This study developed a reinforced steel rod bending machine for rods with diameters of up to 8 mm and annealed wire cutter for up to 5 kg for replacing manual intervention required to bend rods in reinforced concrete columns. This study aims to reduce the physical effort that could lead to occupational diseases, such as tenosynovitis, bursitis, muscle disorders. Clamp manufacturing possesses great risk for workers, who are exposed to injuries while using different cutting devices, such as grinders and electric saws. They also face potential problems such as muscular fatigue due to the nonergonomic and repetitive work positions. The proposed machine features a mechanical dragging and bending systems and manual shears. Additionally, the proposed machine has been designed theoretically and its effectiveness has been assessed through simulations conducted using the SolidWorks CAD software. A bending machine prototype for producing clamps is developed and its machine productivity is measured. Using this machine, approximately 300 clamps can be bent per hour without possessing any risk to the worker.

The topic of this thesis is electrophysiological characterization of Kir2.1 membrane channel. Inward rectifier potassium channel Kir2.1 is located in muscular, heart and nerve cells and its dysfunction causes various diseases. Practical part of this stage is focused on cultivation of the HEK293T cell line that is used to transfection of the plasmid Kir2.1 and subsequent measurement of the ionic current through the electrophysiological method patch-clamp in whole-cell mode.

The transient receptor potential (TRP) are cation channels mostly permeable to both monovalent and divalent cations. ThermoTRP is a specific group of directly thermally activated TRP channels. The vanilloid transient receptor potential 3 (TRPV3) is an ion channel widely expressed in keratinocytes, that is implicated in the regulation of skin homeostasis, thermo- sensing, nociception and development of itch sensation. Our results show the importance of the cytoplasmic inter-subunit interface in the heat sensitivity of TRPV3. As there is a structural analogy within the vanilloid receptors, our hypothesis of the identified important region is supposed to be valid also for other thermally activated TRPV receptors (TRPV1, TRPV2 and TRPV4). We have proved that TRPV3 is a substrate for ERK1/2 protein kinase (kinase regulated by extracellular signal 1 and 2) and we have identified TRPV3 phosphorylation sites that may be direct targets for ERK1/2. Of these residues, threonine 264 has been shown to be the main phosphorylation site responsible for TRPV3 sensitization mediated by ERK kinase. In human keratinocytes, the phosphorylation might be physiologically and pathophysiologically important in processes of TRPV3 sensitization mediated by MAPK signaling pathway. The transient receptor potential ankyrin 1...

Olanzapine, an atypical antipsychotic, alleviates symptoms of schizophrenia while producing fewer side effects compared to first generation antipsychotics. However, chronic usage remains problematic due to the propensity of olanzapine to induce weight gain and metabolic disturbances. Moreover, the cellular mechanisms underlying the metabolic side effects are poorly understood. The central nervous system (CNS) exerts both hormonal and neuronal control over whole body homeostasis. The dorsal motor nucleus of the vagus (DMV) participates in this regulation through modulation of the parasympathetic outflow to subdiaphragmatic organs. We hypothesized that olanzapine disrupts neurotransmission of the DMV, and thus contributes to the dysregulation of metabolism. We used whole-cell patch-clamp recordings from female C57BL/6J to assess the effect of olanzapine on DMV neurons. First, we investigated the effect of acute olanzapine administration on the activity of DMV neurons. Acute application of 10 µM olanzapine on DMV neurons induced both pre- and postsynaptic effects. Voltage-clamp recordings revealed that, in 5 out of 9 DMV neurons, excitatory inputs to DMV neurons were significantly increased by 71.6 ± 22.1%. In addition, in current-clamp mode, olanzapine induced a robust hyperpolarization from -49.00 ± 0.64 mV to -60.82 ± 2.78 mV. The hyperpolarization suppressed action potential firing. As a next step, we investigated the subchronic effect of olanzapine on the activity of DMV neurons. Daily subcutaneous injections were made for 20 days (5 mg/kg/day of olanzapine and vehicle). We did not find significant differences in body weight, blood glucose, and insulin or leptin levels. Subchronic administration of olanzapine generated presynaptic changes in DMV neurons. In treated animals, additional infusion of 10 µM olanzapine on DMV neurons significantly reduced excitatory neurotransmission by 41.0 ± 3.1% in 10 out of 17 neurons. Our findings indicate that olanzapine directly modulates the neuronal activity in DMV neurons, and could thus contribute to the metabolic disturbances seen in long-term treatments. / acase@tulane.edu

