Dissertations / Theses on the topic 'Gating mechanism'

Cys-loop receptors are membrane proteins that are key players for the fast synaptic neurotransmission. Their ion transport initiates new nerve signals after activation by small agonist molecules, but this function is also highly sensitive to allosteric modulation by a number of compounds such as anesthetics, alcohol or anti-parasitic agents. For a long time, these modulators were believed to act primarily on the membrane, but the availability of high- resolution structures has made it possible to identify several binding sites in the transmembrane domains of the ion channels. It is known that ligand binding in the extracellular domain causes a conformational earthquake that interacts with the transmembrane domain, which leads to channel opening. The investigations carried out in this thesis aim at understanding the connection between ligand binding and channel opening. I present new models of the mammalian GABAA receptor based on the eukaryotic structure GluCl co-crystallized with an anti-parasitic agent, and show how these models can be used to study receptor-modulator interactions. I also show how removal of the bound modulator leads to gradual closing of the channel in molecular dynamics simulations. In contrast, simulations of the receptor with both the agonist and the modulator remain stable in an open-like conformation. This makes it possible to extract several key interactions, and I propose mechanisms for how the extracellular domain motion is initiated. The rapid increase in the number of cys-loop receptor structures the last few years has further made it possible to use principal component analysis (PCA) to create low-dimensional descriptions of the conformational landscape. By performing PCA on the crystal structure ensemble, I have been able to divide the structures into functional clusters and sample the transitions between them using various sampling methods. The studies presented in this thesis contribute to our understanding of the gating mechanism and the functional clustering of the cys-loop receptor structures, which both are important to design new allosteric modulator drugs that influence the channel function, in particular to treat neurological disorders.

QC 20160518

Thesis (Ph. D.) -- University of Maryland, College Park, 2005.
Thesis research directed by: Biology. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Computational methods, including homology modelling, in-silico dockings, and molecular dynamics simulations have been used to study the functional dynamics and interactions of K⁺ channels. Molecular models were built of the inwardly rectifying K⁺ channel Kir2.2, the bacterial homolog K⁺ channel KirBac3.1, and the twin pore (K2P) K⁺ channels TREK-1 and TRESK. To investigate the electrostatic energy profile of K⁺ permeating through these homology models, continuum electrostatic calculations were performed. The primary mechanism of KirBac3.1 gating is believed to involve an opening at the helix bundle crossing (HBC). However, simulations of Kir channels have not yet revealed opening at the HBC. Here, in simulations of the new KirBac3.1-S129R X-ray crystal structure, in which the HBC was trapped open by the S129R mutation in the inner pore-lining helix (TM2), the HBC was found to exhibit considerable mobility. In a simulation of the new KirBac3.1-S129R-S205L double mutant structure, if the S129R and the S205L mutations were converted back to the wild-type serine, the HBC would close faster than in the simulations of the KirBac3.1-S129R single mutant structure. The double mutant structure KirBac3.1-S129R-S205L therefore likely represents a higher-energy state than the single mutant KirBac3.1-S129R structure, and these simulations indicate a staged pathway of gating in KirBac channels. Molecular modelling and MD simulations of the Kir2.2 channel structure demonstrated that the HBC would tend to open if the C-linker between the transmembrane and cytoplasmic domain was modelled helical. The electrostatic energy barrier for K⁺ permeation at the helix bundle crossing was found to be sensitive to subtle structural changes in the C-linker. Charge neutralization or charge reversal of the PIP2-binding residue R186 on the C-linker decreased the electrostatic barrier for K⁺ permeation through the HBC, suggesting an electrostatic contribution to the PIP2-dependent gating mechanism. Multi-scale simulations determined the PIP2 binding site in Kir2.2, in good agreement with crystallographic predictions. A TREK-1 homology model was built, based on the TRAAK structure. Two PIP2 binding sites were found in this TREK-1 model, at the C-terminal end, in line with existing functional data, and between transmembrane helices TM2 and TM3. The TM2-TM3 site is in reasonably good agreement with electron density attributed to an acyl tail in a recently deposited TREK-2 structure.

Inwardly-rectifying potassium (Kir) channels comprise a transmembrane domain (TMD) that makes up the conducting pore and a large cytoplasmic domain (CTD) that controls channel gating via ligand-binding. Tissue-specific expression and diverse gating mechanisms endow each Kir family with distinct physiological roles. To better understand channel function and improve treatments for Kir channel-associated diseases, we set out to determine the molecular mechanisms underlying channel gating and block. Inward rectification via voltage-dependent polyamine block is important for maintaining a negative resting membrane potential and permitting the generation of action potentials in excitable cells. Polyamine block was studied in Kir2.1, a prototypical strong rectifier. Using inside-out patch clamp electrophysiology and sterically expanded polyamine blockers, our findings suggest that steep voltage-dependence and external K⁺-dependence of high-affinity block do not require extensive polyamine interactions with the selectivity filter. Furthermore, by introducing MTS moieties into the inner cavity, as well as utilizing a hydrogen bond-deficient blocker and the disease mutant D172N, we determined that hydrogen bonding is crucial to high-affinity binding between spermine and the rectification controller (D172 in Kir2.1). It is important to note that spermine is structurally unique and binds very deep in the inner cavity. This is likely not the case for diamine blockers, which warrants caution when drawing conclusions from them about spermine. A second gating mechanism in Kir channels is regulation by ligand-binding in the CTD. Ligand transduction to the channel gate is key to many physiological roles of Kir channels and can be demonstrated in KATP channels, which are responsible for transducing cellular metabolism to insulin secretion. Using inside-out patch clamp electrophysiology, we screened Kir6.2 slide helix mutants for ATP-sensitivity and voltage-dependent gating kinetics with a “forced gating” background mutation F168E that functionally rescues non-expressing mutants. We found that a highly conserved aspartate in the Kir slide helix is essential for ligand transduction. Further screening of nearby CTD residues revealed several potential interacting partners for this slide helix “aspartate anchor”. However, specific interactions between the aspartate anchor and the CTD remain unclear.

Rhythmic contraction of cardiac myocytes is maintained by precisely controlled Ca2+ efflux from intracellular stores mediated by the cardiac ryanodine receptor (RyR2). Mutations in RyR2 result in perturbed Ca2+ release that can trigger arrhythmias. RyR2- dependent ventricular tachyarrhythmia is an important cause of sudden cardiac death, the mechanistic basis of which remains unclear. RyR2 dysfunction has also been implicated in other cardiovascular disorders such as heart failure and cardiomyopathy, thereby becoming an important target for putative drugs. The massive size of RyR2 (~2.2 MDa) along with its intracellular location poses considerable challenges to studies aimed at understanding the mechanisms underlying channel dysfunction. Single channel studies of reconstituted RyR2 in artificial lipid bilayers have provided important insights into channel behaviour in response to various physiological ligands, toxins, drugs and biochemical modifications. However, the precise mechanisms by which RyR2 is activated by its primary physiological trigger, cytosolic Ca2+, and the structural determinants of channel gating are yet unknown. In this study, I aim to understand the actual physical reality of RyR2 gating behaviour using novel experimental approaches and analytical procedures. I have examined in detail, single channel kinetics of wild type purified recombinant human RyR2 (hRyR2) when activated by cytosolic Ca2+ in a precisely regulated minimal environment where the modulatory influence of factors external to the channel were minimised. This mathematical modelling of hRyR2 single channel behaviour will serve as a future experimental platform upon which the effects of disease causing mutations can be studied, as well as the influence of physiological modulators and potentially therapeutic compounds capable of stabilising mutant channel function. Single channel studies of hRyR2 when modified by its archetypal ligand ryanodine in the absence of Ca2+ have uncovered an unusual voltage sensitive gating behaviour in this ligand-gated channel, providing further insights into the mechanisms underlying channel modification.

