Thesis (Ph. D.)--University of Virginia, 2000. / Spine title: Synaptic role of TYR-phosphatases. Includes bibliographical references (p. 135-172). Also available online through Digital Dissertations.

Thesis (doctoral)--Lund University, 1992. / Added t.p. with thesis statement inserted.

Synapse development is a stepwise process that requires the recruitment of key synaptic components to active zones, followed by continual maintenance of these structures to maintain connectivity and stability throughout the life of the organisms. Early synapse development requires the recruitment of early scaffolding proteins to establish stable connectivity as well as provide sites of recruitment of other vital synaptic proteins. One of the earliest proteins to be localized to the synapse is the conserved protein Syd-1. Syd-1 proteins contain a Rho GTPase activating protein (GAP)-like domain of unclear significance. Here I show that Drosophila Syd-1 interacts with all six fly Rhos and has GAP activity towards RAC1. I then show that lacks GAP activity localizes normally to presynaptic sites and is sufficient to recruit Nrx-1 but fails to cluster Brp normally and genetically interacts with RAC1 in vivo. I conclude that contrary to previous models, the GAP domain of fly Syd-1 is active and required for presynaptic development. Additionally, I’ve identified a previously uncharacterized protein, Vezl, as being critical for retrograde axonal transport and synaptic maintenance. I found that Vezl required for normal neuronal growth and that vezl loss resulted in decreased neuron size and the formation of swollen neuronal terminals that accumulated membrane markers and axonal transport cargo. I found that vezl mutants specifically retrograde transport of cargo and particularly affected signaling endosomes. The signaling endosomes were unable to initiate retrograde transport in vezl mutants and remained stuck within the distal boutons unable to relay their signaling peptides back to the nucleus. I conclude that Vezl is serving a role in attaching retrograde cargo to dynein and the microtubules specifically at neuron tips so that they can undergo retrograde axonal transport. This dissertation includes previously published and unpublished co-authored material. / 2020-09-06

Orientador: Márcia Gallacci / Banca: Marcos Roberto de Mattos / Banca: Vidal Haddad Junior / Resumo: Os acidentes envolvendo as serpentes do gênero Bothrops se destacam no Brasil e em outros países da América Latina, representando 90% das notificações. O envenenamento botrópico é caracterizado por intensa mionecrose local que não é eficientemente neutralizada pelo único tratamento disponível, isto é, a soroterapia. Como conseqüência, em casos graves, este acidente pode levar a amputação de membros, desabilitando a vítima. Os principais responsáveis pelo desenvolvimento da mionecrose são proteínas com estruturas homólogas às enzimas fosfolipases A2 (PLA2s). Entre essas se destacam as variantes cataliticamente inativas que apresentam como característica um resíduo de lisina na posição 49 (Lys49-PLA2s). Tradicionalmente, as Lys49-PLA2s são consideradas miotoxinas não-neurotóxicas, uma vez que não induzem paralisia in vivo. No entanto, em preparações isoladas, tal efeito é observado. Recentemente, sugeriu-se que a paralisia muscular in vitro, da mesma forma que a lesão muscular, resultaria da atividade desestabilizadora de membrana da fibra muscular induzida por estas toxinas. O presente estudo teve como objetivo investigar a relação entre os efeitos miotóxico e paralisante das Lys49-PLA2s e contribuir para o esclarecimento do mecanismo de ação e da relação estrutura/atividade dessas toxinas. Sendo assim, realizaram-se estudos miográficos e morfológicos em preparações neuromusculares de camundongos utilizando a PrTX-I (Lys49-PLA2 isolada do veneno de Bothrops pirajai) e potenciais inibidores vegetais (ácido rosmarínico, ácido caféico e ácido aristolóquico). Os resultados obtidos mostraram as diferentes capacidades dos compostos vegetais em neutralizar os efeitos miotóxico e paralisante da PrTX-I. Assim, o ácido rosmarínico neutralizou ambos os efeitos eficientemente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Accidents caused by Bothrops snake genus stand out in Brazil and other Latin American countries, representing 90% of notifications. Bothropic envenoming is characterized by intense local myonecrosis, not effectively neutralized by serum therapy, the only available treatment. As a result, in severe cases, these accidents can lead to amputation of limbs, disabling the victim. The main responsibles for myonecrosis development are proteins with homologous structures from enzymes phospholipase A2 (PLA2s). Among these stand out catalytically inactive variants that have a characteristic lysine residue at position 49 (Lys49-PLA2s). Traditionally, Lys49-PLA2s myotoxins are considered