New solid phase microextraction (SPME) method was developed for brain tissue bioanalysis on a liquid chromatography mass spectrometry platform. To achieve set objectives, in vivo SPME desorption process was optimized for high throughput analysis through the development of a desorption device. Subsequently, new SPME coatings were developed for the extraction of polar neurotransmitters from biological matrices. In a targeted analysis, in vivo SPME was used to monitor of changes in the concentrations of endogenous compounds (multiple neurotransmitters) and exogenous drugs (carbamazepine and cimetidine) in the striatum of the rat brain extracellular fluid. For the first time, SPME was used for quantitative analysis of neurotransmitters and also study spacial distribution of other drugs in different regions of the brain extracellular fluid. A new approach was developed for improved metabolites coverage in a global non-targeted metabolomics studies. The proposed in vivo method showed how complementary results can be obtained through the combination of microdialysis and SPME for simultaneous sampling of the brain extracellular fluid. Finally, in a clinical application, SPME was used to monitor changes in the concentration of multiple neurotransmitters during deep brain stimulation of the pre-frontal cortex of the brain.

O núcleo acumbens (Acb) é subdividido em core e shell (AcbSh). Há evidências que as divisões do Acb vão além da dicotomia core-shell e que regiões pobres em tirosina hidroxilase (TH) e calretinina (Calr) formam um sistema de corredores no AcbSh. Para detalhar melhor a organização do Acb investigamos a distribuição de TH, Calr, DARPP-32 e do transportador de dopamina (DAT). Em seguida, foi comparada a distribuição destes marcadores com a das subunidades GluA2/3 dos receptores de glutamato do tipo AMPA. Finalmente, exploramos se as aferências pré-frontais são direcionadas à distintos compartimentos do AcbSh. Nossos resultados revelaram que regiões que contém neurônios GluA2/3+ intensamente marcados formam um sistema de corredores no AbSh que coincide com áreas pobres em TH, Calr e DAT. Os experimentos de rastreamento anterógrado indicaram que somente uma pequena parte das aferências pré-frontais é direcionada aos corredores. Nossos resultados delinearam um sistema de corredores no AcbSh que provavelmente constitui um compartimento neuroquímico altamente especializado. / The nucleus accumbens (Acb) is subdivided in core and shell (AcbSh). There is evidence that accumbal subdivisions go beyond this core-shell dichotomy and that regions poor in tyrosine hydroxylase (TH) and calretinin (Calr) form a corridor system in the Acbsh. To better detail accumbal organization, we investigated the distribution of TH, Calr, DARPP-32 and of the dopamine transporter (DAT). Then we compared the distribution of these markers with that of the AMPA-type glutamate receptor subunits GluA2/3. Finally, we explored whether prefrontal afferents are directed to distinct AcbSh compartments. Our findings revealed that regions containing intensely labeled GluA2/3+ neurons form a corridor system in the AcbSh that coincides with regions poor in TH, Calr, and DAT. Anterograde tracing experiments indicated that only a small portion of the prefrontal afferents is specifically related to the corridors. Our findings delineated a complex corridor system in the AcbSh which might constitute a highly specialized neurochemical compartment.

Apprendre de nouvelles séquences d'actions est l'une des fonctions cruciales du système nerveux central. Cet apprentissage est notamment assuré par les ganglions de la base ;ceux-ci jouent un rôle critique dans la sélection d'actions ou la prise de décisions en permettant l'apprentissage et la sélection des programmes moteurs les plus appropriés. Comprendre la physiologie de cette circuiterie neuronale hautement complexe et particulièrement celle du striatum - structure d'entrée des ganglions de la base - est donc d'une importance capitale dans l'amélioration de notre compréhension de ce système neuronal. Dans ce travail, nous nous sommes intéressés au rôle fonctionnel du récepteur NMDA (NMDA-R) dans une sous-population des neurones de projection, les neurones striatopallidaux. Ces récepteurs sont impliqués dans les modifications de l'efficacité synaptique à long terme et jouent donc un rôle central dans le mécanisme d'apprentissage. Notre travail démontre que le NMDA-R des neurones striatopallidaux est un élément essentiel de l'apprentissage au niveau des ganglions de la base. En effet, nous observons que des souris transgéniques, déficientes en NMDA-R spécifiquement dans les neurones striatopallidaux, disposent d'une capacité d'adaptation réduite aux modifications de leur environnement. De plus, nous démontrons que ces souris transgéniques présentent des connexions neuronales affaiblies susceptibles d'expliquer les altérations comportementales