Intracellular Ca2+-release from the sarcoplasmic reticulum (SR) occurs through highly specialised Ca2+-release channels called ryanodine receptors (RyR) in a process known as excitation-contraction coupling (EC-coupling) in striated muscle. The RyR is the main ion-channel mediating Ca2+-release from the SR but the SR also contains many other ion-channels and proteins, with many of unknown physiological role. For example, two SR K+ channels, TRIC-A and TRIC-B, have recently been identified and are thought to play a key role in supporting intracellular Ca2+ movements across the SR. The TRIC double knockout (DKO) mouse dies in embryonic heart failure and the TRIC-B knockout (KO) dies in respiratory failure immediately after birth, highlighting their important roles in different tissue types. The TRIC-A KO mouse survives until adulthood, however, there are SR structural abnormalities and compromised Ca2+ release. I have focused on characterising and comparing the single-channel properties of the native SR K+ channels from WT and TRIC-A KO mouse skeletal muscle. I have investigated if the SR K+ channels can gate in a cooperative manner when multiple SR K+ channels are present in the bilayer. Since TRIC and RyR channels are both located in high abundance in the junctional SR regions, I have examined whether the SR K+ and RyR channels also gate in a cooperative manner when both are present in a bilayer. In addition to voltage-regulation of native SR K+ channels, I have characterised the pH sensitivity of the SR K+ channels from WT and TRIC-A KO tissue. Finally, I investigated if the mouse isoform of TRIC-A could be purified using the Wheat germ cell-free system and incorporated into bilayers without loss of function. In summary, this thesis describes novel mechanisms of regulation of ion-channels that are involved in the process of Ca2+-release from the SR. By focusing on the biophysical properties of SR K+ channels, I have highlighted the complexity of the ionic fluxes that are thought to be required to maintain normal EC-coupling in striated muscle.

Les canaux potassiques (Kir) à rectification entrante sont des protéines transmembranaires impliquées dans des processus physiologiques fondamentaux. Des pathologies existantes sont directement liées à des mutations de ces canaux : des mutations au niveau de Kir2.1 et Kir6.2 provoquent le syndrome d'Andersen et l'hyperinsulinisme respectivement. Cette étude a quatre objectifs: i) comprendre le mécanisme moléculaire d’ouverture et de fermeture de KirBac3.1, ii) comprendre l'impact de la mutation W46R sur le canal, iii) comprendre l'impact de la mutation S129R, une mutation conçue pour stabiliser l’état ouvert du canal, iv) résoudre la structure de Kir2.1 humain par cryo-microscopie. Pour étudier le comportement de ce système, la dynamique moléculaire utilisant la méthode des modes normaux excités (MDeNM) a été employée. Les résultats ont été confrontés à la méthode de HDX-MS et à des expériences d’électrophysiologie pour validation. Pour déterminer la structure de Kir2.1 WT, nous avons utilisé la cryo-microscopie électronique combinée à l’analyse d’images. Des mouvements clés du mécanisme d’ouverture/fermeture du canal ont été détectés comme l'implication du domaine cytoplasmique, des hélices de glissement et transmembranaires. Concernant KirBac3.1 W46R, une modification des interactions entraîne la rupture des interactions entre le bas des deux hélices transmembranaires provoquant le soulèvement de l'hélice de glissement facilitant l’ouverture du canal. La mutation S129R repousse les points de constriction et maintient le canal ouvert avec un rôle surprenant de la mutation R129. L’étude de Kir2.1 par cryo-microscopie a donné une densité électronique de résolution de 7 Å
Inwardly-rectifying potassium (Kir) channels are transmembrane proteins involved in fundamental physiological processes. Pathologies are directly linked to a mutation of these channels: some mutations in Kir2.1 and Kir6.2 cause the Andersen’s syndrome and hyperinsulinism, respectively. This study has four objectives: i) to understand the gating mechanism of the KirBac3.1, ii) to understand the impact of the mutation W46R on the channel (W68R equivalent in Kir6.2 causing hyperinsulinsm), iii) to understand the impact of the mutation S129R on the channel, iv) to resolve the structure of Kir2.1 at an atomic level. To study the dynamical characteristics of this system, Molecular Dynamics using excited Normal Modes (MDeNM) method was used. The results were confronted to HDX-MS Spectrometry and electrophysiological experiments for validation. To determine the structure of the human Kir2.1 WT, we are using cryo-electron microscopy combined with image analysis. Key motions in the gating mechanism were determined like the involvement of the cytoplasmic domain, the slide-helix and the transmembrane helices during the opening of the channel. Concerning W46R, a modification in these interaction at the level of the residue 46 causes the loss of interactions between the bottoms of the two transmembrane helices that triggers the uprising of the slide-helix which opens the channel. The study of KirBac3.1 S129R mutant in the open state explains how the mutation pushes away the point of constriction and keeps the channel open with a surprising role of the mutation R129. We investigated the structure of Kir2.1 by cryo-EM and obtained an electronic density at 7 Å resolution

The cardiac ryanodine receptor (RyR2) contains structural elements within the channel pore that function as gates to regulate the release of intracellular calcium, initiating cardiac muscle contraction. The precise regulation of these gates is critical in maintaining normal cardiac function, and channel dysfunction, resulting in altered calcium handling, underlies the mechanisms of arrhythmia and sudden cardiac death. The enormous size of RyR2 has impeded the gathering of detailed structural information, hence the structural determinants for channel gating remain unknown. Structural modelling studies have revealed similarities between the RyR2 pore and the K+ channel, KcsA, providing a framework in which to test channel gating mechanisms. A region termed the selectivity filter is a gating component in K+ channels, involved in inactivation and flicker closings, and its conformation is maintained by a transient hydrogen-bonding network. This project examined the role of the RyR2 selectivity filter in channel gating by generating mutants at analogous positions to KcsA that either disrupted (Y4813A, D4829A and Y4839A) or maintained (Y4813W, D4829E and Y4839W) a proposed hydrogen-bonding network, and assessed their intracellular Ca2+ release, ryanodine modification and biophysical properties. Y4813A and D4829A had drastic effects on channel function, whereas retaining physicochemical properties of conservative mutations, Y4813W and Y4839W, maintained the functional characteristics of WT RyR2. Flicker closings were affected by Y4839A mutation however, in general, single-channel gating for Y4813W, Y4839A and Y4839W was comparable to WT RyR2. Interestingly, monitoring single-channels for prolonged periods revealed novel insights into channel behaviour, characterised by inherent fluctuations in channel activity under steady-state conditions. This thesis reveals that the selectivity filter region is an important component for RyR2 channel function. However, it remains unclear whether the selectivity filter regulates channel gating, as the proposed hydrogen-bonding network would not be possible due to altered residue distances revealed from recent high-resolution RyR structural models.

