Dissertations / Theses on the topic 'Protéine PARL'

La protéine PARL (Presenilins-Associated Rhomboid-Like protein) est une nouvelle protéine présentant des similarités significatives avec la famille de régulateurs développementaux de Drosophila melanogaster Rhomboid, assurant la protéolyse intramembranaire régulée (RIP). La RIP est un processus par lequel certaines protéines transmembranaires sont clivées à l’intérieur de la membrane pour libérer un domaine cytoplasmique actif. Pour valider l’homologie fonctionnelle entre PARL et Rhomboid-1, nous avons conduit une analyse phylogénétique complète portant sur l’ensemble des membres de la super-famille Rhomboid. Cette étude révèle que cette famille est présente à tous les niveaux évolutifs et que tous ses membres, y compris PARL, sont des sérine-protéases catalysant la RIP. Nos études immunohistochimiques démontrent que le profil d’expression de PARL est régulé dynamiquement au cours du développement postnatal du cerveau de souris. D’autre part, PARL est sélectivement exprimée dans les cellules mitotiques et les neurones indifférenciés, mais pas dans les cellules gliales. De plus, chez l’adulte, l’expression de PARL est restreinte aux zones de neurogenèse active tout au long de la vie de l’animal. L’ensemble de ces résultats indique que l’expression de PARL commence durant la phase proliférative des précurseurs neuronaux, continue dans les étapes précoces de la différenciation neuronale et qu’elle est ensuite régulée négativement au cours de la différenciation. Nous proposons donc que l’activité de RIP-protéase de la protéine PARL joue un rôle essentiel dans l’engagement de la cellule vers la destinée neuronale et dans la neurogenèse.

Le travail présenté dans cette étude a été réalisé au laboratoire du Dr. Luca Pellegrini, qui se concentre sur les mécanismes régulant la dynamique mitochondriale. Dans ce contexte, mon travail s'est focalisé sur la protease rhomboïde Parl, un régulateur essentiel de l'apoptose, de la morphologie mitochondriale et du métabolisme et qui a été impliquée dans la maladie de Parkinson. Au cours de ce travail, nous avons adopté une approche en deux points pour comprendre les bases structurelles et mécanistiques de l'activité protéolytique de Pari et de sa régulation. Dans une première approche, nous avons réalisé une analyse de la structurefonction basée sur un modèle d'homologie pour identifier les déterminants structurels de Pari. L'identification d'un événement de coupure protéique (clivage- Y) qui génère une nouvelle forme de Pari présentant une structure à six segments transmembranaires à partir de la structure classique à 6+1 segments transmembranaires nous a servi de base pour cette étude. Nos résultats montrent des similarités de structure entre la protéine rhomboïde d'origine bactérienne GlpG and Pari. Cependant, Pari semble utiliser une catalyse par triade par opposition à la catalyse par dyade opérée par les rhomboïdes GlpG. Le clivage y perturbe la triade et rend ainsi Pari protéolytiquement inactif. Ainsi, notre étude a identifiée la régulation de la fonction de Pari par l'élimination protéolytique de son activité, ce que nous avons publié dans le journal Cell Death & Differentiation. Dans une seconde approche, nous nous sommes penché sur la validité d'un communiqué publié dans Nature (Chao et al., 2008) qui suppose un rôle pour la protéine HAX1 en tant que protéine présentatrice de substrat pour Pari. En effet, plusieurs faisceaux de preuves n'étaient pas cohérents avec les résultats de cette étude. Nos résultats montrent que Pari et HAX1 sont confinés dans des souscompartiments cellulaires distincts et que l'interaction observée in vitro n'est qu'un artefact. Ainsi in vivo, HAX1 ne peut pas contribuer à la régulation de la protéolyse 2opérée par Pari. Notre étude a contesté les résultats de Chao et al., et a été publiée dans un article de fond dans le journal Cell Death & Differentiation. Enfin, il est important de mentionner qu'au cours de mon doctorat j'ai été le coauteur d'une revue (Jeyaraju et al., BBA 2009) et de deux articles de recherche à travers des collaborations avec les laboratoires du Dr Toth (Lavoie et al., J.Neuroscience, 2011 ) et du Dr Shore (Warr et al., JBC, 2011 ).

Les mitochondries sont des organites essentiels pour la production d'énergie cellulaire grâce à la synthèse d'ATP au cours des étapes de phosphorylations oxydatives (OXPHOS). Les complexes de la chaine respiratoire sont en partie codés par le génome mitochondrial (ADNmt), dont la structure est très sensible aux facteurs exogènes ou endogènes. De nombreuses mutations de l'ADNmt sont associées à des dysfonctionnements de la chaine respiratoire conduisant à des pathologies. La production d'Espèces Oxygénées Réactives (EOR) mitochondriale est la principale source de dommages à l'ADNmt. Une voie de réparation particulière, le système de réparation par excision de bases (BER) est mis en oeuvre dans ce cas. Nous avons, au cours de notre étude, analysé le système BER mitochondrial chez la drosophile. Dans une première approche, nous avons caractérisé de manière globale par une technologie de puces à ADN un ensemble de glycosylases et endonucléases impliquées dans la voie BER mitochondriale et comparé leur variation au cours du vieillissement. Cette étude a été complétée par une analyse transcriptionnelle sur des modèles de drosophiles mutantes pour des enzymes spécifiques de la voie BER, ceci afin de déterminer les éventuelles interactions transcriptionnelles entre les acteurs de cette voie. L'ARNm de Parp présentait de fortes variations dans les différents contextes mutants testés. C'est une molécule essentielle de la réparation BER. Elle a fait l'objet dans un deuxième temps, d'une étude plus approfondie. Dans le modèle des cellules S2, PARP bien que majoritairement nucléaire est également présent dans la mitochondrie. Le comportement différentiel des deux variants ARNm de Parp a pu être mis en évidence lors de stress cellulaires. Les isoformes protéiques de PARP observées dans nos études apparaissent différentes de celles habituellement décrites dans la littérature. Cet aspect a été discuté.

Les modifications post-traductionnelles des protéines de réparation de l'ADN et des facteurs chromatiniens par poly(ADP-ribose)ylation et par phosphorylation sont essentielles pour le maintien de l'intégrité de l'ADN et de la chromatine, en particulier dans la réponse cellulaire aux dommages de l'ADN induits par radiation ionisantes (RI). Parmi les protéines impliquées dans ces deux processus nous trouvons, respectivement, la poly(ADP-ribose) polymérase-1 (PARP-1) et PARP-2, et la kinase dépendante des cyclines Cdk5 : PARP-1 et PARP-2 sont impliqué dans le mécanisme de réparationdes cassures simples brin (CSBs) de l'ADN (Single Strand Break Repair : SSBR ) et la déplétion de Cdk5 a été liée à l'augmentation de la sensibilité des cellules aux inhibiteurs de PARPs. Nous avons montré, en utilisant des cellules HeLa stablement déplété pour Cdk5 ou PARP-2, que ces deux protéines sont impliquées dans les deux sous-voies du SSBR, le short- (SPR) et le long-patch repair (LPR). L'absence de Cdk5 ou PARP-2 entraîne des modifications du fonctionnement du SSBR, notamment en termes de recrutement des protéines de réparation PARP-1 et XRCC1, impliquées dans le SPR, et PCNA, protéine clé du LPR, au site du photo-dommage. PARP-2 et Cdk5 agissent aussi sur la balance du niveau des poly(ADP-ribose) car en absence de Cdk5 une hyper-activation de PARP-1 a été montrée, et en absence de PARP-2 une diminution de l'activité de la protéine poly(ADP-ribose) glycohydrolase (PARG) a été aussi observée. Cependant, malgré ces changements les deux lignées cellulaires dépourvues de Cdk5 (Cdk5KD) ou de PARP-2 (PARP-2KD) réparent de façon normale les CSBs radio-induites, mais, intéressement et contrairement aux cellules PARP-2KD, les cellules Cdk5KDsont sensibles à l'effet létal des RI. De plus nous avons montré que Cdk5, PARP-2 et PARG sont toutes les trois impliquées dans la régulation du recrutement et de dissociation du facteur chromatinien ALC1 suggérant leur implication dans la régulation de la dynamique de la chromatine en réponse aux photo-dommages de l'ADN. Ces résultats avec l'observation de la diminution du recrutement de PARP-1 dans les cellules Cdk5KD et PARP-2KD, montrent l'apparition d'un réseau complexe de phosphorylation et de poly(ADP-ribose)ylation en réponse aux RI qui implique Cdk5 , PARP-1,PARP-2 and PARG et qui est fort probablement initié par l'activité kinase de Cdk5.

La kinase dépendante des cyclines 5 (Cdk5) est un facteur de sensibilité aux inhibiteurs de PARP et aux rayonnements ionisants (RI), elle est nécessaire pour le point de contrôle du cycle cellulaire en phase S. Cependant, elle n’est pas directement impliquée dans la réparation des cassures de brin d’ADN, suggérant un rôle dans les étapes plus précoces de la signalisation des dommages. Nous rapportons ici que des cellules HeLa déplétées pour Cdk5 (Cdk5 KD) montrent une grande sensibilité aux RI surtout lorsqu'elles sont irradiées en phase S, au 5-Fluoro-Uracile, à la 6-Thioguanine et à une exposition chronique à l'hydroxyurée (HU). Les cellules Cdk5 KD montrent une altération de la dynamique de la phase S causée par une vitesse de réplication plus lente et une réduction des origines actives par mégabase d'ADN. En revanche, après un traitement au HU, ces cellules sortent plus rapidement du blocage en phase S. Ceci s’accompagne d’une déficience de la phosphorylation de RPA-32 sur les sérines 29 et 33 et de SMC1 sur la sérine 966 ainsi que d’une réduction du niveau de dommages de l'ADN évalués par le test des comètes alcalines, de l’intensité du signal gamma-H2AX, des foyers RPA, Rad51 et RPA sur les sérines 4 et 8 ainsi que du niveau d'échanges de chromatides sœurs. Des essais kinase in vitro couplés à la spectrométrie de masse ont montré que Cdk5 peut phosphoryler RPA-32 sur ses sérines 23, 29, et 33. De plus, des niveaux inférieurs d’expression de Cdk5 ont été associés à une meilleure survie sans métastases chez des patientes atteintes d’un cancer du sein et à une réduction de la survie des cellules de tumeurs du sein déplétées pour Cdk5 après un traitement aux RI et en présence d’un inhibiteur de PARP. Globalement, ces résultats montrent que Cdk5 est nécessaire pour la réplication basale et l'activation du point de contrôle en phase S en réponse à un stress réplicatif, ouvrant des perspectives cliniques intéressantes pour améliorer la destruction des cellules tumorales dans certaines populations de patientes atteintes de cancer du sein grâce à des agents qui génèrent un stress réplicatif
Cyclin dependent kinase 5 (Cdk5) is a determinant of sensitivity to PARP inhibitors and ionizing radiation (IR) and is required for the intra-S DNA damage checkpoint. It is not however directly implicated in strand break repair suggesting a role in the earlier steps of checkpoint activation. We report here that Cdk5-Depleted (Cdk5-KD) HeLa cells show higher sensitivity to IR when irradiated in S-Phase, and to chronic hydroxyurea (HU) exposure, 5-Fluorouracil and 6-Thioguanine. Cdk5-KD cells show altered basal S-Phase dynamics caused by a slower replication velocity and fewer active origins per megabase of DNA, however they show a faster recovery from an HU block. This was accompanied by impaired RPA-32 priming serine 29 and serine 33 phosphorylations and SMC1-Serine 966 phosphorylation as well as lower levels of DNA damage assessed by the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci and RPA-32 serine 4 and serine 8 phosphorylation and levels of sister chromatid exchanges. In vitro kinase assays coupled with mass spectrometry showed that Cdk5 can phosphorylate RPA-32 on serines 23, 29, and 33. In addition lower Cdk5 levels were associated with longer metastasis free survival in breast cancer patients and lower cell survival in Cdk5 depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and opens up interesting clinical opportunities to enhance tumor cell killing in certain populations of breast cancer patients through agents that generate replication stress

La polarité cellulaire est un processus fondamental qui contrôle les spécificités fonctionnelle et physiologique de la plupart des cellules eucaryotes. Cette asymétrie intracellulaire repose sur l’existence de compartiments membranaires distincts, à la fois dans leur composition en protéines mais également en phosphatidyl-inositols (PIs). Ainsi, la mise en place et le maintien de la localisation asymétrique de modules multi-protéiques associés notamment aux protéines PAR sont essentiels pour l’élaboration des domaines de polarité cellulaire. Durant ma thèse, j’ai étudié les relations entre les protéines de polarité et les PIs dans le contrôle de la polarité cellulaire. Plus particulièrement, en utilisant la chambre ovarienne de Drosophile, j’ai cherché à caractériser la suite d’évènements qui en amont régule l’activité de la PIP5K, Skittles (SKTL), qui produit le PI(4,5)P2 et à caractériser les mécanismes moléculaires qui lient le PI(4,5)P2, SKTL et les protéines PAR dans le contrôle et le maintien de la polarité cellulaire. J’ai contribué à caractériser l’importance de PI(4,5)P2 majoritairement produit par SKTL, dans le maintien de la polarité apico-basale et lors de la morphogenèse des cellules folliculaires de la chambre ovarienne. Le PI(4,5)P2 assure la localisation apicale de PAR3 et le maintien des jonctions adhérentes, sans affecter la localisation de PAR1. Par une méthode de quantification précise, j’ai ensuite démontré dans l’ovocyte que SKTL et le PI(4,5)P2, probablement grâce au trafic vésiculaire, étaient requis pour à la fois l’accumulation à l’antérieur de PAR3 et son exclusion au postérieur qui se fait à partir du stade 9B. L’accumulation antérieure de PAR3 est également dépendante d’un transport Dynéine dépendant et de la kinase IKKε tandis que son exclusion postérieure dépendant des phosphorylations par PAR1. Enfin, j’ai également étudié les modifications post traductionnelles de SKTL et leur importance dans la polarité cellulaire. J’ai identifié la présence de palmitoylation et de phosphorylations dont certaines impliquent la kinase PAR1 et la phosphatase PP1. Ces phosphorylations pourraient avoir un lien avec le rôle de SKTL dans le trafic vésiculaire. Ces résultats permettent donc d’élucider certains mécanismes cellulaires qui contrôlent la mise en place et le maintien de la polarité des cellules en liant les PIs et les protéines PAR
Cell polarity is a fundamental process that controls cell’s functional and physiological specificities. This process relies on membranous compartments differently composed both on proteins and on phosphatidyl-inositols (PIs). Indeed, through their asymmetric localization, polarity proteins, such as the PAR proteins, are essentials to establish and maintain polarity of the cells. During my PhD, I studied the interplay between the polarity proteins and the PIs. Using the Drosophila egg chamber, as a model, I aimed to characterized the upstream events that regulate the PI(4,5)P2 producing kinase (PIP5K), Skittles (SKTL), activity and localization. I also studied the downstream molecular process that link the PI(4,5)P2, SKTL and the PAR proteins in cell polarity. I contributed to the characterization of the importance of PI(4,5)P2, mainly produced by SKTL in maintaining the apical-basal polarity and during the morphogenesis of the follicle cells. The PI(4,5)P2 is ensuring PAR3 and adherens junctions but not PAR1 proper localizations. Next, through a precise quantification method, I showed that SKTL and the PI(4,5)P2, probably via vesicular traffic, were also ensuring PAR3 proper localizations (anterior accumulation and stage 9B posterior exclusion) in the oocyte. PAR3 accumulation also relies on a Dynein mediated transport and the IKKε kinase while its posterior exclusion relies on PAR1 phosphorylation. Finally, I studied SKTL post translational modifications and their relevance on cell polarity. I identified palmitoylation and phosphorylations that are regulated by the kinase PAR1 and the phosphatase PP1. SKTL phosphorylations seem to be related to its role on the vesicular traffic. Altogether these results clarify some mechanisms involving both PIs and PAR proteins in cell polarity maintaining and establishment

A presente tese foi realizada com o objetivo de estudar as relações entre valina (VAL) e isoleucina (ILE), determinar as exigências de valina e a razão pela qual dietas de baixo conteúdo protéico tendem a causar queda no desempenho de frangos de corte. Desta forma, três experimentos foram conduzidos utilizando dietas a base de milho e farelo de soja e linhagens de frangos de corte comercialmente disponíveis. No primeiro estudo foram avaliadas deficiências de VAL e ILE em dietas práticas para frangos de 14 a 35 dias de idade. Tanto VAL quanto ILE foram limitantes para o ganho de peso dos frangos, não sendo possível identificar com precisão a ordem de limitação destes dois aminoácidos, o que sugere uma co-limitação entre eles. No segundo experimento, foram determinadas as exigências de VAL para frangos entre 21 e 42 dias de idade. Os níveis de VAL digestível considerados ideais por meio de regressão linear foram de 0,82 e 0,81% para ganho de peso e conversão alimentar. Entretanto, quando utilizado o método de linha-quebrada o platô para estas mesmas respostas foi obtido com 0,81 e 0,76% de VAL digestível. No terceiro experimento, a suplementação de L-VAL, L-ILE, Glicina (GLY) e/ou L-Ácido glutâmico (L-GLU) (PB) em níveis idênticos aos apresentados por uma dieta controle tida como padrão da indústria indicou influência do nível de nitrogênio sobre o desempenho das aves. Glicina e ácido glutâmico melhoraram o ganho de peso e conversão alimentar das aves. Os benefícios no crescimento devidos ao fornecimento de GLY foram majoritariamente observados em fases iniciais do crescimento das aves enquanto a suplementação de GLU monstrou-se vantajosa ao longo de todo o período experimental. Adições de VAL, ILE, GLU e GLY resultaram em maior deposição de carne de peito em relação as dietas suplementadas somente com valina. Conclui-se que o fornecimento de dietas de reduzido conteúdo protéico fica restrito a utilização de relações precisas entre VAL, ILE e LYS, além do fornecimento adequado de glicina em fases iniciais. O fornecimento de L-GLU como fonte de nitrogênio em dietas de reduzido conteúdo protéico é uma alternativa para a obtenção de desempenho similar ao apresentado por frangos alimentados com dietas de maior conteúdo protéico.
This thesis was carried out to understand the relationships between valine (VAL) and isoleucine (ILE), to determine the requirements of valine and why low protein diets tend to cause drop in broilers performance. Thus, three experiments were conducted using corn and soybean meal diets and broilers strain crosses commercially available. In first study were assessed deficiencies of VAL and ILE in practical diets for broilers from 14 to 35 days of age. VAL and ILE showed limitations on the body weight gain of broilers and it was impossible identify precisely the order of limitation of these two amino acids, suggesting a co-limitation between them. In the second experiment VAL requirements were determined for broilers between 21 and 42 days of age. Ideal digestible levels of VAL considering linear adjustments were 0.82 and 0.81% for body weight gain and feed conversion ratio. However, when used the method of broken-line linear model the plateau for these same responses were obtained with 0.81 and 0.76% of digestible VAL level. In the third experiment, the supplementation of VAL, ILE, Glycine (GLY) and/or Glutamic acid (GLU) as crude protein source at levels similar to those presented by a standard industry diet indicated a strong influence of nitrogen level on the performance of birds. GLY and GLU improved body weight gain and feed conversion of birds. The benefits in growth due to the supply of GLY were mostly observed in early stages of birds growth while the supplementation of GLU showed advantage throughout the experimental period. Additions of VAL, ILE, GLU and GLY showed a higher deposition of breast meat for diets supplemented only with valine. It is concluded that the provision of low protein content diets is restricted to the use of precise relationships between VAL, ILE and LYS, and providing adequate glycine in the early stages. The supply of L-GLU as a source of nitrogen in diets of low protein content is an alternative for obtaining similar performance presented by broilers fed diets of higher protein content.

