The unique native structure is a basic requirement for normal functioning of most proteins. Many diseases stem from mutations in proteins that destabilize the protein structure thereby resulting in impairment or loss of function (Sunyaev et al. 2000). Therefore, it is important from both fundamental and applied points of view, to elucidate the sequence determinants of protein structure and function. With the advent of recombinant DNA techniques for modifying protein sequences, studies on the effect of amino acid replacements on protein structure and function have acquired momentum. It is well established from previous mutagenesis studies that buried residues in a protein are important determinants of protein structure or stability while surface residues are involved in protein function (Rennell et al. 1991; Terwilliger et al. 1994; Axe et al. 1998). Inspite of this, there is no universally accepted definition and probe to distinguish and identify buried residues from exposed residues. A part of this thesis aims to examine the feasibility of using scanning mutagenesis to distinguish between buried and exposed positions in the absence of three-dimensional structure and also to arrive at an experimental definition of the appropriate accessibility cut-off to distinguish between buried and exposed residues. Proline, being an unusual amino acid is usually exploited to determine sites in a protein important for protein stability (Sauer et al. 1992). This thesis also explores the use of proline scanning mutagenesis to make inferences about protein structure and stability. Temperature sensitive mutant proteins, which result from single amino acid substitutions, are particularly useful in elucidating the determinants of protein folding and stability (Grutter et al. 1987; Sturtevant et al. 1989). Temperature sensitive (ts) mutants are an important class of conditional mutants which are widely used to study gene function in vivo and in cell culture (Novick and Schekman 1979; Novick and Botstein 1985). They display a marked drop in the level or activity of the gene product when the gene is expressed above a certain temperature (restrictive temperature). Below this temperature (permissive temperature), the level or activity of the mutant is very similar to that of the wild type. Inspite of their widespread use, little is known about the molecular mechanisms responsible for generating a Ts phenotype. A part of this thesis discusses a set of sequence/structure-based strategies for the successful design and isolation of ts mutants of a globular protein, inferred from saturation mutagenesis of CcdB. The experimental system, CcdB (Controller of Cell Division or Death B protein), is a 101 residue, homodimeric protein encoded by F plasmid. The protein is an inhibitor of DNA gyrase and is a potent cytotoxin in E.coli (Bernard et al. 1993). Crystallographic structures of CcdB in the free and gyrase bound forms (Loris et al. 1999; Dao-Thi et al. 2005) are also available. Expression of the CcdB functional protein results in cell death, thus providing a rapid and easy assay for the protein (Chakshusmathi et al. 2004). This dissertation focuses on understanding the determinants of globular protein stability and temperature sensitivity using saturation mutagenesis of E.coli CcdB. Towards this objective, we attempted to replace each of the 101 residues of CcdB with 19 other amino acids using high throughput mutagenesis tools. A total of 1430 (~75%) of all possible single site mutants of the CcdB saturation mutagenesis library could be isolated. These mutants were characterized in terms of their activity at different expression levels. The correlation between the observed mutant phenotypes with residue burial, nature of substitution and expression level was examined. The introductory chapter (Chapter 1) describes the use of mutagenesis as a tool to understand the relationship between protein sequence, structure and function. It represents an overview of previous large scale mutagenesis studies from the literature. It also addresses the motivation behind this work and problems which we have attempted to address in these studies. Chapter 2 discusses mutagenesis based definitions and probes for residue burial in proteins as derived from alanine and charged scanning mutagenesis of CcdB. Every residue of the 101 amino acid E. coli toxin CcdB was substituted with Ala, Asp, Glu, Lys and Arg using site directed mutagenesis. The activity of each mutant in vivo was characterized as a function of CcdB transcriptional level. The mutation data suggest that an accessibility value of 5% is an appropriate cutoff for definition of buried residues. At all buried positions, introduction of Asp results in an inactive phenotype at all CcdB transcriptional levels. The average amount of destabilization upon substitution at buried positions decreases in the order Asp>Glu>Lys>Arg>Ala. Asp substitutions at buried sites in two other proteins, MBP and Thioredoxin were also shown to be severely destabilizing. Ala and Asp scanning mutagenesis, in combination with dose dependent expression phenotypes, was shown to yield important information on protein structure and activity. These results also suggest that such scanning mutagenesis data can be used to rank order sequence alignments and their corresponding homology models, as well as to distinguish between correct and incorrect structural alignments. When incorporated into a polypeptide chain, Proline (Pro) differs from all other naturally occurring amino acids in two important respects. The  dihedral angle of Pro is constrained to values close to –65o and Pro lacks an amide hydrogen. Chapter 3 describes a procedure to accurately predict the effects of proline introduction on protein stability. 