The goal of this thesis was to develop a method for efficiently synthesizing a large suite of asymmetric oligoester ion channel-forming compounds. A solid-phase organic synthesis (SPOS) approach on Wang resin was used to generate the ion channel candidates. A follow-on goal is to survey the compounds produced to uncover structure-related controls on ion transport activity. Two classes of building blocks were used to generate the oligoesters – head groups and cores. The core building blocks were three omega-hydroxy acid derivatives six, eight and twelve carbons in length and the alcohol protected as a tetrahydropyranyl ether. The head group building blocks were either a glutaric acid monoester derivative of varying lipophilicity (12 to 16 carbon long alkyl tail) or a beta-hydroxy acid derivative; these building blocks used a tert-butyldimethylsilyl ether for alcohol protection. Optimized conditions for building block coupling, deprotection, and product cleavage were first established by the generation of dimeric and trimeric products. The building blocks were coupled using diisopropylcarbodiimide/ dimethylaminopyridine conditions. The deprotection of the tetrahydropyranyl ether group from the alcohol used a dilute acid solution in methanol and dichloromethane. A fluoride solution (from tetrabutylammonium fluoride) in tetrahydrofuran was used to deprotect the tert-butyldimethylsilyl ether group. Cleavage of the product synthesized on Wang resin was achieved by treatment with a trifluoroacetic acid/dichloromethane or ethereal hydrogen chloride solution. The products were then isolated by gel filtration. Mass spectrometry was used to identify the minor impurities which were quantified by proton nuclear magnetic resonance integrations. With the nine building blocks, many tetrameric and pentameric structures can be made, but a directed-library approach was used to address structure-activity related questions. Three pentameric oligoester products were the largest products synthesized to determine the scope and limitations of the SPOS methodology. The oligoester ion channel candidates were tested for ion transport activity using a 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt fluorescence vesicle assay. For each compound a pseudo-first order rate constant was derived at a particular concentration. A more useful normalized rate constant was calculated for an interpolated transporter concentration which allowed for transport activity comparison between compounds. The results from the fluorescence assay showed that some compounds and some isomers were substantially more active than others. There appeared to be an optimal core length and lipophilicity for relatively high activity. The aggregation of the compounds in buffer solution was probed using a pyrene fluorescence experiment. The solid-phase methodology was extended to include coupling of amino acids. A tryptophan derivative was made from one of the most active SPOS oligoester ion channel-forming compounds. The integrity of the molecules synthesized by SPOS which contain the tryptophan group could then be determined by high performance liquid chromatography. The fluorescence of the indole is quenched by acrylamide. By first equilibrating the vesicles with the tryptophan-containing oligoesters and then adding a fluorescence quencher, the resulting indole fluorescence was monitored as a function of quencher concentration. A Stern-Volmer plot was derived based on the quenching data which reported the possible orientations of the tryptophan-containing oligoester within the vesicle.

