Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2017-06-21T17:17:58Z No. of bitstreams: 2 Dissertação - Marília Ferreira Pires - 2016.pdf: 2038317 bytes, checksum: 6bf59849e1b382d63908376fa0df3f69 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Cláudia Bueno (claudiamoura18@gmail.com) on 2017-07-07T19:53:05Z (GMT) No. of bitstreams: 2 Dissertação - Marília Ferreira Pires - 2016.pdf: 2038317 bytes, checksum: 6bf59849e1b382d63908376fa0df3f69 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-07-07T19:53:05Z (GMT). No. of bitstreams: 2 Dissertação - Marília Ferreira Pires - 2016.pdf: 2038317 bytes, checksum: 6bf59849e1b382d63908376fa0df3f69 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-20 / The objective of the present study was to evaluate the effect of adding increasing levels of protected sodium butyrate in the diet of laying hens from 61 weeks of age on performance, egg quality and intestinal parameters. The treatments were 0, 105, 210 and 300 g / t of sodium butyrate protected in the feed. To evaluate the performance and quality of eggs, 320 laying hens were used as Dekalb Whyte lineage (61-76 weeks old) in cages and distributed design in blocks (with the weight of the blocky birds), with four treatments and eight replicates of 10 birds. The experiment was 16 weeks divided into four periods of 28 days each. The 76 weeks of age were separated 48 birds for metabolism trial with four treatments and six replicates of two birds. At the end of the assay was sacrificed a repeat per bird for intestinal development analysis. Was conducted analysis of variance and polynomial regression of the data with the help of the statistical program R. Regarding the performance for the period 73-76 weeks of age was negative linear effect for feed conversion (kg feed / kg eggs). For quality variables eggs were observed increasing linear effect for shell thickness in the periods 61-64 and 65-68 weeks and during the periods 69-72 and 73-76 weeks were observed quadratic effect with maximum point 199 and 174 g / t sodium butyrate protected, respectively. For specific gravity quadratic effect was found with a maximum point at 177 g / t sodium butyrate protected only in the period 73-76 weeks. To the percentage of peeling was found quadratic effect on periods 69-72 and 73 to 76 weeks, with a maximum at point 89 and 172 g / t sodium butyrate protected, respectively. To yolk index found a quadratic effect in the period 69-72 weeks of age with a minimum point at 189 g / t of protected sodium butyrate. When evaluating albume index, found a negative linear effect in the period 69-72 weeks of age. For Haugh unit regression was effective only in the period 65-68 weeks with a quadratic effect, with maximum point at 142 g / t of sodium butyrate. In total period was observed quadratic effect for shell thickness, percentage of peel and breakage resistance, in which maximum points were found in 193; 136 and 198 g / t sodium butyrate protected, respectively. When evaluating yolk index in the total period, found a quadratic effect with minimum point at 181 g / t. For intestinal development variables it was found negative linear effect for the length of the jejunum. For height of villi, it found a linear increase in the duodenum and jejunum. For crypt depth, quadratic effect was found in the jejunum with a minimum point at 151 g / t sodium butyrate protected. For the variable villous / crypt ratio was found linear increase in the duodenum and jejunum, and the ileum quadratic effect, with maximum point at 171 g / t. For BN was found quadratic effect with maximum point at 93 g / t, and for BEE was found increasing linear effect. For EMA and EMAn variables were found increasing linear effect. The addition of protected sodium butyrate in laying hens diet did not influence the performance of the birds, but improved the quality of skin and intestinal parameters (intestinal development and metabolization of nutrients). It is recommended to improve the quality of shell eggs in layers in the final production phase, the level of 150 g / t sodium butyrate protected. For intestinal development and diet metabolizable energy level of 300 g / t of sodium butyrate protected. / Objetivou-se com o presente estudo avaliar o efeito da adição de níveis crescentes de butirato de sódio protegido na ração de poedeiras comerciais, a partir de 61 semanas de idade, sobre o desempenho, qualidade de ovos e parâmetros intestinais. Os tratamentos foram: 0, 105, 210 e 300 g/t de butirato de sódio protegido na ração. Para avaliação do desempenho e qualidade de ovos, foram utilizadas 320 poedeiras comerciais da linhagem Dekalb Whyte (61 a 76 semanas de idade) alojadas em gaiolas e distribuídas em delineamento em blocos ao acaso (sendo o peso das aves blocado), com quatro tratamentos e oito repetições de 10 aves. O experimento teve duração de 16 semanas divididas em