non-neurotoxic, since they do not induce paralysis in vivo. However, this effect is observed in isolated preparations. It was recently suggested that muscle paralysis in vitro, as well as muscle injury, would result from the destabilizing activity of the muscle fiber membrane induced by these toxins. This study aimed to investigate the relationship between paralyzing and myotoxic effects of Lys49-PLA2s and contribute to the elucidation of the mechanism of action and structure/activity relationship of these toxins. Therefore, myographical and morphological studies were performed in neuromuscular preparations of mice using PrTX-I (Lys49-PLA2 isolated from Bothrops pirajai venom) and potential inhibitors from plants (rosmarinic acid, caffeic acid and aristolochic acid) as experimental tools. The results showed the different skills of plant compounds to neutralize myotoxic and paralyzing effects of PrTX-I. Thus, the rosmarinic acid efficiently neutralized both effects, whereas caffeic acid only partially inhibited the... (Complete abstract click electronic access below) / Mestre

Orientador: Maria Julia Marques / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T17:15:31Z (GMT). No. of bitstreams: 1 Taniguti_AnaPaulaTiemi_M.pdf: 2340108 bytes, checksum: d096d9870bc1650ef2e8526da0771778 (MD5) Previous issue date: 2006 / Resumo: A Distrofia Muscular de Duchenne (DMD) é uma miopatia hereditária caracterizada pela falta de distrofina. Camundongos da linhagem mdx, tal como pacientes com DMD, não expressam a distrofina, desenvolvendo distrofia muscular semelhante a DMD. A junção neuromuscular distrófica apresenta alteração no padrão de distribuição dos receptores de acetilcolina (AChRs), provavelmente devida à falta de distrofina. Alterações semelhantes na distribuição dos receptores ocorrem em fibras musculares normais regeneradas e o terminal nervoso tem papel determinante nestas alterações. O presente trabalho teve por objetivo verificar se o terminal nervoso influencia o padrão de distribuição dos AChRs nas fibras musculares regeneradas distróficas. Animais mdx com 01 mês e 06 meses de idade tiveram o músculo esternomastóideo esquerdo desnervado e injetado com cloridrato de lidocaína. O músculo contra- lateral serviu como controle. Após 10 dias, os animais foram sacrificados, os AChRs marcados com rodamina-a-bungarotoxina e observados ao microscópio confocal. Músculos inervados de animais mdx com 01 mês de idade apresentaram os AChRs distribuídos em ilhas em 75,2% das JNMs observadas (n=137), enquanto animais com 06 meses de idade apresentaram 100% das JNMs (n=114) em ilhas. Na ausência da inervação, os AChRs distribuíram-se em padrão desnervado tipo braços contínuos em 79,4% das JNMs observadas (n=90) de animais com 01 mês de idade e em padrão desnervado tipo ilhas em 100% das JNMs (n=100) de animais com 06 meses de idade. Estes resultados sugerem que o terminal nervoso contribui de forma significativa para as alterações no padrão de distribuição dos AChRs de músculos distróficos inervados / Abstract: Changes in the distribution of acetylcholine receptors have been reported to occur at the neuromuscular junction of mdx mice and may be a consequence of muscle fiber regeneration rather than the absence of dystrophin. In the present study, we examined whether the nerve terminal determines the fate of acetylcholine receptor distribution in the dystrophic muscle fibers of mdx mice. The left sternomastoid muscle of young (1 month old) and adult (6 months old) mdx mice was injected with 60 ml lidocaine hydrochloride to induce muscle degeneration-regeneration. Some mice had their sternomastoid muscle denervated at the time of lidocaine injection. After 10 days of muscle denervation, nerve terminals and acetylcholine receptors were labeled with 4-Di-2-ASP and rhodamine-a-bungarotoxin, respectively, for confocal microscopy. In young mdx mice, 75% (n=137 endplates) of the receptors were distributed in islands. The same was observed in 100% (n=114 endplates) of the adult junctions. In denervated-regenerated fibers of young mice, the receptors were distributed as branch- like aggregates in 80% of the endplates (n=90). In denervated-regene rated fibers of adult mice, the receptors were distributed in island-like aggregates in 100% of the endplates (n=100). These findings suggest that nerve-dependent mechanisms are involved in the changes in receptor distribution in young dystrophic muscles. In older dystrophic muscles other factors may play a role in their distribution / Mestrado / Anatomia / Mestre em Biologia Celular e Estrutural

