A preliminary test was conducted evaluating the effect of digestible lysine (dLys) on broiler breeder (BB) male semen quality from forty-one to forty-nine wk of age. Five dietary treatments: corn-soybean meal diet with 1,000 mg dLys/rooser/day (Soy1,000), distillers dried grains with solubles diet with 1,000 (DDGS1,000), 850 (DDGS850), 700 (DDGS700), and 550 (DDGS550) mg dLys/rooster/day. Semen quality was similar except percentage dead sperm which was higher in DDGS550. A second trail was conducted using the same dietary treatments as the preliminary test during twenty to thirty-nine wk of age. Semen quality was similar except percentage dead sperm which was higher in Soy1,000. Soy1,000 exhibited higher body weight (BW), breast weight, and plasma testosterone. In conclusion dLys levels from 1,000 to 700 mg/rooster/day will not adversely affect semen quality of BB males. Attention should be given to BW in BB as it could lead to an increased percentage dead sperm.

The effect of decreasing digestible lysine (dLys) intake by broiler breeder (BB) hens from 35 to 45 wk of age on their reproductive performance and performance of their progeny was evaluated. Two types of diets were fed: a diet from commercially available ingredients consisting of dLys intakes of 1,200 (IDL) and 1,010 mg/hen/day (ID) and a semi-purified diet with dLys intakes of 1,010 (SPL) and 600 mg/hen/day (SP). Hens fed the SPL and SP diets had lower hen-day egg production compared to BB hens fed the IDL and ID diets. Fertility and hatchability of eggs set were lowest (P <0.05) for hens fed the SPL diets. Chick weight at hatch was lower (P<0.05) for those that came from the SP and SPLed hens, but 42 and 56 day body weights (BW) were similar for all treatments. Marginal improvements (P<0.10) in FCR were seen at 42 and 56 days for chicks from IDed hens compared to IDL hens. A decrease in daily intake of dLys appeared to improve BB reproductive performance when hens were fed a semi-purified diet and the same response was not observed in hens fed a diet from commercially available ingredients. Furthermore, the progeny study revealed marginal improvements in some live performance parameters. In a second study, a diet based from corn-soybean meal and formulated to a dLys intake of 1,000 mg/hen/day (CS) and three diets composed primarily of corn, soybean meal, and DDGS with 1,000 (DDGS-1,000), 800 (DDGS-800), and 600 (DDGS-600) mg of dLys/hen/day were fed to evaluate the reproductive performance of BB hens from 24 to 42 wk of age. Feeding diets composed of commercially available ingredients with dLys intake levels below 1,000 mg/hen/day did not impact BB reproductive performance. Reduced BW, carcass and breast weight, and higher (P<0.05) back half weight at 42 days of age was observed from broilers that came from 26 wk old BB hens fed the DDGS-600 diet. Reducing dLys intake in later BB hen ages did not impact progeny performance or carcass characteristics. This suggests that Lys may be in dietary surplus concentrations for commercial breeders under current practical conditions.

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Previous research evaluated the dLys requirement and the impact of varying dLys and AME on Cobb MV x Cobb 500 broilers during the starter and grower phases, leaving the finisher phase to be determined. Experiment 1 estimated the finisher dLys requirement to range from 0.748-1.32% for males and 0.752-1.292% for females. Male broilers were more responsive to increasing dLys than females, though in general, increasing dLys improved performance and processing. Experiment 2 determined the effects of varying finisher dLys and AME on broiler performance and processing. Increasing dLys improved BWG, FCR, ALI, and breast yield while decreasing FI and fat pad yield. Increasing AME consistently reduced FI and FCR while increasing thigh and fat pad yield. Economic analyses for both experiments demonstrated that maximizing performance was not always economical, verifying the importance of considering feed costs and performance to optimize return on investment for Cobb MV x Cobb 500 broilers.

