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Chen, Zhihong. "Modeling Ion Binding in the Chloride Transporter." University of Cincinnati / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1439310689.
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Niemiec, Moritz Sebastian. "Human copper ion transfer : from metal chaperone to target transporter domain." Doctoral thesis, Umeå universitet, Kemiska institutionen, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-100511.
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Many processes in living systems occur through transient interactions among proteins. Those interactions are often weak and are driven by small changes in free energy. Due to the short-living nature of these interactions, our knowledge about driving forces, dynamics and structures of these types of protein-protein heterocomplexes are though limited. This is especially important for cellular copper (Cu) trafficking: Copper ions are essential for all eukaryotes and most bacteria. As a cofactor in many enzymes, copper is especially vital in respiration or detoxification. Since the same features that make copper useful also make it toxic, it needs to be controlled tightly. Additionally, in the reducing environment of the cytosol, Cu is present as insoluble Cu(I). To circumvent both toxicity and solubility issues, a system has evolved where copper is comforted by certain copper binding proteins, so-called Cu-chaperones. They transiently interact with each other to distribute the Cu atoms in a cell. In humans, one of them is Atox1. It binds copper with a binding site containing two thiol residues and transfers it to other binding sites, mostly those of a copper pump, ATP7B (also known as Wilsons disease protein). My work was aimed at understanding copper-mediated protein-protein interactions on a molecular and mechanistic level. Which amino acids interact with the metal? Which forces drive the transfer from one protein to the other? Using biophysical and biochemical methods such as chromatography and calorimetry on wild type and point-mutated proteins in vitro, we found that the copper is transferred via a dynamic intermediate complex that keeps the system flexible while shielding the copper against other interactions. Although similar transfer interactions can be observed in other organisms, and many conclusions in the copper field are drawn from bacterial and yeast analogs, we believe that it is important to investigate human proteins, too. Not only is their regulation different, but also only in humans we find the diseases linked to the proteins: Copper level regulation diseases are to be named first, but atypical copper levels have also been linked to tumors and amyloid dispositions. In summary, my observations and conclusions are of basic research character and can be of importance for both general copper and human medicinal research.
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Senior, Adam Paul. "GerT, an ion transporter homologue in Bacillus cereus and its role in spore germination." Thesis, University of Sheffield, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.425216.
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Nicolson, Tamara Jane. "Type II diabetes and the pancreatic B cell : molecular characterisation of ion channel and transporter polymorphisms." Thesis, Imperial College London, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.516549.
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Lau, Calvin Ho-Fung. "Inorganic ion transport and sensing by the bacterial multidrug ATP-binding cassette transporter LmrA of Lactococcus lactis." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608774.
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Haering, Claudia [Verfasser], Hanns [Akademischer Betreuer] Hatt, and Stefan [Akademischer Betreuer] Wiese. "Characterization of the ion transporter NKCC1 in the field of chemosensation / Claudia Haering. Gutachter: Hanns Hatt ; Stefan Wiese." Bochum : Ruhr-Universität Bochum, 2016. http://d-nb.info/1089005881/34.
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Shawki, Ali. "The Functional Properties and Intestinal Role of the H+-Coupled Divalent Metal-Ion Transporter 1, DMT1." University of Cincinnati / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1448037106.
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Rimington, Tracy L. "Expression, purification and characterisation of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) in Saccharomyces cerevisiae." Thesis, University of Manchester, 2014. https://www.research.manchester.ac.uk/portal/en/theses/expression-purification-and-characterisation-of-the-cystic-fibrosis-transmembrane-conductance-regulator-cftr-in-saccharomyces-cerevisiae(5c8c606b-8925-4627-91dc-67a896b9f286).html.
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Mutations in the eukaryotic integral membrane protein Cystic Fibrosis Transmembrane conductance Regulator (CFTR) cause the hereditary disease cystic fibrosis (CF). CFTR functions as an ion channel at the surface of epithelial cells and regulates the movement of chloride ions and water across the plasma membrane. CFTR is difficult to express and purify in heterologous systems due to its propensity to form insoluble aggregates and its susceptibility to degradation. Obtaining good yields of highly purified CFTR has proven problematic and contributes to our limited understanding of the structure and function of the protein. The most prevalent disease causing mutation, F508del, results in misfolded CFTR which is particularly unstable and is quickly targeted for degradation by the host system and is prevented from being trafficked to the plasma membrane. There are limited treatment options for patients with the F508del mutation and it is therefore of significant interest within CF research. New methods and assays are required to identify potential compounds which could correct the F508del mutation. This thesis investigates the use of Saccharomyces cerevisiae to express and purify codon optimised recombinant CFTR. The use of a green fluorescent protein (GFP) tag enabled quick and simple detection of CFTR in whole cells and after extraction from the plasma membrane. By optimising the culture conditions for CFTR expression and detergent solubilisation conditions, relatively high yields of full-length protein were obtained. When used as a chemical chaperone at the time of inducing CFTR expression, glycerol increased yields of full-length protein. Degradation of CFTR could be limited by inducing expression at an optimal cell density and by harvesting cells within a specific time window. CFTR was extracted by solubilisation in the mild detergent dodecyl-β-D-maltopyranoside (DDM) in the presence of up to 1 M NaCl with up to ~87% efficiency in some cases. Using a gene optimisation strategy in which additional purification tags and a yeast Kozak-like sequence were added, the human CFTR (hCFTR) protein was expressed and purified. Fluorescence microscopy revealed CFTR localisation at the periphery of yeast cells. Immunoaffinity chromatography facilitated by the GFP tag at the C terminus of CFTR produced protein of up to 95% purity. An assessment of the thermal stability of this highly purified CFTR using a fluorescent probe binding assay revealed a denaturation midpoint (Tm) of ~43 degC. The ability of this assay to determine the stability of CFTR is encouraging and there is the potential to further develop it in a high-throughput manner to identify compounds which stabilise the F508del protein and which may hold the key to developing new treatments for CF.
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Murphy-Royal, Ciaran. "Surface diffusion of the astrocytic glutamate transporter glt-1 shapes synaptic transmission." Thesis, Bordeaux, 2014. http://www.theses.fr/2014BORD0113/document.
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Le glutamate est le principal neurotransmetteur excitateur du système nerveux central des vertébrés, et le codage de l’information cérébrale repose en partie sur des modulations de l’amplitude et de la fréquence des transmissions synaptiques glutamatergiques. De ce fait, la résolution spatiale et temporelle de ces transmissions nécessite un contrôle fin de la présence de glutamate dans la fente synaptique. Cette durée de vie du glutamate dans les synapses dépend directement de l’action de transporteurs spécifiques exprimés à la surface des astrocytes, en particulier les transporteurs de type GLT-1, qui retirent le neurotransmetteur et permettent ainsi de « nettoyer » la fente synaptique avant la survenue d’un nouvel épisode de neurotransmissionA classic understanding of neurotransmitter clearance at glutamatergic synapses is that, in order to ensure sufficient glutamate uptake on a fast timescale, it is necessary to have high numbers of glutamate transporters in the vicinity of release sites to compensate for their slow transport kinetics. Using a combination of single molecule imaging and electrophysiological approaches, we now challenge this view by first demonstrating that GLT-1 transporters are not static but highly mobile at the surface of astrocytes, and that their surface diffusion is dependent upon both neuronal and glial cell activities. In the vicinity of glutamate synapses, GLT-1 dynamics are strongly reduced favoring their retention within this strategic location. Remarkably, glutamate uncaging at synaptic sites instantaneously increases GLT-1 diffusion, displacing the glutamate-bound transporter away from this compartment. Functionally, impairment of the transporter lateral diffusion through an antibody-based surface cross linking, both in vitro and in vivo, significantly slows the kinetics of excitatory postsynaptic currents. Taken together, these data reveal the unexpected and major role of the astrocytic surface GLT-1 fast dynamics in shaping glutamatergic synaptic transmission.Keywords:
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Lee, Shernita. "Ironing Out the Host-fungal Interaction in Airway Epithelial Cells." Diss., Virginia Tech, 2014. http://hdl.handle.net/10919/56689.
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Aspergillus fumigatus is a ubiquitous fungus associated with several airway complications and diseases including asthma, allergies, cystic fibrosis, and most commonly invasive aspergillosis. The airway epithelium, a protective barrier, is the first anatomical site to interact with A. fumigatus. Although this host-fungal interaction is often asymptomatic for immunocompetent individuals, for immunocompromised persons, due to a weakened competence of the immune system, they have an increased likelihood of fungal infection.
