Os polinizadores são organismos fundamentais para o funcionamento de ecossistemas naturais e agrícolas, incrementando mais de 75% na produção das culturas agrícolas. Dentre os animais que participam do processo de polinização, as abelhas são consideradas as mais efetivas. Entretanto, um constante declínio na população desses polinizadores vem sendo reportado em diversos países, levando a preocupações ecológicas e econômicas, devido às perdas, tanto na produtividade agrícola como na biodiversidade. Dentre as diversas hipóteses para o declínio, o uso intensivo de agrotóxicos na agricultura tem causado preocupação, já que muitos destes compostos possuem um amplo espectro de ação, atingindo não somente os organismos alvo, mas também insetos benéficos, como as abelhas. Assim, objetivou- se com este estudo avaliar resíduos do inseticida neonicotinoide imidacloprido em amostras de flores de citros e a sua influência na riqueza de espécies de abelhas presentes nos pomares, bem como os possíveis efeitos dessas concentrações na atividade locomotora de abelhas Apis mellifera africanizada, Scaptotrigona postica e Tetragonisca angustula após exposição oral em condições de laboratório. Além disso, estimou-se a concentração letal média (CL50) dos neonicotinoides mais utilizados (acetamiprido, imidacloprido, tiacloprido e tiametoxam) para estas espécies de abelhas, e os níveis de comprometimento motor após exposição de abelhas a essas concentrações. Imidacloprido foi detectado em amostras de flores de todo os pomares, com maior concentração para área em Anhembi/SP (6,26 ng g-1 de imidacloprido) e menor concentração em Barretos/SP (3,24 ng g-1 de imidacloprido). Foram identificadas, no total, 17 espécies de abelhas visitando flores de citros, com predominância de A. mellifera em todos os pomares. No entanto, não houve relação entre a riqueza de espécies e de imidacloprido residual. Alterações significativas na atividade locomotora foram verificadas após exposição às concentrações de 0,006 e 6,26 ng µL-1 de imidacloprido, com maior alteração nas variáveis de duração e frequência de repouso. Além disso, verificaram-se diferentes níveis de toxicidade entre os ingredientes ativos estudados, com menor toxicidade oral aguda para os compostos com radical ciano (acetamiprido e tiacloprido) e maior para os que apresentam radical nitro (imidacloprido e tiametoxam). A locomoção das três espécies de abelhas foi reduzida em relação ao controle após exposição a todos os neonicotinoides. Com menor nível de comprometimento quando expostas ao acetamiprido. Assim, os resultados obtidos neste estudo esclarecem os efeitos dos inseticidas neonicotinoides sobre as espécies de abelhas na região Neotropical, e contribuem para a formulação de estratégias para integrar o controle de pragas e conservação dos polinizadores. / Pollinators are fundamental organisms for the functioning of natural and agricultural ecosystems, increasing more than 75% the production of agricultural crops. Among the animals that participate in the pollination process, bees are considered the most effective. However, a steady decline in the population of these pollinators has been reported in several countries, leading to ecological and economic concerns, due to the losses in both agricultural productivity and biodiversity. Among the several hypotheses for the decline, the intensive use of pesticides in agriculture the fields has caused concern, as many of these compounds possess a broad spectrum of action, reaching not only the target organisms, but also beneficial insects, as bees. Thus, the objectives of this study were to detect and quantify the residual concentration of imidacloprid in samples of citrus flowers and if these concentrations influence the richness of bee species present in the orchards, as well as the possible effects of these concentrations on the locomotion activity of three bee species (Africanized Apis mellifera, Scaptotrigona postica and Tetragonisca angustula) after oral exposure under laboratory conditions. In addition, the median lethal concentration (LC50) the most used neonicotinoids (acetamiprid, imidacloprid, thiacloprid and thiamethoxam) to these bee species was estimated, and the levels of motor impairment after bee exposure to these concentrations. Imidacloprid was detected in flower samples in all orchards, with a higher concentration in Anhembi/SP (6.26 ng g-1 of imidacloprid) and lower concentration in Barretos/SP (3.24 ng g-1 of imidacloprid). A total of 17 species of bees were identified visiting citrus flowers, predominating A. mellifera in all orchards. However, there was no relationship between species richness and residual imidacloprid concentrations in the flowers of each evaluated region. Significant changes in the activity bee locomotion were observed after exposure to concentrations of 0.006 and 6.26 ng µL-1 of imidacloprid, with a higher level of impairment for the variables duration and frequency of rests. In addition, different levels of toxicity were observed among the active ingredients studied, with lower acute oral toxicity for cyano compounds (acetamiprid and thiacloprid) and higher for those with nitro radical (imidacloprid and thiamethoxam). The locomotion of the three bee species was reduced in relation to the control after exposure to all neonicotinoids, with a lower level of impairment when exposed to acetamiprid. Thus, the results obtained in this study shed light on the effects of neonicotinoid insecticides on bee species the Neotropical region, and contribute the formulation of strategies to integrate pest control and pollinator conservation.

