The relationship between leaf physiognomy and precipitation has been explored worldwide in regions under different climate conditions. Unlike the linear relationship established between the percentage of woody dicot species with entire margins and mean annual temperature, precipitation has been reported to correlate to different leaf physiognomic characters depending on the region where the correlation is studied. To investigate if precipitation can be calculated from leaf physiognomic characters on the basis of regional sample sites, data from 50 mesic to humid forests in China were analyzed in this study. With data from Chinese forests, the leaf-area analysis based on linear regression between natural logarithms of leaf size and mean annual precipitation (MAP) shows no significant correlation. Both single and multiple linear regression analyses fail to confirm the correlation between leaf physiognomy and precipitation, which may result from the similarity of modern spatial distribution of temperature and precipitation in China. Our results show that, due to variations in climatic conditions among sampling regions, leaf physiognomic characters that correlate to precipitation are not consistent worldwide, and applications of models without considering regional constraints could mislead our understanding of palaeoclimate. Therefore, when choosing a leaf physiognomic model for palaeoclimate reconstructions, it is important to determine if the leaf physiognomy of the palaeoflora lies within the leaf physiognomic spectrum of the model used.

At the neuromuscular junction, nicotinic acetylcholine receptors (AChRs) convert chemical stimuli into electrical signals. They are heteropentameric membrane protein complexes assembled from four evolutionary related subunits (two α subunits, and one each of the β-, δ-, and ε-subunits), arranged around a central ion-conducting pore, which is regulated by the neurotransmitter acetylcholine. Understanding how the binding of acetylcholine leads to channel opening is of fundamental importance. While it is known that channel opening results from a global conformational change involving the cooperative action of all five subunits, how the subunits achieve this cooperativity is unclear. Our hypothesis is that this subunit cooperation is maintained through coevolution of the subunits, and thus studies of subunit coevolution can provide insight into subunit cooperativity. Using an ancestral reconstruction approach, combined with single-molecule patch clamp electrophysiology, we have begun dissecting the mechanistic consequences of preventing coevolution of the acetylcholine receptor β-subunit. This approach has allowed us to identify new amino acid determinants of acetylcholine receptor function.

DNA encodes the genetic information of the cell, therefore, every single living organism has a precise DNA damage response mechanism to safeguard DNA integrity. Base mismatches are endogenous DNA lesions introduced by the replicative polymerase during DNA replication. The conserved DNA mismatch repair pathway corrects these base mismatches. Mismatch repair initiation is orchestrated by two proteins, MutS and MutL. MutS recognizes and binds to base mismatches and relays the presence of the lesion to MutL. MutL, in turn, interacts with downstream factors to coordinate mismatch excision. The processivity clamp, typically known for its role in tethering the DNA polymerase to DNA during replication, is also involved in several steps of this repair process including MutL endonuclease activation and strand resynthesis. The dynamics of the MutS-MutL and MutL-processivity clamp interactions present one of the bottlenecks to uncovering the spatial and time organization of these protein assemblies. Therefore, little is known about the interactions that orchestrate the early steps of mismatch repair. The biochemical and structural work included in this thesis outlines a precise series of molecular cues that activate MutL. / Thesis / Doctor of Philosophy (PhD)

These experiments were conducted to determine the membrane K+ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K+ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch clamp recordings. Cell depolarization resulted in activation of a Ca2+-dependent outward K+ current, 0.57 ± 0.13 nS/pF at -70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch clamp measurements demonstrated a Ca2+-activated, voltage-dependent K+ channel (K(Ca)) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current and the K(Ca) activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K+ channel, 21 ± 1 pS at positive transmembrane potential (V(m)) and 145 ± 13 pS at negative V(m). Glibenclamide (50 μM), Ba2+ (1 mM) and quinine (100 μM) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K+ channels in inside/out patches in a dose-dependent manner, and the IC50 = 46 μM. The identity of this K+ channel with an ATP-sensitive K+ channel (K(ATP)) was confirmed by its inhibition with ATP (2 mM) and by its activation with diazoxide (100 μM). We conclude that plasma membranes of HTB-9 cells contain the K(Ca) and a lower conductance K+ channel with properties consistent with a sulfonylurea receptor-linked K(ATP).

No description available.

No description available.

No description available.