77 of the 97 non-Pro amino acid residues in the model protein, CcdB, were individually mutated to proline and the in vivo activity of each mutant was characterized. A decision tree to classify the mutation as perturbing or non-perturbing was created by correlating stereochemical properties of mutants to activity data. The stereochemical properties, including main chain dihederal angle and main chain amide hydrogen bonds, were determined from 3D models of the mutant proteins built using MODELLER. The performance of the decision tree was assessed on 74 nsSNPs and 37 other proline substitutions from the literature. The overall accuracy of this algorithm was found to be 89% in case of CcdB, 71% in case of nsSNPs and 83% in case of other proline substitution data. Contrary to previous assertions, Proline scanning mutagenesis cannot be reliably used to make secondary structural assignments in proteins. The studies will be useful in annotating uncharacterized nsSNPs of disease-associated proteins and for protein engineering and design. Mutants of CcdB were also characterized in terms of their activity at two different temperatures (30oC and 37oC) to screen for temperature sensitive (ts) mutants. The isolation and structural analysis of Ts mutants of CcdB is dealt with in Chapter 4. Of the total 1430 single site mutants, 12% showed a ts phenotype and were mapped onto the crystal structure of the protein. Almost all the ts mutants could be interpreted in terms of the wild type, native structure. ts mutants were found at all buried sites and all active sites (except one). ts mutants were also obtained at sites in close proximity to active site residues where polar side-chains were involved in H-bonding interaction with active site residues. Several proline substitutions also displayed a ts phenotype. The effect of expression level on ts phenotype was also studied. 78% of the mutants that showed an inactive phenotype at the lowest expression level and an active phenotype at highest expression level, resulted in a ts phenotype at an intermediate expression level. The molecular determinant responsible for the ts phenotype of buried site ts mutant is suggested to be the thermodynamic destabilization of the protein which results in a reduced steady state in vivo level of soluble, functional protein relative to wild type. The active site ts mutants probably lower the specific activity of the protein and hence the total activity relative to wild type. However these effects might be less severe at lower temperature. Specific structure/function based mutagenesis strategies are suggested to design ts mutant of a protein. These studies will simplify the design of ts mutants for any globular protein and will have applications in diverse biological systems to study gene function in vivo. Chapter 5 represents the structural and sequence correlations of a CcdB saturation mutagenesis library which was obtained by replacing each of 101 amino acid residues with 19 other amino acids. Polar substitutions i.e. Asn, Gln, Ser, Thr and His were poorly tolerated at buried sites at lower expression levels. Aromatic substitutions and Gly were also not well tolerated at buried positions at lower expression levels. Trp was poorly tolerated at residues with accessibility <15%. However, most of the surface exposed residues with accessibility >40% (except functional ones) could tolerate all kinds of substitutions. Chapter 6 deals with the thermodynamic characterization of monomeric and dimeric forms of CcdB. The stability and aggregation state of CcdB have been characterized as a function of pH and temperature. Size exclusion chromatography revealed that the protein is a dimer at pH 7.0, but a monomer at pH 4.0. CD analysis and fluorescence spectroscopy showed that the monomer is well folded, and has similar tertiary structure to the dimer. Hence intersubunit interactions are not required for folding of individual subunits. The oligomeric status of CcdB at pH 7.0 at physiologically relevant low concentrations of protein, was characterized by labeling the protein with two different pairs of donor and acceptor fluorescent dyes (Acrylodan-Pyrene and IAF-IAEDANS) separately and carrying out fluorescence resonance energy transfer (FRET) measurements by mixing them together. CcdB exists in a dimeric state even at nanomolar concentrations, thus indicating that the dimeric form is likely to be the physiologically active form of CcdB. The stability of the dimeric form at pH 7.0 and the monomeric form at pH 4.0 was characterized by isothermal denaturant unfolding and calorimetry. The free energies of unfolding were found to be 9.2 kcal/mol (1 cal=4.184 J) and 21 kcal/mol at 298 K for the monomer and dimer respectively. The denaturant concentration at which one-half of the protein molecules are unfolded (Cm) for the dimer is dependent on protein concentration, whereas the Cm of the monomer is independent of protein concentration, as expected. Although thermal unfolding of the protein in aqueous solution is irreversible at neutral pH, it was found that thermal unfolding is reversible in the presence of GdnCl (guanidinium chloride). Differential scanning calorimetry in the presence of low concentrations of GdnCl in combination with isothermal denaturation melts as a function of temperature were used to derive the stability curve for the protein. The value of Cp (representing the change in excess heat capacity upon protein denaturation) is 2.8 ± 0.2 kcalmol-1K-1 for unfolding of dimeric CcdB, and only has a weak dependence on denaturant concentration. These studies advanced the understanding of protein folding of oligomeric proteins. The concluding section summarizes all the chapters in a nutshell and addresses the future directions provided by these investigations.

