The use of lignocellulosic materials to replace fossil resources for the industrial production of fuels, chemicals, and materials is increasing. The carbohydrate composition of lignocellulose (i.e. cellulose and hemicellulose) is an abundant source of sugars. However, due to the feedstock recalcitrance, rigid and compact structure of plant cell walls, access to polysaccharides is hindered and release of fermentable sugars has become a bottle-neck. Thus, to overcome the recalcitrant barriers, thermochemical pretreatment with an acid catalyst is usually employed for the physical or chemical disruption of plant cell wall. After pretreatment, enzymatic hydrolysis is the preferred option to produce sugars that can be further converted into liquid fuels (e.g. ethanol) via fermentation by microbial biocatalysts. However, during acid pretreatment, several inhibitory compounds namely furfural, 5-hydroxymethyl furfural, phenols, and aliphatic acids are released from the lignocellulose components. The presence of these compounds can greatly effect both enzymatic hydrolysis and microbial fermentation. For instance, when Avicel cellulose and acid treated spruce wood hydrolysate were mixed, 63% decrease in the enzymatic hydrolysis efficiency was observed compared to when Avicel was hydrolyzed in aqueous citrate buffer. In addition, the acid hydrolysates were essentially non-fermentable. Therefore, the associated problems of lignocellulose conversion can be addressed either by using feedstocks that are less recalcitrant or by developing efficient pretreatment techniques that do not cause formation of inhibitory byproducts and simultaneously give high sugar yields. A variety of lignocellulose materials including woody substrates (spruce, pine, and birch), agricultural residues (sugarcane bagasse and reed canary grass), bark (pine bark), and transgenic aspens were evaluated for their saccharification potential. Apparently, woody substrates were more recalcitrant than the rest of the species and bark was essentially amorphous. However, the saccharification efficiency of these substrates varied based on the pretreatment method used. For instance, untreated reed canary grass was more recalcitrant than woody materials whereas the acid treated reed canary grass gave a higher sugar yield (64%) than the woody substrates (max 34%). Genetic modification of plants was beneficial, since under similar pretreatment and enzymatic hydrolysis conditions, up to 28% higher sugar production was achieved from the transgenic plants compare to the wild type. As an alternative to the commonly used acid catalysed pretreatments (prior to enzymatic hydrolysis) lignocellulose materials were treated with four ionic liquid solvents (ILs): two switchable ILs (SILs) -SO2DBUMEASIL and CO2DBUMEASIL, and two other ILs [Amim][HCO2] and [AMMorp][OAc]. viii After enzymatic hydrolysis of IL treated substrates, a maximum amount of glucan to glucose conversion of between 75% and 97% and a maximum total sugar yields of between 71% and 94% were obtained. When using acid pretreatment these values varied between 13-77% for glucan to glucose conversion and 26-83% for total sugar yield. For woody substrates, the hemicellulose recovery (max 92%) was higher for the IL treated substrates than compared to acid treated samples. However, in case of reed canary grass and pine bark the hemicellulose recovery (90% and 88%, respectively) was significantly higher for the acid treated substrates than the IL treated samples. To overcome the inhibitory problems associated with the lignocellulose hydrolysates, three chemical conditioning methods were used 1. detoxification with ferrous sulfate (FeSO4) and hydrogen peroxide (H2O2) 2. application of reducing agents (sulfite, dithionite, or dithiothreitol) and 3. treatment with alkali: Ca(OH)2, NaOH, and NH4OH. The concentrations of inhibitory compounds were significantly lower after treatments with FeSO4 and H2O2 or alkali. Using reducing agents did not cause any decrease in the concentration of inhibitors, but detoxification of spruce acid hydrolysates resulted in up to 54% improvement of the hydrolysis efficiency (in terms of sugar release) compared to untreated samples. On the other hand, application of detoxification procedures to the aqueous buffer resulted in up to 39% decrease in hydrolysis efficiency, thus confirming that the positive effect of detoxification was due to the chemical alteration of inhibitory compounds. In addition, the fermentability of detoxified hydrolysates were investigated using the yeast Saccharomyces cerevisiae. The detoxified hydrolysates were readily fermented to ethanol yielding a maximum ethanol concentration of 8.3 g/l while the undetoxified hydrolysates were basically non-fermentable.

Made available in DSpace on 2014-10-09T12:45:13Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:56:31Z (GMT). No. of bitstreams: 1 07293.pdf: 5734585 bytes, checksum: d14fa6efc5fe260e124df558d69af5bd (MD5) / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Les moustiques génèrent une nuisance importante et sont notamment contrôlés grâce à des traitements insecticides. Aujourd'hui, les gîtes où se développent leurs larves sont souvent pollués par des xénobiotiques environnementaux (hydrocarbures, herbicides, pesticides, toxines naturelles…). Jusqu'à présent, l'impact de ces xénobiotiques sur la capacité des larves de moustiques à résister aux insecticides chimiques reste méconnu. Cette thèse vise à étudier la réponse des larves de d'Aedes aegypti aux xénobiotiques environnementaux et leur impact sur leur tolérance et résistance aux insecticides chimiques. Une première étude, sur le court terme, montre que des larves exposées pendant 24h à divers xénobiotiques deviennent plus tolérantes à vis à vis de différents insecticides chimiques (Poupardin et al. 2008). Des études biochimiques et transcriptomiques suggèrent que l'induction de certaines familles