Τα πενταμερή ιοντικά κανάλια που ενεργοποιούνται από τη δέσμευση ενός προσδέτη (pLGICs-pentameric Ligand Gated Ion Channels) της υπεροικογένειας των υποδοχέων Cys-θηλιάς είναι διαμεμβρανικές γλυκοπρωτεΐνες που εμπλέκονται σε ποικιλία βιολογικών λειτουργιών. Στην παρούσα εργασία παρουσιάζεται η NMR μελέτη της εξωκυττάριας περιοχής ενός προκαρυωτικού pLGIC, της GLIC (Gloeobacter ligand-gated ion channel), που προέρχεται από το κυανοβακτήριο Gloeobacter violaceus και εμφανίζεται ως μονομερές σε διάλυμα. / Pentameric ligand-gated ion channels (pLGICs) of the Cys loop family are transmembrane glycoproteins implicated in a variety of biological functions. Here, we present a solution NMR study of the extracellular domain of a prokaryotic pLGIC homologue from the bacterium Gloeobacter violaceus that is found to be a monomer in solution.

El sistema nervioso consiste en una red muy compleja de billones de neuronas que se comunican entre sí. Las sinapsis químicas son primordiales para la comunicación neuronal rápida y eficiente, y están mediadas por la liberación de neurotransmisores a la hendidura sináptica para unirse a sus receptores específicos ubicados en otras células o neuronas postsinápticas. Entre los receptores de neurotransmisores se encuentran los receptores Cys-loop, que pertenecen a la superfamilia de canales pentaméricos activados por ligando (pLGIC). Son proteínas integrales de membrana que convierten la señal química en una respuesta eléctrica al permitir el paso de iones de un lado a otro de la membrana. Estos receptores se constituyen por 3 regiones fundamentales, el dominio extracelular, donde se encuentra el sitio de unión del neurotransmisor o sitio ortostérico; el dominio transmembrana, que conforma el poro del canal y contiene los sitios de unión para diferentes moduladores alostéricos; y el dominio intracelular que posee sitios de modulación y aminoácidos determinantes de la conductancia del canal. En vertebrados, esta familia de receptores está formada por los receptores nicotínicos de acetilcolina y el receptor de serotonina tipo 3 (5-HT3), los cuales son permeables a cationes, y por los receptores de ácido γ-aminobutírico tipo A (GABAA) y receptores de glicina, permeables a aniones. El repertorio de receptores Cys-loop en invertebrados es más variado y extenso, incluyendo entre ellos un canal aniónico activado por serotonina, denominado MOD-1. Los receptores Cys-loop son ampliamente estudiados por ser blancos moleculares terapéuticos en una extensa variedad de patologías. Es por ello por lo que ampliar el conocimiento de la función y modulación de estos receptores permitirá generar mejores estrategias farmacológicas. En esta Tesis Doctoral se realizaron estudios de la función y modulación de los receptores Cys-loop activados por serotonina humano, 5-HT3, y de Caenorhabditis elegans, MOD-1, para la búsqueda de nuevos fármacos y la generación de estrategias de reposicionamiento de medicamentos de uso clínico. En el Capítulo 1 se estudió la funcionalidad molecular del receptor 5-HT3A humano. Se logró descifrar las diferencias entre la activación ortostérica y la activación y modulación alostérica por terpenoides, utilizando la valiosa técnica de patch-clamp. Se definieron las bases mecanísticas de la activación de 5-HT3A por los agonistas ortostéricos serotonina y triptamina, y de la activación alostérica por los terpenoides timol y carvacrol. Se demostró que triptamina es un agonista de muy baja eficacia y potencia. Mediante registros de whole-cell, se demostró que los terpenoides potencian las corrientes evocadas por serotonina, pero también poseen capacidad para evocar alostéricamente respuestas macroscópicas, con mayor potencia y eficacia que triptamina, aunque de forma más lenta que serotonina. Mediante registros de canal único, se evidenció que dichos compuestos activan al receptor 5-HT3 como agonistas muy eficaces dado que generan episodios de activación más prolongados que serotonina. Nuestro estudio mostró la primera caracterización a nivel de canal único de la activación de 5-HT3A humano por ligandos alostéricos, de creciente interés como herramientas terapéuticas. En el Capítulo 2 se halló un nuevo blanco promisorio para la terapia antihelmíntica, estudiando ampliamente al receptor MOD-1 de C. elegans. Este nematodo es un buen modelo parasitario ya que comparte las características funcionales y farmacológicas con los nematodos parasitarios. Además C. elegans es sensible a la mayoría de los fármacos antiparasitarios que están dirigidos a los receptores Cys-loop de los parásitos. Dado que MOD-1 está ausente en vertebrados, se encuentra limitado al filo, y está presente en nematodos parasíticos, emerge como un blanco farmacológico promisorio. En este capítulo se revelaron las diferencias en la selectividad agonista y en los sitios ago-PAM entre MOD-1 y el receptor de serotonina humano 5-HT3. Se encontró que la triptamina, un agonista muy parcial de 5-HT3A, es un agonista eficaz de MOD-1, y se demostró además que posee una acción antihelmíntica. Por otro lado, se realizó un testeo de una variedad de compuestos en búsqueda de nuevos moduladores de MOD- 1. Se demostró, mediante estudios electrofisiológicos, que muscimol y piperazina (PZE) son antagonistas no competitivos de MOD-1. Mediante ensayos de comportamiento en gusanos se confirmó el efecto de PZE sobre MOD-1, hallando de esta manera un nuevo blanco molecular para este fármaco de uso antiparasitario. Estos