Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The blast and the yellow leaf spot are the major rice and wheat, respectively, both are commonly found as for its destructive potential. Because of this, proving the efficiency of control methods through experiences is of paramount importance, since these results will be used as reference for technicians and producers. The precision is related to the choice of suitable design (local control), the number of repetitions, the sample size in the field, among others. In the case of control experiments involving fungicides, which is the main method of controlling both diseases, leaf sampling in installments is necessary because measuring the entire population becomes unviable. Sampling generates a new error (sampling) within the plot, and this should be minimized by appropriate sampling strategy. Thus, this study aimed to determine sample sizes needed to assess the severity of rice leaf blast and the yellow spot of wheat. For this reason, results of chemical control experiments performed during harvests and 2009/2010 2010/2011 were used. The procedure for collection and analysis of data was identical in all experiments, regardless of culture. In all, the diseases were measured at seven, 14 and 21 days after application of fungicides by sampling 10 leaves in the plots. The variables were disease severity and area under the disease progress curve (AUDPC). Data were subjected to analysis of variance to obtain the experimental and sampling errors, and so, by hypothesis test to check whether the sample dimension and the number of repetitions were adequate. The departure from randomness of variable severity was tested in order to determine whether the methodology used to calculate the sample size was adequate. The departure from randomness test showed that both diseases behaved were distinct, both among treatments between assessments, sometimes distributing randomly in the field, sometimes not. Thus, the combination of theoretical distributions (indicating random or clustered distribution of the disease in the field) to the formula used to calculate the sample size is inadequate. The sample size necessary to measure the average disease severity in the plots was not the same in all treatments and between assessments. This result is expected since, in most of the experiments, the average of the disease was different between treatments and between evaluations. This result leads to a constant change in the relationship between the variance and the mean, which is indicative of the disease in the field of dispersion, this dispersion that is related to the sample intensity. Finally it was observed that, to measure the average AUDPC variable, it is necessary to evaluate fewer leaves in the plots. / A brusone da folha e a mancha amarela são as principais doenças do arroz e do trigo, respectivamente, tanto por serem comumente encontradas quanto pelo seu potencial destrutivo. Devido a isso, a comprovação da eficiência de métodos de controle através de experiências é de suma importância, uma vez que estes resultados serão usados como referência por técnicos e produtores. A precisão experimental está relacionada com a escolha adequada do delineamento (controle local), do número de repetições, do tamanho da amostra na parcela, entre outros. No caso de experimentos de controle envolvendo fungicidas, que é o principal método de controle de ambas as doenças, a amostragem de folhas nas parcelas é necessária, pois mensurar toda a população torna-se inviável. A amostragem gera um novo erro (amostral) dentro da parcela, e este deve ser minimizado através de um dimensionamento amostral adequado. Deste modo, o presente trabalho teve por objetivo determinar tamanhos de amostra necessários para avaliar a severidade da brusone da folha do arroz e da mancha amarela do trigo. Para isso, foram utilizados resultados de experimentos de controle químico realizados durante as safras 2009/2010 e 2010/2011. O procedimento de coleta e análise dos dados foi idêntica em todos os experimentos, independente da cultura. Em todos eles, as doenças foram mensuradas aos sete, 14 e 21 dias após a aplicação dos fungicidas através da amostragem de 10 folhas nas parcelas. As variáveis estudadas foram a severidade das doenças e a área abaixo da curva de progresso das doenças (AACPD). Os dados foram submetidos a análise de variância para a obtenção dos erros experimental e amostral, e assim, através do teste de hipótese, verificar se o dimensionamento amostral e o número de repetições foram adequados. O afastamento da aleatoriedade da variável severidade foi testado com o objetivo de determinar se a metodologia utilizada no cálculo do tamanho da amostra foi adequada. O teste de afastamento da aleatoriedade mostrou que ambas as doenças comportaram-se de foram distintas, tanto entre os tratamentos quanto entre as avaliações, ora distribuindo-se de forma aleatória no campo, ora não. Desse modo, a associação de distribuições teóricas (que indicam distribuição aleatória ou agregada da doença no campo) à formula utilizada no cálculo do tamanho da amostra é inadequado. O tamanho de amostra necessário para mensurar a média da severidade das doenças nas parcelas não foi o mesmo em todos os tratamentos e entre as avaliações. Este resultado era esperado, uma vez que, na grande maioria dos experimentos, a média das doenças foi distinta entre os tratamentos e entre as avaliações. Este resultado levou a uma constante mudança na relação entre a variância e a média, que é um indicativo da dispersão da doença no campo, dispersão esta que esta relacionada com a intensidade amostral. Por fim observou-se que, para mensurar a média da variável AACPD, é necessário avaliar menos folhas nas parcelas. Deste modo, recomenda-se que, sempre que possível, utiliza-se a variável AACPD como forma de comparação entre os tratamentos.

