Bacillus thuringiensis (Bt) é uma bactéria entomopatogênica utilizada como biopesticida contra insetos, principalmente Lepidoptera, Coleoptera e Diptera, e na produção de organismos geneticamente modificados. Com a descoberta da capacidade de Bt se movimentar sistemicamente em plantas, potencialmente atingindo insetos sugadores, surge uma nova possibilidade para o controle de Diaphorina citri Kuwayama, que transmite bactérias associadas ao Huanglongbing, uma séria doença da citricultura. O objetivo do trabalho foi desenvolver sistemas de aquisição de Bt por D. citri, in vitro e in planta, para estudos de patogenicidade. Uma estirpe de Bt transformada com o gene green fluorescent protein (Btk-gfp), cultivada em meio NYSM, foi usada como marcador de aquisição pelo inseto e movimentação nas plantas. Para o sistema de aquisição in vitro, selecionaram-se dietas com base na sobrevivência e atividade alimentar de D. citri. Btk-gfp foi adicionada à dieta selecionada, composta por uma solução de sacarose a 30% em água mineral com corantes alimentícios (verde 0,1% e amarelo 0,4%). A dieta foi acondicionada em um sachê formado por duas membranas de Parafilm®, sobre a parte inferior de uma placa de Petri de 40 mm de diâmetro (gaiola de alimentação). Dez insetos foram confinados em cada gaiola para períodos de acesso à aquisição (PAA) de até 48 h, estabelecendo-se 10 gaiolas por tratamento; como controle, utilizou-se a dieta sem Btk-gfp. Para testes de aquisição in planta, suspensões de Btk-gfp foram inoculadas em ramos novos cortados e no solo, próximo à haste de seedlings de laranja doce [Citrus sinensis (L.) Osbeck] e de murta [Murraya paniculata (L.) Jack], e em diferentes concentrações, avaliando-se a movimentação da bactéria após um período de 48 h. Posteriormente, adultos e ninfas de D. citri foram confinados nestas plantas para avaliar a aquisição de Btk-gfp, usando-se plantas inoculadas apenas com água como controle. Para os bioensaios de patogenicidade a D. citri, 21 estirpes de Bt foram testadas no sistema de aquisição em ramos cortados de murta e 5 estirpes em ramos cortados de laranja para ninfas de 3º. ínstar. In vitro, testaram-se 3 estirpes contra ninfas de 3º. ínstar e 9 contra adultos. Btgfp foi absorvido pelas raízes e ramos novos cortados de seedlings de laranja doce e de murta, e translocado até as folhas, mostrando movimentação sistêmica. Esta estirpe foi adquirida por adultos e ninfas de D. citri que se alimentaram nos ramos novos cortados, mantidos em suspensão bacteriana. O sistema de alimentação in vitro permitiu a aquisição de Bt-gfp (pellet ressuspendido na dieta de sacarose com corantes) por adultos de D. citri com apenas 12 h de PAA, mas 30 h é um período ótimo para exposição do inseto ao sistema. Nenhuma das nove estirpes testadas no sistema in vitro foi patogênica aos adultos de D. citri. Das 21 estirpes testadas contra ninfas de 3º. ínstar em ramos novos de murta, cinco causaram mortalidade de 24 a 45%, em 5 dias. Essas cinco estirpes também foram testadas contra ninfas dem ramos de citros cortados, causando mortalidade de 35 a 75% em 48 h. / Bacillus thuringiensis (Bt) is an entomopathogenic bacterium widely used as a biopesticide against insect pests, mainly Coleoptera, Lepidoptera and Diptera, or for engineering genetically-modified plants. The recent finding that Bt is able to move systemically within plants, potentially targeting piercing-sucking insects, suggests that the bacterium may be effective for microbial control of Diaphorina citri Kuwayama, the vector of Huanglongbing-associated bacteria, a serious citrus disease. The goal of this research was to develop in vitro and in planta acquisition systems of Bt by D. citri for pathogenicity assays. A transformed Bt strain with the green fluorescent protein gene (Btk-gfp), grown in NYSM medium, was used as a marker to demonstrate bacterial acquisition by the insect and movement within the plants. Artificial diets were selected for the in vitro acquisition system based on insect survival and feeding activity. Btk-gfp was added to the selected diet, a 30% sucrose solution in mineral water with green (0.1%) and yellow (0.4%) food coloring. The diet was placed inside a Parafilm® membrane sachet, covering the opening of the lower half of a 40-mm diameter culture plate, and forming the feeding cage. Ten