Orientador: Lea Rodrigues-Simioni / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T14:03:09Z (GMT). No. of bitstreams: 1 Galbiatti_Charlene_M.pdf: 3708573 bytes, checksum: eb349f7f5062bd08e487dc8ae3c13167 (MD5) Previous issue date: 2008 / Resumo: No presente trabalho foram estudados os efeitos neuromusculares da toxina Bmaj-9 isolada do veneno de Bothrops marajoensis no laboratório de química de proteínas da Unicamp. Esta toxina foi ensaiada: nas preparações biventer cervicis de pintainho, nervo frênico diafragma de camundongo e no músculo gastrocnêmio de camundongo. Em preparações de ave, a Bmaj-9 causou bloqueio neuromuscular completo irreversível nas concentrações de 5, 10 e 20 µg/mL, sem afetar as contraturas evocadas pela adição exógena de ACh (110 µM). Além disso mostrou-se pouco ativa na preparação nervo frênico diafragma de camundongo. A Bmaj -9 não interferiu na resposta muscular a estímulos elétricos direto em preparações de biventer cervicis de pintainho previamente curarizadas. Ensaios bioquímicos mostraram que a Bmaj-9 exibiu uma pronunciada atividade fosfolipásica quando comparada a do veneno bruto. Assim, submetendo-se preparações incubadas com a toxina a baixa temperatura (22 °C), observou-se a ausência do seu característico efeito bloqueador neuromuscular. A análise histológica in vitro mostrou que esta fração em baixas concentrações não causou alteração morfológica significativa embora tenha determinado um leve aumento na liberação da enzima creatinoquinase. Em altas concentrações, no entanto, foi observado algum dano muscular na região periférica do músculo. In vivo, a Bmaj-9 causou uma discreta alteração morfológica e também um leve aumento nos níveis de CK após injeção intramuscular, mostrando uma correlação positiva entre os efeitos dos parâmetros estudados - lesão muscular e liberação de CK, esses dados, em nada se comparam aos efeitos de venenos ou toxinas tipicamente botrópicas. O registro do PM mostrou que a Bmaj-9 (10 µg/mL) não produziu despolarização da membrana muscular, e a medida do conteúdo e do tamanho quânticos, em preparação nervo frênico-diafragma de camundongo, mostrou que houve um leve aumento nos valores do conteúdo quânticos após 15 minutos de incubação. Após 60 minutos de incubação, o efeito da Bmaj-9 sobre os valores do conteúdo e tamanho quânticos foram, respectivamente: 70,5 ± 11 e 0,21 ± 0,06 mV. Estes não foram significativamente diferentes quando comparados com os valores controle (68,0 ± 8,9 e 0,17 ± 0,04 mV). Estes resultados sugerem que o bloqueio da transmissão neuromuscular, causado por esta toxina em baixas concentrações, seja muito provavelmente devido a uma ação pré-sináptica, resultado de sua interação com a membrana do terminal nervoso motor. / Abstract: In the present work, the neuromuscular effects of the toxin Bmaj-9 isolated from the venom of Bothrops marajoensis in the laboratory of chemistry of proteins at Unicamp were studied. This toxin was assayed in biventer cervicis preparations of little chicken, phrenic diaphragm nerve of mice and in gastrocnemius muscles of mice. In birds preparation, Bmaj-9 caused irreversible complete neuromuscular blockade in the concentrations of 5, 10 and 20 µg/mL not affecting the contractures caused by the exogenous addition of Ach (110 µM). Moreover, Bmaj-9 was not very active in the phrenic diaphragm preparation of mice. It has not interfered in the muscular response to direct electrical stimuli in biventer cervicis preparations of little chicken previously curarized. Biochemical assays showed that Bmaj-9 exhibited pronounced phospholipase activity when compared to crude venom. Thus, when preparations incubated with the toxin were submitted to low temperatures (22°C), the absence of the characteristic neuromuscular blocking effect of the fraction was observed. The histological analysis in vitro showed that even having determined a low increase on the release of the creatino-kinase enzyme, low concentrations of this fraction did not cause significant morphological alteration. Nevertheless, in high concentrations some muscular damages in the peripheral region of the muscle were observed. In vivo, Bmaj-9 caused a slight morphological alteration and also a discrete increase on the CK levels after intramuscular injection, showing a positive correlation among the effects of the parameters studies - muscular lesion and CK release. These data obtained with Bmaj-9 are extremely inferior to the ones obtained with bothropic venoms or toxins. The registration of the resting membrane potential showed that Bmaj-9 (10 µg/mL) did not produce depolarization of the muscular membrane and the measurement of the content and quantum size, in phrenic diaphragm preparation of mice, showed that a slight increase on the quantum amount was detected after 15 minutes of incubation. After 60 minutes of incubation, the effect of Bmaj-9 over the content and quantum size values was 70.5 ± 11 and 0.21 ± 0.06 mV, respectively; these values were not significantly different when compared to control values (68.0 ± 8.9 and 0.17 ± 0.04 mV). These values suggest that the blockade of the neuromuscular transmission caused by this toxin in low concentrations is probably due to a pre-synaptic action resulting from the interaction of the toxin with the membrane of the motor nerve terminal. / Mestrado / Mestre em Farmacologia

