Through the two influential Protestant reformers Martin Luther´s and JohnCalvin's readings, I intend to explore the view of disability and sin within theChristian tradition. One of the biblical stories that has been read andinterpreted in the light of disability issues is the well-known episode in the NewTestament, which is often called "the paralytic". In both the Old and NewTestaments, there are several places that may indicate a connection betweendisability and sin, and through thoughts of human sin as a common thread Ipresent how the two Bible readers read the pericopes.

Although there are no authenticated records of human illness due to PSP in New Zealand, nationwide phytoplankton and shellfish toxicity monitoring programmes have revealed that the incidence of PSP contamination and the occurrence of the toxic Alexandrium species are more common than previously realised (Mackenzie et al., 2004). A full understanding of the mechanism of uptake, accumulation and toxin dynamics of bivalves feeding on toxic algae is fundamental for improving future regulations in the shellfish toxicity monitoring program across the country. This thesis examines the effects of toxic dinoflagellates and PSP toxins on the physiology and behaviour of bivalve molluscs. This focus arose because these aspects have not been widely studied before in New Zealand. The basic hypothesis tested was that bivalve molluscs differ in their ability to metabolise PSP toxins produced by Alexandrium tamarense and are able to transform toxins and may have special mechanisms to avoid toxin uptake. To test this hypothesis, different physiological/behavioural experiments and quantification of PSP toxins in bivalves tissues were carried out on mussels (Perna canaliculus), clams (Paphies donacina and Dosinia anus), scallops (Pecten novaezelandiae) and oysters (Ostrea chilensis) from the South Island of New Zealand. Measurements of clearance rate were used to test the sensitivity of the bivalves to PSP toxins. Other studies that involved intoxication and detoxification periods were carried out on three species of bivalves (P. canaliculus, P. donacina, P. novaezelandiae), using physiological responses (clearance and excretion rate) and analysis of PSP toxins in the tissues over these periods. Complementary experiments that investigated other responses in bivalves fed with the toxic cells were also carried out. These included byssus production, and the presence of toxic cells in the faeces of mussels, the siphon activity and burrowing depth in clams and the oxygen consumption in scallops. The most resistant species to PSP toxins were the mussel, Perna canaliculus and the clam, Dosinia anus. Both species fed actively on toxic dinoflagellates and accumulated toxins. The intoxication and detoxication rate of the mussel was faster than the other species of bivalve studied (P. donacina and P. novaezelandiae) which confirm mussels as a good sentinel species for early warning of toxic algal blooms. The clearance rate of the clam, Paphies donacina decreased when fed on Alexandrium species but the effect of the PSP toxins on this physiological response was not confirmed. Over the detoxification period of 8 days, this clam did not detoxify which suggests that its ability to retain high level of toxins for an extensive period may be critical for public health management. The scallop, Pecten novaezelandiae was clearly the most sensitive species to the PSP toxins and the clearance rate was significantly lower in the presence of the toxic dinoflagellate A. tamarense. Although the clearance rate was low, the scallops still fed on the toxic dinoflagellate and accumulated PSP toxins in the tissues. The scallops detoxified slowly which would affect the market for this bivalve in the presence of a toxic algal bloom. This bivalve would retain PSP toxins for longer period of time than other species such as mussels. The oyster, Ostrea chilensis, had erratic clearance rate and did not respond clearly to any of the variables tested over the time. Oysters accumulated more toxins than the sensitive species, but they had been exposed to two more days of feeding with A. tamarense; therefore this species may actually have a similar intoxication responses to P. novaezalandiae and P. donacina. The results from this thesis suggest further directions for the aquaculture sector and ongoing research in this field, which in future will lead to a better selection of suitable species for culture as well as species for monitoring of PSP toxins. In the future, research that integrates field and controlled laboratory studies will expand to other species of interest and a more complete record will in time be available in order to manage more efficiently the negative effects that harmful algal blooms may have in New Zealand.