This dissertation aims to investigate the effect of A. fumigatus on the transcriptional response of human airway epithelial cells, focusing on the relationship between innate immunity and iron regulation from the host perspective. The trace element iron is needed by both the fungus and the host for cellular maintenance and survival, but tightly controlled iron regulation in the host is required to prevent oxidative stress and cell death. The research methods in this dissertation employ a systems biology approach, by incorporating mathematical modeling, RNA-seq analysis, and experimental biology techniques to assess the role of airway epithelial cells in the host-fungal interaction. Both the quantitative and qualitative research design allows for characterization of airway epithelial cells and the downstream changes in iron importer genes. This study addresses literature gaps through analysis of the host transcriptome using multiple time points, by performing an extensive evaluation of the effect of cytokines on iron importer genes, and conceptualization of a comprehensive mathematical model of the airway epithelial cell.
The major findings suggest the following: 1) airway epithelial cells avidly respond to A. fumigatus through modification of the expression of immune response related genes at different infection stages, 2) during A. fumigatus co-incubation with airway epithelial cells, the iron importers genes respond in strikingly different ways, and 3) cytokines have a significant effect on the increase in expression of an iron importer gene. We illuminated the role of airway epithelial cells in fungal recognition and activation of the immune response in signaling cascades that consequently modify iron importer genes and hope to use this information as a platform to discover potential therapeutic targets.Ph. D.
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Peterson, Emily. "Proteoliposome Proton Flux Assays Establish Net Conductance, pH-Sensitivity, and Functional Integrity of a Novel Truncate of the M2 Ion "Channel" of Influenza A." BYU ScholarsArchive, 2010. https://scholarsarchive.byu.edu/etd/2420.
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A novel truncate of Influenza A M2 protein (residues 22-62), incorporated into a uniquely tailored proteoliposome proton uptake assay, demonstrated proton flux more characteristic of an ion transporter than a traditional ion "channel." The liposome paradigm was essential for testing the conductance activity of this M2 truncate at a range of extraphysiological pHs appropriate for channel vs. transport function determination. In addition to transporter-typical proton flux, M2(22-62) showed the key characteristics of functional integrity: selective proton uptake into liposomes and block of uptake by amantadine. Two sets of proteoliposome proton flux assays were carried out, Set 1 at pH values of 6.5, 6.0. 5.5, 5.0, and 4.5; Set 2 at pH values of 6.25, 6.0, 5.75, 5.5, 5.25, 5.0, and 4.75. Observed flux rates followed a proton transport saturation curve similar to that observed in mouse erythroleukemia cells1. Proton transport was maximal at pH 5.5 in Set 1 (139 H+/second/tetramer) and at pH 5.75 in Set 2 (43 H+/second/tetramer). Amantadine block was strongest at pH 5.5 in Set 1 and 6.25 in Set 2, and apparent desensitization of the protein severely reduced proton flux and amantadine sensitivity below pH 5.5 in both sets of experiments. Decreased external pH increased proton uptake with an apparent pKa of 6 (Set 1) or 6.5 (Set 2). These data indicate acid activation of M2(22-62) between pH 5.5-6, optimal amantadine block between pH 5.5-6.25, and a loss of peptide functionality between pH 5.9-4.7.
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RIGAMONTI, MARCO. "HYPOTONIC STRESS-INDUCED CALCIUM SIGNALLING IN S. CEREVISIAE INVOLVES A NEW FUNGAL SPECIFIC FAMILY OF TRP-LIKE TRANSPORTERS, HOMOLOGOUS TO HUMAN MUCOLIPIN AND POLYCYSTIC KIDNEY DISEASE RELATED (PKD2) CALCIUM CHANNELS." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2015. http://hdl.handle.net/10281/71811.
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Studi presenti in letteratura hanno dimostrato che nel lievito S.cerevisiae lo stress generato dallo shock ipotonico induce un aumento transiente della [Ca2+]i. Dai nostri studi risulta che lo shock ipotonico è mediato, in cellule cresciute in terreno ricco, da due componenti cineticamente distinte, una molto rapida (circa 5 secondi), leggermente inibita da calcio e dipendente dal calcio extracellulare, e l’altra più lenta (10-20 sec.), fortemente sensibile alla presenza di calcio nell’ambiente esterno, al punto da non essere rilevabile anche a concentrazioni extracellulari di 1 µM. Mentre la prima risposta sembra essere mediata da un trasportatore localizzato sulla membrana plasmaticala seconda è chiaramente dipendente da un rilascio dagli store intracellulari.
Alcuni dati presenti in letteratura sui geni regolati dal calcio ci hanno suggerito di studiare il ruolo nella risposta a shock ipotonico di due geni omologhi, di cui il primo è stata proposto come codificante per un putativo canale per ioni simile ai canali TRP di mammifero: YOR365C e FLC2.
Flc2 è localizzata sulle membrane plasmatica, di Golgi ed ER ed è stato proposto come putativo trasportatore di flavine assieme ai suoi omologhi Flc1 e Flc3. Tuttavia Flc2 era già stato precedentemente identificato come un omologo del canale del calcio pkd2 di S. pombe. Effettivamente in base a nostri allineamenti è risultato evidente come Flc2 appartenga a una sottofamiglia di canali TRP-like tipica dei funghi, a cui appartengono anche le mucolipine umane e le proteine spray fungine, ma non le proteine Pkd2. Solo la mancanza di FLC2 abbatteva completamente il rilascio di calcio dagli store intracellulari in carenza di calcio extracellulare, indicando che questi geni, pur appartenenti alla stessa famiglia, svolgono funzioni molto diverse.
Per caratterizzare in modo efficace i flussi di calcio dovuti allo shock ipotonico abbiamo calcolato la velocità iniziale di incremento della [Ca2+]i (v0) al variare della concentrazione di calcio extracellulare.
Nel ceppo selvatico i dati in nostro possesso non potevano essere interpolati da nessuna equazione di Hill, a causa di una componente additiva presente a concentrazioni submicromolari di calcio extracellulare, mentre prendendo in considerazione i dati rilevati a concentrazioni di calcio superiori a1µM possiamo osservare una perfetta Michaelis-Menten caratterizzata da una KM di 1 µM. A concentrazioni di calcio superiori a 1 µM invece la v0 subisce una forte inibizione dovuta al calcio stesso,con una IC50 di 20 µM.
Al contrario i dati delle v0 calcolati nel ceppo flc2∆ sono interpolati da una curva di Hill anche a concentrazioni submicromolari di calcio,in quanto non si rileva alcuna componente additiva (dovuta probabilmente al rilascio di calcio dall’ER controllato da Flc2) a basse concentrazioni di calcio, né d’altra parte si osserva l’inibizione ad alte concentrazioni. Anche in questo caso la k è di circa 1 µM e la curva mostra un numero di cooperatività di 3.
Questi dati ci portano ad affermare che il flusso di calcio a seguito di uno shock ipotonico è caratterizzato dall’attività di due diversi complessi: uno localizzato sull’ER, attivo solo quando non è disponibile calcio extracellulare e regolato da Flc2, che ha probabilmente funzione di trasporto o di attivazione del complesso; l’altro sulla membrana plasmatica, attivo solo quando è presente calcio nell’ambiente extracellulare a concentrazioni micromolari. La peculiarità di quest’ultimo complesso è che, alla luce della scomparsa dell’inibizione ad alte concentrazioni di calcio osservata nel ceppo flc2∆, anch’esso sembra coinvolgere l’attività della proteina Flc2, che è probabilmente la sub unità regolatoria sensibile al calcio del trasportatore in questione.
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Chounlamountry, Keodavanh. "Les systèmes de capture du glutamate dans le noyau du tractus solitaire. Relations astrocytes-synapses et localisation subcellulaire des transporteurs du glutamate." Thesis, Aix-Marseille 3, 2011. http://www.theses.fr/2011AIX30035.