Orientadores: Lea Rodrigues Simioni, Yoko Oshima Franco / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T21:34:25Z (GMT). No. of bitstreams: 1 Oshima_Mario_M.pdf: 4940483 bytes, checksum: bc15df4a36f3737d47cf0f530205de21 (MD5) Previous issue date: 2009 / Resumo: O polietilenoglicol (PEG) tem sido amplamente utilizado em diversos setores, entre eles, o cosmético, odontológico, farmacêutico (até como carreador de fármacos a órgãos-alvo), em virtude de suas fantásticas propriedades químicas. Por tal razão, é denominada uma molécula bioativa e teve o seu consumo interno aprovado pelo Food and Drug Administration (FDA). O objetivo deste trabalho foi pesquisar os efeitos farmacológicos do PEG 400 sobre a junção neuromuscular; a partir de observações experimentais em preparações de camundongo e pintainho. Concentrações de 3 µM, 20 µM e 100 µM de PEG 400, d-Tc (6 µM), neostigmina (12 µM) e dantrolene (30 µM) auxiliaram na busca do entendimento do mecanismo do PEG sobre a junção neuromuscular. Foram utilizadas em técnicas miográfica, eletrofisiológica e análise morfológica, nas preparações nervo frênico-diafragma (NFD) de camundongos e biventer cervicis (BC) de pintainhos. Especificamente, foi investigada a ação do PEG 400 sobre o terminal nervoso, fenda sináptica, receptores nicotínicos ou fibra muscular. Os resultados obtidos através da técnica eltrofisiológica mostraram que o PEG 400 não exibe ação pré-sináptica, pois não interfere no valor do CQ e nem mesmo apresenta ação anticolinesterásica quando comparado à neostigmina como controle positivo; também não mostrou qualquer efeito em receptores nicotínicos, mas alterou a polaridade do sarcolema causando despolarização das fibras musculares (-80 mV para -16 ± 1,7 mV) para o PEG 400 (100 µM). Além disso, o PEG 400 (20 µM ) em BC, exibiu antagonismo ao dantrolene (inibidor de receptor de rianodina) e impediu seu efeito sobre a contração muscular através do Cálcio. Finalmente, o PEG 400 não causa mionecrose (3 µM e 20 µM), nem mesmo em concentrações maiores (100 µM), nas quais causa bloqueio total das respostas do músculo BC. / Abstract: Polyethyleneglycol has been widely used in several areas such as cosmetic, odontology and pharmaceutical (even as drugs carrier) due to its chemical properties. Thus, it is considered a bioactive molecule being its internal consumption approved by the Food and Drug Administration (FDA). The aim of this study was to analyze the pharmacological effects of PEG 400 over the neuromuscular junction through experimental observations in mice preparations due to the increase of the contractile response amplitude (facilitating effect). Conventional myographic techniques, morphological and electrophysiological analysis were used in mice, phrenic nerve diaphragm preparations (PND) and chick, biventer cervicis preparations (BC). Concentrations of 3 µM, 20 µM and 100 µM of PEG 400, d-Tc 6 µM, neostigmine 12 µM and dantrolene 30 µM helped in the search for the understanding of PEG mechanism. Specifically, the action of PEG 400 was investigated over the nervous terminal, the synaptic cleft, nicotinic receptors or muscle fiber. The results obtained through the electrophysiological technique showed that PEG 400 did not exhibit pre-synaptic action and did not act as an anticholinesterasic, as well as neostigmine. It has not presented action over nicotinic receptors but it presented post-synaptic action, interfering on the sarcolemma polarity and reverting the action of the Calcium antagonist, dantrolene, suggesting an interference of the contraction mechanism through Calcium and the ryanodine receptor. Finally, PEG 400 did not cause mionecrosis (3 µM and 20 µM) even in concentrations high enough to cause total muscle blockade (100 µM). / Mestrado / Mestre em Farmacologia

Orientador: Lea Rodrigues Simioni / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T23:41:00Z (GMT). No. of bitstreams: 1 Monzon_GeorginaSucasaca_M.pdf: 3056250 bytes, checksum: d246d55d0b29698ce7253fb6a9fc3f6a (MD5) Previous issue date: 2008 / Resumo: O veneno botrópico possui uma mistura de substâncias protéicas necessárias para a sobrevivência da serpente, dentre elas, as mais amplamente estudadas são as fosfolipases A2, que hidrolisam glicerofosfolipídios de membrana na posição sn-2, liberando isofosfolipídios e ácidos graxos, que além de desempenhar uma ação biológica no processo de digestão de lipídios, exibem também uma ampla variedade de efeitos farmacológicos. O presente trabalho teve como objetivo, caracterizar farmacologicamente uma nova fosfolipase A2 denominada Bp-13 do veneno de Bothrops pauloensis, serpente endêmica do cerrado brasileiro, avaliando sua ação sobre a junção neuromuscular, efeito miotóxico e atividade edematogênica. A Bp-13 foi purificada utilizando-se o sistema cromatográfico HPLC de fase reversa em coluna µ-Bondapack C18; possuindo uma massa molecular de 14 035 Da e atividade catalítica de 2,43 nmol/min/mg (30-35 ºC), dependente de Ca2+ porém inibida por Mg2+, Mn2+, Cd2+ e Sr2+. A atividade neuromuscular foi avaliada em preparações biventer cervicis (BC) de pintainho, extensor digitorum longus (EDL) e nervo frênico-diafragma (NFD) de camundongo, sob estimulação elétrica indireta durante 120 min a 37 ºC. A Bp-13 (3,56 µM) induziu bloqueio neuromuscular completo e irreversível, nas preparações de mamífero, que se mostraram mais sensíveis à ação da toxina do que as preparações de ave. Em preparação BC de pintainho o bloqueio neuromuscular foi de 28 ± 2 % após 120 min de incubação com a Bp-13 (7,12 µM) e a contratura evocada após a adição exógena de ACh foi parcialmente inibida. Concentrações menores (0,71 e 1,42 µM) foram testadas na preparação NFD de camundongo, induzindo o bloqueio da resposta contrátil em 29 ± 5 % e 55 ± 6 %, respectivamente. Em alguns experimentos, onde o Ca2+ foi substituído pelo Sr2+ na solução de Tyrode houve ausência do bloqueio neuromuscular característico da Bp-13 (3,56 µM); evento semelhante foi observado quando a temperatura do banho foi alterada de 37 ºC para 24 ºC, com bloqueio de 37,4 ± 6 % da resposta contrátil. A Bp-13 (3,56 µM) foi capaz de inibir a resposta contrátil a estímulo elétrico direto em preparações NFD de camundongo previamente curarizadas. Com o estudo eletrofisiológico realizado em preparação músculo hemidiafragma de camundongo, avaliaram-se os potenciais de membrana em repouso (PM). A Bp-13 (3,56 µM) causou uma despolarização progressiva do sarcolema, alcançando valores de -80 ± 1 mV (controle) até -37 ± 3 mV após 120 min de incubação com a toxina. Em preparações músculo diafragma pré-tratadas com d-Tubocurarina (10 µM), observou-se uma redução da despolarização induzida pela da Bp-13 (-37 ± 3 mV para -58 ± 2 mV). A miotoxicidade foi avaliada in vitro através de análise morfológica do músculo diafragma de camundongo e in vivo através da determinação da atividade de creatinoquinase (CK). Na análise morfológica considerou-se fibras normais as que mantiveram íntegro o sarcolema com o formato poligonal e fibras alteradas as que apresentavam hipercontração das miofibrilas, células com lesão tipo delta, edemaciadas e vacuolizadas. A porcentagem de fibras lesadas de preparações incubadas com o veneno de Bothrops pauloensis (100 e 50 µg/mL) foi de 14,4 ± 3 % e 8,7 ± 3 % respectivamente; e com a fração Bp-13 (20 e 50 µg/mL) foi de 8,3 ± 4 % e 30,6 ± 5 %, respectivamente. Quando as preparações foram mantidas em solução de Tyrode, onde o Ca2+ foi substituído pelo Sr2+, as alterações morfológicas foram de 36,7 ± 11 %, ou seja, semelhante aos valores observados com a Bp-13 na solução de Tyrode normal. O aumento nos valores de liberação de creatinoquinase, após 30 min da injeção da Bp-13, revelou o efeito miotóxico in vivo, valores que retornaram ao normal após 6 horas. A dose de 10 µg/pata de Bp-13 injetada em ratos via intraplantar induziram a formação de edema de pata após 15 min de injeção da toxina. Conclui-se, então, que a Bp-13 é uma miotoxina, que inibe a resposta neuromuscular dependente de temperatura e de Ca2+, em preparações isoladas EDL e NFD de camundongo, sugerindo que a atividade catalítica possivelmente contribua com o evento farmacológico, embora o efeito mionecrótico in vitro não seja afetado, indicando uma dissociação entre estes efeitos. / Abstract: Bothropic venom has several substances necessary to serpent survival, among them, one of the most widely studied are phospholipase A2 enzymes, that hydrolise sn-2 from membrane phospholipids, releasing lysophospholipids and fatty acids, which beyond playing a biological action in the lipid digestion process, also exhibit a wide variety of pharmacological effects. The present work had as objective to characterize pharmacologically a new phospholipase A2 denominated Bp-13 from the Bothrops pauloensis (endemic serpent to the Brazilian cerrado) venom, evaluating its action at neuromuscular junction, myotoxic effect and edematogenic activity. Bp-13 was purified through chromatographic system, a reverse phase HPLC on a µ-Bondapack C18 column; it has a molecular mass of 14 035 Da, 2,43 nmol/min/mg (30- 35 ºC) catalytic activity Ca2+ dependent, however it was inhibited by Mg2+, Mn2+, Cd2+ and Sr2+. Neuromuscular activity was evaluated in chick biventer cervicis (BC) preparations, extensor digitorum longus (EDL) and mouse phrenic nerve-diaphragm (PND), under indirect electrical stimulation during 120 minutes, at 37oC. Bp-13 (3,56 µM) induced complete and irreversible neuromuscular block in mammalian preparations, which showed to be more sensitive to toxin action than bird preparations. In chick BC preparation, neuromuscular block was 28 ± 2 % after 120 minutes incubation with Bp-13 (7,12 µM) and evoked contracture after exogenous ACh addiction was partially inhibited. Smaller concentrations (0,71 e 1,42 µM) were tested in mouse PND preparation, inducing block of the contractile response in 29 ± 5 % and 55 ± 6 %, respectively. In some experiments, where Ca2+ was substituted for Sr2+ in Tyrode's solution, there was absence of the Bp-13 characteristic neuromuscular block (3,56 µM); similar event was observed when the bath temperature was altered from 37 ºC to 24 ºC, with a block of 37,4 ± 6 % of the contractile response. Bp-13 (3,56 µM) was able to inhibit contractile response to direct electrical stimulus in previously curarized mouse PND preparations. Through electrophysiological study performed in mouse hemidiaphragm muscle preparation, membrane potentials at rest were evaluated (MP). Bp-13 (3,56 µM) caused a progressive sarcolemma depolarization, reaching values from -80 ± 1 mV (control) until - 37 ± 3 mV after 120 minutes in toxin incubation. In d-tubocurarine (10 µM) pre-treated hemidiaphragm muscle preparations, a depolarization reduction was observed, induced by Bp-13 (-37 ± 3 mV to -58 ± 2 mV). Myotoxicity was evaluated in vitro through morphological analysis of the mouse hemidiaphragm muscle and in vivo through determination of creatinkinase (CK) activity. Regarding morphological analysis, fibers which maintained intact sarcolemma with polygonal shape were considered normal, and fibers which presented hypercontraction of myofibrils, delta type lesion cells, edemaciated and vacuolized, were considered altered fibers. Lesioned fibers from Bothrops pauloensis (100 and 50 µg/mL) venom incubated preparations were 14,4 ± 3 % and 8,7 ± 3 % , respectively; and for Bp-13 fraction (20 e 50 µg/mL) they were 8,3 ± 4 % and 30,6 ± 5 %, respectively. When preparations were Ca2+ was substituted for Sr2+, morphological maintained in Tyrode's solution, where alterations were 36,7 ± 11 %, that is similar to the observed values with Bp-13 in normal Tyrode's solution. The increase in creatinkinase release values after 30 minutes of Bp-13 injection revealed in vivo myotoxic effect, values that returned to normal after 6 hours. Bp-13 concentration of 10 µg injected in rats via intraplantar induced paw edema formation after 15 minutes of toxin injection. In conclusion, Bp-13 is a myotoxin that inhibits temperature and Ca2+ dependent neuromuscular response in mouse EDL and PND isolated preparations, suggesting that its catalytic activity possibly contributes to the pharmacological event, although in vitro myotoxic activity was unaffected by these treatments, indicating a dissociation between these effects. / Mestrado / Mestre em Farmacologia

Orientador: Lea Rodrigues Simioni / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T11:19:56Z (GMT). No. of bitstreams: 1 Moura_PriscilaRandazzode_D.pdf: 2478214 bytes, checksum: bbb891d96754c5530c891d237eeb45c9 (MD5) Previous issue date: 2008 / Resumo: Os efeitos miotóxicos dos venenos botrópicos são inquestionáveis pela exuberante manifestação clínica e são extensivamente relatados na literatura. Já os efeitos neurotóxicos têm sido descritos sob condições in vitro, em preparações neuromusculares, com pouca ou nenhuma evidência clínica. O objetivo do presente estudo foi o de contribuir para o conhecimento sobre a caracterização bioquímica e farmacológica da Bp-12, uma nova miotoxina do veneno de B. pauloensis. O perfil cromatográfico do veneno evidenciou 18 picos, o pico 12 (Bp-12) refere-se a nova fração estudada, uma vez que em ensaios preliminares apresentou efeito sobre a JNM. O perfil eletroforético da Bp-12 mostrou a presença de uma única banda protéica com aproximadamente 14 kDa, confirmado pela espectrometria de massa (13.789,56 Da). A composição de aminoácidos apresentou alto conteúdo dos resíduos, Lys, Tyr, Gly, Pro e Cys, típicos de PLA2 básicas. A Bp-12 possui 122 resíduos de a.a.: SLFELGKMIL QETGKNPAKS LGAFYCYCGW GSQGQPKDAV DRCCYVHKCC YKKITGCNPK KDRYSYSWKD KTLVCGEDNS CLKELCECDK AVAICLRENL NTYNKKYRYF LKPLCKKADA AC, com pI de 8,55 e uma alta homologia com outras PLA2 Lys49 botrópicas (92,6 %). A preparação nervo frênico-diafragma (NFD) de camundongo foi mais sensível à ação da toxina do que a preparação biventer cervicis de pintainho. Em preparações NFD, a Bp-12 (3,6 ?M) induziu 50% de bloqueio em 17 ± 7 min, inibindo completamente a resposta contrátil a estímulos indiretos (120 min, 37 °C); o bloqueio neuromuscular irreversível também foi observado, quando o Ca2+ foi substituído pelo Sr2+ na solução de Tyrode, quando a preparação foi curarizada (estímulo direto) ou quando submetida à baixa temperatura (22 °C). O registro do PM mostrou que a partir de 15 min de incubação com a Bp-12 (3,6 ?M) ocorreu uma progressiva despolarização da membrana, com valores de -20,2 ± 1 mV (90 min, p<0,05). Após 60 min de incubação, o efeito da Bp-12 (3,6 ?M) sobre os valores do conteúdo e tamanho quântico e amplitude média foram respectivamente: 50 ± 8; 0,09 ± 0,01 e 3,0 ± 0,3 mV, estes não foram significativamente diferentes quando comparados com os valores controle (68 ± 9; 0,07 ± 0,01 e 3,3 ± 0,2 mV). Nos estudos de miotoxicidade, após 120 min de incubação com 50 ?g/mL de veneno de B. pauloensis ou de Bp-12, o músculo diafragma apresentou áreas de mionecrose da ordem de 26 ± 2 % e 27 ± 1 % (p<0,05), respectivamente. Os níveis de CK plasmático aumentaram significativamente após a administração intramuscular de Bp-12, sendo: 827 ± 92 U/L (50 ?g) e 225 ± 36 U/L (20 ?g) versus 48,3 ± 8 U/L (controle), entretanto, com a via intravenosa não houve aumento significativo, mostrando que a Bp-12 não produz miotoxicidade sistêmica. A Bp-12 (2,5; 5,0 e 10,0 ?g/pata) induziu edema de pata dependente da dose, caracterizada por apresentar início de ação rápido, em torno de 30 min, sendo: 0,4 ± 0,02 mL; 0,6 ± 0,05 mL e 0,7 ± 0,07 mL, respectivamente; também foi calculada a área sob a curva para cada dose injetada, cujos valores foram (mL.h): 0,5 ± 0,02 (salina); 0,9 ± 0,11; 1,3 ± 0,15 e 1,7 ± 0,19, respectivamente. Conclui-se que a Bp-12 é uma nova PLA2 Lys49 miotóxica, de caráter básico, com baixa atividade catalítica, que induz bloqueio neuromuscular irreversível dependente do tempo e da concentração e que apresenta atividades farmacológica e bioquímica características de miotoxinas botrópicas. / Abstract: The myotoxic effects caused by bothropics venoms are unquestionable by their exuberant clinic manifestation and are extensively related in literature, since the neurotoxic effects have been described in vitro conditions, in nerve-muscle preparations, with little or neither clinic evidence. The aim of this study was to contribute for the knowledge about the pharmacological and biochemical characterization of Bp-12, a new myotoxin from B. pauloensis snake venom. The elution profile of B. pauloensis crude venom after purification in one chromatographic step presented eighteen peaks ranging from Bp-1 to Bp-18. Peak 12 was named Bp-12 and resulted in one small peak with a retention time of 37 min, eluting at 56% of buffer B. The SDS-PAGE gel showed one electrophoretic band, indicating that this toxin was obtained with high homogeneity and a molecular mass of around 14 kDa, confirmed by MALDI-TOF mass spectrometry, which showed that the protein had a molecular mass of 13789.56 Da. The amino acids composition showed that Bp-12 presented high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA2. The sequence of Bp-12 contains 122 amino acid residues: SLFELGKMIL QETGKNPAKS LGAFYCYCGW GSQGQPKDAV DRCCYVHKCC YKKITGCNPK KDRYSYSWKD KTLVCGEDNS CLKELCECDK AVAICLRENL NTYNKKYRYF LKPLCKKADA AC, with a pI value of 8.55 and with a high homology with Lys49 PLA2 from other bothropics venoms. In mouse phrenic nerve-diaphragm (PND), the time needed for 50% paralysis was 17 ± 7 min (3.6 ?M). Bp-12 can induce indirect and directly blocked evoked twitches, even in the preparations in which Ca2+ is replaced by Sr2+ or low temperature (22 °C), being the addition of d-tubocurarine required for direct blocking. The resting membrane potential showed that after 15 min of incubation with Bp-12 (3.6 ?M), progressive despolarization decreased from -83.9 ± 1 mV to -20.2 ± 1 mV (90 min, p<0.05). Intracellular recordings of endplate potentials (EPPs) from mouse diaphragm preparations revealed that Bp-12 did no alter the quantal content (50 ± 8, t60, p>0.05 of the control). Incubation with Bp-12 (50 ?g/mL) or B. pauloensis (50 ?g/mL) for 120 min damaged 26 ± 2 % and 27 ± 1 % (p<0,05), respectively. Bp-12 (20 and 50 ?g/50 ?L) has significantly increased the release of CK levels in the serum after 60 min of i.m. administration, being the values: 827 ± 92 U/L and 225 ± 36 U/L (20 ?g/mL). However, i.v. administration has not presented significant increased. Bp-12 (2.5 - 10.0 ?g/paw) induced local oedema formation in rat paw in vivo at around 30 min, being found the values of: 0.4 ± 0.02 mL (2.5 ?g/paw), 0.6 ± 0.05 mL (5.0 ?g/paw) and 0.7 ± 0.07 mL (10.0 ?g/paw). In conclusion, Bp-12 is a new myotoxic PLA2 (Lys49), with low catalitic activity, that is able to induce irreversible neuromuscular blockade and presents biochemical and pharmacological activities characteristic of bothropics miotoxins. / Doutorado / Doutor em Farmacologia

The mouse nervous system undergoes a vast remodelling of synaptic connections postnatally, resulting in a reduced number of innervating axons to target cells within the first few weeks of life. This extensive loss of connections is known as synapse elimination and it plays a critical role in sculpting and refining neural connectivity throughout the nervous system, resulting in a finely tuned and well-synchronised network of innervation. This process has been well characterised at the mouse neuromuscular junction (NMJ), where synapse elimination takes place postnatally in all skeletal muscles. It has been well studied for the reasons that it is easily accessible for live imaging and post-mortem experimental analysis. Studies utilising this synapse to uncover regulators of synapse elimination have mainly focused on the importance of glial cell lysosomal activity, nerve conduction and target-derived growth factor supply. It is clear that non-axonal cell types play key roles in the success of developmental axon retraction at the NMJ, however the role of glial cells in the regulation of this process has not been fully explored, as lysosomal activity is thought of as a consequence of axon pruning rather than a molecular driver. Previous studies have shown that signals emanating from myelinating glial cells can modulate neurofilament composition and transport within the underlying axons. We know that these changes in neurofilament composition and transport are underway during developmental synapse elimination at the NMJ, so it seems logical to predict that myelinating glial cells may play a role in the regulation of axonal pruning. Myelinating glial cells are found along the entire length of lower motor neurons and form physical interactions with the underlying axons at regions known as paranodes. At the paranode, Neurofascin155 (Nfasc155: expressed by the myelinating glial cell) interacts with a Caspr/contactin complex (expressed by the axon). This site has been proposed as a likely site for axon-glial signalling due to the close apposition of the cell membranes. The main focus of this PhD project was to study the potential role of myelinating glial cells in the success of synapse elimination at the NMJ, using a mouse model of paranodal disruption (Nfasc155-/-). Chapters 3 and 4 show the results of this work. This work has revealed a novel role for glia in the modulation of synapse elimination at the mouse neuromuscular junction, mediated by Nfasc155 in the myelinating Schwann cell. Synapse elimination was profoundly delayed in Nfasc155-/- mice and was found to be associated with a non-canonical role for Nfasc155, as synapse elimination occurred normally in mice lacking the axonal paranodal protein Caspr. Loss of Nfasc155 was sufficient to disrupt axonal proteins contributing to cytoskeletal organisation and trafficking pathways in peripheral nerve of Nfasc155-/- mice and lower levels of neurofilament light (NF-L) protein in maturing motor axon terminals. Synapse elimination was delayed in mice lacking NF-L, suggesting that Nfasc155 influences neuronal remodelling, at least in part, by modifying cytoskeletal dynamics in motor neurons. This work provides the first clear evidence for myelinating Schwann cells acting as drivers of synapse elimination, with Nfasc155 playing a critical role in glial cell-mediated postnatal sculpting of neuronal connectivity in the peripheral nervous system. A small section of the results within this thesis are devoted to the study of axon-glial interactions in a mouse model of childhood motor neuron disease, otherwise known as spinal muscular atrophy (SMA). In SMA, there are reduced levels of the ubiquitously expressed survival motor neuron (SMN) protein. The NMJ is a particularly vulnerable target in SMA, manifesting as a breakdown of neuromuscular connectivity and progressive motor impairment. Recent studies have begun to shed light on the role of non-neuronal cell types in the onset and progression of the disease, presenting SMA as a multi-system disease rather than a purely neuronal disorder. Recent evidence has highlighted that myelinating glial cells are significantly affected in a mouse model of SMA, manifesting as an impaired ability to produce key myelin proteins, resulting in deficient myelination. The final results chapter of this thesis (Chapter 5) is focussed on further exploring the effects that loss of SMN has in Schwann cells including their interactions with underlying axons. This work reveals a disruption to axon-glial interaction, shown by a delay in the development of paranodes, supporting the idea that non-neuronal cell types are also affected in SMA. The results within this thesis reveal a novel role for a glial cell protein, Nfasc155, in the modulation of synapse elimination at the NMJ. Mechanistic insight in to Nfasc155’s role in this process is also uncovered and likely involves axonal cytoskeletal transport systems and the filamentous protein NF-L, which have not previously been implicated in the process of synapse elimination. This work highlights an important role for axon-glial interactions during normal postnatal development of the mouse NMJ. This work also highlights a role for axon-glial interactions in disease states of the NMJ. Using a mouse model of SMA, axon-glial interaction was assessed with the finding of a delay in paranodal maturation due to loss of SMN.