Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Les Escherichia coli pathogènes extra-intestinaux (ExPEC) sont responsables d’une grande variété de maladies. Plus particulièrement, certaines souches ExPEC, du sous-groupe d’E. coli uropathogènes, sont porteuses de fimbriae de type P. Cette famille d’adhésines est soumise à une régulation transcriptionnelle appelée variation de phase; un mécanisme du tout ou rien. Il s’agit d’une compétition entre deux protéines régulatrices : la Dam méthylase et la nucléoprotéine Lrp. Ce mécanisme est aussi soumis à l’influence des régulateurs locaux PapB et PapI, deux régulateurs essentiels. Afin d’étudier PapI et ses homologues ainsi que leur impact sur la variation de phase des fimbriae F1651, Pap et CS31A. Grâce à une fusion chromosomique entre la région régulatrice de clp et les gènes lacZYA, nous avons étudié l’effet, en trans, de PapI et FooI qui ont pu restaurer la variation de phase avec une forte tendance pour la phase OFF. Pour étudier l’action de ces protéines sur foo et pap, nous avons utilisé un système utilisant gfp comme gène rapporteur de l’activité des promoteurs des opérons pap et foo. Cela a permis d’observer la variation de phase au niveau cellulaire par cytométrie en flux et en temps réel par microscopie à fluorescence. Ces expériences ont confirmé que la population de cellules F1651 positives a un phénotype d’expression de F1651 partielle alors que les cellules Pap sont en majorité en phase OFF. PapI et FooI n’ont pas la même influence sur la variation de phase, puisque FooI favorise une plus grande fréquence de variation de phase. / Escherichia coli extra-intestinal pathogenic (ExPEC) are responsible for a wide variety of diseases. Particularly ExPEC strains from the subset called uropathogenic E.coli (UPEC) are carrying fimbriae type P. This adhesin family is subject to transcriptional regulation called phase variation, an all or nothing mechanism. It is a competition between two regulatory proteins: the Dam methylase and the nucleoprotein Lrp. This mechanism is also under the influence of the local regulators PapB and PapI. These two regulators are essential to the phase variation. We therefore sought to investigate PapI and its homologs and their impact on the phase variation of fimbriae F1651, Pap, and CS31A. By means of a chromosomal fusion between the regulatory region of clp gene and lacZYA, we studied the effect in trans of PapI and FooI which could restore the phase variation with a strong tendency to phase OFF. To study the action of PapI and FooI, we used a system with gfp as a reporter gene in operons pap and foo. This allowed the observation of the phase variation at the cellular level by flow cytometry and real-time fluorescence microscopy. These experiments confirmed that the population of F165 positive cells have a partial expression state whereas Pap cells mostly have an OFF expression state. We also confirmed that FooI and PapI do not have the same influence on phase variation and that FooI promotes greater frequency of phase variation.

No description available.