d'enzymes (e.g. P450s et GSTs) par ces xénobiotiques peut être liée à l'augmentation de tolérance des larves vis-à-vis de l'insecticide. Dans le but de mieux caractériser le profil transcriptionnel des précédents gènes candidats, des expérimentations complémentaires ont été faites à différents niveaux (Poupardin et al., 2010). Cette étude a montré que de nombreux gènes étaient préférentiellement transcrits dans des tissus fortement impliqués dans la détoxication de composés exogènes, essentiellement des CYP6. Elle révèle aussi que la transcription de ces P450s varie beaucoup au cours des différents stades de développement et qu'ils étaient induits à des faibles de doses de polluants avec un pic d'induction après 48 et 72 heures d'exposition. Ces études mettent en évidence le rôle potentiel des gènes de détoxication dans la réponse à l'exposition à des xénobiotiques et dans l'augmentation de tolérance aux insecticides chimiques. Concernant l'étude sur le long terme de l'impact des polluants sur la résistance des moustiques aux insecticides, la question est de savoir si les polluants trouvés dans l'environnement influencent la sélection de la résistance aux insecticides et si oui, favorisent-ils la sélection de gènes en particulier? Pour répondre à ces questions, trois souches d'Aedes aegypti ont été sélectionnées à la perméthrine. Ces souches sont exposées ou non à différents polluants avant sélection. Après 10 générations de sélection, des bioessais montrent une résistance de ces 3 souches vis-à-vis de la perméthrine. Aucune différence significative de niveau de résistance n'est observée entre les trois souches sélectionnées pour le moment. Pour identifier les gènes différentiellement transcrits dans ces souches, la puce "Agilent Aedes chip" développée par l'école de médecine tropicale de Liverpool (LSTM) et contenant 14200 transcrits a été utilisée. Les microarrays ont révélé que la présence de polluants ou insecticides résiduels pouvait affecter la sélection des mécanismes de résistance aux insecticides chimiques, notamment par la sélection de gènes particuliers codant pour des enzymes de détoxication (Poupardin et al, en préparation). D'une manière globale, cette thèse permettra de mieux comprendre l'impact de l'environnement chimique sur la résistance des moustiques aux insecticides et fournira de nouvelles pistes afin d'optimiser les traitements insecticides utilisés en démoustication. / Mosquitoes have a major impact on public health due to their capacity to transmit human diseases such as viruses (dengue, yellow-fever, west-Nile, chikungunya…) and parasites (malaria, filariasis…). To control them, insecticides have been heavily used since the 1950's leading to the emergence of insecticide resistance. Today, wetlands where mosquito larvae develop are frequently contaminated by environmental xenobiotics (e.g. residual insecticides, agrochemicals, pollutants and plant allelochemicals) and little is known about the impact of these molecules on the capacity of mosquitoes to resist insecticides. The aim of my thesis is to study the response of mosquito larvae to xenobiotic exposures and the impact of these molecules on the tolerance (single generation) and resistance (multiple generations) of mosquitoes to chemical insecticides. A first ‘short term' study revealed that mosquito larvae exposed for few hours to sub-lethal doses of various xenobiotics become more tolerant to several chemical insecticides (Poupardin et al., 2008, Riaz et al., 2009) and that this increased tolerance is linked with an increase of detoxification enzyme activities. Thanks to the “Aedes detox chip” developed in LSTM, we showed that several detoxification genes, especially P450s, were induced by various xenobiotics which could explain the increased tolerance of mosquito larvae to insecticides. In order to better characterize these genes, their transcription profiles were studied at different life stages and in various organs (Poupardin et al., 2010). We demonstrated that several of these P450s are preferentially transcribed in gastric caeca, midgut and malpighian tubules, known to play an important role in xenobiotic metabolism. Moreover, we found that the transcription levels of these genes vary according to life stages. Finally, several genes were induced by environmental doses of xenobiotics with a maximum induction peak at 48-72h after exposure. Overall, these studies evidenced of the potential role of mosquito detoxification genes to respond to xenobiotic exposure and to affect their tolerance to chemical insecticides. The other aim of my thesis was to understand the ‘long term' (across several generations) impact of xenobiotics on the selection of insecticide resistance mechanisms in mosquitoes. In other words, ‘Do pollutants affect the selection of insecticides resistance mechanism by insecticides treatments' and if yes, ‘are particular genes favoured?' To answer these questions, three strains of the mosquito Aedes aegypti were selected with the pyrethroid insecticide permethrin. Before the selection process, larvae were exposed or not to sub-lethal dose of various pollutants. After 11 generations of selection, the three strains showed elevated resistance to permethrin compared to the susceptible strain. To identify the genes differentially transcribed in these resistant strains, we used the new ‘Agilent Aedes chip' representing more than 14,200 transcripts developed by the LSTM. Microarray results showed that the presence pollutants or residual insecticide can affect the selection of insecticide resistance mechanisms by favouring the selection of particular genes such as those encoding for detoxification enzymes (Poupardin et al., in prep). Globally, this research work will provide a better understanding of the impact of environmental factors on insecticide resistances in mosquitoes and will provide new ways to optimize the control of vectors with insecticides.

