O presente estudo teve como objetivo avaliar o efeito dos níveis de inclusão de polpa cítrica sobre parâmetros sanguíneos de equinos, vinculados ao metabolismo energético dos cavalos. Foram utilizados cinco animais, com idade média de 3,5 anos e peso vivo de 460,6±76,86kg, sem raça definida, arraçoados duas vezes ao dia, às 7:00 e às 16:00. As dietas foram formuladas para atender a demanda de animais em mantença, sendo 60% da energia proveniente do volumoso e 40% do concentrado, contendo níveis crescentes de inclusão de polpa cítrica (0, 7, 14, 21 e 28%). A coleta de sangue ocorreu nos tempos: antes da alimentação, uma, três, seis, nove horas após a alimentação. As amostras de sangue foram coletadas através de venopunção da jugular utilizando tubos coletores a vácuo para determinação de albumina, triglicérides, colesterol, glicose, insulina e ácidos graxos de cadeia curta. Foram calculadas a área abaixo da curva e o pico da glicose e insulina, para as quais não foram observados efeitos (P>0,05). Não houve efeito (P>0,05) da dieta sobre as concentrações sanguíneas dos parâmetros. Não houve efeito (P>0,05) do tempo de coleta sobre as variáveis: ácidos graxos de cadeia curta, colesterol, triglicérides e albumina, porém foi observado efeito (p<0,05) para as concentrações de glicose (y = -0,521x2 + 4,133x + 84,647) e insulina (y = -0,097x2 + 0,795x + 1,616). A polpa citrica é um ingrediente promissor na alimentação de equinos, sem causar alteração nas concentrações sanguineas de glicose, insulina, colesterol, triglicerides e ácidos graxos de cadeia curta. Dietas com alta inclusão de fibra e participação de ingredientes com fibras de fácil fermentação são benéficas a dietas de equinos pois mantem as curvas glicêmicas e insulinêmicas próximas aos valores basais além de postergar a queda glicêmica pós prandial. / The present study aimed to evaluate the effect of levels of citrus pulp on blood parameters of horses tied to energy metabolism of horses. Five animals were used, with an average age of 3,5 years and body weight of 460,6 ± 76,86 kg mongrel, hand fed twice daily at 7:00 and 16:00. Diets were formulated to meet the demand of animals in maintenance, 60% of energy from the forage and 40% concentrate, containing increasing levels of citrus pulp (0, 7, 14, 21 and 28%). Blood collection occurred at times: before eating, one, three, six, nine hours after feeding. Blood samples were collected via jugular venipuncture using vacuum collectors for the determination of albumin, triglycerides, cholesterol, glucose, insulin and short-chain fatty acids tubes. The area under the curve and the peak glucose and insulin, for which no effect (P>0,05) were observed, were calculated. There was no effect (P>0,05) of diet on blood concentrations of parameters. There was no effect (P>0.05 ) and time of collection on the variables: short-chain fatty, cholesterol, triglycerides and albumin acids, but effect ( p<0.05 ) was observed for glucose concentrations (y = - 0.521x2 + 4.133 x 84.647) and insulin (y = -0.097x2 + 0.795 + 1.616 x). The citrus pulp is a promising ingredient in feed for horses without causing changes in blood concentrations of glucose, insulin, cholesterol, triglycerides and short-chain fatty acids. Diets with high fiber inclusion and participation of ingredients with easily fermentable fibers are beneficial because the diets of horses keeps the glucose and insulin curves near the baseline plus delay the postprandial glycemic fall.