Made available in DSpace on 2015-03-26T13:36:45Z (GMT). No. of bitstreams: 1 texto completo.pdf: 381629 bytes, checksum: ba2d77de1fafa6029bbd57e8ee35252d (MD5) Previous issue date: 2013-03-25 / Blast, caused by the fungus Pyricularia oryzae, has become an economically important disease in wheat. The objective of this study was to determine the effect of silicon (Si) on the photosynthetic gas exchange parameters (net CO 2 assimilation rate (A), stomatal conductance to water vapor (g s ), internal CO 2 concentration (C i ), and transpiration rate (E)) and chlorophyll fluorescence parameters (maximum quantum quenching (F v /F m and F v '/F m '), photochemical (q P ) and nonphotochemical (NPQ) quenching coefficients and electron transport rate (ETR)) in wheat plants grown in a nutrient solution containing 0 (-Si) or 2 mM Si (+Si) and inoculated with P. oryzae. The leaf Si concentration significantly increased for the +Si plants compared to the - Si plants and contributed to a decrease in the severity of blast symptoms. For the inoculated +Si plants, A was significantly higher at 72 (14%), 96 (12%) and 120 (58%) hours after inoculation (hai) when compared with their inoculated -Si counterparts. The g s and E were significantly higher by 60 and 42% at 120 hai for the inoculated +Si plants compared with the inoculated -Si plants, respectively. Significant differences between non-inoculated and inoculated plants were observed from 48 to 120 hai for A, g s and E and from 48 to 96 hai for C i . For the inoculated +Si plants, significant differences of F v /F m between the -Si and +Si treatments occurred at 48, 96 and 120 hai and at 72, 96 and 120 hai of F v '/F m '. The values of F v /F m significantly decreased by 1, 3 and 5% at 48, 96 and 120 hai, respectively, in the -Si plants compared with the +Si plants. Significant decreases of 10, 11 and 22% at 72, 96 and 120 hai, respectively, were observed for F v '/F m ' in the -Si plants when compared with the +Si plants. Significant differences between the non-inoculated and inoculated plants occurred from 48 to 120 hai for F v /F m and F v '/F m ', respectively. For the inoculated plants, significant differences between the -Si and +Si treatments occurred at 96 hai for both q P and NPQ and 72 and 120 hai for ETR. Significant differences between the non-inoculated and inoculated plants occurred at 120 hai for q P and at 96 and 120 hai for ETR. The total chlorophyll content (a + b) and the chlorophyll a/b ratio significantly decreased for the -Si plants compared with the +Si plants. The results of this study clearly demonstrate that the severity of blast symptoms decreased in wheat plants supplied with Si. These plants also exhibited improved gas exchange performance and less dysfunctions at the photochemical level. / A brusone, causada pelo fungo Pyricularia oryzae, tornou-se uma doença economicamente importante no trigo. Este estudo teve como objetivo determinar o efeito do silício (Si) sobre os parâmetros das trocas gasosas (taxa de assimilação líquida de CO 2 (A), condutância estomática ao vapor de água (g s ), a concentração interna de CO 2 (C i ) e taxa de transpiração (E)) e parâmetros de fluorescência da clorofila (eficiencia quântica máxima do fotosistema II (F v /F m e F v '/F m '), fotoquímica (q P ) e coeficiente de extinção não-fotoquimico (NPQ) e a taxa de transporte de eletrons (TTE)) em plantas de trigo crescendas em recipiente com solução nutritiva contendo 0 ou 2 mM de silício (Si) e inoculadas com P. oryzae. A