D. citri adults or nymphs were introduced in each cage and allowed acquisition access periods (AAP) of up to 48 h on the diet; as a control, a diet without Btk-gfp was used. For testing the in planta acquisition system, Btk-gfp suspensions were inoculated in young stem cuttings or in the soil, near the stem of Citrus sinensis (L.) Osbeck and Murraya paniculata (L.) Jack seedlings, as well as in different concentrations, and bacterial movement was assessed after 48 h; plants inoculated with water were used as controls. D. citri adults and nymphs were confined on inoculated plants to verify Btk-gfp acquisition. In pathogenicity assays, 21 Bt strains were tested against 3rd instars of D. citri using the acquisition system with stem cuttings of M. paniculata and 5 strains were tested using citrus stem cuttings. The in vitro acquisition system was used to test pathogenicity of 3 and 9 Bt strains against 3rd-instar nymphs and adults, respectively. Bt-gfp absorbed by roots or young stem cuttings of inoculated C. sinensis and M. paniculata was detected in the leaves, showing systemic movement. Bt-gfp was isolated from groups of nymphs and adults that were fed on inoculated stem cuttings or on artificial diets with bacterial suspension, showing that both in planta and in vitro acquisition take place. D. citri adults can acquire Bt-gfp within 12 h of AAP to the artificial diet with bacterium inoculum, but 30 h is the optimum AAP. None of the nine Bt strains assayed in vitro were pathogenic to D. citri adults. Of 21 strains tested for pathogenicity against nymphs using inoculated stem cuttings of M. paniculata, five caused mortality rates varying from 24 to 45% mortality within 5 days. These five Bt strains were also tested against D. citri nymphs using young stem cuttings of C. sinensis, causing mortality rates of 35 to 75% within 48 h.

No description available.

Made available in DSpace on 2014-12-17T14:01:59Z (GMT). No. of bitstreams: 1 DanyheltonDFD.pdf: 1227676 bytes, checksum: c8754b4250e0bb6e5391c5ffbe8423a3 (MD5) Previous issue date: 2009-04-30 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Top-down (grazing) and bottom-up (nutrient, light) controls are important in freshwater ecosystems regulation. Relative importance of these factors could change in space and time, but in tropical lakes bottom-up regulation has to been appointed as more influent. Present study aimed to test the hypothesis that phytoplankton growths rate in Armando Ribeiro reservoir, a huge eutrophic reservoir in semi-arid region of Rio Grande do Norte state, is more limited by nutrient available then zooplankton grazing pressure. Bioassay was conduced monthly from September (2008) to August (2009) manipulating two levels of nutrients (with/without addition) and two level of grazers (with/without removal). Experimental design was factorial 2X2 with four treatments (X5), (i) control with water and zooplankton from natural spot ( C ), (ii) with nutrient addition ( +NP ), (iii) with zooplankton remove ( -Z ) and (iv) with zooplankton remove and nutrient addition ( -Z+NP ). For bioassay confection transparent plastic bottles (500ml) was incubate for 4 or 5 days in two different depths, Secchi`s depth (high luminosity) and 3 times Secchi`s depth (low luminosity). Water samples were collected from each bottle in begins and after incubates period for chlorophyll a concentration analysis and zoopalnktonic organisms density. Phytoplankton growths rates were calculated. Bifactorial ANOVA was performance to test if had a significant effect (p<0,005) of nutrient addition and grazers remove as well a significant interaction between factors on phytoplankton growths rates. Effect magnitude was calculated the relative importance of each process. Results show that phytoplankton growth was in generally stimulated by nutrient addition, as while zooplankton remove rarely stimulated phytoplankton growth. Some significant interactions happening between nutrient additions and grazers remove on phytoplankton growth. In conclusion this study suggests that in studied reservoir phytoplankton growth is more controlled by ascendent factors than descendent / A produ??o prim?ria dos ecossistemas aqu?ticos ? regulada por fatores ascendentes, (disponibilidade de luz e nutrientes), e por fatores descendentes (herbivoria). A import?ncia