Regrowth in the lesioned central nervous system is impeded by inhibitory molecules including myelin-associated inhibitors (MAIs) and chondroitin sulfate proteoglycans (CSPGs). Inhibitory molecules engage neuronal cell surface receptors and activate the small GTPase RhoA in injured neurons to mediate neurite outgrowth inhibition through targeted modifications to the cytoskeleton. Inhibition of RhoA with the ribosyltransferase C3 attenuates neurite outgrowth inhibition in vitro and in vivo but the ubiquitous expression and multifunctionality of RhoA may limit the specificity of therapeutic RhoA antagonists. The hypothesis of the thesis is that molecules that functionally interact with RhoA to mediate myelin-dependent inhibition may represent more specific targets for therapeutic intervention. We have explored the contribution of two RhoA interacting proteins to the neurite outgrowth inhibitory effects of MAIs. In Chapter 2 we describe the contribution of the rho effector, Rho kinase (ROCK) to MAI responses in neurons. In Chapter 3 we identify the cytosolic phosphoprotein CRMP4b (Collapsin Response Mediator Protein 4b) as a novel RhoA binding partner that mediates neuronal responses to CNS inhibitors. By structure function analysis we have developed a molecular antagonist of CRMP4b-RhoA binding that promotes neurite outgrowth on inhibitory substrates in vitro and has the potential to be a potent and specific molecular therapeutic for spinal cord injury. In Chapter 4 we identify glycogen sythase kinase 3b (GSK3b) as an important kinase in the MAI pathway that regulates protein interactions with RhoA. This thesis provides insights into the signal transduction machinery that is engaged in response to CNS inhibitors and suggests several novel therapeutic targets to promote axon regeneration following CNS injury.

The von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome that is transmitted in an autosomal dominant manner. The disease is characterized by the formation of highly angiogenic tumors in many organs but the main causes of mortality are renal cell carcinomas and hemangioblastomas. Mutations in the VHL protein are responsible for the pathogenesis of the disease. VHL associates with elongin Band C to form the VBC complex. The cullin 2 protein (CUL2) and ring box protein 1 (RBX1) also associate with the VBC complex to form an E3 ubiquitin ligase involved in the ubiquitination and subsequent degradation of the hypoxia inducible transcription factor (HIF2alpha). Mutations in VHL that abrogate its E3 ligase activity lead to increased levels ofHIF2alpha and the subsequent accumulation of pro-proliferative and pro-angiogenic HIF2alpha target genes. VHL also has an important function in the regulation of extracellular matrix (ECM) assembly which is independent of its HIF2alpha regulation pathway. VHL's regulation of ECM assembly was shown to have important consequences for tumor angiogenesis and cell invasion. It was shown to be necessary for the proper assembly of a fibronectin matrix and was most recently found to interact with collagen IV alpha 2 (COL4A2). The aim of this thesis is to further characterize the VHL-COL4A2 interaction. VHL was shown to interact directly and specifically to COL4A2 and is necessary for proper COL4A2 matrix assembly. The association of VHL with COL4A2 appears to be independent of its functions as an E3 ubiquitn ligase and CUL2 was identified as part of the VBC complex that associates with collagen IV (COL4). Furthermore, a strategy to identify the binding site of VHL on COL4A2 has been employed and is in progress. These experiments represent the beginning of investigations into the novel interaction between VHL and COL4A2.