concentração foliar de Si incrementou-se significativamente para plantas, contribuindo à dismunição da severidade da brusone. Para plantas inoculadas com +Si, A foi significativamente maior a 72 (14%), 96 (12%) e 120 (58%) hai do que em suas contrapartes inoculadas. A g s e E foram significativamente maiores em 60 e 42%, respectivamente, às 120 hai para as plantas inoculadas +Si em comparação com as plantas inoculadas -Si. Diferenças significativas entre as plantas inoculadas e não inoculadas ocorreu entre as 48 a 120 hai para A, g s e E e entre as 48 a 96 hai para C i . Para as plantas inoculadas +Si, diferenças significativas para F v /F m entre os tratamentos Si e +Si foram encontradas às 48, 96 e 120 dai e às 72, 96 e 120 hai para F v '/ F m '. Os valores de F v /F m diminuiram significativamente em 1, 3 e 5%, respectivamente, aos 48, 96 e 120 hai para plantas -Si, em comparação com as plantas de +Si. Reduções significativas de 10, 11 e 22%, respectivamente, às 72, 96 e 120 hai para F v '/F m ' ocorreu para as plantas -Si, em comparação com as plantas +Si. Diferenças significativas entre as plantas inoculadas e não inoculadas ocorreu às 48 a 120 hai para F v /F m e F v '/F m '. Para as plantas inoculadas, diferenças significativas entre os tratamentos -Si e +Si ocorreu apenas às 96 hai para ambos q P e NPQ e às 72 e 120 hai para TTE. Diferenças significativas entre as plantas inoculadas e não inoculadas só ocorreu em 120 hai para q P e às 96 e 120 hai para TTE. A concentração de clorofila total (a + b), e a razão de clorofil a/b diminuiu significativamente para as plantas -Si, em comparação com as plantas de +Si. Os resultados deste estudo demonstraram claramente que a severidade da brusone diminuiu em plantas de trigo supridas com Si em paralelo a um melhor desempenho das trocas gasosas e menores perdas disfuncionais ao nível fotoquímico.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-08-22T16:13:10Z No. of bitstreams: 1 miguelgontijosiqueirapalmieri.pdf: 16600771 bytes, checksum: 1474cea0351558e2cfb2fcb3834f5ff7 (MD5) / Rejected by Adriana Oliveira (adriana.oliveira@ufjf.edu.br), reason: on 2017-08-24T11:35:30Z (GMT) / Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-08-24T13:34:54Z No. of bitstreams: 0 / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-30T12:31:36Z (GMT) No. of bitstreams: 0 / Made available in DSpace on 2017-08-30T12:31:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-02-03 / O café verde é conhecido principalmente por suas propriedades antioxidantes e antibacterianas. Compostos fenólicos como os ácidos clorogênicos, que são formados através de ácidos hidroxicinâmicos (ácidos cafeico, ferúlico e outros) ligados ao ácido quínico, estão presentes em grandes quantidades e têm sido reconhecidos como antioxidantes naturais. Entretanto, os ácidos hidroxicinâmicos são encontrados principalmente na forma esterificada com ácidos orgânicos, açúcares, lipídeos e covalentemente ligados a parede celular, o que reduz sua biodisponibilidade. Diante disso, o objetivo deste estudo foi realizar a biotransformação da farinha de café verde utilizando o fungo Aspergillus oryzae (CCDCA102604) visando o aumento da atividade antioxidante. A farinha de café verde foi fermentada em estado sólido usando o fungo Aspergillus oryzae a 25 °C por 24, 48 e 72 horas. Após a fermentação os compostos fenólicos foram extraídos utilizando uma solução hidroetanólica. A atividade antioxidante dos extratos foi avaliada pelos métodos