The duodenal epithelium is regularly exposed to HCl, digestive enzymes, bacteria and toxins, and sometimes also to ethanol and drugs. The imbalance of aggressive factors in the intestinal lumen and mucosal barrier function increases the risk of tissue injury and inflammation. The key components of the duodenal barrier function include mucosal permeability, bicarbonate transport and the secretion or absorption of fluids. This thesis aims to elucidate the role of melatonin, neuropeptide S (NPS) and short chain fatty acids (SCFAs) in the regulation of intestinal mucosal barrier function and motility in the anesthetized rat in vivo and in tissues of human origin in vitro. Melatonin was found to reduce ethanol-induced increases in paracellular permeability and motility by a neural pathway within the enteric nervous system involving nicotinic receptors. In response to luminal exposure of ethanol, signs of mild mucosal edema and beginning of desquamation were observed in a few villi only, an effect that was not influenced by melatonin. Melatonin did not modify increases in paracellular permeability in response to luminal acid. NPS decreased basal and ethanol-induced increases in duodenal motility as well as bethanechol stimulated colonic motility in a dose-dependent manner. Furthermore, NPS was shown to inhibit basal duodenal bicarbonate secretion, stimulate mucosal fluid absorption and increase mucosal paracellular permeability. In response to luminal exposure of acid, NPS increased bicarbonate secretion and mucosal paracellular permeability. All effects induced by the administration of NPS were dependent on nitrergic pathways. In rats, administration of NPS increased the tissue protein levels of the inflammatory biomarkers IL-1β and CXCL1. Immunohistochemistry showed that NPS was localized at myenteric nerve cell bodies and fibers, while NPSR1 and nNOS were only confined to the myenteric nerve cell bodies. Perfusing the duodenal segment with the SCFAs acetate or propionate reduced the duodenal mucosal paracellular permeability, decreased transepithelial net fluid secretion and increased bicarbonate secretion. An i.v. infusion of SCFAs reduces mucosal paracellular permeability without any effects on mucosal net fluid flux. However, it significantly decreased bicarbonate secretion. Luminal SCFAs changed the duodenal motility pattern from fasting to feeding motility while i.v. SCFAs was without effect on motility. The systemic administration of glucagon-like peptide-2 (GLP-2) induced increases in mucosal bicarbonate secretion and fluid absorption. An i.v. GLP-2 infusion during a luminal perfusion of SCFAs significantly reduced the duodenal motility. In conclusion, the results in the present thesis show that melatonin, NPS and SCFAs influence the neurohumoral regulation of intestinal mucosal barrier function and motility. Aberrant signaling in response to melatonin, NPS and to luminal fatty acids might be involved in the symptom or the onset of disease related to intestinal dysfunction in humans. / <p>Research funders and strategic development areas:</p><p>- Bengt Ihre Foundation (grant SLS-177521)</p><p>- Socialstyrelsen(grant SLS-176671)</p><p>- Erik, Karin, and Gösta Selanders Foundation</p><p>- Emil and Ragna Börjesson Foundation</p><p>- Uppsala University </p><p>- Ministry of Education of Malaysia</p><p>- Universiti Malaysia Sabah, Malaysia</p>

Les dernières décennies ont été marquées par l’intensification et l’expansion des efflorescences de micro-algues toxiques (HAB). Connues pour perturber les écosystèmes côtiers et pour leur toxicité sur les organismes marins, les HAB sont suspectées d’être à l’origine de défauts de recrutement de bivalves. Cette thèse avait pour objectif d’étudier les conséquences des efflorescences du dinoflagellé toxique Alexandrium minutum, producteur de toxines paralysantes (PST) et des composés bioactifs extracellulaires (BEC), sur la reproduction, le développement et le recrutement de l’huître Crassostrea gigas, une espèce à l’importance économique majeure. Les gamètes libres et les jeunes stades de développement se révèlent être les plus sensibles, en particulier aux BEC produits par A. minutum qui inhibent la fécondation et l’embryogenèse. A. minutum modifie le comportement des larves véligères, provoque une diminution de leur filtration, de leur croissance et du taux de fixation. Une exposition des adultes, pendant la gamétogenèse, affecte le développement des descendants, traduisant des altérations du contenu gamétique et/ou un transfert vertical des PST. Les modalités d’action des PST et des BEC devront être précisées. Nos expérimentations, réalisées à des concentrations de micro-algues rencontrées dans l’environnement, suggèrent que des efflorescences récurrentes d’A. minutum lors des périodes de reproduction et de développement larvaire pourraient, sur le long terme, affecter la structure des populations naturelles et cultivées de C. gigas. / Recent decades have witnessed the intensification and spread of harmful algal blooms (HAB). HAB are known to disrupt coastal ecosystems and to be toxic for marine organisms. These phenomena are also suspected to be responsible for recruitment failures of bivalves. The aim of this PhD was to study the consequences of blooms of toxic dinoflagellate Alexandrium minutum on the reproduction, development and recruitment of the oyster Crassostrea gigas, a species of major economic importance. A. minutum is known to produce paralytic shellfish toxins (PST) and bioactive extracellular compounds (BEC). Gametes and early life stages were the most sensitive, particularly to the bioactive extracellular compounds (BEC) produced by A. minutum, which inhibited fertilization and embryogenesis. A. minutum modified the behaviour of veliger larvae, decreased their filtration, growth and settlement. Exposure of adult oysters during gametogenesis affected the development of offspring, reflecting alterations in gamete content and/or vertical transfer of PST. Mode of action of PST and BEC are to further investigate. These oyster exposures, conducted at environmentally relevant concentrations of microalgae, suggest that recurrent blooms of A. minutum during oyster spawning and larval development could have long-term consequences on the structure of wild and cultured populations of C. gigas.

