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Developmental nicotine exposure (DNE) is associated with increased risk of cardiorespiratory, intellectual, and behavioral abnormalities in neonates, and is a risk factor for apnea of prematurity, altered arousal responses and Sudden Infant Death Syndrome. Alterations in nicotinic acetylcholine receptor signaling (nAChRs) after DNE lead to changes in excitatory neurotransmission in neural networks that control breathing, including a heightened excitatory response to AMPA microinjection into the hypoglossal motor nucleus. Here, we report on experiments designed to probe possible postsynaptic and presynaptic mechanisms that may underlie this plasticity. Pregnant dams were exposed to nicotine or saline via an osmotic mini-pump implanted on the 5th day of gestation. We used whole-cell patch clamp electrophysiology to record from hypoglossal motoneurons (XIIMNs) in thick medullary slices from neonatal rat pups (N = 26 control and 24 DNE cells). To enable the translation of our findings to breathing-related consequences of DNE, we only studied XIIMNs that were receiving rhythmic excitatory drive from the respiratory central pattern generator. Tetrodotoxin was used to isolate XIIMNs from presynaptic input, and their postsynaptic responses to bath application of L-glutamic acid (glutamate) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were studied under voltage clamp. DNE had no influence on inward current magnitude evoked by either glutamate or AMPA. However, in cells from DNE animals, bath application of AMPA was associated with a right shift in the amplitude distribution (P = 0.0004), but no change in the inter-event interval distribution of miniature excitatory postsynaptic currents (mEPSCs). DNE had no influence on mEPSC amplitude or frequency evoked by glutamate application, or under (unstimulated) baseline conditions. Thus, in the presence of AMPA, DNE is associated with a small but significant increase in quantal size, but no change in the probability of glutamate release.

Les récepteurs du glutamate de type AMPA (rAMPA) sont les acteurs principaux de la transmission synaptique excitatrice rapide. Leur abondance au niveau de la densité postsynaptique est essentielle pour l'établissement et le maintien de la fonction synaptique, et est le résultat d'un trafic hautement dynamique. De nombreuses études ont permis de caractériser les mécanismes de diffusion membranaire impliqués dans l’adressage des rAMPA jusqu’à la synapse. Le rôle majeur des protéines auxiliaires des rAMPA dans la modulation de cette étape de trafic a été démontré. Par ailleurs, il est suggéré que la localisation synaptique des rAMPA est aussi régulée lors des phases plus précoces du trafic intracellulaire, c’est-à-dire de l'appareil de Golgi vers la membrane plasmique via les vésicules post-Golgiennes. Cependant le trafic vésiculaire post-Golgien des rAMPA n'a jamais été visualisé et reste donc encore très mal compris. En collaboration avec l'équipe de Guus Smit (Amsterdam), j’ai participé à la caractérisation d’une nouvelle protéine auxiliaire des rAMPA, appelée Shisa6. Dans le cadre de ce projet, j’ai pu étudier le rôle de cette protéine sur la diffusion membranaire des rAMPA en utilisant une technique de suivi de particule unique (Quantum dot) développée au laboratoire. Mon projet de thèse principal a consisté à étudier le trafic vésiculaire post-Golgien des rAMPA par le développement d’une nouvelle méthode d’étude. En effet, l'échec dans la visualisation dynamique du trafic vésiculaire des récepteurs pourrait être expliqué par un faible rapport signal/bruit, conséquence d'une faible concentration vésiculaire en rAMPA combinée à un bruit de fond important dû aux marquages provenant du réticulum endoplasmique (RE) et de la membrane plasmique. Dans le but de surpasser cette difficulté, nous avons mis au point un outil ingénieux (système ARIAD) afin de bloquer les rAMPA dans le RE et contrôler, par l'ajout d'un ligand, leur sécrétion du RE jusqu'à la membrane plasmique. Grâce à cet outil, nous avons non seulement augmenté considérablement la concentration des rAMPA dans les vésicules post-Golgiennes, mais aussi éliminé le bruit de fond membranaire. Par la technique de FRAP nous avons pu éliminer le bruit de fond provenant du RE. Une telle approche, combinée à des techniques d'imagerie sur neurones vivants, nous a permis de visualiser pour la première fois le trafic vésiculaire post-Golgien des rAMPA et de l’étudier. / AMPA-type glutamate receptors (AMPAR) are the main actors of the fast excitatory synaptic transmission. Their abundance at the postsynaptic density is essential for the establishment and maintenance of synaptic function, and is the result of a highly dynamic