Florações de cianobactérias representam grave problema para a qualidade da água de lagos e reservatórios, pela produção de toxinas que podem causar danos a organismos aquáticos e seres humanos. Como implicação, o processo de formação de colônias ou agregados de cianobactérias impede a predação pelo zooplâncton, mas, por outro lado, aumenta a vulnerabilidade das cianobactérias à pressão de herbivoria por peixes filtradores. Estudos experimentais têm quantificado as taxas de ingestão de algas e cianobactérias por Oreochromis (tilápia) em laboratório, porém, pouco se sabe sobre interações de espécies de peixes nativos, como do gênero Geophagus (cará) com cianobactérias. Neste sentido, os objetivos deste estudo foram (1) investigar, experimentalmente, medidas de controle natural de cianobactérias através da utilização de peixes filtradores nativos e exóticos; (2) identificar estruturas relacionadas à eficiência de filtração nestes peixes e (3) avaliar mecanismos de dano-reparo enzimático em peixes que tem sua dieta baseada na ingestão de cianobactérias tóxicas, através da relação de enzimas antioxidantes e a ocorrência de estresse oxidativo devido à possibilidade de produção de cianotoxinas. Experimentos foram realizados avaliando as taxas de consumo das cianobactérias Microcystis aeruginosa e Cylindrospermopsis raciborskii por Oreochromis sp. (espécie exótica) e Geophagus brasiliensis (espécie nativa). Os tratamentos, em triplicata, incluíram peixes de duas classes de tamanho (Tratamento1: peixes de 5-10 cm; Tratamento 2: peixes de 10-15 cm); três concentrações de cianobactérias (10μg/L, 20μg/L e 40 μg/L) e controles contendo somente inóculo algáceo nas concentrações 10μg/L, 20μg/L e 40 μg/L. Com o intuito de evidenciar diferenças entre os tratamentos foi realizada análise da variância (ANOVA). Análises morfológicas nas brânquias de exemplares utilizados nos experimentos foram realizadas através de microscopia eletrônica de varredura (MEV) para verificar a existência de estruturas relacionadas à eficiência de filtração nestes peixes. Por fim, para avaliar os efeitos das cianotoxinas nos exemplares utilizados nos experimentos foram realizadas análises bioquímicas de lipoperoxidação (LPO) avaliando o dano oxidativo e a atividade das enzimas antioxidantes catalase (CAT) e glutationa-s-transferase (GST) no fígado e nas brânquias dos exemplares. Com o intuito de evidenciar diferenças entre os tratamentos foi realizada análise da variância (ANOVA one-way). Para Oreochromis sp. frente à ingestão/filtração de M. aeruginosa, em todas as concentrações foram observadas diferenças significativas (p<0,05). Nos organismos-teste do tratamento 1 ocorreu decréscimo nas concentrações de cianobactérias, já nos organismos-teste do tratamento 2 ocorreu aumento das concentrações de cianobactérias ao longo do tempo. Para Oreochromis sp. frente à ingestão/filtração de C. raciborskii, diferenças significativas (p<0,05) foram observadas principalmente nas concentrações 10μg/L e 20μg/L quanto à redução das concentrações de cianobactérias ao longo do tempo. No experimento utilizando G. brasiliensis frente à ingestão/filtração de M. aeruginosa, não ocorreram diferenças significativas (p>0,05) nas concentrações 10μg/L e 40μg/L, entretanto ocorreu uma diminuição nas concentrações de cianobactérias. Contudo, no experimento avaliando as taxas de ingestão/filtração de C. raciborskii por G. brasiliensis ocorreu redução na concentração de cianobactérias nas concentrações 10μg/L e 20μg/L em ambos os tratamentos (p<0,05). Resultados das análises morfológicas através de microscópio eletrônico de varredura evidenciaram a existência de micro-espinhos branquiais, tanto em Oreochromis sp. quanto em G. brasiliensis, validando a possibilidade de G. brasiliensis poder ser utilizado como controle natural de cianobactérias. Análises do balanço oxidativo revelaram como padrão geral para ambas as espécies de ciclídeos avaliados, a ocorrência de alterações bioquímicas nas condições testadas. Porém nestas condições-teste (espécies de peixes e de cianobactérias, concentrações das cianobactérias avaliadas e tempo de experimento) estes danos foram reparados pela ação das enzimas antioxidantes. Os resultados obtidos neste estudo representam importantes conhecimentos para o manejo e restauração de ecossistemas aquáticos eutrofizados baseado em interações de espécies. / Cyanobacteria blooms represent a serious problem for the quality of water from lakes and reservoirs, the production of toxins to aquatic organisms and humans. As implication, the process of formation of colonies or aggregates of cyanobacteria preventing predation by zooplankton, but on the other hand, increases the vulnerability of the cyanobacteria to filtrating pressure by herbivorous fish. Experimental studies have quantified the ingestion rates of algae and cyanobacteria by Oreochromis (tilapia) in the laboratory, however, little is known about interactions of species of native fish such as the genus Geophagus (cará) with cyanobacteria. In this sense, the objectives of this study were (1) to investigate experimentally measured natural control of cyanobacteria by using filter-native and exotic fishes, (2) identified structures related to the filtration efficiency in these fishes (3) evaluating mechanisms of damage-repair enzyme in fish that have diet based on their ingestion of toxic cyanobacteria, through the relation antioxidant enzymes activity and the occurrence of oxidative stress. Experiments were conducted to evaluate the consumption rates of cyanobacteria Microcystis aeruginosa and Cylindrospermopsis raciborskii by Oreochromis sp. (exotic species) and Geophagus brasiliensis (native species). Were determined treatments in triplicate consisting of fish of two size classes (Treatment 1: fish 5-10 cm; Treatment 2: fish 10-15 cm); three cyanobacterial concentrations (10μg/L, 20μg/L and 40μg/L) and controls containing only algal inoculums concentrations 10μg/L, 20μg/L and 40μg/L. In order to show differences between treatments analysis of variance (ANOVA) was performed. Morphological analysis in the gills of specimens used in the experiments were carried out using scanning electron microscopy (SEM) to verify the existence of the structures related to filtration efficiency in these fishes. To assess the effects of cyanotoxins on the specimens used in the experiments biochemical analyzes were performed in the liver and gill measuring lipoperoxidation (LPO) assessing oxidative damage, and antioxidant enzymes catalase (CAT) and glutathione-s-transferase (GST). In order to show differences between treatments analysis of variance (ANOVA) was performed. Designed to evaluate whether the concentrations and the treatments were effective was an analysis of variance ANOVA two-way and in order to highlight differences between treatments was performed analysis of variance ANOVA. Morphological analysis in the gills of samples used in the experiments were performed using scanning electron microscopy (SEM) to verify the existence of related structures to the filtration efficiency in these fish. To assess the effects of cyanotoxins in the animals used in the experiments were performed biochemical analyzes in the liver and gills of lipoperoxidation (LPO) assessing oxidative damage, and the activity of antioxidant enzymes catalase (CAT) and glutathione-S-transferase (GST). In order to highlight differences between treatments was performed analysis of variance (one-way ANOVA). For Oreochromis sp. the intake/filtration of M. aeruginosa in all concentrations were significant differences (p<0.05) in test organisms in T1 were decreasing concentrations of the cyanobacteria and the test organisms in T2 was increased concentrations of cyanobacteria over time. In the experiment using G. brasiliensis the intake/filtration of M. aeruginosa, no significant difference in the concentration 10g/L and 40g/L (p>0.05), however there was a decrease in cyanobacteria concentrations. For Oreochromis sp. the intake/filtration of C. raciborskii, significant differences were observed mainly in the concentration 10g/L and 20g/L and the reduction of cyanobacteria concentrations over time (p<0.05). However, in the experiment evaluating the intake/filtration rates of C. raciborskii by G. brasiliensis occurred reduction in the concentration of cyanobacteria at 10g/L and 20g/L concentrations in both groups (p<0.05). Results of morphological analysis using a scanning electron microscope revealed the existence of gill microspine, both in Oreochromis sp. as for G. brasiliensis validating the possibility of G. brasiliensis can be used as natural control of cyanobacteria. The analyzes realized in the oxidative balance of the test organisms based diet ingestion of toxic cyanobacteria revealed, as a general standard for both cichlid species evaluated, the occurrence of biochemical changes in the conditions used, but considering these test conditions (fish species, cyanobacteria species, concentrations of cyanobacteria and time trial) such damages were repaired by the action of antioxidant enzymes. These results are important for knowledge management and restoration of eutrophic aquatic ecosystems based on species interactions.