published_or_final_version / Urban Design / Master / Master of Urban Design

F1651, les pili Pap et l’antigène CS31A associé aux antigènes de surface K88 sont tout trois des membres de la famille de type P des facteurs d’adhérence jouant un rôle prépondérant lors de l’établissement d’une maladie causée par des souches Escherichia coli pathogènes, en particulier des souches d’E. coli pathogènes extra-intestinales (ExPEC, Extra-intestinal pathogenic E. coli). Leur expression est sous le contrôle d’un mécanisme de régulation transcriptionnel dépendant de l’état de méthylation de l’ADN, résultant dans l’existence de deux populations définies, l’une exprimant l’adhésine (population ON) et l’autre ne l’exprimant pas (population OFF). Malgré de fortes identités de séquences, ces trois systèmes diffèrent l’un de l’autre, principalement par le pourcentage de cellules ON rencontrées. Ainsi, quand CS31A est systématiquement orienté vers un état considéré comme OFF, F1651 présente une phase ON particulièrement élevée et Pap montre deux états OFF et ON bien distincts, selon le phénotype de départ. La protéine régulatrice sensible à la leucine (Lrp, Leucine-responsive regulatory protein) joue un rôle essentiel dans la réversibilité de ce phénomène épigénétique et il est supposé que les différences de séquences au niveau de la région régulatrice modifient la localisation à ces sites de fixation de Lrp; ce qui résulte, en final, aux différences de phase existant entre CS31A, F1651 et Pap.À l’aide de divers techniques parmi lesquelles l’utilisation de gènes rapporteurs, mutagénèses dirigées et d’analyse des interactions ADN-protéines in vitro, nous montrons dans ce présent projet que la phase OFF prédominante chez CS31A est principalement due à une faible interaction de Lrp avec la région distale de l’opéron clp, et que la présence d’un homologue du régulateur local PapI joue un rôle également clef dans la production de CS31A. Dans le cas de F1651, nous montrons dans cette étude que le taux élevé de cellules en phase ON est dû à une altération dans le maintien de Lrp sur les sites répresseurs 1-3. Ceci est dû à la présence de deux nucléotides spécifiques, situé de part et d’autre du site répresseur 1, qui défavorisent la fixation de Lrp sur ce site précis. Tout comme dans le cas de CS31A, la formation d’un complexe, activateur ou répresseur de la phase ON, dépend également de l’action de du régulatuer local FooI, qui favorise alors le déplacement de Lrp des sites répresseurs 1-3 vers les sites activateurs 4-6. / F1651, the pyelonephritis-associated pili (Pap) and the K88-related surface antigen CS31A are three members of the type P family of adhesive factors that play a key role in the establishment of disease caused by Extra-intestinal Escherichia coli (ExPEC) strains. They are all under the control of methylation-dependent transcriptional regulation that defines the number of fimbriated (ON) and afimbriated (OFF) cells within a clonal population. Despite a high similarity in DNA sequence, these three adhesive systems nonetheless differ in the ratio of ON cells. While CS31A is always turned toward the OFF state, F1651 presents a particularly high level of ON cells and Pap shows two distinct OFF and ON states, depending on the starting phenotype. The leucine-responsive regulatory protein (Lrp) plays an essential role in the reversibility of this epigenetic switch and it is believed that the difference in nucleotides within the regulatory region of each operons could modify the binding of Lrp and, in turn, CS31A, Pap and F1651 phase variation. Using a variety of techniques including gene expression, site-directed mutagenesis, and in vitro protein–DNA interaction analysis, we demonstrate that the preferential OFF state observed in CS31A-positive cells is mainly due to a weak interaction of Lrp with the clp distal region and that the presence of a PapI homologue within the cell plays a key role in CS31A production. For F1651, we show in this study that the high level of ON cells found during F1651 phase variation is due to an altered stability of the DNA complex formed by Lrp at its repressor binding sites 1-3. Again, after each cell cycle, complex formation is modulated by the local regulator FooI (homologue to PapI) which promotes the transit of Lrp toward its activator binding sites 4-6. Furthermore, we identify two nucleotides (T490, G508) surrounding the Lrp-binding site 1 that are critical to maintaining a high OFF to ON switch rate during F1651 phase variation, as well switching Pap fimbriae toward the OFF state.

Thesis (M. Sc.)--University of Hong Kong, 1993. / "Workshop report." Includes bibliographical references.

No description available.

