrophiles to covalently modify reactive cysteines, ejecting structural zinc and disrupting the p300/KDM4A protein fold (Chapter 3). Conjugating electrophilic groups to putative HIF-1α/p300 inhibitors did not replicate the activity of the zinc ejecting HTS hits (Chapter 4). Further work focused on non-covalent inhibitors of the HIF-1α/p300 interaction, first with peptide truncates, and then rationally designed α-helix peptidomimetics. An 11mer truncate showed encouraging activity (IC50 ≈ 70 μM), and corresponded to a key α-helix in the HIF-1α C-terminal transactivation domain. Three distinct double-sided scaffolds were used to imitate up to five hot-spot ampiphilic residues on this α-helix (Chapter 6 and 7). Of the 35 compounds screened, only modest inhibition was observed (IC50 ≈ 200-500 μM). Future work will look to conjugate electrophilic functionality onto the 11mer peptide in an attempt to gain potency from zinc ejection, while maintaining selectivity for p300.

This thesis describes the development of a novel approach for prediction of the three-dimensional structure of transmembrane regions of membrane proteins directly from amino acid sequence and basic transmembrane region topology. The development rationale employed involved a knowledge-based approach. Based on determined membrane protein structures, 20x20 association matrices were generated to summarise the distance associations between amino acid side chains on different alpha helical transmembrane regions of membrane proteins. Using these association matrices, combined with a knowledge-based scale for propensity for residue orientation in transmembrane segments (kPROT) (Pilpel et al., 1999), the software predicts the optimal orientations and associations of transmembrane regions and generates a 3D structural model of a gi ven membrane protein, based on the amino acid sequence composition of its transmembrane regions. During the development, several structural and biostatistical analyses of determined membrane protein structures were undertaken with the aim of ensuring a consistent and reliable association matrix upon which to base the predictions. Evaluation of the model structures obtained for the protein sequences of a dataset of 17 membrane proteins of detennined structure based on cross-validated leave-one-out testing revealed generally high accuracy of prediction, with over 80% of associations between transmembrane regions being correctly predicted. These results provide a promising basis for future development and refinement of the algorithm, and to this end, work is underway using evolutionary computing approaches. As it stands, the approach gives scope for significant immediate benefit to researchers as a valuable starting point in the prediction of structure for membrane proteins of hitherto unknown structure.

In an exploratory study, techniques were developed for isolating bulk plant proteins and measuring the ¹³C isotopic compositions of their constituent amino acids by HPLC-IRMS. Samples of plants expected to be of potential palaeodietary significance in northwestern Europe were selected for investigation. Different tissues of plants, leaves and seeds, may be distinguished from each other by the relative ¹³C isotopic compositions (‘isotopic signatures’) of the amino acids of their constituent proteins. For each tissue type, different plant types may be distinguished in the same way. These signatures can vary slightly according to environment and season, but the variation among types is greater than this. For leaves, isotopic signatures can be used to differentiate (i) nettles, (ii) true grasses, (iii) reeds etc, (iv) trees, (v) legumes, (vi) maize, (vii) freshwater plants and (viii) marine algae. For seeds, these signatures are able to differentiate (i) wheat-type cereals, (ii) barley-type cereals, (iii) C4 cereals, (iv) pseudocereals, (v) legumes and (vi) tree nuts. From investigations using a mixing model, it appears that these signals, particularly those of essential amino acids, are reflected in the tissues of their consumers. Freshwater plants are identified as the base of the food chain for dragonfly larvae, marine algae as the diet of marine molluscs and grass as the diet of archaeological cattle and aurochs. Isotopic ‘marine signals’ identified by previous researchers have been refined using these data and the isotopic signatures of fish muscle. These findings are expected to be of particular value in the study of palaeodiets using proteins from archaeological tissues, especially bone and hair. This approach will also find application in the fields of plant physiology and biochemistry.