La résistance des moustiques aux insecticides pyréthrinoïdes (PYRs) menace les programmes de lutte anti-vectorielle à l'échelle mondiale. Chez le moustique Aedes aegypti, vecteur de la dengue et du Chikungunya, les principaux mécanismes causant cette résistance ont été identifiés. La résistance métabolique joue alors un rôle important et consiste en une biodégradation accrue de l'insecticide par des enzymes de détoxication. Néanmoins, les bases moléculaires de ce mécanisme restent méconnues. La plupart des gènes impliqués dans la résistance métabolique aux PYRs ont été identifiés par des approches transcriptomiques, mais les modifications génomiques à l'origine de leur sur-expression dans les populations résistantes ainsi que les modifications structurales des enzymes en lien avec la résistance restent méconnues. Cette thèse vise alors à utiliser les nouvelles approches de séquençage à haut débit (NGS) pour caractériser les mécanismes moléculaires de la résistance aux PYRs chez le moustique Ae. Aegypti. La première partie de la thèse présente une étude pilote RNA-seq menée sur des populations de laboratoire sélectionnées avec des insecticides. Cette étude a pour objectif d'évaluer les avantages des NGS pour l'étude des mécanismes de résistance chez les moustiques. Le rôle des enzymes de détoxication dans la résistance a ainsi été clairement confirmé. Plusieurs gènes codant pour ces enzymes apparaissent sur-exprimés dans les populations résistantes et un important regroupement de P450 montre une forte empreinte de sélection en lien avec la résistance aux PYRs. La seconde partie de la thèse présente une étude sur des populations naturelles échantillonnées sur divers continents. Cette étude combine les technologies d'enrichissement génomique et de DNA-seq afin d'étudier les variations génomiques liées à la résistance au PYR Deltaméthrine. La comparaison de la couverture de séquençage entre populations résistantes et sensibles a permis d'identifier des variations de nombre de copies (CNVs) de certains gènes de détoxication associées à la résistance à la Deltaméthrine. Des mutations non-synonymes fortement liées au phénotype de résistance ont également été mises en évidence. La comparaison de ces marqueurs de la résistance entre les différentes populations a révélé que les gènes/mutations associés à la résistance à la Deltaméthrine sont peu conservés entre continents, probablement à cause des différences de fond génétique des populations, de leur histoire démographique et des pressions de sélections. La troisième partie de la thèse décrit une étude par RNA-seq portant sur les mêmes populations naturelles, visant à croiser des données de transcriptomique (expression des gènes et polymorphisme des transcrits) avec les données génomiques générées par l'étude précédente. Plusieurs enzymes de détoxications ont été retrouvées sur-exprimées chez les populations résistantes en lien avec les CNVs précédemment identifiées. Des centaines de variations de polymorphisme ont été identifiées par DNA-seq dans les zones cis-promotrices des différents gènes étudiés. Parmi ces variations, plusieurs apparaissent associées à la sur-régulation d'enzymes de détoxication. Enfin, la comparaison des données de polymorphismes obtenues par DNA-seq et RNA-seq a permis d'étudier les phénomènes d'expression d'allèles spécifiques en lien avec la résistance. Cette étude confirme l'intérêt de croiser des données de transcriptomique et de génomique pour caractériser les bases moléculaires de la résistance aux insecticides. D'un point de vue général, cette thèse permet de mieux appréhender les mécanismes de résistance du moustique Ae. aegypti aux PYRs mais aussi d'identifier de nouveaux marqueurs de la résistance potentiellement utilisables pour développer de nouveaux outils moléculaires diagnostiques de la résistance sur le terrain. Ce travail met également en avant les apports des NGS pour l'étude fine des bases moléculaires de l'adaptation d'organismes modèles. / Mosquito control programs worldwide are increasingly threatened by resistance to pyrethroid insecticides (PYRs). In the dengue and chikungunya vector Aedes aegypti, the key resistance mechanisms include modifications in the protein targeted by insecticides (target-site mutations) and metabolic resistance, consisting in an increased insecticide biodegradation by so called detoxification enzymes. However, as opposed to target-site mutations, the molecular basis of metabolic resistance remains poorly understood. Most metabolic resistance genes have been detected by transcriptomic approaches based on their over-expressed in resistant populations, but genomic changes leading to these expression changes as well as structural changes in enzymes potentially involved in resistance remain unknown. In this context, this thesis aims at using next-generation sequencing approaches for characterizing PYR resistance mechanisms in the mosquito Ae. aegypti.The first chapter of this thesis describes a pilot study on laboratory insecticide-selected populations of Ae. aegypti. This study aims at investigating the benefits of next-generation sequencing for studying resistance mechanisms in mosquitoes. This study confirmed that detoxification enzymes play a key role in resistance, with several of them being over-expressed in resistant populations and a large cluster of cytochrome P450 genes showing a selection imprint associated with resistance to PYRs.The second chapter of this thesis describes a study conducted on natural mosquito populations from various continents. Combining genomic target enrichment (targeting about 800 genes potentially involved in resistance) and DNA-seq allowed unravelling genomic changes associated with resistance to the PYR deltamethrin. Comparing normalized sequencing coverage between resistant and susceptible populations identified significant copy number variations (CNVs) in several detoxification genes strongly associated to deltamethrin resistance. Non-synonymous mutations affecting detoxification enzymes associated to the resistance phenotype were also detected. Comparing resistance markers between populations from various continents revealed that genes/mutations associated with deltamethrin resistance are poorly conserved across continents, probably due to differences in the genetic background of populations but also differences in terms of demographic history and selection pressures.The third chapter describes an RNA-seq study performed on the same natural mosquito populations in order to cross-link transcriptomic data (gene expression and transcript polymorphism) with genomic data obtained from the previous study. Multiple detoxification enzymes were found over-transcribed in resistant populations linked with previously identified CNVs. Hundreds polymorphism variations were identified by targeted DNA-seq in cis-promoter regions of detoxification genes. Among them, several were associated with the upper-regulation of detoxification enzymes in resistant populations. Finally, cross-comparing polymorphism data obtain from DNA-seq and RNA-seq allowed investigating allele specific expression (ASE) events related to PYR resistance. Overall, this study confirmed the benefits of combining transcriptomic and genomic NGS approaches for studying the molecular basis of insecticide resistance.As a whole, this thesis not only contributed to better understand PYR resistance mechanisms in the dengue vector Ae. aegypti but also identified novel genomic markers of resistance opening the way for developing new molecular diagnostic to early detect and monitor resistance mechanisms in the field. This work also highlights the benefits of using NGS technologies for unravelling the molecular bases of adaptation in model organisms.