relativa desses fatores pode variar no tempo e no espa?o, mas em lagos tropicais tem sido sugerido que os fatores ascendentes s?o geralmente mais importantes. O presente estudo teve como principal objetivo testar a hip?tese de que o crescimento fitoplanct?nico no reservat?rio Armando Ribeiro Gon?alves, um grande reservat?rio eutr?fico na regi?o semi-?rida do Rio Grande do Norte, ? mais limitado pela disponibilidade de nutrientes do que pela press?o de herbivoria do zoopl?ncton. Bioensaios foram realizados mensalmente entre Setembro (2007) e Agosto (2008), manipulando-se dois n?veis de nutrientes (com/sem adi??o) e dois n?veis de herb?voros (com/sem remo??o). O desenho experimental foi do tipo fatorial 2X2 com quatro tratamentos (x5), (i) controle contendo a ?gua e o pl?ncton natural do ambiente ( C ), (ii) com adi??o de nutrientes ( +NP ), (iii) com remo??o de zoopl?ncton ( -Z ) e (iv) com remo??o de zoopl?ncton e adi??o de nutrientes ( -Z+NP ). Nos bioensaios foram incubadas garrafas pl?sticas transparentes de 500 mL por 4 ou 5 dias em duas profundidades distintas: na profundidade do disco de Secchi (alta luminosidade) e em uma profundidade 3x maior do que a anterior (baixa luminosidade). Amostras de ?gua foram coletadas de cada garrafa no inicio e ap?s o per?odo de incuba??o para an?lises das concentra??es de clorofila a e densidades de organismos zooplanct?nicos. Foi calculada a taxa de crescimento fitoplanct?nico. Uma ANOVA bifatorial foi realizada para testar se houve efeito significativo (p<0,05) da adi??o de nutrientes e da remo??o de herb?voros bem como uma intera??o significativa entre ambos os fatores sobre as taxa de crescimento do fitopl?ncton. A magnitude de cada efeito tamb?m foi calculada para quantificar a import?ncia relativa de cada processo. Os resultados mostram que o crescimento fitoplanct?nico foi geralmente estimulado pela adi??o de nutrientes enquanto a remo??o do zoopl?ncton raramente estimulou o crescimento fitoplanct?nico. Algumas intera??es significativas ocorreram entre os efeitos da adi??o de nutrientes e da remo??o de herb?voros sobre o crescimento fitoplanct?nico. Como conclus?o, esta pesquisa sugere que o crescimento fitoplanct?nico no reservat?rio estudado, ? mais fortemente controlado por fatores ascendentes do que por fatores descendentes

Made available in DSpace on 2014-12-17T14:33:08Z (GMT). No. of bitstreams: 1 LeideAPS_DISSERT.pdf: 986741 bytes, checksum: e18d4b42b9411720a51bd3f7786fa126 (MD5) Previous issue date: 2012-09-25 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Cyanobacterial blooms are common in eutrophic reservoirs in Brazilian northeastern semi-arid. Given this reality, the present study aimed to analyze the effect of potentially toxic cyanobacterial blooms in Gargalheiras reservoir (semi-arid) on the cladocerans Ceriodaphnia dubia and Daphnia gessneri. In vitro chronic bioassays were performed with reservoir water dilutions from August/2011 to May/2012 and the following effects were evaluated on: intrinsic rate of population growth (r), reproductive parameters (age of first reproduction and fecundity per capita) and cladocerans movements. Phytoplankton was dominated by Cylindrospermopsis raciborskii and Planktothrix agardhii and saxitoxin and microcystin were detected in reservoir water. In most months C. dubia showed differences in r between control (absence of cyanobacteria) and treatments, and has shown negative effects on reproductive parameters. In all months paralysis of swimming movements was observed in C. dubia when both C. raciborskii and saxitoxin (cyanotoxin neurotoxic) were present in water. While C. dubia was sensitive to the reservoir water containing cyanobacteria, D. gessneri showed less intense negative effects in r and reproductive parameters. Furthermore, D. gessneri showed no paralysis of swimming movements. These results support the hypothesis in the literature that D. gessneri is resistant to the Cylindrospermopsis effects. The clone&#8223;s life history may be a key to understand the results. The C. dubia clone, isolated from eutrophicated environment, is in the lab for ten years and it is an exotic species in Brazil. D. gessneri is a common species in the country and this clone was isolated from the Gargalheiras reservoir (where there are constant blooms of potentially toxic cyanobacteria) a