observées. Dans ce travail de thèse, nous avons également développé une stratégie expérimentale reposant sur l'utilisation d'outils optogénétiques afin de déterminer le rôle d'une population d'interneurones inhibiteurs du striatum, les "fast spiking interneurons" (FSI). Cette technique a été mise en oeuvre avec succès et nous avons validé son efficacité in vitro. Nous démontrons que cette approche permet un contrôle l'activité électrique des FSI à l'échelle de la milliseconde. Son application in vivo, combinée avec des paradigmes comportementaux, nous permettra d'élucider le rôle spécifique de cette sous-population neuronale, tant au niveau du contrôle moteur que de la prise de décisions, dans des situations physiologiques ou pathologiques. Dans son ensemble, le présent travail ouvre les portes vers une meilleure compréhension de l'orchestration de la microcircuiterie striatale. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

A interleucina-2 (IL-2) atua na modulação da atividade dopaminérgica, que influencia o comportamento maternal. Neste estudo observou-se que o estado lactacional reduziu a atividade geral em campo aberto, porém não alterou os níveis de dopamina e seus metabólitos. A administração de IL-2, tanto sistêmica com diretamente no N.Ac não alterou a atividade geral em campo aberto, indicando a ausência de efeito motor da mesma. Além disso, a administração de IL-2 sistêmica e no N.Ac reduziu as porcentagens de ratas que agrupam os filhotes e de filhotes agrupados por rata. A injeção de IL-2 no N.Ac aumentou as latências de busca do primeiro e segundo filhotes e o comportamento agressivo. A administração sistêmica de IL-2 em ratas virgens reduziu somente do valor absoluto de DOPAC (metabólito de dopamina) após 100 e 120. Este achado corrobora a idéia de que o IL-2 altera a atividade dopaminérgica. Os resultados sugerem que a administração sistêmica da dose de IL-2 estudada não influencia de forma significativa os níveis de dopamina e de seus metabólitos no N.Ac. / Interleukin-2 (IL-2) modulates the dopaminergic neurotransmission, that into the nucleus accumbens (N.Ac) plays a role in maternal behavior. The IL-2 dose used in this study does not have motor effects. Both peripheral and central N.Ac injections decreased the percent of mothers grouping pups together and the number of grouped pups. IL-2 injections into the N.Ac resulted in longer latencies to retrieve first and second pups and increased aggressive behavior. In order to test if these behavioral effects would be related to the IL-2 reduced the DOPAC (dopamine metabolite) concentrations in the N.Ac of virgin rats treated with IL-2. This suggests suggest that the IL-2 dose used in this study does not alter so much the dopaminergic transmission by influencing extracellular levels of this neurotransmitter.

O córtex pré-frontal medial (PFCm) é caracterizado por entradas glutamatérgicas e dopaminérgicas que convergem sobre os mesmos neurônios alvos. Devido à escassa informação sobre as bases anatômicas das interações entre a dopamina (DA) e o glutamato (Glu), mapeamos a distribuição de subunidades (Su) de receptores (Rs) de Glu do tipo AMPA, NMDA e kainato no PFCm e investigamos a sua expressão em neurônios contendo a fosfoproteína DARPP-32 e em interneurônios. Os resultados mostram que as Su GluR2/3 dos Rs do tipo AMPA são as mais amplamente distribuídas no PFCm e expressas em todos os neurônios DARPP-32+. GluR2/3 é também amplamente co-localizado com as Su NMDAR1 dos Rs de Glu do tipo NMDA e GluR5/6/7 dos Rs do tipo kainato. Em contraste, as Su GluR1 e GluR4 são somente fracamente expressos no PFCm e não são co-localizados com DARPP-32, porém com GABA ou parvalbumina. Os resultados indicam que as Su GluR2/3, NMDAR1 e GluR5/6/7 são amplamente expressos em neurônios piramidais DARPP-32+ enquanto GluR1 e GluR4 são predominantemente expressos em interneurônios do PFCm. / The medial prefrontal cortex (PFCm) is characterized by glutamatergic and dopaminergic afferents that converge on the same target neurons. Since there is only limited information about the anatomical bases for interactions between dopamine (DA) and glutamate (Glu), we mapped the distribution of AMPA, NMDA and kainate Glu receptor (Rs) subunits (Su) in the PFcm and investigated their expression in neurons containing the phosphprotein DARPP-32 and in interneurons. Results show that the Su GluR2/3 of AMPA type Rs are the most prominently distributed in the PFCm and expressed in all neurons DARPP-32+. GluR2/3 is also widely co-localized with the NMDA type Su NMDAR1 and the Kainate Su GluR5/6/7. In contrast, the Su GluR1 and GluR4 are only weakly expressed in the PFCm and are not colocalized with DARPP-32 but with GABA or parvalbumin. Results indicate that the Su GluR2/3, NMDAR1, and GluR5/6/7 are prominently expressed in DARPP-32+ pyramidal neurons, whereas GluR1 and GluR4 are predominantly expressed by interneurons in the PFC.