Made available in DSpace on 2014-10-09T12:38:41Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:04:59Z (GMT). No. of bitstreams: 1 06006.pdf: 3042340 bytes, checksum: 62a6ebd206107cfeefee505a16da38f3 (MD5) / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Submitted by Deyse Queiroz (deysequeirozz@hotmail.com) on 2018-05-24T20:13:38Z No. of bitstreams: 1 CLEBIA PEREIRA DE FRANÇA - DISSERTAÇÃO PPGEA 2010..pdf: 11033989 bytes, checksum: 458970c54e3574daebe9028166c5ddeb (MD5) / Made available in DSpace on 2018-05-24T20:13:38Z (GMT). No. of bitstreams: 1 CLEBIA PEREIRA DE FRANÇA - DISSERTAÇÃO PPGEA 2010..pdf: 11033989 bytes, checksum: 458970c54e3574daebe9028166c5ddeb (MD5) Previous issue date: 2010-02 / Este trabalho foi realizado com o objetivo de estudar o potencial da espectrometria NIR e da quimiometria, para classificação de torta e farelo de mamona, submetida a diferentes tratamentos de detoxificação. Utilizaram-se os tratamentos químico com NaCl e Ca(OH)2 e térmico (40, 60, 80 e 100°C) para três cultivares de mamona a partir da torta e do farelo. A torta foi obtida em prensa mecânica das sementes e o farelo através de extração com solvente em Soxhlet. As medidas espectrais na região de 400 a 2500 nm e análise multivariada (PCA e SIMCA) foram empregadas para a identificação de padrões de agrupamento quanto ao processo de detoxificação. As medidas experimentais foram realizadas em duas etapas, em que na primeira foram utilizadas 180 amostras de torta e farelo de mamona da variedade BRS Paraguaçu, para otimização dos modelos quimiométricos. Cada classe foi constituída de 10 amostras representativas, tratadas com NaCl e Ca(OH)2 a 4% (m/m) e nas temperaturas de 40, 60, 80 e 100°, na segunda etapa, 605 amostras foram usadas com 15 unidades para cada classe de tratamento com Ca(OH)2 e NaCl a 1, 2 e 4% (m/m). Os espectros foram registrados em triplicatas autênticas para os tratamentos com 10 repetições para cada amostra. A partir dos espectros obtidos empregaram-se as técnicas multivariadas de PCA e SIMCA. Na PCA, observou-se no gráfico dos escores a formação de classes distintas com separação dos tratamentos com Ca(OH)2 e NaCl, além da sua combinação com incrementos de temperatura de 40, 60, 80 e 100°C. O agrupamento formado com duas componentes principais resultou em uma variância explicada superior a 95%. Com as informações da PCA desenvolveu-se um modelo SIMCA, para o qual foram previstos 100% de acerto para a classe da torta e farelo de mamona detoxificados da variedade Paraguaçu, referentes a primeira etapa, a PCA para as amostras tratadas com Ca( OH)2 e NaCl a 1, 2 e 4% (m/m) permitiu a identificação das amostras consideradasdetoxificadas para as variedades BRS Energia, BRS Paraguaçu e BRS 149 Nordestina. O tratamento a 4% (m/m) se destacou no gráfico dos escores por ser considerado 100% detoxificado, também ocorreu separação entre as classes BRS Paraguaçu e BRS 149 Nordestina em relação à BRS Energia. Com essas observações, a espectrometria NIR e a análise multivariada permitiram a identificação da torta e do farelo de mamona, considerados detoxificados de forma direta, não destrutiva, económica, rápida (30 s), sem o uso de reagentes caros e de geração resíduos químicos. / This work was carried out to study the potential of NIR spectroscopy and chemometrics for the classification of cake and castor meai under different treatments of detoxifícation. We used chemical treatments with NaCl and Ca (OH)2 and heat (40, 60, 80 and 100 °C) for three cultivars from the castor bean cake and meai. The cake was obtained from mechanical pressing of the seeds and bran by solvent extraction in Soxhlet. The spectral measurements in the region from 400 to 2500 nm and multivariate analysis (PCA and SIMCA) were used to identify patterns of grouping as the process of detoxifícation. The experimental measurements were performed in two stages: first stage was used 180 samples of cake and castor oil for the BRS Paraguaçu chemometric optimization. Each class was comprised of 10 representative samples treated with NaCl and Ca (OH)2 to 4% (w / w) and temperatures of 40, 60, 80 and 100 °C. In the second stage, 630 samples were used with 42 units for each class of treatment with Ca (OH)2 and NaCl at 1, 2 and 4% (w / w). The spectra were recorded in triplicate true for treatments with 10 repetitions for each sample. From the spectra obtained were employed multivariate techniques of PCA and SIMCA. In PCA, the graph of the scores observed the formation of separate classes with separate treatments with Ca(OH)2 and NaCl, and combinations of these with temperature increments of 40, 60, 80 and 100 °C. The group formed with two principal components explained variance resulted in a greater than 95%. With the information from the PCA was developed SIMCA model for which predicted 100% correct for the class of the pie and detoxified castor meai variety Paraguaçu on the first step. The PCA for the samples treated with Ca(OH)2 and NaCl at 1, 2 and 4% (w/ w) allowed the identifícation of samples considered detoxified for varieties Energy BRS, BRS 149 and BRS Paraguaçu Northeast. Treatment 4% (w/ w) stood out in the graph of the scores to be considered 100% detoxified. Also there was a separation between the classes Paraguaçu BRS and BRS 149 BRS for Northeast Energy. Given these observations, NIR spectrometry and multivariate analysis allowed the identifícation of the pie and detoxified castor meai considered a direct, non-destructive, inexpensive, rapid (30 s) without the use of expensive reagents and chemical waste generation.

