Thesis (M.Sc. (Pharmacy)) - Rhodes University, 2008.

Orientador: Sergio Marangoni / Tese (mestrado) - Universidade Estadual de Campinas / Made available in DSpace on 2018-08-04T18:57:24Z (GMT). No. of bitstreams: 1 Fagundes_FabioHenriqueRamos_M.pdf: 2584588 bytes, checksum: b35682c6f4cea12ca88485bf389b2262 (MD5) Previous issue date: 2005 / Resumo: Enquanto estudos estruturais de toxinas de veneno de serpentes são feitos usando amostras de veneno, a construção de cDNAs precursores destas toxinas requer o sacrifício do espécime para a extração da glândula de veneno. No presente trabalho nós descrevemos uma técnica simples para isolar mRNAs presentes em amostras de veneno total. Para ilustrar esta técnica nós amplificamos através de RT-PCR um cDNA que codifica uma PLA2 049 (F6) do complexo crotoxina neurotóxica "in vitro" a partir do veneno total da sub espécie Crotalus durissus collilineatus. Estes achados implicam que a aparente ausência do mRNA em matrizes biológicas complexas não são sempre reais e facilitam a maneira de aquisição acelerada de dados micro evolutivos assim como de estrutura-função desta família de proteínas para serem utilizadas como ferramentas moleculares, sem o sacrifício do animal. O cONA que codifica PLA2 F6 do complexo crotoxina presente no veneno total da sub espécie Crotalus durissus collilineatus foi seqüenciada. A seqüência deduzida de aminoácidos deste cDNA codifica a PLA2 (F6) com alto grau de homologia seqüencial desta família de proteínas PLA2 do grupo 11, incluindo os 14 resíduos de cisteina capazes de dar forma a sete ligações dissulfeto que caracterizam este grupo de PLA2. A PLA2, foi isolada a partir do veneno total da sub espécie Crotalus durissus collilineatus através de coluna de cromatografia convencional de baixa pressão (8ephades G75) e por HPLC fase reversa. A massa molecular foi estimada por espectrometria de massa por MALDI - TOF. Para a identificação desta PLA2 (F6) como uma neurotoxina "in vitro", nós usamos a preparação biventer cervicis de pintainho. A análise filogenética da PLA2 (F6) da sub espécie Crotalus durissus collilineatus mostrou que os subtipos estruturais de N6-PLA2s com a substituição F24 ou 824 evoluíram paralelamente, possivelmente descendendo respectivamente das Protobothrops e a Gloydius dos dias de hoje / Abstract: While structural studies of snake venom toxins can be achieved using venom samples, until now the construction of precursor cDNAs required sacrifice of the specimen for dissection of the venom glands. Here we describe a simple and rapid technique that isolation mRNAs present in whole venom samples. To iIIustrate the technique we have RT-PCR amplified the cDNA that it codifies a "in vitro" neurotoxic protein PLA2 049 (F6) from crotoxin complex, from the venom of snake sub specie, Crotalus durissus collilineatus. These findings imply that the apparent absence of mRNA in complex biological matrices is not always real and paves the way for accelerated acquisition of micro evolution as well as of structure-function of this protein family being used as molecular tools, without sacrifice of animal. The cDNA encoding PLA2 F6 from crotoxin complex in the whole venom of sub specie Crotalus durissus collilineatus venom was sequenced. The deduced amino acid sequence of this cDNA encoded PLA2 (F6) with high overall sequence identity of this protein family to the group 11 PLA2, including the 14 cysteine residues capable of forming seven disulphide bonds that characterize this group of PLA2 enzymes. The PLA2, were isolated from the whole venom of sub specie Crotalus durissus collilineatus by the conventional and low pressure chromatography (8ephades G75) column and the reverse phase HPLC. The molecular mass estimated by MALDI-TOF mass spectrometry. For the identified this PLA2 F6 like as a "in vitro" neurotoxin, we use the neuromuscular blocking activities were assayed with the chick biventer cervicis neuromuscular tissue. The sub specie' s Crotalus durissus collilineatus PLA2 (F6) phylogeny analysis showed that two structural subtypes of N6-PLA2s with either F24 or 824 substitution have been evolved in parallel, possibly descended respectively from species related to present-day Protobothrops and Gloydius / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientador: Jose Camillo Novello / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T00:19:49Z (GMT). No. of bitstreams: 1 Damico_DanielaCarladaSilva_D.pdf: 13168744 bytes, checksum: facecc40bd94614d80c42d399206b237 (MD5) Previous issue date: 2006 / Resumo: A subespécie Lachesis m. muta vive em florestas tropicais úmidas de difícil acesso, dificultando sua captura e/ou manutenção em cativeiro (obtenção do veneno). Uma revisão de 20 casos de acidentes ofídicos com humanos ocorridos na Costa Rica, Guiana Francesa, Brasil, Colômbia e Venezuela, confidentemente atribuídos a este gênero de serpente, descrevem sintomas locais de dor, edema, equimose, coagulopatia, sintomas semelhantes aos observados no envenenamento bothrópico. Entretanto, náusea, cólica