L’hypothalamus est une structure cérébrale ayant un rôle clé dans la régulation de la prise alimentaire. Parmi les différentes populations neuronales qui le composent, les neurones produisant la pro-opiomélanocortine (POMC) sont classiquement connus pour diminuer la prise alimentaire et le poids corporel via la libération de neuropeptides produits par le clivage de POMC. Notre étude, grâce à l’utilisation d’approches génétiques, pharmacologiques, électrophysiologiques et moléculaires, remet en question les notions classiques sur la fonction des neurones à POMC dans la balance énergétique, en démontrant qu’il existe deux sous-populations fonctionnellement distinctes de neurones à POMC, qui augmentent ou diminuent la prise alimentaire en fonction du neurotransmetteur qu’elles libèrent, l’acide γ-aminobutyrique (GABA) ou le glutamate. Une troisième population capable de produire aussi bien du GABA que du glutamate a également été identifiée. La régulation des neurones à POMC GABAergiques et glutamatergiques dépend de la voie de la cible de la rapamycine chez les mammifères (mTORC1), qui fonctionne comme un détecteur d’énergie cellulaire, et du système endocannabinoïde (ECS), qui régule la libération de neurotransmetteurs. De plus, nous avons également démontré, via l’utilisation de souris mutantes conditionnelles, l’importance de la protéine p62 ou séquestrome 1 (p62/SQSTM1), qui régule l’activité de mTORC1 et l’autophagie, dans les neurones à POMC dans la régulation de l’homéostasie énergétique. Nos données offrent un nouvel aperçu sur les mécanismes moléculaires impliqués dans la régulation de la balance énergétique. / The hypothalamus is a brain structure with a key role in the regulation of food intake. Among the different neuronal populations of which it is composed, pro-opiomelanocortin (POMC) neurons are classically known to decrease food intake and body weight through the release of neuropeptides produced by the cleavage of POMC. Our study, through the use of genetic, pharmacological, electrophysiological and molecular approaches, challenges conventional notions about POMC neuron function in energy balance by showing that there are two functionally distinct POMC neuronal sub-populations, which increase or decrease food intake depending on which neurotransmitter they release, γ-aminobutyric acid (GABA) or glutamate. A third population capable of producing both GABA and glutamate has also been identified. The regulation of POMC GABAergic and glutamatergic neurons depends on the mechanistic target of rapamycin complex 1 (mTORC1) pathway, which functions as a cellular energy sensor, and the endocannabinoid system (ECS), which regulates neurotransmitters release. In addition, we have also demonstrated through the use of a conditional knockout mice, the importance of the protein p62 or sequestrome 1 (p62/SQSTM1), which regulates mTORC1 activity and autophagy, in POMC neurons for the regulation of energy homeostasis. Our data provide new insights on the molecular mechanisms involved in the regulation of energy balance.

Objective: Previous studies have used acetylcholinesterase (AChE) histochemistry to identify cholinergic nerves in the heart, but this enzyme is not a selective marker for cholinergic neurons. This study maps cholinergic innervation of guinea pig heart using a new antibody to the human high-affinity choline transporter (CHT), which is present only in cholinergic nerves. Methods: Immunohistochemistry was used to localize CHTs in frozen and paraffin sections of heart and whole mount preparations of atrial ganglionated nerve plexus. AChE-positive nerve fibers were identified in sections from separate hearts for comparison. Results: Control experiments established that the antibody to human CHT selectively labeled cholinergic neurons in the guinea pig. CHT-immunoreactive nerve fibers and AChE-positive nerves were very abundant in the sinus and AV nodes, bundle of His, and bundle branches. Both markers also delineated a distinct nerve tract in the posterior wall of the right atrium. AChE-positive nerve fibers were more abundant than CHT-immunoreactive nerves in working atrial and ventricular myocardium. CHT-immunoreactive nerves were rarely observed in left ventricular free wall. Both markers were associated with numerous parasympathetic ganglia that were distributed along the posterior atrial walls and within the interatrial septum, including the region of the AV node. Conclusions: Comparison of labeling patterns for CHT and AChE suggests that AChE histochemistry overestimates the density of cholinergic innervation in the heart. The distribution of CHT-immunoreactive nerve fibers and parasympathetic ganglia in the guinea pig heart suggests that heart rate, conduction velocity, and automaticity are precisely regulated by cholinergic innervation. In contrast, the paucity of CHT-immunoreactive nerve fibers in left ventricular myocardium implies that vagal efferent input has little or no direct influence on ventricular contractile function in the guinea pig.