Florações de cianobactérias representam grave problema para a qualidade da água de lagos e reservatórios, pela produção de toxinas que podem causar danos a organismos aquáticos e seres humanos. Como implicação, o processo de formação de colônias ou agregados de cianobactérias impede a predação pelo zooplâncton, mas, por outro lado, aumenta a vulnerabilidade das cianobactérias à pressão de herbivoria por peixes filtradores. Estudos experimentais têm quantificado as taxas de ingestão de algas e cianobactérias por Oreochromis (tilápia) em laboratório, porém, pouco se sabe sobre interações de espécies de peixes nativos, como do gênero Geophagus (cará) com cianobactérias. Neste sentido, os objetivos deste estudo foram (1) investigar, experimentalmente, medidas de controle natural de cianobactérias através da utilização de peixes filtradores nativos e exóticos; (2) identificar estruturas relacionadas à eficiência de filtração nestes peixes e (3) avaliar mecanismos de dano-reparo enzimático em peixes que tem sua dieta baseada na ingestão de cianobactérias tóxicas, através da relação de enzimas antioxidantes e a ocorrência de estresse oxidativo devido à possibilidade de produção de cianotoxinas. Experimentos foram realizados avaliando as taxas de consumo das cianobactérias Microcystis aeruginosa e Cylindrospermopsis raciborskii por Oreochromis sp. (espécie exótica) e Geophagus brasiliensis (espécie nativa). Os tratamentos, em triplicata, incluíram peixes de duas classes de tamanho (Tratamento1: peixes de 5-10 cm; Tratamento 2: peixes de 10-15 cm); três concentrações de cianobactérias (10μg/L, 20μg/L e 40 μg/L) e controles contendo somente inóculo algáceo nas concentrações 10μg/L, 20μg/L e 40 μg/L. Com o intuito de evidenciar diferenças entre os tratamentos foi realizada análise da variância (ANOVA). Análises morfológicas nas brânquias de exemplares utilizados nos experimentos foram realizadas através de microscopia eletrônica de varredura (MEV) para verificar a existência de estruturas relacionadas à eficiência de filtração nestes peixes. Por fim, para avaliar os efeitos das cianotoxinas nos exemplares utilizados nos experimentos foram realizadas análises bioquímicas de lipoperoxidação (LPO) avaliando o dano oxidativo e a atividade das enzimas antioxidantes catalase (CAT) e glutationa-s-transferase (GST) no fígado e nas brânquias dos exemplares. Com o intuito de evidenciar diferenças entre os tratamentos foi realizada análise da variância (ANOVA one-way). Para Oreochromis sp. frente à ingestão/filtração de M. aeruginosa, em todas as concentrações foram observadas diferenças significativas (p<0,05). Nos organismos-teste do tratamento 1 ocorreu decréscimo nas concentrações de cianobactérias, já nos organismos-teste do tratamento 2 ocorreu aumento das concentrações de cianobactérias ao longo do tempo. Para Oreochromis sp. frente à ingestão/filtração de C. raciborskii, diferenças significativas (p<0,05) foram observadas principalmente nas concentrações 10μg/L e 20μg/L quanto à redução das concentrações de cianobactérias ao longo do tempo. No experimento utilizando G. brasiliensis frente à ingestão/filtração de M. aeruginosa, não ocorreram diferenças significativas (p>0,05) nas concentrações 10μg/L e 40μg/L, entretanto ocorreu uma diminuição nas concentrações de cianobactérias. Contudo, no experimento avaliando as taxas de ingestão/filtração de C. raciborskii por G. brasiliensis ocorreu redução na concentração de cianobactérias nas concentrações 10μg/L e 20μg/L em ambos os tratamentos (p<0,05). Resultados das análises morfológicas através de microscópio eletrônico de varredura evidenciaram a existência de micro-espinhos branquiais, tanto em Oreochromis sp. quanto em G. brasiliensis, validando a possibilidade de G. brasiliensis poder ser utilizado como controle natural de cianobactérias. Análises do balanço oxidativo revelaram como padrão geral para ambas as espécies de ciclídeos avaliados, a ocorrência de alterações bioquímicas nas condições testadas. Porém nestas condições-teste (espécies de peixes e de cianobactérias, concentrações das cianobactérias avaliadas e tempo de experimento) estes danos foram reparados pela ação das enzimas antioxidantes. Os resultados obtidos neste estudo representam importantes conhecimentos para o manejo e restauração de ecossistemas aquáticos eutrofizados baseado em interações de espécies. / Cyanobacteria blooms represent a serious problem for the quality of water from lakes and reservoirs, the production of toxins to aquatic organisms and humans. As implication, the process of formation of colonies or aggregates of cyanobacteria preventing predation by zooplankton, but on the other hand, increases the vulnerability of the cyanobacteria to filtrating pressure by herbivorous fish. Experimental studies have quantified the ingestion rates of algae and cyanobacteria by Oreochromis (tilapia) in the laboratory, however, little is known about interactions of species of native fish such as the genus Geophagus (cará) with cyanobacteria. In this sense, the objectives of this study were (1) to investigate experimentally measured natural control of cyanobacteria by using filter-native and exotic fishes, (2) identified structures related to the filtration efficiency in these fishes (3) evaluating mechanisms of damage-repair enzyme in fish that have diet based on their ingestion of toxic cyanobacteria, through the relation antioxidant enzymes activity and the occurrence of oxidative stress. Experiments were conducted to evaluate the consumption rates of cyanobacteria Microcystis aeruginosa and Cylindrospermopsis raciborskii by Oreochromis sp. (exotic species) and Geophagus brasiliensis (native species). Were determined treatments in triplicate consisting of fish of two size classes (Treatment 1: fish 5-10 cm; Treatment 2: fish 10-15 cm); three cyanobacterial concentrations (10μg/L, 20μg/L and 40μg/L) and controls containing only algal inoculums concentrations 10μg/L, 20μg/L and 40μg/L. In order to show differences between treatments analysis of variance (ANOVA) was performed. Morphological analysis in the gills of specimens used in the experiments were carried out using scanning electron microscopy (SEM) to verify the existence of the