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Le glutamate est le principal neurotransmetteur excitateur du noyau du tractus solitaire (NTS), une structure sensorielle qui reçoit des informations provenant des viscères. Nous avons utilisé l'immunocytochimie et la microscopie électronique pour étudier les systèmes de recapture du glutamate dans le NTS. Nous montrons que le transporteur exprimé par les astrocytes est de type GLT-1 et que la couverture des synapses glutamatergiques par les processus astrocytaires n'est pas complète ce qui autorise des phénomènes de transmission à distance par diffusion du glutamate. Nous montrons aussi que les dendrites des neurones du NTS expriment le transporteur de type EAAC1. Ce transporteur est essentiellement présent sous forme d'un pool intracellulaire. Son expression membranaire pourrait donc être régulée par l'activité. Enfin, dans une dernière partie, nous montrons qu'une inflammation des viscères induit une augmentation de la couverture gliale des synapses glutamatergiques du NTSGlutamate is the main excitatory transmitter in the nucleus tractus solitarii (NTS), a sensory nucleus involved in visceral information processing. Using electron microscope immunocytochemistry, we have investigated neuron to glia relationships and localization of glutamate transporters in the NTS. We show that NTS astrocytes express GLT-1 and that astrocytic wrapping of NTS glutamatergic synapses is incomplete, allowing glutamate to diffuse out of the synaptic cleft. In addition, we demonstrate that NTS neurons express the EAAC1 transporter. EAAC1 is exclusively present in dendrites and mostly located intracellularly. Finally, we show that visceral inflammation increases the glial wrapping of NTS glutamatergic synapses
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Zha, Huiyan, and 查慧艳. "Design, synthesis and characterization of synthetic ion transporters." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hdl.handle.net/10722/206532.
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In recent decades, small molecules have been widely applied to the generation of functional ion transporters. Major discoveries disclosed in this thesis include a self-assembled chloride-dependent potassium channel candidate, a physiological chloride and bicarbonate dual-transporter, and a series of efficient synthetic ion transporters.
In nature, K+ channels play an important role in Ca2+ signaling, volume regulation, secretion, proliferation, and migration. The extracellular K+ concentration (4 mM) is about 40 times lower than the intracellular K+ concentration (160 mM). The opening of K+ channels consequently generates an efflux of positive charge, which hyperpolarizes or repolarizes the cellular membrane. In this research, by using fluorescence assays, NMR and patch clamp experiments, compound ZHY-CM23 was found to self-assemble into a chloride-dependent K+ selective channel mediating K+ transport across lipid bilayers and cell membranes. In addition, the synthetic K+ channel formed by ZHY-CM23 was found to be capable of generating and modulating the membrane potential of liposomes and to significantly hyperpolarize the resting membrane potential of HEK 293 cells. This finding provides new insight into developing drugs for the treatment of severe human diseases caused by K+ channel malfunction, such as arrhythmia, neurological disorders and autoimmune diseases.
Cystic fibrosis is a chronic recessive disease resulting from the loss of function mutations in the gene encoding of the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC family of membrane transporters. Recent findings reveal that restoring bicarbonate transport might be useful for the treatments of the underlying defect in cystic fibrosis. On the basis of fluorescence assays, NMR and short circuit current experiments, the small molecule ZHY-CM11 has been discovered to not only act as a bicarbonate transporter in lipid membranes, but also to induce chloride-dependent bicarbonate secretion in cultured calu-3 epithelia. It is a promising lead compound to be developed for the treatment of cystic fibrosis and other diseases related to chloride and bicarbonate transport defects.
Through structural modifications on the bioactive ion channels and the transporters ZHY-CM23 and ZHY-CM11, some valuable information on the structure-activity relationship has been obtained, and a series of potentially biologically applicable synthetic ion transporters have been discovered.published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy
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Liu, Pengyun, and 劉鵬云. "Design, synthesis, characterization and biological study of ion transporters." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/206419.
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Palo, Renato Miotto [UNESP]. "Passagens de ions peróxido para a superfície dentária externa após clareamento com peróxido de hidrogênio." Universidade Estadual Paulista (UNESP), 2009. http://hdl.handle.net/11449/101632.
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O objetivo deste trabalho foi avaliar a quantidade de peróxido que passa da câmara pulpar para a superfície dentária externa durante o clareamento interno no esmalte, cemento e dentina. Foram utilizados 50 incisivos bovinos extraídos que receberam aberturas coronárias, as raízes foram cortadas a 5 mm da junção amelo-cementária e foi realizado um tampão de 2mm de ionômero de vidro selando a entrada do canal. A extremidade apical dos espécimes foi isolada externamente com resina composta fotoativada. Os dentes foram então impermeabilizados completamente, deixando exposto somente as áreas a serem estudadas. As câmaras coronárias foram preenchidas com peróxido de hidrogênio a 35%. Os dentes foram divididos em três grupos experimentais e dois grupos controles, com 10 espécimes cada. GE – esmalte exposto a ser avaliado; GC – cemento exposto a ser avaliado; GD – dentina exposta a ser avaliada; e dois grupos Controle: GC1 – sem a presença de clareador internamente e sem impermeabilização e GC2 – câmara pulpar preenchida com clareador e impermeabilização total. Cada amostra foi colocado no interior de reservatórios individuais com 1000μl de solução tampão de acetato 2M (pH 4,5). Após 7 dias a 37±1ºC a solução foi transferida para um tubo de ensaio onde foram adicionados 100μl do corante violeta leucocristal e 50 μl de peroxidase, resultando em uma solução de coloração azul. A mensuração da absorbância foi feita em um espectrofotômetro e convertida em μg/ml de peróxido. Para avaliar se houve diferença entre os grupos experimentais e controle, realizou-se os testes de Kruskall-Walis e Dunn-Bunferroni e os resultados mostraram os íon passaram mais pela dentina exposta, seguida da dentina recoberta pelo esmalte e dentina recoberta pelo cemento, sem diferenças estatísticas. Todos os grupos experimentais foram diferentes dos controles...
The aim of this study was evaluated the pulp chamber penetration of peroxide bleaching agent to the root surface during the internal bleach technique in the enamel, dentin and cement. Bovine teeth were sectioned 5mm apical of the cemento-enamel junction and was performed a 2mm cervical seal with glass ionomer cement. The external apical part of samples was filled with composite resin. The teeth were divided into three experimental groups and two control groups: GE – exposition of enamel; GC – exposition of cement; GD – exposition of dentin and control groups: GC1 – no presence of internal agent bleaching e no waterproof and GC2 – pulp chamber filled with bleaching agent and total waterproof. Each sample was placed inside of individual flasks with 1000μl of acetate buffer solution 2M (pH 4.5). After 7 days, the buffer solution was transferred to a glass tube where leuco crystal violet and horseradish peroxidase were added, producing a blue solution. The optical density of the blue solution was determined by spectrophotometer and converted into microgram equivalents of hydrogen peroxide. Data were submitted to Kruskal-Wallis, and Dunn-Bunferroni tests. The results showed that the ions penetration was higher in dentin followed by enamel and cement. All experimental groups presented statistical differences to the control groups.
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Avkiran, Metin. "Pathophysiological roles, pharmacological inhibition and cellular regulation of the cardiac sarcolemmal sodium/hydrogen exchanger." Thesis, University of Bath, 2002. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.601638.
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Al-Salameen, Fadila A. "Strategies for cloning ion transporters in salt-resistant plants." Thesis, University of Glasgow, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266682.
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Velamakanni, Saroj. "Ion transport by ABC multidrug transporters LmrA and BCRP." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613691.
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Carswell, Casey. "The Structural Characterization of Two Prokaryotic Membrane Proteins: CfrA and ELIC." Thesis, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31214.
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This thesis focuses on the structural and functional characterization of two integral membrane proteins; CfrA, an outer membrane TonB-dependent transporter (TBDT) from Campylobacter jejuni, and ELIC, a pentameric ligand-gated ion channel (pLGIC) from Erwinia Chrysanthemi. The spectroscopic characterization of CfrA revealed a fold consistent with the structural and biophysical properties observed for other TBDT. Both a homology model of CfrA and sequence alignments of CfrA with other ferric-enterobactin transporters suggested a unique mode of ligand binding, thus raising the possibility that C. jejuni can be specifically inhibited. To investigate the molecular determinates of binding to CfrA, I set out to crystallize CfrA. Hundreds of crystal trials led to crystals diffracting to 3.6 Å resolution, with a complete data set acquired at 5 Å resolution that led to a structural model of the CfrA β-barrel.
In the second part of this thesis, I reconstituted ELIC into model membranes in order to test the role of intramembrane aromatic interactions in ELIC gating and lipid sensing. ELIC was reconstituted into both asolectin (aso-ELIC) and 1-palmitoyl-2-oleoyl phosphatidylcholine (PC-ELIC), membranes that stabilize the homologous nicotinic acetylcholine receptor (nAChR) in functional coupled versus non-functional uncoupled conformations, respectively. In both membrane environments, ELIC exhibits a mixed α-helical and β-sheet secondary structure, with a thermal denaturation intermediate between those of the nAChR and the close prokaryotic homolog, GLIC, in similar membranes. The data suggest that although ELIC has a decreased propensity to adopt an uncoupled conformation relative to the nAChR, its ability to undergo cysteamine-induced channel gating is sensitive to its lipid environment. The decreased propensity to uncouple may reflect an increased level of aromatics at the interface between the transmembrane α-helices, M1, M3, and M4. To test this hypothesis further, the level or aromatic residues at the M1, M3, and M4 interface in both GLIC and ELIC were varied, and in both cases the levels of intramembrane aromatic interactions correlated with the efficiency of coupling binding to gating. The data provide further evidence for a role of intramembrane aromatics in channel gating and in dictating the propensity of pentameric ligand-gated ion channels to adopt an uncoupled conformation.