This project was undertaken to gain a better understanding of the hydration behaviors of gas phase ions from solutions containing amino acids and peptides. In order to characterize their hydration behavior, the molecules of interest in solutions were first converted into gas phase ions by electrospray ionization (ESI). The completely desolvated ions were then deliberately dispersed into an inert bath gas, usually nitrogen, containing accurately known concentrations of solvent vapor. The resulting mixtures of ions and bath gas were subsequently passed into a vacuum chamber by way of an adiabatic supersonic free jet expansion. The cooling during that expansion caused solvation of the ions, the extent of which was determined by a quadrupole mass analyzer. Mass analysis of the solute ions in the absence of vapor showed peaks with the mass to charge ratios corresponding to the desolvated ions. On the other hand, mass spectrometric analyses of ions in the presence of solvent vapor showed sequences of peaks corresponding to the solvated ions with varying numbers of water molecules. The extent of the ion solvation was controlled by varying the concentration of solvent vapor in the bath gas. Two different scales were proposed for the evaluation of the relative affinities of amino acids for water molecules. One was based primarily on the assumption that the affinities of amino acids for water molecules are directly proportional to their gas phase solvation rate constants (k). An alternative approach produced an affinity scale based on the extent of ion hydration occurred during the free jet expansion. It was found that the addition of a polar solvent vapor to the bath gas at low concentrations substantially enhanced the production of the bare solute ions from the evaporating charged droplets. This remarkable result not only provided a means to increase the ion production and thus detection sensitivity of mass spectrometric analyses, but also yielded important information regarding the ion formation mechanism of ESI. Additional studies revealed that the extent of the increase in ion yield was directly related to the charge state and molecular weight of the solute ions. In sum, this evidence strongly indicated that gas phase ions produced from charged droplets, as in electrospray ionization, must proceed by the sequence of events assumed in the Ion Evaporation Model proposed by Iribarne and Thomson rather than in the Charged Residue Model originally proposed by Malcolm Dole and coworkers. The hydration behaviors of electrospray ions from peptides with similar primary amino acid sequences and capable of forming ions with more than one charge state were also investigated. In a study with dipeptides, the extent of hydration was found to vary widely and to depend not only on the chemical composition of the ions but also on their configurations and charge states. The results obtained with lysine oligomers clearly indicated that the number of charges on an ion played an important role in the solvation process. An exception to this generalization was found in an experiment with multiply protonated pentalysine ions. For example, the quadruply protonated monomers of that species were found to undergo charge reduction via proton exchange with the surrounding water molecules in such a way as to maximize the distance between charges on the molecule, thereby reducing the internal repulsive forces.The hydration study of angiotensin II and III showed that while the former has an additional hydrophilic amino acid on the N-terminus, the latter peptide was more hydrophilic. This result suggests that the hydrophilicities of peptides are not a simple sum of the hydrophilicities of the individual amino acid components. As further evidence of interaction complexity, the Magic Number Clusters containing 21 water molecules were obtained with the doubly protonated angiotensin III, but not with the doubly protonated angiotensin II. Taken together, these observations seem to indicate that the multiply charged ions of angiotensin II and III had different structural conformations.