The structure of the voltage-gated proton (H+) channel Hv1 is homologous to the voltage sensor domain (VSD) of tetrameric voltage-gated Na+, K+ and Ca2+ channels (VGCs), but lacks a pore domain and instead forms a homodimer. Similar to other VSD proteins, Hv1 is gated by changes in membrane potential (V), but unlike VGCs, voltage-dependent gating in Hv1 is modulated by changes in the transmembrane pH gradient (DpH = pHo - pHi). In Hv1, pHo or pHi changes shift the open probability (POPEN)-V relation by ~40 mV per pH unit. To better understand the structural basis of pHo-dependent gating in Hv1, we constructed new resting- and activated-state Hv1 VSD homology models using physical constraints determined from experimental data measured under voltage clamp and conducted all-atom molecular dynamics (MD) simulations. Analyses of salt bridges and calculated pKas at conserved side chains suggests the existence of intracellular and extracellular electrostatic networks (ICEN and ECEN, respectively) that stabilize resting- or activated-state conformations of the Hv1 VSD. Structural analyses led to a novel hypothesis: two ECEN residues (E119 and D185) with coupled pKas coordinately interact with two S4 ‘gating charge’ Arg residues to modulate activated-state pHo sensitivity. Experimental data confirm that pH-dependent gating is compromised at acidic pHo in Hv1 E119A-D185A mutants, indicating that specific ECEN residue interactions are critical components of the ∆pH-dependent gating mechanism. E119 and D185 are known to participate in extracellular Zn2+ coordination, suggesting that H+ and Zn2+ utilize similar mechanisms to allosterically modulate the activated/resting state equilibrium in Hv1.