Adult female Sprague-Dawley rats, age 10 months, were used to estimate the minimum dietary lysine requirement for tissue maintenance. Ten animals were assigned to one of eight treatment groups by weight. The dietary lysine levels ranged from 0.06 to 0.36 % of diet and the feeding period lasted 56 days. Carcass and liver nitrogen and total serum proteins were determined, and a dietary lysine requirement was estimated from the data obtained. Carcass and liver analysis included weight, total nitrogen, percent protein, percent water and percent fat. Using a one-way analysis of variance, results showed no significant differences in carcass or liver composition between the treatment groups. The data indicated that the mature female rat has a dietary lysine requirement lower than 0.06 % of diet, or less than 20.1 mg/day/kg0.75. Since previous investigators used a protein based diet, a possible cause for the insignificant differences between values seen in this study may be a consequence of using a nonprotein, amino acid mix base supplemented with lysine. In future studies for determining the dietary lysine requirement in the adult female rat, dietary lysine levels below 0.06 % of diet must be included when using a nonprotein based diet. / M.S.

La gélatine est un ingrédient utilisé dans de nombreuses industries et sa solubilité influence beaucoup ses propriétés fonctionnelles. Des défauts de solubilité sont parfois constatés, notamment suite à un stockage de la gélatine en grains à température élevée et humidité importante. Cette perte de solubilité pourrait être due à la présence de molécules de haut poids moléculaire. L'objectif de ce travail est d’apporter des éléments de compréhension sur la perte de solubilité observée dans les solutions de gélatine. L'utilisation d’une technique de Fractionnement par Flux-Force couplée à une diffusion de la lumière Multiangulaire, a mis en évidence la présence d’agrégats de haut poids moléculaires dans les solutions de gélatine (de 9.5 à 30.2.105 g.mol-1). Ces agrégats n’avaient jamais pu être identifiés par les techniques classiques d'exclusion stérique car elles sont souvent éliminées dans le volume d'exclusion des colonnes. La quantité d'agrégats formés ne cesse d'augmenter lors d'un traitement thermique à 75 °C, aboutissant à l'insolubilité de la gélatine. La compréhension du mécanisme à l’origine de cette perte de solubilité montre l'implication de la lysine disponible, dans l'apparition des agrégats. La lysine libre réagissant au cours du traitement thermique provoquerait la formation d'agrégats qui modifient le comportement de la gélatine en solution aqueuse. Les paramètres de caractérisation de l’AFlFFF-MALS permettent de discriminer partiellement des échantillons de gélatine dont les comportements en solubilité sont différents. Rajoutés aux paramètres classiques de caractérisation comme la viscosité à 6.67% et au dosage de la lysine disponible, la discrimination devient parfaite. / Gelatin is an important product for several industries and its solubility dramatically influences its functional properties. The lack of solubility observed in gelatine is supposed to be due to the occurrence of molecules with high molecular weights, especially after heat treatments. In order to be able to predict the gelatin behaviour, a new technique for its analysis has been developed with an Asymmetrical Flow Field-Flow Fractionation (AFlFFF-MALS) coupled to a multiangular light scattering. The AFlFFF-MALS analysis showed high molecular weight fractions in gelatin ranging from 9.5 to 30.2 105 g.mol-1 which has not been shown previously with alternative techniques such as size exclusion chromatography. After heat treatment of dry gelatine in an oven at 75°C, some huge aggregates appeared, of which size and density increased and led to partial insolubilisation of gelatin into water. The mechanism responsible for this phenomenon involved lysine residues which plays a very important role in gelatin properties. Quantification of available lysine in gelatin samples by LC-UV has been developed. Thermal treatment during 8 days led to a decrease of free lysine content whereas, at the same time, the molecular weight of gelatin fractions increased and α helixes formation in solution was strongly affected. Intermolecular cross-links led to high molar mass compounds and limited protein chain unfolding. From an industrial point of view, AFlFFF-MALS analysis can help to discriminate gelatine samples in regard to their solubility. If other parameters are added (6.67 % viscosity and free lysine) the discrimination was perfect.