As toxinas do envenenamento paralisante por moluscos (Paralytic Shellfish Poisoning – PSP) são compostos naturais bioativos conhecidos devido ao consumo acidental de frutos do mar contaminados. Estas moléculas, das quais a mais potente é a saxitoxina (STX), são uma classe de alcalóides neurotóxicos que possuem diferentes análogos e diferentes toxicidades, e são produzidas por algumas cianobactérias e algumas espécies de dinoflagelados do gênero Alexandrium, Gymnodinium e Pyrodinium. As toxinas paralisantes são neurotoxinas solúveis em água que agem sobre células nervosas e musculares através do bloqueio dos canais de sódio dependentes de voltagem, desta maneira, impedindo a condução do sinal no neurônio o que leva a uma paralisia muscular. Em casos graves, pode ocorrer morte por insuficiência respiratória. O envenenamento diarréico por moluscos (Diarrhetic Shelfish Poisoning – DSP) é caracterizado por problemas gastrointestinais com sintomas como diarréia, náusea, vômito, dor de cabeça, calafrios e dores abdominais. DSP é conseqüência do consumo de mariscos contaminados que ingeriram dinoflagelados do gênero Dynophysis e Prorocentrun através de sua alimentação por filtração da água. Contaminação de frutos do mar por toxinas PSP ou DSP coloca-se como sério problema para a indústria pesqueira e para a saúde pública. Neste estudo, estabeleceu-se um programa de monitoração para mexilhões (Perna perna) e para peixes (Sardinella brasiliensis, Anchoviella lepidentostole e Brevoortia aurea) coletados em peixarias e entrepostos de pesca no municípios de Caraguatatuba e São Sebastião, São Paulo. Os extratos para PSP foram preparados de duas maneiras: de acordo com a AOAC (Association of Official Analytical Chemists), através do aquecimento por 5 min de uma mistura de 100 g de tecidos homogeneizados com ácido acético 0,1 N; ou a partir da concentração de extratos etanólicos de músculo + pele dos peixes. Os bioensaios com camundongos para PSP consistem na injeção intraperitonial de 1 mL do extrato ácido em cada um dos três camundongos (~ 20 g). O animal é observado quanto aos sintomas clássicos de PSP e o tempo de morte é anotado e então a toxicidade é determinada (em mouse units, MU) pela tabela de Sommer. Para as toxinas causadoras de DSP, os extratos foram preparados pela extração com acetona do homogeneizado das glândulas digestivas, e a determinação da presença destas toxinas é feita através da injeção intraperitonial em camundongos. Nos bioensaios com os extratos preparados segundo o método da AOAC, não houve casos positivos. Para o bioensaio realizado com extratos etanólicos obtiveram-se resultados positivos para 77,8% dos extratos testados. A média de MU de todas as amostras, neste caso, foi de 0,147 MU/g. Nos bioensaios para DSP, três amostras resultaram em sinais que evidenciam a presença destas toxinas, pois os camundongos injetados apresentaram quadro diarréico. Os extratos etanólicos, com positividade para as toxinas de PSP, foram fracionados usando-se colunas Sep-Pak C18. A primeira eluição, com ácido acético 0,1 M, foi analisada usando-se o método de préderivatização e cromatografia líquida de alta eficiência com detecção de fluorescência. As analises em CLAE indicaram a presença de compostos semelhantes às toxinas paralisantes de PSP, confirmando os bioensaios. Portanto, pela primeira vez no Brasil demonstrou-se que as espécies S. brasiliensis, A. lepidentostole e B. aurea são portadoras de toxinas paralisantes, semelhantes às PSP, em