Introdução: As extrassístoles ventriculares e supraventriculares (EV e ESSV) são frequentes e muitas vezes sintomáticas. O íon magnésio (Mg) desempenha um papel importante na fisiologia do potencial de ação transmembrana celular e do ritmo cardíaco. Objetivo: Avaliar se a administração do Pidolato de Magnésio (PMg) em pacientes com EV e ESSV é superior ao placebo (P) na melhora dos sintomas e densidade das extrassístoles(DES). Métodos: Estudo duplo-cego, randomizado, com 90 pacientes sintomáticos consecutivos, com mais de 240/EV ou ESSV ao Holter de 24 horas e selecionados para receber P ou PMg. Para avaliar a melhora da sintomatologia, foi feito um questionário categórico e específico de sintomas relacionado às extrassístoles. Foi considerada significante uma redução de mais de 70% na DES por hora após o tratamento. A dose do PMg foi de 3,0g/dia por 30 dias, equivalente a 260mg do elemento Mg. Nenhum paciente tinha cardiopatia estrutural ou insuficiência renal. Resultados: Dos 90 pacientes estudados, 49 eram do sexo feminino (54,4%). A faixa etária variou de 16 a 70 anos. No grupo PMg, 77,8% dos pacientes tiveram redução maior que 70%, 6,7% deles entre 50% a 70% e, somente 13,3% dos pacientes com redução menor que 50% na DES . No grupo P, 44,4% dos pacientes tiveram melhora de apenas 30% na frequência de extrassístoles (p<0,001). A melhora dos sintomas foi alcançada em 91,1% dos pacientes do grupo PMg, comparada com somente 15,6% do grupo P(p<0,001). Conclusão: A suplementação de Mg por via oral reduziu a DES, resultando em melhora dos sintomas. Estudos clínicos e moleculares são necessários para avaliar o Mg intracelular e orientar quanto às necessidades diárias deste íon, evidenciar as prováveis deficiências e esclarecer melhor como prevenir e tratar pacientes com extrassístoles sintomáticas e sem cardiopatia estrutural. / Introduction: Premature ventricular and supraventricular complexes (PVC and PsVC) are frequent and often symptomatic. The magnesium (Mg) ion plays a role in the physiology of cell membranes and cardiac rhythm. Objective: We evaluated whether the administration of Mg Pidolate (MgP) in patients with PVC and PsVC is superior to placebo (P) in improving symptoms and arrhythmia frequency. Methods: Randomized double-blind study with 90 consecutive symptomatic patients with more than 240 PVC or PsVC on 24-hour Holter monitoring who were selected to receive placebo or MgP. To evaluate symptom improvement, a categorical and a specific questionnaire for symptoms related to PVC and PsVC was made. Improvement in premature complex density (PCD) per hour was considered significant if percentage reduction was >=70% after treatment. The dose of MgP was 3.0 g/day for 30 days, equivalent to 260mg of Mg element. None of the patients had structural heart disease or renal failure. Results: Of the 90 patients, 49 were female (54,4%). Ages ranged from 16 to 70 years old. In the MgP group, 77.8% of patients had a PCD reduction >70%, 6,7% of them from 50% to 70%, and only 13.3% <50%. In the P group, 44,4% showed slight improvement, <30%, in the premature complexes frequency (p<0.001). Symptom improvement was achieved in 91.1% of patients in the MgP group, compared with only 15.6% in the P group (p<0.001). Conclusions: Oral Mg supplementation decreases PCD, resulting in symptom improvement. Clinical and molecular studies are needed to evaluate intracellular Mg and develop better targets for the daily needs of this ion, show probable deficiencies, and explain how to prevent and better treat patients with symptomatic premature ventricular, and supraventricular complexes and no apparent heart disease.