Acrylamide, 2,5-hexanedione, Tri-o-tolyl phosphate and leptophos belong to three fundamentally different chemical classes but all four chemicals cause central-peripheral distal axonopathy. Some of these compounds have been shown to alter brain steady state levels of neurotransmitters or to inhibit the activities of adenosine triphosphatases which are involved in the uptake and storage of biogenic amines. Tests were performed to determine alterations in steady state levels of rat brain norepinephrine and dopamine in response to doses of the above chemicals and of the central nervous system toxin, methyl mercuric chloride sufficient to cause ataxia. Catecholamine synthesis rate constant estimations were performed. Specific activities of tyrosine in brains of control and treatment groups following intravenous injection of labelled tyrosine were compared to determine if passage of tyrosine across the blood-brain barrier were affected by treatments. Levels of the dopamine metabolite, dihydroxyphenylacetic acid were assayed in all cases. Levels of the norepinephrine metabolite, 3-methoxy-4-hydroxymethylethyleneglycol sulfate, were assayed in response to acrylamide administration. Animal weights were recorded at the beginning and end of the treatment period. Rats treated with a cumulative dose of 250 mg/kg acrylamide had significantly lower norepinephrine levels than controls. 2,5-hexanedione administration significantly increased the dopamine synthesis rate constant at a cumulative dose of 210 mg/kg. Cumulative doses of 700 and 2100 mg/kg also appeared to elevate norepinephrine and dopamine synthesis rate constants, but values were not statistically significant. Leptophos caused a slight but significant increase in dopamine levels in rats administered a cumulative dose of 75 mg/kg. Methyl mercuric chloride caused variable effects to norephinephrine synthesis rate and lowered dopamine synthesis rate constant at cumulative doses of 5 to 50 mg/kg. No other alterations were seen in levels of catecholamines or of their metabolites, nor in synthesis rate constants of the catecholamines in response to administration of the five neurotoxic compounds. No evidence of altered blood-brain transport of tyrosine was observed at any level of neurotoxins administered. Rats given the highest cumulative doses of all neurotoxins except tri-o-tolyl phosphate gained significantly less weight than control animals. It was concluded that the four compounds which cause delayed distal neurotoxicity do not alter levels of turnover rates of brain catecholamines in a consistent manner.

Commonly referred to as “red tide”, harmful algal blooms (HABs) formed by Karenia brevis occur frequently in the Gulf of Mexico (GOM). A bloom is defined as cell abundances >105 cells L-1. This thesis will focus primarily on Karenia brevis, formerly known as Gymnodinium breve, in the Gulf of Mexico. K. brevis is harmful because it produces brevetoxin, a ladder-frame polyether that acts as a potent neurotoxin in vertebrates. K. brevis commonly causes fish kills, respiratory irritation in humans, and Neurotoxic Shellfish Poisoning (NSP) if ingested. Blooms of K. brevis occur almost annually along the West Florida Shelf (WFS) in the late summer and early fall, when the coastal current is favorable for bloom initiation. Along the Texas-Louisiana shelf (TLS) however, blooms of K. brevis are infrequent and sporadic. While much is known of the blooms along the WFS due to their frequent presence, little is known of the mechanisms driving the blooms along the TLS due to their inconsistent presence. To understand the stochastic nature of HABs along the TLS, historical data of bloom occurrences from 1996 to present were compared with NOAA station PTAT2 wind, sea-level pressure, air and water temperature data and NCEP NARR-A sea-level pressure data. The difference in the monthly-mean along-shore component of the wind was statistically significant between bloom and non-bloom years in September (p<<0.001) and April (p=0.0015), with bloom years having a strong downcoast current. Monthly mean water temperature values yielded similar results between bloom and non-bloom years. Both March and September monthly-mean water temperature values were lower during non-bloom years with p-values of 0.01 and 0.048, respectively. These results suggest the possibly of forecasting for HABs along the TLS with currently measured, publicly available data.