Folding free energy of a protein is a delicate balance between stabilizing and destabilizing non-covalent itneractions. In this work, we decompose folding free energy into physically meaningful contributions, in which we aim to find general trends. Empirical potential is used to calculate interaction energy between all protein fragments, which are classified based on their dominant term in multipolar expansion. Calculations are done using 1200 non-redundant structures from PDB database. Based on the general trends found in interactions between these fragments, we attempt to better understand relationships between interaction energies calculated using computational chemistry methods and their corresponding free energy contributions on stabilization. 1

Doctor of Philosophy / Department of Animal Sciences and Industry / Barry J. Bradford / The mechanisms behind the metabolic changes observed when transition cows are administered monensin, as well as the effects of supplementing mid-lactation cows with two commercial amino acid products were investigated. Traditionally, the effects of monensin are attributed to increased gluconeogenic precursor supply, but recent research indicated that the effects of monensin extend beyond gluconeogenic flux. Thus, the primary objectives of Experiment 1 were to determine if monensin modulates transition cow feeding behavior, ruminal pH, and/or expression of key metabolic genes. Overall, monensin decreased time between meals prepartum (126 vs. 143 ± 5.0 min; P < 0.03) with a trend appearing postpartum (81.4 vs. 88.8 ± 2.9 min; P < 0.08), which could be related to the smaller ruminal pH standard deviation during the first day cows received the lactation ration (0.31 vs. 0.26 ± 0.015; P < 0.02). Monensin also increased liver mRNA abundance of carnitine palmitoyltransferase 1a (0.15 vs. 0.10 ± 0.002 arbitrary units; P < 0.04), which corresponded to a slower rate of liver triglyceride (TG) accumulation from 7 days before calving through 7 days post calving (412 vs. 128 ± 83 mg TG/g protein over this time period; P = 0.03). No significant effects of monensin supplementation were observed on other metabolic parameters or milk production. Overall, these results confirm that the effects of monensin on transition cows extend beyond altered propionate flux. In Experiment 2, mid-lactation cows consuming a control diet containing 26% wet corn gluten feed (dry matter basis) were compared to cows consuming the same diet supplemented with lysine embedded within Ca salts of fatty acids and the isopropyl ester of 2-hydroxy-4-(methylthio) butanoic acid, a methionine precursor. This trial was conducted because the NRC (2001) model indicated a lysine deficiency prior to supplementation; however amino acid supplementation had no effects. This trial was then extended to decrease dietary CP from 17.9% to 17.1%, and further increase lysine and methionine supply in the treatment diet. No production or intake effects were observed during this period, but MUN was decreased in the treated group (10.8 vs. 12.5 ± 0.2 mg/dL; P < 0.001).

L’objectif de ce travail de thèse est de développer de nouveaux agents tensioactifs, capables de s’auto-assembler sous forme de micelles permettant de solubiliser les principes actifs insolubles, en vue de leur administration par voie intraveineuse. Cette étude a permis la synthèse, la caractérisation physico-chimique ainsi que l’évaluation toxicologique in vitro et in vivo de nouveaux agents tensioactifs d’origine naturelle. Au cours de cette étude, différentes familles de tensioactifs ont été évaluées. Ces nouveaux agents tensioactifs sont composés d’une partie hydrophobe de type cholestérol, sels biliaires ou lipides, associée via une fonction amide à une partie hydrophile dérivée d’acides aminés tels que la lysine, la glutamine ou l’acide glutamique.Ces travaux expérimentaux ont permis d’étudier l’influence de la flexibilité de la partie hydrophobe sur la capacité de solubilisation des tensioactifs. Cette étude a montré que l’efficacité de solubilisation est reliée à la flexibilité de la partie hydrophobe. L’utilisation d’agents tensioactifs composés d’une chaîne lipidique saturée flexible a permis de solubiliser efficacement le