De nombreux processus physiologiques tels que les mouvements stomatiques, l’absorption des nutriments, l’élongation cellulaire et la signalisation cellulaire impliquent des flux d’anions entre les membranes plasmique et vacuolaire des cellules végétales. Ces flux ioniques sont régulés par des canaux et transporteurs membranaires. Les canaux ioniques transportent passivement les ions au travers des membranes selon le gradient électrochimique. Les transporteurs actifs permettent le transport contre le gradient électrochimique de l’ion transporté induisant son accumulation dans un compartiment cellulaire. Dans les cellules végétales, le gradient de H+ entre différents compartiments constitue la principale source d’énergie couplée par les symports et les antiports au transport de NO₃⁻ et Cl⁻. Au cours de ma thèse, j’ai analysé ces flux ioniques avec deux approches. Une première approche a consisté en l’étude fonctionnelle par électrophysiologie de deux protéines membranaires, AtCLCc et AtCLCg impliquées dans le transport d’anions. Dans une deuxième approche, un biosenseur, clopHensor a été exprimé chez A. thaliana et a permis de mesurer simultanément la [NO₃⁻] et le pH cytosoliques in vivo. Les cellules de garde ont été choisies comme modèle cellulaire pour l’étude de la dynamique in vivo de la [NO₃⁻]cyt et du pH. Nous avons mis en évidence que la [NO₃⁻]cyt est influencée par les conditions extracellulaires dans ces cellules. Enfin l’expression de clopHensor en plantes KO pour un antiport NO₃⁻/H⁺ vacuolaire, AtCLCa, et d’un canal anionique de la membrane plasmique, SLAC1, nous a permis d’étudier la contribution de deux membranes dans la régulation de [NO₃⁻] et du pH cytosolique. Les travaux menés ont permis de visualiser l’activité de canaux et de transporteurs d’anions et H⁺ in vivo et de quantifier leur impact sur l’homéostasie du cytosol. / Many physiological processes like stomata aperture, nutrient up-take, cellular elongation and cell signalling involve anion fluxes at the two main membranes, the plasma and vacuolar membranes of plant cells. Specialized membrane proteins form active and passive anion transport systems mediating and regulating anion fluxes. Ion channels are passive transport systems mediating ion fluxes across membranes along the electrochemical gradient. Whereas active transporters work against the electrochemical gradient of the transported ion allowing its accumulation into a cellular compartment. In plant cells, the H⁺ gradient is the main energy source of antiporters and symporters that couple the transport of anions like NO₃⁻ and Cl⁻ to the transport of H⁺. In the presents work, we aimed at analysing anion and H⁺ fluxes at two levels. First, we used an electrophysiological approach to study the functional properties of two anion transport systems acting at the vacuolar membrane, AtCLCc and AtCLCg. We also expressed a biosensor, clopHensor in A. thaliana to dynamically measure in vivo the [NO₃⁻] and pH of the cytosol. We chose stomata guard cells as a cellular model to study these fluxes. Our results illustrate the in vivo dynamics of cytosolic [NO₃⁻] and pH variations in the cytosol of guard cells. Our data show that in guard cells the cytosolic [NO₃⁻] is highly influenced by the extracellular [NO₃⁻]. At last, clopHensor’s expression in plants KO for the vacuolar NO₃⁻/H⁺ antiporter AtCLCa and for the plasma membrane anion channel SLAC1 allowed us to dissect the role of the two membranes in controlling the variation of cytosolic [NO₃⁻] and pH. This work enabled to visualize the activity of an anion channel (SLAC1) and of a NO₃⁻/H⁺ antiporter (AtCLCa) in vivo and to quantify the impact of anion and proton fluxes on cytosolic homeostasis of guard cells.

Proton fluxes through plasma membranes are essential for regulating intracellular and extracellular pH and mediating co-transport of metabolites and ions. Although conventional electrical measurements are highly sensitive and precise for proton current detection, they provide limited specificity and spatial information. My thesis focuses on developing optical approaches to visualize proton fluxes from ion channels and transporters. It has been demonstrated that channel-mediated acid extrusion causes proton depletion at the inner surface of the plasma membrane. Yet, proton dynamics at the extracellular microenvironment are still unclear. In Chapter II, we developed an optical approach to directly measure pH change in this nanodomain by covalently attaching small-molecule, fluorescent proton sensors to the cell’s glycocalyx using glyco-engineering and copper free ‘click’ chemistry. The extracellularly facing sensors enable real-time detection of proton accumulation and depletion at the plasma membrane, providing an indirect readout of channel and transporter activity that correlated with whole-cell proton current. Moreover, the proton wavefront emanating from