year ago. Perhaps the recent contact with cyanobacteria explain the higher resistance presented by this D. gessneri clone. In conclusion, the cladocerans studied have different levels of sensitivity to cyanobacteria, characterizing species-specific responses / Flora??es de cianobact?ras s?o frequentes em reservat?rios eutr?ficos do semi-?rido do nordeste brasileiro. Diante dessa realidade o presente trabalho objetivou analisar o efeito de flora??es de cianobact?rias potencialmente t?xicas do reservat?rio Gargalheiras (semi-?rido brasileiro) sobre os clad?ceros Ceriodaphnia dubia e Daphnia gessneri. Foram realizados ensaios cr?nicos in vitro com dilui??es de ?gua do reservat?rio de agosto/2011 a maio/2012 e foram avaliados efeitos sobre: taxa intr?nseca de crescimento populacional (r), par?metros reprodutivos (idade da primeira reprodu??o e fecundidade per capita) e movimenta??o dos clad?ceros. O fitopl?ncton foi dominado por Cylindrospermopsis raciborskii e Planktothrix agardhii e houve detec??o de saxitoxina e microcistina na ?gua do reservat?rio. Na maioria dos meses C. dubia apresentou diferen?a em r entre controle (aus?ncia de cianobact?rias) e tratamentos, al?m de ter apresentado efeitos negativos nos par?metros reprodutivos. Observou-se ainda um efeito de paralisia dos movimentos natat?rios de C. dubia em todos os meses, concomitante ? presen?a de C. raciborskii e saxitoxina (cianotoxina neurot?xica) na ?gua. Enquanto C. dubia apresentou-se sens?vel ? ?gua do reservat?rio contendo cianobact?rias, D. gessneri apresentou efeitos negativos menos intensos em r e nos par?metros reprodutivos. Al?m disso, esta esp?cie n?o teve os movimentos paralisados. Esses resultados apoiam a hip?tese levantada na literatura de que D. gessneri apresenta resist?ncia aos efeitos da Cylindrospermopsis. A hist?ria de vida dos clones pode ser uma chave para compreens?o dos resultados. O clone de C. dubia, isolado de ambiente eutrofizado, est? em laborat?rio h? dez anos e essa ? uma esp?cie ex?tica no Brasil. D. gessneri ? uma esp?cie comum no pa?s e este clone foi isolado h? um ano do reservat?rio Gargalheiras, onde h? constantes flora??es de cianobact?rias potencialmente t?xicas. A hip?tese mais prov?vel ? que o contato recente deste clone de D. gessneri com cianobact?rias tenha lhe conferido maior resist?ncia. Conclui-se que os clad?ceros estudados apresentam diferentes n?veis de sensibilidade ?s cianobact?rias, caracterizando respostas esp?cie-espec?ficas

Bacillus thuringiensis (Bt) é uma bactéria entomopatogênica utilizada como biopesticida contra insetos, principalmente Lepidoptera, Coleoptera e Diptera, e na produção de organismos geneticamente modificados. Com a descoberta da capacidade de Bt se movimentar sistemicamente em plantas, potencialmente atingindo insetos sugadores, surge uma nova possibilidade para o controle de Diaphorina citri Kuwayama, que transmite bactérias associadas ao Huanglongbing, uma séria doença da citricultura. O objetivo do trabalho foi desenvolver sistemas de aquisição de Bt por D. citri, in vitro e in planta, para estudos de patogenicidade. Uma estirpe de Bt transformada com o gene green fluorescent protein (Btk-gfp), cultivada em meio NYSM, foi usada como marcador de aquisição pelo inseto e movimentação nas plantas. Para o sistema de aquisição in vitro, selecionaram-se dietas com base na sobrevivência e atividade alimentar de D. citri. Btk-gfp foi adicionada à dieta selecionada, composta por uma solução de sacarose a 30% em água mineral com corantes alimentícios (verde 0,1% e amarelo 0,4%). A dieta foi acondicionada em um sachê formado por duas membranas de Parafilm®, sobre a parte inferior de uma placa de Petri de 40 mm de diâmetro (gaiola de alimentação). Dez insetos foram confinados em cada gaiola para períodos de acesso à aquisição (PAA) de até 48 h, estabelecendo-se 10 gaiolas por tratamento; como controle, utilizou-se a dieta sem Btk-gfp. Para testes de aquisição in planta, suspensões de Btk-gfp foram inoculadas em ramos novos cortados e no solo, próximo à haste de seedlings de laranja doce [Citrus sinensis (L.) Osbeck] e de murta [Murraya paniculata (L.) Jack], e em diferentes concentrações, avaliando-se a movimentação da bactéria após um período de 48 h. Posteriormente, adultos e ninfas de D. citri foram confinados nestas plantas para avaliar