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.

[ES] La presente tesis doctoral titulada "Reconocimiento de neurotransmisores basado en nanopartículas de oro y de sílice mesoporosa para aplicaciones de detección y liberación controlada" es una tesis realizada por compendio de artículos la cual se centra en el diseño, preparación, caracterización y evaluación de distintos nanodispositivos para la detección colorimétrica de neurotransmisores y sistemas de liberación controlada que responden a neurotransmisores basados en nanopartículas de oro y nanopartículas de sílice mesoporosa, equipadas con ligandos orgánicos, efectores enzimáticos, puertas moleculares y especies cromofluorogénicas o medicamentos. En el primer capítulo se introduce una visión general de lo que son los neurotransmisores, sus principales características y el importante papel que éstos desempeñan en el funcionamiento de nuestro organismo. Además, se presenta una descripción general de las propiedades y potenciales aplicaciones de las nanopartículas de oro funcionalizadas con ligandos orgánicos como sistemas de detección y las nanopartículas mesoporosas de sílice funcionalizadas con puertas moleculares como sistemas de liberación controlada. A continuación, en el segundo capítulo se presentan los objetivos generales que son abordados en los siguientes capítulos experimentales. En el tercer capítulo, se presentan tres sistemas de detección colorimétrica de neurotransmisores basados en la agregación de nanopartículas de oro doblemente funcionalizadas con ligandos orgánicos. El primer sistema es un sensor capaz de detectar de forma selectiva el neurotransmisor serotonina, utilizando nanopartículas de oro funcionalizadas con ditio-bis(propionato de succinimidilo) y N-Acetil-L-Cisteína. El segundo sistema consiste en un sensor para la detección selectiva del neurotransmisor norepinefrina diseñado a partir de nanopartículas de oro funcionalizadas con 4-(liponiloxi)benzaldehído y ácido 4-mercato fenilborónico. El tercer sistema está compuesto por nanopartículas de oro funcionalizadas con 4-(liponiloxi)benzaldehído y N-Acetil-L-Cisteína, para la detección de normetanefrina, un importante biomarcador del tumor feocromocitoma. Todos estos sistemas se evalúan en medios competitivos como suero sanguíneo u orina. En el cuarto capítulo se muestran dos sistemas de liberación controlados enzimáticamente basados en la apertura de puertas moleculares. El primer sistema de liberación controlada responde a la presencia del neurotransmisor acetilcolina. En concreto, se utilizan nanopartículas de sílice mesoporosa funcionalizadas en su superficie con grupos de ácido fenilborónico y tapadas con la enzima acetilcolinesterasa mediante la formación de ésteres cíclicos de ácido fenilborónico entre las cadenas de oligosacáridos de la enzima y los grupos fenilborónicos de la superficie de las nanopartículas. En este caso la reacción enzimática produce ácido acético que da lugar a la hidrolisis de los ésteres borónicos, destapando los poros y liberando la carga contenida en el interior. Además, se evalúa la capacidad del dispositivo diseñado para liberar el citotóxico doxorubicina en células cancerosas en presencia de acetiltiocolina. El segundo sistema consiste en un nanodispositivo para la liberación controlada en respuesta al neurotransmisor L-glutamato. Para esto se utilizan nanopartículas tipo Janus de oro-sílice mesoporosa funcionalizadas con la enzima L-glutamato oxidasa en la parte del oro y con una puerta molecular autoinmolante de arilboronato en la superficie de la sílice. La liberación controlada se basa en el reconocimiento del L-glutamato por la enzima L-glutamato oxidasa y la posterior formación de peróxido de hidrogeno, que es la especie que induce la escisión de la puerta autoinmolante y la subsecuente apertura de los poros. Finalment es mostra que el sistema dissenyat és capaç d'alliberar un fàrmac citotòxic en cèl·lules de càncer de cervell després de detectar la presència de L-glutamat. / [CA] La present tesi doctoral titulada "Reconeixement de neurotransmissors basat en nanopartícules d'or i de sílice mesoporosa per a aplicacions de detecció i alliberació controlada" és una tesi realitzada per compendi d'articles la qual se centra en el disseny, preparació, caracterització i avaluació de diferents nanodispositius per a la detecció colorimètrica de neurotransmissors i sistemes d'alliberació controlada que responen a neurotransmissors basats en nanopartícules d'or i nanopartícules de sílice mesoporosa equipades