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Rôle du Système de calpaïne/calpastatine dans l'hypertension pulmonaire induite par l'hypoxie chez la souris. Les souris calpaïnes knockout ont montré des effets protecteurs dans l'hypertension pulmonaire (HP) induite par l'hypoxie. Cependant, le modèle animal avec une surexpression de calpastatine (cast) n'a jamais été étudié. Notre objectif est d'utiliser des souris transgéniques CMV-cast qui surexpriment constitutivement la calpastatine intracellulaire sous le contrôle du promoteur CMV dans tous les types cellulaires pour étudier les effects de la calpaïne intracellulaire. Nous utilisons aussi des souris transgéniques CRP-cast qui surexpriment la calpastatine extracellulaire sous le contrôle du promoteur CRP (protéine C-réactive) pour étudier les effets de la calpaïne extracellulaire. Finalement, nous examinons les effets d'un traitement avec le PD150606, inhibiteur de calpaïne, chez des souris C57BL/6j (WT) hypoxiques et SM22-5HTT+ qui dévélopent l'HP spontanément. Nous avons constaté que les protéines calpaïne et calpastatine sont augmentées immédiatement dans un état hypoxique. Les calpaïnes ont ensuite culminé le jour 8 et sont restées élevées jusqu'au jour 18 chez les souris WT hypoxiques, alors que la calpastatine a augmenté du jour 1 au jour 3, retournant au niveau basal jusqu'au jour 18. Les activités des calpaïnes intra- et extra-cellulaires ont augmenté progressivement pour atteindre un sommet au jour 8, restant aux niveaux élevés jusqu'au jour 18 chez les souris WT hypoxiques. En utilisant l'immunofluorescence, nous avons constaté que l'augmentation des calpaïnes sont principalement colocalisés avec les CML vasculaires pulmonaires (α-SMA+). Cependant, chez la souris CMV-cast, la surexpression de la calpastatine a atténué le développement d'HP. Chez les souris CMV-cast hypoxiques les niveaux de calpastatine sont restés plus élevés que ceux des souris WT à tous les moments de l'hypoxie. Les niveaux de calpastatine plus élevés chez les souris CMV-cast ont empêché de manière significative une augmentation des niveaux de protéines calpaïnes et des activités intra- et extra-cellulaires de la calpaïne au cours de l'hypoxie. Les résultats d'immunofluorescence également ont confirmé que moins de calpaïnes colocalisent avec les CML vasculaires pulmonaires (α-SMA+) chez la souris CMV-cast. Après 18 jours hypoxie, CRP-cast mice ont attenué le développement d'HP. En outre, cette surexpression a montré des effets similaires par rapport à celle intracellulaire. Cependant, le PD150606 a eu que des effets supplémentaires chez les souris WT hypoxiques par rapport aux souris CMV-cast et CRP-cast hypoxiques. Chez les souris SM22-5HTT+, les niveaux de calpastatine, de calpaïnes ainsi que des activités intra- et extra-cellulaires de la calpaïne ont été significativement augmentés dans les poumons. Le PD150606 n'a pas modifié les niveaux de calpastatine, mais il a diminué de manière significative des calpaïnes ainsi que des activités intra- et extra-cellulaire de calpaïne. Les niveaux de calpastatine et des calpaïnes ont également paru augmentées dans les vessaux pulmonaires remodelés chez les patients atteints de maladie pulmonaire chronique par rapport à ceux nonremodelés. En résumé, nos résultats indiquent un nouveau rôle des calpaïnes extracellulaires dans la prolifération des CML-AP dans l'HP. Les stratégies d'inhibition des calpaïnes extracellulaires sembleraient être une stratégie thérapeutique dans le traitement de la progression de l'HP. / Targeting the Calpain/Calpastatin System to Protect against Hypoxia-induced Pulmonary Hypertension in Mice. Calpain knockout mice exhibited protective effects against hypoxia-induced PH. Our aim was to study the role of the calpain/calpastatin system on PH development in mice. To this end, we used mice ubiquitously overexpressing intracellular calpastatin (cast) under the control of a CMV promoter (CMV-cast) to explore the effects of intracellular calpains. We also used mice overexpressing extracellular calpastatin under the control of a CRP (C-reactive protein) promoter (CRP-cast) to explore the effect of extracellular calpains. Finally, we examined the effects of treatment with PD150606, an inhibitor of calpain, in WT mice exposed to hypoxia and in SM22-5HTT+ mice with spontaneous PH. During time-course of hypoxia, we found that calpain and calpastatin protein levels increased immediately after hypoxic exposure. Calpain protein levels then peaked on day 8 and remained elevated until day 18 in hypoxic WT mice; however, calpastatin protein levels increased from day 1 to day 3, and returned to basal level until day 18. Both intra- and extra-cellular calpain activities were upregulated gradually and peaked on days 8, and still markedly remained in high levels until day 