estudios ampliaron el conocimiento de receptores Cys-loop de nematodos para el descubrimiento de nuevos compuestos con actividad nematicida. Por lo tanto, se concluye que, derivados de triptamina y PZE podrían ser explorados como nuevos compuestos con potencial actividad antiparasitaria. En el Capítulo 3, se comenzó a plantear estrategias para reposicionar medicamentos de uso clínico, para lograr superar las desventajas de generar un fármaco de novo, acortando los tiempos y recursos necesarios para realizar los estudios farmacológicos, famacocinéticos y de toxicidad requeridos para la producción de nuevos fármacos dirigidos a tratar patologías en las que los receptores estudiados en esta Tesis están involucrados. Con tal fin, se exploró el efecto de fármacos de uso comercial derivados de triptamina y PZE, debido a lo postulado en el capítulo 2, y se encontró que sumatriptan, un fármaco comercializado para tratar la migraña, sería un buen candidato para reposicionar para la terapia antiparasitaria. Además, PZE podría reutilizarse como modulador alostérico negativo de los receptores 5-HT3A humano abordando patologías en las que este receptor está involucrado, tales como náuseas y vómitos relacionados a la quimioterapia, radioterapia y anestesia, así como para mitigar los síntomas del síndrome de intestino irritable y para patologías del sistema nervioso como depresión, ansiedad y esquizofrenia, entre otras en las que este receptor está involucrado. En resumen, aprovechando la potencialidad que brindan los registros de canal único y de corrientes macroscópicas en el estudio de las bases moleculares del funcionamiento y modulación de estos receptores Cys-loop, se logró hallar un nuevo blanco antiparasitario, descifrar por primera vez las diferencias de activación ortostérica y alostérica del receptor 5-HT3A humano por timol y carvacrol, y generar estrategias de reposicionamiento de fármacos de uso clínico mediante la modulación alostérica de estos receptores tanto en C. elegans como en humano. Estos estudios amplían el conocimiento de la función y modulación de los receptores Cys-loop, generando una base para abordar patologías que involucran a estos receptores y para el desarrollo de nuevos y más selectivos fármacos. / The nervous system consists of a highly complex network of billions of neurons that communicate with each other. Chemical synapses are key to the rapid and efficient neuronal communication and are mediated by the release of neurotransmitters into the synaptic cleft to bind to their specific receptors located either on other cells or postsynaptic neurons. Among the neurotransmitter receptors are the Cys-loop receptors, which belong to the superfamily of ligand-gated pentameric channels (pLGIC). They are integral membrane proteins that convert the chemical signal into an electrical response by allowing ions to pass from one side of the membrane to the other. These receptors are made up of 3 fundamental regions: the extracellular domain, where the neurotransmitter binding site or orthosteric site is located; the transmembrane domain, which forms the channel pore and contains the binding sites for different allosteric modulators; and the intracellular domain which contains modulation sites and amino acids that determine channel conductance. In vertebrates, this family of receptors consists of the nicotinic acetylcholine receptors and the serotonin receptor type 3 (5- HT3), both of which are permeable to cations, and the receptors for γ-aminobutyric acid type A (GABAA) and glycine receptors, permeable to anions. The repertoire of Cys-loop receptors in invertebrates is more varied and extensive, including among them, a serotonin-activated anion channel called MOD-1. Cys-loop receptors have been extensively studied as they are therapeutic molecular targets in a wide variety of pathologies. Expanding our knowledge on the function and modulation of these receptors will therefore contribute to generating better pharmacological strategies for the treatment of these disorders. In this Ph. D. thesis studies were carried out on the function and modulation of the Cys-loop receptors activated by human serotonin, 5-HT3, and of Caenorhabditis elegans, MOD-1, for the search for new drugs and the generation of repositioning strategies for drugs for clinical use. In Chapter 1 the molecular functionality of the human 5-HT3A receptor was studied. It was possible to decipher the differences between orthosteric activation and allosteric activation and modulation by terpenoids, using the valuable patch-clamp technique. The mechanistic bases of 5-HT3A activation by the orthosteric agonists serotonin and tryptamine, and allosteric activation by the terpenoids thymol and carvacrol, were defined. It is revealed that tryptamine is an agonist with very low efficacy and potency. Using whole-cell recordings, they show that terpenoids not only potentiate the currents evoked by serotonin but also have the ability to allosterically evoke macroscopic responses, with greater power and efficiency than tryptamine, although more slowly than serotonin. Using single channel recordings, these compounds were shown to activate the 5-HT3A receptor as highly effective agonists, since they generate longer episodes of activation than serotonin. Our study showed the first characterization at the single channel level of the activation of human 5-HT3A by allosteric ligands, of increasing interest as therapeutic tools. In Chapter 