Made available in DSpace on 2014-08-20T14:38:48Z (GMT). No. of bitstreams: 1 dissertacao_veronica_lisboa_santos.pdf: 1521617 bytes, checksum: 22e6fe07f10db8cba96cc690f42e78ed (MD5) Previous issue date: 2012-02-16 / In poultry production systems, molting procedure and the use of substitutes for corn and soybean meal represents good ways to reduce feed costs in poultry production. That s true because farmers skip rearing phase of layers life using molting procedure and the use of grains with similar nutritional requirements as corn and soybean meal are less expensive than those usual used in poultry diets. In this case, whole rice brain (WRB) it s consider a good choice due to its high production and lower market price, justifying the use in animal diets. However, higher levels of WRB in poultry diets show negatives effects due to antinutritional factors as phytic acid and non-starch polysaccharides. Adding enzymes in WRB diets it s a way to increase digestibility and reduce the negative aspects of this factors. During 112 days, divided in four production cycles of 28 days each, performance and egg quality of layers after molting and fed with WRB diets with different energy valorization levels and supplemented with enzymes were evaluated. A total of 480 Hisex Brown layers with 95 weeks old were randomly distributed in groups of five birds per cage in a dark house poultry facility. Treatments consists in 8 different diets, with 12 replicates per treatment, as following: T1: control corn and soybean meal (C+SBM); T2: C+SBM with enzyme supplementation (ES) on top; T3: C+SBM, without ES (100 kcal ME/Kg); T4: C+SBM, with ES (100 kcal ME/Kg); T5: C+SBM, 20% WRB, without ES; T6: C+SBM, 20% WRB, with ES on top; T7: C+SBM, 20% WRB, without ES (100 kcal ME/Kg); T8: C+SBM, 20% WRB, with ES (100 kcal ME/Kg). Performance parameters evaluated were body weight (BW), feed intake (FI), egg production (%EP), feed conversion per dozen (FC/dz) and per mass (FC/m). The parameters used to determine egg quality were egg weight (EW), egg mass (EM), specific gravity (SG), shell thickness (ST), shell weight (SW), Haugh unit (HU), yolk weight (YW), albumen weight (AW) and yolk color (YC). Results showed higher FI, BW, EW and AW in layers fed with WRB diets, which also showed a decrease in YC. Enzyme supplementation and energy valorization (100 kcal ME/Kg) in C+SBM diets improved FC/dz. / Algumas alternativas aparecem em destaque quando o objetivo é diminuir os custos com a ração, entre elas, está a realização da muda forçada e o uso de alimentos alternativos ao milho e farelo de soja. Aves que passam pela prática da muda forçada entram em um segundo ciclo produtivo, dispensando o investimento do produtor em um novo lote. O farelo de arroz integral (FAI) é uma boa alternativa ao milho em função do seu volume de produção, por não competir com outras culturas e por possuir custos de produção relativamente baixos, justificando-se deste modo, sua utilização em rações para animais Entretanto, sua utilização na alimentação animal é limitada, devido a presença de fatores antinutricionais, tais como o ácido fítico e os polissacarídeos não amiláceos. As enzimas são acrescentadas à ração animal com a finalidade de aumentar a sua digestibilidade, remover fatores antinutricionais, melhorar a disponibilidade dos nutrientes, bem como por razões. Durante 112 dias experimentais, divididos em quatro ciclos produtivos, de 28 dias cada, avaliou-se a inclusão de farelo de arroz integral (FAI) e de um complexo enzimático (CE), com diferentes valorizações energéticas, na dieta de poedeiras em segundo ciclo de produção sobre desempenho produtivo e qualidade dos ovos. O estudo contou com 480 poedeiras semipesadas da linhagem Hisex brown, com idade inicial de 95 semanas. As aves foram mantidas em galpão tipo dark house e alojadas cinco a cinco em gaiolas de postura. O delineamento utilizado foi o inteiramente casualizado com oito tratamentos e 12 repetições, sendo: T1: controle milho e farelo de soja (M + FS), sem FAI e sem CE, T2: controle, com CE (sem valorização energética on top), T3: controle, sem CE (100 kcal EM/kg), T4: controle, com CE (100 kcal EM/kg), T5: controle + 20% FAI, sem CE, T6: controle + 20% FAI, com CE (sem valorização energética on top), T7: controle + 20% FAI, sem CE (100 kcal EM/kg) e, T8: controle + 20% FAI, + CE (100 kcal EM/kg). Foram avaliadas as seguintes variáveis de desempenho: peso vivo (PV), consumo de ração (CR), percentual de ovos produzidos (PDOV), conversão alimentar por dúzia (CA/Dz) e conversão alimentar por massa (CA/M); para as variáveis de qualidade externa dos ovos considerou-se: peso dos ovos (POV), massa dos ovos (MOV), gravidade específica (GE), espessura das cascas (EC) e peso da casca(PC) e, para as variáveis de qualidade interna dos ovos: unidade Haugh (UH), pesos da gema (PG), do albúmen (PA) e coloração da gema (CG). Os resultados indicaram que as aves que receberam dietas contendo farelo de arroz integral consumiram mais ração, apresentaram maior peso vivo, aumento no peso do ovo e do albúmen, mas gemas menos pigmentadas. O complexo enzimático valorizado em 100kcal⁄EM adicionado em dietas a base de milho e farelo de soja melhorou a conversão alimentar por dúzia de ovos.