quatro períodos de 28 dias cada. As 76 semanas de idade foram separadas 48 aves para o ensaio metabólico, com quatro tratamentos e seis repetições de duas aves. Ao final do ensaio metabólico foi sacrificada uma ave por repetição para análise de desenvolvimento intestinal. Foi realizado análise de variância e regressão polinomial dos dados com auxílio do programa estatístico R. Com relação ao desempenho, para o período de 73 a 76 semanas de idade houve efeito linear negativo para conversão alimentar (kg ração/kg de ovos). Para as variáveis de qualidade de ovos foram observados efeito linear crescente para espessura da casca nos períodos de 61 a 64 e 65 a 68 semanas, e nos períodos de 69 a 72 e 73 a 76 semanas foram observados efeito quadrático com ponto de máxima em 199 e 174 g/t de butirato de sódio protegido, respectivamente. Para gravidade específica foi encontrado efeito quadrático com ponto de máxima em 177 g/t de butirato de sódio protegido apenas no período de 73 a 76 semanas. Para a porcentagem de casca foi encontrado efeito quadrático nos períodos de 69 a 72 e 73 a 76 semanas, com ponto de máxima em 89 e 172 g/t de butirato de sódio protegido, respectivamente. Para índice de gema encontrou-se efeito quadrático no período de 69 a 72 semanas de idade com ponto de mínima em 189 g/t de butirato de sódio protegido. Ao avaliar índice de albúmen, encontrou-se efeito linear negativo no período de 69 a 72 semanas de idade. Para unidade Haugh houve efeito de regressão apenas no período de 65 a 68 semanas com efeito quadrático, apresentando ponto de máxima em 142 g/t de butirato de sódio. No período total foi observado efeito quadrático para espessura de casca, porcentagem de casca e resistência à quebra, nos quais foram encontrados pontos de máxima em 193; 136 e 198 g/t de butirato de sódio protegido, respectivamente. Ao avaliar índice de gema no período total, encontrou-se efeito quadrático com ponto de mínima em 181 g/t. Para as variáveis de desenvolvimento intestinal, foi encontrado efeito linear negativo para comprimento do jejuno. Para altura de vilos, foi encontrado efeito linear crescente no duodeno e jejuno. Para profundidade de criptas, foi encontrado efeito quadrático no jejuno, com ponto de mínima em 151 g/t de butirato de sódio protegido. Para a variável relação vilo/cripta, foi encontrado efeito linear crescente no duodeno e jejuno, e efeito quadrático no íleo, com ponto de máxima em 171 g/t. Para BN foi encontrado efeito quadrático com ponto de máxima em 93 g/t, e para BEE foi encontrado efeito linear crescente. Para as variáveis EMA e EMAn foram encontrados efeito linear crescente. A adição de butirato de sódio protegido na dieta de poedeiras comerciais não influenciou no desempenho das aves, mas melhorou a qualidade de casca e os parâmetros intestinais (desenvolvimento intestinal e metabolizabilidade de nutrientes). Recomenda-se, para melhorar a qualidade de casca de ovos em poedeiras na fase final de produção, o nível de 150 g/t de butirato de sódio protegido. Para desenvolvimento intestinal e metabolizabilidade da energia da dieta o nível de 300 g/t do butirato de sódio protegido.

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La lutte chimique est la principale méthode utilisée pour contrôler les maladies causées par les champignons phytopathogènes. Dans certains cas, desphénomènes de résistance envers les fongicides se développent au sein despopulations, altérant parfois l’efficacité des molécules. La compréhension du moded’action des fongicides et des mécanismes de résistance sous-jacents participe à élaboreret à adapter des stratégies de management anti résistance ; et ainsi permettre depérenniser la durée de vie des molécules. Le fenhexamid est un fongicide récent (BayerCropScience, 2000), avec un mode d’action unique. Il est le seul fongicide commercialisébloquant l’étape de C4-déméthylation de la biosynthèse de l’ergostérol. Plusieurs typesde résistance (naturelle et acquises) ont été détectées dans les vignobles européens chez lecomplexe d’espèces Botrytis spp. responsable de la pourriture grise de la vigne. Lestravaux développés durant la thèse s’inscrivent dans l’objectif de la caractérisation dumode d’action et de l’élucidation des mécanismes de résistance. Le premier axe s’estattaché à la caractérisation fonctionnelle de deux gènes impliqués dans la C-4déméthylation de la biosynthèse de l’ergostérol : le gène erg27 codant la 3-céto réductase,cible du fenhexamid, et le gène erg28 codant une protéine qui interagirait en partie avecla 3-céto réductase. Concernant la résistance au fenhexamid, il a été démontré que, pargénétique inverse, les mutations détectées dans le gène erg27 de différents types d'isolatsrésistants issus du vignoble (phénotypes de résistance HydR3- et HydR3+) conféraient larésistance. Par ailleurs, une analyse de fitness du phénotype le plus préoccupant(phénotype