2, a promising new target for anthelmintic therapy was found by extensively studying the MOD-1 receptor of C. elegans. This nematode is a good parasitic model since it shares functional and pharmacological characteristics with parasitic nematodes. In addition, C. elegans is sensitive to most antiparasitic drugs that are directed at the Cys-loop receptors of the parasites. Since MOD-1 is absent in vertebrates, limited to the phylum, and present in parasitic nematodes, it emerges as a promising drug target. In this chapter we revealed the differences in agonist selectivity and ago-PAM sites between MOD-1 and the human serotonin 5-HT3 receptor. Tryptamine, a very partial agonist of 5-HT3A, was found to be an effective agonist of MOD-1, and was further shown to have an anthelmintic action. On the other hand, we carried out a test of a variety of compounds in search of new modulators of MOD-1. We demonstrated by means of electrophysiological studies that muscimol and piperazine (PZE) are non- competitive antagonists of MOD-1. Also, through behavioral tests in worms, we confirmed the effect of PZE on MOD-1, thus finding a new molecular target for this drug for antiparasitic use. These studies have considerably expanded the knowledge of nematode Cys-loop receptors for the discovery of new compounds with nematicidal activity. We conclude that tryptamine and PZE derivatives could be explored as new compounds with potential antiparasitic activity. In Chapter 3, we began to propose strategies to reposition drugs for clinical use in order to overcome the disadvantages of generating a new drug, shortening the time and resources necessary to perform the pharmacological, pharmacokinetic, and toxicity studies required for the production of new drugs aimed at treating pathologies in which the receptors studied in this Ph. D. thesis are involved. To this end, the effect of drugs in commercial use derived from tryptamine and PZE was explored based on the results collected in Chapter 2. It was found that sumatriptan, a drug marketed to treat migraine, appears to be a good candidate to reposition for antiparasitic therapy. In addition, PZE could be reused as a negative allosteric modulator of human 5-HT3A receptors, addressing pathologies in which this receptor is involved, such as nausea and vomiting related to chemotherapy, radiotherapy, and anesthesia, as well as to alleviate the symptoms of irritable bowel syndrome and for pathologies of the nervous system, such as depression, anxiety and schizophrenia, among others in which this receptor is involved. Summing up, taking advantage of the potential offered by single channel recordings and macroscopic currents in the study of the molecular bases of the functioning and modulation of the Cys-loop receptors explored in this Ph. D. thesis, it was possible to: i) find a new antiparasitic target, ii) to decipher for the first time the differences of orthosteric and allosteric activation of the human 5-HT3A receptor by thymol and carvacrol, and iii) to generate drug repositioning strategies for clinical use through allosteric modulation of these receptors in both C. elegans and humans. These studies broaden the knowledge of the function and modulation of Cys- loop receptors, generating a basis for addressing pathologies that involve these receptors as well as for developing new and more selective drugs.

Le récepteur à la glycine est un récepteur neuronal qui appartient à la famille des canaux ligand-dépendants «cys-loop». Avec le récepteur ionotrope GABA ils fournissent la neurotransmission inhibitrice rapide dans le SNC des vertébrés grâce à leur perméabilité sélective au Cl-. Les récepteurs à la glycine participent à différents processus physiologiques comprenant le contrôle de l'activité motrice, la respiration, la sensation de douleur inflammatoire, la perception des stimuli visuels et auditifs. Le développement de modulateurs efficaces des récepteurs à la glycine permettra un contrôle précis de leur activité, ce qui est particulièrement important dans le cas des pathologies des récepteurs à la glycine, comme l'hyperekplexie. En utilisant l'analyse électrophysiologique, la mutagenèse dirigée et l'expression de protéines spécifiques dans un système hétérologue, nous avons identifié les acides ginkgoliques et niflumiques comme nouveaux modulateurs de récepteurs de la glycine, caractérisé leur action sur différentes sous-unités du récepteur et déterminé les sites importants pour la potentialisation ou l'inhibition des récepteurs à la glycine par ces composés. Cette approche est très prometteuse et ouvre de nouvelles voies vers des futures actions thérapeutiques. / Glycine receptor is a ligand-gated neuronal receptor that possesses an ion pore permeable for Cl- and represents an important component of inhibitory neurotransmission in CNS of vertebrates. Glycine receptors participate in the control of motor activity, respiration, inflammatory pain sensation, perception of visual and auditory stimuli. Development of efficient modulators of glycine receptors will allow a precise control of their activity, which is especially important in the case of glycine receptor pathologies, such as hyperekplexia. In the present work we have identified ginkgolic and niflumic acids as novel modulators of glycine receptors, characterized their action on different subunits of the receptor and determined the most probable sites of interaction of the compounds with glycine receptors.