La lysine décarboxylase (LDC) appartient à une famille d'enzymes décamériques dépendantes du cofacteur PLP qui sont connus pour catalyser la réaction transformant la L-Lysine en cadavérine tout en consommant un proton. Dans les entérobactéries comme Escherichia coli, nous trouvons deux paralogues, LdcI et LdcC. LdcI permet de faire face à la bactérie au conditions hostile de pH acide lors du passage à travers du tract gastro-intestinale. LdcC est produite pendant la phase stationnaire et aussi quand les bactéries font face aux traitements antibiotiques. La cadavérine produite par les LDCs est connue pour protéger les bactéries du stress oxydant. Cela s’explique par le fait que la cadavérine bloque les porines de la membrane externe, réduisant ainsi la perméabilité des molécules responsables du stress acides et oxydant. L'activité des LDCs chez E. coli est coordonnée avec la réponse stringente qui est mise en place lorsque les microorganismes sont dans des conditions pauvres en nutriments, afin d’éviter l’épuisement intracellulaire de la L-Lysine nécessaire pour la synthèse des protéines. Cependant, cette inhibition peut être levée par la formation d'un complexe en forme de cage avec son partenaire RavA, permettant ainsi aux bactéries de faire face aux stress multiples. Etant donné que la réponse au stress est importante pour que les bactéries puissent exhiber leur pathogénicité, nous nous sommes demandés si la bactérie opportuniste Pseudomonas aeruginosa pourrait employer LdcA pour contrer des conditions de stress qui ont déjà été décrites pour LdcI chez les entérobactéries. Au cours de ma thèse, nous avons abordé cette question en utilisant différentes approches complémentaires. Tout d'abord, nous avons utilisé des fusions promoteur-gène et de l'analyse par Western-blot pour déterminer les conditions dans lesquelles le gène ldcA a été exprimé et sa protéine synthétisée. Nous avons pu observer que ldcA est exprimé sur la phase stationnaire de croissance dans des conditions aérobies en milieux riches et également pendant des conditions anaérobies de respiration avec nitrate. Nous avons également confirmé que l'expression de ldcA est régulée par ArgR et elle est induite complètement lorsque l’acide aminé L-arginine est présente dans le milieu de croissance. Même si nous avons trouvé que les conditions de stress n'induisent pas l'expression de ldcA, nous avons obtenu de nouvelles données suggérant que d'autres mécanismes de régulation tels que le système de quorum sensing dépendant des quinolones (PQS) pourraient être impliqués dans l'expression de ldcA. En utilisant des souches mutantes de ldcA et son complémentée, nous avons évalué si LdcA était impliqué dans la réponse au stress acide et oxydatif. Bien que les données obtenues à l'aide des expériences dans notre laboratoire et des technologies à haut débit (Biolog) aient révélé que LdcA ne présente pas les mêmes fonctions que LdcI, nous avons découvert que la cadavérine produite par LdcA est nécessaire pour la croissance en milieu minimal avec L- Glutamate comme source de carbone. Nous avons également examiné si la présence de LdcA modifie la résistance aux antibiotiques et nous montré que les rends moins persistants face aux carbenicillines. Enfin, en combinant l'analyse phylogénétique et structurelle, nous avons découvert que LdcA appartient à un sous-groupe différent de LDCs bactériennes. Les alignements de séquences montrent que les résidus clés nécessaires pour lier le ppGpp ne sont pas présents dans le site de liaison prédit ce qui a été confirmer par l'analyse biochimique. Notre travail montre que, malgré le fait que LdcA catalyse la même réaction enzymatique et partage les mêmes caractéristiques structurelles que LdcI et LdcC, elle ne joue pas le même rôle que ses homologues. Son rôle est lié aux effets physiologiques de la cadavérine et à la relation entre la L-lysine et le catabolisme de la L-arginine. / The lysine decarboxylase (LDC) belongs to a family of decameric PLP-dependent enzymes that catalyse the reaction transforming L-Lysine into cadaverine while consuming a proton. They are known to be involved in polyamine metabolism and during acid and oxidative stress responses.In enterobacteria like Escherichia coli, two paralogs are present, LdcI and LdcC. LdcI takes part in acid stress response by buffering bacterial cytoplasm. LdcC is produced during stationary phase and also when bacteria face fluoroquinolone treatment. The cadaverine produced by LDCs is known to scavenge reactive oxygen species (ROS) and is capable of blocking outer membrane proteins, thus reducing the permeability of molecules responsible for acid and oxidative stresses. The activity