L’esquizofrènia és una malaltia mental incapacitant que involucra diversos símptomes cognitius, com un filtratge sensoriomotor deteriorat. El filtratge sensoriomotor es pot mesurar mitjançant la inhibició prepols (IPP) de la resposta d’ensurt. Les investigacions en rosegadors sobre l’impacte d’alteracions cerebrals específiques sobre la IPP han estat molt útils per augmentar el coneixement d’aquesta deficiència bàsica de l’esquizofrènia. Aquests estudis mostren que els dèficits en IPP apareixen conjuntament amb altres símptomes, com l’agitació psicomotora, així com alteracions en el circuit cortico-estriato-pallido-talàmic (CEPT). Específicament, tractaments que alteren l’activitat del còrtex prefrontal medial (CPFm), l’hipocamp (HPC) o el nucli accumbens (NAc) redueixen la IPP. És important destacar que la desregulació cortical del balanç excitació-inhibició (E-I) s’ha proposat com el principal substrat subjacent als símptomes cognitius de l’esquizofrènia. A més, aquests estudis mostren que diversos fàrmacs milloren la IPP, com el neuropèptid oxitocina, el qual s’ha proposat com un antipsicòtic natural alternatiu. D’altra banda, els estudis en humans avaluen l’associació entre diferències naturals en la conducta i canvis neurals. En aquesta Tesi Doctoral, amb la idea d’establir un pont entre els estudis en humans i rosegadors, vam explorar si els dèficits naturals en IPP en rates consanguínies i no consanguínies intactes (i) s’associaven amb diferències en altres conductes relacionades amb l’esquizofrènia; (ii) es relacionaven amb diferències funcionals i estructurals en el circuit CEPT; (iii) s’atenuaven per l’administració d’oxitocina. Els nostres subjectes d’estudi van ser les rates consanguínies Romanes d’alta i baixa evitació (RHA i RLA), i les rates no consanguínies HS. Les RHA mostren menor IPP que les RLA, mentre que les HS es van estratificar en subgrups segons els seus nivells d’IPP. Els experiments plantejats també pretenien augmentar la validesa aparent, de constructe i predictiva dels nostres models animals de característiques rellevants per a l’esquizofrènia (RHA i HS-baixa-PPI). Els nostres resultats mostren que l’exploració incrementada en resposta a la novetat s’associa amb dèficits en IPP en rates HS i Romanes. Per estudiar les associacions cerebrals estructurals i funcionals amb la IPP, vam combinar l’ús de ressonància magnètica estructural i expressió de c-Fos després de la IPP. Vam trobar que la baixa IPP s’associa amb baixa activitat del CPFm en rates Romanes i HS i amb un augment d’activitat en el NAc en rates HS. La baixa IPP s’associa també amb una disminució del volum cerebral del CPFm i l’HPC en rates Romanes i HS. A més, emprant immunofluorescència després de la IPP, vam observar un menor percentatge d’activitat d’interneurones inhibitòries GABAèrgiques (parvalbúmina) al CPFm en rates RHA que en RLA. En relació amb l’administració d’oxitocina, vam trobar que l’oxitocina augmentava la IPP en rates HS i RHA, mentre que no afectava la IPP en les RLA. Els valors constitutius d’expressió del gen CD38 (regulador de l’alliberament d’oxitocina) al CPFm eren més baixos en les RHA que en les RLA, mentre que l’administració d’oxitocina va incrementar l’expressió del gen del receptor de oxitocina (OXTR) en ambdues soques. Aquesta Tesi Doctoral mostra un patró consistent d’alteracions conductuals i neurobiològiques en rates HS-baixa-IPP i RHA que incrementa la seva validesa aparent, de constructe i predictiva com a models animals de característiques relacionades amb l’esquizofrènia. És important destacar que els nostres resultats donen suport a la idea que el filtratge sensoriomotor està modulat per estructures cerebrals superiors (CPFm) i el balanç cortical E-I.
La esquizofrenia es una enfermedad mental incapacitante que involucra varios síntomas cognitivos, como un filtraje sensoriomotor deteriorado. El filtraje sensoriomotor se puede medir mediante la inhibición prepulso (IPP) de la respuesta de sobresalto. Los estudios en roedores que analizan el impacto de alteraciones cerebrales específicas sobre la IPP han sido muy útiles para aumentar el conocimiento sobre esta deficiencia básica de la esquizofrenia. Estos estudios muestran que los déficits en IPP aparecen junto a otros síntomas, como agitación psicomotora, y alteraciones en el circuito cortico-estriato-pallido-talámico (CEPT). Específicamente, tratamientos que aumentan o disminuyen la actividad del córtex prefrontal medial (CPFm), el hipocampo (HPC) o el núcleo accumbens (NAc) reducen la IPP. Es importante destacar que la desregulación cortical en el balance excitación-inhibición (E-I) se ha propuesto como el principal sustrato subyacente a los síntomas cognitivos de la esquizofrenia. Además, estos estudios muestran que la IPP mejora con varios fármacos antipsicóticos, como el neuropéptido oxitocina, el cual se ha propuesto como antipsicótico natural alternativo. A diferencia de los estudios en roedores, los estudios en humanos evalúan la asociación entre diferencias comportamentales naturales (diagnóstico, síntomas) y cambios neurales. En esta Tesis Doctoral, nos propusimos contribuir a establecer un puente entre los estudios en humanos y roedores y, para ello, exploramos si los déficits naturales en IPP en ratas consanguíneas y no-consanguíneas intactas (i) se asociaban con diferencias en otras conductas relacionadas con la esquizofrenia; (ii) se relacionaban con diferencias funcionales y estructurales en el circuito CEPT; (iii) se atenuaban por la administración de oxitocina. Usamos las ratas consanguíneas Romanas de alta y baja evitación (RHA y RLA), y las ratas no consanguíneas del stock heterogéneo HS. Las RHA muestran menor IPP que las RLA, mientras que las HS se estratificaron en subgrupos según su IPP. Los experimentos planteados también pretendían aumentar la validez aparente, de constructo y predictiva de nuestros animales modelo de características relevantes para la esquizofrenia (RHA y HS-baja-IPP). En relación con las asociaciones conductuales, nuestros resultados muestran que la exploración incrementada en respuesta a la novedad se asocia con déficits en IPP en ratas HS y Romanas. Para estudiar las asociaciones cerebrales estructurales y funcionales con la IPP, combinamos el uso de resonancia magnética estructural y expresión de c-Fos después de la IPP. Encontramos que la baja IPP se asocia con baja actividad del CPFm en ratas Romanas y HS y con un aumento de actividad en el NAc en ratas HS. La baja IPP se asocia también con una disminución del volumen cerebral del CPFm e HPC en ratas Romanas y HS. Además, mediante el uso de inmunofluorescencia después de la IPP, encontramos un menor porcentaje de actividad de interneuronas inhibitorias GABAérgicas de tipo parvalbúmina en el CPFm en ratas RHA que en RLA. Respecto a la administración de oxitocina, ésta aumentó la IPP en ratas HS y RHA, mientras que no afectó la IPP en las RLA. De acuerdo con el efecto diferencial de la oxitocina sobre la IPP (RHA>RLA), los valores constitutivos de expresión del gen CD38 (regulador de la liberación de oxitocina) en el CPFm fueron más bajos en ratas RHA que en las RLA, mientras que la administración de oxitocina incrementó la expresión del gen del receptor de oxitocina (OXTR) en ambas cepas. Esta Tesis Doctoral muestra un patrón consistente de alteraciones conductuales y neurobiológicas en ratas HS-baja-IPP y RHA que incrementa su validez aparente, de constructo y predictiva como animales modelo de características relacionadas con la esquizofrenia. Nuestros resultados apoyan la idea de que el filtraje sensoriomotor está modulado por estructuras cerebrales superiores (CPFm) y el balance cortical E-I.
Schizophrenia is a debilitating mental disorder that involves several cognitive symptoms, including sensorimotor gating impairments. Sensorimotor gating can be measured via prepulse inhibition (PPI) of the startle response, in which the magnitude of a startle stimulus is attenuated by the presence of a pre-stimulus of lower intensity. Rodent studies evaluating the impact of brain-site specific manipulations on PPI have been very useful to provide insights into this basic schizophrenia-like deficiency. These studies show that PPI deficits are frequently accompanied by other symptoms, including psychomotor agitation, as well as alterations in the cortico-striatal-pallido-thalamic (CSPT) circuit. In particular, treatments that increase or decrease the activity of the medial prefrontal cortex (mPFC), hippocampus (HPC), or nucleus accumbens (NAc) reduce PPI. In this context, a dysfunctional cortical excitatory-inhibitory balance has been proposed as the main neural substrate for cognitive dysfunction in schizophrenia. Moreover, these studies show that PPI deficits can be improved by several antipsychotic drugs, including the neuropeptide oxytocin, which has been suggested as an alternative natural antipsychotic. In contrast to these rodent studies, human studies evaluate the association between natural behavioral differences (diagnosis, symptoms) and neural changes. Thus, in this Doctoral Dissertation, we aimed to contribute to bridge the gap between human and rodent studies by exploring whether spontaneous deficits in PPI in intact inbred and outbred rats are (i) associated with divergences in other schizophrenia-related behaviors, (ii) related to functional and structural differences in the CSPT circuit, and (iii) attenuated by oxytocin. Our subjects of study were the inbred Roman high-avoidance (RHA) and Low-avoidance (RLA) rats, and the outbred heterogeneous stock (HS) rats. RHA rats show lower PPI than RLAs, while HS rats were stratified in sub-groups according to their PPI levels. The present experiments also aimed to provide further face, construct, and predictive validity to our animal models of schizophrenia-relevant symptoms (RHA and HS Low-PPI rats). Regarding behavioral associations, our results show that increased exploration in response to novelty is associated with deficient PPI in HS and Roman rats. Moreover, a high anxious profile was found in rats with increased PPI, while no associations were seen with compulsive-like behavior. In relation to brain structural and functional associations with PPI, we combined structural magnetic resonance imaging and c-Fos expression after PPI in both HS and Roman rats. Our results indicate that lower PPI is associated with decreased mPFC activity in both Roman and HS rats and with increased NAc shell activity in HS rats. Reduced PPI is also associated with decreased mPFC and HPC volumes in Roman and HS rats. Additionally, using immunofluorescence after PPI, we observed a lower percentage of active inhibitory GABAergic parvalbumin interneurons in RHA than RLA rats. Regarding oxytocin administration, we found that oxytocin increased PPI in HS rats, attenuated PPI deficits in RHA rats, and did not affect PPI in RLAs. Consistent with the differential oxytocin effects on PPI (RHA>RLA), constitutive CD38 gen expression (regulator of oxytocin release) was reduced in the mPFC of RHA rats compared to the RLAs, while oxytocin administration increased oxytocin receptor (OXTR) gen expression in both strains. This Doctoral Dissertation shows a consistent pattern of behavioral and neurobiological abnormalities in the HS-Low-PPI rats and RHA rats that increases the face, construct, and predictive validity of these rats as models of schizophrenia-related features. Importantly, our results support the idea that sensorimotor gating is modulated by forebrain structures and highlight the relevance of the mPFC and the cortical excitatory-inhibitory balance in its regulation.
Universitat Autònoma de Barcelona. Programa de Doctorat en Neurociències