trafficking. Many studies have characterized the membrane diffusion mechanisms involved in the AMPAR synaptic localization, and revealed the critical role of the AMPAR auxiliary proteins in the modulation of this trafficking. Furthermore, it is suggested that AMPAR synaptic localization is also regulated during the early steps of the intracellular trafficking, from the Golgi apparatus to the plasma membrane via the post-Golgi vesicles. However, the post-Golgi vesicular trafficking of AMPAR has never been visualized and therefore remains poorly understood. In collaboration with the Guus Smit team (Amsterdam), I participated in the caracterization of a novel AMPAR auxiliary protein called Shisa6. As part of this project, I studied the role of this protein on the AMPAR membrane diffusion, using a method of single particle tracking (Quantum dot) developed in the laboratory. My main thesis project was to study the post-Golgi vesicular trafficking of AMPAR through the development of a new experimental protocol. Indeed, the failure in the dynamic visualization of the receptor vesicular trafficking could be explained by a low signal/noise ratio resulting of a poor AMPAR vesicular concentration, combined with a high background noise due to receptors localized both in the endoplasmic reticulum (ER) and at the plasma membrane. In order to overcome this difficulty, we have used an ingenious tool (ARIAD system) so as to block AMPAR into the ER and, by adding a ligand, control their trafficking from the ER to the plasma membrane. Thanks to this tool we have not only significantly increased the AMPAR concentration in the post-Golgi vesicles, but also eliminated the plasma membrane background noise. The FRAP imaging technique was used in order to remove the ER background noise. Such methodological approach combined with imaging techniques in living neurons, allowed us to clearly visualize for the first time the post-Golgi vesicular trafficking of AMPAR, and to study the mechanisms involved in this trafficking.

Submitted by Cintia Franco Rodrigues Mendonça (cin.franco.unesp@gmail.com) on 2018-08-16T01:04:26Z No. of bitstreams: 1 Dissertação_final.pdf: 2426720 bytes, checksum: 8440ff67ed036967311abb878d2e2139 (MD5) / Approved for entry into archive by Ana Carolina Gonçalves Bet null (abet@iq.unesp.br) on 2018-08-16T11:31:39Z (GMT) No. of bitstreams: 1 mendonça_cfr_me_araiq_int.pdf: 2474970 bytes, checksum: fc7a9704c114879d777e8bb6f3a8ad16 (MD5) / Made available in DSpace on 2018-08-16T11:31:39Z (GMT). No. of bitstreams: 1 mendonça_cfr_me_araiq_int.pdf: 2474970 bytes, checksum: fc7a9704c114879d777e8bb6f3a8ad16 (MD5) Previous issue date: 2018-02-20 / Outra / Atualmente, o glifosato é o herbicida de maior uso para o controle de ervas daninhas, sendo muito utilizado em culturas resistentes, como soja e milho. O glifosato é aplicado diretamente sobre a folhagem das plantas, porém uma parte acaba indo para o solo. Do solo, tanto o glifosato quanto seu metabólito, ácido aminometilfosfônico (AMPA) podem ser lixiviados para águas superficiais ou subterrâneas. Por conta disto, têm se gerado preocupações quanto ao seu potencial tóxico. A presente dissertação teve como objetivo avaliar a contaminação com glifosato e AMPA das águas superficiais de microbacias situadas na Bacia do Paraná 3 (Paraná-BR). Selecionou-se microbacias situadas próximas a regiões com plantios de soja e milho, assim como o período de coleta das amostras foi escolhido dentro do época de plantio destas culturas. Estudou-se dois métodos de preparo das amostras de água: reação de derivatização com cloroformiato de 9- fluorenilmetila (FMOC-Cl) seguida de extração e pré-concentração dos analitos com extração em fase sólida (SPE) utilizando cartuchos C18; pré-concentração dos analitos por liofilização seguida de reação de derivatização com FMOC-Cl. Para a determinação de glifosato e AMPA empregou-se cromatografia líquida de alta eficiência (HPLC) com detector de fluorescência. Os resultados demonstraram que a liofilização apresenta maior eficiência no preparo das amostras. Após a escolha do método de preparo realizou-se sua avaliação de desempenho pelos parâmetros de repetitividade, seletividade, linearidade, limite de detecção de quantificação, sensibilidade e estabilidade. Os resultados demonstraram concentrações na faixa de 0,31 a 8,13, μg L-1 de glifosato, estando abaixo da concentração máxima permitida pelas resoluções brasileiras: 65 μg L-1 para águas doces classe II (CONAMA 357/05) e 500 μg L-1 para águas de consumo (MS 2914/11); com exceção de uma amostra coletada em período chuvoso, apresentando glifosato na concentração de 65 μg L-1. O metabólito AMPA foi encontrado na faixa de 0,32 –14,78 μg L-1, estando abaixo da concentração máxima permitida