pequenas concentrações e que um programa de monitoração é necessário em nosso país para verificação da presença dessas toxinas em organismos que são usados como alimento pela população. / The Paralytic Shellfish Poisoning (PSP) toxins are well-known natural bioactive compounds due to their accidental consumption in contaminated seafood. These molecules, of which the most potent representative is saxitoxin (STX), are a class of neurotoxic alkaloids, having different isoforms and varied toxicities, that are produced by some cyanobacteria and some species of dinoflagellates from the genus Alexandrium, Gymnodinium and Pyrodinium. PSP toxins are water-soluble neurotoxins that act on nerve and muscle cells by blocking sodium channels voltage-dependent, thus preventing the conductance of neuron signal leading to muscular paralysis. In severe cases, death may result due to respiratory failure. Diarrhetic Shellfish Poisoning (DSP) is a gastrointestinal illness with symptoms such as diarrhea, nausea, vomiting, headache, chills and moderate to severe abdominal pain. DSP is usually a consequence of consuming contaminated shellfish that have ingested dinoflagellates of the genera Dinophysis and Prorocentrun through their filter feeding activities. Contamination of seafood by PSP and DSP toxins has posed serious problems to the fisheries industry as well to public health. In this study, was stabilized a monitoring program to shellfish (Perna perna) and finfish (Sardinella brasiliensis, Anchoviella lepidentostole and Brevoortia aurea) collected in fish markets in Caraguatatuba and São Sebastião cities, São Paulo state. The extracts for PSP were prepared by two ways: according to AOAC (Association of Official Analytical Chemists), through the heating for 5 min of blend of 100 g of well mixed sample with 0.1 N HCl; or through of the concentration of ethanolic extracts from finfish’s muscle + skin. The PSP mouse bioassay for PSP toxins involves intraperitonial injection (i.p.) of 1 mL of the acid extract into each of three mice (~ 20 g). The mice were observed for classical PSP symptoms and the time to mouse death was recorded and the toxicity was determinate (in mouse units, MU) from the Sommer’s table. To DSP toxins, the extracts was prepared trough the extraction of digestive glands with acetone, and i.p injection in mice was used to determine the presence of theses toxins. In the mouse bioassay for the extracts prepared by AOAC method no positive results was obtained. For the mouse bioassay with ehtanolic extracts was obtained positive results to 77.8 % of the tested extracts. The media of MU of all samples, in this case, was 0,147 MU/g. To the mouse bioassay for the DSP toxins, three samples gives evidence of presence of the diarrhetic toxins, because the mice showed signal like diarrhea. The ethanolic extracts, that was positive to the PSP toxins, was fractionated by a Sep-Pak C18 cartridge. The first elution, with 0.1 M acetic acid, was analyzed by using prechromatographic oxidation and liquid chromatography with fluorescence detection. The HPLC analysis indicated the presence of the PSP toxins, confirming the bioassays. Therefore, in the first time in Brazil was demonstrated that the species S. brasiliensis, A. lepidentostole and B. aurea are carriers of toxins like PSP in little concentrations and that a monitoring program is necessary in our country to verify the presence of these toxins in organisms that are used as food by the population.