O objetivo do trabalho foi comparar o desempenho de bezerros, não castrados, recriados no período das águas, exclusivamente em pastagens (CONT) ou suplementados com concentrado energético (ENER) ou protéico (PROT). Também foi avaliado o efeito da suplementação, no desempenho posterior destes animais na fase de terminação em confinamento. Foram utilizados 75 bezerros recém desmamados, com 8 meses de idade e 200,08 kg de PV, provenientes do cruzamento de touro Pardo Suíço com vaca F1 Angus x Nelore. Os animais foram mantidos em 22 ha de pastagem de capim Colonião (Panicum maximum), de dezembro de 2004 a maio de 2005. A pastagem foi dividida em 8 piquetes e manejada em sistema rotacionado, com período de descanso variável e adubada com 45 kg de N por pastejo. Os animais pastejavam em lote único e eram separados diariamente em 3 lotes conforme o tratamento experimental para receberem os respectivos suplementos. Os animais do tratamento controle (CONT) eram conduzidos de volta ao pasto logo após a separação. Todos os animais tinham mistura mineral a disposição nos pastos. Os animais dos tratamentos ENER e PROT, recebiam diariamente 0,6% do PV (em matéria natural) dos respectivos suplementos. O suplemento ENER (6,4% de PB na MS) continha polpa cítrica e mistura mineral com monensina sódica. O suplemento PROT (19% de PB na MS), continha polpa cítrica, farelo de algodão e mistura mineral com monensina sódica. O ganho de peso diário (GPD) foi maior (P<0,05) para os animais suplementados, sendo de 0,741; 0,908 e 0,967 kg.animal-1 para os tratamentos CONT, ENER e PROT respectivamente. Não houve diferença de GPD quanto ao tipo de suplemento (P>0,05). Após 158 dias da fase de recria em pasto, os animais foram terminados em confinamento por 127 dias. Todos os animais receberam a mesma ração, com 82% de concentrado e 18% de silagem de milho. Os GPD foram de 1,38, 1,51 e 1,45 kg/cab para os tratamentos CONT, ENER e PROT respectivamente. O GPD do tratamento ENER foi maior (P<0,05) que do CONT. Animais não suplementados durante a recria em pasto, não apresentaram ganho compensatório na fase de confinamento em comparação com os suplementados. O ganho de peso adicional dos animais, obtido com a suplementação na fase de pasto, foi mantido e até ampliado na fase de terminação em confinamento.
The objectives of this trial were to compare the performance of growing cattle grazing tropical pastures without (CONT) or with energy (ENER) or protein (PROT) supplementation. Also, the effects of supplementation during the grazing period on the feedlot finishing period were evaluated. Seventy-five crossbred (Braunvieh x F1 Angus x Nelore) bull calves, averaging 8 months old and 200 kg BW, grazed 22 ha of Guinea grass (Panicum maximum) from December 2004 through may 2005. Pasture area contained 8 paddocks managed on a rotational grazing system, with a variable resting period. Pastures were fertilized with 45 kg of N/ha/grazing cycle. Cattle grazed the pastures as a single group. Every morning, animals were separated according to the 3 experimental treatments, to be fed the respective supplements. The CONT animals were moved back to the pastures while ENER and PROT animals were fed their respective supplements at 0.6% of BW (as fed). ENER supplement (6.4% CP) contained dried citrus pulp and a mineral mix with monensin. PROT supplement (19% CP) contained citrus pulp, cottonseed meal and mineral mix with monensin. Daily weight gain was higher (P<0.05) for ENER (0.908 kg.animal-1) and PROT (0.967kg.animal-1 ) compared to CONT animals (0.74 kg.animal-1). Type of supplement did not affect cattle daily gain (P>0.05). After the 158 days of the grazing period, animals were feedlot finished. All animals were fed the same ration (82% concentrate and 18% corn silage). Average daily gains were 1.38, 1.51 and 1.45 kg.animal-1 for CONT, ENER and PROT treatments respectively and were different (P<0.05). Animals fed energy supplement (ENER) during the grazing period, gained faster (P<0.05) during the feedlot period than no supplemented animals (CONT). Animals not supplemented during the grazing period (CONT) did not present compensatory gain compared to supplemented ones (ENER or PROT) during the feedlot period. Compared to the CONT performance, the extra weight of supplemented animals (ENER or PROT), gained during the grazing period, was maintained or increased during the finishing period.

Le dommage à l’ADN induit une mort cellulaire programmée de type nécrotique à travers les poly(ADP-ribose) polymerases (PARP) et Apoptosis-Inducing Factor (AIF). Suite à l’activation de PARP, AIF est relargué de la mitochondrie et transloque au noyau où il cause la condensation de la chromatine et la fragmentation de l’ADN. Nous avons identifié deux liens moléculaires entre PARP et AIF: les calpaines et Bax. Le dommage à l’ADN induit une mort indépendante de p53 mais impliquant PARP-1 et pas PARP-2. La nécrose due à la sortie d’AIF de la mitochondrie est dépendante des calpaines, mais pas des cathepsines ni des caspases. L’invalidation génétique de Bax, un membre pro-apoptotique de la famille Bcl-2, mais pas de Bak, inhibe à la fois la sortie d’AIF et la mort induite par le dommage à l’ADN. Finalement, nous avons établi l’ordre moléculaire d’activation de PARP-1, les calpaines, Bax puis AIF, et nous démontrons que la sous-expression d’AIF confère une résistance à la nécrose induite par le dommage à l’ADN. Ces études contribuent à l’élucidation des mécanismes régulant la nécrose dépendante d’AIF et impliquent que la nécrose est, comme l’apoptose, une forme de mort cellulaire programmée.

Les modifications post-traductionnelles des protéines par des polymères d’ADP-ribose (PAR) ou par phosphorylation permet l’assemblage des complexes de la réparation de l’ADN à la chromatine endommagée dont les fonctions sont essentielles pour assurer le maintien de la stabilité du génome. En réponse aux lésions de l’ADN, l’activité de synthèse de PAR des protéines PARP1 et PARP2 est fortement stimulée. Les PAR servent de signalisation pour le recrutement de multiples protéines, dont la protéine plateforme XRCC1.Les études menées au cours de cette thèse ont porté sur l’étude de la régulation des fonctions des protéines PARP1, PARP2 dans la réparation des cassures double brins (CDB) et l’étude des modifications de XRCC1 par phosphorylation en réponse à des dommages de l’ADN. En utilisant des substrats permettant de mesurer l’efficacité des différentes voies de réparation des CDB, nous avons démontré que PARP2, et non PARP1, est impliqué dans la régulation du choix des voies de la réparation des CDB. Plus spécifiquement, nous avons montré que PARP2 stimule l’initiation de la résection des extrémités des CDB dépendante de CtIP, indépendamment de son activité catalytique. Par des approches de vidéo-microscopie, nous avons pu déterminer que PARP2 limite l’accumulation de 53BP1 aux sites de dommages induits par micro-irradiation laser. Nous proposons que la protéine PARP2, en limitant le recrutement de la protéine 53BP1 aux sites de dommages, favorise la réparation des CDB dépendante de la résection des extrémités d’ADN, au détriment de la voie canonique de jonction des extrémités. Ces résultats sont les premiers démontrant un rôle de PARP2 dans le choix des voies de réparation des CDB.En parallèle, nous avons analysé comment la phosphorylation régule les fonctions de la protéine XRCC1. Par des approches in vitro et in vivo, nous avons pu déterminer que l’interdomaine 1 de XRCC1 est phosphorylé par la kinase CDK5. En réponse aux dommages induits par un agent alkylant, XRCC1 est activement déphosphorylé in vivo. De plus, nous avons observé que lorsque l’interdomaine 1 ne peut pas être phosphorylé in vitro, l’interaction de XRCC1 avec les PAR synthétisés par PARP1 et PARP2 augmente, et le recrutement de XRCC1 aux sites de dommages de l’ADN est accru. Ces résultats indiquent pour la première fois que la déphosphorylation de XRCC1 en réponse à un stress génotoxique participe activement à son recrutement aux sites de dommages.Dans leur ensemble, ces travaux ont contribué à améliorer nos connaissances fondamentales des réseaux de protéines impliquées dans la prise en charge des dommages de l’ADN. La compréhension de ces mécanismes est essentielle non seulement car ils participent au maintien de la stabilité du génome mais aussi du fait du développement exponentiel de nouvelles stratégies anti-tumorales qui visent à inhiber les voies de la réparation dans la but de cibler spécifiquement les cellules cancéreuses
Post-translational modifications of proteins by polymers of ADP-ribose (PAR) or by phosphorylation allow the assembly of DNA repair protein complexes at damaged chromatin and are crucial to ensure genome stability. In response to DNA insults, the synthesis of PAR by the PARP1 and PARP2 proteins is strongly induced. PAR act as a signaling platform for the recruitment of multiples proteins at the sites of DNA damages, including the scaffold protein XRCC1. Research conducted during this PhD have been focused on studying the regulation of PARP1 and PARP2 functions in double-strands break repair (DSBR), and in investigating the role of XRCC1 modifications by phosphorylation in response to DNA damage.Using DNA repair assay allowing us to assess the accuracy of the different DSBR pathways, we demonstrated that PARP2, and not PARP1, is involved in the regulation of DNA double-strands break repair pathway choice. More precisely, we showed that PARP2 stimulates CtIP dependent initiation of end-resection at DSB, independently of its catalytic activity. By live cell imaging, we were able to determine that PARP2 limit 53BP1 accumulation at DNA damage sites induced by laser-microirradiation. We propose that by limiting 53BP1 accumulation at DNA damage sites, PARP2 stimulate DSB repair pathway that depend on DNA end-resection, thus counteracting the canonical end-joining pathway. These results are the first demonstrating a role for PARP2 in DNA DBSR pathway choice.In addition, we analyzed how the functions of XRCC1 are regulated by phosphorylation. Using in vitro and in vivo approaches, we were able to demonstrate that the linker 1 region of XRCC1 is phosphorylated by the CDK5 kinase. XRCC1 is actively dephosphorylated in response to DNA damage induced by an alkylating agent in vivo. We also observed that when the linker 1 cannot be phosphorylated, the XRCC1 interaction between the PAR synthetized by PARP1 and PARP2 is stimulated, and XRCC1 recruitement at the sites of DNA damage is far more efficient. These evidences indicate for the first time that the dephosphorylation of XRCC1 actively participate in its recruitment at the site of DNA damage. Put together, this work contributed to strengthen our fundamental knowledge of the protein network involved in the DNA damage response. Knowledge of those mechanisms is crucial since they participate in maintaining genome stability, and because new antitumoral drugs targeting DNA repair pathways in the attempt to specifically killed tumor cells are exponentially released

Les aires protégées ont pour but la protection de la nature. Au cours des dernières décennies, certaines stratégies de conservation ont été élaborées afin d’aider l’atteinte de cet objectif, comme l’intégrité écologique, l’aménagement écosystémique, la connectivité, les zones tampons, ou la protection de la mégafaune charismatique. L’ours noir représente un bon modèle pour évaluer l’efficacité de ces stratégies de conservation puisque plusieurs facteurs sont susceptibles d’influencer la démographie de cette espèce, comme la chasse et le piégeage, la perte d’habitat et l’exploitation forestière. La question centrale qui justifie ma thèse est donc la suivante: « Est-ce qu’une aire protégée comme le parc national de la Mauricie peut maintenir l’intégrité écologique d’un grand mammifère comme l’ours noir? ». J’ai utilisé les données du suivi à long terme (1990-2005) de la population d’ours noirs du parc national de la Mauricie. Dans le premier chapitre, j’ai évalué l’influence de la chasse et du piégeage périphériques, de même que l’abattage illégal et le contrôle des animaux nuisibles sur la survie des ours. Les principaux résultats indiquent que les mortalités d’origine anthropique occupent une place importante dans cette population d’ours. J’ai donc entrepris, dans le deuxième chapitre, une analyse de viabilité de cette population. Les taux de croissance démographique estimés à l’aide des données de survie et de reproduction montrent que le nombre de femelles dans la population est relativement stable, mais que le nombre de mâles serait en déclin si la population était isolée. Dans le troisième chapitre, je souligne l’importance de tenir compte de l’erreur d’échantillonnage dans les analyses de viabilité. Finalement, dans le quatrième chapitre, j’ai déterminé l’étendue du grand écosystème pour la gestion de l’ours au parc national de la Mauricie, à partir de l’étude des déplacements des individus. Les différents résultats de cette thèse montrent l’importance du territoire situé à l’extérieur d’une aire protégée. À mon avis, il ne sera pas possible de protéger l’intégrité à long terme d’une population d’ours dans un parc de la taille de celui étudié tant que des objectifs de conservation ne seront pas intégrés dans un plan de gestion d’ensemble du grand écosystème.
Nature conservancy is the main goal of wilderness protected areas. Some conservation strategies, focused on concepts such as ecological integrity, ecosystem management, connectivity, buffer zones, or charismatic megafauna protection have been elaborated in the recent years to reach this goal. The American black bear is a good model to evaluate the efficiency of these conservation strategies since many factors can affect the demography of this species, such as hunting and trapping, habitat loss, and forest exploitation. The central question of my thesis is then the following: “Can a protected area such as La Mauricie National Park of Canada be able to maintain ecological integrity of a large mammal such as the American black bear?”. I used data from the long-term (1990-2005) monitoring of the black bear population in La Mauricie National Park of Canada. In the first chapter, I evaluated the influence of hunting and trapping in the periphery of the park, as well as the influence of poaching and nuisance kills on the survival of bears. Main results indicated that human-caused mortalities have a significant effect on this bear population. In the second chapter, I undertook a population viability analysis. The growth rates estimated with survival and reproduction data indicated that the number of females appears stable in the population, but that the number of males would decline if the population became isolated. In the third chapter, I emphasized the importance of accounting for sampling error in population viability analysis. Finally, in the fourth chapter, I determined the size of the greater ecosystem of La Mauricie National Park of Canada, based on the study of the movements of bears. The results of this thesis show the importance of the territory located outside of a protected area. In my opinion, we will not be able to achieve the long-term protection of the integrity of a bear population in a park of the size of La Mauricie National Park of Canada as long as conservation objectives, supported by the establishment of buffer zones around protected areas, are not integrated in a large-scale greater ecosystem management plan.

Le travail réalisé au cours de cette thèse aborde la thématique complexe de la place des espèces de la faune sauvage dans des habitats qui sont de plus en plus modifiés par les activités humaines. Nous prenons pour modèle d’étude la population de bisons des prairies (Bison bison bison) établie dans le parc national de Prince Albert (Canada) qui utilisent les terres agricoles exploitées bordant le parc. Dans un premier temps, nous démontrons que ce sont les gains énergétiques qui guident principalement la sélection d’habitat des bisons au détriment d’autres facteurs contribuant à la valeur adaptative, tels que le risque de mortalité lié à la chasse. Les parcelles agricoles constituent alors pour cette population un piège écologique, soit un habitat qui est préféré ou également préféré à d’autres habitats disponibles pourtant de meilleure qualité. L’utilisation des parcelles se diffuse parmi la population grâce aux décisions collectives prises au sein du groupe sur la base de l’expérience passée des membres du groupe. La diminution de la taille de la population est concomitante à la diffusion de ce comportement. Nos résultats nous renseignent sur les effets négatifs que peut avoir l’utilisation des milieux anthropisés et sur comment des mécanismes adaptatifs en milieu naturel, comme l’apprentissage social, peuvent se révéler délétères dans des milieux perturbés par l’homme. Nous étudions ensuite les stratégies comportementales mises en œuvre par les bisons pour échapper aux menaces constituées, à la fois, par leur prédateur naturel (le loup gris, Canis lupus) et par les activités humaines. Les bisons réagissent à la proximité du loup gris quand ils sont dans une prairie naturelle uniquement de nuit en écourtant leur temps de résidence. Alors qu’ils sélectionnent les parcelles agricoles principalement la nuit quand les activités humaines sont au plus bas. Ces stratégies divergentes nous renseignent sur les capacités des bisons à intégrer des informations nouvelles et à ajuster leur comportement en fonction de leur expérience passée. Le déclin de la population nous indique toutefois que ce type de stratégie, certainement efficace pour maximiser le temps passé sur les parcelles agricoles tout en minimisant le risque de perturbations, reste largement insuffisant pour prémunir les bisons contre la mortalité induite par la chasse. Pour mieux comprendre les déplacements des bisons et guider la gestion de la population, nous explorons les propriétés du réseau de sentiers créés et entretenus par les bisons pour se déplacer entre les prairies naturelles. Le réseau est très redondant avec de nombreux sentiers indépendants connectant la même paire de prairies. Le nombre de sentiers et de connexions diminue en même temps que le nombre d’individus. Ces résultats nous renseignent sur la capacité de résistance du réseau aux perturbations, mais également sur l’influence de la taille de la population sur la connectivité fonctionnelle. L’utilisation des milieux anthropisés par les espèces de la faune peut également avoir des impacts sur les écosystèmes. Nous décrivons une situation souvent négligée dans le domaine de la conservation: le conflit de conservation, situation au cours de laquelle un objectif de conservation entre en conflit avec un ou plusieurs objectifs d’un autre programme. Les bisons des prairies, espèce au statut vulnérable, transportent un grand nombre de graines d’espèces de plantes non indigènes acquises sur les parcelles agricoles et qui sont potentiellement envahissantes. Ils favorisent l’installation de ces espèces en créant des conditions favorables, comme au niveau des «wallows» (zone dépourvue de végétation en son centre créée par les bisons en se roulant au sol) ou des sentiers créés au cours leurs déplacements. Les bisons en tant que vecteurs d’espèces non indigènes représentent un danger pour la restauration des prairies à fétuques, objectif de conservation prioritaire pour Parcs Canada. En conclusion, ce travail apporte des éléments de compréhension sur le comportement des animaux face à des contraintes émergentes dans les milieux anthropisés et sur les conséquences pour les animaux et les écosystèmes. Il soulève la question de la pérennité de certaines populations animales dans ces milieux anthropisés en l’absence de mesures de gestion adaptées.

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CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico
FAPESPA - Fundação Amazônia de Amparo a Estudos e Pesquisas
O trabalho teve como objetivo desenvolver uma alimentação artificial protéica a base de extrato de soja (saburá artificial), e avaliar o seu efeito sobre a razão sexual, longevidade de operárias e desenvolvimento de colônias recém-divididas de Melipona fasciculata além de verificar a adaptação da espécie dentro de casas de vegetação. O saburá artificial aqui desenvolvido é constituído de 50g de extrato de soja, 20g de saburá fresco e 60ml de xarope de açúcar invertido (60%). Foi utilizado anilina para colorir o saburá artificial na tentativa de rastrear e verificar o consumo pelas operárias dentro das colônias. Foram utilizadas cinco colônias, das quais três receberam somente saburá e duas o saburá artificial. Não houve diferença significativa entre a produção de rainhas e operárias nos dois tratamentos e nos dois casos não houve produção de machos. As operárias que nasceram de caixas alimentadas com saburá artificial apresentaram maior longevidade e menor peso ao nascer. Estas caixas ainda iniciaram o processo de construção de células e postura mais cedo que as caixas alimentadas com saburá, contudo, suas rainhas apresentaram menor taxa de oviposição diária. O alimento a base de extrato de soja (saburá artificial) não afetou negativamente colônias recém divididas de M. fasciculata. Nos primeiros dias, as abelhas passaram a maior parte do seu tempo no topo da casa-de-vegetação tentando fugir, somente após o terceiro dia houve redução na mortalidade das operárias. Não houve diferença significativa, ao longo de cinco dias, entre a mortalidade de operárias em caixas transferidas, durante a noite e durante o dia, para dentro da casa-de-vegetação. A anilina se mostrou uma excelente ferramenta para controlar os alimentos manuseados e consumidos pela colônia.
The study aimed to develop an artificial food of the protein of soybean extract (artificial fermented pollen), and assess its effect on sex ratio, longevity of workers and development of newly divided colonies of Melipona fasciculata, and verify the adjustment the species in greenhouses. The fermented artificial pollen developed here consists of 50g of soybean extract, 20g of fermented pollen and 60mL inverted sugar syrup (60%). Aniline was used to color the fermented pollen artificial in an attempt to trace and verify the use by workers within the colonies. Five colonies were used, three of which received only the fermented pollen and two received artificial fermented pollen. There was no significant difference between the production of queens and workers in both treatments and in both cases there was no production of males. The workers who were born from boxes fed with artificial fermented pollen had greater longevity and lower birth weight. These boxes also have begun to build cells and earlier oviposition that the boxes fed with fermented pollen, however, their queens had lower daily rate of oviposition. The food based on soybean extract not adversely affect newly divided colonies of M. fasciculata. In the early days, the bees spend most of their time at the top of the greenhouse trying to escape, only after the third day there was a reduction in mortality of workers. There was no significant difference, over five days, between the mortality of workers moved in boxes during the night and during the day, inside the greenhouse. The aniline was an excellent tool to track the food handled and consumed by the colony.