abdominal, vômito constante, diarréia e sudorese foram sintomas exclusivos, não reportados em vítimas de outros viperídeos do Brasil. Um dos objetivos deste trabalho foi estudar os efeitos neurotóxico e miotóxico do veneno de L. m. muta em preparações neuromusculares isoladas de ave (biventer cervicis de pintainho) e mamífero (nervo frênico diafragma de camundongo). Em baixa concentração (2 mg/ml), o veneno exibiu potente neurotoxicidade, entretanto, nesta concentração, nenhum efeito miotóxico significativo foi observado em preparação biventer cervicis de pintainho, a miotoxicidade foi observada somente a concentrações superiores, a partir de 10 mg/ml. Ficou claro que o veneno possui componentes ativos farmacologicamente que atuam na junção neuromuscular e nas fibras musculares, o qual a intensidade de ação depende da concentração do veneno e do tipo da preparação neuromuscular (ave ou mamífero) usada. O veneno bruto de L. m. muta também foi estudado quanto a sua ação citotóxica. O veneno mostrou um efeito citotóxico frente à célula tubular epitelial renal (MDCK), induziu a uma diminuição da viabilidade celular, alterou significantemente a resistência elétrica transepitelial através da monocamada e induziu alterações morfológicas e nucleares. Foram purificadas duas novas toxinas (LmTX-I e LmTX-II) a partir do veneno total de L. m. muta, com um alto grau de pureza e homogeneidade molecular, sem perda da atividade biológica. Estas novas toxinas foram caracterizadas como isoformas de PLA2s básicas Asp49, em função das características físico-químicas evidenciadas. Frente a diferentes concentrações de substrato, LmTX-I mostrou um comportamento tipo alostérico. Na ausência de Ca2+ (1 mM) e na presença de íons divalentes como Zn2+ e Cu2+, a atividade PLA2 foi inibida. Foi utilizada preparação biventer cervicis de pintainho para estudar a atividade neurotóxica e miotóxica in vitro da PLA2 (LmTX-I). A toxina produziu um bloqueio irreversível da transmissão neuromuscular em concentrações baixas como 1 mg/ml. Entretanto, nesta concentração, nenhum efeito miotóxico significativo foi produzido pela toxina. O bloqueio neuromuscular não foi acompanhado da inibição da resposta contrátil à adição da acetilcolina (ACh), desta maneira, o bloqueio neuromuscular produzido pela LmTX-I pode ser atribuído preferencialmente por um efeito inibitório na liberação de ACh, sugerindo uma ação présináptica da toxina. Com uma maior concentração (30 mg/ml), LmTX-I afetou a resposta ao KCl (30% de inibição), uma concentração no qual foi observado alterações morfológicas significativas (15% de fibras danificadas), como células com tamanhos heterogêneos, vacuolizadas, ou células com miofibrilas compactadas. Entretanto, nenhum efeito citotóxico significativo, como alteração morfológica e nuclear, redução da viabilidade celular ou indução da liberação de lactato desidrogenase, usando célula tubular epitelial renal (MDCK) e celular muscular esquelética (mioblastos e miotubos) foi observado / Abstract: The subspecies Lachesis m. muta lives in tropical forests of hard access, hindering its capture and/or maintenance in captivity (obtaining of the venom). A review on 20 case-reports that occurred in Costa Rica, French Guiana, Brazil, Colombia and Venezuela, of bites in humans, reliably attributed to this snake genus, described the local symptoms of pain, swelling, blistering, and mild coagulopathy, and as similar to those caused by snakebites of the genus Bothrops and other Latin American pit vipers genera, however, early nausea, abdominal colic, repeated vomiting, watery diarrhea and profuse sweating were distinctive symptoms, not reported in victims of other viperids. The venom of L. m. muta was studied with relationship to the neurotoxic and myotoxic effects in neuromuscular preparations isolated from avian (chick biventer cervicis muscle) and mammalian (mouse phrenic nerve-diaphragm muscle). The venom, in low concentrations (2 mg/ml) exhibited potent neurotoxicity, however, in this concentration, no significant myotoxic effect was observed in avian preparation, the myotoxicity was only observed to high concentrations, up to 10 mg/ml. It became clear that the venom has pharmacologically active components that act on neuromuscular junction and muscle fibers, whose intensity of action will depend on the venom concentration and on the type of nerve-muscle preparation (mouse or chick). The whole venom of L. m. muta also showed a cytotoxic effect in MDCK epithelial cell. The venom induced to a decrease of the cellular viability, altered significant the transepithelial electrical resistance through the monolayer and induced morphologic and nuclear alterations. Through optimized methodologies of purification in HPLC, two new toxins were purified (LmTX-I and LmTX-II) from the venom of L. m. muta, with a high degree of purity and molecular homogeneity, without loss of the biological activity. These new toxins were characterized as basic isoforms of PLA2s Asp49, because they share several chemical and