Cardiac magnetic resonance (MR) imaging can non-invasively assess heart function. Displacement encoding with stimulated echoes (DENSE) is an advanced cardiac MR imaging technique that measures tissue displacement and can be used to quantify cardiac mechanics (e.g. strain and torsion). When combined with clinical risk factors, cardiac mechanics have been shown to be better predictors of mortality than traditional measures of heart function. End-expiratory breath-holds are typically used to minimize respiratory motion artifacts. Unfortunately, requiring subjects to breath-hold introduces limitations with the duration of image acquisition and quality of data acquired, especially in patients with limited ability to hold their breath. Thus, DENSE acquisitions often require respiratory navigator gating, which works by measuring the diaphragm during normal breathing and only acquiring data when the diaphragm is within a pre-defined acceptance window. Unfortunately, navigator gating results in long scan durations due to inconsistent breathing patterns. Also, the navigator echo can be used in different ways to accept or reject image data, which creates several navigator configuration options. Each respiratory navigator configuration has distinct advantages and disadvantages that directly affect scan duration and image quality, which can affect derived cardiac mechanics. Scan duration and image quality need to be optimized to improve the clinical utility of DENSE. Thus, the goal of this project was to optimize those parameters. To accomplish this goal, we set out to complete 3 aims: 1) understand how respiratory gating affects the reproducibility of measures of cardiac mechanics, 2) determine the optimal respiratory navigator configuration, and 3) reduce scan duration by developing and using an interactive videogame to optimize navigator efficiency. Aim 1 of this project demonstrated that the variability in torsion, but not strain, could be significantly reduced through the use of a respiratory navigator compared to traditional breath-holds. Aim 2 demonstrated that, among the configuration options, the dual-navigator configuration resulted in the best image quality compared to the reference standard (traditional breath-holds), but also resulted in the longest scan duration. In Aim 3, we developed an interactive breathing-controlled videogame and demonstrated that its use during cardiac MR can significantly reduce scan duration compared to traditional free-breathing and also led to a small improvement in signal-to-noise ratio of the acquired images. In summary, respiratory navigator gating with DENSE 1) reduces the variability in measured LV torsion, 2) results in the best image quality with the dual-navigator configuration, and 3) results in significantly shorter scan durations through the use of an interactive videogame. Selecting the optimal navigator configuration and using an interactive videogame can improve the clinical utility of DENSE.

博士
國立臺灣大學
生理學研究所
103
The NMDA receptor channel is an obligatory heterotetramer formed by two GluN1 and two GluN2 subunits. However, the differential contribution of the two different subunits to channel operation is not clear. We found that the apparent affinity of glycine to GluN1 (Kgly~0.6 μM) is much higher than NMDA or glutamate to GluN2 (KNMDA~36 μM, Kglu~4.8 μM). The binding rate constant (derived from the linear regression of the apparent macroscopic binding rates) of glycine to GluN1 (~9.8 x 106 M-1s-1), however, is only slightly faster than NMDA to GluN2 (~4.1 x 106 M-1s-1). Accordingly, the apparent unbinding rates of glycine from activated GluN1 (time constant ~2s) are much slower than NMDA from activated GluN2 (time constant ~70 ms). Moreover, the decay of NMDA currents upon wash-off of both glycine and NMDA seems to follow the course of NMDA rather than glycine unbinding. But if only glycine is washed off, the current decay is much slower, apparently following the course of glycine unbinding. The apparent binding rate of glycine to the fully deactivated channel, in the absence of NMDA, is roughly the same as that measured with co-application of both ligands, whereas the apparent binding rate of NMDA to the fully deactivated channel in the absence of glycine is markedly slower. In this regard, it is interesting that the 7th residue in the highly conserved SYTANLAAF motif (A7) in GluN1 and GluN2 are so close that they may interact with each other to control the dimension of the external pore mouth. Moreover, specific mutations involving A7 in GluN1 but not in GluN2 result in channels showing markedly enhanced affinity to both glycine and NMDA and readily activated by only NMDA, as if the channel is already partially activated. We conclude that GluN2 is most likely directly responsible for the activation gate of the NMDA channel, whereas GluN1 assumes a role of more global control, especially on the gating conformational changes in GluN2. Structurally, this inter-subunit regulatory interaction seems to involve the SYTANLAAF motif, especially the A7 residue. Furthermore, the NMDA receptor channel is characterized by its high permeability of Ca2+ ion, and the Ca2+ influxes may play an important role in many cellular physiological and pathophysiological processes. It has been reported that NMDA channel desensitization, an imperative attribute regulating ionic fluxes through the channel pore, could be related to Ca2+ ions flowing through the pore and/or interacting with effector proteins such as calcineurin at the internal pore mouth. Whether extracellular Ca2+ ion itself could directly bind to the NMDA receptor channel to have an effect on the key molecular behaviors of the channel has not been fully characterized. We found that extracellular Ca2+ and Cd2+, an ion with the same charges and ionic radius as Ca2+ and therefore high affinity toward many Ca2+ binding sites, could bind to the closed NMDA channel to affect channel gating as well as ion permeation. Because the activation gate, which is already positioned at the external pore mouth, is not open, this binding site must be located at the very external part of the pore. We also demonstrated that Cd2+ binds to the NMDA channel with a simple bimolecular reaction, and the apparent dissociation constants toward the closed, open, and desensitized states of the channel is ~5, ~2.5, and ~1.2 μM, respectively. The modest but definite differential affinity would indicate that the binding site changes its conformation during the gating process. DRPEER, a motif in the GluN1 but not GluN2 subunit, is located just external to the activation gate of the NMDA channel. Interestingly, the effect of Cd2+ present in either the resting or the activated state is decreased correlatively to the number of charge-neutralization mutations in this motif, with additive free energy changes calculated by double mutant cycle analysis. DRPEER motif therefore very likely constitutes the binding site for Ca2+ or Cd2+, and thus seems to go through a sequential conformational change during channel activation. In this regard, it is intriguing that the inhibitory effect of Cd2+ is also decreased by point mutation T647A located just inside the activation gate in the pore. Moreover, prominent “hooked” current develops after wash-off of Cd2+ (and even more so after wash-off of Ca2+) in this case, suggesting preservation of the channel in the open state (prevention of the channel from entering the desensitized state) and thus an apparently inversed order of affinity of Cd2+ and Ca2+ toward the two states with the point mutation. Desensitization thus seems to involve essential conformational changes in the vicinity of the activation gate in the NMDA channel. Desensitization is an important gating mechanism for the function of N-methyl-D-aspartate (NMDA) receptors. However, the exact modulation of desensitization mechanism remains in question. We found that extracellular tetrapentylammonium (TPentA) binds to the external pore mouth, and induces a strong increase in open probability to disturb the equivalence between open and desensitization states. TpentA seems to prefer to open state of the NMDA channel. Moreover, the A7 residues (at position 7 of the SYTANLAAF motif), shown to mark the activation gate, constitute the key component of the activation gate of the NMDA channel. We further explore whether A7 is also involved in the desensitization. Our data suggest that the desensitization gate should be near the activation gate, A7, in the external pore mouth.