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Wang, Shanshan. "Expression of ion transporters in rat brain vascular endothelial cells." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612129.
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Singh, Himansha. "On the mechanisms of transport and energy coupling in ABC exporters." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/276108.
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The rapid emergence of multidrug resistant bacterial strains represents a major global healthcare issue. Amongst five known classes of membrane transporters, which play a huge role in multidrug efflux, primary-active ATP-binding cassette (ABC) transporters are ATP powered whilst secondary-active transporters utilize electrochemical ion gradients to drive substrate transport. Mechanistic insights into transport by these proteins can help with the design and development of novel therapeutic agents against multidrug resistance, and can increase our understanding of the physiological functions of these transporters. Although available crystal structures illustrate a common alternate access model for transport by ABC transporters, the mechanisms by which metabolic energy is coupled to the transport cycle is still elusive. This thesis presents a series of functional studies using whole cells as well as artificial phospholipid membranes to study the energetics of transport, and the influence of membrane phospholipids on substrate transport by the homodimeric Escherichia coli lipid A/multidrug ABC exporter MsbA. Current alternating access models for ABC exporters involve cycling between conformations with inward- and outward-facing substrate-binding sites in membrane domains (MDs) in response to engagement and hydrolysis of ATP at the nucleotide-binding domains (NBDs). Here we report that MsbA also utilizes another major energy currency in the cell by coupling substrate transport to a transmembrane electrochemical proton gradient. In this thesis, analogous substrate transport reactions are also studied for two other ABC exporters, the MsbA homologue LmrA and the human multidrug transporter ABCG2. The dependence of ATP-dependent transport on proton coupling, and the stimulation of MsbA-ATPase by the chemical proton gradient highlight the functional integration of both forms of metabolic energy. It also raises questions about the role of NBDs in the transport process. Comparisons of drug transport and resistance in cells expressing MsbA-MD (truncated MsbA lacking the NBD) and full length MsbA (MsbA-WT) demonstrate increased transport efficiency of MsbA-WT compared to MsbA-MD. In addition, growth studies using E. coli WD2 cells, which are conditionally defective in MsbA’s essential activity in lipid A transport, show that lipid A transport can be restored by the expression of MsbA-WT but not MsbA-MD or ATP-hydrolysis impaired Walker A mutant (MsbA- ΔK382). Lastly, we also present biochemical experiments with proteoliposomes with a defined phospholipid composition, which suggest that cardiolipin is essential for the transport activity of MsbA. These techniques open the way to further explore lipid-proteins interactions and examine the physiological role(s) of MsbA. In conclusion, this thesis produces new insights in the mechanisms of transport and energy coupling in ABC exporters.
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Gilchrist-Vinatier, Jacqueline Giros Bruno. "Régulation des transporteurs vésiculaires du glutamate chez les rongeurs." Créteil : Université de Paris-Val-de-Marne, 2006. http://doxa.scd.univ-paris12.fr:80/theses/th0232414.pdf.
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Maass, Philipp, Robby Peibst, and Stephan Schott. "Ion diffusion in mixed alkali glasses." Universitätsbibliothek Leipzig, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-195485.
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Gilchrist-Vinatier, Jacqueline. "Régulation des transporteurs vésiculaires du glutamate chez les rongeurs." Paris 12, 2006. https://athena.u-pec.fr/primo-explore/search?query=any,exact,990002324140204611&vid=upec.
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Les transporteurs vésiculaires du glutamate, VGLUT1, -2 et -3, sont des protéines présentes à la membrane des vésicules synaptiques des neurones glutamatergiques. Ils sont responsables de l'accumulation du neuromédiateur dans les vésicules. VGLUT1 et VGLUT2 sont les sous-types majoritaires dans le cerveau et couvrent la totalité des neurones glutamatergiques avec des profils d'expression complémentaires. VGLUT3 est exprimé de façon plus discrète dans des populations de neurones contenant d'autres neuromédiateurs. Au cours de cette thèse, j'ai participé à la description détaillée de la localisation de VGLUT3 dans le cerveau adulte de rat, ainsi qu'à l'étude de l'ontogenèse des trois sous-types. J'ai également réalisé un crible double-hybride avec une partie cytoplasmique de VGLUT1 à la recherche de différences fonctionnelles entre les trois sous-types. J'ai ainsi caractérisé l'interaction entre VGLUT1 et l'endophiline, une protéine impliquée dans l'endocytose des vésicules synaptiques. Cette interaction suggère un lien fonctionnel entre l'accumulation de neuromédiateur et le recyclage des vésicules synaptiques dans la terminaison nerveuseThe vesicular glutamate transporters, VGLUT1, -2 and -3, are proteins present at the membrane of synaptic vesicles in glutamatergic neurons. They are responsible for the accumulation of neurotransmitter into the vesicles. VGLUT1 and VGLUT2 are the majoritary subtypes in the brain and their expression covers all known glutamatergic neurons with complementary expression patterns. VGLUT3 is expressed in a more discrete fashion in neuron populations which were not previously thought to be glutamatergic. During this thesis, I contributed to the detailled description of the localisation of VGLUT3 in the adult rat brain, as well as the ontogenic study of all three subtypes. I also carried out a yeast two-hybrid screen in order to search for putative functionnal differences between the subtypes. Thus, I characterised an interaction between VGLUT1 and endophilin, a protein involved in the endocytosis of synaptic vesicles. This interaction suggests a functional link between the loading in neurotransmitter and the recycling of synaptic vesicles in the nerve terminal
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Sundaresan, Vishnu Baba. "Biological Ion Transporters as Gating Devices for Chemomechanical and Chemoelectrical Energy Conversion." Diss., Virginia Tech, 2007. http://hdl.handle.net/10919/27891.
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This dissertation presents a new class of engineered devices, fabricated from synthetic materials and protein transporters extracted from cell membranes of plants, that use chemomechanical and chemoelectrical energy conversion processes to perform mechanical and electrical work.
The chemomechanical energy conversion concept is implemented in a protein based actuator. The chemical energy is applied as an electrochemical gradient of protons across a membrane assembly formed from phospholipids and SUT4 -a proton-sucrose cotransporter. The membrane assembly forms a physical barrier between two chambers in the actuator. The SUT4 proteins in the membrane assembly balances the applied electrochemical gradient by a concentration gradient of sucrose across the membrane. The sucrose gradient simultaneously generates an osmotic flow which deforms a flexible wall in a constrained chamber of the actuator, thus exhibiting mechanical strain. The sucrose concentration balanced by the protein transporter is used as the control variable for fluid flow through the membrane. The transport properties of the membrane assembly has been characterized for the control variable in the system. The reaction kinetics based model for solute transport through the cotransporter is modified to compute the equilibrium constant for solute binding and fluid translocation rate through the membrane. The maximum initial flux rate through the membrane is computed to be 2.51+/-0.6 ul/ug.cm^2.min for an applied pH4.0/pH7.0 concentration gradient across the membrane. The flux rate can be modulated by varying the sucrose concentration in the actuator. The prototype actuator has been fabricated using the characterized membrane assembly. A maximum deformation of 60microns at steady state is developed by the actuator for 20 mM sucrose concentration in the system.
The chemoelectrical energy conversion concept is based on the electrogenic proton pumps in plasma and vacuolar membranes of a plant cell. A prototype device referred to as a BioCell demonstrates the chemoelectric energy conversion using V-type ATPase extracted from plant cell membranes. The enzyme in the bilayer lipid membrane hydrolyzes ATP and converts the chemical energy from the reaction into a charge gradient across the membrane. Silver-silver chloride electrodes on both the sides of the membrane convert the charge established by the proton pumps into cell voltage. The redox reactions at the surface of the electrodes result in a current through the external load connected to the terminals of the BioCell. The single cell behaves like a constant current power source and has an internal resistance of 10-22kOhms. The specific power from the cell of the membrane assembly is estimated to be around 2microwatts/sq/cm. The demonstration of chemoelectrical energy conversion shows the possibility to use ATP as an alternative source of electrical power to design novel chemo-electro-mechanical devices.Ph. D.
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Silm, Kätlin. "Vesicular glutamate transporters as markers and players in synaptic vesicle trafficking in mouse neurons." Paris 6, 2013. http://www.theses.fr/2013PA066342.