a aquisição de Btk-gfp, usando-se plantas inoculadas apenas com água como controle. Para os bioensaios de patogenicidade a D. citri, 21 estirpes de Bt foram testadas no sistema de aquisição em ramos cortados de murta e 5 estirpes em ramos cortados de laranja para ninfas de 3º. ínstar. In vitro, testaram-se 3 estirpes contra ninfas de 3º. ínstar e 9 contra adultos. Btgfp foi absorvido pelas raízes e ramos novos cortados de seedlings de laranja doce e de murta, e translocado até as folhas, mostrando movimentação sistêmica. Esta estirpe foi adquirida por adultos e ninfas de D. citri que se alimentaram nos ramos novos cortados, mantidos em suspensão bacteriana. O sistema de alimentação in vitro permitiu a aquisição de Bt-gfp (pellet ressuspendido na dieta de sacarose com corantes) por adultos de D. citri com apenas 12 h de PAA, mas 30 h é um período ótimo para exposição do inseto ao sistema. Nenhuma das nove estirpes testadas no sistema in vitro foi patogênica aos adultos de D. citri. Das 21 estirpes testadas contra ninfas de 3º. ínstar em ramos novos de murta, cinco causaram mortalidade de 24 a 45%, em 5 dias. Essas cinco estirpes também foram testadas contra ninfas dem ramos de citros cortados, causando mortalidade de 35 a 75% em 48 h. / Bacillus thuringiensis (Bt) is an entomopathogenic bacterium widely used as a biopesticide against insect pests, mainly Coleoptera, Lepidoptera and Diptera, or for engineering genetically-modified plants. The recent finding that Bt is able to move systemically within plants, potentially targeting piercing-sucking insects, suggests that the bacterium may be effective for microbial control of Diaphorina citri Kuwayama, the vector of Huanglongbing-associated bacteria, a serious citrus disease. The goal of this research was to develop in vitro and in planta acquisition systems of Bt by D. citri for pathogenicity assays. A transformed Bt strain with the green fluorescent protein gene (Btk-gfp), grown in NYSM medium, was used as a marker to demonstrate bacterial acquisition by the insect and movement within the plants. Artificial diets were selected for the in vitro acquisition system based on insect survival and feeding activity. Btk-gfp was added to the selected diet, a 30% sucrose solution in mineral water with green (0.1%) and yellow (0.4%) food coloring. The diet was placed inside a Parafilm® membrane sachet, covering the opening of the lower half of a 40-mm diameter culture plate, and forming the feeding cage. Ten D. citri adults or nymphs were introduced in each cage and allowed acquisition access periods (AAP) of up to 48 h on the diet; as a control, a diet without Btk-gfp was used. For testing the in planta acquisition system, Btk-gfp suspensions were inoculated in young stem cuttings or in the soil, near the stem of Citrus sinensis (L.) Osbeck and Murraya paniculata (L.) Jack seedlings, as well as in different concentrations, and bacterial movement was assessed after 48 h; plants inoculated with water were used as controls. D. citri adults and nymphs were confined on inoculated plants to verify Btk-gfp acquisition. In pathogenicity assays, 21 Bt strains were tested against 3rd instars of D. citri using the acquisition system with stem cuttings of M. paniculata and 5 strains were tested using citrus stem cuttings. The in vitro acquisition system was used to test pathogenicity of 3 and 9 Bt strains against 3rd-instar nymphs and adults, respectively. Bt-gfp absorbed by roots or young stem cuttings of inoculated C. sinensis and M. paniculata was detected in the leaves, showing systemic movement. Bt-gfp was isolated from groups of nymphs and adults that were fed on inoculated stem cuttings or on artificial diets with bacterial suspension, showing that both in planta and in vitro acquisition take place. D. citri adults can acquire Bt-gfp within 12 h of AAP to the artificial diet with bacterium inoculum, but 30 h is the optimum AAP. None of the nine Bt strains assayed in vitro were pathogenic to D. citri adults. Of 21 strains tested for pathogenicity against nymphs using inoculated stem cuttings of M. paniculata, five caused mortality rates varying from 24 to 45% mortality within 5 days. These five Bt strains were also tested against D. citri nymphs using young stem cuttings of C. sinensis, causing mortality rates of 35 to 75% within 48 h.