HydR3+) a été réalisée en conditions contrôlées sur des souches isogéniquesartificielles afin d’apporter une réponse sur la persistance possible de ces souches auvignoble. Une méthode fine de quantification moléculaire de ces mêmes isolats aégalement été mise au point pour faciliter le suivi de leur évolution et de la persistancedes populations naturelles à l’échelle des vignobles. Cette nouvelle méthode, nomméeASPPAA PCR, exploite le polymorphisme nucléotidique du gène erg27, à l’origine de larésistance. Enfin, la résistance naturelle au fenhexamid de l’espèce apparentée à Botrytiscinerea, appelée Botrytis pseudocinerea a été élucidée. La résistance au fongicide de cetteespèce a été expliquée par la combinaison de modifications de cible (mécanismeminoritaire) et d’une dégradation du fongicide par un cytochrome P450 nomméCyp68.4 (mécanisme majeur). Il s’agit de la première identification et caractérisationgénétique d’un mécanisme de résistance à un fongicide conférée par un processus dedétoxification chez un champignon phytopathogène. / Chemical control is the main method used to control diseases caused byphytopathogenic fungi. In some cases, the resistance phenomena towardfungicides occur within fungal populations, which might alter practicalefficiency of molecules. Understanding modes of action of fungicides andunderlying resistance mechanisms participate to the development and adaptationof management strategies against resistance, and thus help to sustain the life ofmolecules. Fenhexamid is a recent fungicide (Bayer CropScience, 2000), with aparticular mode of action. It is the only fungicide marketed blocking the C4-demethylation step of ergosterol biosynthesis. Several types of resistance (naturaland acquired) were detected in European vineyards in the Botrytis spp speciescomplex, causing grey mold disease. This work focused on the characterization ofthe mode of action and the elucidation of resistance mechanisms. The first aspectinvestigated the functional characterization of two genes involved in the C4-demethylation of ergosterol biosynthesis. The erg27 gene potentially encoding the3-keto reductase which is the fenhexamid target and the erg28 gene encoding aprotein that interact in part with the 3-ketoreductase. Concerning fenhexamidresistance, we shown by reverse genetics that mutations detected in the erg27 genefrom different resistant isolates from the vineyards (phenotypes HydR3- andHydR3+) confer resistance. Furthermore, a fitness analysis under controlledconditions on the most worrying resistant phenotype (HydR3+) was performed onisogenic artificial strains in order to predict the possible persistence of these strainsin vineyards. A fine molecular method to quantify these isolates was developed tofacilitate the follow-up of evolution and persistence of resistant populations in thevineyard. This new method, named ASPPAA PCR is based on the nucleotidepolymorphism of the erg27 gene, responsible for fenhexamid resistance. Finally,the natural resistance to fenhexamid of the related species to Botrytis cinerea, B.pseudocinerea, was elucidated. Fungicide resistance of this species is explained bythe combination of target site modifications (minor mechanism) and fungicidedegradation mediated by a cytochrome P450 named Cyp68.4 (major mechanism).This is the first characterization of a genetic resistance mechanism to a fungicideconferred by detoxification in a phytopathogenic fungus.

The Maillard reaction products (MRPs) 3-deoxyglucosone (3-DG) and 5-hydroxymethylfurfural (HMF), which are formed during the thermal processing and storage of food, come into contact with technologically used yeasts during the fermentation of beer and wine. In order for the yeast cells to work efficiently, handling of the stress-inducing carbonyl compounds is essential. In the present study, the utilization of 3-DG and HMF by 13 Saccharomyces yeast strains (7 brewer’s yeast strains, 1 wine yeast strain, 6 yeast strains isolated from natural habitats) was investigated. All yeast strains studied were able to metabolize 3-DG and HMF. 3-DG is mainly reduced to 3-deoxyfructose (3-DF) and HMF is completely converted to 2,5-bishydroxymethylfuran (BHMF) and 5-formyl-2-furancarboxylic acid (FFCA). The ratio of conversion of HMF to BHMF and FFCA was found to be yeast strain-specific and no differences in the HMF stress tolerance of the yeast strains and species were observed. After incubation with 3-DG, varying amounts of intra- and extracellular 3-DF were found, pointing to a faster transport of 3-DG into the cells in the case of brewer’s yeast strains. Furthermore, the brewer’s yeast strains showed a significantly higher 3-DG stress resistance than the investigated yeast strains isolated from natural habitats. Thus, it can be shown for the first time that Saccharomyces yeast strains differ in their interaction of 3-DG induced carbonyl stress.