18 in hypoxic WT mice. By using immunofluorescence, we found that increased calpains were predominantly colocalized with α-SMA positive pulmonary vascular SMCs. In CMV-cast mice, intracellular calpastatin overexpression successfully attenuated PH development. In CMV-cast mice, calpastatin protein levels remained higher than those in WT mice at all time points of hypoxia. The higher calpastatin protein levels in CMV-cast mice did significantly prevent an increase in calpain protein levels and calpain intra- and extra-cellular activities during hypoxia. After 18 days hypoxia, CRP-cast mice exhibited less PH severity. Moreover, extracellular calpastatin overexpression showed similar effects as intracellular calpastatin overexpression. Treatment with PD150606 induced an additional protective effect in hypoxic WT mice but not CMV-cast and CRP-cast mice. In SM22-5HTT+ mice, lung calpastatin and calpain proteins as well as calpain intra- and extra-cellular activities were significantly increased. PD150606 did not alter lung tissue calpastatin. However, it significantly decreased calpain protein levels as well as calpain intra- and extra-cellular activities. In summary, our present results demonstrate that calpain inhibition prevents PH development. Either increasing extracellular calpastatin or increasing both extra- and intra-cellular calpastatin is efficient to attenuate PH. Treatment with PD150606 which inhibits both extra- and intra-cellular calpain activities may be useful in teh setting of PH.

O objetivo deste trabalho foi avaliar o potencial de utilização de marcadores moleculares em genes candidatos da calpaína (CAPN) e calpastatina (CAST) como ferramenta auxiliar para programas de melhoramento de características relacionadas ao crescimento e maciez da carne. Foram avaliados 605 bovinos da raça Nelore, pertencentes à Agropecuária CFM Ltda, com idade média ao abate de 24 meses. Após a extração do DNA de amostras de sangue, por desproteinização em presença de NaCl, a identificação e determinação do polimorfismo para os marcadores moleculares CAPN316, CAPN530, CAPN4751, CAPN4753 e UOGACAST1, foi realizada pelo sistema de detecção TaqManTM utilizando-se PCR em Tempo Real. A análise de maciez da carne, aos 7, 14 e 21 dias de maturação, foi realizada com amostras de carne do Longissimus dorsi, retiradas entre a 12ª e 13ª costela e cisalhadas utilizando-se um Warner Bratzler Shear Force. Nenhum efeito significativo dos marcadores avaliados foi observado para as características de crescimento. Foi verificado efeito significativo, em relação à maciez da carne, para os seguintes polimorfismos: aos 7, 14 e 21 dias de maturação para o marcador CAPN4751; aos 21 dias para o marcador CAPN4753 e aos 14 e 21 dias para o marcador UOGCAST1. Em relação aos efeitos das combinações genotípicas para os marcadores dois a dois, os resultados foram significativos para a combinação CAPN4751/UOGCAST1 nos três tempos de maturação. Para a combinação de marcadores CAPN4753/UOGCAST1 também foram verificados resultados significativos para carnes maturadas aos 14 e 21 dias. Os resultados observados neste trabalho sugerem a possibilidade da utilização de seleção assistida por marcadores (MAS), visando o aumento da qualidade da carne em bovinos da raça Nelore. / The objective of this study was to evaluate the potential utilization of molecular markers on candidate calpain and calpastati n genes as an auxiliary tool for breeding programs on traits related to growth and meat tenderness. A total of 605 Nellore animals, raised by CFM Agro-pecuária Ltda, were used in this study and slaughtered with 24 months in average. After DNA blood samples extraction, by desproteinization in presence of NaCl, the polymorphism (CAPN316, CAPN530, CAPN4751, CAPN4753 and UOGACAST1) identification and determination was realized by TaqManTM detection system using real time PCR. The meat tenderness analysis, at the 7, 14 and 21 days of maturation was realized with Longissimus dorsi meat samples, taken at the 12th and 13th rib interval and Warner Bratzler Peak Shear Force measurements were used. There were no significant effects of molecular markers in growth traits. There were significant effects, regarding to meat tenderness, for following polymorphisms: at 7, 14 and 21 days of maturation, for CAPN4751 marker; at 21 days of maturation, for CAPN4753, and finally, at 14 and 21 days of maturation, for UOGCAST1 marker. In respect to genotypic combination effects analysis for pairwise marker, the results were significant for CAPN4751/UOGCAST1 in three days of maturation. In combination effects for CAPN4753/UOGCAST1 markers, significant effects were also observed for meat tenderness at 14 and 21 days. Theses results suggest that marked selection assisted (MAS) can be used to improve meat quality in Nellore beef cattle.

