Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

La Paralysie Périodique Hypokaliémique de type 1 (HypoPP1) est une myopathie d'origine génétique, autosomique dominante, caractérisées par des épisodes de paralysies musculaires récurrentes pouvant durer quelques heures à quelques jours. Ces crises de paralysies sont accompagnées d'une hypokaliémie responsable d'arythmies cardiaques entraînant la mort dans les cas les plus graves. Les crises peuvent être déclenchées par un stress, une alimentation riche en glucides ou encore suite à un exercice physique intense. L'HypoPP1 est liée à une mutation dans le gène CACNA1S codant la sous-unité principale du canal calcique musculaire (Cav1.1). A l'exception d'une, toutes les mutations HypoPP1 conduisent au remplacement d'une des arginines les plus externes d'un des segments détecteurs de potentiel du canal, nommés S4, par un acide aminé neutre. Des études réalisées dans des modèles d'expression hétérologue de canaux potassiques ou sodiques, dont les structures sont très proches du Cav1.1, ont montré que des mutations similaires à l'HypoPP1 conduisaient à la création d'une voie de passage ionique dite "accessoire" au travers du domaine détecteur de potentiel générant un courant cationique entrant au potentiel de repos. Si une telle voie de passage ionique existe à travers Cav1.1 dans les cellules musculaires des patients souffrant d'HypoPP1, elle pourrait ainsi induire une dépolarisation des cellules musculaires au point de les rendre inexcitables, conduisant ainsi à la paralysie. Mon travail a consisté à étudier les formes R1239H et V876E du Cav1.1 responsables d'HypoPP1 afin de déterminer si une telle voie de passage accessoire est présente dans les canaux calciques mutés. La mutation R1239H correspond à la substitution d'une histidine à la seconde arginine dans le segment S4 du domaine IV tandis que la mutation V876E présente la particularité de ne pas affecter un S4 mais un segment S3 dans le domaine III. Ce travail s'appuie sur l'expression in vivo du gène codant le Cav1.1 humain sain (WT) ou muté R1239H ou V876E dans les muscles des pattes arrière de souris puis sur l'analyse des mouvements ioniques en combinant des techniques d'électrophysiologie et de mesure des concentrations intracellulaires de H+ ou de Na+ par fluorescence sur fibre musculaire isolée. L'étude de la mutation R1239H a montré qu'un influx de protons générant un courant entrant significativement plus important se développait au potentiel de repos dans les fibres exprimant la mutation en comparaison des cellules exprimant la forme WT. L'étude de la deuxième mutation V876E, sur laquelle aucune donnée fonctionnelle n'était disponible à ce jour, a révélé que cette mutation était elle aussi responsable d'un courant ionique accessoire mais dans ce cas-là porté par les ions Na+. Ce résultat est important car il montre que la formation d'un pore ionique accessoire constitue un mécanisme physiopathologique commun à différentes formes d'HypoPP affectant le canal calcique musculaire, y compris lorsque la mutation n'affecte pas directement un segment S4 / The type 1 Hypokalemic Periodic Paralysis (HypoPP1) is a muscle autosomal dominant genetic disease characterized by episodic attacks of paralysis lasting between a few hours and several days. These attacks are associated with hypokalemia which is responsible of cardiac arrhythmias leading to death in worst cases. Attacks are triggered by stress, high carbohydrate diet or during rest following exercise. HypoPP1 is caused by missense mutations in the gene CACNA1S encoding the main subunit of the voltage-gated calcium channel (Cav1.1) of skeletal muscle. In all but one, HypoPP1 mutations lead to the replacement of an outermost arginine in one of the voltage sensor segment, called S4, with a neutral amino-acid. Experiments realized with the closely structurally related voltage-gated K+ and Na+ channels showed that comparable mutations generate an accessory pathtoway called gating pore through which a depolarizing current flows at rest. If such an accessory pathway also exists in the skeletal muscle Cav1.1, it could initiate depolarization of skeletal muscle to the point of inexcitability and lead to paralysis. My work aimed at investigating the properties of HypoPP1-associated mutant Cav1.1s R1239H and V876E in order to determine if such an accessory pathway could be present in mutated Cav1.1. The R1239H mutation corresponds to the replacement of the second arginine in the S4 segment of domain IV by a histidine and the V876E mutation has the particularity to not affect a S4 segment but the S3 segment in domain III. In this work, we used in vivo gene expression of the wild type (WT) or mutated forms of the human Cav1.1in the hind limb muscle of mice and analyzed ions fluxes by combining voltage-clamp and measurements of intracellular H+ or Na+ by fluorescence. The R1239H mutation was shown to induce a significant larger H+ influx giving rise to a larger inward H+ current at rest as compared to WT. The V876E mutation, which had never been investigated so far, was found to induce an elevated inward current at rest but in this case carried by Na+ ions. These results have relevance because they suggest that the presence of an accessory pathway could be a physiopathological mechanism shared by different HypoPPs, even when the mutation does not directly affect a S4 segment