The studies contained in this dissertation are aimed at utilizing chemistry to understand neurobiology and neuronal communication. Chapters 2 and 3 both address the gating of ion channels, describing structure-function studies to shed light on the gating mechanisms of two classes of ion channels. Chapter 2 studies the gating mechanism of the mechanosensitive channel of small conductance (MscS), which is voltage modulated. Elucidating the mechanism of voltage sensation in MscS may provide insight into how voltage-gated channels translate a change in membrane potential to channel gating. The research discussed in Chapter 2 is aimed at elucidating the role of two arginine residues, in the TM1 and TM2 of MscS, in voltage sensing. We generated two MscS mutants, Arg46Ala and Arg74Ala, to evaluate the effects of "neutralizing" the charged side chain on the voltage sensing ability of the channel. The mutants were evaluated using single channel analysis in E. coli spheroplasts. Our preliminary results indicated a potentially significant role for Arg46 in the voltage sensitivity of MscS, however this data set is not extensive due to inconsistency in the spheroplasts preparation.

In Chapter 3, we utilized nonsense suppression to incorporate unnatural amino acids to study the gating of the cation-selective Cys-loop family of ion channel receptors. Specifically, it describes work aimed at elucidating the role of cis-trans isomerization of a proline residue in the gating mechanism of the serotonin-gated 5-hydroxy-tryptamine receptor 3A (5-HT3A) and the nicotinic acetylcholine receptor. A series of proline analgues, of varying cis preference were incorporated at proline 308 in the M2-M3 loop of the 5-HT3A receptor using in vivo nonsense suppression methodology in a Xenopus oocyte expression system. Electrophysiological analysis of the mutant channels revealed a linear relationship between the cis preference of the proline analog and the EC50 of the mutant channel—suggesting that proline 308 may serve as a hinge during the gating 5-HT3A. From these data, we proposed a model of gating for the 5-HT3A receptor. Initial results from similar studies in nAChR suggests that the analogous proline does not play a role in its gating.

Lastly, Chapter 4 addresses the role of fucose-galactose carbohydrates in learning and memory. It aims to identify lectins to fucose-alpha(1-2)-galactose as well as identify the corresponding glycoproteins bearing fucose-alpha(1-2)-galactose. Chemical probes were synthesized and used to study fucose-alpha(1-2)-galactose binding proteins. One of the probes was used to demonstrate the existence of fucose-alpha(1-2)-galactose binding proteins in hippocampal neurons. Furthermore, initial results from experiments with a photoreactive probe suggested that the design of our probe is sufficient to isolate fucose-alpha(1-2)-galactose binding proteins from the brain. Additionally, we were able to use antibodies specific to fucose-alpha(1-2)-galactose epitopes to examine fucose-alpha(1-2)-galactose bearing glycoproteins in the brain. Overall, results from both studies utilizing chemical probes and molecular probes strongly suggest that the modifications of proteins with fucose-alpha(1-2)-galactose epitopes and the expression of fucose-alpha(1-2)-galactose binding proteins are developmentally regulated.

碩士
國立中正大學
電機工程研究所
99
It was getting more and more consumption of the portion of static power in advanced process. In order to decrease the static power consumption, many researches provided the Multi-Threshold CMOS (MTCMOS), one skill of the MTCOMS called Power Gating. There were two problems which affected the circuit’s (system) function when waking up the sleep mode. First, it would turn on the Power/Ground Bounce to affect the stability. Second, it would need longer wake up time may affect the performance. Although the relationship of parameters of the Ground Bounce and Wake up time was an inverse proportion, we suggest the parameters were the smaller the better Recent study provided the mechanism of reducing the rush current and faster Waking up the Power Gating. This mechanism based on calculating the waking process’ working models of the sleep transistor, using the formula of transistor current to calculate limited differential current, which balanced the Ground Bounce range not to over the settings. Using the current integration to determine the change of virtual vdd, to increase integration unit of time in waking process could get the smaller waking time, and hope to smaller the Ground Bounce and rush current at the same time. Besides, we provide a new concept to fix some internal nodes’ voltage of the logic loading block to reach the smaller charge current in waking process. At the end, the recent study used the 0.18um and CCU cell library to tape out the 40 bit ALU with Power Gating circuit. The designed circuit itself contained the Power Gating to control the circuit. Compared with the conventional waking process with the same Ground Bounce, the simulated result indicated the designed circuit decreased 10.23% wake up time, and the designed circuit was tapped out by National chip implementation center already.

碩士
國立中央大學
資訊工程學系在職專班
107
Traditional Chinese Named Entity Recognition based on machine learning usually relies on large amounts of hand-craft features, even dictionaries created by experts specific for entity, and then, uses linear regression and statistical models to gather important features and Chinese semantic rules. However, two obvious flaws can be observed. Firstly, it is extremely time-consuming and complicated to extract features from Chinese texts. Secondly, the usefulness of the models completely depends on the recognition efficiency based on hand-craft features; as a result, it is difficult to improve its accuracy due to semantic confusion that is characteristic in Chinese and unknown vocabularies. In English, spaces are used for word segmentation, and Chinese does not have similar word segmentation. However, Chinese words are highly interdependent and demonstrate semantic differences (homographs, polysemy) based on the context. Therefore, a great challenge as well as a possibility is how to recognize Chinese named entities in large corpora. To provide a solution to the challenge and flaws mentioned above, this study employs deep learning structure to complete Chinese Named Entity Recognition. Firstly, the deep learning model is combined with unsupervised learning to embed a large amount of pre-training words in the vocabulary. Then, the vocabulary is used to numeralize words before using multi-stack convolution to extract textual features. Gating mechanism is also incorporated between layers to generalize features and automatically extract features without employing feature engineering. The purpose of doing so is to reduce the dependency on hand-craft features in Named Entity Recognition and avoid hand-craft Chinese recognition features. This method can be effectively applied to recognizing different types of entities. This study uses documents from SIGHAN Bakeoff-3 and utilizes customized crawler programs to capture internet articles for training data. Electronic files of newspaper articles are used as testing data and form the standard by which the efficiency of different models can be evaluated. The results show that the F1-Measure model proposed by the study reaches outstanding an overall efficiency of 90.76% in SIGHAN and 90.42% in electronic files of newspaper articles.