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Vesicular Glutamate Transporters (VGLUTs) are responsible for filling synaptic vesicles (SV) with the excitatory neurotransmitter glutamate. This activity gives them a key role in assuring functional neurotransmission and makes them extremely faithful markers of excitatory SVs. The VGLUT1Venus knock-in mouse generated in the laboratory takes advantage of these properties and represents a major tool for studying the trafficking of SVs and the cell biology of VGLUT1 in physiological and altered conditions. During the first half of my Ph. D. I took advantage of VGLUT1Venus mice to reveal that a continuous scaling of VGLUT and SV levels at synapses of active networks occurs through the sharing of a super pool of SVs among boutons of an axon. Then, using VGLUT1 knock-out mice, we could identify a probable side function of VGLUT1 in the regulation of SV cluster size and SV mobility. I further investigated the ability of the other isoforms to fulfill the same function and finally started examining the molecular mechanisms behind this novel function of VGLUT(s). Altogether, my work allows to better understand how synaptic vesicles are shared in axons and how the filling of SV may be linked to their exocytosis/endocytosis cycle by means of the dual function of VGLUT1Les transporteurs vésiculaires du glutamate sont responsables du chargement des vésicules synaptiques (SV) en neurotransmetteur glutamate. Cette activité leur confère un role clé pour le déroulement de la neurotransmission excitatrice et ils sont des markeurs extrèmement fiable des SV excitatrices. Les souris knock-in VGLUT1Venus générées par notre laboratoire utilisent avantageusement ces propriétés et représentent un outil de choix pour l’étude du traffic vésiculaire et de la biologie du transporteur VGLUT1 en conditions physiologiques ou altérées. Durant la première moitié de ce travail de thèse, j’ai utilisé les souris VGLUT1Venus pour montrer l’existence d’un régulation dynamique permanente des niveaux de VGLUT1 et des SV aux synapse de réseaux de neurones actifs. Ce phénomène met en jeu les échanges de SV entre les synapses par le super contingent de SV (superpool). Ensuite, en utilisant les souris invalidées pour le gène VGLUT1, j’ai pu montrer une probable fonction secondaire de VGLUT1 dans la régulation de la taille des paquets de SV et de la mobilité des SV dans l’axone. J’ai également tenté de comprendre le rôle des autres transporteurs VGLUT dans la mobilité vésiculaire et de disséquer les mécanismes moléculaire expliquant cette fonction nouvelle. Dans l’ensemble, mon travail permet de mieux comprendre comment les SV sont partagées par les boutons d’un même axone et comment le remplissage des vésicules pourrait être directement lié à leur cycle d’endocytose et exocytose par le biais de cette double fonction de VGLUT1
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Houdinet, Gabriella. "Impact de la symbiose racinaire dans l’adaptation des plantes à la carence potassique : caractérisation et rôle de systèmes de transport membranaires chez le champignon ectomycorhizien Hebeloma cylindrosporum." Thesis, Montpellier, 2019. http://www.theses.fr/2019MONTG067.
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L’un des rôles majeurs concernant les interactions bénéfiques entre les plantes et les champignons est l’amélioration de la nutrition des plantes en échangeant des nutriments, grâce à une meilleure exploration du sol et une meilleure absorption de l’eau et des ions. Par conséquent, la symbiose ectomycorhizienne, établie entre des espèces de plantes ligneuses et des champignons du sol, est cruciale pour que la plante puisse absorber efficacement les minéraux peu disponibles dans les écosystèmes forestiers. Des études physiologiques, des projets de séquençage de génomes récents et des analyses transcriptomiques ont permis de progresser dans l’identification et la caractérisation de transportomes de champignons symbiotiques. Le potassium (K+) est le cation le plus abondant dans les cellules de plantes et il participe à divers processus cellulaires et physiologiques. L’amélioration de la nutrition potassique par la symbiose ectomycorhizienne a été démontrée dans des conditions de carence en K+ en utilisant le couple-modèle Pinus pinaster et Hebeloma cylindrosporum. Des questions sont soulevées pour identifier les systèmes de transport fongiques impliqués dans l’absorption des nutriments du sol et dans leur transfert vers la plante au niveau de l’interface symbiotique entre le champignon et la plante, appelé le réseau de Hartig. Deux types de transporteurs potassiques, Trk et HAK, ont été identifiés comme des candidats pour effectuer l’absorption de K+ à partir du sol à l’aide des hyphes extraracinaires, et deux types de canaux potassiques, Shaker et TOK, qui eux pourraient jouer un rôle dans le relargage du K+ au niveau des hyphes du réseau de Hartig vers l’apoplasme des cellules racinaires. Dans ma thèse, je me suis focalisée sur les trois canaux TOK (« Two-pore Outward K+ ») d’H. cylindrosporum, une famille de canaux spécifiques aux champignons. Ces trois canaux TOK appartiennent à deux sous-familles, ce qui pourrait indiquer des rôles différents dans la nutrition potassique et la symbiose. L’un des trois canaux semble particulièrement intéressant en raison de son induction lors de la mycorhization. Le but de ma thèse a donc été de compléter les précédents résultats sur leur caractérisation, expression, localisation et de participer à la création de nouveaux outils pour analyser leur(s) rôle(s) physiologique(s). En perspective, mes résultats contribueront à une meilleure compréhension des rôles spécifiques des membres de cette famille TOK originale au sein du champignon et au sein de la symbioseA major role of mutualistic interactions between plants and fungi is the improvement of plant nutrition by an exchange of nutrients, due to a better exploration of the soil and a better absorption of water and ions. Therefore, ectomycorrhizal symbiosis, established between woody plants and soil fungi, is crucial for the plant to efficiently take up poorly available nutrients in forest ecosystems. Physiological studies, recent genome sequencing projects and transcriptome analyses have allowed progress towards the identification and characterization of the fungal symbiotic transportome. Potassium (K+) is the most abundant cation in plant cells and is involved in various cellular and physiological processes. Improvement of K+ nutrition by ectomycorrhizal symbiosis has been shown under K+ shortage conditions using the model couple Pinus pinaster and Hebeloma cylindrosporum. Questions are raised to identify the fungal transport systems involved in the uptake of nutrients from the soil and in their transfer towards the plant at the symbiotic fungus-plant interface, called Hartig net. Two types of K+ transporters, Trk and HAK, have been identified as candidates to perform K+ uptake from the soil by extraradical hyphae, and two types of K+ channels, Shaker-like and TOK, that may release K+ by the hyphae of the Hartig net into the plant apoplasm. In my PhD, I focused my research on the three TOK (Two-pore Outward K+) channels of H. cylindrosporum, a channel family specific for fungi. These three TOK channels belong to two different subfamilies, which may imply different roles in potassium nutrition and in symbiosis. One member seems to be especially interesting because of its induction by mycorhization. My PhD aimed to complete the previous results on their functional characterization, expression, localization, and to contribute to the establishment of tools to analyze their physiological roles. In perspective, my results will contribute to a better understanding of the specific roles of these original TOK channel members within the fungus and within the symbiosis
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Spichler, Anne Stambovsky. "Leptospirose letal aguda em Hamster: caracterização de perfis bioquímicos, histopatológicos e celulares renais, relacionada a ensaios terapêuticos." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/5/5148/tde-12022008-133227/.
