La synthèse de molécules possédant une cavité présente un grand intérêt car ces dernières peuvent agir comme récepteurs moléculaires ou comme catalyseurs supramoléculaires et ainsi mimer l'activité des enzymes. Les hémicryptophanes possèdent ainsi des cavités dissymétriques complexes formées à partir d'une unité cyclotrivératrylène et d'une seconde unité de symétrie C3 et présentent des propriétés catalytiques et de reconnaissance particulièrement intéressantes.Les travaux effectués au cours de cette thèse reposent donc sur la synthèse de nouveaux hémicryptophanes et sur leur utilisation pour la reconnaissance moléculaire de composés biologiquement actifs et pour la catalyse supramoléculaire. A ce titre, différentes voies de synthèse ont été abordées et ont permis d'obtenir un panel de nouveaux récepteurs. Ces composés ont alors été appliqués dans un premier temps à la reconnaissance sélective de paires d'ions et de neurotransmetteurs zwitterioniques puis à la reconnaissance stéréosélective de sucres. Les propriétés complexantes de ces composés ont principalement été étudiées par spectroscopie RMN et à l'aide de calculs quantiques. Enfin, la synthèse de complexes supramoléculaires cuivre-hémicryptophane nous a permis de développer l'utilisation de ces composés pour la catalyse biomimétique d'oxydation des alcanes. / Molecular containers are very attractive as they can act as molecular receptors or supramolecular catalysts and so mimic biological entities such as enzymes. Hemicryptophanes are heteroditopic host molecules created from the association of a cyclotriveratrylene unit with another C3-symmetric moiety and which present interesting catalytic and recognition properties. During this thesis, we based our work on the synthesis of new hemicryptophanes and their application, first in molecular recognition of bioactive molecules and then in supramolecular catalysis. Different synthetic paths have been developed to obtain several new receptors. Their binding abilities have then been studied in selective recognition of ion pairs and neurotransmitters and in stereoselective recognition of carbohydrates. These works have been performed mainly thanks to NMR spectroscopy and quantum calculations. Finally, copper-hemicryptophane complexes have been synthesized and used as supramolecular catalysts in C-H oxidation of alkanes.

Toxinas que atuam em canais para Na+ operados por voltagem são as principais responsáveis pelos efeitos tóxicos do envenenamento escorpiônico e podem ser divididas em duas classes: a- e b-neurotoxinas. TsTX-V e TsTX-I da peçonha de Tityus serrulatus (TsV) são, respectivamente, exemplos destas toxinas. Neste trabalho, foram avaliados os efeitos da TsV e destas toxinas sobre a pressão arterial média (PAM) e liberação de catecolaminas em ratos conscientes e não imobilizados, previamente cateterizados, bem como a captação de GABA, dopamina (DA) e glutamato (Glu) em sinaptosomas isolados de cérebro de ratos e a concentração citoplasmática de Ca+2 ([Ca+2 ]C) em células de músculo liso vascular de aorta de ratos. As toxinas foram isoladas por cromatografia de troca iônica (TsTX-I) seguida por CLAE de fase reversa (TsTX-V). As toxinas (15 e 30 g/kg) e TsV (50 e 100 g/kg) foram injetadas intravenosamente. A PAM foi monitorada continuamente através do cateter femoral. Os níveis plasmáticos de adrenalina (ADR) e noradrenalina (NA) foram determinados por CLAE de fase reversa com detector eletroquímico, em 10 min antes e 2,5, 30 e 90 min após os tratamentos. Efeitos pressores máximos foram observados em 2,5?3,5 min. TsV induziu um intenso aumento de longa duração na PAM, bem como a TsTX-I. A TsTX-V mostrou efeitos pressores menores. TsV mostrou os maiores efeitos sobre a liberação de catecolaminas, seguido pela TsTX-I e TsTX-V com um efeito máximo em 2,5 min, seguido por uma gradual redução, permanecendo, todavia, maior que os controles. Embora ambas as classes de toxinas atuem em canais para Na+, TsTX-I mostrou efeitos mais significantes e intensos sobre a liberação de catecolaminas e pressão arterial que a TsTX-V. Parece que a toxicidade da TsTX-V não está somente relacionada à sua capacidade de liberar catecolaminas, indicando que outros neutrotransmissores podem estar envolvidos em sua toxicidade. Nem a TsV ou suas toxinas foram capazes de afetar a captação de 3H-Glu. TsTXI inibiu somente a captação de 3H-DA (IC50 = 28,41 nM). Por outro lado, TsV (0,43ng/mL) inibiu a captação de 3H-GABA e 3H-DA (~50%). TsTX-V mostrou IC50 = 9,37 nM e 22,2 nM para a captação de 3H-GABA e 3HDA, respectivamente. Esses efeitos foram abolidos pelo pré-tratamento com TTX, indicando o envolvimento de canais para Na+ neste processo. Na ausência de Ca+2 e em baixas concentrações de toxinas, a redução não é tão singnificante como na presença de Ca+2. TsTX-V não reduziu a captação de 3H-GABA em células COS-7 expressando os transportadores de GABA, GAT-1 e GAT-3, sugerindo que esta toxina reduz indiretamente o transporte. A redução da captação de 3H-GABA pelos sinaptosomas pode ser devido a rápida e intensa despolarização celular, como revelado por microscopia confocal em células de glioma C6. Assim, TsTX-V causou redução da captação de 3H-GABA e 3H-DA de uma maneira independente de Ca+2, não afetando diretamente os transportadores de GABA, mas em consequencia da despolarização, envolvendo canais para Na+ operados por voltagem. TsV e suas toxinas foram capazes de aumentar a ([Ca2+ ]C , provavelmente por interargir com canais para Na+. Quando comparado aos efeitos despolarizantes do KCl 60 mM (100 %), TsV (100 e 500 g/mL) exibiu um aumento de 49,60 ± 2,58 % e 103,66 ± 5,17 %, respectivamente, enquanto que a TsTX-I e TsTX-V (50 e 100 g/mL de cada) exibiu 43,92 ± 3,06 % e 121,8 ± 8,9 %; 52,56 ± 8,33 % e 79,5 ± 6,1 % de aumento, respectivamente. TsTX-I (100 g/mL) mostrou-se mais potente nesta preparação, visto que uma dose de 100 g/mL causou efeito muito mais intenso do que a TsTX-V na mesma concentração. É possível que as diferenças observadas sobre os efeitos induzidos pela TsTX-I e TsTX-V sejam conseqüência de alterações estruturais entre canais para Na+ presentes em vários tipos de tecidos e inervações. / Voltage-gated Na+ channel toxins are mainly responsible for the toxic effects of scorpion envenoming and can be classified into two classes: a- and b-neurotoxins. TsTX-V and TsTX-I from Tityus serrulatus venom (TsV) are, respectively, examples of these toxins. In this work, were evaluate the effects of TsV and its toxins on mean arterial pressure (MAP) and catecholamines release in conscious unrestrained rats previously catheterized, as well as GABA, dopamine (DA) and glutamate (Glu) uptake in isolated rat brain synaptosomes and cytosolic Ca2+ concentration ([Ca2+ ]C) in vascular smooth muscle cells from rat aorta. Toxins were isolated by ion exchange chromatography (TsTX-I) followed by RP-HPLC (TsTX-V). The toxins (15 and 30 g/kg) and TsV (50 and 100 g/kg) were injected intravenously. MAP was continuously monitored through femoral catheter. Epinephrine (E) and norepinephrine (NE) plasma levels were determined by RP-HPLC with electrochemical detection, at 10 min before and 2.5, 30 and 90 min after treatments. Maximal pressor effects were observed at 2.5 3.5 min. TsV induced intense long lasting increase in MAP, as did TsTX-I. TsTX-V showed the lowest pressor effects. TsV showed the highest effects on catecholamines release, followed by TsTX-I and TsTX-V with maximal effect at 2.5 min, followed by a gradual reduction, however remaining higher than controls. Although both toxins act on Na+ channels, TsTX-I displayed significant and more intense effects on catecholamines release and blood pressure than TsTX-V. It seems that the toxicity of TsTX-V is not related only with its ability to release catecholamines, indicating that other neurotransmitters, may be involved in its toxicity. Neither the TsV or its toxins was capable to affect the 3H-Glu uptake. TsTX-I inhibited only 3H-DA uptake (IC50 = 28.41 nM). On the other hand, TsV (0.43ng/mL) inhibited both 3H-GABA and 3H-DA uptake (~50%). TsTX-V showed IC50 = 9.37 nM and 22.2 nM for 3H-GABA and 3H-DA uptake, respectively. These effects were abolished by pre-treatment with TTX, indicating the involvement of Na+ channels in this process. In the absence of Ca2+ and at low concentrations of toxin, the reduction is not as significant as in the presence of Ca2+. TsTX-V did not reduce 3H-GABA uptake in COS-7 cells expressing GABA transporters GAT-1 and GAT-3, suggesting that this toxin indirectly reduces the transport. The reduced 3H-GABA uptake by synaptosomes could be due to fast and intense cell depolarization as revealed by confocal microscopy of C6 glioma cells. Thus, TsTX-V causes reduction on 3H-GABA and 3H-DA uptake in a Ca2+-independent manner, not affecting directly GABA transporters, but, in consequence of depolarization, involving voltage-gated Na+ channels. TsV and its toxins were able to increase the ([Ca2+ ]C , probably by interact with Na+ channels. When compared to KCl 60 mM depolarizing effect (100 %), TsV (100 and 500 ?g/mL), showed an increase of 49.60 ± 2.58 % and 103.66 ± 5.17 %, respectively, whereas TsTX-I and TsTX-V (50 and 100?g/mL of each) showed 43.92 ± 3.06 % and 121.8 ± 8.9 %; 52.56 ± 8.33 % and 79.5 ± 6.1 %, respectively. TsTX-I (100 ?g/mL) showed most potent effects in this type of preparation, since induced most intense effect that TsTX-V in the same concentration. Thus, it is possible that the differences observed on the effects induced by both toxins are consequence of structural changes among Na+ channels present in several types of tissues and innervations .

La science a avancé depuis le XIX ème siècle. Des nouveaux outils sont apparus : la microscopie optique nous donne la vision des cellules, la microscopie électronique nous entraine au cœur de celles-ci. L’imagerie par résonance magnétique est apparue dans les années soixante-dix. Depuis son évolution, l’IRM nous entraine de plus en plus loin dans les profondeurs secrètes de nos cerveaux. La possibilité d’observer l’activité neuronale à l’aide de l’imagerie fonctionnelle est une grande révolution. Cette thèse montrera la possibilité que l’on a d’observer l’activité d’un neurone individuel sans modification de son réseau grâce à l’imagerie rehaussée au manganèse. L’étude sera effectuée sur des Aplysies à très haut champ (17T). Ces animaux sont des mollusques marins gastropodes qui possèdent une particularité : leurs neurones sont de tailles importantes, ils peuvent atteindre 1 mm de diamètre. Leurs neurones sont regroupés en plusieurs ganglions. Mon étude portera sur le ganglion buccal, qui est le ganglion le plus étudié dans les recherches en électrophysiologie. Avant de réaliser les acquisitions, j’ai dû concevoir plusieurs antennes de tailles microscopiques adaptées à la taille des ganglions. En réduisant la taille des antennes, le rapport signal sur bruit augmente. Dans un deuxième temps, une double antenne a été développée permettant l’acquisition de deux échantillons simultanément. Cette antenne a nécessité de créer des préamplificateurs fonctionnant à 730 MHz. La première série d’expériences a permis d’observer l’évolution de l’activité neuronale selon différents stimulus liés au comportement alimentaire des aplysies in-vivo. J’ai montré grâce à la technique mise en place que l’on peut distinguer par IRM l’activité de chaque neurone face à un stimulus. Par la suite, pour continuer ce travail, une deuxième série d’expériences a été effectuée in-vitro. J’ai étudié le comportement des neurones selon les neuro-stimulateurs perfusés : la dopamine et la sérotonine, tous les deux présents naturellement dans l’aplysie. Globalement les neurones ont été activés mais après les avoir observés individuellement, j’ai remarqué quelques différences selon les neurotransmetteurs. Cette technique peut maintenant être utilisée pour étudier d’autres conduites de l’aplysie comme le comportement compulsif. L’étude sur la mémoire peut être aussi envisagée. Les origines comportementales ont probablement des mécanismes identiques entre les différentes espèces animales et donc avec l’Homme comme l’a démontré les études d’Eric Kandel sur la mémoire. / Science has evolved since the 19th century. New tools have appeared such as optical microscopy which gives us the vision of cells and electronic microscopy which leads us into their hearts. The magnetic resonance imaging appeared in the seventies. Evolving over time, the MRI has taken us farther and farther into the secret depths of our brains. The possibility of observing the neuronal activity thanks to the functional imaging is a major evolution. This thesis will show the possibility we have to observe the activity of a single neuron without modification of its network thanks to the manganese enhanced magnetic resonance imaging technique. The study was done on the Aplysia at very high field magnet (17T). These animals are marine gastropod mollusks with a peculiarity: their neurons are of important size and can reach 1 mm in diameter. Their neurons are grouped into several ganglia. My study concerns the buccal ganglion which is the most studied ganglia in the research in electrophysiology. Before making any acquisitions, I had to conceive several microscopic coils adapted to the size of the ganglions. By reducing the size of the coils, the signal of the noise ratio increases. Then, a double coil allowing the simultaneous acquisition of two samples was built. This antenna required the construction of pre-amplifiers operating at 730 MHz. The first series of experiments helped observe the evolution of the neuronal activity according to different stimuli linked to the eating habits of the Aplysia in vivo. Thanks to the technique implemented, I shall show that, using MRI, it is possible to distinguish the activity of each neuron with respect to a stimulus. Afterwards, to continue this work, a second series of experiments was made in vitro. I studied the behavior of neurons when perfused with neural stimulators: dopamine and serotonin, both naturally present in the Aplysia. Generally, all neurons were activated but when observing them individually, I noticed some differences. Studies in electrophysiology will allow us to get a better understanding and a confirmation of the results of this study. The MEMRI technique can be used in the future to study various disorders such as compulsive behaviors, which are present in the Aplysia, and probably have the same origins as in humans, given that many fundamental processes (such as memory studied by Eric Kandel who he demonstrated that human and Aplysia memories works with the same mechanism) are similar between the two species.

Une partie des recherches actuelles dans le domaine de l’analyse chimique concerne la miniaturisation et l’intégration d’étapes analytiques afin de répondre, entre autres, à des besoins de portabilité, d’automatisation mais aussi d’apporter des solutions pour analyser des échantillons de plus en plus petits. Le développement et la mise en œuvre de colonnes monolithiques d’affinité boronate (µBAMC) couplées « in-line » à des techniques séparatives miniaturisées s’inscrit dans cette démarche. Ce travail de thèse s’est focalisé sur (1) une compréhension des mécanismes de rétention en chromatographie d’affinité boronate (interactions spécifiques avec les composés cis-diols, conditions de reconnaissance, interactions secondaires), (2) le développement de supports monolithiques d’affinité boronate miniaturisés et (3) leur couplage «in-line» avec une séparation électrocinétique et détection conventionnelle dans un format capillaire. Différentes voies d’élaboration de colonnes monolithiques ont été comparées (en termes d’affinité, de nombre de sites boronate actifs et de stabilité). La faisabilité du couplage en ligne de ces supports µBAMC avec une étape de séparation électrophorétique (par CZE et CIEF) a été démontrée vis-à-vis de la purification/préconcentration et séparation de 3 catécholamines contenant des groupements cis-diols (Adrénaline, Noradrénaline et Dopamine) dans l’urine. Les couplages ont été optimisés avec succès permettant l’analyse automatisée et miniaturisée de ces neurotransmetteurs dans l’urine (volume échantillon < 10 µL) avec des limites de détection de l’ordre de la dizaine de ppb et des taux de récupération proches de 100 % / Part of the current research in the field of chemical analysis concerns the miniaturization and the integration of analytical steps in order to meet, among other things, the need of portability and automation but also to provide solutions for analyzing small samples. The development and implementation of monolithic boronate affinity columns (µBAMC) in-line coupled to miniaturized separation techniques is part of this approach. This thesis work focused on (1) an understanding of the retention mechanisms in boronate affinity chromatography (specific interactions with cis-diol compounds, recognition conditions and secondary interactions), (2) the development of miniaturized boronate affinity monolithic supports and (3) their in-line coupling with electrokinetic separation and conventional detection in a capillary format. Different ways of elaboration of monolithic columns were compared (in terms of affinity, number of actives sites and stability). The feasibility of in-line coupling of these µBAMC supports with an electrophoretic separation step (by CZE and CIEF) has been demonstrated in terms of purification / preconcentration and separation of 3 catecholamines containing cis-diol groups (adrenaline, noradrenaline and dopamine) in urine. The couplings have been successfully optimized allowing the automated and miniaturized analysis of these neurotransmitters in urine (sample volume <10 µl) with limits of detection of about the tens of ppb and recovery yields close to 100 %

Made available in DSpace on 2014-12-17T15:16:09Z (GMT). No. of bitstreams: 1 AdrianaJOB_DISSERT.pdf: 1566198 bytes, checksum: 766db20794ef85bc19aa4a81b03784d3 (MD5) Previous issue date: 2010-08-30 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / To the vertebrates, maintain body balance against the gravitational field and be able to orient themselves in the environment are fundamental aspects for survival, in which the participation of vestibular system is essential. As part of this system, the vestibular nuclear complex is the first central station that, by integrating many information (visual, proprioceptive), and the vestibular, assumes the lead role in maintaining balance. In this study, the vestibular nuclear complex was evaluated in relation to its cytoarchitecture and neurochemical content of cells and axon terminals, through the techniques of Nissl staining and immunohistochemistry for neuronal specific nuclear protein (NeuN), glutamate (Glu), substance P (SP), choline acetyltransferase (ChAT) (enzyme that synthesizes acetylcholine-Ach) and glutamic acid decarboxylase (GAD) (enzyme that synthesizes gamma-amino butyric acid-GABA). The common marmoset (Callithrix jacchus) was used as experimental animal, which is a small primate native from the Atlantic Forest in the Brazilian Northeast. As results, the Nissl technique, complemented by immunohistochemistry for NeuN allowed to delineate the vestibular nucleus superior, lateral, medial and inferior (or descending) in the brain of the common marmoset. Neurons and terminals immunoreactive to Glu and ChAT and only immunoreactive terminals to SP and GAD were seen in all nuclei, although in varying density. This study confirms the presence in the vestibular nuclei of the common marmoset, of Glu and SP in terminals, probably from the first order neurons of vestibular ganglion, and of GABA in terminals, presumably from Purkinge cells of the cerebellum. Second-order neurons of the vestibular nuclei seem to use Glu and Ach as neurotransmitters, judging by their expressive presence in the cell bodies of these nuclei in common marmosets, as reported in other species / Para os vertebrados, manter o equil?brio corporal contra o campo gravitacional e ser capaz de orientar-se no ambiente s?o aspectos fundamentais para a sobreviv?ncia, nos quais ? essencial a participa??o do sistema vestibular. Como parte deste sistema, o complexo nuclear vestibular ? a primeira esta??o central que, ao integrar v?rias informa??es (visual, proprioceptiva), al?m da vestibular, assume o papel principal na manuten??o do equil?brio. Neste estudo, o complexo nuclear vestibular do sagui foi avaliado com rela??o a sua citoarquitetura e conte?do neuroqu?mico de c?lulas e terminais ax?nicos, atrav?s das t?cnicas de colora??o de Nissl e imuno-histoqu?mica para prote?na neuronal nuclear espec?fica (NeuN), glutamato (Glu), subst?ncia P (SP), colina acetiltransferase (ChAT) (enzima de s?ntese da acetilcolina-Ach), e descarboxilase do ?cido glut?mico (GAD) (enzima de s?ntese do ?cido gama-amino-but?rico-GABA). Foi utilizado como animal experimental o sagui (Callithrix jacchus), um pequeno primata nativo da Mata Atl?ntica do Nordeste Brasileiro. Como resultados, a t?cnica de Nissl, complementada pela imuno-histoqu?mica para NeuN, permitiu delinear os n?cleos vestibulares superior, lateral, medial e inferior (ou descendente) no enc?falo do sagui. Neur?nios e terminais imunorreativos a Glu e ChAT e apenas terminais imunorreativos a SP e GAD foram vistos em todos os n?cleos, embora em densidade vari?vel. Este trabalho confirma a presen?a nos n?cleos vestibulares do sagui, de Glu e SP em terminais, provavelmente provenientes dos neur?nios de primeira ordem do g?nglio vestibular, e de GABA em terminais, supostamente provenientes das c?lulas de Purkinge do cerebelo. Neur?nios de segunda ordem dos n?cleos vestibulares parecem usar Glu e Ach como neurotransmissores, a julgar pela sua expressiva presen?a em peric?rios destes n?cleos no sag?i, como relatado em outras esp?cies

Made available in DSpace on 2014-12-17T15:36:32Z (GMT). No. of bitstreams: 1 ExpeditoSNJ.pdf: 2182833 bytes, checksum: 1d14b6b7f5a8b99dccd3ec09fd418133 (MD5) Previous issue date: 2007-12-07 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / In this study, two circadian related centres, the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) were evaluated in respect to their cytoarchitecture, retinal afferents and chemical content of major cells and axon terminals with a tract tracer and immunohistochemical techniques in the rock cavy (Kerodon rupestris), a Brazilian caviidae rodent species. The rock cavy SCN is innervated in its ventral portion by terminals from the predominantly contralateral retina. It also contains neurophisin and vasoactive intestinal polypeptide immunoreactive cell bodies and neuropeptide Y and enkephalin immunopositive fibres and terminals and is marked by intense GFAP immunoreactivity. The IGL receives a predominantly contralateral retinal projection, contains neuropeptide Y and nitric oxide synthase producing neurons and enkephalin immunopositive terminals and is characterized by dense GFAP immunoreactivity. This is the first report examining the neural circadian system in a crepuscular rodent species for which circadian properties have been described. The results are discussed comparing with what has been described for other species and in the context of the functional significance of these centres / O sistema de temporiza??o circadiana compreende um conjunto de estruturas neurais diferenciadas, que incluem um marcapasso central, o qual produz ritmicidade na aus?ncia de est?mulos externos; vias de entrada, incluindo as afer?ncias retininas, que permitem a sincroniza??o dos ritmos aos ciclos ambientais; e vias de sa?da, que conectam o marca-passo aos efetores comportamentais. Entre os componentes centrais do sistema de temporiza??o circadiana de mam?feros, destaca-se o n?cleo supraquiasm?tico (NSQ) do hipot?lamo, at? o presente o ?nico marca-passo circadiano formalmente comprovado, e o folheto intergeniculado (FIG) do complexo geniculado lateral do t?lamo, que atua como modulador do marca-passo. Neste estudo, estes dois centros foram avaliados com rela??o a sua citoarquitetura, padr?o de inerva??o retiniana e conte?do neuroqu?mico de c?lulas e terminais ax?nicos, usando um tra?ador neural e t?cnicas imuno-histoqu?micas no moc? (Kerodon rupestris), um roedor nativo do Nordeste Brasileiro, cuja atividade locomotora exibe um padr?o circadiano predominantemente crepuscular. O NSQ do moc? ? inervado em sua por??o ventrolateral por terminais da retina predominantemente contralateral. O NSQ ? dotado de neur?nios contendo neurofisina (NPH) e polipept?deo intestinal vasoativo (VIP) e as prote?nas ligantes de c?lcio calbindina (CB) e cal-retinina (CR), terminais imunorreativos a neuropept?deo Y (NPY) e encefalina (ENK) e ? tamb?m marcado por imunorreatividade ? prote?na ac?dica fibrilar glial (GFAP). O FIG recebe uma proje??o retiniana predominantemente contralateral, cont?m neur?nios produtores de NPY, CB e CR, terminais positivos para ENK e ? marcado por imunorreatividade a GFAP. Este ? o primeiro trabalho a examinar o sistema de temporiza??o circadiana (STC) numa esp?cie de roedor crepuscular, para o qual as propriedades formais dos ritmos circadianos tenham sido descritas. Os resultados s?o comparados com resultados previamente descritos em outras esp?cies diurnas e noturnas e discutidos no seu contexto funcional

Toxinas que atuam em canais para Na+ operados por voltagem são as principais responsáveis pelos efeitos tóxicos do envenenamento escorpiônico e podem ser divididas em duas classes: a- e b-neurotoxinas. TsTX-V e TsTX-I da peçonha de Tityus serrulatus (TsV) são, respectivamente, exemplos destas toxinas. Neste trabalho, foram avaliados os efeitos da TsV e destas toxinas sobre a pressão arterial média (PAM) e liberação de catecolaminas em ratos conscientes e não imobilizados, previamente cateterizados, bem como a captação de GABA, dopamina (DA) e glutamato (Glu) em sinaptosomas isolados de cérebro de ratos e a concentração citoplasmática de Ca+2 ([Ca+2 ]C) em células de músculo liso vascular de aorta de ratos. As toxinas foram isoladas por cromatografia de troca iônica (TsTX-I) seguida por CLAE de fase reversa (TsTX-V). As toxinas (15 e 30 g/kg) e TsV (50 e 100 g/kg) foram injetadas intravenosamente. A PAM foi monitorada continuamente através do cateter femoral. Os níveis plasmáticos de adrenalina (ADR) e noradrenalina (NA) foram determinados por CLAE de fase reversa com detector eletroquímico, em 10 min antes e 2,5, 30 e 90 min após os tratamentos. Efeitos pressores máximos foram observados em 2,5?3,5 min. TsV induziu um intenso aumento de longa duração na PAM, bem como a TsTX-I. A TsTX-V mostrou efeitos pressores menores. TsV mostrou os maiores efeitos sobre a liberação de catecolaminas, seguido pela TsTX-I e TsTX-V com um efeito máximo em 2,5 min, seguido por uma gradual redução, permanecendo, todavia, maior que os controles. Embora ambas as classes de toxinas atuem em canais para Na+, TsTX-I mostrou efeitos mais significantes e intensos sobre a liberação de catecolaminas e pressão arterial que a TsTX-V. Parece que a toxicidade da TsTX-V não está somente relacionada à sua capacidade de liberar catecolaminas, indicando que outros neutrotransmissores podem estar envolvidos em sua toxicidade. Nem a TsV ou suas toxinas foram capazes de afetar a captação de 3H-Glu. TsTXI inibiu somente a captação de 3H-DA (IC50 = 28,41 nM). Por outro lado, TsV (0,43ng/mL) inibiu a captação de 3H-GABA e 3H-DA (~50%). TsTX-V mostrou IC50 = 9,37 nM e 22,2 nM para a captação de 3H-GABA e 3HDA, respectivamente. Esses efeitos foram abolidos pelo pré-tratamento com TTX, indicando o envolvimento de canais para Na+ neste processo. Na ausência de Ca+2 e em baixas concentrações de toxinas, a redução não é tão singnificante como na presença de Ca+2. TsTX-V não reduziu a captação de 3H-GABA em células COS-7 expressando os transportadores de GABA, GAT-1 e GAT-3, sugerindo que esta toxina reduz indiretamente o transporte. A redução da captação de 3H-GABA pelos sinaptosomas pode ser devido a rápida e intensa despolarização celular, como revelado por microscopia confocal em células de glioma C6. Assim, TsTX-V causou redução da captação de 3H-GABA e 3H-DA de uma maneira independente de Ca+2, não afetando diretamente os transportadores de GABA, mas em consequencia da despolarização, envolvendo canais para Na+ operados por voltagem. TsV e suas toxinas foram capazes de aumentar a ([Ca2+ ]C , provavelmente por interargir com canais para Na+. Quando comparado aos efeitos despolarizantes do KCl 60 mM (100 %), TsV (100 e 500 g/mL) exibiu um aumento de 49,60 ± 2,58 % e 103,66 ± 5,17 %, respectivamente, enquanto que a TsTX-I e TsTX-V (50 e 100 g/mL de cada) exibiu 43,92 ± 3,06 % e 121,8 ± 8,9 %; 52,56 ± 8,33 % e 79,5 ± 6,1 % de aumento, respectivamente. TsTX-I (100 g/mL) mostrou-se mais potente nesta preparação, visto que uma dose de 100 g/mL causou efeito muito mais intenso do que a TsTX-V na mesma concentração. É possível que as diferenças observadas sobre os efeitos induzidos pela TsTX-I e TsTX-V sejam conseqüência de alterações estruturais entre canais para Na+ presentes em vários tipos de tecidos e inervações. / Voltage-gated Na+ channel toxins are mainly responsible for the toxic effects of scorpion envenoming and can be classified into two classes: a- and b-neurotoxins. TsTX-V and TsTX-I from Tityus serrulatus venom (TsV) are, respectively, examples of these toxins. In this work, were evaluate the effects of TsV and its toxins on mean arterial pressure (MAP) and catecholamines release in conscious unrestrained rats previously catheterized, as well as GABA, dopamine (DA) and glutamate (Glu) uptake in isolated rat brain synaptosomes and cytosolic Ca2+ concentration ([Ca2+ ]C) in vascular smooth muscle cells from rat aorta. Toxins were isolated by ion exchange chromatography (TsTX-I) followed by RP-HPLC (TsTX-V). The toxins (15 and 30 g/kg) and TsV (50 and 100 g/kg) were injected intravenously. MAP was continuously monitored through femoral catheter. Epinephrine (E) and norepinephrine (NE) plasma levels were determined by RP-HPLC with electrochemical detection, at 10 min before and 2.5, 30 and 90 min after treatments. Maximal pressor effects were observed at 2.5 3.5 min. TsV induced intense long lasting increase in MAP, as did TsTX-I. TsTX-V showed the lowest pressor effects. TsV showed the highest effects on catecholamines release, followed by TsTX-I and TsTX-V with maximal effect at 2.5 min, followed by a gradual reduction, however remaining higher than controls. Although both toxins act on Na+ channels, TsTX-I displayed significant and more intense effects on catecholamines release and blood pressure than TsTX-V. It seems that the toxicity of TsTX-V is not related only with its ability to release catecholamines, indicating that other neurotransmitters, may be involved in its toxicity. Neither the TsV or its toxins was capable to affect the 3H-Glu uptake. TsTX-I inhibited only 3H-DA uptake (IC50 = 28.41 nM). On the other hand, TsV (0.43ng/mL) inhibited both 3H-GABA and 3H-DA uptake (~50%). TsTX-V showed IC50 = 9.37 nM and 22.2 nM for 3H-GABA and 3H-DA uptake, respectively. These effects were abolished by pre-treatment with TTX, indicating the involvement of Na+ channels in this process. In the absence of Ca2+ and at low concentrations of toxin, the reduction is not as significant as in the presence of Ca2+. TsTX-V did not reduce 3H-GABA uptake in COS-7 cells expressing GABA transporters GAT-1 and GAT-3, suggesting that this toxin indirectly reduces the transport. The reduced 3H-GABA uptake by synaptosomes could be due to fast and intense cell depolarization as revealed by confocal microscopy of C6 glioma cells. Thus, TsTX-V causes reduction on 3H-GABA and 3H-DA uptake in a Ca2+-independent manner, not affecting directly GABA transporters, but, in consequence of depolarization, involving voltage-gated Na+ channels. TsV and its toxins were able to increase the ([Ca2+ ]C , probably by interact with Na+ channels. When compared to KCl 60 mM depolarizing effect (100 %), TsV (100 and 500 ?g/mL), showed an increase of 49.60 ± 2.58 % and 103.66 ± 5.17 %, respectively, whereas TsTX-I and TsTX-V (50 and 100?g/mL of each) showed 43.92 ± 3.06 % and 121.8 ± 8.9 %; 52.56 ± 8.33 % and 79.5 ± 6.1 %, respectively. TsTX-I (100 ?g/mL) showed most potent effects in this type of preparation, since induced most intense effect that TsTX-V in the same concentration. Thus, it is possible that the differences observed on the effects induced by both toxins are consequence of structural changes among Na+ channels present in several types of tissues and innervations .

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Indiana University-Purdue University Indianapolis (IUPUI) / Neuropathic pain is a serious health problem that greatly impairs quality of life. The International Association for the Study of Pain (IASP) defines neuropathic pain as ‘pain arising as a direct consequence of a lesion or disease affecting the nervous system’. It is important to note that with neuropathy the chronic pain is not a symptom of injury, but rather the pain is itself a disease process. Novel interactions between the nervous system and elements of the immune system may be key facets to a chronic disease state. One of particular note is the recent finding supporting an interaction between an immune response protein high mobility group box 1 (HMGB1) and Toll like receptor 4 (TLR4). HMGB1 is an endogenous ligand for TLR4 that influences the induction of cytokines in many non-neuronal cells. After tissue damage or injury, HMGB1 may function as a neuromodulatory cytokine and influence the production of pro-nociceptive mediators altering the state of sensory neurons. Very little is known about the HMGB1-TLR4 interaction in sensory neurons and whether chronic changes in endogenous HMGB1 signaling influence the establishment of neuropathic pain. This thesis aims to determine whether a physiologically relevant neuroimmune interaction involving endogenous HMGB1 and TLR4 in the dorsal root ganglia is altered following a tibial nerve injury model of neuropathic pain. I hypothesized that sensitization of sensory neurons following a peripheral nerve injury is dependent on endogenous HMGB1 and TLR4. The studies presented here demonstrate that HMGB1 undergoes subcellular redistribution from the nucleus to the cytoplasm in primary afferent neurons following peripheral nerve injury. Further, the presence of extracellular HMGB1 may directly contribute to peripheral sensitization and injury-induced tactile hyperalgesia. Though thought to be important as a pivotal receptor for HMGB1 activation, neuronal protein expression of TLR4 does not appear to influence the effects of HMGB1-dependent behavioral changes following peripheral nerve injury. Taken together, these findings suggest that extracellular HMGB1 may serve as an important endogenous cytokine that contributes to ongoing pain hypersensitivity in a rodent model of neuropathic pain.