principe actif insoluble avec un taux de charge de 46 % (m/m). Les tensioactifs composés de lipides saturés sont donc plus efficaces en termes de solubilisation que les dérivés de stéroïdes ou de lipides polyinsaturés, moins flexibles. Les études de toxicité ont mis en évidence la relation ente la structure chimique des tensioactifs et leur toxicité, en particulier vis-à-vis des membranes cellulaires. L’introduction de doubles liaisons en configuration cis dans la partie lipidique des tensioactifs permet de diminuer leur interaction avec les membranes cellulaires et donc leur toxicité mais diminue également leur capacité de solubilisation. Le développement de nouveaux agents tensioactifs nécessite donc de trouver un compromis entre la capacité de solubilisation et la toxicité des tensioactifs. / The aim of this thesis was to develop novel surfactants, able to self-assemble into micelles and to solubilize insoluble drugs intented for intravenous injection. Natural-based surfactants were synthesized and their physico-chemical properties were evaluated. In addition, their in vitro and in vivo toxicity were evaluated. Their drug solubilization abitity was also investigated. Three surfactant classes were evaluated. They were composed of a hydrophobic moiety, such as cholesterol, bile salts or lipids, bonded to a hydrophilic moiety, deriving from amino acids, such as lysine, glutamine or glutamic acid, via an amide bond.The influence of surfactant hydrophobic moiety flexibility on drug solubilization ability was evaluated. This study evidenced that solubilization efficiency is related to the surfactant hydrophobic moiety flexibility. The use of surfactants with flexible and saturated lipidic moieties increased drug water solubility with a drug loading of 46 % (w/w). Saturated lipid-based surfactants exhibited a better solubilization efficiency, in comparison with steroid-based surfactants or poly-unsaturated-based surfactants. Toxicity studies evidenced the relation between surfactant chemical structure and their toxicity, in particular with cell membranes. The introduction of double bond in cis configuration in surfactant lipidic moiety decreased their interaction with cell membranes and thus their toxicity. In addition, this chemical modification also decreased their solubilization ability. To develop novel surfactants, it is thus necessary to take into account drug solubilization ability and toxicity of surfactants.

As peçonhas de serpentes contêm uma mistura complexa de substâncias farmacologicamente ativas, como metaloproteases, fosfolipases A2, serino-proteases, L-aminoácido oxidase (LAAO), além de outros importantes compostos sem ação enzimática. LAAOs são flavoproteínas que catalisam a desaminação oxidativa de L-aminoácidos e produzem o -cetoácido correspondente, com a concomitante liberação de amônia e peróxido de hidrogênio. A peçonha de Lachesis muta (L. muta) contém L-aminoácido oxidase, a qual pode contribuir com o envenenamento. Portanto, o objetivo deste trabalho é a purificação da L-amino acido oxidase de peçonha de Lachesis muta (LmLAAO) e a sua caracterização bioquímica, estrutural e funcional. Para isso, foram desenvolvidos dois protocolos distintos de purificação, os quais forneceram LmLAAO com grande pureza. No primeiro protocolo, 20 mg de peçonha bruta de L. muta foram submetidos a uma gel filtração em Sephacryl S100®. Das dez frações obtidas, a primeira fração apresentou atividade L-aminoácido oxidase e foi submetida a mais um passo cromatográfico em Mono Q®. A homogeneidade da fração com atividade L-aminoácido oxidase após a troca iônica foi comprovada por presença de banda única com 60,2 kDa em SDS-PAGE. O segundo protocolo de purificação foi uma sequência de três passos cromatográficos, na qual 200 mg de peçonha bruta de L. muta foram submetidos a gel filtração em Sephacryl S200®, seguido por interação hidrofóbica em Phenyl Sepharose® e Affi- gel Blue Gel®. Da mesma forma, a pureza da enzima obtida depois desses passos cromatográficos foi comprovada por presença de banda única com 64 kDa em SDS-PAGE. Em ambos os protocolos de purificação, LmLAAO manteve sua atividade enzimática. A massa molar de LmLAAO foi determinada por espectrometria de massas (MALDI-TOF) e apresentou valor de 60,85 kDa. Além disso, foi determinado o valor do ponto isoelétrico da LmLAAO como 5,1. A LmLAAO mostrou preferência catalítica por aminoácidos hidrofóbicos (L-Metionina L-Leucina e L-Fenilalanina) e apresentou perda de atividade catalítica quando submetida à altos valores de pH ou de temperatura. Os parâmetros cinéticos foram determinados e a constante de Michaelis-Menten para o substrato L-Leucina foi de 0,9737 mmol/L e a velocidade máxima de reação foi de 0,06345 mol peróxido de hidrogênio/min. A sequência N-terminal dos 40 primeiros resíduos da LmLAAO purificada foi determinada por degração de Edman e a sua estrutura primária completa foi deduzida da sequência do cDNA obtido da glândula de peçonha. Verificou-se uma grande identidade entre as sequências em aminoácidos da LAAO de L. muta e as de outros viperídeos. A estrutura da LmLAAO foi resolvida por substituição molecular usando as coordenadas atômicas da LAAO de Agkistrodon halys pallas (PDB 1REO). As atividades farmacológicas da LmLAAO foram determinadas in vivo e in vitro. A injeção da enzima (10 µg) não induziu edema de pata em camundongos, nem hemorragia (50 µg) e nem toxicidade sistêmica (100 µg). No entanto, provocou uma leve mionecrose (100 µg) e edema em músculo quadríceps de camundongo, aumentando a creatina quinase plasmática. In vitro, foram testadas as atividades citotóxicas da LmLAAO em células de carcinoma. Os dados obtidos mostram IC50 de 22,70 µg/mL, para linhagem AGS (carcinoma de estômago), e IC50 de 1,41 µg/mL linhagem MCF-7 (carcinoma de mama). Para a atividade antiparasitária, foi determinada uma IC50 de 2,22 µg/mL para a forma promastigota de Leishmania brasiliensis. No entanto, a LmLAAO (32 µg/mL) não apresentou toxicidade relevante para a forma tripomastigota de Tripanosoma cruzi. Concluindo, este trabalho descreve o isolamento e a caracterização estrutural e funtional de uma nova LAAO da peçonha de L. muta. A enzima mostrou efeitos antitumorais e leishmanicida, sem toxicidade sistêmica relevante, mas apresentou significativa ação edematogênica e miotóxica local. Este estudo é relevante não apenas por contribuir para uma melhor compreensão do papel da LAAO no envenenamento, mas também por demonstrar seu potencial biotecnológico como agente terapêutico. / Snake venoms comprise a complex mixture of pharmacologically active substances, such as metalloproteases, phospholipase A2, serine proteases and L-amino acid oxidases (LAAOs) other compounds showing no enzymatic activity. LAAOs are flavoproteins catalyzing the oxidative deamination of L-amino acids to produce the corresponding -keto acid with the concomitant release of ammonia and hydrogen peroxide. Lachesis muta (L. muta) venom contains L-amino acid oxidase which may contribute to the envenomation. The aim of this study is the purification of an L-amino acid oxidase from Lachesis muta venom (LmLAAO) and its structural and functional characterization. For that, two purification protocols were performed and both provided highly pure LmLAAO. In the first protocol, 20 mg of crude venom of L. muta were submitted to a gel filtration on Sephacryl S100® and yielded ten fractions, whose were tested for L-amino acid oxidase activity. The first fraction showed L-amino acid oxidase activity and it was submitted to a further chromatographic step on Mono Q®. The homogeneity of the fraction showing L-amino acid oxidase activity after ion exchange was confirmed by the presence of a single band corresponding to 60.2 kDa by SDS-PAGE. The second purification protocol was a sequence of three chromatographic steps, where 200 mg of crude L. muta venom were submitted to gel filtration on Sephacryl S200, followed by hydrophobic interaction on Phenyl Sepharose® and Affi-gel-Blue Gel. For the second protocol, the purity of LmLAAO was confirmed by the presence of a single band with 64 kDa as determined by SDS-PAGE. In both purification protocols LmLAAO kept its enzymatic activity. The molar mass of LmLAAO was determined by mass spectrometry (MALDI-TOF) and showed a value of 60.85 kDa. Moreover, the isoelectric point was 5.1. In addition, LmLAAO showed a catalytic preference for hydrophobic amino acids (L-methionine, L-leucina and