博士
國立臺灣大學
生理學研究所
95
Felbamate (FBM) is a potent nonsedative anticonvulsant whose clinical effect is chiefly ascribable to gating modification (and thus use-dependent inhibition) rather than pore block of N-methyl-D-aspartate (NMDA) channels at pH 7.4. Using whole-cell recording in rat hippocampal neurons, we examine the kinetics of FBM binding to and unbinding from the NMDA channel, as well as the effect of FBM on NMDA and glycine affinity to the channel. We show that FBM modifies NMDA channel gating via a one-to-one binding stoichiometry (one FBM per channel) and has quantitatively the same enhancement effect on NMDA and glycine binding to the NMDA channel. Moreover, the binding rates of FBM to the closed and the open/desensitized NMDA channels are 187.5 and 4.6 × 104 M-1s-1, respectively. The unbinding rates of FBM from the closed and the open/desensitized NMDA channels are 5.4-6.2 × 10-2 and 3.1-3.5 s-1, respectively. From the binding and unbinding rate constants, apparent dissociation constants of ~300 and ~70

abstract: Sensory gating is a process by which the nervous system preferentially admits stimuli that are important for the organism while filtering out those that may be meaningless. An optimal sensory gate cannot be static or inflexible, but rather plastic and informed by past experiences. Learning enables sensory gates to recognize stimuli that are emotionally salient and potentially predictive of positive or negative outcomes essential to survival. Olfaction is the only sensory modality in mammals where sensory inputs bypass conventional thalamic gating before entering higher emotional or cognitive brain regions. Thus, olfactory bulb circuits may have a heavier burden of sensory gating compared to other primary sensory circuits. How do the primary synapses in an olfactory system "learn"' in order to optimally gate or filter sensory stimuli? I hypothesize that centrifugal neuromodulator serotonin serves as a signaling mechanism by which primary olfactory circuits can experience learning informed sensory gating. To test my hypothesis, I conditioned genetically-modified mice using reward or fear olfactory-cued learning paradigms and used pharmacological, electrophysiological, immunohistochemical, and optical imaging approaches to assay changes in serotonin signaling or functional changes in primary olfactory circuits. My results indicate serotonin is a key mediator in the acquisition of olfactory fear memories through the activation of its type 2A receptors in the olfactory bulb. Functionally within the first synaptic relay of olfactory glomeruli, serotonin type 2A receptor activation decreases excitatory glutamatergic drive of olfactory sensory neurons through both presynaptic and postsynaptic mechanisms. I propose that serotonergic signaling decreases excitatory drive, thereby disconnecting olfactory sensory neurons from odor responses once information is learned and its behavioral significance is consolidated. I found that learning induced chronic changes in the density of serotonin fibers and receptors, which persisted in glomeruli encoding the conditioning odor. Such persistent changes could represent a sensory gate stabilized by memory. I hypothesize this ensures that the glomerulus encoding meaningful odors are much more sensitive to future serotonin signaling as such arousal cues arrive from centrifugal pathways originating in the dorsal raphe nucleus. The results advocate that a simple associative memory trace can be formed at primary sensory synapses to facilitate optimal sensory gating in mammalian olfaction.
Dissertation/Thesis
Ph.D. Biology 2012

Mechanosensation in cells is a well known phenomenon that is associated with cellular responses to force. Our knowledge of the trigger mechanism of this phenomenon is, however, limited. Earlier studies in this field have used atomic simulations, which although being accurate, are limited in their feasibility in multi-length scenarios like a mechanosensitive channel that undergoes micro-level changes in the composition of the protein to cause a macro-level change in the state of a biological structure such as the muscle. Finite Element Analysis has been used in various engineering fields to study the mechanical response of complex structures. The current study is a step in utilizing the phenomenal capabilities of Finite Element Analysis in developing and studying a 3D model (Membrane-Channel) of a mechanosensitive channel of large conductance (MscL). A simplified CAD structure of Mycobacterium tuberculosis (TbMscL) was developed in the first stage of this study. The authenticity of this model was tested by applying two types of loading conditions, namely (i) In-plane stretch and (ii) Out-of-plane bending. The results obtained from the first step of analysis are in accordance with previous experimental data, which elucidates the fact that tension within the membrane guides the gating mechanism of the channel and not the curvature of the membrane. The second stage of the analysis involved the use of the same model to study the disease commotio cordis. This was achieved by calculating the loading conditions during the onset of the condition in the human heart and then transferring those conditions to the Membrane-Channel model developed in the first stage. The result showed that although the channel did not fully open but there was a significant change in the channel‟s radius that might cause the flow of ions, thereby changing the state of the channel. It is anticipated that this model will help future research in areas that conventionally have been difficult to model.

碩士
國立臺灣大學
資訊網路與多媒體研究所
106
Attention-based recurrent neural network models for joint intent detection and slot filling have achieved the state-of-the-art performance, while they have independent attention weights. Considering that slot and intent have the strong relationship, this thesis proposes a slot gate that focuses on learning the relationship between intent and slot attention vectors in order to obtain better semantic frame results by the global optimization. The experiments show that proposed model significantly improves the performance compared to the state-of-the-art baselines on benchmark ATIS, Snips and AMI datasets. Furthermore, can we extend the gating mechanism to multi-sentences? In this thesis, we choose the summarization task. Abstractive summarization has been widely studied, while the prior work mainly focused on summarizing single-speaker documents (news, scientific publications etc). In dialogues, there are different interactions between speakers, which is usually defined as dialogue acts. These interactive signals may provide informative cues for better summarizing dialogues. This thesis aims to leverage dialogue acts in a neural summarization model, where a sentence gate is designed to model the relationship between dialogue acts and summaries. The experiments show that proposed model significantly improves the abstractive summarization performance compared to the state-of-the-art baselines on AMI meeting corpus, demonstrating the usefulness of interactive signals.