Today, enzymes are extensively used for many industrial applications, this includes bulk and fine-chemical synthesis, pharmaceuticals and consumer products. Though Nature has perfected enzymes for many millions of years, they seldom reach industrial performance targets. Natural enzymes could benefit from protein redesign experiments to gain novel functions or optimize existing functions. Glutathione transferases (GSTs) are detoxification enzymes, they also display disparate functions. Two Mu-class GSTs, M1-1 and M2-2, are closely related but display dissimilar substrate selectivity profiles. Saturation mutagenesis of a previously recognized hypervariable amino acid in GST M2-2, generated twenty enzyme variants with altered substrate selectivity profiles, as well as modified thermostabilities and expressivities. This indicates an evolutionary significance; GST Mu-class enzymes could easily alter functions in a duplicate gene by a single-point mutation. To further identify residues responsible for substrate selectivity in the GST M2-2 active site, three residues were chosen for iterative saturation mutagenesis. Mutations in position10, identified as highly conserved, rendered enzyme variants with substrate selectivity profiles resembling that of specialist enzymes. Ile10 could be conserved to sustain the broad substrate acceptance displayed by GST Mu-class enzymes. Enzymes are constructed from primarily twenty amino acids, it is a reasonable assumption that expansion of the amino acid repertoire could result in functional properties that cannot be accomplished with the natural set of building blocks. A combination approach of site-directed mutagenesis and chemical modifications in GST M2-2 and GST M1-1 resulted in novel enzyme variants that displayed altered substrate selectivity patterns as well as improved enantioselectivities. The results presented in this thesis demonstrate the use of different protein redesign techniques to modulate various functions in Mu-class GSTs. These techniques could be useful in search of optimized enzyme variants for industrial targets. / biokemi och organisk kemi

M.S. / Biochemistry / The EPR spectrum of the Tyr-Cys· free radical in oxidized apoGAOX has been investigated, using a combination of approaches. Power saturation analysis has been used to resolve two unique spectra through Evolving Factor Analysis (EFA) global fitting, indicating the presence of two distinct free radical species in the sample. The component that dominates at low microwave power arises from the Tyr-Cys· side chain, while the high power component has not yet been assigned. The experimental results show that the EPR spectrum collected at low power includes approximately 7% of the high power component. EPR spectra have been collected for ten different isotope derivatives of GAOX, including ²H-labeled, ¹³C-labeled, 17[superscript]O-labeled, and ³³S-labeled forms. XSophe simulation of the EPR spectra has been performed for the isotopically labeled samples in order to determine the spectroscopic parameters - g-values, hyperfine coupling constants, and linewidths. The g-values and the methylene proton hyperfine coupling constants obtained for the isotopically labeled samples are consistent with the literature values. The magnitude of the hyperfine coupling constants associated with each of the nuclei confirms that significant electron spin density is found on the methylene protons, the alternating carbon atoms within the aromatic π system and the 2p[subscript]z orbital of both sulfur and oxygen. Moreover, the rotation angle of the methylene protons to the phenoxyl ring around the C1-C7 bond has been evaluated based on the experimentally defined hyperfine coupling constants of the two methylene protons.

Haemonchus contortus is a blood feeding parasitic nematode infecting ruminants causing anemia and poor health at great economic cost. The ability to pharmaceutically control infection has been challenged by the rapid development and spread of drug resistance. The discovery of new targets is therefore required for sustainable parasite control. UNC-49 is a nematode ligand-gated ion channel that plays an important role in muscle contraction required for normal locomotion. However, little is known regarding its sensitivity to different agonists and how they interact with the binding site. This thesis describes an investigation into the efficacy of a range of classical GABA receptor agonists on Hco-UNC-49 expressed in Xenopus oocytes. The results of our electrophysiological recordings indicate that there is a size requirement for full agonism of the Hco-UNC-49 binding site. Furthermore, a number of molecules that are known to act on vertebrate GABA receptors have no effect on Hco-UNC-49. This suggests that the binding site of nematode GABA receptors does exhibit some unique properties. These findings could possibly be exploited to develop new drugs that specifically target GABA receptors from parasitic nematodes. / UOIT