Ets-1 est un facteur de transcription, membre de la famille Ets, possédant un domaine de liaison à l’ADN hautement conservé, qui permet de reconnaître un cœur consensus GGAA/T présent dans le promoteur de ses gènes cibles. Ce facteur régule des gènes impliqués dans divers processus physiologiques tels que le développement, l’hématopoïèse et l’angiogenèse ou pathologiques notamment dans la progression et l’invasion tumorale. Malgré les efforts engagés par la communauté scientifique ces dix dernières années, il y a peu de stratégies de ciblage thérapeutique d’Ets-1 qui peuvent être transposées dans un cadre clinique. Compte tenu du fait que ce facteur de transcription est un marqueur de mauvais pronostic pour de nombreux carcinomes, dont entre autres, ceux du sein, des poumons ou encore du colon, la mise en évidence d’une stratégie de ciblage de son activité pro-tumorale pourrait constituer une avancée majeure dans la lutte contre le cancer. Ets-1 n’agit pas seule au niveau de ses promoteurs cibles mais en coopération avec une variété de co-régulateurs transcriptionnels. De plus, ce facteur est ciblé par de nombreuses voies de transduction des signaux cellulaires. L’identification de nouveaux partenaires interagissant avec Ets-1 devrait donc nous permettre de mieux appréhender ses réseaux de régulation afin de mettre au point une stratégie de ciblage de son activité. Dans ce but, nous avons mis en œuvre un système de purification de partenaires basé sur la forte affinité entre la biotine et la streptavidine, appelé « streptavidin pull-down ». Nous avons ainsi identifié de nouveaux partenaires protéiques potentiels. Parmi ceux-ci, nous avons pu confirmer comme partenaires interagissant avec Ets-1, des protéines de réparation de l’ADN tels que le complexe DNA-PK et la PARP-1. La poly(ADP-ribose) polymérase -1 (PARP-1) est une enzyme aux rôles multiples qui catalyse la poly(ADP-ribosyl)ation ou PARylation. Si elle fut identifiée à l’origine comme une protéine de réparation de l’ADN, de nombreux travaux ont montré ces dernières années qu’elle est un co-régulateur majeur des mécanismes de transcription. Nous avons démontré qu’Ets-1 interagit directement avec la PARP-1 et est PARylée par celle-ci. L’utilisation d’inhibiteurs catalytiques de la PARP-1 sur des cellules de lignées cancéreuses, exprimant Ets-1, a pour conséquences l’accumulation massive de ce facteur et une augmentation de son activité transcriptionnelle. Ceci suggère une implication de la PARylation dans la stabilité d’Ets-1 en lien avec sa dégradation par le protéasome. Cependant, sous inhibition de la PARP-1, l’accumulation d’Ets-1 est toxique pour la cellule cancéreuse. En effet, nous avons observé une forte augmentation des dommages à l’ADN qui corrèle avec la mort des cellules tumorales. Nous supposons qu’une activité non régulée d’Ets-1 est néfaste pour le devenir cellulaire d’autant plus quand une enzyme de réparation de l’ADN comme la PARP-1 est inhibée.Ces résultats mettent en évidence un nouveau mécanisme de régulation d’Ets-1 au sein des cellules cancéreuses en liaison avec les protéines de réparation de l’ADN. De plus, l’utilisation d’inhibiteurs de la PARP-1 pourrait constituer une nouvelle stratégie afin de cibler spécifiquement les tumeurs exprimant Ets-1
Ets-1 is a transcription factor, member of the Ets family, having a well-conserved DNA binding domain which recognizes a core consensus sequence, GGAA/T, present in the promoter of target genes. This factor regulates genes involved in various physiological processes such as development, haematopoiesis, and angiogenesis and in pathological processes notably cancer progression and invasion. Despite the efforts of the scientific community during these past 10 years, few strategies for Ets-1 therapeutic targeting can be apply to clinical medicine. Considering the fact that this transcription factor is a poor prognostic marker for numerous carcinomas, such as breast, lung or colorectal cancers, the finding of a new strategy for targeting its activity in tumours could be a new advance in the fight against cancer. Ets-1 does not act alone on its target promoters but with a wide range of transcriptional co-regulators. Moreover, this factor is a target for many signal transduction pathways. Identifying novel proteins that interact with Ets-1 should permit a better understanding of its regulation networks to develop a strategy for targeting its activity. For this purpose, we used a purification system to identify interacting partners based on the strong affinity between biotin and streptavidin, called streptavidin pull-down. We thereby identified new potentials interaction partners. Among those, we could confirm as Ets-1 partners, DNA repair proteins, such as the DNA-PK complex and PARP-1. The poly(ADP-ribose) polymerase-1 (PARP-1) is an enzyme with various roles that catalyses poly(ADP-ribosyl)ation or PARylation. Originally, it was identified as a DNA repair protein. Nevertheless, these past few years, many studies have highlighted its role as a co-regulator in transcription processes. We demonstrated that Ets-1 directly interact with PARP-1 and is PARylated in return. In Ets-1-expressing cancer cells, the catalytic inhibition of PARP-1 caused massive accumulation of this factor and enhanced its transcriptional activity. These results suggest that PARylation is involved in Ets-1 protein stability linked to its proteasomal degradation. Nevertheless, under PARP-1 inhibition, accumulation of Ets-1 is toxic for cancer cells. Indeed, we observed a strong increase in DNA damage that leads to cancer cells death. We assume that an unregulated activity of Ets-1 is harmful to the cellular outcome even more when a DNA repair protein such as PARP-1 is inhibited.These results give new insight into Ets-1 regulation in cancer cells linked to DNA repair proteins. Furthermore, our findings suggest that PARP-1 inhibitors would be useful in a new therapeutic strategy that specifically targets Ets-1-expressing tumours

Cette thèse s'intéresse à trois types différents de cytosquelette impliqués dans la locomotion et la division cellulaires : les cytosquelettes d'actine, de MSP (Major Sperm Protein) et de ParM, dont l'organisation des éléments constitutifs en assemblées macromoléculaires génère des mouvements à grande échelle. L'approche développée repose sur l'utilisation de systèmes minimaux afin de comprendre les paramètres biochimiques et physiques mis enjeu. L'utilisation de ces systèmes expérimentaux dits biomimétiques, et la comparaison du résultat des expériences aux modèles théoriques, permet de discriminer les paramètres les plus pertinents pour décrire ces mouvements. La première partie s'intéresse au cytosquelette d'actine des cellules eucaryotes dans un système biomimétique fait de gouttes d'huile reconstituant la fluidité de la membrane cellulaire. Nous montrons que l'effet global de VASP, molécule présente au niveau du front de migration et des adhésions cellulaires, est d'affaiblir l'attachement entre le réseau d'actine et les activateurs de la polymérisation. Ceci souligne l'importance, dans le contrôle de la motilité par polymérisation de l'actine, de la dynamique de l'attachement du cytosquelette à la membrane cellulaire et du mouvement des activateurs de polymérisation. La seconde partie présente les premières étapes de caractérisation de la machinerie de polymérisation du cytosquelette non conventionnel de MSP, qui assure la migration des spermatozoïdes du ver nématode C. Elegans. La création de la simulation du cytosquelette de ParM, responsable chez Escherichia colide la ségrégation des plasmides R1 avant la division, est exposée dans la dernière partie
This thesis is a study of three different types of cytoskeleton involved in cell migration and division: actin, MSP (Major Sperm Protein) and ParM cytoskeletons. In all three cases, the organization of molecular components into macromolecular assemblies generates large scale movements. The approach used in this thesis involves simplified in vitro Systems, which allow for the controlled study of the biochemical and physical parameters involved in cytoskeleton-based processes. The results obtained with these biomimetic Systems, in conjunction with the comparison with theoretical predictions, enable us to determine which parameters are most relevant. In the first part of this manuscript, we study the eukaryotic actin cytoskeleton using an oil-water interface as a substrate to mimic the fluid properties of the cell membrane. We show that the effect of VASP, a molecule présent at the leading edge of the cell and at adhesion sites, is to decrease the anchoring of the actin cytoskeleton to the cell membrane. This study shows that the dynamics of attachment of the cytoskeleton and the movement of actin polymerization activators on the membrane are key elements controlling actin based movement. The second part presents preliminary results concerning the identification of molecules involved in the polymerization of the non conventional MSP cytoskeleton, which drives the migration of the C. Elegans sperm cells. The third part of this thesis deals with the creation of a simulation of the ParM cytoskeleton, required for R1 plasmid segregation in Escherichia coli before the division of the bacterium

Na intenção de avaliar o efeito da suplementação protéica, uso de subprodutos agroindustriais e o processamento de milho, foram conduzidos quatro experimentos. No experimento I, avaliou-se formas de processamento de milho (milho moído, M; e resíduo de pipoca doce, P) e fontes protéicas (farelo de soja, FS; uréia, U; ou farinha de peixe, FP), combinando-os para formar quatro tratamentos: MFS (M + FS); PFS (P + FS); PFP (P + FS + FP); PU (P + U). Comparado ao milho moído (MFS), o resíduo de pipoca doce (PFS) diminuiu os teores de gordura e proteína no leite. Em relação às fontes protéicas (PFS x PFP x PU), observou-se que a maior produção leiteira foi obtida no tratamento PFS. A farinha de peixe (PFP) levou ao menor teor de gordura no leite, mas aumentou o teor protéico no leite. No experimento II, avaliou-se os efeitos da inclusão de três teores (0,15 e 30% da MS) de farelo de algodão (FA) em substituição ao farelo de soja na dieta. O consumo de MS não foi afetado pelos tratamentos, contudo a inclusão de FA levou a efeitos lineares negativos sobre a produção de leite e teor e produção de proteína e efeitos lineares positivos sobre o teor e produção de gordura no leite. No experimento III, comparou-se os efeitos de uma dieta basal com 16% de PB contra duas outras com 17,5% de PB, sendo esses incrementos obtidos através da adição de uréia (U-17,5, resultando em aumento de proteína degradável no rúmen, PDR) ou farelo de soja e de algodão (FSFA-17,5, resultando em aumento de proteína metabolizável, PM). O aumento do teor de PDR (U-17,5) tendeu a aumentar o consumo de MS em relação à dieta basal. A produção de leite elevou-se com o aumento de PM (FSFA-17,5), não ocorrendo o mesmo com o aumento de PDR (U-17,5). O teor e a produção de proteína no leite também elevou-se com o fornecimento extra de PM. Concluiu-se que a elevação do teor de PM para valores acima das recomendações propostas pelo National Research Council - NRC (2001) para vacas produzindo ao redor de 29 kg/d melhorou as produções de leite e de proteína no leite. O experimento IV consistiu de dois ensaios. No primeiro, os tratamentos continham 0 (RUC-0), 10 (RUC-10) ou 20% (RUC-20) de resíduo úmido de cervejaria (RUC). Todas as dietas continham 1% de uréia. Um quarto tratamento isoprotéico, com 2% de uréia substituindo parcialmente o farelo de soja, sem RUC, foi utilizado. O consumo de MS não foi afetado. A produção de leite e o teor e produção de proteína no leite aumentaram com a inclusão de RUC e foram prejudicados com o fornecimento de 2% de uréia. No ensaio 2, foram comparados o RUC fresco com o RUC ensilado com milho moído. Ambos os tratamentos eram isoprotéicos e continham proporções semelhantes de milho moído e RUC. O consumo de MS, produção de leite e os teores e produções de gordura e proteína no leite não foram afetados pelos tratamentos.
Four studies were conducted to compare protein sources and content, corn processing method and a fibrous by-product for lactating dairy cows. Study 1: corn processing methods (fine ground, M; and popped, P) and protein sources (soybean meal, FS; urea, U; or fishmeal, FP) were compared in 4 treatments: MFS (M + FS); PFS (P + FS); PFP (P + FS + FP); PU (P + U). Compared to fine ground corn (MFS), popped corn (PFS) decreased milk fat and protein content. The 3 protein sources comparisons (PFS x PFP x PU) showed that milk yield was higher for PFS. Feeding fishmeal (PFP) decreased milk fat content, but increased milk protein content. Study 2: the cottonseed meal content in the diet (0, 15 and 30% of DM) in replacement to soybean meal were compared. Dry matter intake was not affected by treatments, but cottonseed meal supplementation had a negative linear effect on milk yield and on milk protein content and yield. Increasing cottonseed meal had a positive linear effect on milk fat content and yield. Study3: basal diet (16% CP) was compared to two diets with 17.5% CP, where crude protein content of diets were increased by feeding extra urea (U-17.5, resulting in increased rumen degradable protein content, RDP) or extra soybean and cottonseed meal (FSFA-17.5, resulting in increased metabolizable protein content, MP). Extra RDP (U-17.5) tended to increase DMI compared to basal diet. Milk yield were increased by extra MP (FSFA-17.5) but not by extra RDP (U-17.5). Milk protein content and yield were also increased by feeding extra MP. In conclusion, increasing diet MP content above National Research Council - NRC (2001) recommendations for cows producing around 29 kg/d improved milk and milk protein yields. Study 4: two trials were conducted. In trial 1, treatments were 0 (RUC-0), 10 (RUC-10) or 20% (RUC-20) wet brewers grains inclusion in diet dry matter. All 3 diets contained 1% urea on a DM basis. A fourth isonitrogenous diet, with additional urea (2% of diet DM) in partial replacement of soybean meal, without brewers grains, was compared to. Dry matter intake were not different. Milk yields and milk protein contents and yields were increased by feeding wet brewers grains and decreased by feeding 2% urea. In trial 2, weekly received fresh wet brewers grains versus wet brewers grains ensiled with ground corn were compared. Both treatments were isonitrogenous diets and contained the same proportions of ground corn and wet brewers grains on a dry matter basis. Dry matter intake, milk yield, milk fat content and yield and protein content and yield were not different.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias.
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O presente estudo teve como objetivo determinar a exigência protéica e correspondente relação energia/proteína (E/P) em dietas para alevinos de piracanjuba, Brycon orbignyanus. Para tanto, seis dietas semi-purificadas isocalóricas, com 3.000 kcal de energia metabolizável (EM)/kg, foram formuladas para conter concentrações de proteína bruta (PB) de 24, 26, 29, 32, 36 e 42%. Para essas concentrações protéicas, as relações E/P foram de 12,3; 11,6; 10,4; 9,2; 8,5 e 7,1 kcal EM/g PB, respectivamente. As fontes de proteína, lipídios e carboidratos digestíveis das dietas foram, respectivamente, caseína/gelatina, óleo de fígado de bacalhau/óleo de soja e dextrina. Após um período de condicionamento de 5 dias, as dietas experimentais foram fornecidas, até a saciedade, em duas refeições diárias. Utilizou-se 162 alevinos de piracanjuba (27 peixes/dieta) com 8,38 ± 0,09 g/peixe de peso inicial, igualmente distribuídos em 18 tanques de fibra-de-vidro de 100 L, conectados a um sistema de recirculação de água. A temperatura média da água foi de 26,3°C, com extremos de 23,7 e 30,2°C. Após 90 dias, a concentração mínima de proteína da dieta que proporcionou ganho de peso máximo aos peixes foi 29% PB com relação E/P igual a 10,4 kcal EM/g PB. As dietas com concentrações de PB acima de 29%, i.e., 32, 36 e 42%, também não mostraram superior conversão alimentar, taxa de eficiência protéica, valor produtivo da proteína e retenção de energia bruta em relação à primeira (P>0,05). A qualidade corporal dos peixes, quanto à deposição de proteína e gordura, não sofreu influência da concentração de PB da dieta.

Chez Arabidopsis, la protéine RISP est détournée par le virus CaMV pour assurer, ensemble avec la protéine virale TAV, la traduction de son ARN polycistronique. RISP a été identifiée comme une cible de la voie de signalisation de TOR et il a été montré que sa phosphorylation est requise pour promouvoir la réinitiation de la traduction activée par TAV. Les résultats que j’ai obtenus suggèrent que RISP, lorsqu’elle n’est pas phosphorylée, intervient ensemble avec eIF3, au niveau du complexe de pré-initiation 43S pour recruter le complexe ternaire grâce à l’interaction entre RISP et la sous-unité b du facteur eIF2. Il s’est avéré que RISP a la capacité, lorsqu’elle est phosphorylée, d’interagir non seulement avec la protéine ribosomique eL24 mais également avec eS6. Nos résultats indiquent que la liaison entre les sous-unités ribosomiques 60S et 40S sous l’effet de RISP, est régulée par la voie de TOR et qu’elle joue un rôle important dans le contrôle de la réinitiation de la traduction
Many factors are required to recruit the tRNAi and a 60S ribosomal subunit to the 40S ribosomal subunit preinitiation complex. This recruitment is normally strictly limited during reinitiation of translation if factors recruited during the primary translation event are shed from 40S. However, factor retention can occur during long ORF translation if the CaMV viral factor TAV is present. RISP is a downstream target of TOR and found either within the 43S preinitiation complex, if bound to eIF3, and/or attached to 60S, if phosphorylated by TOR. We show here that RISP interacts with subunit b of eIF2 before phosphorylation. Critically, TOR activation up-regulates phosphorylation of both RISP and eS6 as well as the binding of both factors. Importantly, eS6-deficient plants are less active in TAV-mediated reinitiation and are thus less susceptible to CaMV infection. It is attractive to propose that eS6 phosphorylation contributes to retention and re-use of 60S during 40S scanning

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Although cassava leaves are rich in proteins, vitamins and minerals and are easily found at low prices, their direct consumption is limited due to the presence of anti-nutritious and/or toxic substances. Aiming to create alternatives to the use of these leaves, the present paper aimed to determine the coefficients of apparent digestibility (AD) from the dry matter (DM), crude protein (CP) and crude energy (CE) of cassava leaf protein concentrate (CLPC) for Nile Tilapia, and to evaluate its inclusion in Nile Tilapia food. The CLPC has been extracted through isoeletric precipitation, method described by CEREDA & VILPOUX (2003). In the CLPC, it has been determined the perceptual of crude protein (CP), ether extract (EE), ashes, dry matter (DM) and crude energy (CE), which have shown 48.42%, 13.57%, 3.48%, 93.01% and 5527 Kcal/kg., respectively. 24 tilapias with an average weight of 86.92 ± 36.70 g have been used. The animals have been submitted to fecal collection methodology, through sedimentation, in tanks with funneled shapes. The DM, CP, and CE of CLPC have presented AD of 33.06%, 66.57% and 30.06%, respectively, thus, showing digestible protein and energy values of 32.23% and 1661.13 kcal of DE/kg. In order to evaluate CLPC inclusion in Nile Tilapia food, 300 larvae at the age of 7 days old have been used, which were distributed in 20 aquariums with capacity for 30 liters of useful volume, in a fully randomized outline with five treatments and four repetitions, pointing out that the experimental unit is consisted of an aquarium with 15 tilapia larvae. Five isoproteic and isoenergetic diets have been elaborated containing 38.6% of digestible protein and 3300 kcal of DE/kg of diets with level of 0, 5, 10, 15 and 20% of CLPC inclusion. At the end of the experimental time, differences have not been noticed (W>0.05) on the final average weight, weight gain, final length and survival of larvae which were fed with diets containing CLPC. It is concluded that CLPC is a protean food and it may be used in diets for tilapia in initial phase in up to 20% of inclusion without causing performance and survival harms.
Folhas de mandioca são ricas em proteínas, vitaminas e minerais e, apesar de serem facilmente encontradas a baixo custo, seu consumo direto fica limitado devido à presença de substâncias antinutritivas e/ou tóxicas. Na busca de criar alternativas para o uso dessas folhas, o presente trabalho objetivou determinar os coeficientes de digestibilidade aparente (CD) da matéria seca (MS), proteína bruta (PB), e energia bruta (EB) do concentrado protéico de folhas de mandioca (CPFM) para a tilápia do Nilo, e avaliar sua inclusão na alimentação da tilápia. O concentrado foi extraído das folhas de mandioca por precipitação isoelétrica, método descrito por CEREDA & VILPOUX (2003). Foram determinados no concentrado protéico de folhas de mandioca (CPFM) o percentual de proteína bruta (PB), extrato etéreo (EE), cinzas, matéria seca (MS) e energia bruta (EB), que apresentaram, 48,42%, 13,57%, 3,48%, 93,01% e 5527 Kcal/Kg, respectivamente. Foram utilizadas 24 tilápias com peso médio de 86,92 ± 36,70g. Os animais foram submetidos à metodologia de coleta de fezes, por sedimentação, em tanques afunilados. A MS, PB e EB do CPFM apresentaram CD de 33,06%, 66,57% e 30,06%, respectivamente. Apresentando valores de proteína e energia digestíveis de 32,23% e 1661,13 Kcal de ED/Kg. Para avaliar a inclusão do CPFM na alimentação da tilápia, foram utilizadas 300 larvas com idade de sete dias, distribuídas em 20 aquários com capacidade para 30 L de volume útil, em um delineamento inteiramente casualizado, com cinco tratamentos e quatro repetições, sendo a unidade experimental constituída por um aquário com 15 larvas de tilápia. Foram elaboradas cinco rações isoprotéicas e isoenergéticas com 38,6% de proteína digestível e 3300 Kcal de ED/Kg de ração com níveis de 0, 5, 10, 15 e 20% de inclusão do CPFM. Ao final do período experimental, não foram observadas diferenças (P>0,05) nas médias de peso final, ganho de peso, comprimento final e sobrevivência das larvas alimentadas com rações, incluindo CPFM. Conclui-se que o CPFM é um alimento protéico e que pode ser utilizado em rações para tilápias em fase inicial em até 20% de inclusão sem causar prejuízos no desempenho e sobrevivência.