La schizophrénie est une pathologie caractérisée par des symptômes positifs (idées délirantes et hallucinations), des symptômes négatifs (émoussement affectif, alogie, apragmatisme, retrait social) et des symptômes de désorganisation (cognitifs et affectifs). Cette pathologie est également associée à des altérations cognitives, perceptuelles et de la qualité de vie. La physiopathologie de la schizophrénie reste mal connue. Récemment, des travaux ont mis en avant le rôle central des processus inflammatoires chroniques dans la physiopathologie de ce trouble psychiatrique. En particulier, il a été montré que la CRP (Protéine C-Réactive), marqueur inflammatoire chronique aspécifique et facile à doser dans une prise de sang, est augmentée dans la schizophrénie. La CRP est reliée à la symptomatologie clinique et aux altérations cognitives des patients souffrant de schizophrénie. Mais le lien entre altérations perceptuelles et de la qualité de vie et CRP reste à explorer. / Schizophrenia is an illness characterized by positive symptoms (delusions and hallucinations), negative symptoms (reduced emotional expression, alogia, apragmatism, reduced social engagement) and disorganized symptoms (cognitive and affective). This pathology is also associated to cognitive and perceptual alterations and to quality of life alterations. The physiopathology of schizophrenia is still unclear. Recently, papers put forward the central role of chronic inflammatory process in pathophysiology of this psychiatric disorder. In particular, CRP (C-Reactive Protein), a nonspecific marker of chronic inflammation and easy to measure with blood sample, was shown to be increased in schizophrenia. CRP is connected to clinical symptomatology and to cognitive alterations in patients with schizophrenia. However the connection between alterations of perception, quality of life and CRP remains to be explored.

Diploma thesis deals with problem of designing the gating systems for wax patterns manufacture by numerical simulation. It was created an optimum numerical simulation of wax blend injection, optimized for a simple test model and then applied to more complex pieces. Based on knowledge of plastic injection and the simulation was designed several gating systems for mass-produced pieces. These findings were subsequently applied in the design of gating systems to produce large and thin wax patterns. By comparing the various proposals were finally selected the best solution in order to verify the optimum numerical simulation in practise.