Neuroinflammation is prevalent in neurodegenerative diseases and plays a significant role in the central nervous system (CNS) innate immunity, which is the body’s first line of defense mechanisms against invading pathogens and injuries to maintain homeostasis. However, in neurodegenerative diseases, neuroinflammation becomes persistent alongside the subsequent damage to nearby neurons and affects CNS-resident immune glial cells, such as microglia and astrocytes. Accumulating evidence suggests that neuroinflammation is mainly characterized by the excessive activation of glial cells, thus causing abnormal changes in their microenvironment and release soluble factors that can promote or inhibit neuroinflammation. Currently, there is no effective treatment to cure these progressive neurological disorders. Therefore, it is critical to understand how neuroinflammation affects astroglia cell function and their biomechanical properties that change their behavior throughout disease progression. Astrocytes are the most predominant glial cell in the CNS and are critical in the development and maintenance of neuroinflammatory disorders. To date, very little is known regarding the role and specific function of Piezo1 mechanosensitive ion channel (MSC) in the CNS. Recently, Piezo1 expression was found to be upregulated in Lipopolysaccharide (LPS)-induced neuroinflammation in mouse astrocyte cultures. However, it is unknown whether the aberrant mechanical environment in astrocytes interplay with the mechanosensory function of Piezo1 and its current activity in neuroinflammatory conditions. In this study, we investigated Piezo1 mechanosensitive ionic currents by performing in vitro patch-clamp electrophysiology and calcium imaging. Our preliminary studies revealed that astrocytes derived from the mouse cerebellum stimulated with LPS or Piezo1 agonist, Yoda1, increased Ca2+ influx and further augmented when treated concurrently. We also found that electrophysiology recordings showed changes in mechanosensitive ionic currents and were comparable with our calcium imaging data indicating that MSCs are involved in neuroinflammation. Therefore, we postulated that Piezo1, a non-selective cation MSC that opens in response to mechanical force is a key mechanosensor involved in neuroinflammation by altered mechanical signals in C8-S astrocytes. Using an in vitro system of Mouse C8-S (Astrocyte type II clone), the goal of this study was to investigate if neuroinflammatory conditions upregulate Piezo1 calcium influx and current activity. We show that astrocytic Piezo1 regulates mechanotransducive release of ATP by controlling the mechanically induced calcium influx and current activation in LPS-induced astrocytes. Additionally, Piezo1 antagonist, GsMTx4 and Piezo1 siRNA significantly reduced the LPS-induced current, indicating that Piezo1 is involved in neuroinflammation. Our findings demonstrate that the activity of Piezo1 stimulated by neuroinflammatory conditions may be significant for the development of therapeutics to prevent or treat neuroinflammatory disorders and diseases.

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El sistema nervioso está formado por una compleja red de billones de neuronas que utilizan señales específicas para comunicarse entre sí. La sinapsis química es una unión funcional entre neuronas en la que el neurotransmisor liberado por una de ellas interactúa específicamente con proteínas de membrana de la otra, los receptores postsinápticos. Los receptores de la familia cys-loop, que incluye al receptor nicotínico de acetilcolina (nAChR, nicotinic acetylcholine receptor), son miembros de la superfamilia de los canales pentaméricos activados por ligando (pLGICs, pentameric ligand-gated ion channels). Están formados por cinco subunidades iguales - receptores homoméricos - o diferentes - receptores heteroméricos -. Los receptores poseen un dominio extracelular, que contiene los sitios de unión al agonista localizados entre dos subunidades adyacentes; un dominio transmembrana, que forma el canal y contiene sitios alostéricos para la acción de moduladores; y un dominio intracelular, de importancia en la conductancia del canal y en su modulación intracelular. El receptor α7 es el prototipo de receptor homopentamérico de la familia de los nAChRs. Es uno de los nAChRs más abundantes en el sistema nervioso, aunque también se encuentra presente en otros tejidos. En neuronas modula la liberación