μ -Calpain is localized to the mitochondrial intermembrane space. Apoptosisinducing factor (AIF), which executes caspase-independent cell death, is also localized to the mitochondrial intermembrane space. Following processing at the N-terminus, AIF becomes truncated (tAIF) and is released from mitochondria. The protease responsible for AIF processing has not been established. The same submitochondrial localization of mitochondrial μ-calpain and AIF gives support to the hypothesis that mitochondrial μ-calpain may be responsible for processing AIF. Atractyloside-induced tAIF release in rat liver mitochondria was inhibited by cysteine protease inhibitor MDL28170, but not by calpain inhibitors PD150606 or calpastatin. Moreover, μ-calpain immunoreactivity was difficult to detect in rat liver mitochondria. In a mitochondrial fraction from SH-SY5Y cells, incubation with 5 mM Ca2+ resulted in the activation of mitochondrial μ-calpain but not in AIF truncation. Finally, in hippocampal neurons calpain activation did not induce AIF processing or nuclear translocation and AIF translocation to nucleus was calpain independent. The localization of μ-calpain to the mitochondrial intermembrane space is suggestive of its possible involvement in AIF processing, but direct experimental evidence supporting such a role has been elusive. We observed that mitochondrial μ-calpain required high Ca2+ for activation. We examined the hypothesis that the endogenous calpain inhibitor, calpastatin, may be present in the neuronal mitochondria. Calpastatin was detected in the mitochondriaenriched fraction obtained from rat cerebral cortex and SH-SY5Y cells. The mitochondrial calpastatin was resistant to proteinase K digestion, indicating localization internal to the outer mitochondrial membrane. Submitochondrial fractionation revealed that the calpastatin was localized to the mitochondrial intermembrane space and mitoplasts (inner mitochondrial membrane and matrix) but not to the mitochondrial outer membrane fraction. Mitochondrial calpastatin was not detected when mitoplasts were incubated with proteinase K, suggesting that calpastatin is not present in the matrix. The N-terminus of XL domain of calpastatin, when fused to GFP and transfected to SH-SY5Y cells showed mitochondrial localization and thus confirmed the presence of a mitochondrial targeting sequence in calpastatin. Together, these results demonstrate the presence of calpastatin in the neuronal mitochondrial intermembrane space, the same submitochondrial compartment as mitochondrial μ-calpain. This finding explains the high Ca2+ requirements for mitochondrial μ-calpain activation.

Calpains are a family of intracellular cysteine proteases activated by calcium. They participate in many processes including cell motility, cell cycle progression and cell death, in response to calcium signaling. Because calpain over-activation as a result of calcium dysregulation is a contributing factor to many disease states, these enzymes are important therapeutic targets. Within the cell, calpains 1 and 2 are regulated by the protein inhibitor calpastatin. This unstructured protein is specific for calpain, binds tightly, and recognizes only the activated form of the enzyme. Detailed kinetic data obtained using surface plasmon resonance allowed the association and dissociation rates of each of the four calpastatin inhibitory domains to be measured. Based on this, inhibitory domain 4 was selected to be co-crystallized bound to calpain 2. The X-ray crystal structure of this complex provided both the first view of the active enzyme, as well as the first view of how it is inhibited. Calpastatin wraps around the enzyme making contact with each domain. It lies in the active site as a contiguous polypeptide chain and escapes cleavage by forming a loop away from the catalytic cysteine. In addition to inhibiting substrate cleavage, calpastatin protects calpain in two ways; it prevents autoproteolysis, and it prevents calcium-dependent aggregation. The crystal structure of the calpastatin:calpain complex revealed no obvious reason for this stabilization. To elucidate how this protection occurs, peptides were synthesized corresponding to conserved subdomains of calpastatin. Surprisingly, each peptide alone was capable of preventing aggregation in vitro, by blocking hydrophobic patches exposed upon activation. The increased hydrophobic surface of the activated enzyme may alter calpain’s affinity for other proteins such as substrates. By binding across many domains of calpain, calpastatin could act to block protein-protein interactions. These studies have characterized calpastatin’s interaction with calpain, which will further our understanding of the enzyme’s regulation and aid in the development of better calpain inhibitors. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2010-04-29 15:27:16.208