physical characteristics: molecular mass, retention time in re-purification on reverse phase HPLC, content of disulfide bonds, isoelectric point and primary structure. In the presence of different substratum concentrations, LmTX-I showed a behavior type allosteric under our experimental conditions. In the absence of Ca2+ (1 mM) and in the presence of divalent ions as Zn2+ and Cu2+, the PLA2 activity was inhibited. Avian muscle preparation was used to study in vitro neurotoxic and myotoxic activity of PLA2 (LmTX-I). The toxin produced an irreversible blockade of the neuromuscular transmission in low concentrations as 1 mg/ml. However, in this concentration, the toxin produced no significant myotoxic effect. The complete neuromuscular blockade of the twitch tension was not accompanied of the inhibition of the response to ACh, this way, the neuromuscular blockade produced by the LmTX-I can be preferentially attributed by an inhibitor effect in the ACh release, suggesting a pre-synaptic action of the toxin. With a high concentration (30 mg/ml), LmTX-I affected the response to KCl (30% of inhibition) after the neuromuscular blockade have been completed, a concentration that was observed significant morphologic alterations (15% of damaged fibers), as cells with heterogeneous sizes, vacuolated, or cells with compacted myofibrils. However, any significant cytotoxic effect, as morphologic and nuclear alteration, reduction of the cellular viability or induction of the release of lactic dehydrogenase was observed using tubular epithelial renal and murine skeletal muscle cells / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Orientador: Sergio Marangoni / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T11:18:09Z (GMT). No. of bitstreams: 1 Ponce-Soto_LuisAlberto_D.pdf: 2824712 bytes, checksum: 7aeb46c8d2e647484f7c95e4df5cfb42 (MD5) Previous issue date: 2005 / Resumo: Através de metodologias otimizadas de purificação em HPLC, foram purificadas novas toxinas a partir do veneno total de Crotalus durissus collilineatus (V-1), Bothrops jararacussus PLA2 D49 Bj-V (isoforma de Bj-IV) obtida da fração BthTX-II e Bothrops alternatus PLA2 K49 Bt II-2; com um alto grau de pureza e homogeneidade molecular, sem perda da atividade biológica. A nova neurotoxina V-1 de Crotalus durissus collilineatus caracterizada físico-quimicamente como desprovida de atividade catalítica, ácida e com uma massa molecular de 9859.45 Da, uma única cadeia polipeptídica, induz uma facilitação contínua no modelo biológico nervo frênico diafragma isolado de camundongo e uma facilitação pronunciada seguida de um bloqueio súbito em biventer cervicis de pintainho, na junção neuromuscular. Os estudos estruturais, neurotóxicos e miotóxicos da PLA2 F6 do complexo crotoxina de Crotalus durissus collilineatus mostram que a PLA2 F6 precisa da crotapotina (estudos de re-associação) para potencializar seu efeito neurotóxico na preparação nervo frênico diafragma isolado de camundongo, mas a PLA2 F6 é capaz de induzir neurotoxicidade in vitro na ausência de crotapotina na preparação biventer cervicis de pintainho. Foram purificadas, a partir da fração BthTX-II, duas PLA2 D49 Bj-IV e V, sendo consideradas como isoformas devido ao fato de que compartilham de várias características físico-químicas, tais como: massa molecular, tempo de retenção na re-purificação em HPLC de fase reversa, análise de composição de aminoácidos, pI e estrutura primária, embora a isoforma Bj-V apresente algumas mutações com respeito à isoforma Bj-IV: W3 -> F3, Q4 -> E4, F5 -> W5, I16 -> N16 e G41 -> 41D; importantes para a atividade catalítica, os efeitos neurotóxicos in vitro foram mantidos e, quando foram inibidas cataliticamente por crotapotinas crotálicas de Crotalus durissus collilineatus (F3 e F4), continuaram diminuindo a resposta contrátil na junção neuromuscular nas preparações nervo frênico diafragma isolado de camundongo e biventer cervicis de pintainho, mostrando que são mais pronunciadas no músculo esquelético de camundongo. A nova toxina Bt II-2 purificada, com um alto grau de pureza e homogeneidade molecular, do veneno de Bothrops alternatus, foi caracterizada como uma PLA2 básica K49, em função das características físico-químicas, evidenciadas: massa de 13898.71 Da, possui caráter básico e uma alta homologia seqüencial na sua estrutura primária, quando comparada com outras PLA2 K49 procedentes de veneno de serpentes.Essa nova PLA2 K49 Bt II-2 revelou um potente efeito neurotóxico in vitro na preparação nervo frênico diafragma isolado de camundongo (1 µg/mL) pré-sináptico, como foi mostrado nos estudos de estímulo indireto via nervo e potencial de membrana ou repouso, diferentemente de qualquer PLA2 neurotóxica botrópica que, para causar um efeito neurotóxico, precisa de dosagem acima de 50 µg/mL. Outros perfis biológicos foram estudados para a Bt II-2, que mostrou uma miotoxicidade local ¿in vivo¿, citotoxicidade em mioblastos e miotubos C2C12, atividade inflamatória e letalidade, corroborando que se enquadra dentro da família