L-phenylalanine) and lost its catalytic activity when subjected to high pH or temperature. The kinetic parameters for LAAO were determined and the Michaelis-Menten constant for the substrate L-leucine was 0.9737 mmol/L, and the maximum reaction velocity was 0.06345µmol hydrogen peroxide/min. Furthermore, the sequence of the first forty residues was determined by Edman degradation and the complete sequence of LmLAAO was resolved by cloning cDNA obtained from the venom glands. The amino acid sequence of LmLAAO showed a great identity with sequences of LAAOs from other viper snakes. The LmLAAO structure was solved by molecular replacement using the the atomic coordinates of the LAAO from Agkistrodon halys pallas (PDB 1REO). In addiction, LmLAAO pharmacological activities were determined in vivo and in vitro. Thus, LmLAAO (10 µg) did not induce paw edema in mice, neither hemorrhage (50 µg) nor systemic toxicity (100 µg). However, it caused a mild myonecrosis (100 µg) and edema in the quadriceps muscles of mice, increasing plasma creatine kinase. In vitro activities of LmLAAO in carcinoma cells was assayed. The IC50 of LmLAAO on AGS cell line (stomach cancer) was 22.70 µg / mL, and the IC50 of LmLAAO on MCF-7 cell line (breast carcinoma) was 1.41 µg/mL. Moreover, antiparasitic activity of LmLAAO was determined on promastigote of Leishmania brasiliensis and an IC50 of 2.22 µg/mL was found, whereas on trypomastigote form Trypanosoma cruzi LmLAAO showed no toxicity at doses of 32 µg/mL. In conclusion, this work reports the isolation and the structural and funtional characterization of a new LAAO from L. muta snake venom. The enzyme showed antitumoral and leishmanicidal effects, without relevant systemic toxicity, but presented a significant local edema inducing and myotoxic action. This study is relevant not only for contributing to a better understanding of LAAO role in envenomation, but also for demonstrating its biotechnological potential as a therapeutic agent.

A otimização do metabolismo e a maximização do desempenho das aves dependem de adequada nutrição. Dentre os nutrientes essenciais a essa nutrição estão o aminoácido lisina e zinco, objetos desta avaliação, sobre a produção, qualidade, composição dos ovos e morfologia intestinal. Setecentos e vinte poedeiras foram submetidas em dois períodos de avaliação. Cada período correspondeu a 12 semanas, sendo a Fase-I de 24 a 36 e a Fase-II de 48 a 60 semanas de idade. Os tratamentos foram distribuídos em delineamento inteiramente casualizado, dispostos em esquema fatorial (5 x 3) aplicados em seis repetições e a unidade experimental foi composta de oito aves/parcela. Os níveis de lisina digestível foram: 0,482; 0,527; 0,582; 0,644 e 0,732% e de zinco: 137, 309 e 655 ppm na forma de quelato. Na Fase-I, houve interação de lisina digestível e zinco nas variáveis: consumo de ração médio diário, conversão alimentar, porcentagem de postura e na massa de ovo. Na Fase-II a interação foi evidenciada no consumo de ração médio diário, peso da casca, composição química e taxas de deposição protéica, lipídica e mineral do ovo. Na maior concentração dietética de zinco o acréscimo de lisina digestível coincidiu com aumento linear no peso da casca. Por outro lado, o acréscimo de zinco independentemente do nível de lisina na dieta, culminou com a redução do peso do ovo e da porcentagem da matéria mineral na gema limitando a eficiência de deposição mineral nessa fração do ovo. Resultado inverso ocorreu no albúmen, quando houve aumento na porcentagem de matéria mineral. Em ambas as fases, a menor concentração de zinco (173ppm) atendeu as necessidades de produção e qualidade das aves. O valor médio estimado de lisina digestível foi de 0,662% ± 0,03 para a Fase-I e de 0,609 ± 0,004 para a Fase-II. / The optimization of the metabolism and poultry performance maximization depend of adequate nutrition. Among the essential nutrients there are lysine and zinc, subjects this evaluation, on egg production, quality, composition and intestinal morphology. Seven hundred and twenty laying were submitted into two study periods. Each period corresponded to 12 weeks, being 24 to 36 Phase I and 48 to 60 weeks of age Phase II. The treatments were allotted to a completely