Les pyréthrinoïdes et les pyréthrines sont des insecticides neurotoxiques auxquels on attribue également des effets néfastes sur les systèmes immunitaire, hormonal et reproducteur. Ils sont abondamment utilisés en agriculture, mais aussi en horticulture, en extermination et dans le traitement d’infestations parasitaires humaines et animales (gale, poux). Il y a donc un intérêt en santé environnementale à connaître l’ampleur de l’exposition humaine à ces pesticides. La mesure des métabolites urinaires des pyréthrinoïdes et des pyréthrines apparaît une approche de choix pour arriver à cette fin puisqu’elle permet, en théorie, d’obtenir un portrait global de l’exposition. Or,traditionnellement et par soucis de simplicité les concentrations volumiques ou ajustées pour la créatinine) de ces biomarqueurs dans des urines ponctuelles sont déterminées, mais l’effet de l’utilisation de ces unités sur la validité des estimations de dose quotidienne absorbée n’a jamais été vérifié. L’objectif général de cette thèse était donc de développer, appliquer et valider une nouvelle stratégie de mesure et d’analyse de biomarqueurs pour améliorer la précision et la fiabilité des évaluations de l’exposition individuelles et populationnelles aux pyréthrinoïdes et pyréthrines. Les objectifs spécifiques étaient : i) de caractériser l’exposition humaine à ces contaminants en région urbaine et rurale au Québec et ii) de comparer la validité de la nouvelle stratégie de mesure et d’analyse de biomarqueurs urinaires proposée avec les autres méthodes plus usuelles utilisées pour estimer l’exposition individuelle et populationnelle et les doses absorbées de pyréthrinoïdes. Des adultes et des enfants, recrutés dans la population de l’Île de Montréal et de la Montérégie ont recueilli leurs urines pendant une période d’au moins douze heures et complété un questionnaire documentant les sources potentielles d’exposition. Les quantités de métabolites de pyréthrinoïdes et pyréthrines (pmol) mesurées dans les urines ont été ajustées pour une période de douze heures exactement et pour le poids corporel. Les quantités excrétées en région urbaine ont été comparées à celles excrétées en région rurale et les données individuelles et populationnelles ont été comparées à celles qui auraient été obtenues si des concentrations volumiques ou ajustées pour la créatinine avaient été mesurées. Les résultats montrent que l’exposition à ces pesticides est ubiquiste puisque plus de 90% des participants excrétaient les principaux métabolites de pyréthrinoïdes et pyréthrines à un niveau supérieur au seuil de détection analytique. Les résultats suggèrent que l’alimentation pourrait être à l’origine de ce niveau de base puisque les autres sources d’exposition connues n’ont été que rarement rapportées. Au Québec, l’exposition en milieu rural apparaissait légèrement plus importante qu’en milieu urbain et certains facteurs d’exposition, comme l’utilisation de pesticides domestiques, ont été rapportés plus fréquemment en milieu rural. Enfin, il a été démontré que la mesure des concentrations volumiques ou ajustées pour la créatinine de biomarqueurs est une approche qui peut entraîner des biais importants (jusqu’à 500% d’erreur et plus) en particulier lors de l’évaluation de l’exposition individuelle. Il est évident que les autorités de santé publique et de santé environnementale employant des biomarqueurs urinaires afin d’évaluer l’exposition aux pyréthrinoïdes et aux pyréthrines (ainsi qu’à d’autres molécules ayant des demi-vies d’élimination similaire)devraient diriger leurs efforts vers la mesure des quantités excrétées pendant une période d’au moins douze heures pour obtenir un portrait le plus valide possible de l’exposition. Il serait aussi souhaitable de mieux documenter la toxicocinétique de ces molécules chez l’humain afin d’établir avec une plus grande confiance la relation entre les quantités excrétées de biomarqueurs et les doses absorbées de ces contaminants. / Pyrethroids and pyrethrins are neurotoxic insecticides also considered to have negative effects on the immune, endocrine and reproductive systems. They are abundantly used for agricultural and horticultural purposes, for pest control and to treat human and animal parasitic infestations (scabies, lice). Consequently, there is in environmental health an interest in evaluating the extent of human exposure to these pesticides. The measurement of pyrethroid and pyrethrin metabolites in urine seems to be the best approach because it provides in theory a global depiction of the exposure. Because of it straightforwardness, it is common practice to use the biomarkers volume-weighted or creatinine-adjusted concentrations in spot urine samples, however the validity of daily doses estimates derived from these units has yet to be assessed. The main goal of this research was to develop, apply and validate a new approach to the measurement and analysis of biomarkers to improve the precision and the reliability of estimates of pyrethroid and pyrethrin exposure at the individual and population levels. The specific objectives were: i) to characterize human exposure to these contaminants in urban and rural populations in Quebec and ii) to assess the validity of this new strategy of measurement and analysis of urinary biomarkers with the biological monitoring strategies generally used to assess individual and population pyrethroid exposure and absorbed doses. Adults and children recruited in the population of the Island of Montreal and of Monteregie collected their urines for at least twelve hours and filled a questionnaire about their potential sources of exposure. The amounts of pyrethroid and pyrethrin metabolites measured in urine (pmol) were adjusted to a fixed twelve hour period and for the body weight. The amounts excreted in the urban area were compared to those from the rural area and individual and population data were compared to those that would have been obtained if volume-weighted or creatinine-adjusted concentrations would have been used. Results show that exposure to these pesticides is very common, with more than 90% of the participants excreting the main pyrethroid and pyrethrin metabolites above the analytical limit of detection. These results also suggest that the diet could be the main contributor to this base level because the other known sources of exposure were rarely reported. In the province of Quebec, the exposure in a rural area seemed slightly more important than in an urban area and some exposure factors, like the use of household pesticides, were reported more frequently in rural area. Finally, it was shown that the measurement of volume-weighted or creatinine-adjusted concentrations is an approach that can lead to an important bias (an error of up to 500% and more) especially for the assessment of individual exposure. It becomes obvious that public health and environmental health authorities using urinary biomarkers to assess pyrethroid and pyrethrin exposure (or other compounds with similar half-lives) should focus their efforts on measuring the amounts excreted during a period of at least twelve hours to obtain the best picture of the exposure. It would also be pertinent to increase the knowledge of the toxicokinetic behaviour of these compounds in humans in order to establish with greater confidence the relation between the excreted amounts and the absorbed doses of these contaminants.