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Fundação de Amparo a Pesquisa do Estado de Minas Gerais
O melhoramento vegetal é um processo longo e caro, de modo que quaisquer procedimentos metodológicos que auxiliem na tomada de decisão durante a seleção das linhagens mais promissoras são extremamente importantes. Este trabalho teve como objetivo avaliar as distribuições das freqüências das populações de retrocruzamento de soja, bem como selecionar as prováveis linhagens promissoras para alto teor de proteína nas sementes. Utilizando-se de um modelo linear misto há possibilidade de se fazer a predição de efeitos aleatórios, na presença de efeitos fixos, por meio dos BLUP's (best linear unbiased prediction) que são de grande valia no melhoramento. Os experimentos foram conduzidos em três municípios do Estado de Minas Gerais, Capinópolis, Oratórios e Viçosa entre os anos de 1999 e 2001. Linhagens de soja foram obtidas pelo método de retrocruzamentos utilizando como progenitores recorrentes as variedades OCEPAR 13, COODETEC 201, COODETEC 202, COODETEC 205, como progenitores doadores para alto teor de proteína nas sementes, as linhagens BARC 8 e BR 80 14887. O método utilizado para determinação do teor de proteína das sementes foi o Kjeldahl. Concluiu-se que as estimativas de componentes de variância, assim como as estimativas de herdabilidade, baseada na máxima verossimilhança restrita, apresentaram-se com magnitudes baixas; selecionando-se somente entre as linhagens que apresentaram maiores valores preditos pelo BLUP, houve aumento do parentesco na população e; a utilização do BLUP para auxiliar seleção de linhagens promissoras mostrou- se satisfatória . As distribuições das observações e, em conseqüência, a natureza da variação genética, foi avaliada por meio da análise da curtose e da assimetria das distribuições nas populações RC 2 F 2 . Observou-se que a média do teor protéico foi deslocada positivamente, indicando potencial para o desenvolvimento de linhagens promissoras, com maior teor de proteína nas sementes; a variabilidade do teor de proteína dentro das populações depende somente da presença de poucos alelos nas diferentes populações; no segundo retrocruzamento, as populações tendem a apresentar distribuição mesocúrtica ou leptocúrtica e, por meio da estimativa de assimetria, foi possível constatar efeito de dominância em algumas das populações avaliadas.
The plant breeding is a long and expensive process, so that any methodological procedures that aid taking decision during the selection of the most promising lines is extremely important. This work had as objective to evaluate the frequencies distributions of soybean backcross populations, as well as to select the probable promising lines for high protein content in the seeds. By using a mixed linear model, there is the possibility to predict the aleatory effects, in the presence of fixed effects, through BLUP's (best lineal unbiased prediction) that are of great value in breeding programs. The experiments were accomplished in three locations of the Minas Gerais State: Capinópolis, Oratórios and Viçosa from 1999 to 2001. The soybean lines were obtained by the backcross method, using as recurrent ancestors the varieties OCEPAR 13, COODETEC 201, COODETEC 202, and COODETEC 205, as donors ancestors for high protein content in the seeds, the lines BARC 8 and BR 80 14887. The method used for determination of the seed protein contents was the Kjeldahl. It was concluded xthat the estimates of variance components, as well as the heritabilities estimates, based on the maxim restrict likelihood, presented low magnitude; selecting only among the lines that presented larger predicted values by BLUP, it was increased the relationship in the population and; the use of BLUP to aid selection of promising lines was shown satisfactory. The distributions of the observations and, in consequence, the nature of the genetic variation, were evaluated through the analysis of the kurtosis and of the asymmetry of the distributions in the RC 2 F 2 populations. It was observed that the protein content average moved positively, indicating potential for the development of promising lines, with higher seed protein content; the variability of the protein content inside the populations depends only upon the presence of few alleles in the different populations; in the second backcross, the populations tend to present mesokurtic or leptokurtic distribution and; through the asymmetry estimate, it was possible to verify dominance effect in some of the appraised populations.

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Tuberculosis is a disease that affects thousands of people in the World. Although Brazil Health Program had achieved the goal of reducing to 50% the rate of death induced by Tuberculosis, this disease continues to be the second cause of death by infectious disease. One of the main problems to control the disease is the low efficacy of BCG vaccine in protecting young adults. The development of new vaccines that induces long lasting immune response or that stimulate the immunity induced by BCG may improve the control of TB spreading. This study evaluated the use of microstructured liposomes containing HspX with or without MPL or CpG DNA adjuvants as vaccine for tuberculosis. The HspX specific humoral and cellular immune responses were compared between the different vaccine formulations. All vaccines containing liposome microparticules and HspX were immunogenic and antigenic. Vaccines formulated with CpG DNA and HspX induced the strongest humoral and cellular immune responses, mainly by generating IFN- and TNF-by both CD4 and CD8 T cells. HspX and MPL mainly induced CD8 T cell activation and humoral specific responses. After protection efficacy evaluation against Mycobacterium tuberculosis challenge, the vaccine formulation that reduced both lung inflammatory lesions and the bacterial load was the microstructured liposome containing HspX and CPG DNA. These results show for the first time the use of microstructured liposome as adjuvant and delivery system in HspX vaccine formulation for tuberculosis.
A tuberculose é uma doença que acomete milhões de indivíduos no mundo. No Brasil esta doença é a segunda causa de morte por doença infectocontagiosa, embora o País tenha reduzido a metade o numero de mortes por tuberculose. Um dos principais problemas para o controle desta doença é a baixa eficácia da vacina BCG em proteger adultos. O desenvolvimento de novas vacinas que induzam uma resposta imune eficaz e duradoura, ou que estimulem a BCG a aumentar a imunidade já existente é de grande valia. O objetivo desta dissertação foi avaliar e comparar se lipossomas microestruturados contendo HspX em diferentes formulações vacinais, associados ou não a adjuvantes conhecidos: CpG DNA ou MPL poderiam influenciar na resposta imune humoral e celular de camundongos contra o Mycobacterium tuberculosis. Nossos resultados mostram que lipossomas microestruturados contendo HspX foram imunogênicos e antigênicos. As formulações vacinais contendo o adjuvante CpG DNA foram as principais indutoras de resposta humoral e celular específica, principalmente com produção de IFN- e TNF- tanto por linfócitos T CD4+ quanto CD8+. Formulações vacinais contendo HspX e MPL induziram resposta imune humoral e ativaram principalmente linfócitos T CD8. A formulação vacinal que melhor reduziu as lesões pulmonares provocadas pelo Mycobacterium tuberculosis, assim como a carga bacilar foi a composta por lipossomas microestruturados contendo HspX e CpG DNA. Estes resultados demonstram pela primeira vez o uso de lipossomas microestruturados em formulações de vacinas com o antígeno HspX para a tuberculose.

Made available in DSpace on 2017-05-12T14:48:23Z (GMT). No. of bitstreams: 1 Priscila Ferri.pdf: 2540438 bytes, checksum: 0e96b497fd0903bba8d9e7c5a5161c00 (MD5) Previous issue date: 2006-07-07
The objective of this work was to evaluate the efficiencies of protein extration for attainment of proteins leaf concentrate cassava (Manihot esculenta Crantz), using seven methods of extration cited by authors CEREDA & VILPOUX (2003), CHAVES (1987), TUPINAMBÁ & VIEIRA (1979) and FASUYI & ALETOR (2005). The experiment was carried through in the dependences of the State University of the West of the Paraná campus Cascavel, in the laboratory of Sanitation of the course of Agricultural Engineering. The cassava leves had been harvested in third part of the plant with age of 12 months in a property of the city of Cascavel. For the protein extration dehydrated leves had been used, with rude protein texts in dry base of 36.55 % and humidity of 11.27%. Methods 1 and 2 cited by CEREDA & VILPOUX (2003), method 5 cited by CHAVES (1987), method 6 cited by TUPINAMBÁ & VIEIRA (1979) and method 7 of FASUYI & ALETOR (2005) had been the ones that had gotten greaters proteins texts in the concentrates, above of 50%. The biggest incomes of extration had been gotten by methods 2, 5, 6 and 7 with incomes of protein extration above of 35%. Methods 1, 2, 4 and 5 had been tested using two consecutive extrations similar to improve the losses of mass and to increase the extration income, however only occurred the reduction of the losses of mass in the process, not being then necessary the use of two phases of extration. Methods 1, 2, 4 and 5 had been compared, already applied with leves dehydrated, using cool leves. The cassava leves (Manihot esculenta Crantz) cool had been harvested in third part of the plant with age of 9 months with rude protein texts in dry base of 27.70% with 72% of humidity. It did not have difference in the extration incomes, being more advantageous to use dehydrated leves due to possess minors toxic factors and greater durability. Methods 2 and 5 had revealed alternative for attainment of cassava proteins leaf concentrates, due to extration easiness, and not to need equipment and materials that can increase the cost of protein extration, thus making possible one better exploitation of cassava leves.
O objetivo deste trabalho foi avaliar as eficiências de extração de proteínas para obtenção de concentrados protéicos de folhas de mandioca (Manihot esculenta Crantz), utilizando sete métodos de extração descritos por: CEREDA e VILPOUX (2003), CHAVES (1987), TUPINAMBÁ e VIEIRA (1979) e FASUYI e ALETOR (2005). O experimento foi realizado nas dependências da Universidade Estadual do Oeste do Paraná, campus de Cascavel, no Laboratório de Saneamento do curso de Engenharia Agrícola. As folhas de mandioca foram colhidas no terço superior da planta com idade de 12 meses em uma propriedade da cidade de Cascavel. Para a extração de proteínas utilizaram-se folhas desidratadas, com teores de proteína bruta em base seca de 36,55 % e umidade de 11,27%. Os métodos 1 e 2 descritos por CEREDA e VILPOUX (2003), o Método 5 descrito por CHAVES (1987), o Método 6 e descrito por TUPINAMBÁ e VIEIRA (1979) e o Método 7 descritos por FASUYI e ALETOR (2005) foram os que obtiveram maiores teores protéicos nos concentrados, acima de 50%. Os maiores rendimentos de extração foram obtidos pelos métodos 2 e 4, com rendimentos de extração de proteína acima de 35%. Foram testados os métodos 1, 2, 4 e 5, utilizando-se duas extrações consecutivas, a fim de melhorar as perdas de massa e aumentar o rendimento de extração, no entanto somente ocorreu a minimização das perdas de massa no processo, não sendo necessária a utilização de duas fases de extração. Foram comparados os métodos 1, 2, 4 e 5, já aplicados com folhas desidratadas, utilizando-se folhas frescas. As folhas de mandioca (Manihot esculenta Crantz) frescas foram colhidas no terço superior da planta com idade de 9 meses com teores de proteína bruta em base seca de 27,70% com 72% de umidade. Não houve diferença nos rendimentos de extração, sendo mais vantajoso utilizar folhas desidratadas, devido ao menor fator tóxico e maior durabilidade. Os métodos 2 e 5 mostraram-se alternativos para obtenção de concentrados protéicos de folhas de mandioca, devido à facilidade de extração e de não necessitarem de equipamentos e materiais que possam aumentar o custo de extração de proteínas, possibilitando assim um melhor reaproveitamento das folhas de mandioca.

Esta tese foi realizada objetivando comparar 4 programas alimentares, em dietas à base de milho e farelo de soja, formuladas com ou sem restrição de proteína bruta (PB), suplementadas ou não com L-Val e L-Ile, e usando diferentes níveis de lisina digestível (dig.). Os programas alimentares (PRG) foram: PRG 1, PB limitada a um mínimo (22,4, 21,1, 19,8, 18,4% para as fases pré-inicial, inicial, crescimento e final, respectivamente, com relações de AA:Lis definidos apenas para Met+Cis (0,72) e Tre (0,65); PRG 2, a PB não foi restrita e as relações estendidas para Val (0,77) e Ile (0,67); PRG 3, mesmas restrições do PRG 2 e suplementadas com L-Val; PRG 4, mesmas restrições do PRG 3, e suplementadas com L-Ile. Dois experimentos foram realizados, um com 1.800 e outro com 4.800 pintos machos Cobb x Cobb 500 de 1 dia de idade. No primeiro experimento, as rações foram formuladas usando os 4 PRG e níveis de Lis dig. de 1,324%, 1,217%, 1,095% e 1,006% ou 5% maiores, para cada fase, totalizando 8 tratamentos e 9 repetições cada. Para o segundo experimento, os 4 PRG foram utilizados até 1-21 d, totalizando 4 tratamentos com 48 repetições. E de 22 a 42 dias, cada PRG foi subdividido nos 4 PRG, passando cada PRG da fase anterior a receber todos os 4 PRG para as fases de crescimento e final, totalizando 16 tratamentos com 12 repetições cada. Não houve interação entre os tratamentos em ambos os experimentos, com exceção para o ganho de peso (GP) e conversão alimentar (CA) de 36 a 43d, no primeiro experimento, onde aves alimentadas com PRG 2 demonstraram melhorias no GP e CA quando alimentadas com um aumento de 5% no nível de Lis. Também, o GP e a CA acumulados aos 35 e 43d foram melhores quando as aves foram alimentadas com o PRG 2, sem diferença estatística para o PRG 3 e 4. Um aumento em 5% no nível de Lis dig. resultou na melhoria da CA acumulativa aos 43d. A gordura abdominal, como porcentagem da carcaça eviscerada aos 43d, foi maior para as aves alimentadas com a PRG 1. No segundo experimento, as aves alimentadas com PRG 2 tiveram melhor CA de 22 a 42d e de 1 a 42d, mas sem diferença estatística para os PRG 3 e 4. As aves alimentadas com PGR 1 de 1 a 42d apresentou o menor GP e o pior CA sem diferenças entre os PGR 2 e 3, e os PGR 3 e 4, respectivamente. Os dados do presente estudo demonstram que a suplementação de dietas para frangos de corte com fontes sintéticas dos cinco primeiros AA limitantes permitiram resultados de desempenho semelhantes a uma dieta com PB restrita a um valor mínimo.
This thesis was carried out to compare four corn-soy feeding programs formulated with or without crude protein (CP) restrictions supplemented with or without L-Val and L-Ile, and using different digestible (dig.) Lys levels. Feeding programs (PRG) were: PRG 1, CP was restricted to a minimum (22.4, 21.1, 19.8, 18.4% for pre starter, starter, grower and finisher phases, respectively) with AA to Lys ratios set only for TSAA (0.72) and Thr (0.65); PRG 2, CP was not restricted while minimum ratios were also extended to Val (0.77) and Ile (0.67); PRG 3, restrictions were as in PRG 2 but with L-Val added; PRG 4, restrictions were as in PRG 3, but with L-Ile added. For this two experiments were conducted, one using 1,800 and the other using 4,800 oneday- old Cobb 500 male broiler chicks. For the first experiment, feeds had formulated using the 4 PRG and dig. Lys as 1.324%, 1.217%, 1.095% and 1.006% or 5% higher, for each phase, totaling 8 treatments with 9 replicates each. For the second experiment, the 4 PRG were used from 1 to 21 d, totaling 4 treatments with 48 replicates. From 22 to 42d, each PRG was subdivided in 4 PRG having the same rational as it was done to 21d, where each of the 4 PRG began receiving all 4 PRG for grower and finisher phases, totaling 16 treatments with 12 replicates each. No interaction was found between treatments in both experiments, with one exception for body weight gain (BWG) and feed conversion ratio (FCR) from 36 to 43d, in the first experiment, with birds fed PRG 2 demonstrating improvements in BWG and FCR when fed the 5% increasing in dig. Lys. And also, cumulative BWG and FCR at 35 and 43d and in each individual feeding phases showed broilers from PRG 2 having the best BWG and FCR; however, mean separations using Tukey showed no difference from birds fed PRG 3 and 4. Feeding a dietary program with 5% increase in dig. Lys resulted in improvement in cumulative FCR at 43d. Abdominal fat, as a percentage of the eviscerated carcass at 43d, was highest for birds fed the PRG 1. For the second experiment, birds fed with PRG 2 led to the best FCR from 22 to 42d and from 1 to 42d, but without statistical difference from PRG 3 and 4. Birds fed PGR 1 from 1 to 42d showed the lowest BWG and the highest FCR without statistical differences from PGR 2 and 3 and PGR 3 and 4, respectively. Data from the current study demonstrate that the supplementation of broiler diets with crystalline sources of the first five limiting AA allowed performance results similar to those produced when CP is restricted to a minimum.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2013-11-06T19:57:37Z No. of bitstreams: 1 Leonardo Vicitini Arruda Desenvolvido de plasmídio para expressão proteíca....pdf: 2125843 bytes, checksum: cf2e5fe050fd36b4ebba0206f40d6e3a (MD5)
Made available in DSpace on 2013-11-06T19:57:37Z (GMT). No. of bitstreams: 1 Leonardo Vicitini Arruda Desenvolvido de plasmídio para expressão proteíca....pdf: 2125843 bytes, checksum: cf2e5fe050fd36b4ebba0206f40d6e3a (MD5) Previous issue date: 2013
Universidade Federal da Bahia. Faculdade de Medicina da Bahia. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil.
Protozoários do gênero Leishmania provocam uma ampla gama de doenças, e passam por um processo de diferenciação entre o inseto vetor e a forma intracelular no hospedeiro mamífero. Apesar das diferenças entre as formas do ciclo de vida, não estava descrita ferramenta para expressar proteínas de modo estágio específico. Neste trabalho apresentamos um plasmídio para Leishmania que expressa proteína recombinante de forma estágio específica. Testamos uma possível construção que usava as região 3’ UTR do gene amastina para controlar a expressão de uma proteína fluorescente e a expressar exclusivamente na fase amastigota. Também utilizamos a região 3’ UTR de uma tubulina para obter uma fluorescência homogênea em todos os estágios do ciclo de vida do parasita. Assim como esperado, obtivemos uma fluorescência exclusiva para a fase amastigota quando utilizamos a região 3’ UTR da amastina, e uma fluorescência constitutiva quando a expressão foi regulada pela região 3’ UTR da tubulina. O plasmídio descrito neste trabalho é versátil, pois a droga de seleção ou a proteína a ser expressa podem ser substituídas com grande facilidade. Adicionalmente, como o plasmídio pFL expressou um gene repórter exclusivamente no estágio amastigota ou constitutivamente, acreditamos que este plasmídio pode também ser utilizado para expressar proteínas de forma restrita aos estágios promastigota metacíclico e procíclico. Alguns possíveis usos desta nova ferramenta também são discutidos nesta tese
The parasitic protozoan Leishmania causes a wide spectrum of diseases and passes through differentiation between the sand fly and the intracellular form. Despite distinction between life-cycle forms of the parasite, there is no described tool to express a protein in a specific stage. Here, we present a plasmid for Leishmania that can express recombinant proteins in a specific life-cycle stage. We tested one possible construction that used the 3’ UTR region of the amastin gene to control the expression of a fluorescent protein exclusively in the amastigote stage. We also used the 3’ UTR of a tubulin to obtain a homogeneous fluorescence in all stages of the parasite life cycle. As expected, was observed a fluorescence exclusive to the amastigote phase with the 3’ UTR amastin construction, and a constitutive fluorescence when the expression were regulated by the 3’ UTR of a tubulin. The plasmid described here is versatile since the drug that will be used for selection or the protein that will be expressed can be easily changed. Moreover, plasmid pFL have expressed a reporter gene exclusively in the amastigote stage or constitutively, we believe that this plasmid can also be used to express proteins in metacyclic and procyclic promastigote stages only. Some possible uses of this new tool are also discussed.

Submitted by (edna.saturno@ufrpe.br) on 2017-02-06T16:53:46Z No. of bitstreams: 1 Albino Luciani Goncalves Leal.pdf: 575820 bytes, checksum: 4cc0fd6d3bf7fe9198563d35eae04f53 (MD5)
Made available in DSpace on 2017-02-06T16:53:46Z (GMT). No. of bitstreams: 1 Albino Luciani Goncalves Leal.pdf: 575820 bytes, checksum: 4cc0fd6d3bf7fe9198563d35eae04f53 (MD5) Previous issue date: 2007-02-15
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Aquaculture requires high-quality feeds with high protein content. So, the determination of less-expensive sources of protein which provides good growth is advantageous. Shrimp wastes have been identified as an animal protein source with great potential. Shrimp protein hydrolysate (SPH), a derived product obtained from shrimp wastes, was considered an excellent alimentary source and may serve as an useful source of protein and flavorants in food formulations. This work aimed to evaluate the nutritional quality of SPH through growth performance of juvenile Nile tilapia and its protein utilization. SPH was included in isonitrogenous diets at levels of 0, 5, 10 and 20% of fish meal protein replacement (SPH0, SPH5, SPH10 and SPH20) and offered to juvenile Nile tilapia (1.7±0.4 g) stocked in 40-L glass aquaria in a 45-day feeding trial. The inclusion of SPH did not produce statistical differences (P≥0.05) on final weight (27.18, 29.46, 26.02 and 25.19 g), survival (100%), relative weight gain (1,571, 1,624, 1,388 and 1,301%), average daily gain, ADG (0.57, 0.62, 0.54 and 0.52 g day-1), specific growth rate, SGR (7.15, 7.38, 6.85 and 6.73 % day-feed conversion ratio, FCR (1.15, 1.09, 1.13 and 1.17) and protein efficiency ratio, PER (2.26, 2.33, 2.20 and 2.14), respectively. The inclusion of SPH in diets for Nile tilapia statistically affected (P<0.05) the final fish body composition. Protein and ash contents decreased and fat content increased with SPH inclusion levels. This study clearly demonstrates that SPH could be included in diets for Nile tilapia without adverse effects on growth and protein utilization.
A produção aqüícola requer rações de alta qualidade, com alto conteúdo protéico. Assim, a determinação de fontes protéicas de menor custo e que promovam bom crescimento é benéfica. Resíduos de camarão têm sido identificados como uma fonte de proteína animal de grande potencial. Hidrolisado protéico de camarão (HPC) foi considerado como uma excelente fonte alimentar e pode servir como uma adequada fonte de proteína e flavorizante em formulações alimentares. Este trabalho objetivou avaliar a qualidade nutricional do HPC através do desempenho em crescimento de juvenis da tilápia do Nilo e sua utilização protéica. SPH foi incluído em dietas isoprotéicas em níveis de 0, 5, 10 e 20% de substituição da proteína advinda da farinha de peixe (HPC0, HPC5, HPC10 e HPC20) e ofertadas aos peixes (1,7±0,4 g) estocados em aquários de 40 L, por um período experimental de 45 dias. A inclusão do HPC não produziu diferenças estatísticas (P≥0,05) no peso final (27,18, 29,46, 26,02 e 25,19 g), sobrevivência (100%), ganho de peso relativo (1.571, 1.624, 1.388 e 1.301%), ganho de peso diário, GPD (0,57, 0,62, 0,54 e 0,52g dia-1), taxa de crescimento específico, TCE (7,15, 7,38, 6,85 e 6,73 % dia-conversão alimentar, CA (1,15, 1,09, 1,13 e 1,17) e eficiência protéica, EP (2,26, 2,33, 2,20 e 2,14), respectivamente. A inclusão do HPC nas dietas para a tilápia do Nilo afetou estatisticamente (P<0,05) a composição final dos peixes. Os teores de proteína e cinzas diminuíram e o teor de gordura aumentou com os níveis de inclusão do HPC. Este estudo claramente demonstra que o hidrolisado protéico de camarão pode ser incluído em dietas para a tilápia do Nilo sem efeitos adversos em crescimento e utilização protéica.