Ion channels are membrane proteins that facilitate electrical signaling in important physiological processes, such as the rhythmic contraction of the heart. KCNQ1 is the pore-forming subunit of a voltage-gated potassium channel that assembles with the β-subunit KCNE1 in the heart to generate the IKs current, which is critical to cardiac action potential repolarization and electrical conduction in the heart. Mutations in IKs subunits can cause potentially lethal arrhythmia, including long QT syndrome, short QT syndrome, and atrial fibrillation. Each channel consists of four voltage-sensing domains and a central pore through which ions permeate. Voltage-dependent gating occurs when movement of voltage sensors cause pore opening/closing through coupling mechanisms. Although KCNQ1 by itself is able to form a voltage-dependent potassium channel, its assembly with KCNE1 is essential to generating the physiologically critical cardiac IKs current, characterized by a delay in the onset of activation, an increase in current amplitude, and a depolarizing shift in the current-voltage relationship. KCNE1 is thought to have multiple points of contact with KCNQ1 that reside within both the voltage-sensing domain and the pore domain, allowing for extensive modulation of channel function. Atrial fibrillation is the most common cardiac arrhythmia and affects more than 3 million adults in the United States. Much rarer, genetic forms of atrial fibrillation have been associated with gain-of-function mutations in KCNQ1, such as two adjacent mutations, S140G and V141M. Both mutations drastically slow channel deactivation, which underlies their pathophysiology. Deactivation slowing causes accumulation of open channels in the context of repeated stimulation, which abnormally increases the repolarizing K+ current, excessively shortens the action potential duration, and predisposes to re-entry arrhythmia such as atrial fibrillation. Although both mutations are located in the voltage-sensing domain, their mechanisms of action remain unknown. Understanding the gating mechanisms underlying deactivation slowing may provide key insights for the development of mechanism-based pharmacologic therapies for arrhythmias associated with KCNQ1 mutations. In addition to gain-of-function mutations, molecular activators of KCNQ1 can slow deactivation and increase channel activity. An existing problem in the pharmacologic treatment of arrhythmia is that many antiarrhythmic drugs do not have specific targets and cause undesired side effects such as additional arrhythmia. Thus, developing mechanism-based therapies may optimize clinical treatment for patients with specific forms of channel dysfunction. Two KCNQ1 activators, ML277 and R-L3, have been previously shown to slow current deactivation, but the underlying gating mechanisms remain known. Although these modulators are unlikely to serve directly as antiarrhythmic therapy, investigating their mechanisms will likely provide fundamental insights on channel modulation and guide future efforts to develop personalized therapies for arrhythmia, such as congenital long QT syndrome. Given the central importance of deactivation slowing in both pathophysiology and pharmacology, we focused on investigating gating mechanisms that underlie deactivation slowing. To this end, we utilized voltage clamp fluorometry, a technique that simultaneously assays for voltage sensor movement and ionic current through the channel pore. In Chapter 1, we begin our study by examining the gating mechanisms of KCNQ1 atrial fibrillation mutations in the absence of KCNE1. We show that S140G slows voltage sensor deactivation, which indirectly slows current deactivation. On the other hand, V141M neither slows voltage sensor nor current deactivation. This is followed by Chapter 2, where we examine the gating mechanisms underlying deactivation slowing by atrial fibrillation mutations in the presence of KCNE1. We show that both S140G and V141M slow IKs deactivation by slowing pore closing and altering voltage sensor-pore coupling. Based on these findings, we proposed a molecular mechanism in which both mutations disrupt the orientation of KCNE1 relative to KCNQ1 and thus impede pore closing, implying that future efforts to modulate KCNQ1 function can benefit from targeting the β-subunit. Finally, in Chapter 3, we explore the gating mechanisms underlying deactivation slowing for two small-molecule activators of KCNQ1. We show that ML277 predominantly slows pore transitions, whereas R-L3 slows voltage sensor deactivation, which indirectly slows current deactivation. Taken together, these studies guide future efforts to develop mechanism-based therapies for arrhythmia.

The successful fabrication and characterization of two chemiresistive platforms for biomolecule detection was demonstrated by this work. The Si/Silica based single walled nanotube thin film (SWNTTF) platform was developed to understand the effect of device geometry on pH and M13 bacteriophage sensing capabilities as well as the underlying mechanisms governing SWNTTF chemiresistive biosensors. The dominant mechanism of sensing switched from direct chemical doping to electrostatic gating when the target analyte changed from H+/OH- ions in pH testing to whole viruses. The experimental limit of detection for M13 for this platform was 0.5pM and an increased sensitivity as well as variability was observed in devices with smaller channel widths. Preliminary device calibration was completed in order to correlate a resistance response to a bulk M13 concentration. The polyethylene terephthalate (PET) based SWNTTF platform was developed to demonstrate the commercial potential of SWNTTF chemiresistive biosensors by detecting relevant concentrations of brain natriuretic peptide (BNP) on economically viable substrates. The pH response of these chemiresistors confirmed that chemical doping was the cause for resistance change in the SWNTTFs. The preliminary results demonstrated successful BNP detection at 50pg/mL using both aptamers and antibodies as recognition elements. Using SWNTTFs as the transducing element of chemiresistors allowed for further understanding of electrical mechanisms of sensing as well as achieving sensitive, real-time and reproducible electrical virus and biomolecule detection. Although these platforms do not achieve ultrasensitive limits of detection, they demonstrate the commercial potential of platforms using SWNTTFs as the transducing element of electrical biomolecule sensors.

Heteropolymeric O antigen (O-Ag)-capped lipopolysaccharide is the principal constituent of the Gram-negative bacterial cell surface. It is assembled via the integral inner membrane (IM) Wzx/Wzy-dependent pathway. In Pseudomonas aeruginosa, Wzx translocates lipid-linked anionic O-Ag subunits from the cytoplasmic to the periplasmic leaflets of the IM, where Wzy polymerizes the subunits to lengths regulated by Wzz1/2. The Wzx and Wzy IM topologies were mapped using random C-terminal-truncation fusions to PhoALacZα, which displays PhoA/LacZ activity dependent upon its subcellular localization. Twelve transmembrane segments (TMS) containing charged residues were identified for Wzx. Fourteen TMS, two sizeable cytoplasmic loops (CL), and two large periplasmic loops (PL3 and PL5 of comparable size) were characterized for Wzy. Despite Wzy PL3–PL5 sequence homology, these loops were distinguished by respective cationic and anionic charge properties. Site-directed mutagenesis identified functionally-essential Arg residues in both loops. These results led to the proposition of a “catch-and-release” mechanism for Wzy function. The abovementioned Arg residues and intra-Wzy PL3–PL5 sequence homology were conserved among phylogenetically diverse Wzy homologues, indicating widespread potential for the proposed mechanism. Unexpectedly, Wzy CL6 mutations disrupted Wzz1-mediated regulation of shorter O-Ag chains, providing the first evidence for direct Wzy–Wzz interaction. Mutagenesis studies identified functionally-important charged and aromatic TMS residues localized to either the interior vestibule or TMS bundles in a 3D homology model constructed for Wzx. Substrate-binding or energy-coupling roles were proposed for these residues, respectively. The Wzx interior was found to be cationic, consistent with translocation of anionic O-Ag subunits. To test these hypotheses, Wzx was overexpressed, purified, and reconstituted in proteoliposomes loaded with I−. Common transport coupling ions were introduced to “open” the protein and allow detection of I− flux via reconstituted Wzx. Extraliposomal changes in H+ induced I− flux, while Na+ addition had no effect, suggesting H+-dependent Wzx gating. Putative energy-coupling residue mutants demonstrated defective H+-dependent halide flux. Wzx also mediated H+ uptake as detected through fluorescence shifts from proteoliposomes loaded with pH-sensitive dye. Consequently, Wzx was proposed to function via H+-coupled antiport. In summary, this research has contributed structural and functional knowledge leading to novel mechanistic understandings for O-Ag biosynthesis in bacteria.
Bookmarks within the document have been provided for ease of access to a particular section in the body of the thesis. Each entry in the Table of Contents, List of Tables, and List of Figures has been "linked" to its respective position and as such can be clicked for direct access to the entry. Similarly, each in-text Figure or Table reference has been "linked" to its respective figure/table for direct access to the entry.
1.) Canadian Institutes of Health Research (CIHR) Frederick Banting and Charles Best Canada Graduate Scholarship doctoral award, 2.) CIHR Michael Smith Foreign Study Award, 3.) Cystic Fibrosis Canada (CFC) doctoral studentship, 4.) University of Guelph Dean's Tri-Council Scholarship, 5.) Ontario Graduate Scholarship in Science and Technology, 6.) Operating grants to Dr. Joseph S. Lam from CIHR (MOP-14687) and CFC