randomly, disposed randomized design under factorial scheme (5 x 3) with six replications and eight bird per experimental unit. The lysine levels were: 0.482; 0.527; 0.582; 0.644 e 0.732% and zinc: 173, 309 and 655 ppm in chelate form. At Phase I, there was interaction of digestible lysine and zinc in variables: average feed intake, feed: gain, laying percentage and egg mass. At Phase II the interaction was evidenced at the average feed intake, shell weight, chemical composition, proteic, lipidic and mineral depositionn rates egg. In the higher dietetic zinc concentration the digestible lysine accretion coincide with linear increase in shell weight. On the other hand, the zinc increase, of independently from diet lysine level, coincide with egg weight and yolk mineral percentage decrease, however limiting the mineral accretion efficiency in this egg fraction. In both Phases, the smallest zinc concentration (173ppm) attended the poultry production and quality needs. The estimate value of digestible lysine was 0.662% ± 0.03 for Phase I and 0.609% ± 0.004 for Phase II.

The aim of this thesis was to evaluate the feeding and nutrition of tropical farmed fish and shrimp, targeting its applicability to aquaculture farming. The study of the actual panorama of aquafeed quality for tilapia Oreochromis niloticus and shrimp Litopenaeus vannamei farmed in Brazil showed that the proximate compositions between the analyzed feeds were mostly consistent with the declared values from the manufacturers, however, the feed water stability showed the opposite; the in vitro pH-stat species-specific method to determine the protein degree of hydrolysis (DH) showed to be a useful tool to evaluate feed quality; and NIRS technique can be used in many applications throughout the aquafeed industry, being an efficient tool for rapidly assessing feed quality in terms of DH. A second study evaluated the acid-insoluble ash (AIA) and chromic oxide (Cr2O3) as inert markers and feed processing method (industrial extruded vs. laboratory cold pelleted) to determine apparent digestibility coefficient (ADC) of dry matter and crude protein of juvenile L. vannamei. The AIA showed to be an effective natural endogenous marker for digestibility trials with L. vanammei, however, for commercial feeds attention should be paid to feed AIA level; the extruded feed showed better animal performance than the cold pelleted feed, however, ADC of both feeds were not significantly different from each other. Further, two feeding trials were conducted with juvenile cobia Rachycentron canadum: (1) feeding trial conducted within floating net cages to test the nutritional efficacy of different dietary feeding regimes ranging from the use of trashfish, in-house formulated feeds, to dry commercial extruded marine fish feed; and (2) feeding trial conducted within indoor water-recirculated tanks to test the nutritional efficiency of different potential dietary fishmeal replacers within dry in-house prepared diets. Generally, fish performance was superior in the net-cage feeding trial compared with the indoor water-recirculated tank trial; overall, the fish growth and performance of the experimental diets were very similar, showing that the alternative ingredients could be included and replace part (50%) of the fishmeal component; the results from both trials concluded that the cobia requires practices diets with high levels of crude protein and lipid, and the inclusion of alternative plant-based and terrestrial animal protein sources was possible; the quantitative essential amino acid (EAA) requirement values estimated by the protein accretion method was highly correlated to the average of each of the EAA requirement for the species of carnivorous fish reported in the literature, and could be recommended for formulation of commercial feed for cobia R. canadum. Finally, a tentative to quantify the total sulfur amino acid requirement of juvenile Florida Pompano Trachinotus carolinus, was performed using combinations of various soybean protein products in order to develop cost-effective and environmentally-friendly diets. Although there was an apparent tendency in the results, the range of methionine levels employed in this study may