Les abeilles domestiques subissent des pertes de colonies sans précédents, ce qui pourrait à terme avoir un fort impact économique compte tenu de leur apport à l’agriculture en service de pollinisation. Ces pertes sont définies comme causées par des stress multifactoriels. Cependant, l’impact de ces stress est en général testé séparément sur les individus ou les colonies. Les colonies sont connues pour concentrer les pesticides provenant de l’environnement à travers le pollen et le nectar récoltés mais aussi les pathogènes. L’étude de la co-exposition entre le thiaméthoxam, un insecticide néonicotinoïde, et deux virus de l’abeille, le DWV et le CBPV, a mis en évidence l’existence d’interactions entre chacun de ces virus et le pesticide. Diverses méthodes de transmission des virus ont été testées afin de s’approcher au mieux des conditions naturelles et une méthode de transmission du CBPV par contact répétable a été développée. Une cinétique de métabolisation du thiamethoxam a été effectuée pour la première fois, renseignant sur la forte probabilité d’excrétion du métabolite en conditions naturelles. Lors d’expérimentations en cagettes la co-exposition entre le CBPV et le thiaméthoxam à fortes doses chroniques a causé des mortalités synergiques, ou une augmentation des charges virales atteignant le seuil en nombre de copies virales connu pour déclencher des infections apparentes chez les abeilles. Les différences de résultats entre des abeilles provenant de colonies dans diverse conditions a souligné un effet de tolérance aux stress encore non étudié chez l’abeille domestique. En parallèle, la co-exposition au sein de la colonie entre le thiaméthoxam à une dose sublétale aigüe et le DWV a causé des premières sorties de butinage extrêmement précoces chez les abeilles, ainsi que de fortes proportions de premiers vols sans retour, ce qui pourrait à terme avoir un effet dévastateur sur les colonies. L’étude de la transcription de gènes suggère que les effets obtenus sur les charges virales en CBPV seraient dus à un effet du thiaméthoxam sur dorsal-1a, un facteur de l’immunité, et les sorties précoces à une baisse significative de la transcription de la vittelogénine, entrant en jeu dans les mécanismes de vieillissement, due au DWV. Le développement de clones moléculaires du DWV a été amorcé, ce qui pourra à terme permettre des études de génétique inverse sur ce virus, qui pourront expliquer ses modes de transmission et d’infections encore inconnus à ce jour. / Domestic honeybees are suffering from unprecedented colony losses, which could at term have a strong economic impact, considering their part in crop pollination, notably. These losses are defined as caused by multifactorial stresses. However, the impact of these stresses is usually measured separately, on individuals or colonies. Colonies are known to concentrate pesticides from their environment through collection of pollen and nectar, but they also concentrate pathogens. The study of a co-exposure between thiamethoxam, a neonicotinoid insecticide, and two honeybee viruses, DWV and CBPV, has shed light on the existence of interactions between each of these viruses and the pesticide. Different virus transmission methods have been tested to be as close as natural conditions, and a repeatable CBPV transmission method through contact has been developed. Metabolisation kinetics of thiamethoxam have been obtained for the first time, underlining the strong probability for excretion of the metabolite in natural conditions. During caged experiments, the co-exposure experiments between thiamethoxam at high chronic doses and CBPV cause synergistic mortalities, or an increase in viral loads which reached the number of viral copies threshold associated with clinical signs in bees. The varying results between bees from colonies kept in different conditions underlined a stress tolerance effect yet to be studied in the honeybee. In parallel, co-exposure in colonies between sublethal doses of thiamethoxam and DWV caused extremely precocious first foraging trips in bees, as well as large proportions of bees never returning to the hive after their firs exit, which both could in the end have a devastating effect on the colonies. The study of gene transcripts suggests that the obtained effects on CBPV viral loads could be due to a negative effect of thiamethoxam on dorsal-1a, an immune factor, and the precocious trips to a significant down-regulation of vitellogenin, which takes part in honeybee aging, cause by DWV. The development of molecular clones of DWV and a DWV recombinant was initiated, which will in the future allow for reverse genetics studies on this virus, which will help explain their transmission and infection mechanisms that are still unknown to this day.