DPIDI-1• (2,2-difenil-1-picril-hidrazila) e poder redutor e a determinação dos teores de fenólicos totais foi realizada utilizando o método de Folin-ciocateu. Foi realizada também a quantificação de ácido clorogênico (5-ACQ), ácido cafeico e cafeína nos extratos utilizando a cromatografia líquida de alta eficiência. De acordo com os resultados ocorreu um aumento significativo de 115,7% e 66,4% da atividade antioxidante dos extratos de farinha de café verde fermentados por 24 horas em relação ao extrato de café não fermentado, determinada pelos métodos DPPI-1• e poder redutor, respectivamente. Além disso, o processo de fermentação pelo fungo A. oryzae durante 24 horas também promoveu um aumento de 68,6% na concentração de compostos fenólicos em relação aos extratos não fermentados. Os extratos fermentados por 24 horas apresentaram um aumento significativo nos teores de ácido clorogênico e ácido cafeico quando comparados aos extratos não biotransformados. O aumento da atividade antioxidante não foi observado nos extratos fermentados por 48 e 72 horas. Os resultados deste estudo demostraram que o processo de biotransformação é uma estratégia para a obtenção de um extrato enriquecido de compostos antioxidantes em suas formas livres com potencial aplicação nas indústrias alimentícias, de suplementos alimentares e cosmética. / Green coffee is known mainly for its antioxidant and antibacterial properties. Phenolic compounds such as chlorogenic acids, which are formed through hydroxycinnamic acids (caffeic, ferulic and other acids) linked to quinic acid, are present in large quantities in green coffee and have been recognized as natural antioxidants. However, hydroxycinnamic acids are found mainly in the esterified form with organic acids, sugars, lipids and are covalently bound to the cell wall, which reduces their bioavailability. Therefore, the objective of this study was to perform the biotransformation of the green coffee flour using the Aspergillus oryzae fungus aimed at increasing the antioxidant activity. The green coffee flour was fermented in solid state using the fungus Aspergillus oryzae (CCDCA102604) at 25 °C for 24, 48 and 72 hours and after fermentation the phenolic compounds were extracted using a hydroethanolic solution. The antioxidant activity of the extracts was evaluated by the DPPH• and reducing power methods, and the determination of total phenolic contents was performed using Folin-ciocateu method. Quantification of chlorogenic acid (5-ACQ), caffeic acid and caffeine in the extracts was also performed using high performance liquid chromatography. The results showed a significant increase of 115.7% and 66.4% of the antioxidant activity of the green coffee flour extracts fermented for 24 hours in relation to the unfermented coffee extract, measured by the DPPI-1• and reducing power methods, respectively. In addition, the fermentation process by the A. oryzae fungus for 24 hours also promoted a 68.6% increase in the content of phenolic compounds in relation to the unfermented extracts. The extracts fermented for 24 hours showed an increase in the content of chlorogenic acid and caffeic acid when compared to the non-biotransformed extracts. The increase in antioxidant activity was not observed in the extracts fermented for 48 and 72 hours. The results of this study showed the biotransformation process as a strategy to obtain an enriched extract of antioxidant compounds in their free forms with potential application in the food, supplementation and cosmetic industries.