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No. of bitstreams: 1 enriquezvalencia_ce_dr_jabo.pdf: 1054537 bytes, checksum: 7dec2790aa8a6f7dc9e2010c1e059d9f (MD5) Previous issue date: 2016-06-24 / Pró-Reitoria de Extensão Universitária (PROEX UNESP) / Com o intuito de estudar o potencial de aplicação de genes candidatos na seleção animal para o melhoramento genético da qualidade de carne de bovinos Nelore (Bos indicus), o presente trabalho teve como objetivos principais: (1) Avaliar em bovinos Nelore e cruzamentos Nelore x Bos taurus a ocorrência de associações entre o SNP g.98535683A>G:BTAU7 do gene CAST e as características da carne produzida e (2) quantificar a expressão gênica e proteica da cadeia pesada da miosina em bovinos Nelore com características fenotípicas contrastantes de crescimento e maciez da carne. Para o desenvolvimento do primeiro objetivo, 500 animais foram genotipados para o SNP g.98535683A>G:BTAU7 e fenotipados para força de cisalhamento (FC), índice de fragmentação miofibrilar (MFI), área de olho de lombo (AOL), espessura de gordura subcutânea (EGS) e lipídeos totais (LIP). Associação significativa entre o SNP e a maciez da carne foi observada. O genótipo AA apresentou os melhores valores em relação a esta característica. As diferenças encontradas entre os genótipos AA e AG (AA - AG) foram de - 0,19 kg e 5,31 para FC e MFI, respectivamente. O SNP não mostrou associação significativa com AOL, EGS e LIP. Para a execução do segundo objetivo, 90 bovinos Nelore terminados em confinamento foram utilizados. Levando em consideração o peso final ao abate (PF) e a FC dos 90 animais, 24 indivíduos com características contrastantes de peso e maciez da carne foram selecionados e divididos em quatro grupos experimentais (6 animais/grupo). Os grupos corresponderam a animais leves com carne macia (PF = 493,50 kg e FC = 3,76 kg), animais leves com carne dura (PF = 515,66 kg e FC = 8,06 kg), animais pesados com carne macia (PF = 604,33 kg e FC = 3,99) e animais pesados com carne dura (PF = 605,33 kg e FC = 7,83 kg). Em cada grupo, foram avaliadas características fenotípicas como MFI, AOL, EGS, LIP, índice de marmorização (IM), coloração instrumental (L*, a*, b*) e perdas por cozimento (PT). A análise de expressão dos genes MYH7, MYH2 e MYH1 foi feita por PCR em tempo real (RT-qPCR). A caracterização e quantificação proteica das isoformas da MyHC (MyHC-I, MyHC-IIa e MyHC-IIx/d) foi realizada por eletroforese SDS-PAGE. Em todas as análises (qualidade da carne e expressão gênica e proteica) não foi observada interação significativa entre crescimento e maciez da carne. Nas características AOL, EGS, IM e LIP não foi observado efeito significativo do crescimento e a maciez da carne. Porém, os animais pesados apresentaram maiores valores de L*, a* e b* em relação aos animais leves. Na maciez, o grupo de carne macia mostrou maiores valores de L* (32,29 ± 2,78) que o grupo de carne dura (29,10 ± 2,61). Por outro lado, maiores PT foram observadas em animais com carne dura em relação a animais com carne macia. Nos resultados de expressão gênica, o gene MYH7 não mostrou diferenças entre animais leves e pesados. Entretanto, a expressão dos genes MYH2 e MYH1 foi significativamente menor nos animais pesados em relação aos animais leves. Quanto à maciez da carne, a expressão dos três genes não diferiram significativamente entre grupos de carne dura e macia. Na expressão proteica, as três isoformas (MyHC-I, IIa e IIx), não mostraram diferença significativa entre animais leves e pesados. No entanto, as proporções da MyHC-I foram significativamente maiores no grupo de carne dura (16,51 ± 5,47) em relação ao grupo de carne macia (9,12 ± 3,16) e pelo