La stabilité du génome est contrôlée par une machinerie complexe qui répare les dommages causés à l'ADN, le principal responsable du cancer et des maladies associées au vieillissement. En effet, Les cellules humaines sont exposées à différentes sources de stress oxydatif qui entraînent des dommages de l'ADN, notamment les cassures simple brin et les cassures double brin. Les sources de cassures de l’ADN peuvent être exogènes (lumière ultraviolette, pollution, rayonnements ionisants) ou endogènes (métabolisme cellulaire et inflammation). Après un stress génotoxique, les cellules de mammifères déclenchent une cascade d'événements qui commencent par le recrutement des membres de la famille de protéines (ADP-ribosyl) transférase, PARP-1 et -2, aux dommages de l'ADN. Lorsque les PARP se lient à un ADN endommagé, une activité catalytique est activée pour synthétiser un polymère négativement chargé appelé poly (ADP-ribose) ou PAR. Les polymères PAR résultants sont liés de manière covalente aux PARP eux-mêmes (autoPARylation) ou également à de nombreuses autres protéines. Les polymères de PAR liés de manière covalente aux protéines accepteurs peuvent ensuite être hydrolysés pour former du PAR libre ou du mono (ADP-ribose) par la PAR glycohydrolase (PARG). Un niveau excessif de PAR endogène dû à une hydrolyse altérée du PAR induit également la présence de nucléoles supplémentaires présentant des anomalies ultrastructurales. La synthèse et la dégradation des polymères de PAR sont parmi les événements clés de la réponse cellulaire aux dommages de l'ADN chez les eucaryotes supérieurs et sont impliqués dans de nombreux aspects de la réponse au stress, tels que le remodelage chromosomique, la régulation de la transcription et l'assemblage de granules de stress.Le polymère PAR a une charge hautement négative et peut donc concurrencer efficacement l'ARN pour la liaison aux protéines de liaison à l'ARN, ce qui permet de redistribuer les protéines de liaison à l'ARN dans la cellule. En accord avec cela, la synthèse de PAR après un endommagement de l'ADN entraîne la relocalisation nucléaire de protéines de liaison à l'ARN telles que THRAP3, un facteur d'épissage, TAF153, et la translocation nucléocytoplasmique de HuR, une protéine de liaison à l’ARN, après une longue exposition au lipopolysaccharide. Récemment, une accumulation de certaines protéines de liaison à l'ARN dans des régions du noyau exposées à un faisceau laser a été détectée, ce qui soulève des questions quant à leur rôle présumé dans la réparation de l'ADN. En particulier, le FUS a retenu l’attention du fait qu’il s’agit de l’une des protéines de liaison à l’ARN nucléaire la plus abondante et la plus hautement PARylée. Cependant, les rôles de l'accumulation de FUS et de sa PARylation au niveau de l'ADN endommagé dans la réponse cellulaire au stress génotoxique restent à découvrir.Par une approche combinée cellulaire, biochimique et de molécule unique, nous montrons que la liaison du FUS au PAR induit la formation de compartiments dans lesquels l’ADN endommagé est concentré. Des assemblages moléculaires transitoires déclenchés par le FUS lors de l'activation de PARP-1 peuvent faciliter la réparation de l'ADN par la compartimentation des sites de dommages de l'ADN. Cette fonction dans la réparation de l'ADN était précédemment attribuée au poly (ADP-ribose) lui-même, mais la présence de protéines de liaison à l'ARN pourrait être nécessaire pour augmenter la capacité du PAR à recruter des facteurs de réparation de l'ADN en formant des compartiments regroupant l'ADN endommagé. L'hydrolyse de ces compartiments par PARG permet la réversibilité de l'ensemble du processus en dissociant ces compartiments. PARG permet ainsi la translocation du FUS du noyau au cytoplasme. Dans le cytoplasme, FUS peut participer à la réponse traductionnelle au stress ou s’accumuler dans les agrégats, comme dans les neurones de patients atteints de maladies neurodégénératives spécifiques
The stability of the genome is controlled by a complex DNA repair machinery that counteracts DNA damage, the major guilty in cancer and ageing related diseases. Human cells are exposed to different sources of oxidative stress that lead to DNA damages including single strand breaks (SSBs) and double strand breaks (DSBs). Sources of the damaging reactive oxygen species can be exogenous (UV light, pollution, ionizing radiation) or endogenous (cellular metabolism and inflammation). After genotoxic stress, mammalian cells trigger a cascade of events that start from the recruitment of the members of the (ADP-ribosyl) transferase protein family, PARP-1 and -2, to DNA damage. Upon PARPs binding to damaged DNA, a catalytic activity is turned on to synthesize a highly negatively charged polymer called poly(ADP-ribose) or PAR using NAD+ as substrate. The resulting PAR polymers are covalently attached to PARPs itself (autoPARylation) or also to many other acceptor proteins. The protein poly(ADP-ribosyl)ation (PARylation) is a reversible post-translational modification. PAR polymers covalently attached to acceptor proteins and can be subsequently hydrolyzed to form free PAR or mono(ADP-ribose) by PAR glycohydrolase (PARG). An excess level of endogenous PAR due to an impaired PAR hydrolysis also induces the presence of additional nucleoli with ultrastructural abnormalities in PARG knockout flies. Synthesis and degradation of PAR polymers is among the key events in the cellular response to DNA damages in higher eukaryotes and is involved in many aspects of the response to stress like chromosome remodelling, transcription regulation and stress granule assembly.PAR polymer has a highly negative charge density and thus can effectively compete with nuclear DNA- and RNA-binding proteins for the binding to nucleic acids, which provides a mean to redistribute RNA-binding proteins in the cell. In agreement with this, PAR synthesis after DNA damage leads to the nuclear relocation of RNA-binding proteins such as THRAP3, a splicing factor, TAF153, and the nucleocytoplasmic shuttling of HuR, a 3’UTR mRNA-binding protein, after long lipopolysaccharide exposure. Recently, a PAR-dependent accumulation in DNA regions damaged by short laser beam exposures has been detected for some RNA binding proteins, raising issues about their putative role in DNA repair. In particular, FUS (FUS/TLS, Fused in Sarcoma) has received some attention since it is one of the most abundant and most highly PARylated nuclear RNA-binding protein. However, the roles of the PAR dependent FUS accumulation and its PARylation at damaged DNA in the cellular response to genotoxic stress remain unclear.By a combined cellular, biochemical, and single molecule approach, we show that the binding of FUS to PAR induces the formation of compartments in which damaged DNA is concentrated. Transient molecular assemblies triggered by FUS upon PARP-1 activation may facilitate DNA repair through compartmentalization of DNA damage sites. This function in DNA break repair was previously attributed to poly(ADP-ribose) itself but the presence of RNA-binding proteins might be required to increase the capacity of PAR to recruit DNA repair factors by formation of DNA-damaged rich compartments. The hydrolysis of these compartments with PARG provides reversibility of the whole process by dissociating these compartments. PAR hydrolysis by PARG then enables the shuttling of FUS from the nucleus to the cytoplasm. In the cytoplasm, FUS can participate in the translational response to stress or accumulate in the aggregates, as found in neurons of patients affected by specific neurodegenerative diseases

La poly(ADP-ribosylation) est une modification post-traductionnelle des protéines régulée essentiellement par la poly(ADP-ribose) polymérase I (PARP-1), synthétisant le poly(ADP-ribose), et la poly(ADP-ribose) glycohydrolase (PARG), responsable du catabolisme. Tandis que PARP-1 joue un rôle central dans la réparation de l'ADN, la stabilité génomique, la transcription et l'apoptose, les fonctions biologiques de PARG sont inconnues. Le but de notre étude est de mieux comprendre le rôle physiologique de PARG et du catabolisme du poly(ADP-ribose). Par ciblage de gène, nous avons invalidé PARG chez la souris. Ces animaux sont viables et sans phénotype apparent, mais ont un catabolisme du poly(ADP-ribose) réduit ainsi qu'une sensibilité accrue aux traitements génotoxiques et aux chocs septiques. Cette étude fournit de nouveaux éléments quant aux fonctions biologiques de PARG, et contribuera au développement de stratégies thérapeutiques pour le traitement de l'inflammation

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Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco
Nos últimos anos, a indústria pesqueira brasileira tem vivenciado um período importante de desenvolvimento. Mas, juntamente com o crescimento na produção, temse o aumento da quantidade de resíduos gerados no processamento do pescado, que usualmente são descartados no ambiente, causando poluição. Os resíduos do processamento são constituídos por cabeças, carcaças e vísceras de peixes, cabeças e cascas de crustáceos e fauna acompanhante, e podem representar de 20 a 70% do pescado comercializado. O aproveitamento dos resíduos incrementa a economia do setor industrial pesqueiro e permite que essa atividade seja mais ecologicamente sustentável e economicamente viável. O mal uso de subprodutos do processamento de pescados é considerado por muitos um desperdício de moléculas biativas valiosas. As cabeças de camarão representam um dos principais resíduos do processamento de pescados e constituindo em até 50% do peso total do animal. Esse material é utilizado atualmente como fonte de proteína e na produção de quitina das cascas, matéria prima para produção comercial de quitosana. Diversos métodos são empregados para a recuperação de hidrolisados protéicos e quitina, dentre eles a hidrólise utilizando proteases exógenas. Entretanto, o emprego destas enzimas encarece o processo tornando-o pouco adequado para o uso industrial. Nas cabeças de camarões estão contidas as vísceras (parte do trato digestivo) que possuem enzimas hidrolíticas que podem ser aplicadas na solubilização dos tecidos e deproteinização das cascas, facilitando as etapas de extração e purificação dos produtos. No presente trabalho é descrito um novo método para obtenção de vários compostos a partir de cabeças de camarão (Litopenaeus vannamei) submetidas à autólise. No processo, foi obtido hidrolisado protéico, carotenóides, quitina e quitosana e glicosaminoglicanos sulfatados. A partir de 1kg de cabeças obteve-se 1,46L de hidrolisado protéico (solução a 9%) e 194mg de carotenóides totais, sendo 111,96mg de carotenóides não identificados e 82,56mg de astaxantina. A partir de 53g de carapaça foram obtidas cerca de 25g de quitina e 17g de quitosana. Quitina e quitosana foram analisadas por espectroscopia de 13C-RMN e FT-IR para comprovar a efetiva desacetilação dos resíduos de Nacetilglucosamina. O grau de desacetilação foi calculado por titulação potenciométrica, análise elementar e FT-IR para quitosana produzidas a partir uma e duas desacetilações bem como a quitosana purificada. Os valores obtidos encontram-se ente 60-80%. O conteúdo de glicosaminoglicanos obtidos do sedimento após extração de carotenóides e lipídios foi de 23,32 μg kg-1 de subproduto e mostraram migração eletroforética semelhante aos padrões de mamífero. As frações precipitadas foram suscetíveis à ação das heparitinases I e II, o que sugere a presença de um heparam sulfato. As biomoléculas recuperadas a partir de cabeças de camarão possibilitam um amplo espectro de aplicações como na suplementação e formulação de rações animais, em abordagens biomédicas e biotecnológicas

O objetivo deste trabalho foi verificar os efeitos da suplementação energético-protéica na contagem de células T e de seus subtipos CD4 e CD8. Os sujeitos eram praticantes de atividade física de carga alta e diária. Foram avaliados em dois momentos: repouso (antes da atividade) e agudo (após a atividade). A contagem de células foi feita por citômetro de fluxo. A atividade física alterou os parâmetros escolhidos e a mesma, associada com a suplementação, alterou mais ainda. Observou-se que a suplementação reduziu a contagem das células T e de seus subtipos, no momento de repouso, indicando possível ação redutora na mobilização celular gerada pelo exercício. Foram levantados indícios que apontam para o fato da proteína de soja ter uma ação potencialmente melhor em relação às proteínas do soro do leite, para os parâmetros estudados. A suplementação com proteína e maltodextrina não se mostrou mais eficaz do que a suplementação com proteínas somente.
The objective of this research was to evaluate the effects of energetic-proteic supplementation on T cells counting as well as its CD4 and CD8 subtypes. The subject individuals studied were practitioners of daily high-load physical activities. The evaluations took place in two different moments and respective conditions: at rest (prior to the development of activities) and peak (immediately after performing activities). The physical activity modified the chosen parameters as long it was more effective as associated with the supplementation. At the rest moment, it was observed that the supplementation reduced the counting of T cells and its subtypes, thus indicating possible reducing action in the cellular mobilization generated by the exercise. For the studied parameters, indications were detected that point to the fact of the soy protein as having a potentially better action in relation to whey proteins. The supplementation with protein and maltodextrin did not prove to be more efficient than the supplementation only with proteins.

Os isolados protéicos de soja são utilizados como ingredientes em diversos alimentos e sua utilização vêm aumentando juntamente com o aumento das pesquisas sobre os metabólitos secundários da soja, as isoflavonas. Alguns efeitos benéficos vem sendo associados às isoflavonas, entre estes a sua ação antioxidante, a redução ao risco de câncer, doenças cardiovasculares e osteoporose. O objetivo deste estudo foi o de otimizar as condições de extração das isoflavonas e de suas formas conjugadas a partir da farinha desengordurada de soja, visando o preparo de isolado protéico de soja. Os resultados mostraram que a obtenção de isolados protéicos de soja com teor aumentado de isoflavonas depende da utilização de condições brandas de centrifugação para a separação do precipitado isoelétrico, assim como da utilização de água acidificada na sua lavagem. A presença de isoflavonas no isolado resulta de três fatores, o primeiro referindo-se à associação entre isoflavonas e proteínas através de interações hidrofóbicas, eletrostáticas, e pontes de hidrogênio; o segundo à menor solubilidade das isoflavonas presentes na farinha desengordurada de soja no pH isoelétrico; e o último ao processo de carreamento (físico) das isoflavonas pelas proteínas insolubilizadas.
Soy protein isolates are used as ingredients in several food products and their use is increasing together with the increase of the researches on the secondary metabolites of soy, the isoflavones. Some beneficial effects have been associated to the isoflavones, among these their antioxidant action, prevention of cancer, cardiovascular diseases and osteoporosis. The objective of this study was to optimize the extraction conditions of the isoflavones from the defatted soy flour, seeking the preparation of soy protein isolates. The results showed that the obtention of soy protein isolates with increased content of isoflavones depends on the use of mild conditions of centrifugation for the separation of the isoelectric precipitate, as well as on the use of water acidified in the washing step. The presence of isoflavones in the isolates resulted from three factors, the first refers to the association between isoflavones and proteins through hydrophobic; and electrostatic interactions, and hydrogen bonding; the second to the decreased solubility of the isoflavones extracted from the defatted soy flour in the isoelectric pH; and the last to the carrying process (physical) of isoflavones by the precipitating proteins.

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Com o objetivo de avaliar níveis de valina em rações de baixo nível protéico para codornas japonesas na fase de postura, foram utilizadas 648 codornas, com 154 dias de idade, taxa de postura inicial média de 86,26% distribuídas em delineamento experimental em blocos ao acaso, constituídos por seis tratamentos e seis repetições de 18 aves por unidade experimental. Os tratamentos consistiram de ração basal com 16% de PB correspondente ao nível de valina de 0,686% e suplementada com valina (0,197; 0,344; 0,491; 0,638% na ração), em substituição ao ácido glutâmico, em equivalente protéico, correspondendo aos níveis de valina de 0,833; 0,980; 1,127; 1,274%. As dietas experimentais foram comparadas a uma dieta controle contendo 20% PB, totalizando seis tratamentos e os níveis dos demais nutrientes também de acordo com Silva (2009). Os parâmetros estudados foram: consumo de ração, de proteína bruta e de valina, porcentagem de postura, porcentagem de ovos íntegros, peso médio dos ovos, massa de ovos, conversão alimentar por dúzia e por quilograma de ovos produzidos, viabilidade, gravidade específica, porcentagens de gema, albúmen e casca, espessura da casca, resistência da casca à quebra, cor da gema, sólidos totais dos ovos e nitrogênio nas excretas das aves. Os resultados foram submetidos à análise de variância e os efeitos dos níveis de valina foram submetidos a análise de regressão. O tratamento testemunha (20% PB) foi comparado com cada um dos demais aplicando o teste de Dunnett a 5% de probabilidade. Foi observado aumento linear para consumo de valina com elevação dos níveis de valina das dietas e efeito quadrático para o conteúdo de sólidos totais nos ovos. Pela comparação das médias de cada combinação de níveis de valina para dietas com 16% PB com o tratamento controle 20%PB, verificou-se...
Aiming to assess levels of valine in diets of low protein level for Japanese quails posture. A total of 648 quails were used, with 154 days of age, laying rate initial average 86.26% distributed in experimental design in blocks, consisting of six treatments and six replicates of 18 birds each. Treatments consisted of a basal level corresponding to 0.686% of valine and supplemented with valine (0.197, 0.344, 0.491, 0.638% in diet), replacing the glutamic acid in protein equivalent, corresponding to the levels of valine (0.833 ; 0.980, 1.127, 1.274%). The experimental diets were compared to a control diet containing 20% CP, totaling six treatments and the levels of other nutrients also according to Silva (2009). The parameters studied were: feed intake, crude protein and valine, egg production, percentage of intact eggs, average egg weight, egg mass, and feed conversion ratio per dozen and per kilogram of produced eggs, viability, specific gravity, percentages of yolk, albumen and shell, shell thickness, the breaking strength of the shell, yolk color, egg total solids and nitrogen in poultry excreta. Results were subjected to analysis of variance and the effects of levels of valine were subjected to regression analysis. The control treatment (20% CP) was compared with others (16% CP) using the Dunnett test at 5% probability. We observed a linear increase in consumption of valine with higher levels of valine diets and quadratic effect of total solids content in eggs. By comparing the averages of every combination of levels of valine for diets with 16% CP with the control 20% CP, it was found that the consumption of PB, intake valine, egg weight, nitrogen excretion and yolk color were significantly influenced. For Japanese quail fed diets with 16% CP, valine level of... (Complete abstract click electronic access below)