La caractérisation des propriétés électriques et diélectriques des membranes d’UF et de NF constitue une étape essentielle pour la compréhension de leurs performances de filtration. Une nouvelle approche a été développée pour déterminer les propriétés diélectriques d’une membrane de NF par spectroscopie d’impédance. Celle-ci repose sur l’isolement de la couche active du support membranaire et sur l’utilisation de mercure comme matériau conducteur, ce qui nous a permis de prouver que la constante diélectrique de la solution à l’intérieur des nanopores est inférieure à celle de la solution externe. Deux configurations de cellule (fibres immergées dans la solution d’étude ou fibres noyées dans un gel isolant) ont été étudiées pour la réalisation de mesures électrocinétiques tangentielles sur fibres creuses et la solution autour des fibres s’est avérée influencer à la fois le courant d’écoulement et la conductance électrique de la cellule. De plus, la contribution importante du corps poreux des fibres au courant d’écoulement ne permet pas de convertir cette grandeur en potentiel zêta luminal. Les propriétés intéressantes de ces membranes ont finalement été mises à profit pour décontaminer des solutions contenant des ions métalliques. Les performances de dépollution, en termes de rétention des polluants et d’impact écotoxicologique, ont été étudiées sur des solutions synthétiques et un rejet issu de l’industrie du traitement de surface. Bien que les performances de rétention aient été remarquables, la toxicité de l’effluent réel n’a pu être totalement annihilée. Une étude approfondie de la rétention des polluants non métalliques s’avère donc nécessaire. / The characterization of electrical and dielectric properties of UF and NF membranes is an essential step to understand their filtration performance. A new approach has been developed to determine the dielectric properties of a NF membrane by impedance spectroscopy. This is based on the isolation of the membrane active layer and the use of mercury as conductive material, which allowed us to prove that the dielectric constant of the solution inside nanopores is lower than that of the external solution. Two cell configurations (fibers immersed in the solution or fibers embedded in an insulating gel) were investigated for the implementation of tangential electrokinetic measurements with hollow fibers and the solution around the fibers was found to influence both streaming current and cell electrical conductance. Moreover, the important contribution of the fiber porous body to the streaming current does not allow the conversion of the latter to luminal zeta potential. The advantageous properties of these membranes were finally used to decontaminate solutions containing metal ions. Decontamination performances in terms of both pollutant retention and ecotoxicological impact were studied on synthetic solutions and a discharge water from surface treatment industry. Although retention performances were remarkable, the toxicity of the real effluent could not be totally annihilated. A thorough study of the retention of non-metallic contaminants is thus required.