contrário, as proporções da MyHC-IIa foram maiores no grupo de carne macia (73,78 ± 4,45) em relação ao grupo de carne dura (65,45 ± 8,79). A isoforma MyHC-IIx/d não mostrou efeito significativo sobre a maciez. Os resultados deste trabalho mostraram o potencial de aplicação do SNP g.98535683A>G:BTAU7 do gene CAST e da isoforma MyHC-IIa na seleção de animais para o melhoramento da maciez da carne de bovinos Nelore, além de, manifestar o diferencial de expressão dos genes MYH1 e MYH2 entre animais pesados e leves. / In order to study the potential of application of candidate genes in animal selection for breeding meat of Nellore cattle, this work had as main objectives: (1) Assess in Nellore cattle (Bos indicus) and crosses Nellore x Bos taurus the occurrence of associations between the g.98535683A>G:BTAU7 SNP in the CAST gene and traits of the meat produced and (2) quantify the gene and protein expression of myosin heavy chain in Nellore cattle with contrasting phenotypic traits of growth and meat tenderness. For the development of first objective, 500 animals were genotyped for the g.98535683A>G:BTAU7 SNP and phenotyped for shear force (SF), myofibrillar fragmentation index (MFI), ribeye area (RA), backfat thickness (BT) and total lipids (TL). Significant association between the SNP and meat tenderness was observed. The AA genotype showed the best values in relation to this trait. The differences found between genotypes AA and AG (AA - AG) were – 0.19 kg and 5.31 for SF and MFI, respectively. The SNP did not show significant association with RA, BT and TL. For the execution of the second aim, 90 feedlot Nellore cattle were used. Considering the final slaughter weight (FW) and the SF of the 90 animals, 24 individuals with contrasting characteristics of weight and tenderness of meat were selected and divided into four experimental groups (6 animals / group). The groups corresponded to lightweight animals with tender meat (FW = 493.50 kg and SF = 3.76 kg), lightweight animals with tough meat (FW = 515.66 kg and SF = 8.06 kg), heavy animals with tender meat (FW = 604.33 kg and SF = 3.99) and heavy animals with tough meat (FW = 605.33 kg and SF = 7.83 kg). In each group, were evaluated phenotypic characteristics as MFI, RA, BT, TL, marbling índex (MI), instrumental color (L*, a*, b*) and cooking losses (CL). The expression analysis of the MYH7, MYH2 and MYH1 genes was performed by Real-Time PCR (RT-qPCR). The characterization and quantification of the MyHC isoforms (MyHC-I, MyHC-IIa and MyHC-IIx/d) was carried out by SDS-PAGE. In all analyzes (meat quality and gene and protein expression) there was no significant interaction between growth and meat tenderness. In the traits MFI, RA, BT, IM and TL it was not observed significant effect of the growth and the tenderness of meat. However, the heavy animals showed higher values of L*, a* and b* than lightweight animals. In the tenderness, the tender meat group showed higher values of the L* (32.29 ± 2.78) than the tough meat group (29.10 ± 2.61). On the other hand, higher CL were observed in animals with tough meat than animals with tender meat. In the results of gene expression, the MYH7 gene did not show differences between lightweight and heavy animals. However, the expression of the MYH2 and MYH1 genes was significantly lower in heavy animals than lightweight animals. As for meat tenderness, the expression of the three genes did not differ significantly between tough and tender meat groups. In the protein expression, the three isoforms (MHC-I, IIa and IIx), did not show significant difference between lightweight and heavy animals. Nevertheless, the MyHC-I proportions were significantly higher in the tough meat group (16.51 ± 5.47) than tender meat group (9.12 ± 3.16) and on the contrary, the proportions of the MyHC-IIa were higher in tender meat group (73.78 ± 4.45) than tough meat group (65.45 ± 8.79). The MyHC-IIx isoform did not show significant effect on tenderness meat. The results of this work showed the potential of application of the g.98535683A>G:BTAU7 SNP in gene CAST and the MyHC-IIa isoform in animal selection to improve meat tenderness of Nellore cattle, in addition, it showed the differential expression of MYH1 and MYH2 genes between heavy and lightweight animals.