Orientador: Edivaldo Antônio Garcia
Banca: Adriano Sakai Okamoto
Banca: Otto Mack Junqueira
Banca: Lúcio Francelino Araújo
Banca: Ricardo de Albuquerque
Resumo: Com o objetivo de avaliar níveis de valina em rações de baixo nível protéico para codornas japonesas na fase de postura, foram utilizadas 648 codornas, com 154 dias de idade, taxa de postura inicial média de 86,26% distribuídas em delineamento experimental em blocos ao acaso, constituídos por seis tratamentos e seis repetições de 18 aves por unidade experimental. Os tratamentos consistiram de ração basal com 16% de PB correspondente ao nível de valina de 0,686% e suplementada com valina (0,197; 0,344; 0,491; 0,638% na ração), em substituição ao ácido glutâmico, em equivalente protéico, correspondendo aos níveis de valina de 0,833; 0,980; 1,127; 1,274%. As dietas experimentais foram comparadas a uma dieta controle contendo 20% PB, totalizando seis tratamentos e os níveis dos demais nutrientes também de acordo com Silva (2009). Os parâmetros estudados foram: consumo de ração, de proteína bruta e de valina, porcentagem de postura, porcentagem de ovos íntegros, peso médio dos ovos, massa de ovos, conversão alimentar por dúzia e por quilograma de ovos produzidos, viabilidade, gravidade específica, porcentagens de gema, albúmen e casca, espessura da casca, resistência da casca à quebra, cor da gema, sólidos totais dos ovos e nitrogênio nas excretas das aves. Os resultados foram submetidos à análise de variância e os efeitos dos níveis de valina foram submetidos a análise de regressão. O tratamento testemunha (20% PB) foi comparado com cada um dos demais aplicando o teste de Dunnett a 5% de probabilidade. Foi observado aumento linear para consumo de valina com elevação dos níveis de valina das dietas e efeito quadrático para o conteúdo de sólidos totais nos ovos. Pela comparação das médias de cada combinação de níveis de valina para dietas com 16% PB com o tratamento controle 20%PB, verificou-se... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Aiming to assess levels of valine in diets of low protein level for Japanese quails posture. A total of 648 quails were used, with 154 days of age, laying rate initial average 86.26% distributed in experimental design in blocks, consisting of six treatments and six replicates of 18 birds each. Treatments consisted of a basal level corresponding to 0.686% of valine and supplemented with valine (0.197, 0.344, 0.491, 0.638% in diet), replacing the glutamic acid in protein equivalent, corresponding to the levels of valine (0.833 ; 0.980, 1.127, 1.274%). The experimental diets were compared to a control diet containing 20% CP, totaling six treatments and the levels of other nutrients also according to Silva (2009). The parameters studied were: feed intake, crude protein and valine, egg production, percentage of intact eggs, average egg weight, egg mass, and feed conversion ratio per dozen and per kilogram of produced eggs, viability, specific gravity, percentages of yolk, albumen and shell, shell thickness, the breaking strength of the shell, yolk color, egg total solids and nitrogen in poultry excreta. Results were subjected to analysis of variance and the effects of levels of valine were subjected to regression analysis. The control treatment (20% CP) was compared with others (16% CP) using the Dunnett test at 5% probability. We observed a linear increase in consumption of valine with higher levels of valine diets and quadratic effect of total solids content in eggs. By comparing the averages of every combination of levels of valine for diets with 16% CP with the control 20% CP, it was found that the consumption of PB, intake valine, egg weight, nitrogen excretion and yolk color were significantly influenced. For Japanese quail fed diets with 16% CP, valine level of... (Complete abstract click electronic access below)
Doutor

CORDEIRO JÚNIOR, Evandro Lima. Concentrado protéico de soja e óleo de soja em rações experimentais para o camarão marinho Litopenaeus vannamei. 2011. 54 f. : Dissertação (mestrado) - Universidade Federal do Ceará, Centro de Ciências Agrárias, Departamento de Engenharia de Pesca, Fortaleza-CE, 2011
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Currently, there are many studies to replace total or part of fishmeal in the artificial feeds by plant protein sources in order to lower costs and greater predictability of production. The study aimed to evaluate the growth performance of juvenile shrimp Litopenaeus vannamei (1.59 ± 0.46 g) reared in the laboratory under controlled conditions for 72 days. They were stored in 50 polyethylene circular tanks of 500 L at the density of 40 shrimp/tank (70 shrimp/m2). Soybean protein concentrate was the main substitute for fish meal and the soybean oil was the main substitute for fish oil. Eight experimental feeds were formulated combining four levels of inclusion of fish meal and two levels of fish oil (120/20, 120/10, 85/20, 85/10, 50/20, 50/10, 0//20 and 0/10). The first number refers to the inclusion of fishmeal (g kg-1) and the second number refers to the inclusion of fish oil (g kg-1). Differences were significant in weekly growth, weight gain, final weight, total feed intake, FCR, yield and PER based on the percentage of inclusion of fishmeal in diets. Survival and ANPU were not significant. Results showed that it is possible to reduce the level of inclusion of fishmeal in the diet of 120 g kg-1 to 85 g.kg-1 and this doesn’t compromise the shrimp growth only if the diet contains at least 10 g.kg -1 of fish oil. If the level of inclusion of fish oil decreases to 10 g kg-1, the minimum inclusion of fish meal, without prejudice, will be subject to 85 g kg-1. There was a reduction in growth rate of L. vannamei when it was removed completely from the fish meal diet. This fact occurred regardless of inclusion level of tested fish oil (20 or 10 g kg-1). Further work is required to evaluate other levels of inclusion, cheap production techniques, alternative ingredients to fish meal etc
Atualmente, há grande esforço de pesquisa no sentido de se substituir, parcial ou totalmente, a farinha de peixe das rações artificiais por fontes protéicas vegetais, visando menores custos, e maior previsibilidade de produção. O presente trabalhou objetivou avaliar o desempenho zootécnico de juvenis do camarão marinho Litopenaeus vannamei (1,59 ± 0,46 g) cultivados em laboratório sob condições controladas durante 72 dias, sendo estocados em 50 tanques circulares de polietileno de 500 L, na densidade de 40 camarões/tanque (70 camarões/m²). No estudo, o concentrado protéico de soja foi o principal substituto da farinha de peixe, e o óleo de soja o principal substituto do óleo de peixe. Oito rações experimentais foram formuladas combinando quatro níveis de inclusão de farinha de peixe e dois níveis de inclusão de óleo de peixe (120/20, 120/ 10, 85/20, 85/10, 50/20, 50/10, 0/20 e 0/10), o primeiro número se refere à inclusão de farinha de peixe em g kg-1 e o segundo número se refere à inclusão de óleo de peixe em g kg-1. Houve diferença significativa no crescimento semanal, peso final, consumo total de ração, FCA, produtividade e TEP em função do percentual de inclusão de farinha de peixe nas dietas, já para sobrevivência e VPP não houve diferença significativa. Pode ser concluído que é possível diminuir o nível de inclusão de farinha de peixe na dieta de 120 g.kg- 1 para 85 g.kg-1 sem comprometer o crescimento dos camarões somente se a dieta contiver, no mínimo, 10 g.kg-1 de óleo de peixe. Se o nível de inclusão de óleo de peixe cair para 10 g kg-1, a inclusão mínima de farinha de peixe sem prejuízo zootécnico será para 85 g kg-1. Houve perda significativa na taxa de crescimento do L. vannamei quando se retirou por completo a farinha de peixe da dieta, independentemente do nível de inclusão de óleo de peixe testado (20 ou 10 g kg-1). Trabalhos futuros são requeridos para avaliar outros níveis de inclusão, técnicas de produção baratas, ingredientes alternativos à farinha de peixe etc

Le collagene v, membre de la famille des collagenes fibrillaires est un constituant essentiel de la matrice extracellulaire. Largement implique dans le controle du diametre des fibres, les differentes donnees connues sur ce collagene laissent a penser qu'il joue un role crucial dans d'autres processus. C'est pourquoi une cartographie de ses domaines fonctionnels a ete entreprise. Deux domaines restreints, tous deux presents sur la chaine 1 du collagene v ont ete analyses : le module parp localise dans une partie non-collagenique et le site d'interaction a l'heparine, dans la region helicoidale. Les collagenes possedent une structure modulaire. Parp est un module pour lequel ni la structure fine ni la fonction ne sont connues. Surproduit en fusion avec 6 histidines, parp s'est avere prisonnier des corps d'inclusion bacteriens, donc insoluble. Apres un long travail de mise au point, parp a pu finalement etre renature avec formation d'un pont disulfure intrachaine caracteristique de la molecule native, qui a permis l'obtention d'anticorps specifiques. Ces anticorps serviront a determiner le devenir de ce module dans les tissus sous sa forme libre, i. E. Apres maturation en n-terminal du collagene v pour essayer de degager un role commun pour ce module present dans d'autres proteines matricielles. Le site de liaison a l'heparine sur la triple helice de collagene v est un site conformationnel qui se situe, comme montre en microscopie electronique, exactement au niveau du site localise sur la chaine 1 (v) individuelle. Les trois formes moleculaires du collagene v ont une affinite pour l'heparine qui augmente avec le nombre de chaine 1(v) incorporee dans la molecule. L'homotrimere, 1(v) 3, produit dans un systeme eucaryote, a permis pour la premiere fois l'etude de cette forme moleculaire d'un point de vue fonctionnel. Afin de caracteriser plus precisement la sequence impliquee dans cette interaction, un fragment de 12 kda (i 8 2 4p 9 5 0), hep v, a ete designe et surproduit dans un systeme procaryote. Il presente la meme affinite pour l'heparine que la chaine 1(v) native. Si l'on reduit la sequence a un peptide synthetique comprenant le site minimal putatif, l'affinite pour l'heparine est perdue. La modelisation moleculaire de cette sequence revele cependant une repartition amphiphile des acides amines qui est une caracteristique commune aux sites de liaison connus pour des proteines non-collageniques. La mutation de residus basiques du fragment hepv, au niveau du site minimal putatif, a permis de cibler precisement les residus impliques. Enfin, hepv est capable de promouvoir l'adhesion de cellules. Cette interaction est inhibee par de l'heparine sulfate montrant qu'hepv se comporte comme un site adhesif pouvant interagir avec les proteoglycanes a heparine sulfate membranaires. Ces domaines fonctionnels specifiques pourraient conferer au collagene v, bien que minoritairement represente dans les tissus, un role majeur dans de nombreux processus physiologiques et pathologiques.

Ce travail a permis de démontrer que l’avenir de la conservation des ressources biologiques ne se joue pas seulement dans les aires protégées, mais aussi que l’intégration des zones non protégées dans un plan de gestion rationnelle du territoire pourrait être un gage de conservation à long terme. En outre, il a aussi démontré aux différents acteurs que la présence d’une aire protégée dans une localité n’est pas sans effets sur les zones périphériques. Pour ce faire, 65 impacts des parcs nationaux sur les zones périphériques ont été répertoriés dont 28 impacts positifs soit 43% contre 37 impacts négatifs soit 57%. Les impacts socio-économiques sont les plus nombreux suivis des impacts culturels et des impacts écologiques.L’analyse critique et comparative, qui s’est inscrite dans la perspective du paradigme de développement durable, a révélé que beaucoup d’impacts écologiques se manifestent à long terme. Les impacts socio-économiques et culturels négatifs sont mieux gérés dans une approche de gestion participative par opposition à une gestion trop centralisée et exclusive. Les facteurs extrinsèques qui ont influencé l’intensité des impacts sont d’ordre socio-politique, démographique, climatique, les systèmes de cultures locales et l’empreinte écologique des centres urbains situés à proximité. Cette influence témoigne de la difficulté à dissocier les impacts imputés aux créations des espaces protégés de ceux générés par d’autres facteurs. Ce qui justifie à suffisance que non seulement les impacts sont multiformes mais s’expriment différemment selon les contextes. En outre, les approches participatives permettent de faire accepter les impacts négatifs sans pouvoir les atténuer totalement
This work has enabled to show that the future of biological resources safeguarding is not only to be performed on the protected areas but also the integration of non protected areas in the rational management framework of the territory could be a guarantee of a sustainable safeguarding. Moreover, it has also shown to different actors that the presence of a protected area in a location is not without impact on surrounding areas. In this case, 65 national parks impacts on the surrounding areas have been identified meaning there are 28 positive impacts representing 43% against 37 negative impacts representing 57%. Socio-economic impacts are the most numerous followed by cultural impacts and ecological impacts.In order to get these results, two case studies have been performed on Manda and Sena Oura national parks in Chad, all located in the southern area. These are two national parks of the differents generations. In the study framework, 152 people have been surveyed among which there are 19 civil servants, 11 development representatives and 122 members of local communities adjacent to national parks. The satellite images have completed the analysis agenda.The critical and comparative analysis which is included in the perspective of the sustainable development paradigm has revealed that many ecological impacts will occur in long term. The negative socio economic and cultural impacts are best managed in a participative management approach as opposed to a too centralized and exclusive management. The intrinsic factors which have influenced the intensity of impacts are of socio political, demographic, climatic order, the local culture systems and ecological footprints of urban centers nearby. This influence witnesses the difficulty to separate impacts attributed to the creation of protected areas from those generated by other factors

A carne mecanicamente separada de frango (CMSF), material oriundo do processamento de carcaças na indústria avícola, foi empregada como fonte protéica para a produção do hidrolisado utilizando enzimas proteolíticas. As condições de hidrólise foram otimizadas usando metodologia de superfície de resposta (MSR). A influência das variáveis temperatura (T), pH e concentração de enzima (E) foram avaliadas em função do Índice de Hidrólise (IH). As condições ótimas encontradas para a enzima Alcalase e Flavourzyme foram T de 50 °C, pH de 7,5 e (E) de 2,5 % e T de 50 °C, pH de 6,0 e (E) de 3,5%, respectivamente. Alcalase obteve uma recuperação protéica de 89 % e a Flavourzyme obteve 66,5 %. Alcalase foi utilizada para a produção do hidrolisado, posteriormente seco em spray-drier, o qual obteve um conteúdo de proteína total de 57,90 %. O hidrolisado apresentou boa qualidade microbiológica e o perfil molecular indicou a presença de pequenas proteínas e peptídeos na faixa de 12 000 a 5807 Da. A avaliação biológica mostrou elevada digestibilidade e NPR para a proteína hidrolisada quando comparada com a caseína, contrastando com os baixos valores encontrados para metionina e cistina na análise de aminoácidos.
Mechanically deboned chicken meat (MDCM), obtained from surplus carcasses in the poultry process industry, was used as a source to the production of protein hydrolysates using proteolytic enzymes. Hydrolysis conditions were optimized using the response surface methodology (RSM). The influence of the process variables temperature (T ºC), pH and enzyme concentration, (E) was studied with regard to the hydrolysis index (HI). The optimum values for Alcalase and Flavourzyme were T of 50 ºC, pH of 7.5 and (E) of 2.5 % and T of 50 ºC, pH of 6.0 and (E) of 3.5 %, respectively. Alcalase recovered 89 % of protein and Flavourzyme 66.5 %. The Alcalase was used to produce the spray-dried hydrolysate and obtained 57.90 % of total protein. The spray-dried hydrolysate presented good microbiological quality and molecular weight ranging from 5,807 to 12,000 Da. The biological evaluation showed high digestibility and NPR to protein hydrolysate when compared with casein, contrasted with the low values of methionine and cystine found in amino acid analysis.