O objetivo deste trabalho foi investigar os efeitos do palmitato (16:0) e palmitoleato (16:1 <font face=\"Symbol\">w7) sobre o metabolismo e sobre a produção de peróxido de hidrogênio (H2O2) em músculos sóleos de ratos. Os músculos foram incubados durante 1 ou 4 horas com 500 <font face=\"Symbol\">mM de palmitato (PA) ou palmitoleato (PO) ou 30 <font face=\"Symbol\">mM do antioxidante ebselen (Eb). A produção de H2O2 foi mensurada pelo método de oxidação da sonda Amplex<font face=\"Symbol\">&#210; e a captação de glicose foi avaliado por meio da captação de 2-desoxi-[2,6-3H]-D-glicose. O PA aumentou a produção de H2O2 e a captação de glicose após 1 hora de incubação e estes efeitos foram abolidos pelo antioxidante Eb. O PO não alterou esses parâmetros. Após 4 horas, o PA reduziu a captação de glicose e este efeito foi abolido pelo Eb. O PO não alterou a captação de glicose. O tratamento com estes ácidos graxos não alterou o metabolismo de proteínas. Tais resultados sugerem que o aumento na produção de H2O2 está envolvido na regulação do metabolismo de glicose em músculo sóleo. / The objective of this study was to investigate the effects of palmitate (16:0) and palmitoleate (16:1 <font face=\"Symbol\">w7) on the metabolism and the production of hydrogen peroxide (H2O2) in soleus muscles of rats. The muscles were incubated for 1 or 4 hours with 500 <font face=\"Symbol\">mM palmitate (PA) or palmitoleate (PO) or 30 <font face=\"Symbol\">mM of the antioxidant ebselen (Eb). The production of H2O2 was measured by the Amplex<font face=\"Symbol\">&#210; oxidation method and glucose uptake was assessed by uptake of 2-deoxy-[2,6-3H]-D-glucose. PA increased production of H2O2 and glucose uptake after 1 hour of incubation and these effects were abolished by Eb antioxidant. The PO did not alter these parameters. After 4 hours, PA decreased glucose uptake and this effect was abolished by Eb. PO did not alter glucose uptake. Treatment with these fatty acids did not affect protein metabolism. These results suggest that the increased production of H2O2 is involved in the regulation of glucose metabolism in soleus muscle.

O estudo consistiu de dois experimentos com o objetivo de avaliar o efeito metabólico, custos e viabilidade econômica de dietas com diferentes fontes nitrogenadas e teores proteicos. No primeiro experimento avaliou-se o efeito de dois teores de proteína bruta (PB) (130 e 148g/kg de MS) e duas fontes nitrogenadas (farelo de algodão 38 e grão de soja cru integral) na dieta de vacas leiteiras com cana de açúcar como volumoso, sobre as frações nitrogenadas do leite, balanço de nitrogênio e perfil metabólico. Foram utilizadas 12 vacas da raça Holandesa com 155 (±65) dias em lactação, agrupadas em três quadrados latinos 4x4 contemporâneos, com período experimental de 21 dias, sendo 14 dias para adaptação às dietas e os sete últimos para a realização das coletas. As vacas foram alojadas em baias individuais e alimentadas ad libitum. As amostras de leite para análise do balanço nitrogenado e frações nitrogenadas foram coletadas no 15° dia de cada período. O consumo e balanço de nitrogênio foram maiores para vacas alimentadas com dietas com 148 g PB/kg de MS. Por outro lado, vacas alimentadas com dietas contendo farelo de algodão apresentaram maior excreção de nitrogênio no leite do que vacas alimentadas com grão de soja cru integral. A relação entre caseína e proteína verdadeira no leite foi maior em vacas alimentadas com grão de soja cru integral. Houve interação entre fonte nitrogenada e teor de PB da dieta sobre as concentrações de nitrogênio ureico no leite (NUL) e nitrogênio não proteico (NNP). A concentração de NUL foi maior em vacas alimentadas com farelo de algodão e com maior teor de PB, em contrapartida houve menor excreção de NUL em vacas alimentadas com grão de soja com maior teor proteico. Não houve efeito das dietas sobre os teores de proteína do leite, nitrogênio não caseinoso (NNC), caseína e proteína do soro. Conclui-se que o uso de dietas com 130g