La première partie du travail présenté ici se concentre sur l’optimisation d’une cathode à oxygène. Tout d’abord, l’utilisation d’une nouvelle enzyme (la BOD de Magnaporthe oryzae) permet de multiplier le courant de réduction de l’oxygène en eau jusqu’à neuf fois. Ensuite la synthèse d’un polymère rédox adapté a permis d’améliorer le coefficient de diffusion des électrons dans l’hydrogel résultant en l’augmentation de la densité de courant générée. Enfin nous sommes passés d’uneélectrode de carbone en 2 dimensions à une fibre d’or poreuse tridimensionnelle. Après modification de cette fibre avec l’hydrogel rédox à base de BOD de M. oryzaenous avons évalué sa biocompatibilité : in vitro les tests ont montré l’absence totale de cytotoxicité et seule une très faible réponse inflammatoire ; in vivo aucune infection ne s’est déclarée pendant les 8 semaines d’implantation dans les souris etla formation d’une capsule fibrotique autour de l’électrode traduit sa bonne intégration dans les tissus de l’animal. La seconde partie concerne la biopile dans son intégralité, construite à partir de la cathode optimisée et d’une anode adaptée à base de GDH. Elle permet de générer jusqu’à 240 μW.cm-2 dans du tampon Pipes/CaCl2 à 5mM de glucose. La biopile a ensuite été testée dans du sang humain total. Un maximum de 129 μW.cm-2 a été obtenu dans un échantillon avec une glycémie de 8,2 mM sous air. De plus nous avons constaté que la densité de puissance délivrée augmente proportionnellement avec la glycémie des différents échantillons de sang testés, faisant de la biopile à la fois une source d’électricité et un biocapteur à glucose ce qui n’avait jamais été démontré auparavant. / The first part of the work presented here focuses on the optimization of an oxygen cathode. First, the use of a new enzyme (BOD from Magnaporthe oryzae) permit to increase the current of reduction of oxygen into water by a factor nine. Then the synthesis of a suitable redox polymer greatly improved the diffusion coefficient of electrons in the hydrogel, resulting in an increase of the current density. Finally we switched from a two-dimensional carbon electrode to a three-dimensional porous gold fiber. After modification of the fiber with the redox hydrogel based on BOD from M. oryzae, we assessed its biocompatibility: in vitro the tests showed the total absence of cytotoxicity and only a very low inflammatory response; in vivo noinfection appeared during the 8 weeks of implantation in mice and the formation of afibrotic capsule around the device reflects its successful integration into the animal tissues.The second part concerns the full biofuel cell, elaborated from the optimized cathode and an adapted GDH-based anode. It could generate up to 240 μW.cm-2 at 5mMglucose in Pipes/CaCl2 buffer. The biofuel cell was then tested in whole human blood. A maximum of 129 μW.cm-2 was obtained in a sample with 8,2 mM glycaemiaunder air. In addition we observed that the delivered power density increased proportionally with the glycaemia of the different blood samples tested, making the biofuel cell both a power source and a glucose biosensor at the same time which had never been shown before.

Nous avons montré les performances et les limitations en électrochimie des deuxnouvelles BODs de Bacillus pumilus et de Magnaporthe oryzae. La BOD de M.oryzae commence à réduire l’O2 à un potentiel de + 0,50 V vs. Ag/AgCl et B. pumilusà + 0,44 V vs. Ag/AgCl. La BOD de M. oryzae est peu sensible à la concentration dephosphate de sodium dans l’hydrogel rédox mais est sensible au chlore, à l’urate etaux fortes température. La BOD de B. pumilus a une activité élevée en présence dechlore et à 50 °C mais est sensible à la concentration de phosphate dans l’hydrogel.Cette sensibilité est compensée par une meilleure stabilité en présence d’urate, ainsielle ne perd que 9 % d’activité après 3 heures dans le sérum de veau. La BOD de M.oryzae immobilisée sans médiateur est plus performante que B. pumilus. Sonutilisation dans des nouveaux carbones poreux contenant des nanoparticules d’or amis en évidence l’effet des conditions de séchage des enzymes et de la méthode desynthèse des nanoparticules. Les