Made available in DSpace on 2014-12-17T15:07:05Z (GMT). No. of bitstreams: 1 JoaquimADO_DISSERT.pdf: 965951 bytes, checksum: e69351da4d366a13722fe0777dc7cdd6 (MD5) Previous issue date: 2013-06-07 / This work has the general proposal of studying the composition of the trevisanian fictional universe, the aesthetic expression engineered to formulate this universe, and last, the representations of reality that emanates from this aesthetic-narrative construction. It seemed to us, however, that it would be impossible to cover, even superficially, so vast and voluminous set of texts as the trevisanian. Therefore, so that we could accomplish this task, and still do it in a way that was, at the same time, plausible and thorough, it was necessary an objective cut within that library. Then we selected a book, representative of the whole trevisanian work, which served as the object of study: Cemit?rio de Elefantes, 1964, one of the first books of short stories by this author. Within this one, we focused even more our sight, then moving towards what we call a reality forked in paralytic world and world in violence . Supported by the theories and methodologies of Auerbach (2011), Candido (2002, 2006), Adorno (2003), Gennete (1995), Friedman (2002), and still moved by the spirit of Russian Formalism (1973), we built our criticism. Our approach was based then in three steps: first we are dedicated to comment the general context of the trevisanian work, supported strongly by reading two of the biggest critics of his literature, Miguel Sanches Neto and Berta Waldman, secondly, we turn us to comment on the detailed formal construction of language and narrative elements of the trevisanian prose; finally we raised our criticism to the level of reading representations of reality, and then we started to comment the construction of the trevisanian fictional universe, focusing in splitting this universe in paralytic world and world in violence / O presente trabalho tem como proposta geral estudar a composi??o do universo ficcional trevisaniano, a express?o est?tica engendrada para formular esse universo, e, em ?ltima inst?ncia, as representa??es da realidade que emanam dessa constru??o est?tico-narrativa. Pareceu-nos, no entanto, que seria imposs?vel dar conta, mesmo que superficialmente, de t?o vasto e volumoso conjunto de textos como o ? o trevisaniano. Portanto, para que pud?ssemos realizar tal tarefa, e ainda faz?-lo de um modo que fosse, a um s? tempo, plaus?vel e mais aprofundado, fez-se necess?rio um recorte objetivo dentro dessa biblioteca. Selecionamos ent?o um livro, representativo de toda a obra trevisaniana, que nos serviu como objeto de estudo: Cemit?rio de elefantes, de 1964, um dos primeiros livros de contos desse autor. Dentro desse, ainda focalizamos mais o nosso olhar, voltando-nos ent?o para o que denominamos de uma realidade bifurcada em mundo paral?tico e mundo em viol?ncia . Amparados pelas teorias e metodologias de Auerbach (2011), Candido (2002, 2006), Adorno (2003), Gennete (1995), Friedman (2002), e ainda movidos pelo esp?rito do Formalismo russo (1973), constru?mos nossa cr?tica. Nossa abordagem fundamentou-se, ent?o, em tr?s passos: primeiramente nos dedicamos a comentar o contexto geral da obra trevisaniana, suportados fortemente pela leitura de dois dos maiores cr?ticos da literatura desse autor, Miguel Sanches Neto e Berta Waldman; em segundo lugar, voltamo-nos ao coment?rio minucioso sobre a constru??o formal da linguagem e dos elementos narrativos da prosa trevisaniana; por fim, al?amos nossa cr?tica ao n?vel da leitura das representa??es da realidade, e passamos a comentar a constru??o do universo ficcional trevisaniano, focando na biparti??o desse universo em mundo paral?tico e mundo em viol?ncia

As toxinas do envenenamento paralisante por moluscos (Paralytic Shellfish Poisoning – PSP) são compostos naturais bioativos conhecidos devido ao consumo acidental de frutos do mar contaminados. Estas moléculas, das quais a mais potente é a saxitoxina (STX), são uma classe de alcalóides neurotóxicos que possuem diferentes análogos e diferentes toxicidades, e são produzidas por algumas cianobactérias e algumas espécies de dinoflagelados do gênero Alexandrium, Gymnodinium e Pyrodinium. As toxinas paralisantes são neurotoxinas solúveis em água que agem sobre células nervosas e musculares através do bloqueio dos canais de sódio dependentes de voltagem, desta maneira, impedindo a condução do sinal no neurônio o que leva a uma paralisia muscular. Em casos graves, pode ocorrer morte por insuficiência respiratória. O envenenamento diarréico por moluscos (Diarrhetic Shelfish Poisoning – DSP) é caracterizado por problemas gastrointestinais com sintomas como diarréia, náusea, vômito, dor de cabeça, calafrios e dores abdominais. DSP é conseqüência do consumo de mariscos contaminados que ingeriram dinoflagelados do gênero Dynophysis e Prorocentrun através de sua alimentação por filtração da água. Contaminação de frutos do mar por toxinas PSP ou DSP coloca-se como sério problema para a indústria pesqueira e para a saúde pública. Neste estudo, estabeleceu-se um programa de monitoração para mexilhões (Perna perna) e para peixes (Sardinella brasiliensis, Anchoviella lepidentostole e Brevoortia aurea) coletados em peixarias e entrepostos de pesca no municípios de Caraguatatuba e São Sebastião, São