O objetivo deste trabalho foi avaliar o potencial de utilização de marcadores moleculares em genes candidatos da calpaína (CAPN) e calpastatina (CAST) como ferramenta auxiliar para programas de melhoramento de características relacionadas ao crescimento e maciez da carne. Foram avaliados 605 bovinos da raça Nelore, pertencentes à Agropecuária CFM Ltda, com idade média ao abate de 24 meses. Após a extração do DNA de amostras de sangue, por desproteinização em presença de NaCl, a identificação e determinação do polimorfismo para os marcadores moleculares CAPN316, CAPN530, CAPN4751, CAPN4753 e UOGACAST1, foi realizada pelo sistema de detecção TaqManTM utilizando-se PCR em Tempo Real. A análise de maciez da carne, aos 7, 14 e 21 dias de maturação, foi realizada com amostras de carne do Longissimus dorsi, retiradas entre a 12ª e 13ª costela e cisalhadas utilizando-se um Warner Bratzler Shear Force. Nenhum efeito significativo dos marcadores avaliados foi observado para as características de crescimento. Foi verificado efeito significativo, em relação à maciez da carne, para os seguintes polimorfismos: aos 7, 14 e 21 dias de maturação para o marcador CAPN4751; aos 21 dias para o marcador CAPN4753 e aos 14 e 21 dias para o marcador UOGCAST1. Em relação aos efeitos das combinações genotípicas para os marcadores dois a dois, os resultados foram significativos para a combinação CAPN4751/UOGCAST1 nos três tempos de maturação. Para a combinação de marcadores CAPN4753/UOGCAST1 também foram verificados resultados significativos para carnes maturadas aos 14 e 21 dias. Os resultados observados neste trabalho sugerem a possibilidade da utilização de seleção assistida por marcadores (MAS), visando o aumento da qualidade da carne em bovinos da raça Nelore. / The objective of this study was to evaluate the potential utilization of molecular markers on candidate calpain and calpastati n genes as an auxiliary tool for breeding programs on traits related to growth and meat tenderness. A total of 605 Nellore animals, raised by CFM Agro-pecuária Ltda, were used in this study and slaughtered with 24 months in average. After DNA blood samples extraction, by desproteinization in presence of NaCl, the polymorphism (CAPN316, CAPN530, CAPN4751, CAPN4753 and UOGACAST1) identification and determination was realized by TaqManTM detection system using real time PCR. The meat tenderness analysis, at the 7, 14 and 21 days of maturation was realized with Longissimus dorsi meat samples, taken at the 12th and 13th rib interval and Warner Bratzler Peak Shear Force measurements were used. There were no significant effects of molecular markers in growth traits. There were significant effects, regarding to meat tenderness, for following polymorphisms: at 7, 14 and 21 days of maturation, for CAPN4751 marker; at 21 days of maturation, for CAPN4753, and finally, at 14 and 21 days of maturation, for UOGCAST1 marker. In respect to genotypic combination effects analysis for pairwise marker, the results were significant for CAPN4751/UOGCAST1 in three days of maturation. In combination effects for CAPN4753/UOGCAST1 markers, significant effects were also observed for meat tenderness at 14 and 21 days. Theses results suggest that marked selection assisted (MAS) can be used to improve meat quality in Nellore beef cattle.

No description available.

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No. of bitstreams: 2 Tese - Leonardo Guimarães de Oliveira - 2016.pdf: 2541166 bytes, checksum: 5b0501be9f076d03111ca00fcf4980a7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-03-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The protein profile of hornless Nellore cattle from a segregating population for meat tenderness of extremes shear force, low extreme group (M) and extreme high (D) group, showed differentially expressed proteins. They had greater relative abundance of proteins of the glycolytic process in group M and proteins of the oxidative metabolism evidenced more expressed in group D, fact that probably is correlated to the final tenderness of the meat. Only identified in group D, the cytochrome c protein indicates induction of the apoptotic process in this group of animals. Structural proteins were identified in group M, indicating a possible greater proteolysis. Calpastatin was only identified in group D, this protein is highly related to the final meat tenderness because it is a natural inhibitor of the calpain. Separation of calpastatin by ion exchange column chromatography of two different muscles described two peaks of calpain inhibitory activity: peak 1 (CAST1) and peak 2 (CAST2). CAST 1 activity increased during the post-mortem period in Triceps brachii and showed no difference between days in Longissimus dorsi and on the other hand, total activity of calpastatin