de neurotransmisores e induce respuestas estimulatorias, contribuyendo a la cognición, el procesamiento de la información sensorial y la memoria. En tejidos no neuronales está involucrado en inmunidad, inflamación y neuroprotección. Debido a sus múltiples funciones, emerge actualmente como nuevo blanco terapéutico para desórdenes neurológicos e inflamatorios. En el primer capítulo de este trabajo de tesis exploramos el rol funcional de una subunidad truncada del receptor α7 específica de humanos, dupα7. Dicha subunidad, que carece de una región del dominio N-terminal extracelular que comprende parte del sitio de unión al ligando, se encuentra asociada con desórdenes neurológicos e inmunomodulación. Utilizando expresión heteróloga en células de mamífero en conjunto con microscopía de fluorescencia y registros de electrofisiología mediante la técnica de patch clamp, determinamos que: i) dupα7 no es capaz de formar homopentámeros funcionales activables por acetilcolina o por un agonista alostérico; ii) La subunidad dupα7 puede combinarse con la subunidad α7 para formar heteropentámeros de diferentes estequiometrías, con características cinéticas similares a las del receptor α7; iii) Es necesaria la presencia de al menos dos subunidades α7 ubicadas en forma consecutiva en el heteropentámero para que los receptores sean funcionales; iv) La expresión conjunta de dupα7 y α7 disminuye la disponibilidad de sitios de unión al agonista, reduciendo la sensibilidad de los receptores. En forma global, nuestros resultados muestran que la subunidad dupα7 posee un rol modulador negativo sobre la actividad del receptor α7. En el segundo capítulo, se evaluó la modulación de los péptidos β-Amiloide 1-40 y 1-42 sobre el receptor α7. Dichos péptidos poseen un rol fundamental en la enfermedad de Alzheimer, dado que su acumulación excesiva en el cerebro provoca la formación de placas seniles, a partir de las cuales se desarrolla un proceso de neurodegeneración e inflamación. Sin embargo, evidencias más recientes sugieren que son las formas oligoméricas de β-Amiloide las especies más neurotóxicas. Empleando estudios espectrofluorimétricos y la técnica de patch clamp demostramos que: i) Los oligómeros β-Amiloide provocan cambios conformacionales en el receptor α7 que pueden ser detectados por la sonda conformacional cristal violeta; ii) Los oligómeros de péptidos β-Amiloide son capaces de activar al receptor α7 en concentraciones del orden picomolar o nanomolar bajo; iii) Los oligómeros β-Amiloide reducen la potenciación de α7 por moduladores alostéricos positivos (PAMs, positive allosteric modulators) a concentraciones del orden nanomolar o micromolar bajo; iv) La reducción de la potenciación causada por los péptidos β-Amiloide no es específica del tipo de modulador alostérico positivo. Estos resultados demuestran un rol dual, dependiente de la concentración, de los oligómeros de β-Amiloide como agonistas y como moduladores negativos de α7. El efecto inhibitorio podría contribuir al deterioro cognitivo asociado a la enfermedad de Alzheimer. Por último, en el tercer capítulo se evaluó la acción del ion Ca2+ como modulador alostérico positivo de α7. Existen reportes sobre cationes divalentes que actúan como moduladores de los pLGICs, variando su efecto según el tipo de receptor. Sobre α7, el ion Ca2+ actúa como un PAM, pero la base mecanística de esta acción no ha sido explorada. Combinando registros de corrientes macroscópicas y de canal único en las configuraciones cell-attached e inside-out de la técnica de patch clamp demostramos que: i) La presencia del ion Ca2+ extracelular potencia la respuesta macroscópica a acetilcolina y a colina dependiendo de la concentración de agonista; ii) La ausencia de Ca2+ en la solución extracelular disminuye la frecuencia de apertura del canal y aumenta levemente la corriente unitaria; iii) El mecanismo por el cual el ion Ca2+ potencia la respuesta al agonista es compatible con el aumento de la probabilidad de apertura del canal. De este modo, identificamos el mecanismo asociado a la acción moduladora de calcio sobre el receptor α7. Nuestros resultados contribuyen al entendimiento de la modulación del receptor α7 por una subunidad proteica asociada con enfermedades neurológicas, por péptidos amiloides producidos en patologías neurodegenerativas y por el catión Ca2+, todos procesos de relevancia en la señalización colinérgica en el sistema nervioso central. / The nervous system is formed by a complex net of billions of individual neurons that use specific signals to communicate with each other. The chemical synapse is a functional union between neurons in which the neurotransmitter released by one neuron interacts specifically with integral membrane proteins of the