Paulo. Os extratos para PSP foram preparados de duas maneiras: de acordo com a AOAC (Association of Official Analytical Chemists), através do aquecimento por 5 min de uma mistura de 100 g de tecidos homogeneizados com ácido acético 0,1 N; ou a partir da concentração de extratos etanólicos de músculo + pele dos peixes. Os bioensaios com camundongos para PSP consistem na injeção intraperitonial de 1 mL do extrato ácido em cada um dos três camundongos (~ 20 g). O animal é observado quanto aos sintomas clássicos de PSP e o tempo de morte é anotado e então a toxicidade é determinada (em mouse units, MU) pela tabela de Sommer. Para as toxinas causadoras de DSP, os extratos foram preparados pela extração com acetona do homogeneizado das glândulas digestivas, e a determinação da presença destas toxinas é feita através da injeção intraperitonial em camundongos. Nos bioensaios com os extratos preparados segundo o método da AOAC, não houve casos positivos. Para o bioensaio realizado com extratos etanólicos obtiveram-se resultados positivos para 77,8% dos extratos testados. A média de MU de todas as amostras, neste caso, foi de 0,147 MU/g. Nos bioensaios para DSP, três amostras resultaram em sinais que evidenciam a presença destas toxinas, pois os camundongos injetados apresentaram quadro diarréico. Os extratos etanólicos, com positividade para as toxinas de PSP, foram fracionados usando-se colunas Sep-Pak C18. A primeira eluição, com ácido acético 0,1 M, foi analisada usando-se o método de préderivatização e cromatografia líquida de alta eficiência com detecção de fluorescência. As analises em CLAE indicaram a presença de compostos semelhantes às toxinas paralisantes de PSP, confirmando os bioensaios. Portanto, pela primeira vez no Brasil demonstrou-se que as espécies S. brasiliensis, A. lepidentostole e B. aurea são portadoras de toxinas paralisantes, semelhantes às PSP, em pequenas concentrações e que um programa de monitoração é necessário em nosso país para verificação da presença dessas toxinas em organismos que são usados como alimento pela população. / The Paralytic Shellfish Poisoning (PSP) toxins are well-known natural bioactive compounds due to their accidental consumption in contaminated seafood. These molecules, of which the most potent representative is saxitoxin (STX), are a class of neurotoxic alkaloids, having different isoforms and varied toxicities, that are produced by some cyanobacteria and some species of dinoflagellates from the genus Alexandrium, Gymnodinium and Pyrodinium. PSP toxins are water-soluble neurotoxins that act on nerve and muscle cells by blocking sodium channels voltage-dependent, thus preventing the conductance of neuron signal leading to muscular paralysis. In severe cases, death may result due to respiratory failure. Diarrhetic Shellfish Poisoning (DSP) is a gastrointestinal illness with symptoms such as diarrhea, nausea, vomiting, headache, chills and moderate to severe abdominal pain. DSP is usually a consequence of consuming contaminated shellfish that have ingested dinoflagellates of the genera Dinophysis and Prorocentrun through their filter feeding activities. Contamination of seafood by PSP and DSP toxins has posed serious problems to the fisheries industry as well to public health. In this study, was stabilized a monitoring program to shellfish (Perna perna) and finfish (Sardinella brasiliensis, Anchoviella lepidentostole and Brevoortia aurea) collected in fish markets in Caraguatatuba and São Sebastião cities, São Paulo state. The extracts for PSP were prepared by two ways: according to AOAC (Association of Official Analytical Chemists), through the heating for 5 min of blend of 100 g of well mixed sample with 0.1 N HCl; or through of the concentration of ethanolic extracts from finfish’s muscle + skin. The PSP mouse bioassay for PSP toxins involves intraperitonial injection (i.p.) of 1 mL of the acid extract into each of three mice (~ 20 g). The mice were observed for classical PSP symptoms and the time to mouse death was recorded and the toxicity was determinate (in mouse units, MU) from the Sommer’s table. To DSP toxins, the extracts was prepared trough the extraction of digestive glands with acetone, and i.p injection in mice was used to determine the presence of theses toxins. In the mouse bioassay for the extracts prepared by AOAC method no positive results was obtained. For the mouse bioassay with ehtanolic extracts was obtained positive results to 77.8 % of the tested extracts. The media of MU of all samples, in this case, was 0,147 MU/g. To the mouse bioassay for the DSP toxins, three samples gives evidence of presence of the diarrhetic toxins, because the mice showed signal like diarrhea. The ethanolic extracts, that was positive to the PSP toxins, was fractionated by a Sep-Pak C18 cartridge. The first elution, with 0.1 M acetic acid, was analyzed by using prechromatographic oxidation and liquid chromatography with fluorescence detection. The HPLC analysis indicated the presence of the PSP toxins, confirming the bioassays. Therefore, in the first time in Brazil was demonstrated that the species S. brasiliensis, A. lepidentostole and B. aurea are carriers of toxins like PSP in little concentrations and that a monitoring program is necessary in our country to verify the presence of these toxins in organisms that are used as food by the population.