En la comunicación neuronal, las transmisiones sinápticas son mediadas por canales iónicos activados por ligandos (LGIC), receptores que intervienen en procesos fisiológicos claves en el sistema nervioso central. La función esencial de estos receptores es acoplar la unión del neurotransmisor a la apertura del canal. Dado su papel esencial en la transmisión sináptica, los LGICs son blancos de agentes farmacológicos y numerosas patologías se asocian a su mal funcionamiento de estos receptores. Dentro de esta superfamilia de LGIC se encuentra la llamada familia de receptores Cys-loop que incluye a los receptores excitatorios nicotínico (AChR), de serotonina 5-HT3 y receptores inhibitorios GABAA y de glicina. Desde hace un tiempo, se ha discutido la posibilidad de que los campos magnéticos estáticos (CME) o electromagnéticos (CEM) resulten dañinos para la salud. Debido a los rápidos avances en las tecnologías de comunicación, la población está cada vez más expuesta a campos magnéticos. Esto aumenta la preocupación sobre los potenciales efectos para la salud derivados de la exposición a los mismos. A nivel celular, se ha propuesto que los campos probablemente inician sus efectos a través de los canales iónicos. En el presente trabajo de tesis doctoral hemos estudiado la influencia de campos magnéticos estáticos (CME) y campos electromagnéticos (CEM) sobre dos miembros de la familia de receptores Cys-loop, el receptor de acetilcolina muscular adulto (AChR) y el receptor de serotonina homopentamérico tipo 3A (5-HT3AR). El AChR es considerado el receptor modelo, tanto estructural como funcional, para todos los miembros de esta familia. En primer lugar, estudiamos los efectos de CME sobre el AChR. Encontramos que un CME de intensidad entre 80-180 mT, a temperatura ambiente, no induce cambios sobre las constantes macroscópicas, así como tampoco en las propiedades cinéticas a nivel de canal único. Dado que las propiedades diamagnéticas de la membrana celular se modifican por encima de una temperatura crítica, se realizaron estudios electrofisiológicos en un rango de temperatura de 5 °C a 50 °C, donde encontramos nuevamente que los LGIC no son sensibles a CME a otras temperaturas. En segundo lugar, caracterizamos la influencia del CEM sobre los AChR y 5-HT3AR. Los ensayos electrofisiológicos de corrientes macroscópicas mostraron que la amplitud de corriente disminuye en función de la frecuencia del CEM aplicado. La constante de decaimiento temporal no resulta modificada, mientras que el tiempo de activación aumenta significativamente. A nivel de canal único, encontramos que la exposición a los CEM no afecta la amplitud ni las constantes cinéticas de apertura y cierre. Sin embargo la frecuencia de episodios de activación (clusters), disminuye en función de la frecuencia. Es decir, que la presencia del CEM induce un nuevo estado no conductor, resultando en la disminución del pico de corriente y de la frecuencia de eventos de activación de clusters. Si bien los cambios cualitativos del CEM fueron equivalentes para AChR y 5-HT3AR, el receptor 5-HT3A mostró mayor sensibilidad a los efectos del campo. El trabajo desarrollado en esta tesis revela que los CEM pueden modificar la actividad de LGIC y abre puerta para entender los mecanismos moleculares y bases estructurales por los cuales los CEM modifican éstos receptores. / In neuronal communication, synaptic transmission is mediated by Ligand-Gated Ion Channels (LGICs), which are involved in fundamental physiological processes in the central nervous system. The essential function of these receptors is to couple neurotransmitter binding to channel opening. Given its essential role in synaptic transmission, the LGICs are targets of pharmacological agents and many diseases are associated with their incorrect function. The Cys-loop receptor family belongs to the LGIC superfamily and it includes the excitatory receptors, nicotinic and serotonin 5-HT3, and inhibitory receptors, GABAA and glycine receptors. For a while, it has been discussed the possibility that static magnetic fields (SMF) or electromagnetic (EMF) result harmful to health. Due to the rapid advances in communication technologies, the public is increasingly exposed to magnetic fields. This has raised concern about potential health effects resulting from exposure to them. At the cellular level, it is has been proposed that magnetic fields probably initiate their effects through ion channels. In the present thesis we studied the influence of static magnetic fields (SMF) and electromagnetic fields (EMF) on two members of the Cys-loop family receptors, the nicotinic acetylcholine receptor (AChR) and the homopentameric serotonin type 3A receptor (5-HT3AR). The AChR has been the structural and functional model for all members of this family. First, we studied the effects of SMF on the AChR. We found that a strong SMF of 80-180 mT, at room temperature, does not induce any changes on macroscopic currents response to the agonist or in the kinetic properties at the single channel level. Since diamagnetic properties of the cellular membrane are modified above a critical temperature, electrophysiological studies were carried out at a temperature range of 5°C to 50 °C. Again, we found that the LGIC receptor is not sensitive to SMF at a range of temperatures. Secondly, we characterized the influence of EMF on the AChR and 5-HT3AR. The electrophysiological recordings of macroscopic currents showed that the amplitude of the current decreases as a function of the EMF frequency applied. The temporal decay constant is not modified, whereas the rise time increases significantly. At the single channel level, we found that the exposure to EMF does not affect the amplitude or channel kinetics. However, the frequency of activation episodes elicited by agonist (cluster) is reduced significantly. Thus, the reduction of the peak current together with the frequency of channel suggests that EMF induces and stabilizes a new closed, non conductive state. Receptors not affected, or leaving this state, do not show changes in activation kinetics. The EMF affects the functionality of both AChR and 5-HT3AR being this influence on the 5-HT3AR is steeper that on the AChR. The work in this thesis contributes to understand how the EMF can modify the activity of LGIC and obtain the molecular mechanisms by which EMFs alter the kinetics of these receptors.