Made available in DSpace on 2015-03-26T13:30:23Z (GMT). No. of bitstreams: 1 texto completo.pdf: 308576 bytes, checksum: 4517ced7e005a18483bc835f89df8a70 (MD5) Previous issue date: 2009-02-17 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) is the most important pest of tomato Lycopersicon esculentum Mill in Brazil. Their larvae consume the leaf mesophyll, attack stems, flowers and fruits of tomato, reducing the productivity of this culture and consequently the profitability of farmers. For reducing this insect attack, farmers use insecticides intensively, and often the efficacy control is not reached as desired. One possible reason for the low efficiency is the resistance development to insecticides in populations of this insect. Therefore, tomato producers need search for detecting and monitoring of the T. absolute populations resistance to insecticides. The objective of this study was identified resistance to insecticides in T. absoluta populations from major regions tomato producing of Brazil, and verified the control failures of this pest. The populations were from Cerrado, Southeast and Northeast of Brazil. Ten insecticides were used, six neurotoxic, three insects growth regulators and one which has the bacterium Bacillus thuringiensis as active ingredient. In populations from Cerrado and Southeast the abamectin, spinosad, deltamethrin, triazophos, indoxacarb and bifenthrin were in ascending order, the insecticides that showed the lowest level of resistance among the products tested. In Northeast s population the same resistance was observed, except for bifenthrin, which was replaced by teflubenzurom. The insecticides diflubenzurom, permethrin, triflumurom and the B. Thuringiensis had larger numbers of resistant populations to insecticides in all regions. The T. absoluta population from Viçosa-MG is the most appropriate to be used as susceptibility pattern in resistance study to insecticides of this pest. In recommended doses by industries, the abamectin, spinosad, deltamethrin and triazophos insecticides had not control failures of the T. absolute populations, while the bifenthrin has control failures of populations from Uberlândia, Paulínea and Camocim de São Felix, the indoxacarb had control failures of populations from Uberlândia, São João da Barra and Viçosa, teflubenzurom had control failures of populations from Goianápolis, São João da Barra, Viçosa and Santa Tereza, diflubenzurom, permethrin, triflumurom and B. thuringiensis had control failures in all the T absolute populations which were tested. The correct management of this pest, through the practices which contribute for increasing of control efficacy, can help prevent, delay or reverse the resistance development of this insect pest to insecticides. / A Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) é a praga mais importante do tomateiro Lycopersicon esculentum Mill no Brasil. As larvas desse inseto-praga consomem o mesófilo foliar, bloqueiam os ponteiros, os botões florais, as flores e frutos do tomateiro, reduzindo a produtividade dessa cultura, comprometendo a lucratividade dos tomaticultores. Com o intuito de minimizar o efeito desse inseto, os agricultores fazem o uso intensivo de inseticidas, que muitas vezes não alcançam a eficiência de controle desejada. Uma das possíveis razões para a baixa eficiência esta o desenvolvimento da resistência a inseticida em populações desse inseto. Dessa forma, a tomaticultura carece de trabalhos que visem à detecção e o monitoramento adequado de populações de T. absoluta resistentes a inseticidas. Assim, o objetivo desse trabalho estudar e identificar a ocorrência de resistência em populações de T. absoluta a inseticidas nas principais regiões produtoras de tomate do Brasil, e verificar falhas no controle desta praga. As populações foram provenientes do Cerrado, Sudeste e Nordeste. Foram utilizados dez inseticidas, sendo seis neurotóxicos, três reguladores de crescimento e um que tem como ingrediente ativo a bactéria Bacillus thuringiensis. Para as macrorregiões do Cerrado e Sudeste, abamectina, espinosade, deltametrina mais triazofós, indoxacarbe e bifentrina foram, em ordem crescente, os inseticidas que apresentaram o menor nível de resistência dentre os produtos testados. Para o Nordeste observou-se o mesmo padrão, com exceção da bifentrina que foi substituída pelo teflubenzurom. Os inseticidas diflubenzurom, permetrina, o triflumurom e o B. thuringiensis apresentaram números maiores de populações resistentes a inseticidas em todas as macrorregiões. A população de Tuta absoluta de Viçosa-MG é a mais adequada a ser utilizada como padrão de suscetibilidade em estudos de resistência a inseticidas. Nas doses recomendadas pelos fabricantes, os inseticidas abamectina, espinosade, deltametrina mais triazofós não apresentaram falhas no controle das populações de T. absoluta, já a bifentrina apresentou falhas no controle das populações de Uberlândia, Paulínea e Camocim de São Felix, o indoxacarbe apresentou falhas no controle das populações de Uberlândia, São João da Barra e Viçosa, o teflubenzurom apresentou falhas no controle das populações de Goianápolis, São João da Barra, Viçosa e Santa Tereza, o diflubenzurom, permetrina, triflumurom e o a base de B. thuringiensis apresentaram falhas no controle de todas as populações de T absoluta testadas. O manejo correto dessa praga através de práticas que contribuam para um controle mais eficiente pode ajuda a prevenir, retardar ou reverter à evolução da resistência deste inseto-praga a inseticidas.