and CAST 2 decreased during post-mortem aging. The 115 kDa band of calpastatin decreased its intensity during post-mortem aging in both muscles with more than 70% of the change occurring on the first day. The mitochondrial ATP synthase beta subunit proteins increased and Succinyl CoA ligase decreased after aging and Adenylate kinase isoenzyme decreased on day 7. Calpastatin peaks had weak phosphorylated bands and had different IP and MW patches than 2D-SDS -PAGE. During the purification process of the calpastatin peaks the activity per mg of protein increased but lost half of the total activity presented during the first purification step. For peak 2 of calpastatin the specific activity increased 139.8 times and at the end of this process 36% of the total activity remained. Purified calpastatin was identified on the second-size gel having a molecular weight similar to the western blot. Spots from calpastatin peak 1 and two from calpastatin peak 2 were identified as peptides belonging to the calpastatin molecule. Sequence of peptides identified in Spot from purified peak 1 as part of the inhibitory domain III and IV and of the C-terminus and from the purified peak 2 a sequence of peptides identified as part of the inhibitory domain I, II and III. These results lead us to believe that both peaks, in this case, are degradation products of the intact molecule, and probably the small peptides are broken down during the process. The results of the present study show that purification of distinct forms of active calpastatin is possible, however, the intact form of calpastatin was not present in this purification. The presence of peptides was not conclusive to determine the origin and composition of each active peak. / O perfil protéico de animais da raça nelore mocho de uma população segregante para a maciez da carne de extremos valores de força de cisalhamento, grupo extremo baixo (M) e grupo extremo alto (D), apresentou proteínas diferentemente expressas as quais houve maior abundância relativa de proteínas do processo glicolítico no grupo M e proteínas do metabolismo oxidativo evidenciadas mais expressas no grupo D, fato que provavelmente está correlacionado à maciez final da carne. Apenas identificada no grupo D, a proteína citocromo c indica indução do processo apoptótico neste grupo de animais. Proteínas estruturais foram identificadas no grupo M, indicando uma possível maior proteólise. A calpastatina foi somente identificada no grupo D, esta proteína está altamente relacionada com a maciez final da carne por ser inibidora natural das calpaínas. A separação de calpastatina por cromatografia em coluna de troca iônica de dois músculos diferentes descreveu dois picos de actividade inibitória de calpaínas: pico 1 (CAST1) e pico 2 (CAST2). Atividade de CAST 1 aumentada durante o período post mortem no Triceps brachii e não apresentou diferença entre os dias em Longissimus dorsi e por outro lado, a atividade total de calpastatina e CAST 2 diminuiu durante o envelhecimento post mortem. A banda de 115 kDa da calpastatina diminuiu sua intensidade durante o envelhecimento post mortem em ambos os músculos com mais de 70% da alteração ocorrendo no primeiro dia. As proteínas mitocondriais da subunidade ATP sintase beta aumentaram e a Succinil-CoA ligase diminuiu após o envelhecimento e a Adenilato quinase isoenzima diminuiu no dia 7. Os picos de calpastatina apresentavam faixas fosforiladas fracas e apresentavam manchas em IP e MW diferentes do que 2D-SDS-PAGE. Durante o processo de purificação dos picos de calpastatina a actividade por mg de proteína aumentou mas perdeu metade da atividade total apresentada durante a primeira etapa de purificação. Para o pico 2 de calpastatina a actividade específica aumentou 139,8 vezes e no final deste processo permaneceu 36% da atividade total. A calpastatina purificada foi identificada no gel de segunda dimensão com um peso molecular semelhante ao western blot. Spots do pico 1 da calpastatina e dois do pico 2 da calpastatina foram identificados como peptídeos pertencentes à molécula da calpastatina. Sequêcia de peptídeos identificados em Spot a partir do pico 1 purificado como parte do domínio inibidor III e IV e do terminal C e do pico 2 purificado uma sequêcia de peptideos identificados como parte do domínio inibidor I, II e III. Estes resultados levam-nos a crer que ambos os picos, neste caso, são produtos de degradação da molécula intacta e, provavelmente, os pequenos peptideos são quebrados durante o processo. Os resultados do presente estudo mostram que é possível a purificação de formas distintas de calpastatina activa, contudo a forma intacta de calpastatina não estava presente nesta purificação. A presença de peptídeos não foi conclusiva para determinar a origem ea composição de cada pico ativo.