pela resolução MS 2914/11: 500 μg L-1. / Today , glyphosate is the herbicide of greatest consumption for the control o f weeds, widely used in resistant crops such as soybean and corn . Glyphosate is applied directly to the foliage of plants, but part of it ends up going to the soil. From the soil, both glyphosate and its metabolite , aminomethylphosphonic acid ( AMPA ) can be carried to surface water or under ground water. Because of this, concerns have been raised about its toxic potential. In this dissertation, the objective is to evaluate the contamination of surface waters of watersheds located in the Paraná Basin 3 (Paraná - BR) with glyphosate and AMPA. Microbasin located near regions with soybean and corn plantations were chosen, as well as the period of sampling was chosen within the time of planting of these crops . Two methods of preparation of the water samples were stud ied: 9 - Fluorenylmethyl chloroformate ( FMOC - Cl ) derivatization reaction and extraction and pre - concentration of analytes with s olid p hase e xtraction (SPE) using C 18 cartridges; pre - concentration of the analytes with lyophilization followed by derivatization reaction with FMOC - Cl. High p erformance l iquid c hromatography (HPLC) with fluorescence detector was used f or glyphosate and AMPA analysis . The results showed that lyophilization has a better performance in the preparation of the samples. After the choice of the preparation method, its performance evaluation was performed based on evaluation of repeatability, selectivity, linearity, sensitivity and stability were evaluated. The results showed concentrations in the range of 0.31 to 8.13, μg L - 1 of glyphosate , being below the maximum concentration allowed by the Brazilian resolutions: 65 μg L - 1 for Class II (CONAMA 357/05) and 500 μg L - 1 for drinking water (MS 2914/11); with the exception of a sample collected in the rainy season, presenting glyphosate at the concentration of 65 μg L - 1 . The AMPA metabolite was found in the range of 0.32 - 14.78 μg L - 1 , being below the maximum concentration allowed by resolution MS 2914/11: 500 μg L - 1 . / Fundação Parque Tecnológico de Itaipu (FPTI): 901696/2016

Indiana University-Purdue University Indianapolis (IUPUI) / Background Compulsive alcohol drinking is a defining characteristic of alcohol use disorder and the dorsolateral striatum (DLS) is implicated in regulating this inflexible behavior. AMPA receptors have been implicated in both goal-directed (dorsomedial striatal dependent) and DLS dependent inflexible behaviors with antagonism in the DLS and general DLS inhibition altering inflexible behavior including habit and compulsion. Discrepancies exist in the preclinical models used to investigate compulsive-like alcohol. The purpose of these experiments was to establish a robust model of compulsive-like quinine adulterated alcohol (QuA) drinking in C57BL/6J male and female mice, assess associated AMPA receptor protein expression in the dorsal striatum, and to antagonize DLS AMPA receptors during compulsive-like QuA drinking using a model of binge-like alcohol drinking, Drinking-in-the-Dark (DID). Methods In aim 1, C57BL/6J mice were allowed free access to 20% (v/v) alcohol (alcohol history), or water (water history) for two hours each day beginning three hours into the dark cycle for 23 days. On days 15 and 22 mice were given QuA to test for compulsive-like QuA drinking. 24-hours following the last DID session brain slices were taking for DLS and DMS AMPA receptor western blot. In aim 2, C57BL/6J mice were given a total of 21 days alcohol history, to establish a compulsive-like phenotype, or water history, prior to infusion. On days 22 and 24 mice were given a bilateral infusion of one of three concentrations of NBQX, an AMPA receptor antagonist, into the DLS, immediately prior to DID where the DID solution was either alcohol or QuA. Results We found that three weeks, not two, is sufficient to produce robust compulsive-like QuA drinking in C57BL/6J mice. We failed to replicate our compulsive-like DID model in aim 2 and found that infusion of NBQX reduced 2-hour alcohol drinking and reduced 2-hour QuA drinking when QuA was the second solution presented on infusion days in male water history mice only. We also found that NBQX reduced 20-minute front-loading in female alcohol history mice on alcohol intake and trended toward QuA intake. Overall locomotor activity was affected by drug infusions. Conclusions Together, these data suggest that compulsive-like alcohol drinking can be achieved following three-weeks DID and DLS infusion of NBQX reduces both alcohol and QuA drinking in a sex and drinking history dependent way, and these effects may be reliant on an initial single QuA or alcohol exposure.