Orientadores: Lea Rodrigues Simioni, Yoko Oshima Franco / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-09T01:04:42Z (GMT). No. of bitstreams: 1 Ferreira_SandroRostelato_M.pdf: 1413633 bytes, checksum: 6030097384697994f16895f8fb1a4c92 (MD5) Previous issue date: 2007 / Resumo: O veneno de Bothrops jararacussu (Bjssu) e sua miotoxina bothropstoxina-I (BthTX-I), induzem neurotoxicidade e miotoxicidade. Como o tratamento com o antiveneno é pouco eficaz contra a miotoxicidade, muitos estudos têm sido realizados utilizando substâncias que neutralizem a atividade miotóxica induzida pelo veneno, entre elas, a heparina. Os objetivos deste trabalho foram: 1) verificar o efeito da heparina sobre a miotoxicidade induzida pelo veneno e toxina, utilizando-se uma baixa concentração de heparina, porém capaz de impedir o bloqueio neuromuscular e, 2) esclarecer o papel protetor da heparina contra Bjssu. Controles foram realizados com antiveneno botrópico (AVB) comercial ou solução nutritiva de Tyrode ou salina. Para avaliar a neurotoxicidade empregou-se técnica miográfica convencional em preparações nervo frênico-diafragma de camundongos (in vitro) e nervo ciático poplíteo externo-tibial anterior de ratos (in vivo); para avaliar a miotoxicidade in vitro empregou-se a técnica histológica (microscopia óptica) e in vivo a dosagem bioquímica da creatinoquinase (CK); para avaliar o papel protetor da heparina empregou-se a protamina, um antagonista farmacológico. Os resultados obtidos in vitro mostraram que a resposta contrátil de 12 ± 2% (n=6) frente à incubação com Bjssu (40 µg/mL) por 120 min foi aumentada para 79,6 ± 5,9% (n=6) quando pré-incubado com heparina (5 UI/mL) e 68,3 ± 6,2% (n=6) quando pré-incubado com AVB (120 µL/mL); na mesma situação a BthTX-I (2,9 µM) passou de 5 ± 1,3% (n=8) para 78,8 ± 6,8% (n=8) com heparina e 62,3 ± 6,1% (n=6) com AVB. A média da quantificação do dano morfológico (leitura de três diferentes observadores) mostrou que o veneno provocou lesões de 27% e a toxina de 40%, que passaram para níveis de 5% e 9%, respectivamente, quando tratadas com heparina e 11% e 3% quando com AVB. Os pré-tratamentos não apresentaram diferença significativa em relação ao controle Tyrode. Os resultados in vivo (em ratos) mostraram que as mesmas concentrações de veneno e toxina utilizadas nos ensaios in vitro não provocaram alterações na resposta contrátil; contudo, quando injetados no músculo gastrocnêmio de camundongos, apresentaram níveis plasmáticos de CK (U/L) de: 1454 ± 185 (Bjssu, n=6) diminuindo (P<0,05) para 236 ± 40 (com heparina, n=6) e 47 ± 5 (com AVB, n=6); 1531 ± 166 (BthTX-I, n=5) diminuindo (P<0,05) para 900 ± 149 (com heparina, n=5) e 935 ±135 (com AVB, n=5). A adição de protamina (0,8 UI/mL) aos 15 minutos de incubação da mistura heparina + veneno causou o bloqueio neuromuscular característico do veneno em preparações in vitro. Conclui-se que a heparina é mais eficaz (mas pode ser totalmente bloqueada pela protamina) que o AVB quanto a sua capacidade de impedir a neurotoxicidade in vitro causada por Bjssu e BthTX-I, e que nas mesmas concentrações a heparina demonstrou nenhuma neurotoxicidade in vivo (ratos) e que ela é tão eficiente quanto o AVB na miotoxicidade in vitro, mas menos eficaz in vivo em relação ao veneno bruto / Abstract: Bothrops jararacussu venom (Bjssu) and its myotoxin bothropstoxin-I (BthTX-I) induce neurotoxicity and myotoxicity. Since the treatment with the antivenom is weakly efficient against the myotoxicity, many reports concentrate on studies utilizing substances that neutralize the myotixicity activity induced by the venom, including heparin. The objectives of this work were: 1) to examine the effect of heparin on the myotoxicity induced by venom and toxin, using a low heparin concentration, capable to prevent the neuromuscular blockade and, 2) to examine the protective role of heparin against Bjssu. Control experiments were performed with commercial bothropic antivenom (CBA), Tyrode solution or saline. To examine the neurotoxicity, a conventional myoghraphic technique was used in studies with mouse phrenic nerve-diaphragm preparations (in vitro) and rat popliteal external nerve/muscle anterior tibialis (in vivo). Histological technique (light microscopy) and biochemical measurement of creatine kinase (CK) were used to examine the myotoxicity in vitro e in vivo, respectively. Protamine (a pharmaceutical antagonist) was used to evaluate the protective role of heparin. The results in vitro showed that the twitch-tension of 12 ± 2% in the presence of Bjssu (40 µg/mL; n=6) after 120 min was increased to 79.6 ± 5.9% when preincubated with heparin (5 UI/ml; n=6) and 68.3 ± 6.2% when preincubated with CBA (120 µL/mL; n=6). Similarly, the BthTX-I (2.9 µM) - induced responses amounted to 5 ± 1.3% (n=8) and 78.8 ± 6.8% with heparin (n=8) and 62.3 ± 6.1% with