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A leptospirose é uma zoonose de importância mundial, causada por leptospiras patogênicas. Aproximadamente 5 a 10% das infecções humanas cursam com a forma grave. A Doença de Weil é a forma mais comum de doença grave e pode apresentarse com duas formas de evolução, aguda progressiva monofásica ou de curso prolongado. A doença grave se caracteriza por uma combinação de hemorragia, mais comumente pulmonar, icterícia e insuficiência renal, com letalidade de 5 a 15%. O rim é um órgão muito acometido na Leptospirose. Clinicamente o envolvimento renal ocorre de 16 a 40%, com manifestações peculiares como poliúria, hipocalemia, e perda de sódio. A disfunção tubular renal, é característica da leptospirose forma grave, com envolvimento dos transportadores renais de sódio ao longo do néfron, levando às manifestações observadas. A terapêutica antimicrobiana é recomendada na Leptospirose, porém com controvérsias à sua indicação após o quarto dia de doença. Quando da instalação da lesão não haveria benefícios com a utilização de antibióticos. O tratamento pode diminuir a morbidade e letalidade, assim como interferir no envolvimento renal e na expressão dos transportadores renais de sódio. A patogênese pode estar relacionada a efeitos diretos da leptospira ou a resposta inflamatória assim como o estresse oxidativo. A utilização de antioxidantes, pode ser considerada como terapia adjuvante. Nós avaliamos a expressão no túbulo proximal do trocador Na+-H+ (NHE3) e na porção espessa da medula ascendente o cotransportador Na+-K+-2Cl- (NKCC2), no modelo de hamster com as duas formas de evolução de doença grave, mimetizando a doença de humanos realizados em dois experimentos. Os experimentos envolveram animais infectados não tratados e tratados com ampicilina associado ou não ao antioxidante, N-acetilcisteína. A presença de antígenos de Leptospira e a expressão dos transportadores foram avaliadas por imunohistoquímica, e o ácido tiobarbitúrico, marcador de estresse oxidativo, (TBARS) foi quantificado.Hamsters infectados, apresentaram altas quantidades de antígenos nos tecidos-alvo, enquanto que a expressão de ambos os transportadores apresentou-se diminuída. O tratamento com ampicilina esteve associado com mínima detecção ou ausência de antígenos, restabelecimento da expressão dos transportadores nos respectivos locais e redução dos níveis de TBARS. O tratamento precoce e tardio com ampicilina restabeleceu os defeitos tubulares na leptospirose forma grave em ambos experimentos, sem benefícios com a utilização da N-acetilcisteína.Leptospirosis is a zoonosis of worldwide distribution. About 5-10% of all human infections presents with severe forms. Weil\'s syndrome, the most common presentation of severe forms of leptospirosis, may courses either as a single monophasic disease or as a disease with prolonged course, characterized by a combination of hemorrhage, particularly in the lung, renal failure, and jaundice, with fatality rates ranging from 5 to 15%. The kidney is an important target organ in leptospiral infection. Clinically, renal involvement in leptospirosis occurs in 16% to 40% of cases and is unique because of the atypical presentation of polyuria, hypokalemia, and sodium wasting, suggestive of a special form of tubular dysfunction related to the major renal sodium transporters expressed along the nephron. A wide range of antimicrobial therapy for leptospirosis was described and benefits have been disputed for cases with more than four days of clinical disease, because after a threshold of leptospiremia, the delayed use of antibiotics is unlikely to reduce fatality. Antimicrobial therapy is thought to interfere on fatality, renal involvement, and renal sodium transporters expression during severe disease. The pathogenesis may be related to direct effects of leptospiral compounds or inflammatory response due to oxidative stress. Antioxidant could be considered for adjunctive therapy.We evaluated the expression of proximal tubule type 3 Na+/H+ exchanger (NHE3) and thick ascending limb Na+-K+-2Cl- cotransporter (NKCC2) in infected non treated and treated hamsters reproducing the two forms of clinical human presentations of Weil\'s syndrome divided in two experiments. Animals were treated or not with ampicillin and/or N-acetyl-cysteine (NAC). Leptospiral antigen/s and expression of renal transporters were evaluated by immunohistochemistry, and serum thiobarbituric acid (TBARS) was quantified. Infected hamsters had high amounts of detectable leptospiral antigen/s in target tissues while renal expression of NHE3 and NKCC2 decreased. Ampicillin treatment was associated with minimal or no detection of leptospiral antigens, normal expression of NHE3 and NKCC2 transporters, and reduced levels of TBARS. Early and late ampicillin treatment rescued tubular defects in leptospirosis severe disease in both experiments, and there was no evidence of benefit from antioxidant therapy.
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Cattani, Adriano. "Genesis of recurrent epileptic paroxysmal burst in newborn rats after inhibition of glutamate transporters." Aix-Marseille 2, 2009. http://www.theses.fr/2009AIX22045.
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Les Cell-surface transporteurs u glutamate contrôle de manière efficace les activités du réseau dans la maturation du cerveau des rongeurs : leur inhibition par le DL-TBOA conduit in vitro, en tranches coritcal (néocortex de l'hippocampe) à la génèse de depolarizations recurrent et salves de potentiels d'action dans les cellules pyramidales et les interneurones en alternance avec période de faible activité, une tendance que nous avons fait référence en tant que slow entwok oscilliations (SNOs). In vivo, l'inhibation génère des crises partielles et récurrentes paroxystiques rafales alteranant avec des périodes de silence, un modèle qui correspond à "suppression burst" (SB). Cette activité orticale est particulièrement intéressant car il est observé chez les nouveaux-nés souffrant de certaines formes de début de l'encéphalopathie épileptique ce qui soulève la possibilité que les transporteurs du glutamate pourrait être impliquée dans cette maladie. Les deux SNOs et SB impliqué l'activation des récepteurs NMDA, car ils ont tous deux été supprimés par les antagonistes des récepteurs NMDA suggérant qu'ils partagent les mêmes mécanismesCell-surface glutamate transpoters control efficiently entwork activities in maturing rodent brain : their inhibition by DL-TBOA leads in vitro, in cortical slices (neocortex and hippocampus) to the genesis of recurrent depolarizations and burst of action potentials in interneurons and pyramidal cells alternating with period of low activity, a pattern that we referred as slow network oscilliations (SNOs). In vivo, their inhibition generates partial seizures and also recurrent paroxylasmal bursts alternating with silent periods, a pattern that corresponds to "suppression burst" (SB). This cortical activity is particulary interestind since it is observed in neonates suffering forms of early epilecptic encephalopathy thus raising the possibility that glutamate transporters could be implated in this disease. Both SNOs and SB involved the activation of NMDA receptors since they were both abolished by NMDA receptor antagonists suggesting that they share similar mechanisms
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Cameron, Lynn Michele. "The design and synthesis of macrocycles for use as components of ion transporters." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0014/NQ32736.pdf.
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Maass, Philipp, Robby Peibst, and Stephan Schott. "Ion diffusion in mixed alkali glasses." Diffusion fundamentals 2 (2005) 30, S. 1-2, 2005. https://ul.qucosa.de/id/qucosa%3A14360.
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Heitjans, Paul. "Diffusion in lithium ion conductors – from fundamentals to applications." Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-181798.
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Martin, Manfred. "Oxygen and cation diffusion processes in oxygen ion conductors." Universitätsbibliothek Leipzig, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-193656.
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We discuss oxygen and cation diffusion processes in oxygen ion conductors. While the high oxygen diffusivity determines the proper oxygen ion conductivity, slow cation diffusion processes are important for sintering and degradation processes. In the first part of the paper we discuss an analytical model for the ionic conductivity of a strongly acceptor doped, fluorite-type oxygen ion conductor, i.e. a concentrated solution of AO2 and BB2O3. The model can be applied, e.g., to yttria doped zirconia (YSZ) and gives a qualitative
explanation of the observed maximum of the conductivity as a function of the dopant fraction. The model considers nearest neighbor interactions between oxygen vacancies and dopant cations, which may be negligible, attractive or repulsive, and jump barriers that depend on the nature of the cation-cation edge that has to be crossed during a jump between adjacent oxygen sites. In the second part we discuss cation diffusion processes in doped lanthanum gallates (LSGM). The experimental results of nearly identical cation
diffusion coefficients in the A- and B-sublattices of the perovskite LSGM can be explained by a bound defect cluster mechanism containing cation vacancies of both the Aand the B- sublattice and anion vacancies.
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Chen, Ichia. "Structural basis for the dual transport-channel functions of SLC1A transporters." Thesis, The University of Sydney, 2021. https://hdl.handle.net/2123/26804.
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The excitatory amino acid transporters (EAATs) play a vital role in the maintenance of glutamatergic neurotransmission within the synapse, involved in fundamental brain functions such as learning and memory. These secondary active transporters enable rapid glutamate reuptake into the surrounding glial cells and neurons by coupling to pre-existing electrochemical gradients of sodium ions, potassium ions and protons. In addition to this conventional transporter function, the EAATs also possess a unique ability to conduct chloride ions in a channel-like process that is thermodynamically uncoupled from substrate transport. Aberrant glutamatergic neurotransmission caused by dysfunction of the EAATs has been associated with excitotoxity-mediated cell death and the pathogenesis of multiple debilitating neurological disorders. In particular, an increased chloride conductance via an EAAT1 mutant has been directly linked to a neurological disease, episodic ataxia type 6. This thesis describes a body of work that investigates the interplay between the elevator mechanism of substrate transport and chloride permeation in the archaeal homolog and sodium-dependent aspartate transporter, GltPh, using X-ray crystallography and single particle cryo-EM. It presents crystal structures of GltPh mutants locked in the outward-facing and inward-facing states, and a novel cryo-EM structure of a GltPh protomer in the chloride conducting state, which fills in the final missing puzzle required to map the complete substrate translocation pathway. This chloride conducting state was further confirmed by a combination of computational and functional analysis, which unveiled two clusters of hydrophobic residues at either side of the membrane that gate the channel. Furthermore, this thesis also explore pathogenic mutations (P206R and L90R in GltPh) associated with episodic ataxia type 6 structurally in an attempt to explain their functional consequences. Together, this thesis enhance our mechanistic understanding of the dual functions conserved in the SLC1A transporter family, opening doors for alternative approaches to treat neurological disorders associated with transporter dysfunctions.