de PB/Kg na MS não altera o balanço de nitrogênio e de composição do leite de vacas leiteiras em comparação com teores de 148g/Kg de PB na MS, e resultam em menor excreção de nitrogênio no ambiente. No segundo experimento foram calculados os custos e margens totais de dietas com cinco fontes nitrogenadas principais (ureia, farelo de soja, farelo de algodão, grão de soja cru integral e farelo de glúten de milho) e cinco teores proteicos (130, 145, 148, 157 e 160 g/kg de MS) com cana de açúcar como volumoso para vacas em lactação. Os dados foram provenientes de três estudos conduzidos com a finalidade de coleta de dados produtivos e respostas metabólicas. A análise econômica foi realizada com base nos preços históricos deflacionados (corrigidos do efeito da inflação) praticados durante o período 2002 a 2012, e no cálculo dos custos de alimentação, em função do consumo de alimento, da produção de leite e do teor de proteína bruta no leite. Dietas com cana de açúcar com teor proteico de 14,5% com ureia como fonte nitrogenada principal apresentaram a maior margem bruta (diferença entre a receita da venda do leite e do custo da dieta) com valor médio anual de R$1,85.vaca-1.dia-1. A dieta com 14,8% de PB com grão de soja cru integral apresentou a menor margem bruta de R$ 2,16.vaca-1.dia-1. / The study consisted of two experiments to evaluate the N balance and economical analysis of diets with different nitrogen sources and crude protein levels. On the first experiment it was evaluated the effect of two crude protein (CP) levels (130 e 148 g/kg DM) and two nitrogen sources (cottonseed meal 38% and whole raw soybean) in diets of dairy cows using sugar cane as forage on nitrogen in milk, nitrogen balance and metabolic parameters. Twelve Holstein cows with 155 (±65) days in lactation were distributed into three contemporary 4x4 Latin squares, with experimental period of 21 days, 14 days for diet adaptation and the remaining seven days for sampling. The cows were housed in individual pens and fed ad libitum. Milk samples for nitrogen balance and milk nitrogen fractions analysis were collected on the 15th day of each experimental period. Nitrogen intake and nitrogen balance were higher for the cows fed diets with 148 g CP/kg DM. However cows fed diets with cottonseed meal had higher nitrogen excretion in milk than cows fed diets with whole raw soybean. The casein: true milk protein ratio was higher in cows fed diets with whole raw soybean. There was interaction between the nitrogen source and the diet CP content on the milk urea nitrogen and non-protein nitrogen. Milk urea nitrogen was higher in cows fed diets with cottonseed meal and higher CP concentrations, however lower milk urea nitrogen was observed in cows fed diets with whole raw soybean and higher CP concentration. The concentration of crude protein, noncasein protein, casein and whey protein in milk did not differ between diets. In conclusion the use of low concentrations of protein (130g/Kg in MS) does not affect the performance of dairy cows and provides lower excretion of nitrogen in the environment. On the second experiment, the costs and the gross margin were calculated for diets formulated with five nitrogen sources (urea, soybean meal, cottonseed meal, whole raw soybean and corn gluten meal) and five protein levels (130, 145, 148, 157 e 160 g/kg DM) using sugar cane as forage. Performance data were obtained from three experiments conducted previously. Economical analysis were performed based on historical prices adjusted for the effect of inflation during the period between 2002 and 2012 and based on the feed costs, cow intake, milk prodution and milk protein levels. The higher gross margin (difference between the income from milk sale and diet costs) were obtained for 145 g/kg of CP in DM diets and urea as main nitrogen source, with mean of R$1,85.vaca-1.dia-1. The lower gross margin were observed in the 148 g/kg of CP in DM diet and whole raw soybean as nitrogen source, with mean of R$ 2,16.vaca-1.dia-1.