CBA (n=6). The quantification of morphological changes showed that the venom induced a damage of 27% and the toxin of 40%, which were reduced to 5% and 9%, when treated with heparin and 11% and 3% with CBA, respectively. The pre-treatment did not cause significant differences compared to Tyrode solution. The results in vivo showed that the same concentrations of venom and toxin utilized in in vitro assays did not induce alteration in twitch-tension. However, when injected in mouse gastrocnemius muscle, plasma levels of CK (U/l) of 1454 ± 185 (in the presence of Bjssu, n=6) were decreased to 236 ± 40 (heparin, n=6) and 47 ± 5 (CBA, n=6). Similarly, a value of 1531 ± 166 in the presence of BthTX-I (n=5) was decreased to 900 ± 149 (heparin, n=5) and 935 ±135 (CBA, n=5). The addition of protamine (0.8 UI/ml) at 15 min incubation of the mixture heparin+venom, induced a neuromuscular blockade similar to the venom in in vitro preparations. We conclude that heparin is more efficient (although totally antagonized by protamine) than CBA with respect to the in vitro neurotoxicity induced by Bjssu and BthTX-I, which did not cause myotoxicity in vivo (rats). Heparin is as efficient as CBA in myotoxicity in vitro, but less efficient in vivo compared to the crude venom / Mestrado / Mestre em Farmacologia

Dehydroepiandrosterone, a C-19 steroid, is found endogenously with the highest circulating serum levels. It is converted to important steroids such as the sex hormones oestrogen and testosterone. DHEA has come under the spotlight as a purported “fountain of youth” due to its well-characterised age-related decline. The supplementation of DHEA in both the elderly and those with a pathophysiological deficiency has been shown to be of benefit, particularly with regard to wellbeing and depression. The role of DHEA in the periphery has not been elucidated beyond its role as a precursor hormone in sex steroid biosynthesis, though it has been established as a neuroactive neurosteroid, capable of exerting neuroprotective effects in the brain. Since the importance of free radicals in aging and neurodegeneration is well established, investigations were conducted on the ability of DHEA to inhibit free radical generation or scavenge existing free radicals. DHEA was able to significantly inhibit quinolinic acid-induced lipid peroxidation, a measure of membrane damage, over a range of concentrations, although the reduction did not appear to be dose-dependent. This was observed in both in vitro and in vivo studies. Thus, the ability of a compound to reduce the degree of lipid peroxidation may indicate its value as a neuroprotectant. However, DHEA did not significantly reduce cyanide induced generation of the superoxide free radical, suggesting that DHEA is not an effective free radical scavenger of the superoxide anion and that the reduction in lipid peroxidation does not occur through a scavenging mechanism. Apoptosis is a physiological process which is necessary for development and homeostasis. However, this form of programmed cell death can be initiated through various mechanisms and too much apoptotic cell death results in deleterious effects in the body. DHEA was shown not to induce apoptosis. Even the lowest concentration of DHEA investigated in this thesis shows a remarkable decrease in the degree of apoptosis caused by intrahippocampal chemical insult by the neurotoxin quinolinic acid. Cresyl violet was used to visualise tissue for histological examination which revealed that DHEA is able to preserve the normal healthy morphology of hippocampal cells which have been exposed to quinolinic acid. Cells maintained their integrity and showed little evidence of swelling associated with necrosis. Organ culture studies were performed by assessing the impact of DHEA on several pineal metabolites. The study revealed that DHEA exerted an effect on the metabolism of indoleamines in the pineal gland. Melatonin, the chief pineal hormone, did not appear to be affected while the concentrations of N-acetylserotonin, serotonin and methoxytryptamine showed significant alterations. Thus, the neuroprotective mechanism of DHEA does not appear to be mediated by an increase in the presence of melatonin. The biological importance of metal ions in neurodegeneration is also well established and thus the potential interaction between DHEA and metal ions was considered as a mechanism of action. Spectroscopic and electrochemical analyses were performed to determine whether DHEA is able to interact with metal ions as a ligand. These reveal that DHEA does not form a strong bond with the metals investigated, namely copper (II) and iron (III), but that a weak interaction is evident. These investigations were conducted in a rodent model, which has neither large amounts of endogenous DHEA, nor the enzymatic infrastructure present in humans. Thus, the theory that DHEA exerts its effects through downstream metabolic products is unlikely. However, these investigations reveal that there is merit in the statement that DHEA itself is a neuroprotective molecule, and confirm that the further investigation of DHEA is an advisable strategy in the war against neurodegeneration and aging.