Les récepteurs du glutamate de type AMPA (rAMPA) sont les récepteurs ionotroniques responsables de la majeure partie des courants excitateurs rapides lors de la transmission synaptique dans le système nerveux central. Le nombre de rAMPA stabilisés à la synapse est responsable en partie de l’intensité de la transmission synaptique et de nombreux phénomènes de plasticité synaptique. Les rAMPA se répartissent en trois populations en équilibre dynamique: les récepteurs intracellulaires, les récepteurs extra-synaptiques, et les récepteurs synaptiques stabilisés au niveau de la densité post-synaptique. L’implication des protéines transmembranaires régulatrices des rAMPA (TARP) dans la stabilisation des rAMPA est établie, et repose au moins en partie sur la liaison de la protéine TARP γ-2 avec la protéine d’échafaudage PSD-95. Dans l’hippocampe, siège de nombreux phénomènes de plasticité, l’isoforme γ-8 est particulièrement enrichie. La TARP γ-8 a pour particularité de posséder un domaine C-terminal plus long que son homologue γ-2 et de s’exprimer au niveau synaptique et extra-synaptique. Mon travail de thèse à consisté à étudier les mécanismes moléculaires mis en jeu dans la régulation de la liaison des protéines TARP γ-2 et γ-8 avec la protéine PSD-95, ainsi que l’implication respective des deux isoformes dans la régulation de la mobilité latérale des rAMPA. Les résultats majeurs de cette étude sont : a) l’interaction entre γ-2 et PSD-95 est régulée par la longueur apparent du domaine C-terminal de γ-2 modulée par la phosphorylation; b) γ-8 lie PSD-95 dans les compartiments synaptiques et extra-synaptiques, toutefois cette interaction n’est pas corrélée avec une immobilisation des rAMPA. Ces résultats suggèrent que γ-2 et γ-8 jouent des rôles bien distincts dans l’adressage des rAMPA à la synapse. / AMPA type glutamate receptors (AMPARs) are ionotropic receptors responsible for most excitatory transmission in the central nervous system. The number of stabilized AMPARs in front of glutamate release sites determines in large part the strength of synaptic transmission and variation in this number is thought to underlie numerous forms of synaptic plasticity. AMPARs are present in three main subcellular pools between which they are in a dynamic equilibrium by processes of trafficking: intracellular receptors, extrasynaptic receptors, and synaptic receptors stabilized at the postsynaptic density (PSD). Transmembrane AMPAR regulatory proteins (TARPs) are known to be implicated in AMPAR stabilization at the synapse through the interaction of TARP γ-2/8 with the scaffolding protein PSD-95. In the hippocampus, a structure exhibiting various synaptic plasticity patterns, γ-8 is the most abundant TARP. This isoform is characterized by a longer C-terminal fragment than γ-2 and a synaptic and extrasynaptic localization. During my Ph.D, I studied the molecular mechanisms involved in the regulation of TARP γ-2 and γ-8 binding to PSD-95 and their respective roles in regulating AMPAR lateral mobility. The main results are: a) γ-2 interaction with PSD-95 is regulated by the apparent length of its C-terminus domain that is modulated by phosphorylation; b) γ-8 binds PSD-95 in synaptic and extrasynaptic compartment however this interaction is not correlated with AMPAR immobilization. Altogether, those results suggest that those two TARP isoforms have independent functional roles.