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Universidade Federal de Viçosa
The present work was developed based on four experiments. The two firsts were conducted aiming to evaluate the fecal dry matter production using total fecal collection or estimated by chromic oxide (Cr2O3), titanium dioxide (TiO2), isolated, purified and enriched lignin of Eucalyptus grandis (LIPE®), indigestible neutral and acid detergent fiber (NDFi and ADFi, respectively), in two periods of sample collection (three or five days); to evaluate the use of Cr2O3 and TiO2 to estimate the individual intake of concentrate, and the use of NDFi and ADFi to estimate the individual intake of roughage; and to determine the dry matter intake, the digestibilities coefficients of dry matter and of the nutrients and to determine the microbial protein production in Nellore cattle of three genders. In the first experiment, four heifers were fed with sugar cane plus 1,0% of urea/ammonium sulfate on as-fed basis, ad libitum and 1,0% of live weight in concentrate, fed separate from roughage. Each animal was considered as sub-block (main plots), in a splitplitplot design, being the methods to estimate fecal dry matter the sub-plot and the sampling days the sub-sub-plot. The Cr2O3 and TiO2 were mixed in the concentrate (10 g/animal), and the LIPE® was introduced via cannula (0,5 g/animal). In the second trial, eighteen animals were used, allotted to a complete randomized design, in factorial design 2x3, being two levels of concentrate offer and three genders. The diets had 12,5% CP in the dry matter and the roughage used was corn silage. The ruminal synthesis of nitrogen compounds was estimated using the purine derivatives. All markers estimated properly the fecal dry matter production, with no effects in the sample period, excepting ADFi. There was not difference between Cr2O3 and TiO2 to estimate the individual concentrate intake (P>0,05). There was no significant difference (P>0,05) between genders and levels of concentrate in dry matter intake and nutrient digestibilities, excepted for ether extract. The average microbial efficiency was 133,42 g of microbial CP/kg of TDN. Therefore, it is recommended to use three days of sample collection period because it s cheaper and less labor intensive. The final decision when choosing between Cr2O3 and TiO2 to estimate concentrate intake has to take in account the availability, its cost and laboratorial analysis. The NDFi can be used to estimate the roughage intake. There were not differences in the dry matter intake between Nellore bulls, steers and heifers. The gender didn t influence the microbial protein production. The third experiment was developed aiming to estimate the individual intake of dry matter, concentrate and roughage of animal fed in group. To evaluate the dry matter intake (DMI), daily weight gain (DWG) and feed conversion (FC) by animals of three genders, fed individually or in group, fed two levels of concentrate offer; and determine the effect of two levels of concentrate offer (1, 0% of live weight or 1,25% of live weight) and the genders effect in carcass traits of confined Nellore cattle. It was also the objective to validate the Hankins and Howe (1946) equations, to estimate the carcass physic composition, and the Paulino (2002) equations, to estimate the empty body weight macromineral composition of Nellore cattle; determine the metabolized requirements of energy for maintenance and liquid requirements of crude protein, energy and macromineral for gain, and also the efficiency of conversion of liquid protein requirements into metabolized protein requirements in Nellore cattle. Forty five Nellore (15 bulls, 15 steers and 15 heifers) were used, being nine animals (three of each genders) slaughtered at the beginning performing the reference group. The 36 animals remaining (12 of each gender) were allotted in two levels of concentrate offer (1,0% and 1,25% of live weight) and two schemes of feeding (individually or in group), in a factorial design 2x2x3. The trial lasted 112 days. To estimate the individual DMI of the animal fed in group, the LIPE® was used to estimate the fecal dry matter production, the Cr2O3 and TiO2 to estimate the individual concentrate intake and NDFi and ADFi were used to estimate the individual roughage intake. At the end of the experiment all animals fed individually were slaughtered to determine the empty body weight and corporal composition, being the right carcass divided in five commercial cuts. From left carcass the HH section was obtained. The liquid requirements for gain were obtained by deriving the equation of corporal content of each nutrient, in function of log of empty body weight. The metabolized energy requirements for maintenance were estimated by a regression of retained energy and metabolized energy intake. The efficiency of conversion into metabolized protein was estimated by regressing the protein retained and metabolized protein intake. There were not effects (P>0,05) of concentrate level and feed scheme in DMI, DWG and FC. Bulls were superior to steers in some weights of the commercial cuts and steers were superior to females. Paulino s (2002) equation was efficient to estimate Ca concentration in the empty body weight. For all macromineral were proposed news equations to estimate its contents in empty body weight using the HH section. The equations obtained for retained energy were: RE = 0,0559*EBW0,75*EBWG1,1037 (bulls); RE = 0,0738*EBW0,75* EBWG0,9688 (steers); RE = 0,1020*EBW0,75*EBWG1,0408 (females). The feed scheme did not influence the DMI and DWG of Nellore cattle of three genders. Bulls grow 24% faster than steers and steers grow 22% faster than females. The concentrate offer between 1,0 and 1,25% of live weight did not influence carcass traits in Nellore cattle. Bulls were more efficient in meat deposition and had higher yield in some commercial cuts. Liquid energy requirements for gain increase with higher live weights while liquid protein requirements for gain decrease with higher live weights. The metabolized energy requirements for maintenance were higher for females and the efficiency of conversion into metabolized protein is about 50%. It can be concluded that is possible to estimate the macromineral composition using the HH section in Nellore cattle. The fourth experiment was developed aiming to determine the fraction A, B and kd of dry matter and crude protein of 27 feeds used for ruminants in Brazil. It was also the objective of the trial to determine the intestinal digestibility of rumen undegraded protein by the three steps and the mobile nylon bag techniques. Four Nellore females, cannulated in rumen and duodenum, were fed ad libitum with corn silage and concentrate. The Orskov and McDonald (1979) model was used to obtain the rumen degradability of DM and CP, and the times of incubation were 0, 2, 4, 6, 12, 16, 24, 48 and 72 hours, being after that determined the DM and total nitrogen in the residues of incubation. To determine the intestinal digestibility (ID) of CP was used the three steps and the mobile nylon bag techniques. The ruminal degradation of DM and CP data were similar to literature. The three step technique did not estimate properly the ID of all feed evaluated, excepted for the proteic feeds. The soybean meal (93,95%) had the higher ID of CP using the mobile nylon bag technique, and corn (85,58%), cotton seed meal 46% of CP (84,93%), corn gluten (82,98%) comes after. It can be concluded that the most feeds have total CP digestibility higher or next to 90%, excepting for soybean, coffee and cacao hulls and elephant-grass and corn silage. It is recommended to use the equation IDCP (%) = 5,1906 + 1,1053*X, to correct the ID obtained by the three steps technique for non-proteic feeds, being X the ID obtained by the three steps technique.
O presente trabalho foi desenvolvido a partir de quatro experimentos. Os dois primeiros foram conduzidos com os objetivos de avaliar a produção de matéria seca fecal (MSF) obtida a partir de coleta total ou estimada com o uso de óxido crômico (Cr2O3), dióxido de titânio (TiO2), lignina isolada, purificada e enriquecida de Eucalyptus grandis (LIPE®), fibra em detergente ácido (FDAi) e neutro (FDNi) indigestíveis, adotando-se dois períodos de coleta (três ou cinco dias); avaliar o uso de Cr2O3 ou TiO2 para estimar o consumo de concentrado, e da FDAi e FDNi para estimar o de volumoso; e determinar o consumo de matéria seca (CMS), os coeficientes de digestibilidade da matéria seca e dos nutrientes e a produção de proteína microbiana em animais Nelore de três classes sexuais. No primeiro experimento, foram utilizadas quatro novilhas mestiças, alimentadas com cana-de-açúcar, corrigida com 1,0% de uréia/sulfato de amônio na base da matéria natural, à vontade e oferta de concentrado na base de 1,0 % do peso vivo, fornecido separado do volumoso. Utilizou-se delineamento em sub-blocos, em esquema de parcelas sub-sub-divididas, tendo nas subparcelas os métodos para estimar a MSF e nas sub-sub-parcelas os dias de coleta. O Cr2O3 e o TiO2 foram administrados ao concentrado, na dose de 10 g/animal, e a LIPE® foi colocada via fístula ruminal, na quantidade de 0,5 g/animal. No segundo experimento, foram utilizados 18 animais, distribuídos em delineamento inteiramente casualizado, em esquema fatorial 2x3, sendo dois níveis de oferta de concentrado e três classes sexuais. As dietas foram isoprotéicas (12,5% PB) e o volumoso utilizado foi a silagem de milho. A síntese ruminal de compostos nitrogenados foi estimada pela excreção urinária de derivados de purinas. Todos os indicadores estimaram adequadamente a produção de MSF, independente do período de coleta, com exceção da FDAi. Não houve diferença entre o Cr2O3 e o TiO2 para estimar o consumo individual de concentrado (P>0,05). Não houve diferença (P>0,05) entre as classes sexuais e o nível de oferta de concentrado para o CMS e as digestibilidades dos nutrientes, com exceção do extrato etéreo. A eficiência microbiana média encontrada foi de 133,42 g de PB microbiana/kg de NDT. Portanto, recomenda-se utilizar três dias para coletas de fezes, por ser mais barato e menos trabalhos. A escolha entre Cr2O3 e o TiO2 para estimar o consumo de concentrado dependerá de sua disponibilidade e preço, e da facilidade de análises. A FDNi pode ser utilizada para se estimar o consumo individual de volumoso. Não houve diferenças para os consumos de matéria seca entre machos inteiros (MI), machos castrados (MC) e fêmeas (FE) da raça Nelore. A classe sexual não influenciou a produção de proteína microbiana. O terceiro experimento foi conduzido com os objetivos de estimar o consumo individual de matéria seca, de concentrado e de volumoso, de animais alimentados em grupo; avaliar o CMS, o ganho médio diário (GMD) e a conversão alimentar (CA) de animais de três classes sexuais, alimentados individualmente ou em grupo, recebendo dois níveis de oferta de concentrado; e determinar o efeito de níveis de oferta de concentrado (1,00 ou 1,25% do peso vivo) e de classe sexual sobre as características de carcaça de animais Nelore, terminados em confinamento. Também foi objetivo validar as equações de Hankins e Howe (1946), para estimação da composição física da carcaça, e de Paulino (2002) para estimação da composição de macrominerais no corpo vazio de bovinos Nelore; determinar as exigências de energia metabolizável para mantença e exigências líquidas de proteína, energia e macrominerais para ganho, além da eficiência de transformação das exigências líquidas de proteína em exigências de proteína metabolizável, em bovinos Nelore. Foram utilizados 45 bovinos Nelore (15 MC, 15 MI e 15 FE), sendo que nove animais (três de cada classe sexual) foram abatidos ao início do experimento para constituir o grupo referência. Os 36 animais restantes, sendo 12 de cada classe sexual, foram distribuídos em dois esquemas de alimentação (individual ou em grupo), alimentados com dois níveis de oferta de concentrado (1,00 ou 1,25% do PV), perfazendo um esquema fatorial 2x2x3. O experimento teve a duração de 112 dias. Para a estimativa do CMS individual dos animais alimentados em grupo foi utilizada a LIPE® para estimar a produção fecal, o Cr2O3 e o TiO2 para estimar o consumo de concentrado e a FDNi e FDAi para estimar o consumo de volumoso. Ao final do experimento, todos os animais alimentados individualmente foram abatidos para determinação do peso de corpo vazio e composição corporal, sendo que suas meias-carcaças direitas foram divididas em cinco cortes comerciais. Da meia-carcaça esquerda foi retirada a seção HH. As exigências líquidas para ganho de peso foram obtidas derivando-se a equação de predição do conteúdo corporal de cada nutriente, em função do logaritmo do peso de corpo vazio (PCVZ). As exigências de energia metabolizável para mantença foram estimadas a partir da regressão linear da energia retida (ER) em função do consumo de energia metabolizável. A eficiência de conversão das exigências líquidas de proteína em exigências de proteína metabolizável foi obtida pela equação de regressão entre a proteína retida e o consumo de proteína metabolizável. Não houve efeito (P>0,05) do nível de concentrado e do esquema de alimentação sobre o CMS, o GMD e a CA dos animais. Os machos inteiros foram superiores aos MC no rendimento de alguns cortes básicos e estes foram superiores às FE. A equação proposta por Paulino (2002) foi eficiente para se estimar a concentração de Ca no corpo vazio. Para todos minerais estudados foram propostas novas equações para estimar o conteúdo corporal a partir da sua concentração na seção HH. As equações obtidas para estimativa da energia retida foram: ER = 0,0559*PCVZ0,75*GPCVZ1,1037 (MI); ER = 0,0738*PCVZ0,75* GPCVZ0,9688 (MC); ER = 0,1020*PCVZ0,75*GPCVZ1,0408 (FE). O esquema de alimentação não alterou o CMS nem o GMD de animais Nelore de diferentes classes sexuais. Os machos inteiros ganharam aproximadamente 24% a mais de peso do que MC. Os machos castrados ganharam aproximadamente 22% a mais de peso do que FE. Ofertas de concentrado entre 1,00 e 1,25% do PV não influenciaram as principais características de carcaça e seu rendimento, em bovinos Nelore. Os machos inteiros foram mais eficientes que MC e FE, na deposição de carne e apresentaram maiores rendimentos em alguns cortes básicos. As exigências líquidas de energia para ganho aumentaram na medida em que se aumentou o peso vivo dos animais enquanto as exigências líquidas de proteína para ganho diminuíram na medida em que se aumentou o peso vivo dos animais. As exigências líquidas de energia para mantença foram maiores em fêmeas em relação à machos e a eficiência de conversão das exigências líquidas de proteína em exigências de proteína metabolizável foi aproximadamente 50%. Conclui-se que é possível estimar a composição de macrominerais no corpo vazio de bovinos Nelore a partir da seção HH. O quarto experimento teve o objetivo de determinar as frações A e B bem como a taxa de degradação ruminal (Kd), da matéria seca e da proteína bruta, de 27 alimentos utilizados na alimentação de bovinos. Além disso, foi objetivo do trabalho determinar a digestibilidade intestinal da proteína não degradada no rúmen pelas técnicas do saco de náilon móvel e de três estágios. Foram utilizadas quatro fêmeas da raça Nelore, fistuladas no rúmen e no duodeno, e mantidas em sistema de alimentação à vontade, à base de silagem de milho e concentrado. Para a obtenção da degradabilidade ruminal da MS e da PB, os alimentos foram incubados nos tempos 0, 2, 4, 6, 12, 16, 24, 48 e 72 horas, sendo posteriormente determinadas a MS e N total dos resíduos da incubação. As degradabilidades ruminais da PB foram calculadas utilizando-se o modelo proposto por Ørskov e McDonald (1979). Para determinação da digestibilidade intestinal (DI) da proteína bruta foram utilizadas as técnicas do saco de náilon móvel e de três estágios. A maioria dos dados de degradação ruminal da matéria seca e da proteína bruta obtidos estão de acordo com dados encontrados na literatura. A técnica dos três estágios não estimou satisfatoriamente a DI de todos os alimentos avaliados, com exceção dos alimentos protéicos. O farelo de soja foi o alimento que apresentou maior DI da PB (93,95%), seguido pelo milho (85,58%), farelo de algodão 46% de PB (84,93%) e glúten de milho (82,98%), quando se utilizou a técnica do saco de náilon móvel. Concluiu-se que a maioria dos alimentos avaliados possui digestibilidade total da PB maior ou próxima de 90%, com exceção das casca de soja, de café e de cacau e das silagens de milho e de capim-elefante. Recomenda-se a utilização da equação DIPB (%) = 5,1906 + 1,1053*X, para corrigir a DI obtida pela técnica dos três estágios para alimentos não protéicos, sendo X a DI obtida pela técnica de três estágios.

Depuis le sommet mondial sur le développement durable de Johannesburg 2002 et le Congrès Mondial des Aires Protégées (Afrique du Sud, 2003), les Aires Marines Protégées (AMP) sont perçues comme des outils de conservation et de développement durable. Leur contribution dans la conservation de la biodiversité marine et la reconstitution des stocks halieutiques est jugée déterminante pour une protection fondée sur la gouvernance participative qui associe les populations locales au projet de conservation et le développement durable. C'est pourquoi, les AMP comme le Parc National du Banc d'Arguin (PNBA) ont pris une importance majeure pour l'atteinte de ces objectifs. Le PNBA est une zone de reproduction des poissons et oiseaux et de frayère d'une grande importance. Malgré ses atouts, l'espace du PNBA est exposé à de multiples défis. On assiste depuis quelques années à une raréfaction progressive des ressources halieutiques et le développement de l'effort de pêche. La spécificité du PNBA est dû aussi à sa population résidante ; les Imraguen, qui sont les seules autorisés à y pratiquer la pêche à l'aide de planches à voile latine de type canarienne. L'objectif de cette recherche est ainsi de questionner les modalités participatives des différents acteurs (Direction du PNBA, populations locale Imraguen et la FIBA). Il essaie de s'interroger sur l'amélioration de la gouvernance participative dans le Parc National du Banc d'Arguin (PNBA). Les résultats de cette thèse mettent en exergue les principales contraintes qui empêchent l'amélioration de la gouvernance dans cet espace. Ainsi, nous proposons une reconfiguration de cette gouvernance par l'amélioration du fonctionnement de ces espaces publiques de concertation, mais aussi une meilleure collaboration entre les différents acteurs et notamment par une meilleure implication des Imraguen dans la réalisation des actions de conservation et de développement durable dans le PNBA
Since the World Summit on Sustainable Development in Johannesburg in 2002 and the World Congress on Protected Areas ( South Africa, 2003) , Marine Protected Areas (MPAs ) are seen as tools for conservation and sustainable development. Their contribution to the conservation of marine biodiversity and the recovery of fish stocks is considered crucial for protection based on participatory governance which involves local people in the project of conservation and sustainable development. That is why the AMP as the National Park of Banc d'Arguin (PNBA), took a major importance for achieving these objectives. The PNBA is a breeding ground for fish and birds and spawning of great importance. Despite its strengths, space PNBA is exposed to multiple challenges. The last few years have seen a gradual depletion of fishery resources and the development of fishing effort. The specificity of the PNBA is the also its resident population: Imraguen, which are only allowed to go fishing with lanches Canarian lateen sail types. The objective of this research was thus questioning participatory manner different actors (Directorate of PNBA, Imraguen local populations and FIBA ). He tries to examine how improving participatory governance in the National Park of Banc d'Arguin (PNBA). The results of this thesis highlight the main constraints to improving governance in this space. Thus, we propose a reconfiguration of this governance by improving the functioning of these public spaces for dialogue , but also a better collaboration between different stakeholders and in particular through better involvement of Imraguen in achieving conservation action and sustainable development in the PNBA

La protease de hiv-1 constitue une cible therapeutique interessante. Afin d'eviter les inconvenients d'une production virale, un gene synthetique a ete assemble puis exprime chez e. Coli. Deux versions du gene ont ete construites. L'une correspond a la forme mature de l'enzyme (pr100), l'autre code pour un precurseur (pr107). L'expression de la protease a ete optimisee grace a un outil genetique mis au point au laboratoire: la serie de plasmides para, inductibles a l'arabinose. D'apres une estimation realisee a l'aide de fusions avec lacz, 0,4mg a 4mg de protease soluble et directement active dans les conditions physiologiques peuvent etre obtenus par gramme de cellules. La purification de l'enzyme a donc ete entreprise. La sequence n-terminale supplementaire de la forme pr107 presente un effet amplificateur d'expression lorsqu'elle est placee en amont du gene de la protease mais aussi de lacz. Ce phenomene depend du promoteur et semble intervenir lors de la traduction. La protease recombinante est capable de s'automaturer in vivo des cotes n et c-terminaux a partir de ses formes fusionnees avec la beta-galactosidase. Des substitutions realisees sur les residus p4 et p5 de la jonction protease/beta-galactosidase, ainsi qu'une mutagenese systematique de l'acide amine en position p3 ont montre que la nature de ces residus n'etait pas determinante pour la proteolyse. Quelques inhibiteurs ont ete testes en conditions complexes et en presence du substrat naturel de la protease, la polyproteine gag. Des mutations ponctuelles dans l'enzyme ont revele l'importance des residus p(9), g(86) et n(87), particulierement conserves dans les proteases retrovirales

La plupart des traitements anticancéreux, comme la chimiothérapie ou la radiothérapie, sont cytotoxiques et causent des dommages à l'ADN dans le but d’induire la mort des cellules tumorales. Cependant, l’efficacité d’activité de réparation de l'ADN des tumeurs entraine des résistances intrinsèques et acquises aux traitements. L'une des étapes précoces de la réparation de l’ADN est le recrutement de protéines au niveau du site de dommage. Ce recrutement est coordonné par une cascade de modifications et est contrôlé par des protéines senseurs telles que la protéine kinase ADN dépendante (DNA-PK) et / ou la poly (ADP- ribose) polymérase (PARP). Dans ce manuscrit, nous avons identifié et caractérisé le mécanisme d'action de petites molécules d'ADN (les siDNA), mimant des cassures double brin (appelé Dbait) ou simple brin (appelé Pbait), dans l’inhibition des voies de réparation des cassures simple brin (SSBR/BER). Nous démontrons que les molécules Dbait recrutent et activent à la fois PARP et DNA-PK, contrairement aux molécules Pbait qui ne recrutent que la PARP. L'étude comparative de ces deux molécules permet d'analyser les rôles respectifs des deux voies de signalisation: les deux molécules recrutent les protéines impliquées dans la voie de réparation des cassures simple brin (comme PARP, PCNA et XRCC1) et empêchent leurs recrutements aux niveaux des lésions chromosomiques. Les molécules Dbait inhibent par ailleurs le recrutement des protéines impliquées dans la voie de réparation des cassures double brin (NHEJ et HR). Pbait et Dbait désorganisent la réparation de l’ADN et sensibilisent les cellules tumorales aux traitements. L’inhibition de la réparation des cassures simple brin semble dépendre d’un piégeage des protéines directement sur les siDNA ou indirectement sur les polymères PAR. L’inhibition des voies de réparation des cassures double brin (DSB) semble par contre se faire de façon indirecte ; cette inhibition résulterait plutôt de la phosphorylation des protéines de réparation des DSB de part l’activation de DNA-PK. Les molécules Dbait et Pbait induisent un effet de létalité synthétique des cellules tumorales BRCA mutées. Cependant, la mutation BRCA semble être suffisante mais non nécessaire pour induire la sensibilité des cellules tumorales aux traitements Dbait. En effet, nous avons démontré que les molécules Dbait peuvent aussi sensibiliser les cellules ne présentant pas de mutation BRCA mais ayant toutefois une forte instabilité génétique. Nous avons trouvé une corrélation entre le niveau basal de protéines de réparation de l'ADN (ɣH2AX, PARP et PAR), le taux basal de cassures à l’ADN, la présence de micronoyaux (MN) et la sensibilité des cellules tumorales au traitement Dbait. Nous avons émis l’hypothèse que cette instabilité génétique, déterminé par la quantification de MN dans des biopsies tumorales, pourrait être un biomarqueur prédictif de l’effet du Dbait, non seulement dans les cancers du sein, mais aussi dans les glioblastomes, les mélanomes, les mélanomes uvéaux et les cancers du côlon
Most conventional cancer treatments, such as chemotherapy or radiotherapy, are cytotoxic and cause DNA damages in the tumoral treated cells, which ultimately lead to their death. However, several intrinsic and acquired resistances of tumors to these treatments are due to the tumor efficient DNA repair activities. One of the major early steps of DNA repair is the recruitment of repair proteins at the damage site and this is coordinated by a cascade of modifications controlled by sensor proteins such as DNA-dependent protein kinase (DNA-PK) and/or poly (ADP-ribose) polymerase (PARP). In this manuscript, we identify and characterize the mechanism of action of short interfering DNA molecules (siDNA), mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) in Single Strand Break Repair pathway (SSBR/BER) inhibition. We demonstrate that Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. The comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both molecules recruit proteins involved in single-strand break repair (such as PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break (DSB) repair. By these ways, Pbait and Dbait disorganized DNA repair, thereby sensitizing cells to treatments. SSB repair inhibition depends upon a direct trapping of the main proteins on both molecules and an indirect trapping in PAR polymers. DSB repair inhibition may be indirect, resulting from the phosphorylation of DSB repair proteins by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations tumoral cell lines. However, BRCA mutation could be sufficient but not necessary to induce breast cancer cell lines and tumors sensitivity to Dbait treatment. In fact, we demonstrate that Dbait molecules could also have a stand-alone effect in BRCA wild type cells with a high genetic instability. We found a correlation between DNA repair proteins basal level (ɣH2AX, PARP and PAR), DNA break basal level, presence of micronucleus (MN) and tumoral cell lines sensitivity to Dbait treatment. We hypothesis that this genetic instability, determined by MN in tumor biopsies, could be a predictive biomarker of Dbait stand-alone effect, not only in breast cancer treatment, but also in glioblastoma, melanoma, uveal melanoma and colon cancer treatment

Le cancer du sein de phénotype « Basal-like » triple négatif (BLTN) est particulièrement agressif et de mauvais pronostic. Il est insensible aux traitements hormonaux laissant pour seule stratégie de traitement la chimiothérapie conventionnelle. De ce fait, de nouvelles thérapeutiques ciblées sont en développement, tels que les inhibiteurs de la Poly-ADP-Ribose-Polymerase (PARP). Dans ce contexte, nos travaux de recherche ont été orientés sur l’optimisation du traitement des cancers du sein BLTN en modélisant l’action d’un anti-PARP modèle, l’Olaparib sur la lignée SUM1315 de phénotype BLTN. Dans un premier temps, l’étude de la coexpression de la BCRP et de la P-gp, deux protéines « Multidrug Resistance » (MDR) majeures en présence de 50 µM d’Olaparib® a montré une induction de leurs expressions chez les cellules SUM1315, avec une réponse de type relais. La BCRP établirait une première ligne de défense cellulaire et son action serait ensuite relayée par la P-gp durant 24h de traitement. Ce mécanisme est en corrélation avec la concentration intracellulaire d’Olaparib mesurée par HPLC. L’ensemble de nos résultats suggère qu’il serait possible de contourner le mécanisme de résistance induit par les protéines MDR si une concentration stable en Olaparib est maintenue dans les cellules à long terme. Nous avons ensuite étudié la potentialisation de l’action de l’Olaparib en le combinant avec un traitement par radiothérapie à basse et haute énergie, sur la viabilité des cellules de la lignée SUM1315. La comparaison des résultats avec un traitement Olaparib seul ou irradiation seule et ceux des traitements combinés Olaparib/radiothérapie a alors mis en évidence un effet synergique des deux traitements sur la viabilité cellulaire. L’effet synergique de cette combinaison fonctionne même avec de faibles doses d’Olaparib. De cette manière, il serait possible de réduire les doses d’anti-PARP utilisées tout en gardant les bénéfices du traitement. Enfin, nous avons développé deux techniques de cultures cellulaires en trois dimensions (i) « hanging drop » et (ii) « liquid overlay », permettant de mimer plus fidèlement les conditions des tumeurs in vivo. L’observation en microscopie électronique à transmission et à balayage des sphéroïdes obtenus par ces deux techniques a permis de démontrer l’intégrité des cellules au sein des sphéroïdes ainsi que la formation de jonctions cellulaires. Cependant, les sphéroïdes obtenus en « liquid overlay » ont montré une meilleure intégrité ultra-structurale
« Triple Negative Basal-Like » (BLTN) breast cancer is particularly aggressive and of poor prognosis. It is insensitive to hormone-targeted therapies leaving conventional chemotherapy as the only treatment strategy. Therefore, new promising targeted therapies are being developed, such as Poly-ADP-Ribose-Polymerase inhibitors (anti-PARPs). In this context, our research has been directed towards optimizing the treatment of BLTN breast cancer by modelling the action of an anti-PARP model, Olaparib®, on BLTN cell line SUM1315. Firstly, the study of the co-expression of BCRP and P-gp, two major “Multidrug Resistance” proteins (MDR) in the presence of 50 µM Olaparib® showed an induction of their expression in SUM1315 cells, with a relay-type response. BCRP would establish a first line of cellular defense and its action would then be taken over by P-gp, for 24h of treatment. This mechanism is correlated with the intracellular concentration of Olaparib® measured by HPLC. All of our results suggest that it would be possible to circumvent the induced MDR resistance mechanism if a stable concentration of Olaparib® is maintained in cells in the long term. Secondly, we studied the potentiation of the action of Olaparib® combining it with low and high-energy radiations on the viability of SUM1315 cells. Comparison of the results with single Olaparib®, single irradiation, or the combination of Olaparib®/radiotherapy then demonstrated a synergistic effect of the two treatments when delivered concomitantly, on cell viability. The synergistic effect of this combination works even with low doses of Olaparib®. In this way it would be possible to reduce the anti-PARP doses while maintaining the benefits of this treatment. Finally, we have developed two techniques of cell culture in three dimensions: (i) "hanging drop" and (ii) "liquid overlay", in order to mimic more accurately the conditions of tumours in vivo. Observations of spheroids obtained by these two techniques by transmission and scanning electron microscopy demonstrated the integrity of cells within as well as the formation of cell junctions. However, the spheroids obtained by "liquid overlay" showed better ultra-structural integrity