Orientador: Antonio Francisco Godinho / Resumo: Nas últimas décadas os níveis de contaminação ambiental por metais pesados e inseticidas vêm aumentando significativamente, acompanhando exponencialmente o rápido e crescente desenvolvimento industrial levando à exposição simultânea a vários agentes químicos com ação sobre o sistema nervoso central (SNC). Sob o ponto de vista toxicológico, co-exposição a substâncias químicas tende a piorar de forma qualitativa e quantitativa os danos produzidos e a irreversibilidade dos processos de neurotoxicidade. Um dos mais prevalentes tipos de desordens sobre o SNC é o da ansiedade. O objetivo deste trabalho foi estudar a co-exposição gestacional ao metal pesado cádmio (Cd) e ao inseticida organofosforado dimetoato (DM) e sua influência sobre o comportamento de ansiedade na prole de ratos em desenvolvimento. Fêmeas prenhes foram expostas durante toda a gestação, por via oral, a solução de Cd (10mg/l), DM (4mg/kg, gavage) e Cd+DM. Após o nascimento dos filhotes foram avaliados: desenvolvimento físico e sensório-motor dos filhotes durante os primeiros dias de idade e o comportamento de ansiedade nas idades de 30 e 80 dias, utilizando arena de campo aberto (ACA), labirinto em cruz elevado (LCE) e caixa claro-escura (CCE). Também foram quantificadoso Cd e o DM tecidual e a atividade da enzima acetilcolinesterase (AChE) sanguínea e cerebral. Os resultados obtidos mostram que: a co-exposição aos agentes alterou o desenvolvimento físico dos filhotes; Cd, DM e Cd+DM aumentaram a ansiedade da pro... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In the last decades the levels of environmental contamination by heavy metals and insecticides increased significantly, following exponentially the fast and crescent industrial development leading to the simultaneous exposition to some chemical agents with action on the central nervous system (SNC). Under the toxicological point of view, co-exposure enhances qualitatively and quantitatively the damages produced and the irreversibility of the neurotoxicity processes. One of the most prevalent types of disorders on the SNC is the anxiety. The objective of this work was to study the gestational co-exposure to heavy metal cadmium (Cd) and organophosphate insecticide dimethoate (DM), and its influences on anxiety behavior of rats offspring during its development. Pregnant females were exposed during gestational period, by oral view, to the Cd solution (10 mg/l), DM (4 mg/kg, gavage), or Cd+DM. After birth were evaluated: offspring's physical and sensory-motor development in the first days of age, and anxiety behavior in the ages of 30 and 80 days, using open field arena (OF), elevated plus maze (EPM) and light-dark box (LDB) apparatus. Also were quantified Cd and DM in tissues and activity of the acetylcholinesterase enzyme (AChE) in blood and brain. The obtained results show that: co-exposition to chemical agents modified the physical development of the young rats; Cd, DM and Cd+DM increased the anxiety of the offspring in the young and adult age however, the increase of anxiety ... (Complete abstract click electronic access below) / Mestre

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Paints occupational exposure is an important health problem due to the wide variety of chemicals and xenobiotics present in their constitution. Organic solvents and heavy metals are among the main chemical constituents of paints. In the view of the variety of substances found in paints, can be noted that painters are simultaneously exposed to various xenobiotics, suggesting a case of co-exposure or exposure mixed. Biological monitoring is an essential tool for assessing the risk to health and occupational health practices. Moreover, it is known that there is a close relationship between the xenobiotics constituents of paints and oxidative stress, such as toxicological mechanism. In this study was evaluated oxidative damage by quantification of blood biomarkers of oxidative stress such as malondialdehyde (MDA), reduced glutathione (GSH), δ-aminolevulinate dehydratase enzyme (ALA-D), superoxide dismutase (SOD) and catalase (CAT ), in individuals