meilleures performances sont obtenues pour unséchage à 25 °C sous vide et une synthèse séquentielle des nanoparticules. Nousavons combiné ces deux BODs dans une nouvelle cathode bi-enzymatique. Au ratiooptimal de 50 %v de chaque BOD, elle opère à + 0,50 V vs. Ag/AgCl avec un courantde -0,86 ± 0,01 mA.cm-2 dans les conditions physiologiques. Elle a une forte activité àhaute température et en présence de chlore et une stabilité intermédiaire enprésence d’urate. Dans les mêmes conditions nous avons réalisé une cathode bienzymatiqueavec B. pumilus et la laccase de Podospora anserina. Elle estégalement plus performante que les cathodes mono-enzymatiques correspondantes. / Here we showed the performances and the limits in electrochemistry of the two newBODs from Bacillus pumilus and Magnaporthe oryzae. The onset potential for theoxygen reduction with the BOD from M. oryzae is + 0.50 V vs. Ag/AgCl and with B.pumilus is + 0.44 V vs. Ag/AgCl. The BOD from M. oryzae is not sensitive to theconcentration of sodium phosphate in redox hydrogel but is sensitive to chloride,urate and high temperatures. The BOD from B. pumilus has a high activity in thepresence of chloride and at 50 °C, but is sensitive to the concentration of phosphatein the hydrogel. This sensitivity is offset by an improved stability in the presence ofurate, so it loses only 9 % of activity after 3 hours in calf serum. The BOD from M.oryzae immobilized without mediator outperforms B. pumilus. Its use in new porouscarbon materials containing gold nanoparticles showed the effect of enzymes dryingconditions of the synthesis method of the nanoparticles. The best performance isobtained for a drying at 25 °C under vacuum and a sequential synthesis ofnanoparticles. We combined these two BODs in a new bi-enzymatic cathode. At theoptimal ratio of 50 %v of each BOD, it operates at + 0.50 V vs. Ag/AgCl with a currentdensity of -0.86 ± 0.01 mA.cm-2 under physiological conditions. It has a high activityat high temperatures and in the presence of chloride and an intermediate stability inthe presence of urate. Under the same conditions we conceived a bi-enzymaticcathode with B. pumilus and laccase Podospora anserina. It is also more efficientthan the single-enzymatic corresponding cathodes.

Single cell proteins such as that of edible filamentous fungal biomass are considered as a promising sustainable source of animal feed supplementation. Filamentous fungi can be cultivated on different organic substrates including volatile fatty acids (VFAs) such as acetic, propionic, and butyric acids. These VFAs can be generated through the famous waste valorisation approach of anaerobic digestion (AD) as intermediate metabolites. This project investigates a sustainable approach for the production of animal feed supplementation through cultivation of fungal biomass on waste derived VFAs along with the in vitro analysis of fungal biomass digestibility as ruminant feed. In this regard, optimum conditions for the production of Aspergillus oryzae biomass on different VFAs effluents derived from anaerobic digestion process of food waste plus chicken manure (FWCKM) and potato protein liquor (PPL) at different pH, nitrogen sources, and feed mixture was studied. Accordingly, analyses showed that PPL has the highest biomass yield with 0.4 (g biomass/g consumed VFAs) based on the volatile solids (VS) by adjusting pH to 6.2. Furthermore, the digestibility of the produced fungal biomass is analysed by using three different in vitro digestion methods including Tilley and Terry (TT) method, Gas Production Method (GPM), and Nylon Bag Method (NBM) and the results are compared with the conventional feed (silage and rapeseed meal). Results obtained from different digestibility methods illustrate that different A. oryzae fungal biomass had approximately 10-15 % higher dry matter digestibility fraction compared to silage and rapeseed meal (reference feeds). Hence, these results revealed that A. oryzae fungal biomass can grow on VFAs effluents and produce protein-rich fungal biomass while this biomass has better digestibility compared to conventional feeds and confirmed the initial hypothesis of the study.