El cerebro humano está formado por una compleja red de células nerviosas que utilizan diversas señales para comunicarse entre ellas. La propagación de señales tiene lugar en la sinapsis química en donde el neurotransmisor liberado por la neurona presináptica interacciona con un receptor postsináptico específico. Los canales iónicos activados por ligandos (LGIC) median respuestas rápidas en dichas sinapsis. El rol vital de los mismos es convertir una señal química en un impulso eléctrico. Para generar una respuesta adecuada los LGIC deben ser capaces de activarse en presencia del neurotransmisor y cerrarse en su ausencia. Estos receptores están involucrados en el aprendizaje, la memoria, el movimiento y en enfermedades genéticas, y son blancos de numerosos fármacos. Los receptores pentaméricos Cys-loop son LGIC que intervienen en sinapsis químicas rápidas. La duración, amplitud y frecuencia de una respuesta sináptica es gobernada por la cinética de apertura, cierre y desensibilización del canal. Los mecanismos moleculares de estos procesos no se conocen todavía. Los receptores poseen un dominio extracelular, unidor del neurotransmisor, y una región transmembranal, formadora del poro iónico. Uno de los objetivos de este trabajo de Tesis fue dilucidar el rol funcional de la interfase entre ambos dominios. Con este fin utilizamos receptores homopentaméricos con interfases con secuencias de 7 y 5-HT3A en los diferentes loops que las componen y evaluamos los tiempos de apertura y desensibilización de los receptores formados. Esta estrategia nos permitió determinar la contribución de cada loop y las consecuencias funcionales de la interacción entre ellos. Determinamos que la interacción entre los distintos loops de la interfase permite el acoplamiento de la unión del agonista con la apertura del poro iónico y gobierna la cinética de apertura y desensibilización de los receptores Cys-loop, controlando de esta manera la duración de la respuesta sináptica y el período refractario. Los nematodos parásitos tienen importancia médica y veterinaria ya que afectan la salud del hombre y del animal. Los fármacos antihelmínticos son esenciales para controlar los nematodos parásitos. Los agentes levamisol, pirantel, morantel y oxantel, ejercen su acción actuando sobre los nAChRs de los helmintos. En los últimos años se ha demostrado que la acción de estos fármacos depende del subtipo de receptor nACh. Exploramos las bases estructurales de dichas diferencias estudiando cómo estos agentes activan a los receptores nACh muscular y 7 de mamífero utilizando la técnica electrofisiológica de Patch-clamp. Encontramos que todas estas drogas son agonistas débiles del receptor nACh muscular adulto de mamífero. Por el contrario, pirantel y morantel cambian su comportamiento a agonistas completos y más potentes que la ACh en el receptor 7. Determinamos que la posición 57, localizada en el lado complementario del sitio de unión de agonistas, es responsable de la diferente activación de los receptores nACh muscular y 7 por morantel y pirantel. Esta posición no altera la activación de ACh o de los fármacos oxantel y levamisol. El conocimiento de la activación de los nAChRs por antihelmínticos contribuirá al diseño de terapias más selectivas contra los parásitos y a comprender como éstos desarrollan resistencia a estos fármacos. / The human brain is a vast and complicated network, where billions of nerve cells use signals to communicate with each other. At chemical synapses, neurotransmitters are released from the presynaptic cell. They interact with ligand-gated ion channels (LGIC) at the postsynaptic cell that convert signals from chemical to electrical in less than one millisecond. The channels close as the neurotransmitter dissociates to terminate the synaptic event. These receptors are involved in learning, memory, movement and disease processes, and are targets for clinically relevant drugs. The pentameric Cys-loop receptors are LGIC involved in fast chemical synapsis. Following the neurotransmitter release and binding to Cys-loop receptors, the post-synaptic response is governed by the kinetics of channel activation, deactivation and desensitization. The molecular mechanisms of these processes are still unknown. Cys-loop receptors have an extracellular domain, which contains the agonist binding sites, and a transmembrane domain where the ion pore is located. One of the goals of this Thesis was to determine the functional role of the interfacial region between extracellular and transmembrane domains. We generated homomeric chimeric receptors carrying sequences of 7 or 5-HT3A at the different loops of the interface and evaluated the open channel lifetime and rate of desensitization. This strategy allowed us to determine the functional contribution of each loop and the consecuences of structural mismatching among them. We concluded that the network of loops at the binding-pore interface of homomeric receptors is essential for coupling agonist binding to channel opening and also for dictating the kinetics of gating and desensitization. Thus, this region controls the duration of the refractory period and the synaptic response. Parasitic nematodes are of medical and veterinary importance, affecting human and animal health. Anthelmintic drugs are essential to control nematode parasites. These agents, such as levamisol, pyrantel, morantel and oxantel, exert their action at nAChRs in nerve and muscle of nematodes. In the last years, it has been demonstrated that the actions of these drugs depend on nAChR subtypes. To understand the structural basis of the differential activation of anthelmintics among nAChR subtypes, we studied the activation of mammalian muscle and 7 nAChRs by these agents at the single-channel and macroscopic-current levels. We showed that anthelminitic agents are low efficacious agonists of mammalian muscle AChRs. By contrast, morantel and pyrantel are high-efficacious and more potent agonists than ACh of 7 receptor. Also, we determined that position 57, located at the complementary face of the binding site, is a main determinant of the differential activation of mammalian muscle and 7 nAChRs by morantel and pyrantel. This position is not involved in ACh, oxantel or levamisol activation. These results provide new information for further progress in drug design and help to understand how parasites develop resistance to these drugs.