of the LDCs from E. coli is coordinated with the stringent response (nutrient starvation) in order to prevent intracellular L-Lysine depletion. The stringent response signal molecule ppGpp is able to bind directly to LDCs and inhibit their enzymatic activity. However, the inhibition of the LdcI can be prevented by the formation of a cage-like complex with its partner RavA allowing bacteria to face the challenge of both acid and nutrient stresses.Since mechanisms allowing bacteria to counter stress challenges are important for displaying full virulence, we wondered if the opportunistic bacterium Pseudomonas aeruginosa could be using LdcA to counter stress conditions that have already been described for LdcI in enterobacteria. During my PhD, we addressed this question by using different but complementary approaches.First of all, we used promoter-gene fusions and western-blot analysis to determine the conditions in which ldcA was expressed and its product synthesized. We could observe that ldcA is expressed on stationary phase under aerobic conditions in rich media and also during nitrate-respiring anaerobic conditions. As previously described in literature, we also confirmed that ldcA expression is regulated by ArgR and fully induced when L-Arginine is present in the growth medium. Even though we found out that acid and oxidative stress conditions do not induce the expression of ldcA, we obtained new data suggesting that other regulation mechanisms such as the quinolone signal system (PQS) could be involved in ldcA expression.In paralell, we constructed an ldcA mutant and its complemented strain to understand whether LdcA was involved in acid and oxidative stress response. Although the data obtained by using manual screenings and high-throughput technologies (Biolog) revealed that LdcA is not displaying the same functions as LdcI, we discovered that the cadaverine produced by LdcA is needed for full growth fitness when growing in minimal medium using L-glutamate as carbon source. Since slow growing phenotypes are linked to heightened bacterial persistence and because cadaverine has been shown to reduce the persisters population, we also examined if the presence of LdcA is modifying the amount of persisters during carbenicillin treatment. Our data has confirmed that this is indeed the case.Finally, by combining phylogenetic and structural analysis, we discovered that LdcA belongs to a different subgroup of bacterial LDCs. Sequence alignments show that key residues needed for binding ppGpp are not present in the predicted binding site which also suggests that the enzymatic activity is not inhibited by this molecule. And biochemical analysis has confirmed that this is indeed the case as it is the case for Arginine decarboxylases.Our work shows that, in spite of the fact that LdcA catalyses the same enzymatic reaction and shares the same structural fold than LdcI and LdcC, it is not implicated in acid stress or oxidative stress responses. Its role is linked to physiological effects of cadaverine and to the relationship between L-lysine and L-Arginine catabolism.

Orientador: Paulo Arruda / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T11:56:55Z (GMT). No. of bitstreams: 1 Serrano_GuilhermeCoutinhodeMelo_M.pdf: 1314215 bytes, checksum: f2ea574317887a52e797252a637d3649 (MD5) Previous issue date: 2010 / Resumo: A lisina é considerada um aminoácido essencial, pois é componente fundamental de proteínas e não pode ser sintetizado por animais, sendo necessário ingeri-lo em sua forma final. Sua concentração é baixa em cereais, principal fonte de alimento animal e sua carência pode trazer sérios danos ao organismo, principalmente relacionados ao sistema nervoso. Por outro lado, seu excesso, causado pela deficiência na degradação, também é danoso podendo levar ao retardo no desenvolvimento mental. A síntese da lisina em plantas e bactérias é realizada principalmente pela via do ácido aspártico, que além desse aminoácido é responsável pela produção de treonina, metionina e isoleucina. A degradação da lisina em animais e plantas ocorre principalmente pela via da sacaropina. Essa via, por sua vez, é utilizada para a síntese de lisina em fungos. Assim, tanto a síntese como a degradação de lisina em diferentes organismos possuem arquiteturas metabólicas particulares que, durante o processo evolutivo, foram selecionadas para adequar-se as necessidades do metabolismo, diferenciação e desenvolvimento. Até o momento não existia indício da existência e da funcionalidade da via do ácido aspártico em animais e nem da via da sacaropina em bactérias. O presente trabalho apresenta um conjunto de resultados que sugerem a existência da via