La presente tesis doctoral titulada “Materiales de sílice funcionales para la liberación controlada, detección y eliminación de moléculas de interés” se centra en el diseño y desarrollo de materiales híbridos orgánico-inorgánicos mediante la aplicación de los conceptos de Química Supramolecular. Durante el desarrollo de la presente tesis doctoral se han preparado y caracterizado diferentes materiales de base silícea para distintas aplicaciones. La primera parte de la tesis se centra en el desarrollo de materiales de base silícea capaces de variar su comportamiento fluorescente en función de la presencia o ausencia de un cierto analito en el medio. Estos materiales emplean como soporte nanopartículas de sílice que se funcionalizan superficialmente con dos unidades diferentes, una coordinante y una indicadora (un fluoróforo). La interacción del analito de interés (en nuestro caso aniones) con la unidad coordinante modificará las propiedades emisivas del fluoróforo. Así, se han preparado dos materiales en los cuales el grupo fluorescente es rodamina mientras que el grupo coordinante es un imidazolato o una sal de guanidinio respectivamente. Una vez caracterizados ambos materiales se estudió su comportamiento frente a diferentes especies aniónicas a diferentes concentraciones resultando selectivos a la presencia de benzoato (el material funcionalizado con imidazolatos), dihidrógeno fosfato e hidrógeno sulfato (el material funcionalizado con sales de guanidinio). El tercer capítulo de la tesis se centra en la aplicación de materiales híbridos orgánico-inorgánicos para la detección y eliminación de especies altamente tóxicas como son los agentes neurotóxicos. Estos son compuestos organofosforados capaces de causar graves lesiones en el sistema nervioso central. En una primera aproximación se emplea el concepto de puerta molecular para la detección de agentes neurotóxicos. Para ello, se utiliza como soporte inorgánico un material mesoporoso de sílice (MCM-41) cuyos poros se cargan con un colorante que actúa de indicador mientras que la superficie externa del mismo se funcionaliza con una molécula capaz de reaccionar con dichos agentes neurotóxicos. Dicha molécula es capaz de interaccionar entre sí (mediante enlaces de hidrógeno) formando una red que mantiene bloqueada la salida de los poros. En presencia de DCP (dietilclorofosfato, un simulante de agente neurotóxico), y después de que este reaccione con dicha molécula, se produce una reorganización espacial que permite la liberación del colorante. De este modo, la presencia de los agentes neurotóxicos está señalizada mediante un cambio de color. En una segunda aproximación se aborda el uso de soportes inorgánicos de tipo MCM-41 como materiales para la eliminación de agentes neurotóxicos. Para ello se modificaron químicamente las superficies de estos materiales silíceos mediante tratamiento con distintas bases. Como consecuencia de este tratamiento básico los silanoles de la superficie se desprotonan dando lugar a los correspondientes silanolatos (nucleófilos fuertes). Estos silanolatos son capaces de reaccionar con los agentes neurotóxicos descomponiéndolos y favoreciendo su eliminación de un medio contaminado. Por último, se estudia la aplicación de materiales híbridos orgánico-inorgánicos funcionalizados con puertas moleculares en aplicaciones de liberación controlada, concretamente, en liberación controlada intracelular de fármacos de interés. El material híbrido consta de un soporte de sílice mesoporosa cuyos poros se cargan con un compuesto citotóxico (camptotecina) y su superficie externa se funcionaliza con una gluconamida. La presencia de una monocapa densa de gluconamidas por el exterior del material inhibe la liberación del compuesto citotóxico. Al añadir enzimas con capacidad para hidrolizar enlaces amida (amidasa y pronasa) se produce la liberación de la camptotecina. El correcto funcionamiento del material se comprobó in vitro e in vivo (en células HeLa). / Candel Busquets, I. (2014). Functional silica materials for controlled release, sensing and elimination of target molecules [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/39101 / TESIS

Methylenedioxymethamphetamine (MDMA) popularly known as "Ecstasy" continues to gain popularity as a recreational drug that has been shown to increase serotonin and dopamine levels. The present study has demonstrated that repeated exposure to MDMA produces long-term damage to serotonergic and dopaminergic neurons in various regions of the rat brain.