amb lligands orgànics, efectors enzimàtics, portes moleculars i espècies cromofluorogénicos o medicaments. En el primer capítol s'introdueix una visió general del que són els neurotransmissors, les seves principals característiques i l'important paper que aquests tenen en el funcionament del nostre organisme. A més es presenta una descripció general de les propietats i potencials aplicacions de les nanopartícules d'or funcionalitzades amb lligands orgànics com a sistemes de detecció, i de les nanopartícules mesoporoses de sílice funcionalitzades amb portes moleculars com a sistemes d'alliberament controlat. A continuació, en el segon capítol es presenten els objectius generals que són abordats en els següents capítols experimentals. En el tercer capítol, es presenten tres sistemes de detecció colorimètrica de neurotransmissors basats en l'agregació de nanopartícules d'or doblement funcionalitzades amb lligands orgànics. El primer sistema és un sensor capaç de detectar de forma selectiva el neurotransmissor serotonina, utilitzant nanopartícules d'or funcionalitzades amb ditiobis (propionat de succinimidilo) i N acetil-L-cisteïna. El segon sistema consisteix en un sensor per a la detecció selectiva de neurotransmissor norepinefrina dissenyat a partir de nanopartícules d'or funcionalitzades amb 4- (liponiloxi) benzaldehid i Àcid 4-mercatofenilborònic. El tercer sistema està compost per nanopartícules d'or funcionalitzades amb 4- (liponiloxi) benzaldehid i N acetil-L-cisteïna, per a la detecció de normatanefrina un important biomarcador del tumor feocromocitoma. Tots aquests sistemes s'avaluen en mitjans competitius com sèrum sanguini u orina. En el quart capítol es mostren dos sistemes d'alliberament controlats enzimàticament basats en l'obertura de portes moleculars. El primer sistema d'alliberament controlat respon a la presència del neurotransmissor acetilcolina. En concret, s'utilitzen nanopartícules de sílice mesoporosa funcionalitzades en la seva superfície amb grups d'àcid fenilborònic i tapades amb l'enzim acetilcolina esterasa mitjançant la formació d'èsters cíclics d'àcid fenilborònic entre les cadenes d'oligosacàrids de l'enzim i els grups fenilborónicos de la superfície de les nanopartícules. En aquest cas, la reacció enzimàtica produeix àcid acètic que dóna lloc a la hidròlisi dels èsters borònics, destapant els porus i alliberant la càrrega continguda a l'interior. A més, s'avalua la capacitat del dispositiu dissenyat per alliberar el citotòxic doxorubicina en cèl·lules canceroses en presència d'acetiltiocolina. El segon sistema consisteix en un nanodispositiu per alliberació controlada en resposta al neurotransmissor L-glutamat, per al que s'utilitzen nanopartícules tipus Janus d'or-sílice mesoporosa funcionalitzades amb l'enzim L-glutamat oxidasa en la part de l'or i amb una porta molecular autoimmolant d'arilboronat a la superfície de la sílice. La alliberació controlada es basa en el reconeixement de L-glutamat per l'enzim L-glutamat oxidasa i la posterior formació de peròxid d'hidrogen, que és l'espècie que indueix l'escissió de la porta autoimmolant i la subseqüent obertura dels porus. Finalment es mostra que el sistema dissenyat és capaç d'alliberar un fàrmac citotòxic en cèl·lules de càncer de cervell després de detectar la presència de L-glutamat. / [EN] This doctoral thesis entitled "Neurotransmitters recognition based on gold and mesoporous silica nanoparticles for sensing and controlled release applications" it is a thesis carried out by compendium of articles, which is focused on the design, preparation, characterization and evaluation of nanodevices for the colorimetric sensing of neurotransmitters and controlled delivery systems responsive to neurotransmitters, based on gold nanoparticles and mesoporous silica nanoparticles equipped with organic ligands, enzymatic effectors, molecular gates and chromo-fluorogenic species or drugs. The first chapter introduces an overview about what neurotransmitters are, their main characteristics and the important role they play in the functioning of our body. In addition, a general description of the properties and potential applications of gold nanoparticles functionalized with organic ligands as detection systems and mesoporous silica nanoparticles functionalized with molecular gates as controlled delivery systems is presented. In the second chapter, the general objectives that are addressed in the following experimental chapters are presented. In the third chapter, three colorimetric detection systems of