do ácido aspártico em insetos e a via da sacaropina em bactérias. Foram identificados em Anopheles gambie e Aedes aegypti, respectivamente 6 e 5 genes dos 9 que compõem a via do ácido aspártico em bactérias. A análise de similaridade de sequências sugere que os genes da via do ácido aspártico encontrados nos insetos pode ter se originado a partir de bactérias endosimbióticas. A necessidade da via de síntese de lisina nesses insetos pode estar ligada a fonte de alimentação, em alguns casos deficiente em aminoácidos essenciais. Quanto à degradação da lisina, os resultados sugerem a existência, em alguns grupos de bactérias, dos genes que codificam as enzimas lisinacetoglutarato redutase e sacaropina desidrogenase (lkr e sdh), responsáveis pelos passos iniciais da via em plantas e animais. Em alfa-proteobactérias, encontramos os genes lkr e sdh ligados em tandem na forma de um operon. Em plantas e animais esses genes também são ligados e codificam um polipeptídio bifuncional contendo as atividades da LKR e de SDH. O operon da lkr e sdh encontrado em alfaproteobactérias contém também os genes que codificam as enzimas glutationa-Stransferase (GST) e fosfogliceraldeído mutase (FM). Essa estrutura genômica sugere que a LKR e a SDH podem fazer parte de um conjunto de enzimas importantes na defesa contra estresse oxidativo, além da degradação da lisina como pode ser demonstrado pela atividade enzimática de LKR e SDH nesse organismo. A análise da estrutura genômica dos genes da via da sacaropina em bactérias revelou um fato curioso. Em plantas existe um peptídeo de aproximadamente 100 aminoácidos intercalando os domínios polipeptídicos da LKR e da SDH. Esse peptídeo, denominado de interdomínio (ID) é exclusivo de plantas, não tendo sido encontrado em nenhum dos genomas de eucariotos seqüenciados até o momento. Seqüências similares ao ID foram encontradas em cianobacterias e algumas archeobacterias, porém em nenhum caso esses IDs puderam ser associados com seqüências codificadoras de LKR e/ou SDH. Neste trabalho buscamos remontar uma possível história evolutiva da via da sacaropina e apresentamos uma hipótese evolutiva para a origem dos genes LKR e SDH de plantas e animais / Abstract: Lysine is an amino acid that is not synthesized by animals and therefore need to be ingested in its final form. Lysine concentration is low in cereals and the lack of this amino acid in diet can cause severe damage to the organism specially associated to the nervous system. On the other hand its excess, caused by the deficiency in the degradation of lysine, can also lead to organism disorder as mental development retardation. Lysine is mainly synthesized trough the aspartate pathway in plant and bacteria and the same pathway can also lead to synthesis of threonine, methionine and isoleucine. Lysine degradation occours trough the saccharopine pathway in animal and plant, but other pathways are known for bacteria and fungi. Until now there was not an evidence of the existence of any of the pathways. Here we present new data of the existence of the aspartate pathway in animals and the saccharopine pathway bacteria respectively. We were able to demonstrate the existence of most of the genes that compose the aspartate pathway in Anopheles gambie e Aedes aegypti and sequence clustering suggest a possible origin of these genes from endosymbiontic bacteria. Concerning the lysine degradation, we have gathered data that indicates the existence of genes responsible for producing the proteins for two first enzymatic steps, lysine ketoglutarate redutase (LKR) and saccharopine dehidrogenase (SDH), in bacteria. Exclusively in Proteobacteria we found these genes in the exact same structure of animals and plants and we were able to detected enzymatic activity for LKR and SDH. Curiously these genes are inside a single operon that also has glutatione s transferase and phosphoglyceraldehide mutase, both genes previously associated with defense against oxidative stress. This may suggest a possible new role for LKR/SDH in preventing oxidative stress. The analysis of amino acid sequence and domains of LKR/SDH in plant, animals and fungus, shows that there is a region of approximately 110 aa between LKR and SDH domains that is exclusive to plants, we called this region the inter domain (ID). Interestedly we found only in Cyanobacterial this typical plant domain, the ID. After these novel results we attempted to reconstruct the evolutionary history of pathway, suggesting an explanation for the origin of plants and animals LKR and SDH / Mestrado / Genetica Vegetal e Melhoramento / Mestre em Genética e Biologia Molecular

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