structures related to filtration efficiency in these fishes. To assess the effects of cyanotoxins on the specimens used in the experiments biochemical analyzes were performed in the liver and gill measuring lipoperoxidation (LPO) assessing oxidative damage, and antioxidant enzymes catalase (CAT) and glutathione-s-transferase (GST). In order to show differences between treatments analysis of variance (ANOVA) was performed. Designed to evaluate whether the concentrations and the treatments were effective was an analysis of variance ANOVA two-way and in order to highlight differences between treatments was performed analysis of variance ANOVA. Morphological analysis in the gills of samples used in the experiments were performed using scanning electron microscopy (SEM) to verify the existence of related structures to the filtration efficiency in these fish. To assess the effects of cyanotoxins in the animals used in the experiments were performed biochemical analyzes in the liver and gills of lipoperoxidation (LPO) assessing oxidative damage, and the activity of antioxidant enzymes catalase (CAT) and glutathione-S-transferase (GST). In order to highlight differences between treatments was performed analysis of variance (one-way ANOVA). For Oreochromis sp. the intake/filtration of M. aeruginosa in all concentrations were significant differences (p<0.05) in test organisms in T1 were decreasing concentrations of the cyanobacteria and the test organisms in T2 was increased concentrations of cyanobacteria over time. In the experiment using G. brasiliensis the intake/filtration of M. aeruginosa, no significant difference in the concentration 10g/L and 40g/L (p>0.05), however there was a decrease in cyanobacteria concentrations. For Oreochromis sp. the intake/filtration of C. raciborskii, significant differences were observed mainly in the concentration 10g/L and 20g/L and the reduction of cyanobacteria concentrations over time (p<0.05). However, in the experiment evaluating the intake/filtration rates of C. raciborskii by G. brasiliensis occurred reduction in the concentration of cyanobacteria at 10g/L and 20g/L concentrations in both groups (p<0.05). Results of morphological analysis using a scanning electron microscope revealed the existence of gill microspine, both in Oreochromis sp. as for G. brasiliensis validating the possibility of G. brasiliensis can be used as natural control of cyanobacteria. The analyzes realized in the oxidative balance of the test organisms based diet ingestion of toxic cyanobacteria revealed, as a general standard for both cichlid species evaluated, the occurrence of biochemical changes in the conditions used, but considering these test conditions (fish species, cyanobacteria species, concentrations of cyanobacteria and time trial) such damages were repaired by the action of antioxidant enzymes. These results are important for knowledge management and restoration of eutrophic aquatic ecosystems based on species interactions.

Florações de cianobactérias representam grave problema para a qualidade da água de lagos e reservatórios, pela produção de toxinas que podem causar danos a organismos aquáticos e seres humanos. Como implicação, o processo de formação de colônias ou agregados de cianobactérias impede a predação pelo zooplâncton, mas, por outro lado, aumenta a vulnerabilidade das cianobactérias à pressão de herbivoria por peixes filtradores. Estudos experimentais têm quantificado as taxas de ingestão de algas e cianobactérias por Oreochromis (tilápia) em laboratório, porém, pouco se sabe sobre interações de espécies de peixes nativos, como do gênero Geophagus (cará) com cianobactérias. Neste sentido, os objetivos deste estudo foram (1) investigar, experimentalmente, medidas de controle natural de cianobactérias através da utilização de peixes filtradores nativos e exóticos; (2) identificar estruturas relacionadas à eficiência de filtração nestes peixes e (3) avaliar mecanismos de dano-reparo enzimático em peixes que tem sua dieta baseada na ingestão de cianobactérias tóxicas, através da relação de enzimas antioxidantes e a ocorrência de estresse oxidativo devido à possibilidade de produção de cianotoxinas. Experimentos foram realizados avaliando as taxas de consumo das cianobactérias Microcystis aeruginosa e Cylindrospermopsis raciborskii por Oreochromis sp. (espécie exótica) e Geophagus brasiliensis (espécie nativa). Os tratamentos, em triplicata, incluíram peixes de duas classes de tamanho (Tratamento1: peixes de 5-10 cm; Tratamento 2: peixes de 10-15 cm); três concentrações de cianobactérias (10μg/L, 20μg/L e 40 μg/L) e controles contendo somente inóculo algáceo nas concentrações 10μg/L, 20μg/L e 40 μg/L. Com o intuito de evidenciar diferenças entre os tratamentos foi realizada análise da variância (ANOVA). Análises morfológicas nas brânquias de exemplares utilizados nos experimentos foram realizadas através de microscopia eletrônica de varredura (MEV) para verificar a existência de estruturas relacionadas à eficiência de filtração nestes peixes. Por fim, para avaliar os efeitos das cianotoxinas nos exemplares utilizados nos experimentos foram realizadas análises bioquímicas de lipoperoxidação (LPO) avaliando o dano oxidativo e a atividade das enzimas antioxidantes catalase (CAT) e glutationa-s-transferase (GST) no fígado e nas brânquias dos exemplares. Com o intuito de evidenciar diferenças entre os tratamentos foi realizada análise da variância (ANOVA one-way). Para Oreochromis sp. frente à ingestão/filtração de M. aeruginosa, em todas as concentrações foram observadas diferenças significativas (p<0,05). Nos organismos-teste do tratamento 1 ocorreu decréscimo nas concentrações de cianobactérias, já nos organismos-teste do tratamento 2 ocorreu aumento das concentrações de cianobactérias ao longo do tempo. Para Oreochromis sp. frente à ingestão/filtração de C. raciborskii, diferenças significativas (p<0,05) foram observadas principalmente nas concentrações 10μg/L e 20μg/L quanto à redução das concentrações de cianobactérias ao longo do tempo. No experimento utilizando G. brasiliensis frente à ingestão/filtração de M. aeruginosa, não ocorreram diferenças significativas (p>0,05) nas concentrações 10μg/L e 40μg/L, entretanto