other neuron: the postsynaptic receptors. The receptors of the cys-loop family, that includes the nicotinic acetylcholine receptor (nAChR), are members of the superfamily of the pentameric ligand-gated ion channels family (pLGICs). They are formed by five identical - homopentameric receptors - or different subunits – heteropentameric receptors -. The receptors have an extracellular domain, which contains the agonist binding sites that are located between two adjacent subunits; a transmembrane domain that forms the channel and contains allosteric sites for the action of modulators; and an intracellular domain, important for the conductance of the channel and modulation. The α7 receptor is a homopentamer of the nAChRs family. It is one of the most abundant nAChRs in the nervous system and is also present in cells of other tissues. In neurons, it modulates the neurotransmitters release and induces stimulatory responses, thus contributing to cognition, sensory information processing and memory. In non-neuronal tissues, it is involved in immunity, inflammation and neuroprotection. Because of its multiple functions, it is emerging as a new therapeutic target for neurologic and inflammatory disorders. In the first chapter of this thesis we evaluated the functional role of a human-specific truncated subunit of the α7 receptor, dupα7. This subunit, which lacks part of the N-terminal extracellular ligand-binding domain, is associated with neurological disorders and immunomodulation. Using heterologous expression of heteropentamers in mammalian cells combined with fluorescence microscopy and patch clamp recordings, we determined that: i) dupα7 is not able to form functional receptors activated by acetylcholine or an allosteric agonist; ii) dupα7 subunits can combine with α7 subunits to form heteropentamers of different stoichiometries, with similar kinetic properties to those of α7; iii) the presence of at least two α7 subunits located consecutively in the heteropentamer forming an agonist binding site is necessary for functional heteropentamers; iv) the co-expression of dupα7 and α7 decreases the availability of agonist binding sites, reducing the sensibility of the receptors. Overall, our results show that dupα7 has a negative modulatory role on the activity of the α7 receptor. In the second chapter, we evaluated the modulation of α7 receptor by Amyloid- β 1-40 and 1-42 peptides. These peptides play a fundamental role in Alzheimers’ disease since their excessive accumulation in the brain causes the formation of senile plaques, from which a process of neurodegeneration and inflammation develops. More recent evidence suggests that the oligomeric forms of amyloid- β are the most neurotoxic species. Using spectrofluorimetric and electrophysiological studies we demonstrated that: i) The amyloid-β peptides cause conformational changes on the α7 receptor that can be sensed by the crystal violet conformational probe; ii) The oligomers of the amyloid-β peptide are capable of activating the α7 receptor at picomolar or low nanomolar concentrations; iii) The oligomers of the amyloid-β peptide reduce α7 potentiation by positive allosteric modulators (PAMs) at nanomolar or low-micromolar concentrations; iv) The reduction in the potentiation caused by the amyloid-β peptides is not specific of the PAM type. Our results demonstrate a dual role of the amyloid- β oligomers as agonists and negative modulators of α7, depending on the concentration. The inhibitory effect could contribute to the cognitive impair associated to Alzheimer’s disease. Last, in the third chapter we evaluated the action of the Ca2+ cation as a PAM of α7. Divalent cations have been reported to modulate different pLGICs, varying their effects with the receptor type. On α7, Ca2+ acts as a PAM, but the mechanistic basis of this action has not been explored yet. Combining macroscopic and single-channel current recordings in the cell-attached and inside-out patch clamp configurations, we demonstrated that: i) Extracellular Ca2+ potentiates the macroscopic responses to ACh and choline and the level of potentiation is dependent on the agonist concentration; ii) The absence of extracellular calcium diminishes the frequency of channel opening and slightly increases the unitary current; iii) The mechanism by which Ca2+ enhances the response to the agonist is compatible with an increase of the channel opening probability. Our results contribute to the understanding of the molecular actions at α7 of a truncated protein subunit associated with neurological diseases, of amyloid peptides produced in neurodegenerative pathologies and of the Ca2+ cation, which are all relevant modulatory processes of the cholinergic pathway at the central nervous system.