「風險決策」行為非常普遍的存在於吾人之日常生活中，而選項所帶來的風險和獎勵是吾人進行決策時的重要考量因素。風險選擇的適當與否，對於個體的生存扮演著相當重要的角色。在以往的文獻中，對於決策的行為歷程已有所關注及探討，但對於風險選擇行為的神經生理機制迄今未明。本研究藉由大白鼠於T字迷津中，選擇確定之低酬賞或高不確定性之高酬賞的行為表現，進行風險選擇行為的探討。本研究中以兩項主要實驗，探討風險選擇行為之神經行為機制。實驗1a中，確定之低酬賞端固定呈現1顆食物粒，而高不確定性之高酬賞端則同時操弄酬賞物機率(50%、25%及12.5%)以及酬賞物的量(2、4及8顆)，以系統性地檢驗期望值(0.5、1和2)於此風險選擇行為中扮演的角色。行為結果顯示當風險較低時，大白鼠會選擇高不確定性之高酬賞端；而風險較高時，則轉為選擇確定之低酬賞端。實驗1b中，系統性地施打不同劑量之安非他命，探討多巴胺系統在此風險選擇行為中之機制。實驗結果顯示施打安非他命後，大白鼠表現出相對地追求風險之行為，亦即選擇高不確定之高酬賞端之比例顯著高於控制組。實驗2中，藉由毀除大腦特定部位(依核、背外側之紋狀體、眶前額皮質、內側之前額皮質)，檢驗風險選擇行為之神經基礎。毀除後之結果顯示，僅有依核受到毀除之大白鼠表現出相對地趨避風險之選擇行為。綜合以上結果，本研究建立之風險選擇行為與多巴胺有關，而依核在此行為歷程中扮演重要的調節角色。 / Many decisions people make every day involve uncertainty where both risks and rewards associated with each option need to be considered. Behavioral performance associated to risk-based choice appears wildly over the lifespan, and the fitness of risky choice behavior plays an important role in individual survival. Despite a growing body of research has focused to investigate the neurobiology of decision making, little is known about the neurobehavioral mechanisms of risky choice behavior. Based on a pilot work, this study used a T-maze to study decision under a probability-based risk in the rat. The subject was assessed on making choice to obtain either a large reward associated with risk of non-reward “empty” or a small reward ensured for every entry. Two experiments were conducted in this project to investigate neurobehavioral mechanisms of probabilistic risky choice behavior. In Experiment 1a, probabilistic risky choice behavior was systemically assessed under three expected values (0.5, 1.0, and 2.0) by manipulating the probabilities of reward presence (50%, 25%, and 12.5%) and the reward magnitude (2, 4, or 8 pellets) in the probabilistic high reward (PHR) arm. Behavioral data showed that the subject chose the probabilistic high reward in a lower risk condition but would shift to the choice of certain low reward (CLR) as the risk is increased. In Experiment 1b, the dose effects of amphetamine on this probabilistic risky choice task was tested to verify whether the dopaminergic mechanism was involved. Amphetamine, presumably activating brain dopamine systems, produced a relatively risk-seeking effect on the present behavioral task. In Experiment 2, the excitoneurotoxic lesion was conducted in the nucleus accumbens, the dorsolateral striatum, the orbitofrontal cortex, and the medial prefrontal cortex to examine the neural substrates for this probabilistic risky choice behavior. The results showed that the lesion of the nucleus accumbens significantly produced a relatively risk-averse effect on the present behavioral task, as compared to the lesions made on the other three brain areas. In conclusion, the probabilistic risky choice behavior established in the present study is dopamine dependent. And, the nucleus accumbens plays a major role of mediating this behavioral processing.