Glycine receptors (GlyRs) are ligand-gated ion channels (LGICs) that, along with other members of the cys-loop superfamily of receptors, mediate a considerable portion of fast neurotransmission in the central nervous system (CNS). GlyRs are pentameric channels, organized quasi-symmetrically around an ion-conducting pore. Opening of the integral ion pore depends on ligand binding and transduction of this binding signal to the channel gate. Research presented in this dissertation describes a number of critical electrostatic interactions that play a role in conserving the closed-state stability of the receptor in the absence of ligand, ensuring that receptor activation occurs only upon neurotransmitter binding. These amino acids, aspartic acid at position 97 (D97), lysine 116 (K116), arginine 119 (R119) and arginine R131 (R131) are charged residues that interact with one another through electrostatic attraction. When D97 is replaced with any other amino acid this destabilizes the closed state of the channel and causes spontaneous GlyR channel opening. I show that restoration of this electrostatic interaction in GlyR bearing double mutations in which the charges are swapped (D97R/R119E and D97R/R131D) markedly decreases this spontaneous current. In addition, I investigate how these residues that interact at the interfaces between receptor subunits affect the efficacies of GlyR partial agonists. My work shows that the partial agonist taurine is converted into a full agonist at both D97R and R131D receptors. Furthermore, I analyzed the structure of the more extracellular part of the transmembrane (TM) 2 segment that lines the ion channel pore, showing that it is unlikely that this fragment (stretching from T13’ to S18’) is constrained in a true alpha helical conformation. From this work, using disulfide trapping and whole cell electrophysiology, I conclude that a significant level of flexibility characterizes this part of the TM2 domain. This segment includes residue S267, previously shown to be significant for alcohol and anesthetic actions, as well as residue Q266 that, when mutated, produces a hyperekplexia-like phenotype. The range of movement of residues in this region may therefore play an important role not only in channel gating but also in how modulators of GlyR function exert their actions. / text

Glutathione (GSH) is an important biological thiol, it performs significant biological functions such as serving an antioxidant which protect cells from oxidative stress by trapping free radicals which damage DNA and RNA. It is known that abnormal plasma levels of GSH have been linked to various human diseases. Therefore, the rapid, sensitive and highly selective detection of GSH is of great importance for investigating its functions in diseases diagnosis. Interestingly, we found in cetyl trimethylammonium bromide (CTAB) medium, the resorufin-based probe shows an extremely fast, highly selective response to GSH. The result indicates that this dye can be employed to detect GSH in biological samples such as human plasma. Cysteine (Cys) is another important biological thiol which is involved in a variety of significant cellur functions, including protein synthesis, detoxication, and metabolic process, etc. Abnormal levels of Cys are related to many diseases, such as slowed growth, Alzheimer's disease and cardiovascular disease. Thus, the detection and quantification of Cys in physiological media is of great importance. In this thesis, I am going to present two organic fluorescent probes (Resorufin-based probe and SNF probe) for the detection and quantification of Cys. In addition, we prove that they can directly quantify Cys in human plasma. The chemical mechanisms involved in the detection of Cys are discussed.

Dentro de la superfamilia de los canales iónicos activados por ligando (LGIC) se encuentra la familia de los receptores Cys-loop que incluye a canales catiónicos, como los receptores nicotínicos de acetilcolina (nAChRs) y los receptores 5-HT3 de serotonina; y canales aniónicos, como los activados por GABA (GABAA) y glicina. Estos receptores revisten importancia tanto en la fisiología normal como así también en diversas y muy variadas patologías, entre las que se pueden mencionar las enfermedades neurodegenerativas como la enfermedad de Alzheimer (EA). Este hecho los posiciona como blancos moleculares importantes para el desarrollo de nuevos fármacos, y es por esto que el conocimiento de su función y modulación resulta un aspecto clave. Los receptores Cys-loop son proteínas transmembrana, por lo que su función se encuentra condicionada por su entorno lipídico y estímulos propios de la membrana celular, además de por distintos estímulos o factores externos a ella. Están formados por 5 subunidades organizadas pseudo-simétricamente alrededor de un poro o canal central. En estos receptores pueden distinguirse un dominio extracelular (ECD, del inglés extracellular domain), donde se encuentran los sitios de unión al agonista y/o antagonistas, un dominio transmembrana (TMD, del inglés transmembrane domain), que contiene el canal iónico y que se encuentra en extenso contacto con los lípidos de la membrana, y un dominio pequeño intracelular (ICD, del inglés intracellular domain), que contiene distintos sitios de regulación y señalización intracelular. En esta Tesis Doctoral se profundizó en el estudio de la función y modulación de 2 receptores de neurotransmisores pertenecientes a los receptores Cys-loop: los nAChRs, muscular y α7 neuronal, y los receptores 5-HT3. En el capítulo I se estudió la modulación del nAChR muscular por el entorno lipídico de la membrana en la que se encuentra. Se hizo foco especialmente en el colesterol (Col), lípido clave para el correcto funcionamiento del nAChR y que presenta distintas asimetrías laterales y transmembrana en diferentes momentos de la vida y en condiciones de salud o enfermedad. Se trabajó con distintos sistemas modelo: vesículas unilamelares grandes (LUVs), vesículas unilamelares gigantes (GUVs), membranas ricas en nAChR de T. californica o células BOCSC 23 expresando heterologamente el nAChR muscular. A cada uno se le realizaron distintos tratamientos para modificar las condiciones de Col con el fin de obtener sistemas que presenten: i) un aumento del contenido de Col, ii) un aumento de esteroles en la hemicapa externa (por agregado de colesterol hemisuccinato (CHEMS)), o iii) transformación de parte del Col a colestenona (por la enzima colesterol oxidasa (Cox)). Mediante estudios de fluorescencia utilizando las sondas DPH, TMA-DPH y Laurdan se determinó que el orden de membrana aumentó cuando se incorporó CHEMS. Mediante estudios de espectrofotometría utilizando la sonda cristal violeta (CrV) y la técnica de patch-clamp se detectaron, respectivamente, modificaciones conformacionales en el nAChR y cambios en la activación del mismo cuando los diferentes sistemas habían sido incubados con CHEMS o Cox, no así con Col. Tratando LUVs con Tritón, y posterior centrifugación y separación por SDS-PAGE, se determinó que luego de la incorporación de Col o CHEMS al sistema, el nAChR se localizó mayoritariamente en fracciones ordenadas, mientras que cuando se produjo la transformación del Col a colestenona el receptor se localizó principalmente en fracciones desordenadas. Por último, estudios de microscopía confocal utilizando GUVs mostraron que alteraciones en el Col causaron cambios significativos en la presencia y distribución de dominios ordenados y desordenados; y que la presencia de un componente proteico en el sistema, como es un péptido representativo del segmento transmembrana yTM4 del nAChR, alteró las características de la membrana, llevando a todo el sistema a un mayor orden. De acuerdo con estos resultados, distintas modificaciones del Col en la membrana llevan a cambios de su orden y asimetría, y al mismo tiempo a cambios en la localización del nAChR en un determinado entorno, hecho que repercute directamente en su funcionalidad. Su localización en dominios ordenados ocasiona una disminución de su actividad, mientras que su presencia en dominios más desordenados lleva a un aumento de su función. De esta manera, la localización del nAChR en uno u otro dominio podría explicar los cambios en la señal colinérgica reportados en situaciones de envejecimiento y/o patologías como la EA. Existen numerosas evidencias de interacciones funcionales entre los péptidos β-amiloide (Aβ), involucrados en la enfermedad de Alzheimer, y el nAChR α7. En el capítulo II se estudió la modulación del nAChR α7 neuronal por oligómeros de Aβ1-40 y Aβ1-42. Se ha postulado que la acumulación excesiva de estos péptidos en el cerebro provoca la formación de placas seniles, directamente relacionadas con el proceso de neurodegeneración e inflamación característico de esta enfermedad. Sin embargo, evidencias más recientes sugieren que las especies más neurotóxicas serían las formas oligoméricas de Aβ. Mediante estudios de fluorescencia utilizando la sonda CrV se demostró que los oligómeros de Aβ provocan cambios conformacionales en el receptor α7. Utilizando la técnica de patch clamp se determinó que dichos oligómeros son capaces de activar al receptor α7 en muy bajas concentraciones y que, a concentraciones más altas, disminuyen la potenciación de α7 por moduladores alostéricos positivos (PAMs, positive allosteric modulators). Estos resultados muestran un rol dual de los oligómeros de Aβ dependiente de su concentración, como agonistas y como moduladores negativos de α7. El efecto agonista, potenciador de la señal colinérgica, podría tener un rol importante en la fisiología normal del individuo, mientras que el efecto inhibitorio, dado por su exacerbada producción, podría contribuir a la deficiencia en la memoria y cognición asociada a la EA. Teniendo en cuenta las características multifactoriales de la EA, la estrategia de búsqueda de nuevas moléculas apunta al descubrimiento y desarrollo de “multitarget drugs”, es decir de moléculas activas en varios blancos moleculares, por lo que el capítulo III se centró en el descubrimiento y optimización de nuevas moléculas bifuncionales que sean capaces de activar al nAChR e inhibir a la acetilcolinesterasa (ACE). Partiendo de un screening de extractos obtenidos de distintas plantas medicinales, mediante técnicas cromatográficas y de resonancia magnética nuclear se identificó a la molécula de cafeína como una molécula sumamente interesante para la EA. Además de confirmar su ya conocida actividad inhibidora de la ACE, utilizando técnicas de espectrofotometría con la sonda fluorescente CrV y de patch-clamp, en este trabajo se postula su actividad agonista sobre los nAChRs muscular y α7 neuronal. Caracterizada la actividad de la cafeína sobre el sistema colinérgico, se obtuvieron luego 5 análogos sintéticos de esta molécula mediante diseño racional. Se determinó que todos los análogos presentan actividad sobre ambos blancos moleculares, la ACE y los nAChRs, y cuya potencia es mayor a la de la cafeína. Mediante estudios de modelado molecular y docking se identificaron los sitios de inhibición de los análogos de la cafeína en la ACE y de interacción con el nAChR permitiendo postular a estos compuestos como agonistas parciales. Estos hallazgos resultan prometedores en la búsqueda de nuevos fármacos como una estrategia terapéutica para la EA. El otro receptor estudiado en este trabajo de Tesis fue el receptor 5-HT3, uno de los menos conocidos de la familia de los Cys-loop, el cual a nivel presináptico participa de la finalización de la señal nerviosa, tornándose un blanco molecular novedoso tanto en la EA como en otras patologías neurodegenerativas que requieran una potenciación de dicha señal. En el capítulo IV, utilizando el receptor de alta conductancia 5-HT3AHC, se descifraron por primera vez las diferencias entre la activación ortostérica del mismo y su activación alostérica por timol y carvacrol. Mediante la técnica de patch clamp, combinando registros de corrientes macroscópicas en la configuración whole-cell y de canal único en la configuración cell-attached, se caracterizó en primer lugar la activación del receptor humano 5-HT3AHC por los