occupationally exposed to paints (n = 48) and non-exposed subjects (n = 30). Biological monitoring of toluene was performed by quantification of different biomarkers of exposure, urinary hippuric acid and ortho-cresol, and blood toluene. For xylene, styrene, ethylbenzene and lead were quantified urinary levels of methylhippuric acid, mandelic acid, phenylglyoxylic acid and blood lead, respectively. Despite all IBEs were below the biological limit exposure (BEL), significant changes in oxidative stress biomarkers were found in exposed group. Plasma levels of MDA and blood antioxidant enzymes (SOD and CAT) had shown increased in the group of painters when compared with non-exposed group; this increase was accompanied by GSH levels depletion and enzyme ALA-D inhibition. It was also observed several correlations between the oxidative stress biomarkers and biomarkers of exposure to xenobiotics present in paints. By statistical tests were evaluated which of the paints constituents played the greatest influence on changes in oxidative stress biomarkers, in this case of co-exposure. Among the exposure biomarkers tested, blood toluene was suggested as the main responsible for increased lipid peroxidation; in addition, it was appointed as a new and important inhibitor of the enzyme ALA-D. Thus, it is suggested that a joint evaluation of biomarkers of exposure and oxidative stress biomarkers might be useful to ensure, in longterm, the worker s health. / A exposição ocupacional a tintas representa um importante problema de saúde devido à ampla variedade de substâncias químicas e xenobióticos presentes na sua constituição. Solventes orgânicos e metais pesados encontram-se entre os principais compostos químicos constituintes das tintas. Tendo em vista a variedade de substâncias presentes na composição das tintas, pode-se constatar que pintores encontram-se simultaneamente expostos a diferentes xenobióticos, caracterizando um processo de coexposição ou exposição mista. A monitorização biológica é uma ferramenta essencial para a avaliação do risco à saúde e práticas de saúde ocupacional. Por outro lado, sabe-se que existe uma estreita relação entre os xenobióticos constituintes de tintas e o estresse oxidativo, como mecanismo toxicológico. Neste estudo foi avaliado o dano oxidativo, através da quantificação de biomarcadores sanguíneos do estresse oxidativo, como alondialdeído (MDA), glutationa reduzida (GSH), enzima δ-aminolevulinato desidratase (ALA-D), superóxido dismutase (SOD) e catalase (CAT); em indivíduos expostos (n=48) e não expostos (n=30) ocupacionalmente a tintas. A monitorização biológica do tolueno foi realizada através da quantificação dos diferentes biomarcadores de exposição, ácido hipúrico e orto-cresol urinários, e tolueno sanguíneo. Para xileno, estireno, etilbenzeno e chumbo foram quantificados os níveis urinários dos ácidos metil-hipúrico, mandélico, fenilglioxílico, e chumbo sanguíneo, respectivamente. Apesar de todos os IBEs se encontrarem abaixo dos índices biológicos máximos permitidos (IBMP), alterações significativas nos biomarcadores do estresse oxidativo foram encontradas no grupo de expostos. Os níveis plasmáticos de MDA e das enzimas sangüíneas antioxidantes (SOD e CAT) se mostraram aumentados no grupo de pintores quando comparados com o grupo não exposto; esse aumento foi acompanhado pela depleção nos níveis de GSH e inibição da enzima ALA-D. Foram observadas ainda várias correlações entre os biomarcadores do estresse oxidativo e biomarcadores de exposição aos xenobióticos presentes nas tintas. Através de testes estatísticos foram avaliados quais dos constituintes das tintas desempenhavam maior influência sobre as alterações nos biomarcadores de estresse oxidativo, nesse caso de co-exposição. Dentre os biomarcadores de exposição analisados, o tolueno sanguíneo foi sugerido como o principal responsável pelo aumento da peroxidação lipídica; além de ser apontado como um novo e importante inibidor da enzima ALA-D. Dessa forma, sugere-se que a avaliação conjunta de biomarcadores de exposição e biomarcadores do estresse oxidativo possa ser útil para assegurar, em longo, prazo a saúde do trabalhador.