Os resultados obtidos nesta tese podem ser distribuídos em três grupos: composição química da hemolinfa e do casulo coletivo e determinação química de alguns componentes principais do corpo gorduroso e túbulos de Malpighi ao longo do desenvolvimento. Os principais resultados referentes à química da hemolinfa de larvas maduras são: 1) A hemolinfa possui uma densidade 1,043, pH = 7,27, osmolaridade = 216 miliosmoles e corresponde a 37% do pêso-úmido do animal e 26% de seu pêso-sêco. A hemolinfa não se coagula e possui um volume de células correspondente a 0,3% de seu volume total. 2) A análise química realizada deu conta de 88% do peso-sêco total da hemolinfa e revelou que entre os componentes presentes mais importantes em massa estão as proteínas, seguidas dos aminoácidos livres, enquanto que os osmóticamente mais ativos são os aminoácidos livres seguido de Mg++ e Na+. Entre os aminoácidos é notável a presença de ornitina e cistationina em concentrações relativamente elevadas e a ausência, mesmo em traços, de cisteína e/ou cistina e citrulina. 3) Os peptídeos ocorrem em concentrações elevadas, mas em pequeno número, e são compostos de 2 a 3 resíduos de aminoácidos em média; o mais abundante dos quais deve ser um dipeptídeo de histidina e ácido aspártico. 4) Citrato é o ânion mais importante da hemolinfa, seguido dos fosfatos orgânicos, enquanto que trealose é o principal carboidrato presente. 5) Existem pelo menos 6 carotenóides ligados de forma não covalente a proteínas da hemolinfa e uma cromoglicoproteína, de côr amarelo-limão, fluorescente, de natureza desconhecida. Os carotenóides mais importantes quantitativamente são: &#946;-caroteno e um similar ao astaceno. A composição em massa do casulo coletivo na véspera da muda pupal é a seguinte, em números inteiros: 44% de CaCO3; 34% de proteínas e 10% de carboidratos, ambos insolúveis em todos solventes utilizados (SDS 10%, uréia 6M, ácido fórmico 50%, HCl 2N, KOH 1M e Na HCO3 1M); 10% de material hidrossolúvel 4% de cinzas não identificadas. O material hidrossolúvel foi parcialmente identificado como: proteína (4%), carboidratos (2%), enquanto que 4% ainda permanece desconhecido. O correlacionamento da análise química da hemolinfa, casulo, túbulos de Malpighi e corpo gorduroso ao longo do desenvolvimento, possibilitou ainda as seguintes conclusões: 1) A fração insolúvel do casulo (proteínas e carboidratos) corresponde à sêda secretada pelas glândulas salivares da larva, enquanto que o CaCO3 presente provém dos túbulos de Malpighi. 2) As proteínas do casulo devem originar-se, pelo menos em parte, das proteínas da hemolinfa, enquanto que seus carboidratos devem provir do glicogênio do corpo gorduroso. Os resultados são considerados em têrmos de seus possíveis significados metabólicos e eventualmente fisiológicos. Ênfase é dada na discussão do papel metabólico dos componentes orgânicos e inorgânicos da hemolinfa, assim como dos mecanismos de secreção da sêda e CaCO3 e sua possível regulação hormonal. / The results of this thesis are concerned to three main lines of research: the chemical composition of the hemolymph, of the communal cocoon, and the chemical determination of some components in the rat body and Malpighian tubules along larval development. The chief results on the hemolymph chemistry of mature larvae are: 1) The hemolymph has a density = 1.043, pH = 7.27, osmolarity = 2l6 miliosmols and corresponds to 37% of the larva (Wet-Weight) or to 26% of the larva (dry weight). The hemolymph does not clott and the volume of the hemocytes is 0.3% of blood total volume. 2) The chemical analysis dealt with 88% (dry-weight) of the hemolymph and showed that proteins are the most important component in mass, while free amino acids, Mg++ and Na+ are the most osmotically active substances. Among the amino acids is remarkable the titres of ornithine and cystathionine, and the complete absence of cysteine and/or cystine and citrulline. 3) There are few peptides, but present in high titres. They are built of two to three amino acids residues, and the most concentrated of them must be a dipeptide of histidine and aspartic acid. 4) Citrate and organic phosphates are the most important anions in the hemolymph. Trehalose is the chief carbohydrate present. 5) There are at least 6 non-covalentely protein-bound carotenoids and one lemon-yellow, fluorescent chromoglycoprotein of unknown nature. The chemical composition of the communal cocoon of R. americana expressed in percentage of its total dry weigth (numbers rounded to the nearest unity) are: 44% of CaCO3; 34% of proteins and 10% of carbohydrates both insoluble in all solvents used (10% SDS, 6M urea, 50% formic acid, 2N HCl, 1M KOH and 1M NaHCO3 ); 10% of water soluble material and 4% of unknown nature. The water soluble material was identified in part as: protein (4%) and carbohydrates (2%), while 4% remained unknown. The interrelationship among the results of the chemical analysis of the hemolymph, cocoon, Malpighian tubules and fat body during larval development was used to draw the conclusions: 1) The insoluble fraction of the cocoon proteins and carbohydrates) corresponds to the silk produced by the larval salivary glands, while CaCO3 comes from Malpighian tubules. 2) The cocoon proteins must come, at least in part, from the hemolymph proteins, while its carbohydrates must come from the fat body glycogen. The results are discussed in terms of its possible metabolic and eventually physiological meanings. Emphasis is given in the discussion of the metabolic role of the inorganic and organic components of the hemolymph, as yet in the the mechanisms of the silk secretion and CaCO3 deposition and their possible hormonal regulation.