agonistas ortostericos 5-HT y triptamina, y luego la activación alostérica mediada por 2 terpenoides, carvacrol y timol. Se confirmó que tanto el carvacrol como el timol se comportan macroscópicamente como agonistas menos eficaces que 5-HT. Sin embargo, a nivel de canal único, estos 2 terpenoides activan a los receptores 5-HT3A con mayor eficacia que 5-HT. Más aún, se determinó que además de actuar como agonistas, el carvacrol y el timol actúan como modulares alostéricos positivos del receptor 5-HT3AHC humano, y que el efecto potenciador es evidente a concentraciones menores que el efecto agonista. De esta manera, los resultados de este capítulo permitieron definir las bases mecanísticas que subyacen a las diferencias entre la activación y potenciación alostérica u ortostérica del receptor humano 5-HT3A. En resumen, en este trabajo de Tesis se profundizó en el conocimiento de distintas formas de modulación de los nAChRs y del receptor 5-HT3, ya sea por moléculas exógenas o por el entorno lipídico en el que se encuentran, brindando bases moleculares para el entendimiento de su función, su modulación y su afección en determinadas patologías como la EA. En este trabajo se lograron avances significativos, con resultados de gran relevancia, ya sea para el estudio o tratamiento de esta enfermedad. / The Cys-loop receptor family, which belongs to the Ligand-gated ion channel (LGIC) superfamily, includes cation channels, such as nicotinic acetylcholine receptors (nAChRs) and serotonin 5-HT3 receptors, and anion channels, such as those activated by GABA (GABAA) and glycine. These receptors are important in normal physiology and in various pathologies, among which neurodegenerative diseases, such as Alzheimer's disease (AD), are included. They are therefore important molecular targets for the development of new drugs, this being the reason why the knowledge of their function is indeed a very interesting aspect worth exploring. Cys-loop receptors are integral membrane proteins whose function is conditioned by their lipid environment as well as by different extrinsic factors. These receptors are formed by five subunits organized pseudo-symmetrically around a central pore. Three different domains can be distinguished: an extracellular domain (ECD), where the agonist and/or antagonist binding sites are localized, a transmembrane domain (TMD), which contains the ion pore and is in extensive contact with membrane lipids, and a small intracellular domain (ICD), which is important for the conductance of the channel and modulation. In this Doctoral Thesis, the function and modulation of two neurotransmitter receptors that belong to the Cys-loop receptor family, nAChRs and 5-HT3 receptors, were explored by using a large variety of fluorescence, confocal microscopy and electrophysiological approaches. In Chapter I, modulation of the muscle nAChR by its lipid environment was studied. Special attention was put on cholesterol (Chol), which displays different lateral and transmembrane asymmetries all throughout life as well as under health and disease conditions. This lipid is crucial for the proper functioning of the nAChR. Different model systems were used: large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs), T. californica nAChR rich membranes and BOSC 23 cells heterologously expressing the muscle nAChR. The experimental systems were subjected to different treatments to modify their Chol conditions resulting in: i) an increment in Chol content, ii) an increment in sterols in the outer layer (after addition of cholesteryl hemisuccinate (CHEMS)), and iii) conversion of part of the Chol to cholestenone (through the enzyme Cholesterol Oxidase (ChOx)). Through fluorescence studies using DPH, TMA-DPH and Laurdan probes it was determined that the membrane order increased when CHEMS was incorporated. By using the fluorescent probe Crystal Violet (CrV) and patch-clamp recordings, conformational changes of the nAChR associated to changes in its activation properties were detected after incubation with either CHEMS or ChOx, but not with Chol. By treating LUVs with Triton, and subsequent centrifugation and separation by SDS-PAGE, it was determined that after incorporation of Chol or CHEMS to the system the nAChR was located mostly in ordered fractions, while after the conversion of Chol to cholestenone the receptor was found in disordered fractions. Confocal microscopy studies using GUVs showed that Chol alterations caused significant changes in the presence and distribution of ordered and disordered domains, and that the mere presence of a protein component in the system, specifically a peptide corresponding to the forth transmembrane segment of the nAChR (M4), led to a higher order of the entire system. According to our results, we conclude that modifications of membrane Chol lead to changes in membrane order and asymmetry, and, at the same time, in the location of the nAChR, a fact that would directly affect its functionality. The location of the nAChR in ordered domains causes a decrease in its activity whereas its location in more disordered domains enhances its function. In this way, the location of the nAChR in one or another domain fine-tunes its function and could explain the changes in the cholinergic signal reported in situations of aging and/or pathologies, such as AD. There is increasing evidence of functional interactions between β-Amyloid (Aβ) peptides, involved in Alzheimer's disease, and α7 nAChR. In Chapter II, modulation of α7 nAChR by Aβ1-40 and Aβ1-42 oligomers was studied. It has been postulated that the accumulation of these peptides in the brain causes the formation of senile plaques, which are directly related to the neurodegeneration