Chez les plantes, le nitrate est un élément essentiel mais sa disponibilité dans le sol est fluctuante. Il est donc stocké dans la vacuole grâce à un échangeur nitrate/proton appelé AtClCa. La famille de protéines ClCs comporte à la fois des échangeurs mais aussi des canaux suggérés comme issus de l’évolution des échangeurs par une conversion mécanistique. Chez Arabidopsis thaliana, seuls des ClCs échangeurs assurent la gestion du nitrate. Deux glutamates très conservés, E203 et E270 dans AtClCa, sont essentiels pour le transport des protons chez les ClCs échangeurs. La mutation du résidu E203 en une alanine, un acide aminé non protonable (E203A) a permis de produire artificiellement une telle conversion mécanistique. Afin de mieux comprendre l’importance physiologique du mécanisme d’échange, une analyse a été conduite sur des plantes exprimant la forme mutée d’AtClCa pour ce glutamate. Chez ces plantes, le stockage vacuolaire est fortement réduit au profit d’une importante assimilation accroissant la teneur en protéines. En dépit de cela, elles présentent un défaut de production de biomasse résultant en grande partie d’une perturbation de l’homéostasie hydrique. Elles sont également plus sensibles aux stress hydrique et probablement azoté. La conservation d’un échangeur est donc requise pour croitre en dépit des fluctuations environnementales. En parallèle, la mutation E270A a été introduite en plante afin d’étudier son importance sur la physiologie d’Arabidopsis. Une analyse préliminaire de la biomasse et des contenus en nitrate et eau de plantes exprimant la forme mutée de ce glutamate est donc présentée dans la seconde partie de cette thèse. / Nitrate is a major element for plant but its availability is very fluctuant in soils. Then, it is stored in vacuoles thanks to a nitrate/proton exchanger named AtClCa. In ClCs, exchangers but also channels were identified, the latest were suggested to be evolved from exchanger in which a mechanistic switch happened. In Arabidopsis thaliana, only exchangers are involved in nitrate management. Two conserved glutamate, E203 and E270 in AtClCa, are essential for protons transport in ClCs exchangers. The mutation of E203 into an alanine, a non-protonable amino acid (E203A) artificially produces such a mechanistic switch. To better understand the physiological importance of this exchange mechanism, a study was conducted in plants expressing the mutated form of AtClCa for this glutamate. In those plants, the vacuolar storage is highly restricted whereas the assimilation is favoured and the protein content increased. Despite that, the biomass production is decreased mostly because of a hydric homeostasis disruption. Those plants are also more sensitive to hydric and probably nitrogenous stress. The exchanger conservation is then required for plant growth whatever the environmental fluctuations. In parallel, the mutation E270A was introduced in planta to study its physiological importance. A preliminary analysis of plant biomass and nitrate and water contents was then performed in plants expressing the E270A mutated form of AtClCa and the results are presented in the second part of the manuscript.

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.

Respiratory motion during PET acquisition degrades image quality. It is mainly the area around the thorax and abdomen which is affected. External devices do provide respiratory gating solutions but are time-consuming to set up on patients and may not always be available. A data-driven gating (DDG) method based on principal component analysis (PCA) was found to provide a reliable respiratory gating signal, discriminating the need for external gating systems with FDG, but it remains to be investigated how well it performs with other PET tracers. The HER2-targeting radiotracer 68Ga-ABY-025 is currently in phase 3 development and is aimed to develop methods to select breast cancer patients that benefit from HER2-targeted treatment. Hence, absolute quantification is important. Respiratory motion correction will be important for improved quantitative accuracy since many patients have metastases in the lower part of the lungs or the liver.  DDG was applied to PET/CT list mode data retrospectively using quiescent period gating. Gated images were then compared to reconstructions without gating with a matched number of coincidences. Two iterative reconstructions were evaluated, TOF OSEM (3 iterations, 16 subsets, and a 5 mm gaussian postprocessing filter) and TOF BSREM β 400. Images were evaluated for standardized uptake value (SUV) changes for well-defined lesions in thorax and abdomen where respiratory motion is prevalent. Respiratory motion was detected in a mean 2.1 bed positions per examination. DDG application resulted in a mean increase of 12.7% in SUVmax for TOF OSEM reconstruction (p=0.0156).

Increased neuroinflammation has been shown in individuals diagnosed with schizophrenia (SCHZ). This study evaluated a novel immune modulator (PD2024) that targets the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) to alleviate sensorimotor gating deficits and microglial activation employing two different rodent models of SCHZ. In Experiment 1, rats were neonatally treated with saline or the dopamine D2-like agonist quinpirole (NQ; 1 mg/kg) from postnatal day (P) 1-21 which produces increases of dopamine D2 receptor sensitivity throughout the animal's lifetime. In Experiment 2, rats were neonatally treated with saline or the immune system stimulant polyinosinic:polycytidylic acid (Poly I:C) from P5-7. Neonatal Poly I:C treatment mimics immune system activation associated with SCHZ. In both experiments, rats were raised to P30 and administered a control diet or a novel TNFα inhibitor PD2024 (10 mg/kg) in the diet from P30 until P67. At P45-46 and from P60-67, animals were behaviorally tested on auditory sensorimotor gating as measured through prepulse inhibition (PPI). NQ or Poly I:C treatment resulted in PPI deficits, and PD2024 treatment alleviated PPI deficits in both models. Results also revealed that increased hippocampal and prefrontal cortex microglial activation produced by neonatal Poly I:C was significantly reduced to control levels by PD2024. In addition, a separate group of animals neonatally treated with saline or Poly I:C from P5-7 demonstrated increased TNFα protein levels in the hippocampus but not prefrontal cortex, verifying increased TNFα in the brain produced by Poly I:C. Results from this study suggests that that brain TNFα is a viable pharmacological target to treat the neuroinflammation known to be associated with SCHZ.