not have been broad enough to accurately measure the dietary concentration necessary to estimate the total sulfur amino acid requirement; in addition, high variation results among the three replicates per diet did not provide sufficiency robustness for its estimation; this study within 45 days should not have been enough to show significant differences among the treatments. Long term feeding trials would be recommended from fingerling/juvenile to market size with full nutritional and economic evaluation of results. / O objetivo dessa tese foi avaliar a alimentação e nutrição de peixes e camarões tropicais cultivados, visando a sua aplicação à aquicultura. O estudo do atual panorama da qualidade das rações para tilapia e camarão cultivados no Brasil mostrou que a maioria das composições proximais das rações analisadas foram consistentes com os valores declarados pelos fabricantes, porém, a estabilidade das na água mostrou-se o oposto; o método in vitro pH-stat com enzimas espécie-específica, para determinação do grau de hidrólise da proteína (DH), mostrou ser uma ferramenta útil para avaliar a qualidade das rações; a técnica da espectrofotometria do infravermelho próximo (NIRS) pode ser usada em várias aplicações na indústria de alimentos aquáticos, sendo uma ferramenta eficiente para avaliar rapidamente a qualidade dos alimentos em termos de DH. Um segundo estudo avaliou a cinza insolúvel em ácido (AIA) e óxido de cromo (Cr2O3) como marcadores inertes, além de métodos de processamento de alimentos (extrusado industrial vs. peletizada a frio no laboratório) para determinar o coeficiente de digestibilidade aparente (ADC) da matéria seca e proteína bruta de juvenis de L. vannamei. O AIA mostrou ser um marcador endógeno natural eficaz para ensaios de digestibilidade com L. vanammei, no entanto, atenção especial deve ser dada aos níveis de AIA nas rações comerciais; a ração extrusada teve a melhor performance dos animais, porém, os valores de ADC entre as rações não foram diferentes significantemente. Além disso, dois experimentos de alimentação foram conduzidos com juvenis de beijupirá Rachycentron canadum: (1) experimento realizado em tanques-rede flutuantes para testar a eficácia nutricional de diferentes regimes alimentares, variando entre rejeito de pesca, rações preparadas em laboratório, e ração comercial extrusada; e (2) um experimento realizados em tanques com recirculação de água para testar a eficiência nutricional de diferentes potenciais substitutos de farinha de peixe, com dietas completadas preparadas em laboratório. Em geral, o desempenho dos peixes foi superior no experimento nos tanques-rede, comparado com o experimento nos tanques de recirculação de água; no geral o crescimento e performance dos peixes das dietas experimentais foram bem similares, concluindo que ingredientes alternativos podem ser incluídos e substituírem parte (50%) da farinha de peixe das rações; os resultados de ambos os estudos concluíram que o beijupirá requer dietas práticas com alto teores de proteína bruta e lipídeos, e a inclusão de fontes proteicas de origem vegetal e animal foi possível. Os valores quantitativos das exigências de amino acido essenciais (EAA) estimados pelo método de acreção de proteína na carcaça foram altamente correlacionados com as médias de exigência de cada EAA das as espécies de peixes carnívoros encontrados na literatura, e pode ser recomendado para a formulação de ração comercial para beijupirá R. canadum. Por ultimo, uma tentativa de quantificar as exigências de amino ácidos sulforosos totais para Florida Pompano Trachinotus carolinus, foi realizada utilizando combinações de vários ingredientes proteicos a base de soja, a fim de desenvolver dietas de baixo custo e ecologicamente sustentáveis. Embora houve uma tendência clara nos resultados, os intervalos dos níveis de metionina utilizados nesse estudo podem não ter sido amplos o suficiente para medir com precisão a concentração alimentar necessária para estimar a exigência dos de amino ácidos sulforosos totais; além disso, a alta variação entre as replicas não forneceu uma estimativa robusta; este estudo de 45 dias não deve ter sido suficiente para mostrar diferenças significativas entre os tratamentos.