and inflammation observed in AD. However, more recent evidence suggests that the most neurotoxic species are the oligomeric forms of Aβ. Fluorescence studies using the CrV probe demonstrated that Aβ oligomers cause conformational changes in the α7 receptor, which were dependent on the concentration. Accordingly, single channel recordings revealed that whereas at very low concentrations (pM to nM range) these oligomers are capable of activating the α7 receptor, at higher concentrations, which are compatible with those found in AD patients, they decrease the enhancement of α7 by positive allosteric modulators (PAMs). These results show a dual role of Aβ oligomers depending on their concentration, as agonists and as negative modulators of α7. The agonist effect, which enhances the cholinergic signal, could have an important role in normal physiology, while the inhibitory effect, given by the exacerbated production of Aβ, could contribute to the failure in memory and cognition associated with AD. Taking into account that Alzheimer’s is a multifactorial disease, the search for new molecules aims at discovering and developing “multitarget drugs”. Chapter III is focused on the discovery and optimization of new bifunctional molecules capable of activating the nAChR and inhibiting the AChE. Starting from a screening of extracts obtained from different medicinal plants and using chromatographic techniques and nuclear magnetic resonance, the caffeine molecule was identified as a highly interesting molecule for AD. In addition to its already known AChE inhibitory activity, using CrV fluorescence probe and patch-clamp techniques we postulated its agonist activity in the muscle and α7 nAChRs. After having characterized the activity of caffeine in the cholinergic system, five synthetic analogs of this molecule were obtained by rational design. It was determined that all the analogs have activity in both molecular targets, the AChE and the nAChRs, and that they have a greater potency than caffeine. Through docking studies, the sites of inhibition of caffeine analogs in the AChE and the sites of interaction with the nAChRs were proposed. These compounds are promising as new drugs for a therapeutic strategy for AD. The other receptor explored in this Thesis was the 5-HT3 receptor, one of the least known of the Cys-loop family, which has been recently proposed as a novel molecular target both in AD and in other neurodegenerative pathologies. In Chapter IV, by taking advantage of the high-conductance receptor 5-HT3AHC, differences between orthosteric and allosteric activation of the human 5-HT3A receptor were deciphered for the first time. By using the patch clamp technique, and combining macroscopic current recordings in the whole-cell configuration and single channel recordings in the cell-attached configuration, the activation of the human 5-HT3AHC receptor by the orthosteric agonists, 5-HT and tryptamine, and the allosteric activation, by carvacrol and thymol, were described. Both carvacrol and thymol were less effective agonists than 5-HT at the macroscopic level. However, at the single channel level, these two terpenoids activated 5-HT3A receptors more efficiently than 5-HT. Furthermore, it was demonstrated that in addition to acting as agonists, carvacrol and thymol behave as positive allosteric modulators of the human 5-HT3AHC receptor at low concentrations. In this way, these results made it possible to define the mechanistic bases underlying the differences between orthosteric and allosteric activation and potentiation of the human 5-HT3A receptor. Summing up, in this Doctoral Thesis we deepened into the knowledge of modulation of the nAChRs and the 5-HT3 receptor, either by extrinsic molecules or by the lipid environment in which they are located. Our findings provide molecular bases for the understanding of the function and modulation of these receptors and their implication in certain pathologies, such as AD. In this work, significant advances with highly relevant results were made for the study or treatment of this disease.

Dans la nature, les plantes sont constamment exposées à des modifications de leur environnement générant un stress oxydant auquel elles doivent s’adapter du fait de leur immobilité. Les végétaux ont donc développé un grand nombre de mécanismes antioxydants permettant de protéger leurs fonctions vitales. L’étude d’une enzyme de type peroxyrédoxine (PRX) localisée dans le chloroplaste montre que son activité antioxydante est importante pour la croissance et la tolérance des végétaux aux contraintes environnementales. De plus, cette PRX pourrait interagir avec d’autres protéines impliquées notamment dans des mécanismes antioxydants et dans le métabolisme du carbone. Parmi elles, une protéine de type fibrilline participant à la protection des structures photosynthétiques, pourrait avec la PRX protéger la photosynthèse, fonction propre aux végétaux et essentielle pour la vie sur terre. / In nature, plants are constantly exposed to environmental changes leading to oxidative stress, to which they must adapt due to their immobility. Plants have developed many antioxidant systems allowing them to maintain their vital functions. The study of a peroxiredoxin, enzyme (PRX) localized in chloroplasts, shows that its direct antioxidant activity is essential for growth and tolerance of plants to environmental constraints. In addition, this PRX could interact with other proteins especially involved in antioxidant mechanisms and carbon metabolism. Among them, fibrillin proteins, which participate in the protection of the photosynthetic structures, could preserve with the PRX the plant photosynthesis, which is essential for the life on earth.