Das streng regulierte Gleichgewicht zwischen CO2-Aufnahme und Transpiration ist für Pflanzen essentiell und hängt von kontrollierten Turgoränderungen ab, die durch die Aktivität verschiedener Anionen- und Kationenkanäle verursacht werden. Diese Kanäle sind Teil von Signalkaskaden, die z. B. durch Phytohormone wie ABA (Abscisinsäure) und JA (Jasmonat) ausgelöst werden, die beide bei Trockenstress in den Schließzellen wirken. Darüber hinaus ist bekannt, dass JA an der Reaktion der Pflanze auf Pathogenbefall oder Verwundung beteiligt ist. GORK (guard cell outward rectifying K+ channel) ist der einzige bekannte, auswärts gleichrichtende K+-Kanal in Schließzellen und somit für den K+-Efflux beim Schließen der Stomata verantwortlich. Im Rahmen dieser Arbeit konnte nachgewiesen werden, dass GORK ein wesentlicher Bestandteil des JA-induzierten Stomatschlusses ist. Dies gilt für beide Auslöser, sowohl die Blattverwundung als auch die direkte Anwendung von JA. Patch-Clamp-Experimente an Protoplasten von Schließzellen untermauerten dieses Ergebnis, indem sie GORK-K+-Auswärtsströme als direktes Ziel von JA-Signalen entlarvten. Da bekannt ist, dass zytosolische Ca2+-Signale sowohl bei ABA- als auch bei JA-Signalen eine Rolle spielen, wurde die Interaktion von GORK mit Ca2+-abhängigen Kinasen untersucht. Eine antagonistische Regulation von GORK durch CIPK5-CBL1/9-Komplexe und ABI2 konnte durch DEVC (double electrode voltage clamp) sowie Protein-Protein-Interaktions-Experimente identifiziert und durch in-vitro Kinase-Assays untermauert werden. Patch-Clamp-Aufzeichnungen an Protoplasten von Schließzellen der cipk5-2 Funktions-Verlust-Mutante zeigten die Bedeutung von CIPK5 für den JA-induzierten Stomaschluss via Aktivierung von GORK. Die Interaktion verschiedener CDPKs (Ca2+-abhängige Proteinkinasen) mit GORK wurde ebenfalls untersucht. Neben der Ca2+-Signalübertragung ist auch die Produktion von ROS (reaktive Sauerstoffspezies) für die ABA- und MeJA-Signalübertragung von Bedeutung. In DEVC-Experimenten konnte ein reversibler Effekt von ROS auf die GORK-Kanalaktivität nachgewiesen werden, was ein Teil der Erklärung für diese ROS-Effekte bei ABA- und MeJA-Signalen sein könnte. / Maintaining the balance between CO2 uptake and transpiration is important for plants and depends on tightly controlled turgor changes caused by the activity of various anion and cation channels. These channels are part of signaling cascades triggered, for example, by phytohormones such as ABA (abscisic acid) and JA (jasmonate), both of which act during drought stress in guard cells. In addition, JA is known to be involved in the plant's response to pathogen attack or wounding. GORK (guard cell outward rectifying K+ channel) is the only known outward rectifying K+ channel in guard cells and therefore responsible for K+ efflux during stomatal closure. In the course of this work it could be demonstrated by stomatal aperture assays, that GORK is an essential part of JA-induced stomatal closure. This is true for both triggers, leaf wounding as well as direct MeJA (methyl jasmonate) application. Patch clamp experiments on guard cell protoplasts backed this finding by revealing GORK K+ outward currents as a target of JA signaling in guard cells. As cytosolic Ca2+ signals are known to be involved in both ABA as well as JA signaling, the interaction of GORK with Ca2+-dependent kinases was examined consequently. An antagonistic regulation of GORK by CIPK5-CBL1/9 complexes and ABI2 was identified by DEVC (double electrode voltage clamp) and protein-protein interaction experiments and backed up by in vitro kinase assays. Patch-clamp recordings on guard cell protoplasts of cipk5-2 kinase loss-of-function mutant revealed the importance of CIPK5 for JA-triggered stomatal closure via activation of GORK. The interaction of different CDPKs (Ca2+-dependent protein kinases) with GORK was also investigated. Besides Ca2+ signaling also ROS (reactive oxygen species) production is essential in ABA and MeJA signaling. In DEVC experiments a reversible effect of ROS on GORK channel activity could be demonstrated, which could be one piece in the explanation of those ROS effects in ABA and MeJA signaling.