Bothrops jararaca é uma das principais serpentes responsáveis por acidentes ofídicos em São Paulo. O efeito do envenenamento pode ser local ou sistêmico, os quais são mediados por uma variedade de componentes do veneno. Considerando que, em animais vertebrados, o fígado desempenha atividades metabólicas essenciais, além de ser o principal órgão responsável pela síntese de proteínas do plasma, a obtenção do transcriptoma deste é de extrema importância para o estudo destas proteínas. Assim, o objetivo deste trabalho foi analisar o perfil de expressão de RNA no fígado de B. jararaca. Para isto foram obtidas 1700 sequências nucleotídicas denominadas Expressed Sequences Tags (ESTs). As sequências de nucleotídeos foram reunidas em 260 contigs, que foram submetidos ao banco de dados NCBI GenBank usando os algorítimos Blastx e Blastn. dos transcritos tiveram hit com banco de dados NR enquanto 30,5% das ESTs não tiveram homologia. De acordo com a análise do Gene Ontology (GO), os transcritos foram designados para processo biológico, componente celular e função molecular. A maioria dos transcritos foram agrupados em processo biológico, distribuídos em processo metabólico, processo celular e regulação biológica. No entanto, as atividades de ligação proteica, catalítica e de atividade regulatória enzimática foram as principais categorias relacionadas à função molecular. A análise do componente celular apresentou maior número de transcritos envolvidos com a parte celular, região extracelular e restrito a membrana de organela. O maior grupo de transcritos foi relacionado a inibidores de metaloproteases, inibidores de serinoproteases e inibidores de PLA2. Estudos de expressão gênica de alguns alvos selecionados na biblioteca de cDNA de B. jararaca foram realizados para comparação da expressão entre serpentes jovens e adultas. Esses resultados fornecem dados que poderão auxiliar nos estudos filogenéticos entre as diferentes espécies de serpentes e investigar a diferença no padrão da expressão gênica, fornecendo dados importantes sobre a biologia desses animais, contribuindo assim para a elucidação da fisiologia desses animais. Além disso, será útil na identificação de moléculas que possam ser candidatas a alvos terapêuticos. / Bothrops jararaca is the main responsible for snake bites in São Paulo. There are both local and systemic envenomation effects, which are mediated by a variety of venom components. Considering that in vertebrate animals the liver is an important organ responsible for synthesis of plasma proteins, it would be valuable to get transcriptomic information about it. Thus, the aim of this work was to analyze expression profile at RNA level in B. jararaca liver. For this purpose, we sequenced 1700 Expressed Sequence Tags (ESTs) from a cDNA library of B. jararaca liver. Nucleotide sequences were assembled into 260 contigs, which were submitted to the GenBank NCBI database using BLASTX and BLASTN algorithms. Transcripts showed 43% hits with NR database while 30.5% of ESTs had no homology. According to Gene ontology (GO) analysis, transcripts were assigned for biological process, cellular component and molecular function. Majority of transcripts were classified in biological process category distributed in metabolic process, cellular processes and biological regulation, whereas binding and catalytic activities were the main category in molecular function. Cellular component analysis identified transcripts related to cell part, extracellular region and membrane-bounded organelle. The major group of transcripts was related to metalloproteinase inhibitors, followed by serine proteinase inhibitors and phospholipase A2 inhibitors. Studies of gene expression of some selected targets in the cDNA library of B. jararaca, by real time PCR were performed to compare expression in juvenile and adult snake specimens. Our results will help in studies of phylogenetic relationships between different snake species, and investigate differences in gene expression pattern. In addition, our findings are also helpful in the identification of active compounds for development of improved therapeutics for snake bites.