De nos jours, l’évaluation de la qualité d’un milieu naturel repose traditionnellement sur des analyses ciblant des molécules déjà connues pour être biologiquement actives. Cependant ces molécules ne reflètent que partiellement les effets biologiques observés et la complexité des matrices environnementales. Par ailleurs, les contaminants dans l’environnement subissent des phénomènes de dégradation (hydrolyse, biodégradation, photodégradation). Ces processus peuvent conduire à la génération de produits de transformation qui sont encore trop peu pris en compte dans l’évaluation du risque, alors qu’ils sont susceptibles d’occasionner des effets toxiques plus importants que les molécules mères. L’impact écotoxicologique de ces mélanges nécessite préalablement l’obtention d’une vision globale quant à la présence et au devenir de ces molécules.Dans ce contexte, ces travaux appliquent une méthodologie pluridisciplinaire afin d’identifier des molécules préoccupantes et pertinentes à surveiller. Pour cela, ce travail s’appuie sur des analyses ciblées ultra-traces, des outils bioanalytiques, et des analyses non ciblées, qui ont été appliqués sur des échantillons environnementaux ou issus d’études en laboratoire mimant l’évolution des matrices environnementales. Au final, cette étude a permis : (1) d’identifier des molécules préoccupantes en estuaire de Seine sur la base de leur occurrence et de leur persistance, (2) d’identifier de nouveaux produits de transformation et (3) d’identifier les molécules responsables d’activités biologiques dans des matrices très complexes. / To date, environmental risk assessment is based on a restricted number of molecules assessed by targeted chemical analyses. However, this approach give a partial picture of co-occurrence of known and unknown compounds. Moreover, in the aquatic environment, chemicals are not completely mineralized and are subject to abiotic and biotic processes. Transformation products (TPs) can be more toxic and more persistent than the parent compound. However, TPs are not typically included in classical monitoring and risk assessment. Because of complexity and variability of these matrices and the restricted number of molecules focused in targeted chemical analyses, selection of relevant molecules for environmental monitoring is often laborious.In this context, an integrative approach was used in order to identify chemicals of concern for a classical monitoring. This work has been realized on environmental complex samples and on laboratory-generated samples. A combination of targeted, non-targeted analyses and in vitro bioassays was performed and allowed to: (1) identify polar chemical of concern in the Seine estuary on the basis of occurrence and persistence, (2) identify new transformation products and (3) identify compounds responsible of biological activity observed in complexes matrices.

<p>The rapid analysis of complex samples using mass spectrometry (MS) provides valuable information in both point-of-care (e.g. drug testing) and laboratory-based applications, including the generation of spectral libraries for classification of biosamples, the identification of biomarkers through large-scale studies, as well as the synthesis and bioactivity assessments of large compound sets necessary for drug discovery. In all these cases, the inherent speed of MS is attractive, but rarely fully utilized due to the widespread use of sample purification techniques prior to analysis. Ambient ionization methodologies can help circumvent this drawback by facilitating high-throughput qualitative and quantitative analysis directly from the complex samples without any need for work-up. For instance, the use of swabs or paper substrates allows for rapid identification, quantification, and confirmation, of drugs of abuse from biofluids or surfaces of forensic interest in a matter of minutes, as described in the first two chapters of this dissertation. Faster analysis can be achieved using an automated desorption electrospray ionization (DESI) platform which allows for the rapid and direct screening of complex-sample microarrays with throughputs better than 1 sample per second, giving access to rich spectral information from tens of thousands of samples per day. The development of the bioanalytical capabilities of this platform, particularly within the context of drug discovery (e.g. bioactivity assays, biosample analysis), is described across most other chapters of this dissertation. The use of DESI, a contactless ambient ionization method developed in our laboratory and whose 20 years of history are overviewed in the introduction of this document, provides an additional advantage as the secondary microdroplets generated through the DESI process act as reaction vessels that can accelerate organic reactions by up to six orders of magnitude, facilitating on-the-fly synthesis of new compounds from arrays of starting materials. Unique implications of this microdroplet chemistry in the prebiotic synthesis of peptides and spontaneous redox chemistry at air-solution interfaces, together with its practical applications to the synthesis of new drug molecules, are also overviewed. The success obtained with the first automated DESI-MS system, developed within the DARPA Make It program, led to increased interest in a new-generation platform which was designed over the past year, as overviewed in the last section of this dissertation, and which is currently being installed for validation prior to the transfer of the technology to NCATS, where we anticipate it will make a significant impact through the consolidation and acceleration of the early drug discovery workflow.</p>