This dissertation develops a modeling and simulation methodology for design, verification, and testing (DVT) power supply system using a virtual prototype. The virtual prototype is implemented before the hardware prototyping to detect most of the design errors and circuit deficiencies that occur in the later stage of a standard hardware design verification and testing procedure. The design iterations and product cost are reduced significantly by using this approach. The proposed modeling and simulation methodology consists of four major parts: system partitioning, multi-level modeling of device/function block, hierarchical test sequence, and multi-level simulation. By applying the proposed methodology, the designer can use the virtual prototype effectively by keeping a short simulation CPU time as well as catching most of the design problems. The proposed virtual prototype DVT procedure is demonstrated by simulating a 5 V power supply system with a main power supply, a bias power supply, and other protection, monitoring circuitry. The total CPU time is about 8 hours for 780 tests that include the basic function test, steady stage analysis, small-signal stability analysis, large-signal transient analysis, subsystem interaction test, and system interaction test. By comparing the simulation results with the measurements, it shows that the virtual prototype can represent the important behavior of the power supply system accurately. Since the proposed virtual prototype DVT procedure verifies the circuit design with different types of the tests over different line and load conditions, many circuit problems that are not obvious in the original circuit design can be detected by the simulation. The developed virtual prototype DVT procedure is not only capable of detecting most of the design errors, but also plays an important role in design modifications. This dissertation also demonstrates how to analyze the anomalies of the forward converter with active-clamp reset circuit extensively and facilitate the design and improve the circuit performances by utilizing the virtual prototype. With the help of the virtual prototype, it is the first time that the designer is able to analyze the dynamic behavior of the active-clamp forward converter during large-signal transient and optimize the design correspondingly. / Ph. D.