O consumo alimentar residual (CAR) é uma medida de eficiência alimentar independente do crescimento e do peso à maturidade. O melhoramento genético para CAR pode reduzir o custo de alimentação de bovinos, porém uma melhor compreensão dos processos biológicos relacionados ao CAR é necessária. Além disso, associações entre CAR e qualidade de carcaça têm sido pouco investigadas em raças zebuínas. Desta forma, o objetivo com este estudo foi avaliar o metabolismo protéico, a composição corporal, as características de carcaça e a qualidade de carne em bovinos zebuínos com alto e baixo CAR. Adicionalmente, foi testada a hipótese da existência de interações entre CAR e peso vivo ao abate para características de carcaça e composição corporal. Setenta e dois novilhos da raça Nelore (16 a 21 meses de idade, 334±19 kg de peso vivo inicial [PV]) foram mantidos em confinamento e alimentados ad libitum (74,5% NDT; 14,3%PB) por 70 dias. O consumo de matéria seca (CMS) e o ganho médio de peso (GMD) diários foram medidos individualmente. Os 12 novilhos com maior CAR e os 12 com menor CAR foram classificados como grupos de alto e baixo CAR, respectivamente (fase de seleção) e foram alimentados até quando alcançassem pesos vivo ao abate de 460, 490, 520 e 550 kg (fase de terminação). Antes do abate, foi realizada colheita total de urina para a determinação da excreção diária de 3-metil-histidina e da taxa fracional de degradação miofibrilar. A composição química corporal foi estimada pelo método de diluição isotópica utilizando óxido de deutério. A maciez objetiva da carne e a atividade de proteases cálcio-dependentes foram determinadas no músculo Longissimus. Na fase de seleção, novilhos com baixo CAR tiveram menores CMS, conversão alimentar, CAR e ganho de gordura sobre a garupa que novilhos com alto CAR, mas nenhuma diferença foi observada no GMD, no PV final, na gordura subcutânea e na área do Longissimus. Na fase de terminação, nenhuma interação foi observada entre CAR e PV ao abate. Não houve diferenças entre animais mais e menos eficientes quanto ao peso e rendimento de carcaça, gordura renal, pélvica e inguinal, vísceras, área de Longissimus, gordura subcutânea, marmorização, aparas e porção comestível. Novilhos com baixo CAR apresentaram menos gordura sobre o trato gastrintestinal (TGI) que novilhos com alto CAR. Não foram observadas diferenças quanto ao índice de fragmentação miofibrilar, força de cisalhamento e atividade do sistema calpaína. As taxas fracionais de degradação, síntese e acréscimo protéico foram similares entre os grupos de CAR. Novilhos Nelore com baixo CAR depositaram menos gordura subcutânea na carcaça em pesos vivos entre 340 e 460 kg. Em pesos mais elevados (460-550 kg), as características de carcaça e a composição corporal não foram influenciadas pelo CAR, mas indivíduos menos eficientes apresentaram maior massa de gordura visceral. A seleção de bovinos zebuínos para baixo CAR pode diminuir a ingestão de alimentos e melhorar sua eficiência alimentar, sem comprometer a qualidade da carne. / Residual feed intake (RFI) is a feed efficiency trait that is independent of growth rate and mature weight. Genetic improvement in RFI may reduce the costs of feeding cattle, however a better understanding of biological processes underlying variation in RFI is necessary. Moreover, associations between RFI and carcass quality have been poorly investigated in Zebu breeds. Therefore, this study aimed to evaluate protein metabolism, body composition, carcass traits and meat quality in high- and low-RFI Zebu cattle. In addition, the hypothesis that there are interactions between RFI and harvest body weight for carcass traits and body composition was investigated. Seventy-two Nellore steers (16 to 21 month-old, 334±19 kg initial body weight [BW]) were fed a finishing ration (74.5% TDN, 14.3%CP) on an ad libitum basis, for 70 days. Daily dry matter intake (DMI) and body weight gain (ADG) were measured individually. The 12 lowest and the 12 highest RFI steers were classed as low- and high-RFI groups, respectively (selection phase), and were fed until reaching slaughter BW of 460, 490, 520 and 550 kg (finishing phase). Before slaughter, total urine was collected for determination of daily 3-methylhistidine excretion and myofibrillar protein breakdown rates. Body chemical composition was estimated by the isotope dilution method using deuterium oxide. Objective tenderness and Ca+2-dependent protease activities were measured on Longissimus muscle. In the selection phase, low-RFI steers had lower DMI, feed:gain, RFI, and ultrasound rump fat thickness gain than high-RFI cattle, but no differences were observed for ADG, final BW, ultrasound fat thickness and Longissimus area. In the finishing phase, no interactions were observed between RFI and slaughter BW. No differences between more and less efficient cattle were observed for hot carcass weight, dressing percentage, kidney, pelvic and inguinal fat, visceral mass, Longissimus area, backfat thickness, marbling score, trimmings and retail product yield. Low-RFI steers presented less fat on the gastrointestinal tract (GIT) than high-RFI cattle. No differences were observed for myofibrillar fragmentation index, Warner-Bratzler shear force and calpain system activities. Fractional rates of protein degradation, synthesis and accretion were similar between high- and low-RFI cattle. Low-RFI Nellore steers may store less subcutaneous carcass fat at body weights ranging from 340 and 460 kg. At higher body weights (460-550 kg), carcass traits and body composition are not affected by RFI, but least efficient cattle present greater visceral fat mass. Breeding zebu cattle for improved RFI may decrease feed intake and improve feed efficiency without compromising meat quality.