O desenvolvimento humano pode ser influenciado pelo ambiente. Estímulos recebidos ao longo da vida determinam seu progresso e sucesso. Estímulos positivos levam ao desenvolvimento de habilidades, melhorando funções cognitivas e da memória, enquanto estímulos negativos podem predispor a patologias como o estresse. Eventos estressantes durante a infância aumentam a predisposição para o desenvolvimento de transtornos psiquiátricos ao longo da vida. A separação materna, modelo animal de estresse pós-natal, promove diversas alterações comportamentais e encefálicas. Animais submetidos à separação materna apresentam comportamentos que mimetizam doenças psiquiátricas humanas. Por outro lado, diversos trabalhos sugerem que o enriquecimento ambiental pode ter efeito benéfico na reversão ou atenuação de modificações comportamentais e encefálicas promovidas por modelos animais de depressão, esquizofrenia, ansiedade e hiperatividade. Esses aspectos motivaram-nos a estudar se as alterações causadas por estresse podem ser revertidas ou atenuadas pelo enriquecimento do ambiente. Há evidências que sugerem um importante envolvimento de células gliais e de transportadores de glutamato presentes nessas células em modelos animais de transtornos psiquiátricos. Sendo assim, investigamos a expressão de mRNA e proteínas de dois transportadores de glutamato gliais e um neuronal, do receptor de glutamato AMPA, de marcadores gliais GFAP, S100?, glutamina sintase (GS) e do marcador de neurônios maduros NeuN na camada molecular e granular do giro denteado do hipocampo de ratos de 60 dias. Observamos que a separação materna diminui a expressão das proteínas GLAST, GLT-1, GS e NeuN, reduz a expressão dos genes Gria1 (AMPA) e S100?, e aumenta a expressão da proteína EAAC1 no giro denteado. Nossos dados sugerem uma reversão das alterações causadas pela separação materna em relação ao gene Gria1/AMPA e às proteínas GLAST, GLT-1 e EAAC1 após o enriquecimento ambiental. Portanto, o enriquecimento ambiental pode reverter as modificações causadas pela separação materna nas vias glutamatérgicas. Esses efeitos benéficos podem ser investigados para auxiliar no tratamento de transtornos psiquiátricos relacionados à separação materna. / Human development can be influenced by the environment. Stimuli received throughout life determine its progress and success. Positive stimuli lead to development of skills, improving cognitive and memory functions, while negative stimuli may predispose to pathologies such as stress. Stressful events during childhood increase the predisposition to psychiatric disorders throughout life. Maternal separation, an animal model of postnatal stress, promotes several behavioral and encephalic changes. Animals submitted to maternal separation stage behaviors associated with psychiatric diseases in humans. On the other hand, some researches have suggested that environmental enrichment may have some beneficial effects on the reversal or attenuation of behavioral and encephalic modifications promoted by animal models of depression, schizophrenia, anxiety and hyperactivity, which motivates us to study if these changes, stirred by this kind of stress, can be reversed or mitigated by environmental enrichment. There are evidences suggesting the involvement of glial cells and glutamate transporters existent in these cells in psychiatric disorders and animal models of these disorders. Therefore, we investigated mRNA and protein expression of two glial and one neuronal glutamate transporters, AMPA glutamate receptor, glial markers GFAP, S100?, glutamine synthase (GS), and the NeuN neuronal marker in the molecular and granular layer of the hippocampal gyrus in sixty-days-old rats. We observed that maternal separation decreases expression of GLAST, GLT-1, GS and NeuN proteins, reduces Gria1 (AMPA) and S100? gene expression, and increases EAAC1 protein expression in the dentate gyrus. After environmental enrichment, our data suggests a reversal of the maternal separation changes in the Gria1/AMPA gene and the GLAST, GLT-1 and EAAC1 proteins. Therefore, environmental enrichment may reverse the maternal separation changes in the glutamatergic pathways. These beneficial effects may be investigated to aid in the treatment of psychiatric disorders related to maternal separation.

No Brasil é sabido que o uso de insumos agrícolas para o melhoramento da produtividade é crescente. Dentre esses insumos, pode-se destacar o uso de pesticidas. Neste trabalho foi desenvolvida e validada metodologia para análise de deltametrina (um piretróide usado como inseticida) por CLAE/UV. Nessas amostras também foram avaliadas a presença de glifosato (uma glicina substituída usada como herbicida) e de seu metabólito, o AMPA, via CG/EM. Essas metodologias foram aplicadas para análise de amostras reais de água, solo e sedimento no município de São Carlos, a fim de avaliar a contaminação por esses xenobióticos. Para melhor elucidação do potencial contaminante e de lixiviação destes, avaliou-se sua interação com ácidos húmicos, além de estudos de adsorção em latossolo vermelho, onde se verificou a grande influência da matéria orgânica na retenção de xenobióticos no solo. A fim de propor um método de remediação de solos em casos de contaminação por derramamento de formulações comerciais, testes de toxicidade (mortalidade, biomassa e reprodução) envolvendo minhocas Eisenia foetida com adição de 3% de vermicomposto foram executados. Todo o trabalho foi desenvolvido respeitando os conceitos de Gestão de Qualidade contempladas na norma ISO 17025. / In Brazil it is known that the use of agricultural inputs to improve productivity has been increasing. Among these inputs, we can highlight the use of pesticides. This work developed and validated a methodology for deltamethrin analysis (a pyrethroid used as insecticide) by HPLC/UV. These samples were evaluated in the presence of glyphosate (a substituted glycine used as herbicide) and its metabolite, AMPA, by GC/MS. These methodologies were applied to analyze real samples of water, soil and sediment in São Carlos in order to evaluate the contamination by these xenobiotics. To elucidate the potential contaminant and leaching of these, we also evaluated the interaction with humic acids, and adsorption studies were performed in red latosol where there was a great influence of organic matter on xenobiotics retention in the soil. So as to propose a method for remediation of soils in cases of contamination by shedding of commercial formulations in soil, three toxicity tests (mortality, biomass and reproduction) involving Eisenia foetida earthworms with the addition of 3% of hummus were performed. All the study was developed in compliance with the Quality Management concepts covered in ISO 17025.

Descreve-se o desenvolvimento de um método de análise por injeção sequencial para determinação de glifosato em formulações comerciais por voltametria de onda quadrada em eletrodo de gota de mercúrio. O glifosato foi derivatizado em modo estacionário com ácido nitroso para produção de N-nitroso glifosato, que é eletroativo. Os limites de detecção (LD) e quantificação (LQ) foram 0,7 e 2,4 mg L-1 (4 e 14 µmol L-1), respectivamente. O método foi aplicado a três formulações, obtendo resultados que diferiram do valor indicado pelo fabricante por -2,6, -0,8 e -28%. Em seguida descreve-se o desenvolvimento de um método de injeção seqüencial para a determinação fluorimétrica de glifosato, Esse método baseou-se em uma primeira etapa de conversão de glifosato a glicina por reação com hipoclorito de calcio, seguida por reação com o-ftaldialdeído na presença de 2-mercaptoetanol (OPA-MCE) em tampão borato (pH> 9) para produzir o composto fluorescente (2\'-hidroxietiltio)-2-N-alkilisoindol (excitação em 340 nm e emissão em 450 nm). O método apresentou resposta linear para concentrações entre 0,25 e 25,0 µmol L-1, com LD e LQ de 0,08 e 0,25 µmol L-1 (14 e 42 µg L-1), respectivamente, e frequência de amostragem de 18 análises por hora. O método foi aplicado para estudar as propriedades de adsorção / dessorção em um solo e em uma amostra de sedimento. Os dados das isotermas de adsorção e dessorção foram tratados pelas equações de Freundlich e Langmuir, que permitiram estimar uma capacidade de adsorção de 1384 ± 26 e 295 ± 30 mg kg-1 para as amostras de solo e sedimento, respectivamente. A determinação de glifosato e AMPA em amostras de água foi realizada por cromatografia por injeção sequencial com detecção fluorimétrica explorando as mesmas reações com hipoclorito e OPA-MCE. A solução transportadora com papel de fase móvel foi composta por mistura metanol: tampão fosfato 10 mmol L-1 (pH 6,8) na proporção 25:75 (v v-1). A frequência de amostragem foi de 6 análises por hora, com LD e LQ de 0,14 e 0,42 µmol L-1 para AMPA e 0,11 e 0,33 µmol L-1 para glifosato, respectivamente. Testes de adição e recuperação em amostras de águas naturais enriquecidas com 0,50 µmol L-1 desses compostos levaram a taxas de recuperação de 82 a 140% para AMPA e de 84 a 96% para glifosato. Investigou-se o efeito do herbicida em cultivos de duas espécies de microalgas marinhas - a Chlorophyta, Tetraselmis gracilis e a diatomácea, Phaeodactilum tricornutum. Para isso adicionou-se diferentes concentrações de glifosato (2 a 1000 mg L-1) a cultivos crescendo em fase exponencial. Doses de 2 e 50 mg L-1 provocaram leve aumento de crescimento algal quando comparados com o cultivo controle sem glifosato. O rendimento quântico da fotossíntese aumentou levemente em alguns dias no cultivo de P. tricornutum em concentrações baixas de glifosato. Na concentração de glifosato de 200 mg L-1 houve declínio do crescimento e acentuada queda do rendimento quântico da fluorescência da fotossíntese. Perfis dos aminoácidos aromáticos, tirosina, triptofano e fenilalanina apresentaram diferenças nas algas. / We describe a development of a voltammetric method for determination of glyphosate in commercial formulations using sequential injection analysis to carry the sample to a flow cell adapted to the capillary of a mercury drop electrode. Glyphosate is batch-derivatized with nitrous acid in hydrochloric acid to produce N-nitroso glyphosate, which is electroactive. The limits of detection (LOD) and quantification (LOQ) were 0.7 and 2.4 mg L-1, respectively. The method was applied to three formulations, obtaining results that differed from the value indicated by the manufacturer by -2.6, -0.8 and -28%. Next, the thesis describes the development of a sequential injection method to automate the fluorimetric determination of glyphosate based on a first step of oxidation to glycine by hypochlorite, followed by reaction with o-phthaldialdehyde in presence of 2-mercaptoethanol (OPA-MCE) in borate buffer (pH>9) to produce a fluorescent 1-(2´-hydroxyethylthio)-2-N-alkylisoindole. The proposed method exhibited a linear response for glyphosate concentrations between 0.25 and 25.0 µmol L-1, with LOD and LOQ of 0.08 and 0.25 µmol L-1, respectively. The sampling rate of the method was 18 samples per hour. The method was applied to study adsorption/desorption properties in a soil and in a sediment sample. Adsorption and desorption isotherms were properly fitted by Freundlich and Langmuir equations, leading to adsorption capacities of 1384 ± 26 and 295 ± 30 mg kg-1 for the soil and sediment samples, respectively. Determination of glyphosate and AMPA in water samples was performed by sequential injection chromatography (SIC) with fluorimetric detection by exploiting the same reactions with hypochlorite and OPA-MCE. The carrier solution plays a role of mobile phase, being composed of 25:75 (v v-1) methanol : 10 mmol L-1 phosphate buffer (pH 6.8). The chromatographic step enhanced the method selectivity. The sampling throughput was 6 analyzes per hour with LOD and LOQ of 0.14 and 0.42 µmol L-1 for AMPA and 0.11 and 0.33 µmol L-1 for glyphosate, respectively. Recovery tests in natural water samples enriched with 0.50 µmol L-1 in the compounds led to recovery rates from 82 to 140% for AMPA and 84 to 96% for glyphosate. We investigated the effect of the herbicide on cultivation of two species of marine microalgae - the Chlorophyta, Tetraselmis gracilis and the diatomaceous Phaeodactilum tricornutum. To achieve this goal, different concentrations of glyphosate (2 to 1000 mg L-1) were added to cultures growing in exponential phase. Doses between 2 and 50 mg L-1 caused a slight increase in algal growth compared to the control culture without glyphosate. The quantum yield of photosynthesis increased slightly in some days in the cultivation of P. tricornutum at low concentrations of glyphosate. The glyphosate concentration of 200 mg L-1 decreases the cell growth and caused a marked decline in the fluorescence quantum yield of photosynthesis. Profiles of aromatic amino acids, tyrosine, tryptophan and phenylalanine differed in the algae