neurotransmitters based on the aggregation of gold nanoparticles doubly functionalized with organic ligands are presented. The first system is a sensor capable of selectively detecting the neurotransmitter serotonin, using gold nanoparticles functionalized with dithio-bis(succinimidyl propionate) and N acetyl-L-cysteine. The second system consists of a sensor for the selective detection of the neurotransmitter norepinephrine designed from gold nanoparticles functionalized with 4- (liponyloxy)benzaldehyde and 4-mercaptophenylboronic acid. The third system is composed of gold nanoparticles functionalized with 4-(liponyloxy)benzaldehyde and N-Acetyl-L-Cysteine, for the detection of normetanephrine, an important biomarker of the pheochromocytoma tumor. All these systems are evaluated in competitive media such as blood serum or urine. In the fourth chapter, two enzymatic controlled delivery systems based on the opening of molecular gates are developed. The first controlled delivery system responds to the presence of the neurotransmitter acetylcholine. Specifically, it consists of mesoporous silica nanoparticles functionalized on their surface with phenylboronic acid groups and capped with the enzyme acetylcholinesterase, via the formation of cyclic phenylboronic acid esters between the oligosaccharide chains of the enzyme and the phenylboronic groups on the nanoparticles surface. In this case, the enzymatic reaction produces acetic acid that induces the hydrolysis of the boronic esters, uncapping the pores and releasing the entrapped payload. In addition, the ability of the nanodevice to release the cytotoxic doxorubicin in cancer cells in the presence of acetylthiocholine is evaluated. The second delivery system consists of a nanodevice responsive to the neurotransmitter L-glutamate. It is based on Janus gold-silica mesoporous nanoparticles functionalized with the enzyme L-glutamate oxidase in the gold part and with a self-immolative arylboronate molecular gate on the surface of the silica. Controlled delivery is based on the recognition of L-glutamate by the enzyme L-glutamate oxidase and the subsequent formation of hydrogen peroxide, which results in the cleavage of the self-immolative gate and the uncapping of the pores. Finally, it is shown that the designed system is capable of releasing a cytotoxic drug in brain cancer cells after detecting the presence of L-glutamate. / The authors acknowledge financial support from the Spanish Government (Projects MAT2015-64139-C4-1-R, MAT2015-64139-C4-4-R (MINECO/FEDER) and Project AGL2015-70235-C2-2-R) and the Generalitat Valenciana (Projects PROMETEOII/2014/047 and PROMETEO/2018/024). T. Godoy-Reyes is grateful to Generalitat Valenciana for her Santiago Grisolía fellowship. A. García-Fernández is grateful to the Spanish Government for her FPU fellowship. A. Llopis-Lorente thanks “La Caixa” Foundation for his PhD grant. SCSIE (Universitat de València) is gratefully acknowledged for all the equipment employed. / Godoy Reyes, TM. (2020). Neurotransmitters recognition based on gold nanoparticles and mesoporous silica nanoparticles for sensing and controlled release applications [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/158420

Title: Computer modeling of ion protein interactions: Allosteric effects of phenolic ligands and ions on insulin hexamer structure Author: Vladimír Palivec Department: Department of Physical and Macromolecular Chemistry Faculty of Science UK Advisor: prof. RNDr. Pavel Jungwirth, DSc., IOCB AS CR, v.v.i. Advisor's email address: pavel.jungwirth@uochb.cas.cz Abstract: Insulin hexamer is an allosteric protein capable of undergoing conformational changes between three states: T6, T3R3, and R6. Transitions between them, as well as the formation of insulin hexamers, are mediated through binding of phenolic ligands or ions. This thesis presents a molecular dynamics study of allosteric behavior of insulin using empirical force fields. Two effects are closely inspected - cation (Zn2+ , Ca2+ , K+ , and Na+ ) binding to the insulin hexamers and a possible binding of two neurotransmitters - dopamine and serotonin to the phenolic pocket. The results show that high charge density cations (Zn2+ and Ca2+ ) are mostly localized in the B13 glutamate cavity, slow- down diffusion, while preventing other cations from entering. In contrast, low charge density cations (Na+ and K+ ) do not have this effect. Concerning neurotransmitters, dopamine does not bind to the phenolic pocket whereas serotonin binds in a similar way like...