Doctor of Philosophy / Department of Grain Science and Industry / Praveen V. Vadlani / The purpose of this study was to investigate micro- and macro-scale aspects of solid state fermentation (SSF) for production of cellulolytic enzymes using fungal cultures. Included in the objectives were investigation of effect of physicochemical characteristics of substrate on enzymes production at micro-scale, and design, fabrication and analysis of solid-state bioreactor at macro-scale. In the initial studies response surface optimization of SSF of soybeans hulls using mixed culture of Trichoderma reesei and Aspergillus oryzae was carried out to standardize the process. Optimum temperature, moisture and pH of 30ºC, 70% and 5 were determined following optimization. Using optimized parameters laboratory scale-up in static tray fermenter was performed that resulted in production of complete and balanced cellulolytic enzyme system. The balanced enzyme system had required 1:1 ratio of filter paper and beta-glucosidase units. This complete and balanced enzyme system was shown to be effective in the hydrolysis of wheat straw to sugars. Mild pretreatments– steam, acid and alkali were performed to vary physicochemical characteristics of soybean hulls – bed porosity, crystallinity and volumetric specific surface. Mild nature of pretreatments minimized the compositional changes of substrate. It was explicitly shown that more porous and crystalline steam pretreated soybean hulls significantly improved cellulolytic enzymes production in T. reesei culture, with no effect on xylanase. In A. oryzae and mixed culture this improvement, though, was not seen. Further studies using standard crystalline substrates and substrates with varying bed porosity confirmed that effect of physicochemical characteristics was selective with respect to fungal species and cellulolytic activity. A novel deep bed bioreactor was designed and fabricated to address scale-up issues. Bioreactor’s unique design of outer wire mesh frame with internal air distribution and a near saturation environment within cabinet resulted in enhanced heat transfer with minimum moisture loss. Enzyme production was faster and leveled within 48 h of operation compared to 96 h required in static tray. A two phase heat and mass transfer model was written that accurately predicted the experimental temperature profile. Simulations also showed that bioreactor operation was more sensitive to changes in cabinet temperature and mass flow rate of distributor air than air temperature.

Doctor of Philosophy / Department of Plant Pathology / William W. Bockus / James P. Stack / Wheat blast, caused by the Triticum pathotype of Magnaporthe oryzae (MoT), is a serious disease of wheat causing yield failures and significant economic losses during epidemic years in Brazil, Paraguay, and Bolivia. Although outbreaks occur only sporadically, wheat blast is considered a major disease affecting wheat production in South America and may be a threat to the wheat crop in the United States. Wheat is a major crop in the U.S. and wheat exports from the U.S. are important to food security of several countries around the World. Thus, it is important to understand the potential for MoT entry and establishment into the U.S. and to test U.S. wheat cultivars for susceptibility to MoT. The hypotheses of this research project were a) importing wheat grain from Brazil does not pose a risk for MoT establishment in the U.S., and b) resistance to MoT head infection does not exist in U.S. hard red winter wheat elite cultivars. Quantitative pathway analysis models were used to estimate the risk of MoT entry and establishment, in the coterminous U.S. and in a more targeted area within southeast North Carolina, via the importation of wheat grain from Brazil. The pathway model predicted that significant risk for MoT entry and establishment exists in some areas of the U.S. However, in approximately 60% of the coterminous U.S. winter wheat production areas the risk of MoT establishment was estimated to be zero. With respect to winter wheat growing areas in the U.S., conditions for MoT establishment and wheat blast outbreak occur only in small, restricted geographic areas. A higher resolution pathway analysis based on a ground transportation corridor in North Carolina indicated that conditions for MoT establishment exist seven out of ten years. Among U.S. cultivars tested, a continuum in severity to head blast was observed; cultivars Everest and Karl 92 were highly susceptible with more than 90% disease severity, while cultivars PostRock, Jackpot, Overley, Jagalene, Jagger, and Santa Fe showed less than 3% infection.