ocorreu uma diminuição nas concentrações de cianobactérias. Contudo, no experimento avaliando as taxas de ingestão/filtração de C. raciborskii por G. brasiliensis ocorreu redução na concentração de cianobactérias nas concentrações 10μg/L e 20μg/L em ambos os tratamentos (p<0,05). Resultados das análises morfológicas através de microscópio eletrônico de varredura evidenciaram a existência de micro-espinhos branquiais, tanto em Oreochromis sp. quanto em G. brasiliensis, validando a possibilidade de G. brasiliensis poder ser utilizado como controle natural de cianobactérias. Análises do balanço oxidativo revelaram como padrão geral para ambas as espécies de ciclídeos avaliados, a ocorrência de alterações bioquímicas nas condições testadas. Porém nestas condições-teste (espécies de peixes e de cianobactérias, concentrações das cianobactérias avaliadas e tempo de experimento) estes danos foram reparados pela ação das enzimas antioxidantes. Os resultados obtidos neste estudo representam importantes conhecimentos para o manejo e restauração de ecossistemas aquáticos eutrofizados baseado em interações de espécies. / Cyanobacteria blooms represent a serious problem for the quality of water from lakes and reservoirs, the production of toxins to aquatic organisms and humans. As implication, the process of formation of colonies or aggregates of cyanobacteria preventing predation by zooplankton, but on the other hand, increases the vulnerability of the cyanobacteria to filtrating pressure by herbivorous fish. Experimental studies have quantified the ingestion rates of algae and cyanobacteria by Oreochromis (tilapia) in the laboratory, however, little is known about interactions of species of native fish such as the genus Geophagus (cará) with cyanobacteria. In this sense, the objectives of this study were (1) to investigate experimentally measured natural control of cyanobacteria by using filter-native and exotic fishes, (2) identified structures related to the filtration efficiency in these fishes (3) evaluating mechanisms of damage-repair enzyme in fish that have diet based on their ingestion of toxic cyanobacteria, through the relation antioxidant enzymes activity and the occurrence of oxidative stress. Experiments were conducted to evaluate the consumption rates of cyanobacteria Microcystis aeruginosa and Cylindrospermopsis raciborskii by Oreochromis sp. (exotic species) and Geophagus brasiliensis (native species). Were determined treatments in triplicate consisting of fish of two size classes (Treatment 1: fish 5-10 cm; Treatment 2: fish 10-15 cm); three cyanobacterial concentrations (10μg/L, 20μg/L and 40μg/L) and controls containing only algal inoculums concentrations 10μg/L, 20μg/L and 40μg/L. In order to show differences between treatments analysis of variance (ANOVA) was performed. Morphological analysis in the gills of specimens used in the experiments were carried out using scanning electron microscopy (SEM) to verify the existence of the structures related to filtration efficiency in these fishes. To assess the effects of cyanotoxins on the specimens used in the experiments biochemical analyzes were performed in the liver and gill measuring lipoperoxidation (LPO) assessing oxidative damage, and antioxidant enzymes catalase (CAT) and glutathione-s-transferase (GST). In order to show differences between treatments analysis of variance (ANOVA) was performed. Designed to evaluate whether the concentrations and the treatments were effective was an analysis of variance ANOVA two-way and in order to highlight differences between treatments was performed analysis of variance ANOVA. Morphological analysis in the gills of samples used in the experiments were performed using scanning electron microscopy (SEM) to verify the existence of related structures to the filtration efficiency in these fish. To assess the effects of cyanotoxins in the animals used in the experiments were performed biochemical analyzes in the liver and gills of lipoperoxidation (LPO) assessing oxidative damage, and the activity of antioxidant enzymes catalase (CAT) and glutathione-S-transferase (GST). In order to highlight differences between treatments was performed analysis of variance (one-way ANOVA). For Oreochromis sp. the intake/filtration of M. aeruginosa in all concentrations were significant differences (p<0.05) in test organisms in T1 were decreasing concentrations of the cyanobacteria and the test organisms in T2 was increased concentrations of cyanobacteria over time. In the experiment using G. brasiliensis the intake/filtration of M. aeruginosa, no significant difference in the concentration 10g/L and 40g/L (p>0.05), however there was a decrease in cyanobacteria concentrations. For Oreochromis sp. the intake/filtration of C. raciborskii, significant differences were observed mainly in the concentration 10g/L and 20g/L and the reduction of cyanobacteria concentrations over time (p<0.05). However, in the experiment evaluating the intake/filtration rates of C. raciborskii by G. brasiliensis occurred reduction in the concentration of cyanobacteria at 10g/L and 20g/L concentrations in both groups (p<0.05). Results of morphological analysis using a scanning electron microscope revealed the existence of gill microspine, both in Oreochromis sp. as for G. brasiliensis validating the possibility of G. brasiliensis can be used as natural control of cyanobacteria. The analyzes realized in the oxidative balance of the test organisms based diet ingestion of toxic cyanobacteria revealed, as a general standard for both cichlid species evaluated, the occurrence of biochemical changes in the conditions used, but considering these test conditions (fish species, cyanobacteria species, concentrations of cyanobacteria and time trial) such damages were repaired by the action of antioxidant enzymes. These results are important for knowledge management and restoration of eutrophic aquatic ecosystems based on species interactions.

Made available in DSpace on 2014-10-09T12:38:41Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:04:59Z (GMT). No. of bitstreams: 1 06006.pdf: 3042340 bytes, checksum: 62a6ebd206107cfeefee505a16da38f3 (MD5) / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP