Organophosphates (OPs), such as nerve agents and insecticides, potently inhibit acetylcholinesterase (AChE). Oximes, such as the currently FDA approved oxime 2-PAM, remove the OP from the inhibited enzyme. 2-PAM is effective against select OPs and cannot effectively pass the blood-brain barrier to attenuate OP induced CNS damage. Our laboratory has synthesized a series of substituted phenoxyalkyl pyridinium oximes (Patent number: 9,227,937) that have demonstrated increased survival rates compared to 2-PAM. This research investigated 1) in vitro oxime reactivation of rat, human, and guinea pig serum BChE after inhibition by nerve agent and insecticidal OPs; 2) in vitro determination of reactivation kinetic rate constants for OP inhibited human and rat serum BChE and electric eel AChE after inhibition by a sarin surrogate and paraoxon; 3) intranasal delivery of oximes to reactivate brain AChE in vivo after inhibition by a sarin surrogate. Novel oxime 15 demonstrated significant broad spectrum reactivation of OP-inhibited rat serum BChE while novel oxime 20 demonstrated significant broad spectrum reactivation of OP-inhibited human serum BChE. All tested oximes were poor reactivators of OP-inhibited guinea pig serum BChE. Kinetic analysis of reactivation for NIMP and paraoxon human and rat serum BChE and electric eel AChE demonstrated differences in the second order rate constants. Oxime 20 demonstrated reactivation efficiency for both NIMP and paraoxon inhibited rat and human serum BChE and electric eel AChE more effectively than 2-PAM. Intranasal delivery of either oxime 20 or 2-PAM showed attenuation of NIMP-inhibited brain AChE inhibition in select brain regions and select time points. Oxime 20 demonstrated a larger window of effectiveness but neither oxime attenuated brain AChE inhibition in the hindbrain for any time point or for any brain region at the ten minute time point. These data suggest that reactivation of OP-inhibited BChE may be contributing to the observed increases in survival seen with our oximes. Novel oxime 20 demonstrated reactivation efficacy towards both BChE and AChE inhibited enzyme and a rapid entry into the brain after intranasal delivery. Having an oxime that can be effective in a multitude of ways would be of great value to medical and military personnel.

Os efluentes contendo agrotóxicos são a causa de grande preocupação uma vez que estes compostos apresentam toxicidade, persistência no meio ambiente e muitos são recalcitrantes. Dentre os compostos orgânicos utilizados no combate à pragas destacam-se os inseticidas organofosforados, os quais apresentam menor toxicidade quando comparados aos compostos organoclorados. Dois inseticidas organofosforados vêm sendo amplamente utilizados no Brasil, são eles: metil paration (MP) e clorpirifós (CP). Dentre os métodos de tratamento de efluentes, ressaltam-se os processos eletroquímicos com a utilização de eletrodo de diamante dopado com boro, que apresenta alta eficiência na remoção de compostos orgânicos com baixa capacidade de adsorção. Este estudo propõe um método de tratamento para efluente contendo MP e CP (em separado) utilizando eletrodos de diamante dopados com boro (DDB) com diferentes níveis de dopagem (15.000 e 30.000 ppm da relação B/C, designados de DDB15 e DDB30, respectivamente) em meio ácido. Os eletrodos foram caracterizados utilizando técnicas morfológicas e eletroquímicas, tais como Microscopia Eletrônica de Varredura, Microscopia Óptica Digital, Espectroscopia Raman, Difração de Raios X, Voltametria Cíclica e Microscopia Eletroquímica de Varredura. A eficiência no processo de degradação foi acompanhada por técnicas analíticas como espectroscopia na região do UV-Vis, Cromatografia Líquida de Alta Eficiência (CLAE) utilizando detectores de espectroscopia na região do UV e detector de arranjo de diodos (DAD) e analisador de carbono orgânico total (COT). A eficiência do processo foi relacionada com o nível de dopagem do eletrodo DDB bem como a natureza dos compostos. As imagens MEV mostraram o crescimento homogêneo dos filmes sobre o substrato titânio e os espectros Raman confirmam o crescimento do carbono com ligações sp3 (diamante) e a incorporação de átomos de boro. As análises de Difração de Raios X mostram a cristalinidade dos filmes DDB bem como a presença de impurezas. As caracterizações eletroquímicas mostram o amplo intervalo de potencial de ambos os eletrodos e a transferência eletrônica do sistema Fe(CN)63-/4- mostrou a maior reversibilidade com o eletrodo mais dopado. O mesmo comportamento foi obtido com a técnica de SECM. Os ensaios de degradação do inseticida MP mostraram que o eletrodo mais dopado foi mais eficiente na diminuição da concentração (valor máximo de 86%) e remoção da matéria orgânica (valor máximo de 72%) com menor consumo de energia elétrica. Em relação à remoção do inseticida CP, não foi observado diferença expressiva nas respostas de remoção do inseticida bem como na mineralização da matéria orgânica utilizando os dois eletrodos DDB. Entretanto, a cinética de remoção foi mais rápida quando utilizou-se o eletrodo DDB30. Deste modo, pode-se dizer que os eletrodos DDB altamente dopados são eficientes na remoção da matéria orgânica, mas a eficiência do aumento de dopagem deve ser associada ao composto a ser degradado. / Effluents containing pesticides are the cause of great concern because these compounds exhibit toxicity, environmental persistence and most are recalcitrant. Among the organic compounds used to combat pests highlight the organophosphate insecticides, which have lower toxicity when compared to organochlorine compounds. Two organophosphate insecticides have been widely used in Brazil, they are: methyl parathion (MP) and chlorpyrifos (CP). Among the methods of wastewater treatment, it emphasizes the electrochemical data with the use of boron-doped diamond electrode, which shows high efficiency in removing organic compounds with low adsorption capacity processes. This study proposes a method of treatment for wastewater containing MP and CP (separately) using electrodes of boron-doped diamond (BDD) with different doping levels (15,000 to 30,000 ppm B/C ratio, designated by BDD15 and BDD30, respectively) in acid medium. The electrodes were characterized using morphological and electrochemical techniques, such as Scanning Electron Microscopy, Digital Optical Microscopy, Raman Spectroscopy, X-Ray Diffraction, Cyclic Voltammetry and Scanning Electrochemical Microscopy. The efficiency of the degradation process was monitored by analytical techniques such as UV-Vis spectroscopy, High Performance Liquid Chromatography (HPLC) using spectroscopic detectors in the UV region and diode array detector (DAD) and the total organic carbon analyzer (COT). The process efficiency is related to the doping level of the BDD electrode and the compounds. The SEM images showed smooth growth of the film on the substrate titanium and the Raman spectra confirm the growth of carbon with sp3 bond (diamond) bonds and the incorporation of boron atoms. Analyses of X-Ray Diffraction showed the crystallinity of the BDD. Electrochemical characterizations showed the wide potential range of both electrodes and the electronic transfer system Fe(CN)63-/4- showed greater reversibility with more doped electrode. The same result was obtained with the SECM technique. MP insecticide assays showed that the most doped electrode was more effective in decreasing the concentration (maximum 86%) and removal of organic matter (maximum 72%) with lower electric consumption. Regarding the insecticide CP removal, no significant difference was observed in the pesticide removal and mineralization of organic matter using two BDD electrodes. However, the kinetics of removal was faster when the BDD30 electrode was used. Thus, it can be stated that highly doped electrodes are efficient in the organic matter removal, but the increase the efficiency of doping should be associated with the compound to be degraded.

Os efluentes contendo agrotóxicos são a causa de grande preocupação uma vez que estes compostos apresentam toxicidade, persistência no meio ambiente e muitos são recalcitrantes. Dentre os compostos orgânicos utilizados no combate à pragas destacam-se os inseticidas organofosforados, os quais apresentam menor toxicidade quando comparados aos compostos organoclorados. Dois inseticidas organofosforados vêm sendo amplamente utilizados no Brasil, são eles: metil paration (MP) e clorpirifós (CP). Dentre os métodos de tratamento de efluentes, ressaltam-se os processos eletroquímicos com a utilização de eletrodo de diamante dopado com boro, que apresenta alta eficiência na remoção de compostos orgânicos com baixa capacidade de adsorção. Este estudo propõe um método de tratamento para efluente contendo MP e CP (em separado) utilizando eletrodos de diamante dopados com boro (DDB) com diferentes níveis de dopagem (15.000 e 30.000 ppm da relação B/C, designados de DDB15 e DDB30, respectivamente) em meio ácido. Os eletrodos foram caracterizados utilizando técnicas morfológicas e eletroquímicas, tais como Microscopia Eletrônica de Varredura, Microscopia Óptica Digital, Espectroscopia Raman, Difração de Raios X, Voltametria Cíclica e Microscopia Eletroquímica de Varredura. A eficiência no processo de degradação foi acompanhada por técnicas analíticas como espectroscopia na região do UV-Vis, Cromatografia Líquida de Alta Eficiência (CLAE) utilizando detectores de espectroscopia na região do UV e detector de arranjo de diodos (DAD) e analisador de carbono orgânico total (COT). A eficiência do processo foi relacionada com o nível de dopagem do eletrodo DDB bem como a natureza dos compostos. As imagens MEV mostraram o crescimento homogêneo dos filmes sobre o substrato titânio e os espectros Raman confirmam o crescimento do carbono com ligações sp3 (diamante) e a incorporação de átomos de boro. As análises de Difração de Raios X mostram a cristalinidade dos filmes DDB bem como a presença de impurezas. As caracterizações eletroquímicas mostram o amplo intervalo de potencial de ambos os eletrodos e a transferência eletrônica do sistema Fe(CN)63-/4- mostrou a maior reversibilidade com o eletrodo mais dopado. O mesmo comportamento foi obtido com a técnica de SECM. Os ensaios de degradação do inseticida MP mostraram que o eletrodo mais dopado foi mais eficiente na diminuição da concentração (valor máximo de 86%) e remoção da matéria orgânica (valor máximo de 72%) com menor consumo de energia elétrica. Em relação à remoção do inseticida CP, não foi observado diferença expressiva nas respostas de remoção do inseticida bem como na mineralização da matéria orgânica utilizando os dois eletrodos DDB. Entretanto, a cinética de remoção foi mais rápida quando utilizou-se o eletrodo DDB30. Deste modo, pode-se dizer que os eletrodos DDB altamente dopados são eficientes na remoção da matéria orgânica, mas a eficiência do aumento de dopagem deve ser associada ao composto a ser degradado. / Effluents containing pesticides are the cause of great concern because these compounds exhibit toxicity, environmental persistence and most are recalcitrant. Among the organic compounds used to combat pests highlight the organophosphate insecticides, which have lower toxicity when compared to organochlorine compounds. Two organophosphate insecticides have been widely used in Brazil, they are: methyl parathion (MP) and chlorpyrifos (CP). Among the methods of wastewater treatment, it emphasizes the electrochemical data with the use of boron-doped diamond electrode, which shows high efficiency in removing organic compounds with low adsorption capacity processes. This study proposes a method of treatment for wastewater containing MP and CP (separately) using electrodes of boron-doped diamond (BDD) with different doping levels (15,000 to 30,000 ppm B/C ratio, designated by BDD15 and BDD30, respectively) in acid medium. The electrodes were characterized using morphological and electrochemical techniques, such as Scanning Electron Microscopy, Digital Optical Microscopy, Raman Spectroscopy, X-Ray Diffraction, Cyclic Voltammetry and Scanning Electrochemical Microscopy. The efficiency of the degradation process was monitored by analytical techniques such as UV-Vis spectroscopy, High Performance Liquid Chromatography (HPLC) using spectroscopic detectors in the UV region and diode array detector (DAD) and the total organic carbon analyzer (COT). The process efficiency is related to the doping level of the BDD electrode and the compounds. The SEM images showed smooth growth of the film on the substrate titanium and the Raman spectra confirm the growth of carbon with sp3 bond (diamond) bonds and the incorporation of boron atoms. Analyses of X-Ray Diffraction showed the crystallinity of the BDD. Electrochemical characterizations showed the wide potential range of both electrodes and the electronic transfer system Fe(CN)63-/4- showed greater reversibility with more doped electrode. The same result was obtained with the SECM technique. MP insecticide assays showed that the most doped electrode was more effective in decreasing the concentration (maximum 86%) and removal of organic matter (maximum 72%) with lower electric consumption. Regarding the insecticide CP removal, no significant difference was observed in the pesticide removal and mineralization of organic matter using two BDD electrodes. However, the kinetics of removal was faster when the BDD30 electrode was used. Thus, it can be stated that highly doped electrodes are efficient in the organic matter removal, but the increase the efficiency of doping should be associated with the compound to be degraded.

Of the 800,000 suicides recorded globally every year, over a third are due to pesticide ingestion, the majority of which occur in rural Asia with organophosphorus (OP) compounds. These anticholinesterase pesticides cause an acute cholinergic syndrome characterised by decreased consciousness, excessive airway secretions and respiratory failure. A combination of these clinical features is the most common cause of death. Up to 30% of OP pesticide poisoned patients are admitted to the Intensive Care Unit (ICU) for tracheal intubation and lung ventilation, but up to half die. It is not understood why the case fatality for intubated poisoned patients is so high, but one hypothesis is that the patients, when unconscious, aspirate their stomach contents (including the OP and the solvent present in its agricultural formulation) causing a toxic lung injury which contributes to the observed high mortality. In this PhD, I aimed to characterise the lung injury caused by OP pesticide self-poisoning through both indirect (ingestion) and direct (aspiration) means. To achieve this, I analysed data from previous toxicological minipig work and designed and conducted a specific minipig pulmonary aspiration study which was complemented by an experimental OP poisoning ex vivo lung perfusion model and human data from pesticide poisoned patients in Sri Lanka. I first investigated the pulmonary pathophysiology resulting from orogastric administration of OP pesticide without aspiration. Analysis of my group’s Gottingen minipig in vivo work demonstrated that orogastric placement of agricultural OP (dimethoate EC40) produced lung injury via exposure to blood-borne pesticide. Pathological lung changes consisted of alveolar and interstitial oedema, pulmonary haemorrhage and modest neutrophilia with increased concentrations of protein, IL-6 and IL-8 when compared with controls, but with low concentrations of TNF-α and IL-10 in bronchoalveolar lavage fluid (BALF). In a second study, OP poisoned minipigs had increased concentrations of BALF protein, neutrophils, IL-8 and CRP six hours after orogastric poisoning when compared with their baseline values. Electron microscopy images of both studies demonstrated damage to the alveolar capillary membrane secondary to systemic OP poisoning. Prior to conducting the main pulmonary aspiration study in minipigs, there was considerable refinement of the processes involved through use of: (i) pilot aspiration and dose ranging studies; (ii) the development of a specific pulmonary histopathological scoring system; and (iii) employment of modern human anaesthetic equipment and intensive care patient management protocols. After this period of model development, an in vivo 48 hour study using Gottingen minipigs (n=26) was conducted to investigate the pulmonary pathophysiology in animals given either sham bronchoscopy (sham control) or 0.5 mL/kg of: saline (saline control), porcine gastric juice [GJ], OP (dimethoate EC40) + GJ [OP+GJ], or solvent (cyclohexanone) + GJ [Solv+GJ] into the right lung under bronchoscopic guidance. The results showed that in a minipig model OP and GJ placed into one lung created a direct (right) and indirect (left) lung injury significantly different to controls, and in some respects worse than GJ alone 48 hours after poisoning. The direct lung injury caused by OP+GJ was characterised by significantly worse pathology (p=0.0003) in terms of: pulmonary neutrophilia, alveolar haemorrhage, necrosis, oedema and fibrin deposition, when compared with sham controls at 48 hours. Lungs injured directly with OP+GJ also had significantly higher concentrations of BALF neutrophils (p≤0.01), protein (p≤0.05), IL-6 (p≤0.01), IL-8 (p≤0.01) and CRP (p≤0.05) at 24 hours, and BALF protein (p≤ 0.01), and CRP (p≤ 0.05) when compared with sham controls at 48 hours. The BALF from OP+GJ minipigs at 48 hours also had higher numbers of aerobic bacteria than other groups, suggesting the development of pneumonia could be a source of additional lung injury. Lung damage might also have resulted from a reduction in the surfactant component responsible for the lowering of alveolar surface tension. Direct lung injury with OP+GJ caused a proportional reduction of beneficial pulmonary surfactant phosphatidylcholine (PC) species 16:0/16:0 [29(±4) % vs. 38(±4) %] when compared with sham controls at 48 hours. Unlike the other groups, OP+GJ (direct and indirectly-injured) lungs had type 2 alveolar cell ultrastructural morphological differences in the lamellar bodies that stored the surfactant. The lamellar bodies were more numerous and more dense in the OP+GJ lungs compared with other groups and could signify a failure of surfactant release or some other pathology pertinent to OP aspiration lung injury. Computed tomography analysis showed that direct lung injury with OP+GJ caused significantly more lung tissue to be poorly or non-aerated [77 (±13) % ; p≤0.0001 when compared with sham] as opposed to 62 (±27) % in GJ, 53(±13)% in sham and 47(±0.2)% in saline control animals by 47.5 hours and was mainly due to pulmonary haemorrhage and oedema fluid. The key differences between aspiration of OP+GJ versus GJ alone was that the majority of inflammatory markers (e.g. BALF protein, IL-6 and CRP) appeared to increase from 24-48 hours in OP+GJ treated animals, but decreased in GJ pigs, possibly signifying resolution. Treatment with GJ alone produced less severe histopathological damage, bacterial BALF numbers and percentage of poorly and non-aerated lung tissue. Importantly, there was less evidence of indirect lung injury within the GJ pigs when compared with animals treated with OP+GJ. Solvent placed into the lung seemed to offer some form of protection from the effects of GJ aspiration. This was dramatically demonstrated by the histopathology scores, proportional percentage of beneficial phosphatidylcholine (PC) species 16:0/16:0 and the percentage of poorly and non-aerated lung tissue all approaching control animal levels by 48 hours in minipigs that had Solv+GJ placed in the directly-injured (right) lung. Further evidence of benefit was provided by statistically significant reductions (p≤ 0.05) in BALF concentrations of IL-8, IL-6 and CRP in minipigs which had aspirated Solv+GJ when compared with OP+GJ and/or GJ minipig groups at 24 hours. The pathophysiology of aspirated OP+GJ was also investigated in a pilot ovine ex vivo lung perfusion (EVLP) model (n=4). Lungs directly-injured with OP+GJ had higher concentrations of total protein (4300 mg/L vs. 350 mg/L) with a proportional reduction of beneficial pulmonary surfactant phosphatidylcholine species 16:0/16:0 (27% vs.34%) when compared with control lungs. Analysis of toll-like receptor (TLR) lung tissue expression in the OP+GJ directly and indirectly-injured lungs indicated that inflammatory mechanisms might also involve upregulation of TLR 3 and 5, unlike other lung injuries e.g. those induced with lipopolysaccharide, which typically upregulates TLR 2 and 4. To compare OP-induced lung injury in humans and the minipigs, a small feasibility study was conducted in the ICUs of the University of Peradeniya hospital, Sri Lanka. Unfortunately, ethics review and recruitment proved more difficult than expected and we failed to recruit to target. We did however find raised BALF concentrations of IL-6, IL-8 and CRP and low concentrations of TNF, IL-1β, IL-10 in intubated OP poisoned patients at 24 hours when compared with controls. We also found that two plasma micro-RNA biomarkers thought to be involved in inflammation and lung injury, MiR-21 and MiR-146a, had significantly reduced expression in OP-poisoned patients with aspiration compared to non-intubated control patients from the UK (p=0.008 and p=0.0083 respectively). The work from this thesis has allowed the characterisation of both indirect and direct lung injuries caused by OP pesticide ingestion and aspiration. The minipig model showed that at 48 hours the lung injury created by aspiration of OP+GJ appeared more severe than GJ alone, but the addition of the solvent cyclohexanone seemed protective and even beneficial in the context of GJ aspiration. The cytokine expression profiles from both the human and minipig work, combined with the preliminary TLR lung tissue analysis from the EVLP model, suggest that OP+GJ aspiration is unlike normal GJ aspiration and classic ARDS. / Increased concentrations of aerobic bacteria in the minipig OP+GJ lungs at 48 hours and evidence of suppression of plasma miR-21 and miR-146a in OP poisoned patients could be linked, and may involve cholinergic immune system modulation. These molecular mechanisms need to be investigated further in both in vitro and in vivo models. These discoveries indicate the complex nature of the pulmonary injury that occurs after OP pesticide poisoning, and suggests that damage is not caused by gastric contents alone. Preliminary findings indicate that aspiration of OP+GJ could create favourable conditions for the development of aspiration or ventilator-associated pneumonia but this would need confirmation in larger clinical studies. The potential roles of micro RNA as a biomarker of OP poisoning and lung injury, and solvent as a therapy for aspiration should be explored in further pre-clinical studies.

Organophosphates (OPs) are used in agriculture via pesticides, and warfare and terrorism via nerve agents. OPs can inhibit acetylcholinesterase (AChE) activity in the nervous system, leading to the buildup of acetylcholine (ACh), and overstimulation of the nervous system and eventual asphyxiation and death. The development of novel blood-brain barrier () penetrating pyridinium oxime reactivators have previously demonstrated efficacy towards treatment of OP poisoning after exposure of rats to a sarin or a VX surrogate, nitrophenyl isopropyl methylphosphonate (NIMP) and nitrophenyl ethyl methylphosphonate (NEMP), respectively. An effective oxime antidote capable of penetrating the and restoring nervous system activity after exposure to a cyclosarin surrogate, nitrophenyl cyclohexyl methylphosphonate (NCMP), has yet to be determined. In Chapter 2, in vitro testing of the efficacy of 17 total novel oxime candidates to utilize against NCMP was conducted with a modified Ellman’s AChE assay. Pools of naïve adult male rat brains were utilized as the AChE source. The first variable investigated was the measurement of AChE activity after inhibition with NCMP and subsequent reactivation with one of the oximes. The second variable investigated restoration of AChE activity after simultaneous oxime and NCMP incubation. The final variable investigated the restoration of AChE activity after simultaneous 2-PAM, oxime and NCMP incubation. A thorough kinetic analysis of our best oximes has yet to be accomplished. In Chapter 3, the best oxime antidotes for NEMP and NIMP were used for kinetic analysis with a modified 96-well plate Ellman’s AChE assay. Protein concentrations were analyzed with a modified Lowry protein tube assay to ensure consistent analytical concentrations. The sources of AChE included pooled rat brain and skeletal muscle, and rat and human erythrocytes and plasma. Butyrylcholinesterase (BChE) activity was also measured in the rat and human plasma samples. The results of these studies strengthen the argument that our oxime antidotes can be used as potential therapeutic drugs for OP poisoning. The kinetic data provided critical information to help propose, for Chapter 4, a dynamic pharmacokinetic based model to predict human AChE or BChE activity after exposure to nerve agent surrogates (NEMP and NIMP) and the oximes (44.08 and 44.25).

Obesity is a prevalent disease in which, when compared to individuals of normal weight, obese individuals have noted pathophysiological alterations including increased adiposity and fat mass which may alter toxicokinetics of xenobiotics and therefore alter their toxicities. However, the effects of obesity and the altered pathophysiology accompanying this condition on the toxicity of many widely utilized pesticides have not been established. Organophosphate (OP) pesiticides, including chlorpyrifos (CPS), have historically been widely used for agricultural purposes. CPS and several other notable OPs are no longer registered for household use but still used mainly in agriculture by registered users. CPS is bioactivated to form chlorpyrifos-oxon (CPO) in liver by cytochrome P450 isozymes. Acute toxicity of CPS exposure is inhibition of acetylcholinesterase (AChE) in the central and peripheral nervous systems. The other targets following CPS exposure are the noncholinergic serine hydrolase enzymes: carboxylesterase (CES), fatty acid amide hydrolase (FAAH), and monoacylglycerol lipase (MAGL). Therefore the current study was designed to: 1) determine the time and concentration-dependent effects of CPS on noncholinergic endpoints of OP toxicity utilizing hepatoma cells under normal and steatotic conditions 2) determine if obese phenotype altered the toxicity of CPS, including both cholinergic and non-cholinergic endpoints and 3) determine the effects of high fat diet on CPS bioaccumulation and detoxication by determining CPS and 3,5,6-trichloro-2-pyridinol (TCP) levels at different time points. The current in vitro studies determined that CES is more sensitive to CPS mediated inhibition in normal and steatotic conditions compared to FAAH and MAGL. The in vivo studies determined that CPS (2 mg/kg) exposure produced a greater inhibitory effect on hepatic CES and FAAH in obese animals compared to lean animals which indicates that the obese animals may be at greater risk for CPS mediated alterations in hepatic lipid metabolism upon chronic exposure. Our toxicokinetic studies using a higher dose of CPS (25 mg/kg) did not inhibit AChE in CNS and did not alter the overall CPS levels between normal and obese animals. Peak levels of TCP were decreased in liver of obese animals at 3 hours and overall hepatic TCP levels were significantly decreased in obese animals.

Les composés organophosphorés sont des molécules importantes aussi bien dans le domaine de l'industrie nucléaire que pour les systèmes vivants. En effet, plusieurs extractants à la base de procédés du cycle de retraitement du combustible sont des molécules organophosphorées (TBP, HDEHP par exemple) et dans le domaine biologique les nucléotides sont des organophosphates qui jouent un rôle très important dans différents processus métaboliques.Si la littérature concernant les interactions des phosphates inorganiques avec les actinides est abondante, les études publiées avec des composés organophosphates se limitent généralement à des approches macroscopiques et/ou physiologiques. L'objectif de cette thèse est d'étudier la structure des plusieurs molécules organophosphorées avec des actinides pour affiner la compréhension et développer de nouveaux édifices spécifiques. La famille de molécules retenue pour cette approche est composée de 3 nucléotides d'adénine mono, bi et triphosphate (AMP, adénosine monophosphate – ADP, adénosine diphosphate - ATP, adénosine triphosphate) et d'un aminoalkylphosphate (AEP, O-phosphoryléthanolamine). La synthèse en milieu aqueux et faiblement acide (2,8-4) de plusieurs lanthanides représentants des actinides(III) (Lu, Yb, Eu) et d'actinides (U(VI), Th(IV) et Am(III)) a été réalisée. Plusieurs techniques analytiques et spectroscopiques ont été employées pour décrire la structure des complexes obtenus: les analyses spectrométriques réalisées par FTIR et RMN ont permis d'identifier les groupes fonctionnels impliqués dans la complexation, les analyses par ESI-MS et par titrage pH-métrique ont permis d'affiner la spéciation en solution et les analyses par EXAFS réalisées sur la ligne MARS du synchrotron SOLEIL, ont permis de décrire l'environnement proche des cations, tant pour des composés solides que pour des solutions. Quelques approches théoriques par DFT pour identifier des structures stables ont complété les approches expérimentales.Tous les complexes solides (AMP, ADP, ATP et AEP) présentent des organisations polynucléaires, alors que les complexes d'ATP en solution sont mononucléaires. Dans tous les complexes synthétisés, l'interaction prépondérante entre les cations et les groupes phosphates des ligands a été démontrée. Les complexes avec les ligands monophosphatés (Lu-AMP, Lu-AEP et Th-AMP) s'organisent de manière identique avec des phosphates pontants indiquant que la partie organique n'a pas un effet important sur leurs structures.Les complexes solides d'ADP et d'ATP (avec les deux ions métalliques sphéroïdes Lu et Th) présentent beaucoup de similitudes au niveau de l'environnement local indiquant que la présence d'un troisième groupe phosphate n'a pas d'effet important sur l'organisation des complexes au niveau local. La structure fine de ces complexes n'a cependant pas pu être déterminée précisément, malgré les approches théoriques qui ont été menées.Les complexes de lanthanides et d'actinides avec l'ATP ont un comportement similaire au niveau macroscopique suggérant une structure identique au niveau moléculaire.Avec l'uranyle, le complexe U-AMP à pH acide montre une organisation moléculaire différente de celui préparée à pH basique mais avec les mêmes sites de coordination: phosphates et hydroxyles du sucre. / Organophosphorus compounds are important molecules in both nuclear industry and living systems fields. Indeed, several extractants of organophosphorus compounds (such as TBP, HDEHP) are used in the nuclear fuel cycle reprocessing and in the biological field, the nucleotides are organophosphates which play a very important role in various metabolic processes. If the literature on the interactions of actinides with inorganic phosphate is abundant, published studies with organophosphate compounds are generally limited to macroscopic and / or physiological approaches. The objective of this thesis is to study the structure of several organophosphorus compounds with actinides to refine a better understanding and develop new specific buildings blocks. The family of the chosen molecules for this approach consists of three adenine nucleotides mono, bi and triphosphate (AMP, adenosine monophosphate - ADP, adenosine diphosphate - ATP, adenosine triphosphate) and an aminoalkylphosphate (AEP O-phosphorylethanolamine). Complexes Synthesis was conducted in aqueous medium and weakly acidic (2.8-4) for several representatives of lanthanides considered as actinides (III) (Lu, Yb, Eu) and actinides (U (VI), Th (IV) and Am (III)). Several analytical and spectroscopic techniques have been used to describe the organization of the synthesized complexes: spectrometric analysis performed by FTIR and NMR were used to identify the functional groups involved in the complexation, analysis by ESI-MS and pH-metric titration were used to determine the solution speciation and EXAFS analyzes were performed on Mars beamline of the SOLEIL synchrotron, have described the local cation environment, for both solution and solid compounds. Some theoretical approaches of DFT were conducted to identify stable structures in purpose of completing the experimental approaches. All solid complexes (AMP, ADP, ATP and AEP) have polynuclear structures, while soluble ATP complexes are mononuclear. For all synthesized complexes, it has been demonstrated that the dominant interaction is between the cations and the phosphate groups of the ligands. Complexes with monophosphate ligands (AMP-Lu, Lu-Th-AEP and AMP) show similar organizations with bridging phosphates indicating that the organic part does not have a significant effect on their structures. ADP and ATP solid state complexes (with two spheroid metal ions: Lu and Th) show several similarities in terms of local environment indicating that the occurrence of a third phosphate group has no significant effect on the local organization of the complex. However, despite the theoretical approaches that have been conducted, the fine structure of these complexes has not been accurately determined, Complexes of lanthanides and actinides with ATP behave similarly at macroscopic level suggesting an identical structure at the molecular level for these complexes.With uranyl, U-AMP complexe synthesized at acidic pH show different behavior at molecular level than that observed at alkaline pH but the same coordination sites (phosphates and hydroxyls ribose groups) have been demonstrated for both complexes.

Made available in DSpace on 2017-07-25T19:31:03Z (GMT). No. of bitstreams: 1 Fernanda Wagner.pdf: 2201127 bytes, checksum: 1f084a627bdbb8e6aed48d44eac35a00 (MD5) Previous issue date: 2017-04-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Soybean is one of the most cultivated crops in the country, and the stink bugs of the largest losses in productivity. The Neotropical brown stink bug Euschistus heros cause severe damage to soybeans, however the green belly stink bug Dichelops melacanthus cause severe damage to corn and wheat, and can also attack soybeans. For its control, seed treatment can be performed, as in the case of D. melacanthus, or sprays with insecticides. The objective of this work was to determine the feasibility of conducting ingestion biossays in E. heros and D. melacanthus, as well as to determine the mode of action of the insecticides used to control the E. heros stink bug. The feasibility of ingestion bioassays on E. heros and D. melacanthus with systemic inseticides was investigated, as well a topical bioassay with synergistic substances on E. heros to determine the mode of action of the most common recommended insecticides. The insects used in the bioassays were collected in Cândido Mota-SP, Palmital-SP, Pedrinhas Paulista-SP and Sertaneja-PR. The data obtained were analyzed by ANOVA and Probit analysis. The CL50 for E. heros for the thiametoxam + lambda cyhalothrin mixture was (0.038 + 0.028 μg iamL-1) and for D. melacanthus (0.219 + 0.164 μg de i.a.mL-1). In ingestion trials, this mixture achieved high mortality compared to the other active ingredients tested. When inoculation with the insecticide thiametoxam and the synergist piperonyl butoxide (PBO) agent was performed, the mortalities were significantly higher in the four populations tested indicating that the monooxygenases would be detoxifying the insecticide. Likewise, imidacloprid caused higher mortality when applied with the same synergist, indicating detoxification by monooxygenase enzymes. In the same way, treatments with pyrethroids (lambda-cyhalothrin and beta-cyfluthrin) and PBO caused higher or faster mortalities than the insecticide applied alone. When treatments with acephate and acephate plus diethyl maleate were performed, E. heros mortalities were faster with the synergist diethyl maleate, indicating detoxification of the insecticide by glutathione-S-transferases enzymes, mainly in the population of Sertaneja-PR. / A soja é uma das culturas mais cultivadas no país, sendo os percevejos da família Pentatomidae responsáveis por parte das maiores perdas em produtividade. O percevejo marrom, Euschistus heros, causa danos severos em soja. Por outro lado, o percevejo barriga-verde, Dichelops melacanthus, causa danos intensos em milho e trigo, podendo atacar também a soja. Para seu controle, podem ser realizadas pulverizações com inseticidas ou o tratamento de sementes, como no caso de D. melacanthus. O objetivo deste trabalho, foi determinar a viabilidade de realizar bioensaios de ingestão em E. heros e D. melacanthus, assim como determinar o modo de ação dos inseticidas utilizados para o controle do percevejo E. heros. Os insetos utilizados para o ensaio de ingestão eram provenientes do laboratório e nos ensaios de aplicação tópica, foram realizadas coletas de campo em: Cândido Mota, SP, Palmital, SP, Pedrinhas Paulista, SP e Sertaneja, PR. Os dados obtidos foram analisados por ANOVA e análise de Probit. A CL50 de E. heros para a mistura thiametoxam + lambda cialotrina calculada foi de (0,038 + 0,028 μg de i.a.mL-1) e para D. melacanthus (0,219 + 0,164 μg de i.a.mL-1), respectivamente. Nos ensaios de ingestão, essa mistura causou mortalidade elevada em comparação aos outros ingredientes ativos testados. Quando foi realizada a inoculação com o inseticida thiametoxam e o agente sinergista butóxido de piperonila (PBO) as mortalidades foram significativamente maiores nas quatro populações testadas indicando que as enzimas monooxigenases estariam realizando a detoxificação do inseticida. Da mesma forma, o imidacloprido causou maior mortalidade quando aplicado com o mesmo sinergista, indicando detoxificação por enzimas monooxigenases e/ou esterases. Os tratamentos com os piretróides (lambda-cialotrina e beta-ciflutrina) e o PBO causaram mortalidades superiores ou mais rápidas que o inseticida aplicado isoladamente. Quando foram realizados os tratamentos com acefato e acefato mais dietil maleato, as mortalidades de E. heros foram mais rápidas com o sinergista dietil maleato, indicando o envolvimento de enzimas glutationa-S-transferases, no processo de detoxificação, principalmente com níveis mais elevados na população de Sertaneja-PR.

I present a simulation model that should be a useful tool for risk assessment of the impact of insecticide inhibitors of cholinesterase (ChE) applied in irrigated agricultural fields on non-target wildlife. I developed the model as a compartment model based on difference equations (Δt = 1 hour) and programmed with Stella® VII software. Conceptually the model is compartmentalized into six submodels describing the dynamics of (1) insecticide application, (2) insecticide movement into floodable soil, (3) irrigation and rain, (4) insecticide dissolution in water, (5) foraging and insecticide intake from water, and (6) ChE inhibition and recovery. To demonstrate application of the model, I simulate historical, current, and “worst-case” scenarios, that examined the impact of ChE-inhibiting insecticides on white-winged doves (WWDO - Zenaida asiatica) in the Lower Rio Grande Valley of Texas (LRGV), USA. To my knowledge, there are no field data verifying that the cause of ChE deprivation in WWDO is due to the ingestion of ChE-inhibiting insecticide residues dissolved in drinking water. I parameterized the model to represent a system composed of fields of cotton, sorghum, corn, citrus, and brushland that encompasses the activity range of a WWDO in the LRGV. I simulated situations representing the typical scenario of WWDO using irrigated crop fields in the absence and in the presence of rain. I also simulated “worst case” scenarios in which methyl parathion was applied at high rates and high frequency. Based on results of the simulations, I conclude that it is unlikely that WWDO are seriously exposed to ChE-inhibiting insecticides by drinking contaminated water. Only in rare cases, for example, when a rain event occurs just after the application of insecticides, are levels of ChE inhibition likely to approach diagnostic levels (20 %). The present simulation model should be a useful tool to predict the effect of ChE-inhibiting insecticides on the ChE activity of different species that drink contaminated water from irrigated agricultural fields. It should be particularly useful in identifying specific situations in which the juxtaposition of environmental conditions and management schemes could result in a high risk to non-target wildlife.

Pesticides are natural or artificial molecules aimed to kill, or mitigate any harmful organism. Although their use in agriculture provides us with an increased crop yield, remains of chemicals on the products creates health concerns in society. Organophosphates and carbamates are two groups of insecticides. Although they are far more lethal against insects and small animals, they can also cause poisoning in humans through the inhibition of acetylcholinesterase enzyme (AChE) that plays an important role in human nervous system. Therefore, the detection of these compounds is crucial. The conventional methods for the detection of these compounds are expensive, time-consuming and need expertise. In this study, a fast, disposable, cheap and accurate acetylcholinesterase biosensor was developed to detect organophosphate and carbamate-based pesticide residues. By means of adsorption method, AChE, the chromophore 5,5&#039 / -Dithio-bis(2-nitrobenzoic acid) (DTNB) and artificial substrate acetylthiocholine (ATCh) were immobilized on the supporting material. In optimization studies / from 3 to 15U/mL concentrations were experimented for AChE, 1 to 5mM DTNB and 1 to 5mM ATCh concentration gradients were used. v As a result of the optimization studies 12U/mL ACHE concentration, 5mM DTNB concentration and 5mM ATCh concentration were determined for constructing a pesticide biosensor. Detection limit of malathion, an organophosphate-based insecticide was found as 2.5ppm in 5% methanol solution. The biosensor conserved its integrity between pH 4 and 8, and gave false positive results after pH 10. Stability studies showed that, biosensor retained its activity for at least 60 days at 4&deg / C to discrimnate between positive and negative controls.