La maladie alcoolique du foie (MAF) regroupe l’ensemble des lésions qui apparaissent suite à une consommation excessive et chronique d’alcool. A consommation d’alcool égale, les patients n’évolueront pas tous vers les formes sévères de la maladie. Le microbiote intestinal est un cofacteur déterminant dans la sévérité de la MAF. Parmi les métabolites fécaux entre des souris recevant le microbiote intestinal de patients avec des lésions sévères ou non, les acides biliaires ont été identifiés comme les plus discriminants. Le récepteur aux acides biliaires TGR5, exprimé sur les cellules de Kupffer, favorise leur profil anti-inflammatoire.Nous avons évalué l’impact du récepteur TGR5 dans la MAF chez des souris déficientes pour ce récepteur. La déficience en TGR5 aggrave la MAF, sans passer par une modulation de la cellule de Kupffer. C’est en fait le microbiote intestinal qui est impacté chez les souris déficientes pour TGR5, et qui médie cette aggravation.Par ailleurs, afin de moduler le microbiote intestinal au cours de la MAF, nous avons évalué le rôle de la pectine, qui favorise la croissance de certaines bactéries et peut chélater les acides biliaires. Malgré ses propriétés chélatantes, ce sont bien les modifications du microbiote intestinal induites par la pectine qui jouent un rôle protecteur et curatif dans la MAF.Ces différentes études devraient permettre d’identifier des cibles thérapeutiques potentiellement applicables chez des patients alcooliques et basées sur la modulation du microbiote intestinal. / Alcoholic liver disease (ALD) includes all the liver injuries occurring as a result of excessive and chronic alcohol consumption. Nevertheless, among heavy drinker, only a subset of individuals will develop severe liver injury. Intestinal microbiota was identified as a major player in the mechanisms involved in ALD. Moreover, bile acids were the most discriminant faecal metabolites between mice with or without liver injury. The bile acids receptor TGR5, which is expressed on Kupffer cells, promotes their anti-inflammatory profile.We assessed the role of bile acids receptor TGR5 in ALD using TGR5-deficient mice. TGR5-deficiency worsens ALD, but without modulating the Kupffer cells profile. However, intestinal microbiota is impaired in TGR5-deficient mice, and this is responsible for ALD worsening.Furthermore, in order to modulate the intestinal microbiota during ALD, we assessed the role of pectin, which is known to promote the growth of certain bacteria and that is a bile acids sequestrant. Despite its sequestrant properties, pectin-induced changes in intestinal microbiota play a protective and curative role in ALD.These studies will allow the identification of new therapeutic targets that could be used for alcoholic patients, using intestinal microbiota modulation.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22288号 / 理博第4602号 / 新制||理||1660(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)講師 嶋田 知生, 教授 鹿内 利治, 教授 長谷 あきら / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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L’objectif de cette thèse est d’encapsuler dans une matrice polyosidique des probiotiques sous forme de biofilm en utilisant comme modèle Lactobacillus paracasei ATCC334. La matrice d’encapsulation (billes) est obtenue par gélification ionique d’une pectine amidée et faiblement méthylée en présence d’ions calcium. Nous avons remarqué que le réseau de pectine dans ces billes d’un diamètre d’environ 500 µm permet la formation de microcolonies sphériques (biofilm-like) avec une taille de 25 µm de diamètre. Une augmentation de la concentration bactérienne de L. paracasei ATCC334 de 3 Log d’UFC après 24 h de croissance des bactéries immobilisées est observée pour atteindre au sein de ces billes une concentration supérieure à 10 Log d’UFC/g humide. Nous avons également noté une distribution homogène des microcolonies dans les billes et une organisation structurée des bactéries au sein des microcolonies. En effet, une adhésion des bactéries à la matrice pectine est observée ainsi que la présence de substances polymériques permettant de lier les bactéries les unes aux autres. Les résultats suggèrent donc des phénomènes d’interaction entre le réseau formé par la pectine et les bactéries dans les billes. Nous avons montré que L. paracasei ATCC334 formulée en biofilm dans ces billes de pectine présente une résistance accrue à un stress gastrique (pH 2) et au séchage par lyophilisation. La capacité d’adhésion des bactéries formulées à des cellules épithéliales est conservée et la pectine semble stimuler cette adhésion aux cellules de l’hôte. Des résultats in-vivo utilisant un modèle murin d’inflammation intestinale montrent que les biofilms de L. paracasei ATCC334 sont libérés au niveau intestinal où ils s’implantent notamment dans le côlon. Des microcolonies de tailles approchant les 20 µm sont retrouvées au niveau du côlon suggérant que les billes de pectine ont libéré les bactéries sous forme de biofilm. Par ailleurs, l’administration aux souris de la formulation à base de pectine avec des biofilms de probiotiques a entrainé chez les souris ayant reçu un traitement DSS (une molécule capable de déclencher une inflammation intestinale) : une perte de poids moindre, un état de santé général amélioré, une muqueuse colique moins altérée ainsi qu’une diminution de la réponse inflammatoire. / The aim of this thesis is to encapsulate biofilm probiotics in a polysaccharide matrix using Lactobacillus paracasei ATCC334 as a model. The encapsulation matrix (beads) is obtained by the ionotropic gelation of amidated low-methoxylated pectin with calcium ions. We noticed that the pectin network in these beads with a diameter of approximately 500 microns, allow the formation of spherical microcolonies (biofilm-like) with a diameter of 25 microns. An increase of 3 Log CFU in the bacterial