Endocytosis is both an ancient and a diverse feature of the eukaryotic cell. Studying how it evolved can provide insight into the nature of the last common eukaryotic ancestor, and the diversification of eukaryotes into the known extant lineages. In this thesis, I present two studies on the evolution of endocytosis. In the first part of the thesis I report results from a large-scale, phylogenetic and comparative genomic study of clathrin-mediated endocytosis (CME). The CME pathway has been studied to a great level of detail in yeast to mammal model organisms. Several protein families have now been identified as part of the complex set of protein-protein and protein-lipid interactions which mediate endocytosis. To investigate how such complexity evolved, first, I defined the modular nature of the CME interactome (CME-I) by literature review, and then I carried out a systematic phylogenetic and protein domain architecture analysis of the proteins involved. These data were used to construct a model of the evolution of the CME-I network, and to map the expansion of the network's complexity to the eukaryotic tree of life. In the second part of the thesis, I present results from evolutionary and functional studies of the eisosome, a protein complex which has been proposed to regulate the spatial distribution of endocytosis in S. cerevisiae. The phylogeny of eisosomes components Pil1 and Lsp1 reported here, suggests that eisosomes are likely to have originated at the base of the fungi, and then diversified significantly via multiple gene duplications. I thus studied the localisation and function of Pil1 and Lsp1 homologues in Magnaporthe oryzae to investigate the role of eisosomes in filamentous fungi. Results suggests that eisosomes are linked with septal formation and integrity in M. oryzae, and that the septal specific Pil2 paralogue was lost in budding yeasts. Together, the data presented in this thesis describe the evolutionary history of a complex biological system, but also highlights the problem of asymmetry in the understanding of endocytic diversity in the eukaryotes.

La pyriculariose, maladie causée par le champignon phytopathogène Magnaporthe oryzae, affecte gravement le riz qui constitue l'aliment de base de plus de la moitié de la population mondiale. La connaissance des mécanismes de résistance est nécessaire pour guider la sélection variétale. Au cours de ce travail, une synthèse de la littérature a permis de recenser plus de 60 gènes régulateurs du riz impliqués dans la résistance du riz à différents agents pathogènes. Nous avons complété ces données en étudiant le rôle in planta de huit de ces régulateurs. Un rôle central du facteur de transcription OsWRKY28 a pu être établi et le rôle du récepteur CEBiP a été démontré. Ce travail a aussi exploré l'éventuelle implication d'une nouvelle famille de récepteurs, les Wall-Associated Kinases (WAK) dans la résistance chez le riz. Ce travail montre l'implication des WAK dans la résistance à M. oryzae. Alors que la transcription de la plupart de ces gènes est induite au cours de l'infection, celle du gène WAK112d est réprimée. La régulation transcriptionnelle précoce observée pour certains gènes WAK est déclenchée par la chitine et sous contrôle partiel du récepteur CEBiP et d'OsWRKY28. L'étude de mutants d'insertion et de lignées de surexpression a permis de montrer le rôle positif de trois gènes WAK et le rôle négatif du gène WAK112d dans la résistance. Des approches biochimiques seront nécessaires pour comprendre le mode de fonctionnement de ces récepteurs et pour les relier aux autres systèmes de défense connus. / Rice blast disease, caused by the fungus Magnaporthe oryzae, is one of the most serious diseases on rice which is the staple food of more than the half of the world population. Improving our knowledge of resistance mechanisms is necessary to guide breeding programs. In this study, we reviewed over 60 rice gene regulators involved in resistance against various pathogens. We completed these data by analyzing the role of eight of these regulators. A pivotal role for the transcription factor OsWRKY28 has been established and the role of the CEBiP receptor in planta has been demonstrated. This work also shows the implication of some WAKs in rice blast resistance. Whereas the transcription of most of these genes is induced, transcription of the OsWAK112d gene is repressed upon infection. The early transcriptional regulation observed for some OsWAK genes is triggered by chitin and partially under CEBiP and OsWRKY28 regulation. Analysis of insertion mu tants and over-expressor lines revealed a positive role for three OsWAK genes and a negative role for OsWAK112d gene in rice blast resistance. Biochemical studies will be essential to understand how these receptors work and to connect them to other known defense systems.