In dieser Arbeit wird ein durch eine passive Matrix angesteuertes Sensorarray vorgestellt. Das Sensorarray besteht aus zwei parallel zueinander angeordneten Leiterplatten mit jeweils vier Leiterbahnen, die als Arbeits- und Gegenelektroden verwendet werden. Die Leiterbahnen kreuzen sich in einem Winkel von 90°, wobei an jedem Kreuzungspunkt ein Sensorelement gebildet wird. Ein selektives Auslesen der Sensorelemente wird durch eine mechanische oder auf Kapillarkräften basierenden Unterteilung des Elektrolyten sowie eine spezielle elektrotechnische Auslesemethode erreicht. Durch die Verwendung einer aus Preußisch Blau bestehenden kombinierten Gegen- und Referenzelektrode können in dem Zwei Elektrodensystem Bedingungen, die denen eines Drei Elektrodensystems sehr nahe kommen, realisiert werden.Mit diesem einfach aufzubauenden Sensorarray konnte die Lücke zwischen den, in der Größe limitierten Sensoren mit Einzeladressierung und den wesentlich aufwändigeren, aber eine hohe Packungsdichte aufweisenden CMOS Sensoren geschlossen werden. Die Funktionalität dieses Sensorarrays wurde anhand von zwei unterschiedlichen Anwendungsbeispielen aus dem Bereich der Kombinatorischen Chemie unter Beweis gestellt.

Indiana University-Purdue University Indianapolis (IUPUI) / Voltage-gated sodium channels (VGSCs) are dynamic membrane-spanning proteins crucial for determining the electrical excitability in nerve and muscle. VGSCs transition, or gate, between opened, closed, and inactivated states, in response to changes in transmembrane potential. Altered VGSC gating can affect electrical communication and is implicated in numerous channelopathies. Nav1.7, a VGSC isoform highly expressed in the peripheral nervous system, plays a unique role in pain perception as evidenced by single point missense mutations causing a spectrum of pain syndromes (inherited erythromelalgia; IEM and paroxysmal extreme pain disorder; PEPD) and nonsense mutations resulting in human insensitivity to pain (CIP). These studies indicate Nav1.7 is critical in pain transduction and, as such, structural perturbations to Nav1.7 affecting conformational stability and response to changes in transmembrane potential have the potential to cause pain. Therefore, the aims of this dissertation were to (1) examine the effects of PEPD mutations on the voltage-dependent properties Nav1.7; (2) investigate the effects Nav1.7 alternative splicing has on the impact of IEM and PEPD mutations; (3) evaluate the effects channelopathies, resulting from slowed inactivation, have on modulating an unusual type of sodium current that flows during membrane repolarization; and (4) determine the structural components involved in stabilizing Nav1.7 inactivation. Standard patch-clamp electrophysiology was used to study changes in channel properties. Results from this dissertation demonstrate that (1) PEPD mutations significantly shift the voltage-dependent properties of Nav1.7 channels, destabilize an inactivated state in a residue specific manner, and render nociceptive neurons hyperexcitable; (2) alternative splicing can functionally impact PEPD; (3) channelopathies, resulting from slowed inactivation in neuronal and muscle VGSC isoforms, increase an unusual sodium conductance that flows during repolarization; and (4) specific residues located in distinct regions of Nav1.7 serve as docking sites to stabilize inactivation at different membrane potentials. Overall, this dissertation answers key questions regarding the molecular mechanics required during inactivation and the biophysical consequences of Nav1.7 mutations implicated in painful disorders. The results of this dissertation are important for a more detailed understanding of pain perception and validate the applicability of studying Nav1.7 for discovery of therapeutic targets for treatment of pain. – Theodore R. Cummins, Chair