one cell was readily visible as it crossed over nearby cells. The transport of monocarboxylates, such as lactate and pyruvate is critical for energy metabolism and is mainly mediated by proton-coupled monocarboxylate transporters (MCT1-MCT4). Although pH electrodes and intracellular fluorescent pH sensors have been widely used for measuring the transport of proton-coupled MCTs, they are unable to monitor the subcellular activities and may underestimate the transport rate due to cell’s volume and intracellular buffering. In Chapter III, we used the Chapter II approach to visualize proton-coupled transport by MCT1-transfected HEK293T cells and observed proton depletion followed by a recovery upon extracellular perfusion of L-lactate or pyruvate. In addition, we identified a putative MCT, CG11665/Hrm that is essential for autophagy during cell death in Drosophila. The results demonstrate that Hrm is a bona fide proton-coupled monocarboxylate transporter that transports pyruvate faster than lactate. Although the approach developed in Chapter II enables visualization of proton fluxes from ion channels and transporters, it’s not applicable in some cell types which cannot incorporate unnatural sialic acid precursors into their glycocalyx, such as INS-1 cells and cardiomyocytes. To address this, in Chapter IV we developed a pH-sensitive, fluorescent WGA conjugate, WGA-pHRho that binds to endogenous glycocalyx. Compared to the results in Chapter II and III, cell surface-attached WGA-pHRho has similar fluorescent signals in response to proton fluxes from proton channel Hv1, omega mutant Shaker-IR R362H and MCT1. With WGA-pHRho, we were able to label the plasma membrane of INS-cells and cardiomyocytes and visualized the transport activity of MCT1 in these cells. Taken together, these findings provide news insights into proton dynamics at the extracellular environment and provide new optical tools to visualize proton fluxes from ion channels and transporters. Moreover, the modularity of the approaches makes them adaptable to study any transport events at the plasma membrane in cells, tissues, and organisms.

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A well documented laboratory setup, leech preparation process, and bio-potential data recording process are needed. Repeatability and quality data recordings are essential and thus dictate the requirements of the laboratory setup and processes listed above. Advances in technology have both helped and hindered this development. While very precise equipment is required to record the low voltage bio-potentials, noisy electronic equipment and wires surrounding the work area provide high levels of interference. Proper laboratory setup and data recording processes, however, limit the unwanted interference. Quality data can only be recorded from a properly handled and prepared leech subject. Proper setup and procedures result in quality recordings which lend a clean signal for furthering the understanding of nerve functionality. The electrophysiology lab at California Polytechnic State University in San Luis Obispo is an example of a proven lab setup for high quality signal capture.

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Yes / Skin pigmentation is directed by epidermal-melanin units, characterized by long-lived and dendritic epidermal melanocytes (MC) that interact with viable keratinocytes (KC) to contribute melanin to the epidermis. Previously we reported that MC:KC contact is required for melanosome transfer, that this can be enhanced by filopodial and by UVR/UVA irradiation, which can up-regulate melanosome transfer via Myosin X-mediated control of MC filopodia. Both MC and KC express Ca2+-dependent E-cadherins. These homophilic adhesion contacts induce transient increases in intra-KC Ca2+, while ultraviolet radiation (UVR) raises intra-MC Ca2+ via calcium selective ORAI1 ion channels; both are associated with regulating melanogenesis. However, how Ca2+ triggers melanin transfer remains unclear, and here we evaluated the role of E-Cadherin in UVR-mediated melanin transfer in human skin cells. MC and KC in human epidermis variably express filopodia-associated E-Cadherin, Cdc42, VASP and β-catenin, all of which were upregulated by UVR/UVA in human MC in vitro. Knockdown of E-cadherin revealed that this cadherin is essential for UVR-induced MC filopodia formation and melanin transfer. Moreover, Ca2+ induced a dose-dependent increase in filopodia formation and melanin transfer, as well as increased β-catenin, Cdc42, Myosin X, and E-Cadherin expression in these skin cells. Together these data suggest that filopodial proteins and E-Cadherin, which are upregulated by intracellular (UVR-stimulated) and extracellular Ca2+ availability, are required for filopodia formation and melanin transfer. This may open new avenues to explore how Ca2+ signalling influences human pigmentation.