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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
The aim of this study was to characterize the chemical composition of the roasted baru almond, which is derived from different native plants in the west of the State of Goiás, and to compare its protein quality to that of the peanut, cashew nut and Brazil nut. Standardized methods were used to determine centesimal composition, amino acid profile, fatty acids and mineral content. The experiment was carried out with 42 male weanling Wistar rats. The animals were randomly assigned into seven groups. The experiment lasted fourteen days. The diets were formulated according to AIN-93G, six diets with 10% protein: CAS7 (7% lipid casein), CAS15 (15% lipid casein), BA (baru almond), PN (peanut), CN (cashew nut), BN (Brazil nut) and a protein-free diet. The consumption was controlled according to the energy value of the diets. The protein value was estimated using NPR (Net Protein Ratio) and PDCAAS (Protein Digestibility-Corrected Amino Acid Score). The seeds and nuts are sources of proteins (16.3 to 32.3g.100g-1) and lipids (42.7 to 57.9g.100g-1). The protein content of baru almonds varied significantly (Tukey, p<0.05) between plants (24.2 to 31.9g.100g-1). The amino acid profile of baru almonds also varied significantly between plants. Lysine (Limiting Essential Amino Acid - LEAA of 43% to 72%) and sulfur amino acids (LEAA of 78% to 90%) were the limiting amino acids, according to the WHO/FAO/UNU standard for school children. The best 6: 3 fatty acid ratio (9: 1) was found in the baru almond, which is rich in iron (3.6 to 4.7mg.100g-1) and zinc (2.8 to 4.1mg.100g-1). The cashew nut had a relative NPR of 81%, followed by the Brazil nut (68%), peanut (61%) and baru almond (45%). The PDCAAS varied significantly between baru plants (32% to 68%), and between the other samples (63% for the BN, 69% for the PN and 88% for CN). There are differences in the nutritional value of baru almonds from different plants in the same Cerrado subpopulation and between different seeds and nuts. These foods present a high density of nutrients such as proteins, lipids, minerals and dietary fiber. For this reason, their inclusion in the diet and use in home and industrial food processing should be encouraged.
Este trabalho teve como objetivo caracterizar quimicamente a amêndoa de baru torrada, oriunda de diferentes plantas nativas da região Oeste do estado de Goiás e avaliar sua qualidade protéica, comparando-a com o amendoim, castanha-de-caju e castanha-do-pará. Determinou-se a composição centesimal, perfil de aminoácidos, ácidos graxos e conteúdo mineral, conforme métodos padronizados. Foi realizado experimento com 42 ratos Wistar, machos, recém-desmamados, distribuídos em sete grupos segundo delineamento por blocos casualizados, durante catorze dias. As dietas foram formuladas segundo AIN-93G, sendo seis dietas com 10% de proteína: CAS7 (caseína 7% de lipídios), CAS15 (caseína 15% de lipídios), AB (amêndoa de baru), AMD (amendoim), CJ (castanha-de-caju), CP (castanha-dopará) e uma dieta aprotéica. O consumo foi controlado segundo valor energético das dietas. O valor protéico foi estimado por meio do NPR (Net Protein Ratio) e PDCAAS (Protein Digestibility-Corrected Amino Acid Score). As sementes e nozes são fontes de proteínas (16,3 a 32,3g.100g-1) e lipídios (42,7 a 57,9g.100g-1). O conteúdo protéico das amêndoas de baru variou significativamente (Tukey, p<0,05) entre plantas (24,2 a 31,9g.100g-1). O perfil de aminoácidos das amêndoas de baru também variou significativamente entre plantas, sendo a lisina (Escore de Aminoácidos Essenciais - EAE de 43% a 72%) e os sulfurados (EAE de 78% a 90%) os aminoácidos limitantes, segundo padrão WHO/FAO/UNU para escolares. Constatou-se melhor relação de ácidos graxos 6: 3 (9:1) na amêndoa de baru, que é rica em ferro (3,6 a 4,7mg.100g-1) e zinco (2,8 a 4,1mg.100g-1). A castanha-de-caju apresentou NPR relativo de 81%, seguida pela castanha-do-pará (68%), amendoim (61%) e amêndoa de baru (45%). O PDCAAS variou significativamente entre as amêndoas de baru (32% a 68%), e entre as demais amostras (63% para a CP, 69% para o AMD e 88% para a CJ). Existe diferença no valor nutricional de amêndoas de baru de diferentes plantas de uma mesma subpopulação do Cerrado, assim como entre diferentes sementes e nozes. Esses alimentos apresentam alta densidade de nutrientes como proteínas, lipídios, minerais e fibras alimentares. Portanto, a inclusão na dieta e o uso em formulações culinárias e industrializadas desses alimentos devem ser estimulados.

Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós–Graduação em Aqüicultura, Instituto de Oceanografia, 2011.
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Como resultado da estagnação das pescarias industriais, a aquacultura surge como uma alternativa ao setor pesqueiro. Porém, a alta dependência da farinha de peixe utilizada como principal fonte protéica nas rações de muitas espécies na aquacultura, mostra que a pressão sobre os estoques de pequenos peixes pelágicos continua alta. Além disso, a produção de farinha de peixe se mantém estabilizada nas últimas décadas, com os preços variando de acordo com a oferta e procura. Assim, muitos esforços têm sido direcionados na busca por ingredientes protéicos alternativos mais baratos e mais disponíveis para o uso em rações, como é o caso dos produtos de base vegetal e dos subprodutos da pecuária e da avicultura. O farelo de soja oferece uma fonte econômica e nutricionalmente viável de proteína, porém fatores antinutricionais, baixa palatabilidade e deficiência de aminoácidos e ácidos graxos essenciais podem limitar sua utilização em rações para L. vannamei. Por outro lado, o concentrado protéico de soja (CPS) apresenta características superiores ao farelo, como razoável perfil de aminoácidos, altos valores protéicos, energéticos e de digestibilidade. Porém, a utilização desses ingredientes depende em grande parte do requerimento nutricional da espécie, dos preços relativos à farinha de peixe e de regulamentações ambientais dos sistemas de produção. Outro ingrediente que vem obtendo sucesso como substituto da farinha de peixe em rações para L. vannamei são os flocos microbianos formados em ambientes de criação super-intensiva de peixes e camarões. Além de possuir inúmeras vantagens do ponto de vista nutricional, traz também benefícios ambientais com a reutilização dos efluentes. A aquacultura apenas poderá exercer seu papel na contribuição para a demanda mundial de proteína se reduzir a utilização de peixes na alimentação e adotar práticas de manejo mais ecológicas.
Aquaculture is an alternative to the fishing sector due to industrial fisheries stagnation. However, the pressure on small pelagic fish stocks remains high because for fish meal production, main protein source used in diets for many aquaculture species. Moreover, fish meal production is stabilized in recent decades, with prices varying according to supply and market demand. Thus, many efforts have been done on the search for cheaper and more available protein ingredients for animal feed, like plant-based products and by-products of livestock and poultry production. The soy bean meal offers an economically and nutritionally viable source of protein. However, problems such deficient levels of indispensable amino acids and fatty acids, anti-nutrients and poor palatability can restrict its use in L. vannamei feeds. On the other hand, soy protein concentrate presents superior characteristics than soy bean meal, like suitable amino acids profile, high protein and energetic values and good digestibility. Nevertheless, the use of these ingredients depends largely on: species nutritional requirement, fish meal prices and environmental regulations for production systems. Another ingredient that has being used successfully as a substitute for fishmeal in diets for L. vannamei are microbial flocs formed in super-intensive fish and shrimp farming. Besides having numerous profits on nutrition, it also brings environmental benefits because of the effluent reuse. Aquaculture can only play its role in contributing to the global protein demand if we start to reduce fish meal use and adopt environmental friendly practices.

PEREIRA, Fábia de Mello. Desenvolvimento de rações protéicas para abelhas apis mellifera utilizando produtos regionais do nordeste brasileiro. 2005. 191 f. Tese (doutorado em zootecnia)- Universidade Federal do Ceará, Fortaleza-CE, 2005.
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The objective of this research was to develop a protein diet for honeybees Apis mellifera using regional products of NE Brazil that are of easy access and reduced costs for beekeepers. Experiments were carried out between March 2001 and January 2005 in the “Núcleo de Pesquisa com Abelhas” (NUPA) of the Embrapa Meio-Norte in Teresina (5°05’ S and 42°49’ W) and in Castelo do Piauí (5º20’ S and 41º34’ W), Piauí, Brazil. Products selected at the beginning of the research were cassava hay (Manihot esculenta), leucaena hay (Leucaena leococephala), mesquite pod meal (Prosopis juliflora), “bordão-de-velho” pod meal (Pithecellobium cf. saman), babassu bran (Orbygnia martiana) and succedaneums for calfskin from Purina®. The performance of honeybees that consumed these components was compared with those that consumed pollen obtained from COORPEPÓLEN, Cooperativa de Pólen do Brasil, located in Canavieiras, Bahia, Brazil. Pollen fed to bees was predominantly Palmae pollen. Initial components selected were tested about toxic effects for honeybees; contents of crude protein, total soluble sugars, free amino acids, and contents of glycin, alanine, valine,leucine, isoleucine, phenylalanine, tyrosine, serine, methionine, asparagines, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, asparagines and -aminobutyric acid. Results showed that the high content of sugars in the flour of “bordão-de-velho” does not allow its use for feeding honeybees, considering that it was observed an early mortality in the honeybees feeding with this meal. The other substances studied did not show any toxic effect, but only the leucaena hay have the contents of essential amino acids demanded by the honeybees. These results permitted the formulation of four diets: (T01) - 260 g of cassava hay, 140 g of mesquite pod meal, 437,39 g of sugar syrup and 0,96 g of vanilla essence; (T02) - 68 g of cassava hay, 332 g of babassu bran, 643,90 g of sugar syrup and 1,32 g of vanilla essence; (T03) - 304 g of babassu bran, 96 g of succedaneums for calfskin, 507,73 g of sugar syrup and 1,08 g of vanilla essence and (T04) – 500 g of pollen and 254,79 of sugar syrup. The feeds were tested for consumption, colony development and digestibility. Results showed that the three diets formulation did not show the same consumption and brood maintenance that pollen. However, by the end of the experiment all colonies were in better conditions than in the beginning, with higher food area and colony weight. The higher digestibility observed in the tests of digestibility could be attribute to the high consumption of sugar syrup and water. Results give evidence that all diets were efficient in maintaining the colonies strong and can be used by the beekeepers to maintain colony strength over the leanest period of the year. However, when the diet was the single source of protein, it was necessary to search for other alternative.
A pesquisa foi realizada com o objetivo de desenvolver uma ração para abelhas Apis mellifera usando produtos regionais do Nordeste de fácil acesso e baixo custo para o produtor. Os experimentos foram conduzidos entre março de 2001 e janeiro de 2005 no Núcleo de Pesquisa com Abelhas (NUPA) da Embrapa Meio-Norte com sede em Teresina (5°05’ S de latitude e 42°49’ W de longitude) e nos apiários experimentais em Castelo do Piauí (5º20’ S de latitude e 41º34’ W de longitude). Levando-se em consideração os alimentos fornecidos às abelhas pelos apicultores, a facilidade dos mesmos serem colhidos, produzidos ou encontrados comercialmente na região e a preferência natural das abelhas, medida por observações empíricas, iniciou-se o trabalho com feno das folhas de mandioca (Manihot esculenta); feno das folhas de leucena (Leucaena leococephala); farinha de vagem de algaroba (Prosopis juliflora); farinha de vagem de bordão-de-velho (Pithecellobium cf. saman); farelo de babaçu (Orbygnia martiana) e sucedâneo do leite para bezerros da marca Purina®. O desempenho das abelhas alimentadas com estes componentes foi comparado com o desempenho das abelhas alimentadas com pólen adquirido da COORPEPÓLEN, Cooperativa de Pólen do Brasil, localizada na cidade de Canavieiras, Bahia, havendo predominância do pólen de Palmae. Inicialmente os componentes selecionados foram testados quanto à toxicidade e analisados quanto aos teores de proteína bruta, açúcares livres totais, aminoácidos totais e teores de glicina, alanina, valina, leucina, isoleucina, fenilalanina, treonina, serina, metionina, aspargina, glutamina, aspartato, glutamato, lisina, arginina, histidina, asparagina e -aminobutirato. Os resultados demonstram que o alto teor de açúcares contido na farinha de bordão-de-velho não permite que a mesma seja fornecida às abelhas na forma in natura, uma vez que houve uma mortalidade precoce das abelhas alimentadas com esta farinha. Os demais alimentos não se mostraram tóxicos, porém, somente o feno da leucena contém os teores de aminoácidos essenciais requeridos pelas abelhas Apis mellifera. Com base nos dados obtidos foram formuladas as seguintes composições de rações: (T01) - 260 g de feno de mandioca, 140 g de farinha de algaroba, 437,39 g de xarope e 0,96 g de essência de baunilha; (T02) - 68 g de feno de mandioca, 332 g de farelo de babaçu, 643,90 g de xarope e 1,32 g de essência de baunilha; (T03) - 304 g de farelo babaçu, 96 g de sucedâneo do leite, 507,73 g de xarope e 1,08 g de essência de baunilha e (T04) – 500 g de pólen apícola e 254,79 g de xarope. As rações foram testadas quanto ao consumo, desenvolvimento das colônias e digestibilidade. Os resultados demonstraram que apesar de nenhuma das três rações ser tão palatável e nem tão eficiente quanto o pólen na manutenção das crias, contribuíram para que as colônias ao final do experimento estivessem em condições melhores do que as iniciais, com maior área de alimento e maior peso. Os resultados do teste de digestibilidade demonstraram que os alimentos fornecidos tiveram altos índices de digestbilidade, o que pode ser atribuído ao alto consumo de xarope invertido e de água. Com os resultados obtidos pode-se recomendar as rações formuladas a apicultores como suplementação alimentar para manutenção dos enxames fortes, entretanto, em situações em que as rações passam a ser a única fonte protéica fornecida às abelhas seria necessária a busca de novas alternativas.

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Universidade Federal de Sao Carlos
Enzymatic hydrolyzed proteins present some advantages over the pool of amino acids obtained after a chemical hydrolysis: lower osmolality (easier absorption by the organism) and better sensorial characteristics. The removal of phenylalanine (Phe) from these hydrolysates allows their use in the diet of phenylktonuria (PKU) patients. This Thesis studies the sequential hydrolysis of cheese whey proteins. This process aims at producing a protein hydrolysate with low contents of Phe, after two reaction stages: in the first one, concentrated cheese whey proteins are hydrolyzed by the endoprotease chymotrypsin, immobilized on agarose gel. The substrate of the second stage is the product of the first one. This reaction is the central focus of this Thesis, and consists in a hydrolysis using the exoprotease carboxipeptidase A (CPA), immobilized on the same matrix. Since the scope of this work was a process for obtaining a protein hydrolysate with low contents of Phe for phenylketonurics, another stage had to be incorporated: ultrafiltration, to separate the amino acids released by the action of CPA, mainly Phe (and other amino acids that inhibit the reaction). With this purpose, an enzymatic membrane reactor (EMR) is proposed. It should be noticed that, to the best of our knowledge, the reactor design presented here has not been published yet. The EMR had spherical agarose particles within the reaction space, retained by a stainless steel mesh (400 Tyler). Hence, the ultrafiltration membrane was in charge only of the separation of the amino acids in the hydrolysate, which was continuously recycled to the flask containing the immobilized enzyme. Assays to characterize the membranes were performed. These membranes were then used in the integrated process (reaction with immobilized enzyme coupled to the ultrafiltration), for different experimental conditions. The substrates where Prato and Minas Frescal cheese whey. Two set-ups of the ultrafiltration unit were tested: flat plate and hollow fiber, both with 1 kDa cut-off. The reactor volume/membrane area ratios were 7.5×10-2 cm and 76.9×10-2 cm, respectively. Finally, the performance of three systems was compared: EMR with flat plane and hollow fiber and a batch reactor, followed by diafiltration. The hollow fiber EMR showed the best performance (85% conversion, productivity of 275.2×10-7 ghidrolisado/mgPhe/UH-PHE/h and only 1% retention of Phe in the membrane, after assays of 10 h). The resulting product was appropriate for phenylketonurics, with high protein contents (53.4 g/L), mainly constituted by small peptides (≤ 5.8 kDa) and with 19.9 mgPhe/gProteína, within the admissible range for PKU
Os hidrolisados protéicos via enzimática apresentam algumas vantagens, em relação à mistura de aminoácidos livres obtida pela hidrólise química: menor osmolalidade (e, portanto, maior facilidade de absorção pelo organismo) e características sensoriais mais agradáveis. Por sua vez, a remoção da fenilalanina (Phe) desses hidrolisados permite a utilização dos hidrolisados na dieta de fenilcetonúricos. Nesta Tese foi estudada a hidrólise enzimática seqüencial de proteínas de soro de queijo, processo proposto para obtenção de um hidrolisado protéico, com reduzido teor de Phe, e constituído de duas etapas reacionais: na primeira, as proteínas do soro de queijo concentrado são hidrolisadas pela endoprotease quimotripsina, imobilizada em gel de agarose. A segunda etapa, cujo substrato é o produto da primeira proteólise, é o foco central desta Tese, e consiste na hidrólise do substrato prehidrolisado utilizando-se a exoprotease carboxipeptidase A (CPA), imobilizada na mesma matriz. Como o escopo do trabalho era o processo para obtenção de um hidrolisado protéico com baixo teor de Phe para pacientes fenilcetonúricos foi necessário incorporar outra etapa: ultrafiltração, para separar os aminoácidos liberados por ação da CPA, principalmente Phe (além de outros aminoácidos inibidores da reação). Para isso, propõe-se trabalhar com reator enzimático de membrana (REM). Destaque-se que a concepção de reator aqui apresentada é, até onde vai nosso conhecimento, inédita. Estudou-se um reator de membrana com a enzima imobilizada em partículas esféricas de gel de agarose, retidas no volume reacional propriamente dito por meio de um filtro de aço (400 Tyler). Assim, a membrana de ultrafiltração ficou a cargo apenas da separação dos aminoácidos presentes no hidrolisado, que era reciclado continuamente para o frasco contendo a enzima imobilizada. Foram realizados ensaios para caracterização das membranas utilizadas, para posteriormente serem realizados ensaios em diferentes condições experimentais no processo integrado (reação com enzima imobilizada acoplada à ultrafiltração), utilizando como substratos soro de queijo tipo Prato e Minas Frescal. Foram testadas duas configurações de unidades de ultrafiltração: placa plana e fibra oca, ambas com corte de 1 kDa As razões volume de reator/área de membrana testadas foram de 7,5×10-2 e 76,9×10-2 cm, rescpectivamente. Foi desenvolvido um modelo matemático do REM, e suas predições foram comparados com dados experimentais de hidrólise com CPA para ambos os sistemas. Finalmente, foi comparado o desempenho de três sistemas: reator com membrana de placa plana, reator com membrana de fibra oca e reator em batelada, seguido por diafiltração com membrana de fibra oca. O REM tipo fibra oca apresentou o melhor desempenho (85% de conversão, uma produtividade de 275,2×10-7 ghidrolisado/mgPhe/UH-Phe/h e apenas 1% de retenção de Phe na membrana, em ensaios de 10 h. O produto resultante desse sistema apresentou características próprias para ser utilizado como complemento protéico para pacientes fenilcetonúricos, com um alto conteúdo protéico (53,4 g/L) constituído majoritariamente por pequenos peptídeos (≤ 5,8 kDa) e com 19,9 mgPhe/gProteína valor que se encontra dentro da faixa tolerada para pacientes de fenilcetonúria