de proteínas PLA2 homólogas K49, mas com características únicas nos estudos de neurotoxicidade in vitro. A reprodutibilidade da atividade biológica, através dos efeitos farmacológicos, só é possível com a utilização de frações quimicamente homogêneas que mantenham a integridade da função biológica. Essas frações são obtidas com metodologias de alta eficiência: HPLC, LC, espectrometria de massa, cujos resultados podem ser associados com sua atividade biológica, eliminando a subjetividade causada por veneno total ou frações impuras. Essa abordagem pode ser aplicada nos estudos bioquímicos, estrutura-função, fisiológicos e farmacológicos, podendo revelar mecanismos ainda desconhecidos na relação estrutura-função das PLA2 procedentes de veneno de serpentes. A presença de um sítio farmacológico distinto do sítio catalítico presente nas PLA2 pode ser usada como ferramenta molecular para o reconhecimento dos receptores de membrana desconhecidos em células ou tecidos / Abstract: Through optimized methodologies of purification in HPLC, new toxins were purified from total venom of Crotalus durissus collilineatus (V-1), Bothrops jararacussus PLA2 D49 Bj-V (Bj-IV isoform) obtained from the fraction BthTX-II and Bothrops alternatus PLA2 K49 Bt II-2; with a high level of purity and molecular homogeneity, with no loss of biological activity. The new neurotoxin V-1 of Crotalus durissus collilineatus, chemically and physically characterized as unproved of catalytic, acid activity and with a molecular mass of 9859.45 Da, an only polypeptide sequence, induces to a continual facilitation in the biological isolated mouse phrenic nerve diaphragm model of mouse and a pronunciated facility of a subital block in chick biventer cervicis preparation en, in the neuromuscular junction. The structural, neurotoxic and miotoxic studies on PLA2 F6 from crotoxin complex of Crotalus durissus collineatus show crotapotin is necessary (re-association studies) to increase its neurotoxic effect in nerve phrenic diaphragm isolated preparation of mouse, but the PLA2 F6 is able to induce neurotoxity in vitro in ausency of crotopatin in the preparation chick biventer cervicis. Two PLA2 D49 Bj-IV and V were purified from the fraction BthTX-II and considered isoform because they share several chemical and physical characteristics, such as molecular mass, retention time in re-purification in reverse phase HPLC, analyses of amino acid composition, pI and primary structure, although the Bj-V isoform shows some mutations regarding Bj-IV: W3 -> F3, Q4 -> E4, F5 -> W5, I16 -> N16 e G41 -> 41D isoform. Important for the catalytic activity, neurotoxic in vitro effects were maintained and, when catalytically inhibited by crotapotin crotalic of Crotalus durissus collilineatus (F3 e F4), they continued dimishing the contractile reply in neuromuscular junction in nerve phrenic diaphragm isolated preparations of mouse and preparation chick biventer cervicis, which shows they are more pronunciated in the skeleton muscle of the mouse. The new and purified BtII-2 toxin of venom of Bothrops alternatus, with a high level of purity and molecular homogeneity, was characterized as a PLA2 basic K49, because its chemical and physical evidenced characteristics, mass of 13898.71 Da, basic character and a high sequential homology in its primary structure, when it is compared with other PLA2 K49 from venom of serpents. This new PLA2 K49 Bt II-2 has revealed a potent neurotoxic effect in vitro in neuromuscular junction in nerve phrenic diaphragm isolated preparation of mouse (1 µg/mL) ante-synaptic, as it was proved by studies on indirect stimulus through nerve and potential of membrane or rest, differently of some neurotoxic botropic PLA2 that needs a dose above 50 µg/mL to cause a neurotoxic effect. Other biological profiles were studied for the Bt II-2, and the conclusion was a local myotoxic ¿living¿, a cytotoxic in myoblasts and myotubes C2C12, imflammatory and letality activity, showing they are fitted in the family of proteins PLA2 homologous K49, but with particular characteristics in studies on neurotoxic in vitro. The reprodubility of biological activity, through pharmacological effects, is just possible with the utilization of chemically homogenous fractions that maintain the integrity of biological function. These fractions were obtained with high efficient methodologies as HPLC, LC, and mass spectrometry, whose results may be associated with their biological activities, eliminating the subjectivity caused by total venom or impure fractions. This approximation may be applied in the biochemical studies, structure-function, physiological and pharmacological, and it may reveal still unknown mechanisms in the relation structure-function of PLA2 from the venom of serpents. The presence of a pharmacological site different of the catalytic one in the PLA2 may be used as a molecular instrument to the recognition of the receptors of unknown membranes in cells or tissues / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a profound loss of dopaminergic neurons from the substantia nigra (SN). Among the many pathogenic mechanisms thought to be responsible for the demise of these cells, dopamine (DA)-dependent oxidative stress and oxidative damage has taken center stage due to extensive experimental evidence showing that DA-derived reactive oxygen species (ROS) and oxidized DA metabolites are toxic to SN neurons. Despite its being the most efficacious drug for symptom reversal in PD, there is concern that levodopa (LD) may contribute to the neuronal degeneration and progression of PD by enhancing DA concentrations and turnover in surviving dopaminergic neurons. The present study investigates the potential neurotoxic and neuroprotective effects of DA in vitro. These effects are compared to the toxicity and neuroprotective effects observed in the rat striatum after the administration of LD and selegiline (SEL), both of which increase striatal DA levels. The effects of exogenous LD and/or SEL administration on both the oxidative stress caused by increased striatal iron (II) levels and its consequences have also been investigated. 6-Hydroxydopamine (6-OHDA) is a potent neurotoxin used to mimic dopaminergic degeneration in animal models of PD. The formation of 6-OHDA in vivo could destroy central dopaminergic nerve terminals and enhance the progression of PD. Inorganic studies using high performance liquid chromatography with electrochemical detection (HPLC-ECD) show that hydroxyl radicals can react with DA to form 6-OHDA in vitro. SEL results in a significant decrease in the formation of 6-OHDA in vitro, probably as a result of its antioxidant properties. However, the exogenous administration of LD, with or without SEL, either does not lead to the formation of striatal 6-OHDA in vivo or produces concentrations below the detection limit of the assay. This is despite the fact that striatal DA levels in these rats are significantly elevated (two-fold) compared to the control group. The auto-oxidation and monoamine oxidase (MAO)-mediated metabolism of DA causes an increase in the production of superoxide anions in whole rat brain homogenate in vitro. In addition to this, DA is able to enhance the production of hydroxyl radicals by Fenton chemistry (Fe(III)-EDTA/H2O2) in a cell free environment. Treatment with systemic LD elevates the production of striatal superoxide anions, but does not lead to a detectable increase in striatal hydroxyl radical production in vivo. The co-adminstration of SEL with LD is able to prevent the LD induced rise in striatal superoxide levels. It has been found that the presence of DA or 6-OHDA is able to reduce lipid peroxidation in whole rat brain homogenate induced by Fe(II)-EDTA/H2O2 and ascorbate (Fenton system). However, DA and 6-OHDA increase protein oxidation in rat brain homogenate, which is further increased in the presence of the Fenton system. In addition to this, the incubation of rat brain homogenate with DA or 6-OHDA is also accompanied by a significant reduction in the total GSH content of the homogenate. The exogenous administration of LD and/or SEL was found to have no detrimental effects on striatal lipids, proteins or total GSH levels. Systemic LD administration actually had a neuroprotective effect in the striatum by inhibiting iron (II) induced lipid peroxidation. Inorganic studies, including electrochemistry and the ferrozine assay show that DA and 6-OHDA are able to release iron from ferritin, as iron (II), and that DA can bind iron (III), a fact that may easily impede the availability of this metal ion for participation in the Fenton reaction. The binding of iron (III) by DA appears to discard the involvement of the Fenton reaction in the increased production of hydroxyl radicals induced by the addition of DA to mixtures containing Fe(II)-EDTA and hydrogen peroxide. 6-OHDA did not form a metal-ligand complex with iron (II) or iron (III). In addition to the antioxidant activity and MAO-B inhibitory activity of SEL, the iron binding studies show that SEL has weak iron (II) chelating activity and that it can also form complexes with iron (III). This may therefore be another mechanism involved in the neuroprotective action of SEL. The results of the pineal indole metabolism study show that the systemic administration of SEL increases the production of N-acetylserotonin (NAS) by the pineal gland. NAS has been demonstrated to be a potent antioxidant in the brain and protects against 6-OHDA induced toxicity. The results of this study show that DA displays antioxidant properties in relation to lipid eroxidation and exhibits pro-oxidant properties by causing an increase in the production of hydroxyl radicals and superoxide anions, as well as protein oxidation and a loss of total GSH content. Despite the toxic effects of DA in vitro, the treatment of rats with exogenous LD does not cause oxidative stress or oxidative damage. The results also show that LD and SEL have some neuroprotective properties which make these agents useful in the treatment of PD.

The purpose of the present study was to determine the degree of relationship between neurotoxic chemicals in the blood of chemically sensitive patients and psychoneurological functioning. Blood samples were drawn from 30 patients being treated for environmental illness. All patients were administered a standardized intermediate psychoneurological examination. Results indicate a significant positive relationship between psychoneurological (cognitive neurobehavioral) functioning and the number of and total parts per billion of certain environmental toxins (solvents) in the blood of the subjects. The symptoms most commonly exhibited included deficits in short-term memory, problems with coordination and motor sequencing, somatosensory deficits, and cognitive dysfunction.

Orientadores: Lea Rodrigues Simioni,Yoko Oshima Franco / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T13:41:21Z (GMT). No. of bitstreams: 1 Bueno_LilianGobbodeFreitas_M.pdf: 986839 bytes, checksum: 048ec75c1469d547de311f325aab2cc3 (MD5) Previous issue date: 2006 / Resumo: Manganês, um bloqueador neuromuscular com atividades pré e pós-sinápticas, foi utilizado neste trabalho com o objetivo de estudar a neurotoxicidade e miotoxicidade induzida pelos venenos das serpentes Crotalus durissus terrificus (Cdt) e Bothrops jararacussu (Bjssu) em preparações biventer cervicis de pintainho (BCp). O pré-tratamento das preparações com Mn2+ (0,66 e 1,6 mM) não impediu o bloqueio neuromuscular induzido pelo veneno de Cdt, reduziu parcialmente a resposta contraturante à adição exógena de ACh, mas não à de KCl. Embora o bloqueio neuromuscular induzido pelo veneno de Bjssu seja irreversível, o pré-tratamento com ambas as concentrações de Mn2+ (0,66 e 1,6 mM) tornou o bloqueio parcialmente reversível após a lavagem das preparações com solução de Krebs, e somente nas preparações pré-tratadas com 1,6 mM Mn2+ houve redução do bloqueio da resposta contraturante à adição exógena de ACh (p<0,05). Os tipos de miotoxicidade induzidos pelos venenos de Cdt e Bjssu interferiram diferentemente na resposta contrátil. Em preparações pré-tratadas com Mn2+ (1,6 mM) houve redução parcial da porcentagem de dano muscular e da atividade miotóxica (CC, U/L) induzida por ambos os venenos. Os resultados desta pesquisa indicam que: Mn2+ interfere na contratura induzida pela ACh nos receptores nicotínicos; Mn2+ não evitou a neurotoxicidade do veneno de Cdt, mas reduziu parcialmente sua miotoxicidade in vitro, por sua ação estabilizante da membrana muscular; Mn2+ reduziu parcialmente a miotoxicidade e o bloqueio neuromuscular induzido pelo veneno de Bjssu. A dupla ação do Mn2+, pré- e pós-sináptica, é considerada útil para o estudo dos venenos de serpentes, uma vez que a maioria desses venenos apresenta também estas ações. Além disso, com a utilização deste cátion foi possível resgatar a coerência entre a interpretação dos resultados experimentais e clínicos / Abstract: In this study, we examined the effects of Mn2+, a neuromuscular blocker with pre and postsynaptic actions, on the neurotoxicity and myotoxicity induced by Crotalus durissus terrificus and Bothrops jararacussu venoms in chick biventer cervicis preparations. Pretreating the preparations with Mn2+ (0.66 or 1.6 mM) did not affect the blockade induced by C. d. terrificus venom or KCl-induced contractures, but partially reduced ACh-induced contractures. On the other hand, both concentrations of Mn2+ partially prevented the blockade induced by B. jararacussu venom (seen after washing the preparations with Krebs solution), whereas only 1.6 mM Mn2+ significantly restored ACh-induced contractures. Pretreatment with Mn2+ (1.6 mM) partially prevented the muscle damage and the release of creatine kinase induced by both venoms. These results show that Mn2+ did not prevent the neurotoxicity of Cdt venom, but partially reduced its myotoxicity, whereas this metal partially attenuated the myotoxicity and neuromuscular blockade caused by B. jararacussu venom. The pre- and postsynaptic actions of Mn2+ may be useful for studying snake venoms that show one or both of these activities / Mestrado / Mestre em Farmacologia

O desenvolvimento de um equipamento de eletroforese capilar com detecção condutométrica sem contato e hifenizado a um sistema de amostragem de gases é apresentado. Ele foi construído a partir de chapas de poli(metacrilato de metila) por ablação a laser de CO2, mesclando métodos convencionais com os de microfabricação. Este protótipo traz como inovações: (1) a refrigeração em estado sólido da coluna capilar, (2) a inclusão de eletrólise separada em microdispositivos e (3) a montagem de uma válvula embutida no microchip, baseada em servomotor. O equipamento é portátil (31 × 29 × 25 cm) e projetado para ser utilizado em bancada ou embarcado num robô para análises remotas de agentes neurotóxicos e outros gases ionizáveis em contato com a fase aquosa. O sistema de termostatização permitiu o controle da temperatura interna do capilar de sílica fundida entre 12 e 50 °C utilizando uma elemento Peltier a a partir da temperatura ambiente de 21 °C. A amostragem de gases é feita utilizando-se uma membrana tubular porosa acoplada ao sistema microfluídico. Ela possui 10 cm de comprimento com 300 micrometros de diâmetro externo e 3,1 microlitros de volume interno. Como prova de conceito, foram feitas amostragens de ar contendo ácidos fórmico, acético e propiônico, separação por eletroforese capilar dos respectivos ânions e detecção com relação sinal-ruído de 414, 150 e 115, respectivamente. A amostragem foi realizada por 30 s e a injeção eletrocinética, por 2 s a 1 kV. A corrida eletroforética foi concluída em 4 min. / The development of a capillary electrophoresis equipment with contactless conductivity detection coupled to a gas sampling system is described. It was constructed from poly(methyl methacrylate) by carbon dioxide laser ablation, using conventional methods and microfabrication. The innovations are: (1) solid-state cooling system of the silica capillary, (2) the inclusion of separate electrolysis on a microdevices, and (3) a valve assembly embedded in the microchip based servomotor. It is portable (31 × 29 × 25 cm) and designed to be used on a workbench or embedded in a robot for remote analysis of nerve agents and other ionized gases. The thermostating system allowed the temperature control of the fused silica capillary between 12 and 50 °C using a Peltier chip from ambient temperature of 21°C. Gas sampling is performed using a tubular porous membrane coupled to the microfluidic system. It is 10 cm long with outer diameter of 300 micrometers and 3.1 microliters of internal volume. Air containing formic, acetic, and propionic acids were sampled, and the corresponding anions were separated and detected in the equipment as a proof of concept. The signal-to-noise ratio were, respectively, 414, 150, and 115, after sampling by 30 s and electrokinetic injection by 2 s at 1 kV. The electrophoretic run was accomplished in 4 min.

O desenvolvimento de um equipamento de eletroforese capilar com detecção condutométrica sem contato e hifenizado a um sistema de amostragem de gases é apresentado. Ele foi construído a partir de chapas de poli(metacrilato de metila) por ablação a laser de CO2, mesclando métodos convencionais com os de microfabricação. Este protótipo traz como inovações: (1) a refrigeração em estado sólido da coluna capilar, (2) a inclusão de eletrólise separada em microdispositivos e (3) a montagem de uma válvula embutida no microchip, baseada em servomotor. O equipamento é portátil (31 × 29 × 25 cm) e projetado para ser utilizado em bancada ou embarcado num robô para análises remotas de agentes neurotóxicos e outros gases ionizáveis em contato com a fase aquosa. O sistema de termostatização permitiu o controle da temperatura interna do capilar de sílica fundida entre 12 e 50 °C utilizando uma elemento Peltier a a partir da temperatura ambiente de 21 °C. A amostragem de gases é feita utilizando-se uma membrana tubular porosa acoplada ao sistema microfluídico. Ela possui 10 cm de comprimento com 300 micrometros de diâmetro externo e 3,1 microlitros de volume interno. Como prova de conceito, foram feitas amostragens de ar contendo ácidos fórmico, acético e propiônico, separação por eletroforese capilar dos respectivos ânions e detecção com relação sinal-ruído de 414, 150 e 115, respectivamente. A amostragem foi realizada por 30 s e a injeção eletrocinética, por 2 s a 1 kV. A corrida eletroforética foi concluída em 4 min. / The development of a capillary electrophoresis equipment with contactless conductivity detection coupled to a gas sampling system is described. It was constructed from poly(methyl methacrylate) by carbon dioxide laser ablation, using conventional methods and microfabrication. The innovations are: (1) solid-state cooling system of the silica capillary, (2) the inclusion of separate electrolysis on a microdevices, and (3) a valve assembly embedded in the microchip based servomotor. It is portable (31 × 29 × 25 cm) and designed to be used on a workbench or embedded in a robot for remote analysis of nerve agents and other ionized gases. The thermostating system allowed the temperature control of the fused silica capillary between 12 and 50 °C using a Peltier chip from ambient temperature of 21°C. Gas sampling is performed using a tubular porous membrane coupled to the microfluidic system. It is 10 cm long with outer diameter of 300 micrometers and 3.1 microliters of internal volume. Air containing formic, acetic, and propionic acids were sampled, and the corresponding anions were separated and detected in the equipment as a proof of concept. The signal-to-noise ratio were, respectively, 414, 150, and 115, after sampling by 30 s and electrokinetic injection by 2 s at 1 kV. The electrophoretic run was accomplished in 4 min.

This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Thesis: Ph. D., Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Department of Biology; and the Woods Hole Oceanographic Institution), 2019 / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references. / Harmful algal blooms (HABs) can produce potent neurotoxins that accumulate in seafood and affect human health. One HAB toxin of concern is domoic acid (DomA), a glutamate analog produced by the marine diatom Pseudo-nitzschia spp. Current regulatory limits are designed to prevent acute neurotoxicity in adult humans. However, research shows that low-level exposure during early life can lead to long-term changes in behavior, neural connectivity, and brain morphology. To determine the underlying mechanisms of developmental toxicity, this dissertation used zebrafish as a tool to: i) Establish the developmental window of susceptibility for DomA toxicity, ii) Characterize the behavioral consequences of exposures, and iii) Identify the cellular targets and processes perturbed by DomA. I found that DomA exposure particularly at 2 days post fertilization (dpf) led to altered startle response behavior, myelination defects, and the downregulation of axonal and myelin structural genes. / Using vital dyes and immunolabeling, I assessed DomA-induced alterations in cells required for the startle response. I found no differences in the number of sensory neuromasts or in the sensory cranial ganglia structures that detect the acoustic stimuli. However, the majority of DomA-treated larvae lacked one or both Mauthner cells - hindbrain neurons critical for fast startle responses. DomA-treated larvae also had oligodendrocytes with fewer and shorter myelin sheaths, and appeared to aberrantly myelinate neuronal cell bodies. The loss of the Mauthner neurons and their axons may lead to a cellular environment where oligodendrocytes myelinate neuronal cell bodies in the absence of adequate axonal targets. Indeed, pharmacological treatment that reduced the oligodendrocyte number also led to the reduction in the number of these aberrant, myelinated cell bodies. / These results indicate that exposure to DomA at a particular period in neural development targets specific cell types, disrupts myelination in the spinal cord, and leads to prolonged behavioral deficits. These mechanistic insights support hazard assessments of DomA exposures in humans during critical periods in early development. / "Funding for my research came from the Ocean Ventures Fund, Hill family foundation, Woods Hole Sea grant NA14OAR4170074, and the Woods Hole Center for Oceans and Human Health (COHH), which is jointly funded by the National Institutes of Health (P01ES02192, P01ES028938), and the National Science Foundation (OCE-1314642, OCE-1840381). My funding came from the National Institutes of Health (NIH) P01ES021923-04S1, the Ocean Ridge Initiative Fellowship, the Von Damm Fellowship, and the MIT/WHOI Joint Program Academic Programs Office"--Page 5 / by Jennifer Martinez Panlilio. / Ph. D. / Ph.D. Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Department of Biology; and the Woods Hole Oceanographic Institution)