O manejo pré-abate de suínos representa grande desafio para produtores e indústrias de carne suína e seus derivados. A reatividade dos animais ao manejo pode introduzir variação na resposta ditando a magnitude e extensão das mudanças metabólicas musculares em resposta ao estresse, com implicações para atributos de qualidade da carne. O objetivo deste trabalho foi verificar a influência de diferentes tipos de manejo durante a condução de suínos no período pré-abate, em animais com graus de reatividade divergentes, sobre atributos de qualidade da carne. As características de qualidade avaliadas no músculo Longissimus dorsi foram: pH, índice de fragmentação miofibrilar (MFI), força de cisalhamento (FC), cor, perda por cozimento (PPC) e perda por gotejamento (PPG). O delineamento inteiramente ao acaso foi utilizado envolvendo 48 animais divididos em dois tipos de manejo pré-abate (estressante-E e tranquilo-T), dois grupos de reatividade divergentes (reatividade alta-RA e baixa-RB) e sexos (fêmeas e machos), caracterizando um arranjo fatorial 2 (manejo pré-abate) x 2 (reatividade) x 2 (sexo). O pH diferiu (P<0,05) apenas entre os tempos de amostragem pós-abate, com valores médios de 6,24±0,11 e 5,80±0,16, para 3 e 24 horas pós-abate, respectivamente. Os valores de MFI encontrados diferiram (P<0,05) apenas entre os períodos de maturação de 1, 4 e 6 dias, com valores de 29,5±0,91, 50,3±0,91 e 70,3±0,91, respectivamente. O manejo E resultou em FC de 3,86kgf±0,16, que superou (P<0,05) os 3,46kgf±0,15 obtidos para manejo T dos animais. A carne de animais RA apresentou 20,3%±0,70 de PPC, sendo inferior (P<0,05) aos 23,1%±0,72 observados para carne de suínos RB. Houve interação reatividade*manejo para PPG, com as menores perdas (P<0,05) para carne dos animais RA/E (8,05%±0,42) comparada à de animais RA/T (9,28%±0,42) e RB/E (9,33%±0,45), sendo a carne de animais RB/T (8,75%±0,42) similar a todos os outros tipos. A coloração não apresentou diferenças entre os tratamentos. Embora os valores de pH, MFI e cor não tenham sido modificados pelos diferentes manejos ou reatividades, as diferenças observadas em FC, PPC e PPG indicam que manejo, reatividade e a interação entre estes fatores podem influenciar de maneiras diversas os atributos de qualidade de carne. / The pre-slaughter handling of pigs represents a great challenge for growers and pork and its derivatives. The reactivity of animals to land use may introduce variation in the response dictating the magnitude and extent of changes in muscle metabolic response to stress, with implications for quality attributes of meat. The objective of this study was to evaluate the influence of different types of management during the conduct of pigs in the pre-slaughter in animals with differing degrees of reactivity on quality attributes of meat. The quality characteristics evaluated in the Longissimus dorsi were: pH, myofibrillar fragmentation index (MFI), shear force (FC), color, cooking loss (PPC) and drip loss (PPG). The completely randomized design was used involving 48 animals divided into two types of pre-slaughter (stressful-E and relaxed-T), two groups of different reactivity (high reactivity-RA and low-RB) and gender (female and male), featuring a factorial 2 (pre-slaughter) x 2 (reactivity) x 2 (gender). The pH differed (P<0.05) between sampling times post-slaughter, with average values of 6.24±0.11 and 5.80±0.16, for 3 and 24 hours after slaughter, respectively. MFI values found differ (P<0.05) between the periods of maturation of 1, 4 and 6 days, with values of 29.5±0.91, 50.3±0.91 and 70.3±0.91, respectively. The management and resulted in FC of 3.86kg±0.16, which exceeded (P<0.05) to 3.46±0.15kg obtained for T management of animals. The meat of RA showed 20.3%±0.70 PPC, being lower (P<0.05) to 23.1%±0.72 observed for beef and pork RB. There was interaction reactivity*management for PPG, with the lowest losses (P<0.05) for meat from animals RA/E (8.05%±0.42) compared to animals RA/T (9.28%±0.42) and RB/E (9.33%±0.45), and the meat of animals RB/T (8.75%±0.42) similar to all other types. The color did not differ between treatments. Although the pH values, MFI and color have not been modified by different managements or reactivity, the differences observed in CF, PPC and PPG indicate that management, reactivity and the interaction between these factors may influence differently the quality attributes of meat.

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