concentration of L. paracasei ATCC334 is observed after 24 hours of growth of the immobilized bacteria, while the observed concentration in these beads reaches more than 10 Log of CFU/g wet. We also noticed that the microcolonies within the beads are homogeneously distributed and the bacteria within the microcolonies are well structured. Moreover, a bacterial adhesion to the pectin matrix is observed as well as the presence of polymeric substances that bind the bacteria to each other. Our results suggest that interaction phenomenon may take place between pectin network and bacteria within beads. We also showed that biofilms of L. paracasei ATCC334 formulated in these pectin beads exhibit increased resistance to the gastric stress (pH 2) and to the freeze-drying process. In addition, the adhesion capacity of the formulated bacteria to epithelial cells is conserved and pectin seems to stimulate this adhesion to host cells. In-vivo results using a murine model presenting intestinal inflammation showed that L. paracasei ATCC334 biofilms are released in the intestinal level and are specifically implanted in the colon. Moreover, microcolonies of sizes approaching 20 μm are found in the colon suggesting that the bacteria are released in their biofilm form. In addition, the administration of pectinate beads containing probiotic biofilms to mice which have received a DSS treatment (inducing intestinal inflammation) resulted in: less weight loss of mice, improved their overall health status, less injured colonic mucosa and a decrease in the inflammatory response.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23524号 / 農博第2471号 / 新制||農||1087(附属図書館) / 学位論文||R3||N5355(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 髙野 義孝, 教授 寺内 良平, 教授 吉田 健太郎 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Rac/Rop GTPases are molecular switches in plants that control the growth of polarized cells such as pollen tubes and root hairs, differentiation, development, actin dynamics, production of reactive oxygen species (ROS) and disease resistance. These small GTPases are activated by guanine nucleotide exchange factors (GEFs) that replace GDP for GTP and are referred to as RopGEFs in plants. To identify upstream components of the RopGEF regulated signaling pathways, GEF1 from Arabidopsis thaliana was used as a bait to screen a seedling cDNA library in a yeast two-hybrid system. This yielded members of a small family of the Catharanthus roseus Receptor-like kinase (CrRLK), referred to as the Feronia-like Receptor-like kinase (FlRLK) family as the potential GEF interactors. A synergid cell-expressed member of this family, FERONIA/SIRENE (RLK-10), regulates pollen tube reception by the female gametophyte and along with the other two plasma-membrane bound receptors of this family, THESEUS (RLK-3) and HERCULES (RLK-4) promote cell elongation in Arabidopsis. I have chosen two of these FlRLKs for my studies: RLK-5, the most abundant pollen expressed member of the family and RLK10. My studies with RLK-5 suggest that it plays a significant role in pollination or fertilization since homozygous rlk-5 was never recovered from selfed heterozygous rlk-5 mutant. In addition to this, the reciprocal crosses with RLK-5/rlk-5 and wild type resulted in severe male transmission defect indicating that the rlk-5 mutation induces male sterility. This observation was found consistent with the pollen-specific expression pattern of RLK-5 suggested by microarray data and confirmed by histochemical GUS staining analysis of the RLK5p-GUS transgenic Arabidopsis. The RLK-5/rlk-5 pollen displayed no pollen-viability defects and the pollen tube growth in-vivo appears normal. Despite the unaffected pollen viability and apparent normal in-vivo pollen tube growth, the RLK-5/rlk-5 plants formed low seed set suggesting compromised fertilization. Additional analysis will be required to determine the basis of male deficiency and reduced seed sets in RLK5/rlk5 mutants. Loss of function mutations of RLK-10 (FERONIA/SIRENE) gene show failure in pollen tube growth arrest upon penetration of the female gametophyte, supernumerary pollen tube penetration of the ovule and reduced female fertility. Our studies with RLK-10 suggest that it is important for mediating a proper oxido-reductive condition within the ovule necessary for pollen tube rupture and fertilization of the female gametophyte. The multiple pollen tube entry phenotype of the Arabidopsis knockout mutant, rlk-10 correlated with decreased ROS level and de-esterified pectin in the filiform apparatus lining the synergid cell of its female gametophyte. These results establish an important signaling link between RLK-10 and ROS and pectin in a GEF-Rac/Rop regulated pathway in pollen tube-ovule interaction.

Pectin has a long history as a food additive. However, elucidation of its fine structural and property relationships remains elusive. Recent research has focused on pectin's ability to complex with divalent heavy metals to aid in characterizing it. Commercial pectins of unknown composition were obtained from local grocers. Purified pectin samples from orange peel, lemon peel, and apple pomace, each of low and high levels of methyl esterification and of unknown distribution pattern were also purchased. Instead of metal complexation, several highly absorbing dyes such as Ruthenium Red, Nile Blue, and Acridine Orange were used to complex with the pectins and their resulting UV-Vis spectral patterns were employed to determine if one can characterize the different pectins. Chemometric methods are also included to aid in distinguishing them apart.

No description available.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23251号 / 農博第2458号 / 新制||農||1085(附属図書館) / 学位論文||R3||N5341(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 阪井 康能, 教授 小川 順, 教授 井上 善晴 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM