Submitted by Ana Damasceno (ana.damasceno@unipampa.edu.br) on 2016-09-13T20:01:43Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Investigação bioquímica e eletrofisiológica da atividade neurotóxica da urease de Canavalia Ensiformis sobre o sistema nervoso de mamíferos.pdf: 4036702 bytes, checksum: 7c0fc1ca67b3af15c724790f838de2ed (MD5) / Made available in DSpace on 2016-09-13T20:01:43Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Investigação bioquímica e eletrofisiológica da atividade neurotóxica da urease de Canavalia Ensiformis sobre o sistema nervoso de mamíferos.pdf: 4036702 bytes, checksum: 7c0fc1ca67b3af15c724790f838de2ed (MD5) Previous issue date: 2016 / A urease de Canavalia ensiformis (Jack Bean Urease, JBU) possui uma cadeia polipeptídica de 90.770 kDa, com 840 resíduos de aminoácidos. Do ponto de vista biotecnológico, já foram caracterizadas suas ações fungicida e inseticida. Quando administrada na corrente sanguínea de mamíferos, esta, induz convulsões que culminam em morte dos animais, um efeito cujo mecanismo de ação ainda carece de investigação. O objetivo desse trabalho foi investigar os padrões eletrofisiológicos da JBU no sistema nervoso central e periférico de roedores in vivo e in vitro, e as correlações bioquímicas em termos de viabilidade celular e exocitose do L-glutamato. Nos ensaios bioquímicos, verificou-se aumento na liberação de L-glutamato em sinaptossomas corticais de ratos na concentração de 100 nM. Os ensaios de viabilidade celular por MTT em fatias de hipocampo de camundongos não demonstraram alterações. Nos ensaios eletrofisiológicos, verificou-se redução significativa na amplitude do potencial de ação composto (CAP) de nervo ciático de camundongo, nas doses de 1 e 10 nM. Nos ensaios eletroencefalográficos em ratos a injeção da urease (10nM) no hipocampo induziu um traçado de espícula-onda. A redução na amplitude do CAP provavelmente está relacionada a uma inibição dos canais de sódio voltagem-dependentes, já que a administração posterior de tetrodotoxina, não aumentou o nível de bloqueio da condução. Nossos resultados demonstram um efeito excitatório da JBU do tipo crise de ausência sobre o sistema nervoso central de mamíferos. Este resultado sugere o envolvimento dos canais de Ca2+ do tipo T nos efeitos excitatórios da JBU. O bloqueio da condução do nervo ciático de camundongos sugere o envolvimento de canais de Na+ voltagem dependentes, o qual corroboraria a atividade excitatória da urease sobre o sistema nervoso central de mamíferos. / The Canavalia ensiformis urease (Jack Bean Urease) has a 90,770 kDa, polypeptide containing 840 aminoacid residues. JBU is known to exhibit insecticidal and fungicidal activities. When administered endovenously in mammalians, it induces tonic clonic convulsions culminating in the death of the animals. This mechanisms involved in the excitatory activity of JBU has not been elucidated so far. In this work, we sought to investigate the central and peripheral electrophysiological patterns of Jack Bean Urease in rodents, in vivo and in vitro, as well as the biochemical correlation of cell viability and glutamate release. In the biochemical assays, JBU induced increase in L-Glutamate release in rat cortical sinaptossomes, with no alteration of mice hippocampal cell viability. The electrophysiological assays, showed that JBU induce a significant decrease on mice sciatic nerve compound action potentials (CAP), and spike-wave discharges (SWD) similar to “petit mal” seizures when injected directly in the hippocampus (10 nM). The decrease in CAP amplitude is related to a blockage of voltage-gated sodium channels, since it was not affected by the concomitant application of tetrodotoxin. Our results show that JBU exerts an effect of spike wave discharges-like activity over the mammalian central nervous system. This later result suggests an involvement of T-type voltage gated calcium channels in the excitatory activity of JBU. The blockade of mouse sciatic nerve compound action potential conduction corroborates the excitatory activity of the urease upon the mammalian central nervous system.

L’expression anormale du canal sodique Nav1.5 dans le cancer du sein est corrélée au développement métastatique et à une mortalité augmentée. Le canal Nav1.5 est localisé dans les invadopodes des cellules cancéreuses mammaires humaines MDA-MB-231 et augmente leur activité protéolytique par une modulation allostérique de l’échangeur NHE-1 et l’activation de protéases acides. In vivo, dans un modèle de xénogreffe sur souris NMRI nude, l’expression de Nav1.5 potentialise la colonisation des poumons par les cellules cancéreuses mammaires humaines. Cette colonisation métastatique est inhibée par un traitement à la ranolazine, un inhibiteur pharmacologique des canaux Nav1.5. La sous-unité β4, auxiliaire des canaux Nav, voit son expression diminuer au cours de la progression cancéreuse, ce qui est associé in vitro à une augmentation de l’invasivité cellulaire. Cette augmentation d’invasivité semble indépendante du canal Nav1.5 et pourrait être associée à une transition des cellules vers un phénotype amiboïde. En conclusion, l’expression de Nav1.5 et la perte d’expression de β4 semblent jouer des rôles complémentaires dans l’invasivité des cellules cancéreuses. / The abnormal expression of sodium channel Nav1.5 in breast cancer is correlated with metastatic development and an increased mortality. The Nav1.5 channel is located in invadopodia in human breast cancer cells MDA-MB-231, where it increases proteolytic activity by allosteric modulation of exchanger NHE-1 and activation of acidic proteases. In vivo, in a xenograft model in nude NMRI mice, the expression of Nav1.5 potentiates lung colonization by human breast cancer cells. Metastatic colonization is inhibited by treatment with ranolazine, a pharmacological inhibitor of Nav1.5. The β4 subunit, an auxiliary subunit of Nav channels, is expressed at low levels or lost when tumors are more aggressive, and its suppression in vitro increases celI invasiveness. This increase seems to be independent of Nav1.5 and could be associated with the transition of cells to an amoeboid phenotype. In conclusion, Nav1.5 expression and the loss of β4 expression seem to play complementary roles in the invasiveness of cancer cells.

A diabetes mellitus (DM) pode levar a distúrbios da motilidade gastrointestinal que podem se tornar clinicamente relevantes em alguns pacientes. Modelos de Dm em roedores indicam anormalidades morfofuncionais do sistema nervoso entérico nessa doença. Neste trabalho, avaliamos se o DM experimental pode levar a alterações na eferência colinérgica excitatória, densidade neuronal e expressão dos canais de sódio voltagemdependente (Nav) no plexo mioentérico do íleo. Hiperglicemia induzida por estreptozotocina em ratas foi desenvolvida durante oito semanas. Experimentos de imunofluorescência de tripla marcação revelaram que a densidade de neurônios por área do íleo no DM foi significativamente reduzida quando comparada ao controle. Foram observadas reduções médias de 52,2% do total de neurônios (p=0,0001); 50,0% dos neurônios colinérgicos (p=0,0068) e 54,8% dos neurônios nitrérgicos (p=0,0042). Também foram observadas reduções significativas de neurônios por área de gânglio (28,2% do total, p=0,0002; 27,7% de colinérgicos, p= 0,0002 e 32,1% de neurônios nitrérgicos, p=0,0016). A densidade de fibras colinérgicas na superfície do músculo longitudinal também foi significativamente reduzida (controle: 41 ± 2, DM: 24 ± 3%; p=0,003), embora a análise western-blot não indicasse redução na expressão de ChAT no DM. grupo. A isoforma Nav1.6 foi detectada em diferentes neurônios mioentéricos e a DM tendeu a mudar o padrão de imunomarcação para neurônios de tamanho maior (297,4 ± 10 vs. 372,5 ± 8,4 µm2), mas esse efeito não foi significativo (p=0,3). Os dados de RT-qPCR não sugeriram uma alteração dos transcritos para ChAT, nNOS, Nav1.3, Nav1.6 ou Nav1.7. Nossos dados confirmam a visão de que o DM crônico leva à redução das fibras colinérgicas excitatórias e da densidade neuronal. No entanto, mudanças no perfil de expressão do canal de sódio, que poderiam levar à disfunção neuronal, não foram detectadas. / Diabetes mellitus (DM) may lead to gastrointestinal motility disorders that may become clinically relevant in some patients. Rodent models of DM indicate morpho-functional abnormalities of the enteric nervous system in this disease. In this work, we have evaluated whether experimental DM can lead to changes in excitatory cholinergic efference, neuronal density and voltage-gate sodium channels (Nav) expression in the myenteric plexus of ileum. Streptozotocin-induced hiperglycemia in female rats was allowed to develop during eight weeks. Triple immunofluorescence labeling experiments revealed that the density of neurons per area of ileum in DM was significantly reduced when compared to the control. It were observed average reductions of 52.2% of total neurons (p=0.0001); 50.0% of cholinergic (p=0.0068) and 54.8% of nitrergic neurons (p=0.0042). Significant reductions of neurons per area of ganglion were also observed (28.2% of total, p=0.0002; 27.7% of cholinergic, p=0,0002 and 32.1% of nitrergic neurons, p=0.0016). The cholinergic fibers density at the surface of longitudinal muscle was also significantly reduced (control: 41 ± 2, DM: 24 ± 3%; p=0.003), although western-blot analysis did not indicate a reduction in the expression of ChAT in DM group. The Nav1.6 isoform was detected in different myenteric neurons and DM tended to shift the immunolabeling pattern towards neurons of bigger size (297.4 ± 10 vs. 372.5 ± 8.4 m2), but this effect was not significant (p=0.3). RT-qPCR data did not suggest an alteration of transcripts for ChAT, nNOS, Nav1.3, Nav1.6 or Nav1.7. Our data support the view that chronic DM leads to reduction in excitatory cholinergic fibers and neuronal density. However, changes in sodium channel expression profiling, which could lead to neuronal dysfunction, were not detected. / Fundação de Amparo à Pesquisa do Estado de Minas Gerais

Le segment initial de l’axone (SIA) est un sous-domaine fonctionnel du neurone localisé dans l’axone proximal, qui assure deux fonctions : l’initiation du potentiel d’action et le maintien de l’identité axonale. Le maintien et la stabilité du SIA sont des éléments fondamentaux de l’excitabilité du neurone et la nature dynamique de l’organisation fonctionnelle du SIA a été mise en évidence. Les objectifs de mes travaux de thèse ont été d’étudier les mécanismes responsables du maintien du SIA, en condition physiologique ou pathologique et d’identifier de nouveaux acteurs impliqués dans ces mécanismes. Dans un premier temps, nous avons identifié et caractérisé l’expression d’une nouvelle protéine au SIA : la protéine Scrib1. En utilisant une approche par ARN interférent nous avons montré que Scrib1 est nécessaire au maintien de la morphologie du SIA. Les conséquences fonctionnelles de l’absence de Scrib1 sont une diminution de l’excitabilité neuronale. Dans un second temps, nous nous sommes intéressés aux mécanismes pouvant être à l’origine de l’expression ectopique du canal Nav1.8 observée dans certaines pathologies démyélinisantes. Nous avons montré que Nav1.8 possède un site d’interaction à l’ankyrine G. Ce motif d’interaction est suffisant pour adresser un canal chimérique au SIA et perturber l’expression des Nav1 endogènes. A l’inverse des Nav1 du système nerveux central, l’interaction entre Nav1.8 et l’ankyrine G n’est pas régulée par la CK2. Cette interaction constitutive entre Nav1.8 et l’ankyrine G pourrait expliquer son expression ectopique dans le système nerveux central. / The axonal initial segment (AIS) is a unique sub-domain that plays a central role in the physiology of the neuron, as it orchestrates both electrogenesis and the maintenance of neuronal polarity. The maintenance and the stability of the AIS after assembly ensure a reliable generation of action potentials. However, new mechanisms affecting AIS protein-protein interaction and composition have been shown to modulate the electrogenesis of the neuron. Moreover, recent findings highlight that the AIS is capable of homeostatic plasticity through an activity–dependent change either in its location along the proximal axon or in its length. The objectives of my thesis were to study the mechanisms responsible for AIS maintenance in physiological or pathological condition and to identify new players involved in these mechanisms.First we identified and characterized the expression of a novel protein in AIS: the protein Scrib1. Using an shRNA approach we showed that Scrib1 is necessary to maintain the AIS morphology. The functional consequence of the absence of Scrib1 is a decreased of neuronal excitability.Second, we are interested in the mechanisms that cause the ectopic expression of Nav1.8 channel observed in demyelinating diseases. We found that Nav1.8 constitutively interacts with ankG in contrast to Nav1.2, which requires CK2 phosphorylation to bind ankG. Furthermore, when Nav1.8 ankyrin-binding domain was expressed in hippocampal neuron, it clustered at the AIS where it acted as a dominant negative for endogenous Nav1. This constitutive interaction between Nav1.8 and ankG could explain the ectopic expression of Nav1.8 in the central nervous system.

Le syndrome de Brugada (BrS) est une cardiopathie héréditaire à transmission autosomique dominante, se manifestant par une anomalie de l'ECG et un risque accru de mort subite. Les mutations retrouvées dans la sous-unité α du canal sodique cardiaque Nav1.5 chez certains patients entraînent un défaut d'adressage membranaire de ces canaux. Ceux-ci restent alors séquestrés dans des compartiments intracellulaires. L'étude de ces mutants se réduisant souvent à l'utilisation de traitements correcteurs, les mécanismes de rétention impliqués restent encore méconnus. L'objectif de ce travail est d'étudier des mutants Nav1.5 présentant un défaut d'adressage en tenant compte non seulement de l'hétérozygotie des patients BrS mais également de la présence de la sous unité régulatrice β1 prédominante dans le cœur. Des études fonctionnelles et biochimiques mettent en évidence un effet dominant négatif exercé par les mutants R1432G, L325R et S910L sur la densité de courant INa sauvage (WT). Cet effet nécessite la présence de la sous-unité β1 et passe par l'altération de l'adressage membranaire des formes WT. Ceci est la conséquence d'une interaction physique entre des sous-unités α mutantes et WT. D'autre part, les mutants étudiés présentent un profil de maturation lié aux N-glycosylations qui différent de celui des canaux WT. Nos données suggèrent que ces canaux peuvent emprunter (i) la voie classique d'adressage dans leur forme mature (ii) la voie dite non conventionnelle lorsqu'ils sont partiellement glycosylés. En conclusion, ces travaux mettent en évidence le rôle de la sous-unité β1 ainsi que l'implication des N-glycosylations dans la modulation de l'adressage des canaux Nav1.5 / Brugada syndrome (BrS) is an inherited autosomal dominant cardiac channelopathy characterized by abnormal ECG pattern and an increased risk of sudden cardiac death. Several mutations on the cardiac sodium channel Nav1.5 which are responsible for BrS lead to misfolded proteins that do not traffic properly to the plasma membrane and are instead retained in intracellular compartments. Although pharmacological rescue is commonly used to characterize misfolded mutants, underlying cellular retention mechanisms remain unclear. The aim of this work is to investigate trafficking defective Nav1.5 mutants considering BrS patient heterozygosity and the presence of the regulatory β1-subunit which is largely expressed in cardiac tissue. By combining electrophysiology and biochemical approaches, we show that three distinct mutants, R1432G, L325R and S910L, exert a strong dominant negative effect upon wild-type (WT) sodium current density. Our data indicate that this effect requires the presence of the β1-subunit and is mediated by disruption of membrane trafficking of WT channels. Co-immunoprecipitation experiments demonstrate a physical interaction between mutant and WT α-subunits occurring only when the β1-subunit was present. Furthermore, we investigate the maturation pattern of Na channels. Our data show distinct N-glycosylated states between WT and mutant channels, suggesting that Nav1.5 α-subunits traffic (i) via unconventional secretion pathway as a partially glycosylated product, (ii) through the classical secretory pathway for mature fully-glycosylated form. This work highlights that β1-subunit and N-linked glycosylation process play key roles in modulating Nav1.5 trafficki

A natural phenomenon characterized by dense aggregations of unicellular photosynthetic marine organisms has been termed colloquially as red tides because of the vivid discoloration of the water. The dinoflagellate Karenia brevis is the cause of the Florida red tide bloom. K. brevis produces the brevetoxins, a potent suite of neurotoxins responsible for substantial amounts of marine mammal and fish mortalities. When consumed by humans, the toxin causes Neurotoxic Shellfish Poisoning (NSP). The native function of brevetoxin within the organism has remained mysterious since its discovery. There is a need to identify factors which contribute to and regulate toxin production within K. brevis. These toxins are produced and retained within the cell implicating a significant cellular role for their presence. Localization of brevetoxin and identification of a native receptor may provide insight into its native role as well as other polyether ladder type toxins such as the ciguatoxins, maitotoxins, and yessotoxins. In higher organisms these polyether ladder molecules bind to transmembrane proteins with high affinity. We anticipated the native brevetoxin receptor would also be a transmembrane protein. Photoaffinity labeling has become increasingly popular for identifying ligand receptors. By attaching ligands to these photophors, one is able to activate the molecule after the ligand binds to its receptor to obtain a permanent linkage between the two. Subsequent purification provides the protein with the ligand directly attached. A molecule that is capable of fluorescence is a fluorophore, which upon excitation is capable of re-emitting light. Fluorescent labeling uses fluorophores by attaching them covalently to biologically active compounds. The synthesis of a brevetoxin photoaffinity probe and its application in identifying a native brevetoxin receptor will be described. The preparation of a fluorescent derivative of brevetoxin will be described and its use in localizing the toxin to an organelle within K. brevis. In addition, the general utility of a synthesized photoaffinity label with other toxins having similar functionality will be described. An alternative synthetic approach to a general photoaffinity label will also be discussed whose goal was to accelerate the preparation and improve the overall synthetic yields of a multifunctional label.

Indiana University-Purdue University Indianapolis (IUPUI) / The voltage-gated sodium channel Nav1.7 has gained traction as a pain target with recognition that loss-of-function mutations in SCN9A, the gene encoding Nav1.7, are associated with congenital insensitivity to pain, whereas gain-of-function mutations produce distinct pain syndromes due to increased Nav1.7 activity. Selective inhibition of Nav1.7 is fundamental to modulating pain via this channel. Understanding the regulation of Nav1.7 at the cellular and molecular level is critical for advancing better therapeutics for pain. Although trafficking of Nav1.7 remains poorly understood, recent studies have begun to investigate post-translational modifications of Navs and/or auxiliary subunits as well as protein-protein interactions as Nav-trafficking mechanisms. Here, I tested if post-translational modifications of a novel Nav1.7-interacting protein, the axonal collapsin response mediator protein 2 (CRMP2) by small ubiquitin-like modifier (SUMO) and phosphorylation could affect Nav trafficking and function. Expression of a CRMP2 SUMOylation incompetent mutant (CRMP2-K374A) in neuronal model CAD cells, which express predominantly Nav1.7 currents, led to a significant reduction in huwentoxin-IV-sensitive Nav1.7 currents. Increasing deSUMOylation with sentrin/SUMO-specific protease SENP1 or SENP2 in wildtype CRMP2-expressing CAD cells decreased Nav1.7 currents. Consistent with reduced current density, biotinylation revealed significant reduction in surface Nav1.7 levels of CAD cells expressing CRMP2-K374A or SENP proteins. Diminution of Nav1.7 sodium current was recapitulated in sensory neurons expressing CRMP2-K374A. Because CRMP2 functions are regulated by its phosphorylation state, I next investigated possible interplay between phosphorylation and SUMOylation of CRMP2 on Nav1.7. Phosphorylation of CRMP2 by cyclin dependent kinase 5 (Cdk5) was necessary for maintaining Nav1.7 surface expression and current density whereas phosphorylation by Fyn kinase reduced CRMP2 SUMOylation and Nav1.7 current density. Binding to Nav1.7 was decreased following (i) loss of CRMP2 SUMOylation, (ii) loss of CRMP2 phosphorylation by Cdk5, or (iii) gain of CRMP2 phosphorylation by Fyn. Altering CRMP2 modification events simultaneously was not synergistic in reducing Nav1.7 currents, suggesting that Nav1.7 co-opts multiple CRMP2 modifications for regulatory control of this channel. Loss of either CRMP2 SUMOylation or Cdk5 phosphorylation triggered Nav1.7 internalization involving E3 ubiquitin ligase Nedd4-2 as well as endocytosis adaptor proteins Numb and Eps15. Collectively, my findings identify a novel mechanism for regulation of Nav1.7.

No description available.

[ES] El síndrome de Dravet es una epilepsia severa rara causada por mutaciones en el gen SCN1A, el cual codifica para la proteína Nav1.1, subunidad α de los canales de sodio regulados por voltaje. En esta tesis se ha generado mediante recombinación homóloga, una nueva mutación en el gen para que hemos denominado paraKO, el cual cumple la misma función en Drosophila melanogaster. Estas moscas han mostrado un fenotipo epiléptico inducido por altas temperaturas, y muerte súbita en el caso de las crisis de larga duración. También se han observado alteraciones musculares en ensayos de geotaxis negativa, vuelo y locomoción. Del mismo modo, han presentado problemas cognitivos como la ansiedad y dificultades en el aprendizaje. El uso de imanes como terapia contra el fenotipo epiléptico ha tenido buenos resultados retrasando la aparición de las crisis y disminuyendo su duración y la cantidad de moscas que las padecen. El perfil metabolómico de las cabezas de estas moscas mostró un incremento en la concentración de aminoácidos, succinato y lactato, alteraciones que se pueden relacionar con la epilepsia y la disfunción mitocondrial. El neurotransmisor GABA, principal implicado en el síndrome de Dravet, mostró niveles superiores en el modelo generado. El análisis electrofisiológico de las corrientes de sodio de las motoneuronas aCC en estadío de larva señaló aumentos en las corrientes persistentes de sodio y su ratio con las transitorias, lo cual podría justificar las crisis epilépticas. Además, la excitabilidad y el tamaño de estas células fueron menores. Todos estos cambios presentes en los mutantes KO generados hacen de estas moscas un buen modelo para estudio de la epilepsia en general, y del síndrome de Dravet en particular. Este modelo ofrece nuevas herramientas para entender la patofisiología de la enfermedad y la búsqueda de biomarcadores y tratamientos. Finalmente la búsqueda de modificadores genéticos a través de ensayos de supervivencia, tiempo de recuperación a crisis y vuelo empleando el modelo parabss1 obtuvo buenos resultados con los genes nAchRα4 y KCNQ. El gen toy por el contrario resultó ser intensificador. La variabilidad en los resultados obtenidos en este apartado cuestiona la manera de llevar a cabo este tipo de estudios en modelos animales y pacientes del síndrome de Dravet. / [CA] La síndrome de Dravet és una epilèpsia severa rara causada por mutacions en el gen SCN1A, el qual codifica para la proteïna Nav1.1, subunitat α dels canals de sodi regulats por voltatge. En aquesta tesis s'ha generat, mitjançant recombinació homòloga, una nova mutació en el gen para, anomenada paraKO, el qual té la mateixa funció en Drosophila melanogaster. Aquestes mosques han mostrat un fenotip epilèptic induït por altes temperatures, y mort súbdita en el cas de les crisis de llarga duració. També s'han observat alteracions musculars en assajos de geotaxis negativa, vol y locomoció. De la mateixa manera, han presentat problemes cognitius como l'ansietat i dificultats en l'aprenentatge. L'ús d'imants com teràpia contra el fenotip epilèptic ha tingut bons resultats endarrerint l'aparició de les crisis i disminuint la seua durada i la quantitat de mosques que les pateixen. El perfil metabolòmic dels caps d'aquestes mosques mostrà increments en la concentració d'aminoàcids, succinat i lactat, alteracions les quals es poden relacionar amb l'epilèpsia y la disfunció mitocondrial. El neurotransmissor GABA, principal implicat en la síndrome de Dravet, mostrà nivells superiores en el model generat. L'anàlisi electrofisiològic de las corrents de sodi de les motoneurones aCC en estadi de larva assenyalà augments en les corrents persistents de sodi y el seu ràtio amb las transitòries, lo qual podria justificar las crisis epilèptiques. A més a més, l'excitabilitat y el tamany d'aquestes cèl·lules va ser menor. Todos aquests canvis presents en els mutants KO generats fan d'aquestes mosques un model per a l'estudi de l'epilèpsia en general, i de la síndrome de Dravet en particular. Aquest model ofereix noves ferramentes per a entendre la patofisiología de la malaltia i la recerca de biomarcadors y tractaments. Finalment la recerca de modificadors genètics a través d'assajos de supervivència, temps de recuperació a crisis y vol mitjançant el model parabss1 va obtindre bons resultats amb els gens nAchRα4 y KCNQ. El gen toy pel contrari resultà ser intensificador. La variabilitat en els resultats obtinguts en aquest apartat qüestiona la manera de fer aquest tipus d'estudis en models animals i pacients de la síndrome de Dravet. / [EN] Dravet syndrome is a severe rare epileptic disease caused by mutations in the SCN1A gene coding for the Nav1.1 protein, a voltage-gated sodium channel alpha subunit. In this thesis we have made a new mutation in a gene called paraKO through homologous recombination, the single Drosophila melanogaster gene encoding this type of protein. These flies showed a heat-induced seizing phenotype, and sudden death in long term seizures. In addition to seizures, neuromuscular alterations were observed in climbing, flight and locomotion tests. Moreover, they also manifested some cognitive alterations such as anxiety and difficulties in learning. Using magnets as a therapy for epileptic phenotype, seizures start was delayed, and its duration and the quantity of flies affected was lower. Metabolomic profile of these flies' brains showed an increase in the amount of aminoacids, succinate and lactate, alterations that could be related with epilepsy and mitochondrial dysfunction. GABA, the main neurotransmitter involved in Dravet syndrome, was higher in the paraKO model. Electrophysiological sodium current analysis from aCC motoneurons in larvae stage revealed an increase in persistent currents and their ratio with transients, which is a symptom for epileptic seizures. Cell size and excitability were lower in these cells too. All these changes in the paralytic knock-out flies indicate that this is a good model for epilepsy and specifically for Dravet syndrome. This model could be a new tool to understand the pathophysiology of the disease and to find biomarkers, genetic modifiers and new treatments. Finally, a search for genetic modifiers through survival, recuperation time and flight using parabss1 flies obtained good results with nAchR¿4 y KCNQ. Otherwise, toy gene was an enhancer. However, variability observed in these type of assays dispute how modifiers search is made with model animals and Dravet syndrome patients. / Tapia González, A. (2022). Generación de nuevos modelos y búsqueda de modificadores para el Síndrome de Dravet en Drosophila Melanogaster [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/186189 / TESIS

Cerebral ischaemia is a leading cause of disability and death world-wide. The only effective treatments are thrombolytic therapy (plasminogen activator; tPA) and hypothermia (33?C). However, tPA has limited clinical application due to its short therapeutic time window and its specific application in thrombo-embolic stroke. Moderate hypothermia (33?C) is only being used following cardiac arrest in comatose survivors. Hence more treatments are urgently required. The first step in developing new treatments is the identification and characterisation of a potential therapeutic target. Since brain damage following cerebral ischaemia is associated with disturbances in intracellular calcium homeostasis, the sodium-calcium exchanger (NCX) is a potential therapeutic target due to its ability to regulate intracellular calcium. Currently, however there is uncertainty as to whether the plasma membrane NCX has a neuroprotective or neurodamaging role following cerebral ischemia. To address this issue I compared hippocampal neuronal injury in NCX3 knockout mice (Ncx3-/-) and wild-type mice (Ncx3+/+) following global cerebral ischaemia. In order to perform this study I first established a bilateral common carotid occlusion (BCCAO) model of global ischaemia in wild-type C57/BlHsnD mice using controlled ventilation. After trials of several ischaemic time points, 17 minutes was established as the optimum duration of ischaemia to produce selective hippocampal CA1 neuronal loss in the wild-type mice. I then subjected NCX3 knockout and wild-type mice to 17 minutes of ischaemia. Following the 17 minute period of ischaemia, wild-type mice exhibited 80% CA1 neuronal loss and 40% CA2 neuronal loss. In contrast, NCX3 knockout mice displayed > 95% CA1 neuronal loss and 95% CA2 neuronal loss. Following experiments using a 17 minute duration of global ischaemia, a 15 minute duration of ischaemia was also evaluated. Wild-type mice exposed to a 15 minute period of ischaemia, did not exhibit any significant hippocampal neuronal loss. In contrast, NCX3 knockout mice displayed 45% CA1 neuronal loss and 25% CA2 neuronal loss. The results clearly demonstrate that mice deficient for the NCX3 protein are more susceptible to global cerebral ischaemia than wild-type mice. My findings showing a neuroprotective role for NCX3 following ischaemia, suggest that the exchanger has a positive role in maintaining neuronal intracellular calcium homeostasis. When this function is disrupted, neurons are more susceptible to calcium deregulation, with resultant cell death via calcium mediated pathways. Therefore, improving NCX activity following cerebral ischaemia may provide a therapeutic strategy to reduce neuronal death.