Lors des efflorescences de micro-algues productrices de toxines paralysantes (PST), les bivalves filtreurs peuvent bioaccumuler une grande quantité de toxines et devenir à leur tour toxiques, notamment pour l’homme. La quantité de toxines PST accumulée d’un individu à l’autre s’avère être très variable au sein même d’une population de bivalves. Ainsi, dans nos conditions expérimentales, la quantité de PST accumulées par des huîtres creuses, Crassostrea gigas, d’un même lot, exposées au dinoflagellé toxique Alexandrium minutum, variait d’un facteur 450. L’origine de cette variabilité est inconnue jusqu’alors mais l’une des hypothèses pour l’expliquer serait l’existence de plusieurs formes de canaux sodium voltage-dépendant (NaV), cible des PST, qui confèreraient aux bivalves des sensibilités différentes aux PST. L’objectif principal de cette thèse était de comprendre s’il existe une sensibilité individuelle aux PST différente entre les huîtres et si cette variabilité pouvait être due à des formes différentes de NaV.Une première partie a permis de caractériser le NaV chez C. gigas par une approche de biologie moléculaire. Deux gènes NaV ont été mis en évidence chez C. gigas : CgNaV1, codant un canal sodium et CgNaV2 codant un canal potentiellement sélectif du sodium et du calcium. L’épissage alternatif de CgNaV1 produits trois variants (A, B et C) avec des profils d’expression différents : au niveau des jonctions neuromusculaires pour CgNaV1A, dans les cellules nerveuses pour CgNaV1B et dans les deux pour CgNaV1C. L'acide aminé Q, observé dans le site de liaison aux PST (domaine II) de la séquence CgNaV1 pour les 3 variants et chez tous les individus des 4 populations étudiées, pourrait conférer aux huîtres une certaine résistance aux PST. Ainsi, les variants issus du génotypage/épissage de CgNaV1 ne seraient donc pas le point déterminant du niveau de bioaccumulation des huîtres.Une deuxième partie a permis d’étudier la sensibilité aux PST des nerfs de l’huître creuse C.gigas en relation avec l’accumulation de PST par une approche d’électrophysiologie. La sensibilité à la STX des nerfs cérébroviscéraux d'huîtres a été évaluée en étudiant leur potentiel d'action (CNAP).Il a été montré que les nerfs de C. gigas possédaient une sensibilité à la STX de l’ordre du micromolaire, ce qui leur confère une sensibilité intermédiaire parmi les bivalves. Cette sensibilité des nerfs peut varier selon la période à laquelle les huîtres ont été prélevées et potentiellement selon leur condition physiologique. Une pré-exposition des huîtres à A. minutum semble augmenter la résistance des nerfs à la STX. Cependant, aucune corrélation significative n'a été observée entre la sensibilité nerveuse à la STX et la charge en PST dans la glande digestive des huîtres.Il apparait donc que la variabilité de l’accumulation des PST par les huîtres résulterait plutôt d’une plasticité physiologique, en terme de filtration, d’ingestion et d’assimilation, que d’une sensibilité différentielle des NaV. / During bloom of microalgae producing paralytic shellfish toxins (PST), filtering bivalves can bio-accumulate a large quantity of toxins and become toxic for human consumption. The amount of accumulated PST can greatly vary from one individual to another within a bivalve population. Indeed, under our experimental conditions, the amount of accumulated PST by Pacific oysters, Crassostrea gigas, exposed to the toxic dinoflagellate Alexandrium minutum, varied by a factor of 450. To explain such variability we hypothesized the existence of several forms of voltage-gated sodium channel (NaV), target of the PST, resulting in different sensitivities to PST. The main objective of this thesis was to understand whether there are relationships between nerve sensitivity to PST, the different forms of NaV and the amount of accumulated PST.The NaV was first characterized in C. gigas by a molecular biology approach. Two NaV genes were reported in C. gigas: CgNaV1, encoding a sodium channel and CgNaV2 encoding a channel potentially selective for sodium and calcium. Alternative splicing of CgNaV1 produced three variants (A, B and C) with different expression profiles: at the neuromuscular junctions for CgNaV1A, in the nerve cells for CgNaV1B and in both for CgNaV1C. The amino acid Q observed in the binding site of PST (domain II), of the sequence CgNaV1 for the 3 variants and in all individuals from the 4 studied populations possibly provide some PST resistance to oysters. Thus, the variants resulting from the genotyping/splicing of CgNaV1 would not therefore be the determining factor of the level of bioaccumulation in oysters.A second part allowed studying the nerve sensitivity to PST of C. gigas oyster in relation to the accumulation of PST by an electrophysiology approach. The sensitivity to saxitoxin (STX, a PST) of the cerebro-visceral nerves from oysters was assessed by studying their action potential (CNAP). C.gigas nerves have been shown to have sensitivity to STX of the micromolar range, which gives them intermediate sensitivity among bivalves. This nerve sensitivity may vary depending on the period at which the oysters were collected and potentially according to their physiological condition. A preexposure of oysters to A. minutum appears to increase nerve resistance to STX. However, there was no significant correlation between STX nerve sensitivity and PST content in the oyster digestive gland.Overall, it appears that the variability of the PST accumulation by oysters would result rather from a physiological plasticity, in terms of filtration, ingestion and assimilation, than from a differential sensitivity of the NaV.

Alexandrium tamarense is a harmful algal species that can produce saxitoxins, a suite of powerful neurotoxins that bioaccumulate up the food chain and can have severe economic and health impacts. With harmful algal blooms increasing temporally and spatially, it is important for us to understand the relationship between harmful algal blooms and nutrients, particularly nitrogen from anthropogenic sources. To this end, the stable nitrogen isotopic composition (δ¹⁵N) of medium nitrate, algal cells and toxin in both nitrogen-replete and nitrogen-limited batch cultures of A. tamarense were measured in order to assess the potential for using the δ¹⁵N of the toxin as a tracer of the nitrogen source used for growth. A. tamarense cells grown under nitrate-replete conditions were depleted by 1.5‰ relative to the growth medium, and saxitoxin was depleted by 1.5‰ relative to the whole cells. Under nitrate-limiting conditions, the isotopic difference between cells and saxitoxin changed as nitrate in the growth medium was depleted, indicating uncoupling of toxin synthesis and cell growth rates under changing external nutrient conditions. Determination of the absolute magnitude of the isotopic differences between the medium nitrate and either the cells or the saxitoxin was confounded by 1) using two different nitrate sources – one nitrate source was used to grow the inoculum and a different nitrate source was used for the experimental medium - with different ‰ values and 2) the presence of an unidentified, isotopically-light, nitrogen blank in the low-nitrate medium samples. I conclude that STX nitrogen isotope values have the potential to be used as nitrogen source indicators. However, overall fractionation between whole cells and STX is unknown due to the uncoupling between cell growth and STX synthesis observed during my nitrogen-limited experiment. Based on previous research on cell growth and toxin production dynamics under different nutrient regimes, it is also reasonable to assume that the observed results here may differ if a different nitrogen source was utilized by the cells for STX production. Further research could include isotope analysis of cultures grown on different nitrogen sources, such as ammonium and urea; isotopic analysis of additional compounds, such as amino acids; or use of additional stable isotopes, such as C or O. / text

Proper neuronal function requires the preservation of appropriate neural excitability. An adaptive increase in neural excitability after exposure to agents that depress neuronal signaling blunts the sedative drug effects upon subsequent drug exposure. This adaptive response to drug exposure leads to changes in drug induced behaviors such as tolerance, withdrawal and addiction. Here I use Drosophila melanogaster to study the cellular and neuronal components which mediate behavioral tolerance to the anesthetic benzyl alcohol. I demonstrate that rapid tolerance to benzyl alcohol is a pharmacodynamic mechanism independent of drug metabolism. Furthermore, tolerance is a cell autonomous response which occurs in the absence of neural signaling. Using genetic and pharmacological manipulations I find the synapse to play an important role in the development of tolerance. In addition, the neural circuits that regulate arousal and sleep also alter benzyl alcohol sensitivity. Beyond previously described transcriptional mechanisms I find a post-translational role of the Ca2+-activated K+-channel, slowpoke in the development of tolerance. Finally, I explore a form of juvenile onset tolerance, which may have origins that differ from rapid tolerance. The implications of this study go beyond tolerance in Drosophila melanogaster to benzyl alcohol and can shed light on human drug tolerance, withdrawal and addiction. / text