<p>Canines can detect different diseases simply by smelling different biological sample types, including  urine,  breath  and  sweat.  This  has  led  researchers  to  try  and  discovery  unique  volatile  organic compound (VOC) biomarkers. The power of VOC biomarkers lies in the fact that one day they may be able to be utilized for noninvasive, rapid and accurate diagnostics at a point of care using  miniaturized  biosensors.  However,  the  identity  of  the  specific  VOC  biomarkers  must  be  demonstrated before designing and fabricating sensing systems. Through  an  extensive  series  of  experiments,  VOCs  in  urine  are  profiled  by  solid  phase  microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS) to identify biomarkers for breast cancer using murine models. The results from these experiments indicated that  unique  classes  of  urinary  VOCs,  primarily  terpene/terpenoids  and  carbonyls,  are  potential  biomarkers  of  breast  cancer.  Through  implementing  chemometric  approaches,  unique  panels  of  VOCs  were  identified  for  breast  cancer  detection,  identifying  tumor  location,  determining  the  efficacy of dopaminergic antitumor treatments, and tracking cancer progression. Other diseases, including COVID-19 and hypoglycemia (low blood sugar) were also probed to identify volatile biomarkers present in breath samples.  VOC biomarker identification is an important step toward developing portable gas sensors, but  another  hurdle  that  exists  is  that  current  sensors  lack  selectivity  toward  specific  VOCs  of  interest.  Furthermore,  testing  sensors  for  sensitivity  and  selectivity  is  an  extensive  process  as  VOCs  must  be  tested  individually  because  the  sensors  do  not  have  modes  of  chromatographic  separation or compound identification. Another set of experiments is presented to demonstrate that SPME  fibers  can  be  coated  with  materials,  used  to  extract  standard  solutions  of  VOCs,  and  analyzed  by  GC-MS  to  determine  the  performance  of  various  gas  sensing  layers.  In  the  first  of  these  experiments,  polyetherimide  (PEI)  was  coated  onto  a  SPME  fiber  and  compared  to  commercial polyacrylate (PAA) fibers. The second experiment tuned the extraction efficiency of polyvinylidene fluoride (PVDF) - carbon black (CB) composites and showed that they had higher sensitivity  for  urinary  VOC  extraction  relative  to  a  polydimethylsiloxane  (PDMS)  SPME  fiber.  These results demonstrate SPME GC-MS can rapidly characterize and tune the VOC adsorption capabilities of gas sensing layers. </p>

Studies in the USA have reported that species of meiobenthic copepods can be used as bioindicators of sediment-associated contaminants. The main objective of this research project was to develop and validate methods to assess the effects of estuarine pollution, using the marine benthic copepod Robertsonia propinqua as a bioindicator of environmental health in New Zealand intertidal / estuarine areas. Cultures of R. propinqua were set up and maintained in the laboratory and individuals used in 96h acute and full life-cycle chronic bioassays using the pre-selected contaminants atrazine and zinc sulphate. From the 96h acute experiments it was found that the lethal doses at which 50% mortality occurred (LC50) for exposed nauplii and adult individuals were 7.5 mg/L and 31.8 mg/L, respectively for atrazine and 1.7 mg/L and 2.7 mg/L, respectively for zinc sulphate. This indicated that the nauplii life stage was more sensitive than were the adult life stages for exposure to both contaminants. Based on the 'trigger' values reported (atrazine = 0.013 mg/L, zinc = 0.015 mg/L) in the Australian and New Zealand guidelines for fresh and marine water quality, which provide values at which concentrations of contaminants can occur in the environment before they begin causing effects on aquatic fauna, it is unlikely that the calculated LC50s in the current research will induce biological effects in exposed copepods in the short-term. The calculated LC50 results were then used to further investigate the effects of chronic exposure of sediment-associated contaminants on the complete life-cycle (egg-reproductive adult) of R. propinqua. In a laboratory-based full life-cycle toxicity test, field-collected sediments from polluted sites in the Auckland and Bay of Plenty regions reduced reproductive output (nauplii and copepodite production) of R. propinqua individuals, but the number of males and females, gravid females, clutch size per female and the number of eggs produced were not affected by either the polluted or non-polluted (reference) sediment samples from both field regions. Field investigations of meiofauna community composition in polluted and non-polluted field sites were carried out in 2004 in the Auckland and Bay of Plenty field regions in New Zealand. Greater sediment organic content and a correspondingly deeper redox potential discontinuity layer occurred in all polluted field sites compared with the non-polluted sites. However, species composition could not be used to characterise polluted and non-polluted sites, as there were no dominant taxa which were representative of these sites. The results presented in this thesis indicate that R. propinqua has strong potential to be a good candidate species as a bioindicator of environmental contamination. Furthermore, the full life-cycle toxicity test could be used as a rapid test to detect immediate changes in individual reproduction and development as well as long-term population effects. The technologies developed as part of this research may eventually provide additional tools for commercial environmental consultancies and may compliment existing standard operating procedures for environmental assessments involving pollution of estuarine ecosystems.