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Ouahid, Soumia. "Transport facilité du glucose à travers une membrane échangeuse d'anions avec l'ion borate comme transporteur." Rouen, 1994. http://www.theses.fr/1994ROUES029.
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Par couplage diffusion-réaction, le transport d'un substrat non ionique à travers une membrane échangeuse d'ions, peut être facilité en choisissant un contre-ion apte à réagir réversiblement avec le substrat. Dans ce travail, nous avons réalisé le transport facilité à travers une membrane échangeuse d'anions. L'ion borate était utilisé comme transporteur et sa teneur dans la membrane était fixée par l'activité de l'acide borique. Nous avons mené simultanément la modélisation et l'expérimentation du transport. Par des études d'équilibre et de conductivité, nous avons déterminé les paramètres physicochimiques, tels que le coefficient de stabilité du complexe glucose-borate et le coefficient de diffusion des différentes espèces présentes. Nous avons, de plus, mis en évidence en présence de glucose, deux aspects originaux: la variation de l'accessibilité des sites membranaires et la variation de leurs interactions avec certains contre-ions. Dans la majorité des cas, nous avons choisi une activité d'acide borique relativement faible afin que les polyborates soient minoritaires devant les autres contre-ions. Dans une première série de mesures, nous avons réalisé le transport facilité du glucose en fonction de son activité, en maintenant constante celle de l'acide borique. Dans une seconde série, le transport a été réalisé en fonction de l'activité du transporteur. A partir des paramètres préalablement déterminés, nous avons élaboré un modèle théorique qui permet de comparer les valeurs théoriques et expérimentales des flux du glucose. Nous parvenons à un bon accord lorsque le transport a été réalisé en imposant pour l'acide borique une activité pour laquelle les paramètres du modèle ont été caractérisés
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Yau, Kwok-hei, and 邱國禧. "Small molecule-based synthetic ion channels modulate smooth muscle contraction and epithelial ion transport." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hdl.handle.net/10722/196079.
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In living systems, ion channels are membrane transport proteins that provide pathways for the passive diffusion of ions through lipid membranes. The flow of ions across membranes is the basis of many important physiological processes, including but not limited to the regulation of membrane potential, transepithelial transport and cell volume. While many efforts have been made to understand the biological roles of natural ion channels, the biological activities of artificial ion channels remain largely unknown. Recently, it was reported that a small molecule 1, which forms synthetic chloride (Cl–) channels in membranes via self-assembly, is capable of modulating vascular functions. In this thesis, novel small molecules that are structurally similar to 1 are shown to form artificial ion channels in membranes. Together with 1, the effects of these small molecules on the contractile activities of smooth muscles and epithelial ion transport are explored. The therapeutic implications of the findings are also discussed.
A collection of small molecules was screened using liposome-based fluorescence assays. In these assays, the ability of the synthetic compounds to modulate membrane potential was monitored. The screening yielded compound 3 that formed synthetic potassium (K+) channels in liposomal membranes, although the liposome-based fluorescence experiments suggested that 3 also transported Cl–. Two derivatives of 3, namely, compounds 2 and 4 were also examined. Single-channel recording experiments suggested that 2 forms synthetic Cl– channels in liposomal membranes.
The effects of compounds 2 and 3 on the functions of the vascular smooth muscle are explored. Using confocal imaging, it was shown that both 2 and 3 counteracted the effects of high-K+ depolarizing solution on membrane potential and intracellular Ca2+ concentration ([Ca2+]i) in cultured vascular smooth muscle cells. 2 and 3 also relaxed mice aortic rings pre-contracted with high-K+ solution. These observations can be explained in terms of the Cl– transporting functions of 2 and 3.
To determine the potential for developing the compounds into bronchodilators, the effects of compounds 1 and 3 on the contractile activities of the airway smooth muscle (ASM) were explored using organ bath technique. The contractile activities of the trachea isolated from Sprague-Dawley (SD) rats were first characterized. Among the contractile agents used, only potassium chloride (KCl), cholinergic agonists, serotonin and endothelin-1 were contractile to the SD rat trachea. 1 and 3 relaxed the ASM pre-contracted with KCl, whereas the contractions induced by other agonists were not affected.
The ability of compounds 2, 3 and 4 to modulate ion transport across cultured epithelia was tested by the short-circuit current measurement technique. It was shown that the compounds were capable of inducing Cl– secretion when applied to the apical side of airway and colonic epithelia. Importantly, the synthetic compounds induced apical Cl– secretion in immortalized cystic fibrosis (CF) bronchial epithelia. This suggests that the synthetic compounds may be used to correct the anion transport defect in CF epithelia.
In summary, the small-molecule based synthetic ion channels demonstrated two important general functions of natural ion channels, namely, the regulation of membrane potential and epithelial ion transport.published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy
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Walters, Rhodri J. "Ion channel regulation in small intestinal crypts." Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318409.
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De, Souza Roger A., and Manfred Martin. "Secondary ion mass spectrometry and its application to diffusion in oxides." Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-186567.
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Heitjans, Paul. "Diffusion in lithium ion conductors – from fundamentals to applications." Diffusion fundamentals 20 (2013) 19, S. 1-2, 2013. https://ul.qucosa.de/id/qucosa%3A13583.
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Martin, Manfred. "Oxygen and cation diffusion processes in oxygen ion conductors." Diffusion fundamentals 6 (2007) 39, S. 1-16, 2007. https://ul.qucosa.de/id/qucosa%3A14216.
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We discuss oxygen and cation diffusion processes in oxygen ion conductors. While the high oxygen diffusivity determines the proper oxygen ion conductivity, slow cation diffusion processes are important for sintering and degradation processes. In the first part of the paper we discuss an analytical model for the ionic conductivity of a strongly acceptor doped, fluorite-type oxygen ion conductor, i.e. a concentrated solution of AO2 and BB2O3. The model can be applied, e.g., to yttria doped zirconia (YSZ) and gives a qualitative
explanation of the observed maximum of the conductivity as a function of the dopant fraction. The model considers nearest neighbor interactions between oxygen vacancies and dopant cations, which may be negligible, attractive or repulsive, and jump barriers that depend on the nature of the cation-cation edge that has to be crossed during a jump between adjacent oxygen sites. In the second part we discuss cation diffusion processes in doped lanthanum gallates (LSGM). The experimental results of nearly identical cation
diffusion coefficients in the A- and B-sublattices of the perovskite LSGM can be explained by a bound defect cluster mechanism containing cation vacancies of both the Aand the B- sublattice and anion vacancies.
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Zhang, Hao. "Chemoelectromechanical Actuation in Conducting Polymer Hybrid with Bilayer Lipid Membrane." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/3074.
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Biological and bio-inspired systems using ion transport across a membrane for energy conversion has inspired recent developments in smart materials. The active mechanism in bioderived materials is ion transport across an impermeable membrane that converts electrochemical gradients into electrical and mechanical work. In addition to bioderived materials, ion transport phenomenon in electroactive polymers such as ionomeric and conducting polymers produces electromechanical coupling in these materials. Inspired by the similarity in transduction mechanism, this thesis focuses on integrating the ion transport processes in a bioderived material and a conducting polymer for developing novel actuation systems. The integrated membrane has a bilayer lipid membrane (BLM) formed on a conducting polymer, and the proteins reconstituted in the BLM regulate ion transport into the conducting polymer. The properties of the polymer layer in the integrated device are regulated through a control signal applied to the bioderived layer and hence the hybrid membrane resembles an ionic transistor. Due to the bioderived nature of this device, it is referred to as a ‘bioderived ionic transistor’. The research carried out in this thesis will demonstrate the fabrication, characterization and design limitations for fabricating a chemoelectromechanical actuator using the BIT membrane. The BIT membrane has been fabricated using BLM (DPhPC) reconstituted with protein (alamethicin) to gate Na$^+$ transport into conducting polymer membrane (PPy(DBS)). In this membrane, the bioderived layer is fabricated with proteins by vesicle fusion method and conducting polymer is fabricated by electropolymerization. The bioderived layers, the conducting polymer layers and the hybrid membrane are characterized using electrochemical measurements such as cyclic voltammetry, chronoamperometry, and electrochemical impedance spectroscopy. The fabrication, characterization and design effort presented in this thesis focuses on the integration of ion transport through the bioderived membrane into volumetric expansion and bending actuation. The characterization efforts are supported by empirical and physics-based models to represent the input-output relationship for both PPy(DBS) actuator and bioderived membrane, and design rules for the proposed actuation platforms are specified. The electropolymerized PPy(DBS) actuator is anticipated to be used in a bicameral device with the chambers kept separated by the DPhPC-alamethicin bioderived membrane. The relationship between the gradient potential, ionic current through the gate, ion concentration, ion transport coefficient in the conducting polymer layer, and the induced tip displacement in the polymer has been concluded from experiments and fitted to the actuation system model. This thesis will also address future directions for this research and anticipated applications for this hybrid actuation concept, such as artificial muscle, drug delivery.
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Giovanola, M. "MOLECULAR INSIGHTS IN ION ACCESS, DEPENDENCE AND SELECTIVITY IN THE NSS/SLC6 TRANSPORTERS KAAT1 AND GAT1." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/232725.
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In this thesis we have investigated some aspects of the molecular physiology of the amino acid transporter KAAT1 and of the GABA transporter GAT1 both belonging to the Neurotransmitter:Sodium Symporter (NSS) family. Wild type as well mutants of these proteins were investigated after expression in Xenopus laevis oocytes and functionally analyzed by radiochemical and electrophysiological assays.
In the first chapter a general overview of the biological importance of membrane proteins is provided jointly to a description of the Xenopus oocytes as expression system for this kind of proteins. The second part of the chapter is presenting the general physiological features of KAAT1 and GAT1 while the last part collects information regarding the thermodynamic aspects of secondary active transport as well as the characteristics of the bacterial amino acid transporter LeuT and of the insect dopamine transporter DAT, the two structural models of the NSS family. The last part of the chapter presents one of the most intriguing and cutting edge aspects in the field of membrane transporters: the role of symmetry in the process of ferrying substrates through the lipid bilayer.
In chapter 2 are reported the results of our investigation of a highly conserved glycine triplet in KAAT1; this sequence is conserved at the Extracellular Loop 1 (EL1) in almost all the members of the NSS family but the data available at the beginning of our research for GAT1 and for the serotonin transporter SERT were not exhaustive and not in full agreement one to each other. We found that in KAAT1 the flexibility that these amino acids provide to the EL1 is of fundamental importance for the cation access to the extracellular vestibule of the protein. The role that we propose could justify the high degree of conservation showed by this stretch of residues in order to allow the driver ion to get access to its binding site.
Different aspects of KAAT1 molecular physiology are addressed in the chapter 3. With our experiments we were able to link the potassium selectivity that characterizes KAAT1 to the polarity of Na1 site and to the dimensional flexibility that is provided in Na2 site by a KAAT1 specific residue of glycine. Beside cation selectivity, we explored the weak chloride dependence of KAAT1 providing new evidences of the fact that its interaction with chloride occurs in an almost unique fashion. The analysis of the 3D homology model of KAAT1 allowed us to identify Thr67 as a residue that we proved experimentally to be a key molecular hinge for the coupling mechanism of ion and substrate flux realized by KAAT1. Furthermore this residue is involved in the initial stereochemical selection that the transporter operates on its substrates as well in the chloride dependence of the transport mechanism.
Chapter 4 will briefly resume some preliminary data concerning the possibility to combine the insect Sf9 cell line from Spodoptera frugiperda with the highly efficient Baculovirus expression system with the aim of obtaining the adequate amount of purified protein for KAAT1 crystallization.
The last chapter gathers the results of our analysis regarding the influence of internal chloride in the reverse operational mode of the GABA transporter GAT1. Our results provides a link between the oscillations of the intracellular concentration of this anion with the calcium independent GABA release that is described in different pathological and physiological conditions.
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Stavrinidou, Eleni. "Understanding and engineering ion transport in conducting polymers." Phd thesis, Ecole Nationale Supérieure des Mines de Saint-Etienne, 2013. http://tel.archives-ouvertes.fr/tel-00968227.
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Many organic electronic and bioelectronics devices rely on mixed (electronic and ionic) transport within a single organic layer. Although electronic transport in these materials is relatively well understood, a fundamental understanding of ion transport is missing. I developed a simple analytical model that describes ion transport in a planar junction between an electrolyte and a conducting polymer film. The model leads to predictions of the temporal evolution of drift length of ions and current. These predictions are validated by numerical simulations and by using realistic parameters, I show that the analytical model can be used to obtain the ion mobility in the film. Furthermore, I developed an experimental method which allows the application of the analytical model and leads to a straightforward estimation of the ion drift mobilities in conducting polymers. PEDOT:PSS was found to support efficient transport of common ions, consistent with extensive swelling of the film in water. Crosslinking the film decreased its swelling and the ion mobility. Understanding the high correlation of hydration and ionic conductivity enables us to engineer materials with high and defined ion mobilities. As an example tuning of ion mobility by adjusting the relative ratio of the hydroscopic phase to PEDOT:TOS is presented. Finally I performed electrochemical impedance spectroscopy during a moving front experiment, in order to give a physical interpretation of the impedance spectra at a conducting polymer/electrolyte junction.
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Wohlmuth, Dominik, Viktor Epp, Ute Bauer, Anna-Maria Welsch, Harald Behrens, and Martin Wilkening. "7 Li ion diffusion in isotope-diluted glassy Li 2 Si 3 O7." Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-184004.
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Engevik, Melinda A. "Ion Transport and the Gut Microbiota." University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1397466973.
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Harley, Rachel. "Ion transport physiology and its interaction with trace element accumulation and toxicity in inanga (Galaxias maculatus)." Thesis, University of Canterbury. School of Biological Sciences, 2015. http://hdl.handle.net/10092/10738.
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Inanga (Galaxias maculatus) are a culturally and economically important fish species in New Zealand and abroad. However, very little is known about their ability to deal with trace element contamination. As a scaleless fish with the ability to survive in relatively extreme environments, they may not fit toxicity models (such as the biotic ligand model; BLM) based on other fish species. The aim of this study was to determine how this fish responds to elevated trace elements in both the laboratory and field in order to determine the applicability of these toxicity models.
In order to determine the impacts of stress on ion transport and subsequent metal toxicity, inanga were exposed to handling stress and measures of ion uptake were collected. Handling stress was shown to result in increased ventilation rates, resulting in stimulated sodium (Na+) efflux. A compensatory increase in Na+ influx was also measured as a result of this stress. Inanga largely recovered from this ionoregulatory stress within 2 hours, with full recovery after 24 hours. This was indicative of a rapid homeostatic response for maintaining ion balance. Enhanced Na+ uptake in response to this stress resulted in increased copper (Cu) uptake in Cu-contaminated water, suggesting stressed fish will accumulate more Cu (and likely other Na+ mimics) than an unstressed fish. These results suggest a heightened vulnerability of inanga to this type of contaminant as a result of exercise stress during migrations.
A combination of field and laboratory studies was used in order to measure trace element accumulation in inanga. In situ field studies showed changes to aluminum (Al) and iron (Fe) body burdens when inanga were placed in streams of varying trace element concentrations along the West Coast of the South Island. However, other trace elements measured did not alter over the period of exposure (9-10 days). Biochemical biomarker analysis showed no changes in the activity of Na+/K+-ATPase (NKA), but a marker of lipid peroxidation (thiobarbituric acid reactive substances; TBARS) was elevated in one stream. Analysis suggested that stream pH was the major driver of this effect, whether directly or via changes to metal bioavailability. Subsequent laboratory exposures (96 h) of inanga to 1.2, 2.7, 10.8, and 44 µg L-1 dissolved Fe and 5.6, 23.3, 60.7, and 128.7 µg L-1 dissolved zinc (Zn) showed no difference in whole body trace element accumulation, ammonia excretion, ion influx (Ca2+ and Na+), and TBARS. There were significant differences in oxygen consumption (MO2) after Fe exposures, with increases in the 2.7 and 44 µg L-1 dissolved Fe exposures. Laboratory exposure results suggest inanga are relatively insensitive to short-term Fe and Zn exposures.
Both in vivo (whole body partitioning) and in vitro (Ussing chamber) techniques were used to determine the influence of cutaneous ion transport on preventing trace element accumulation. Results suggest inanga use their skin as an additional site of calcium (Ca2+) and Na+ uptake. This is the first study to confirm these ion transport capabilities in inanga, and revealed that up to 48% of Na+ uptake may occur across the skin. Pharmacological inhibition of Ca2+ uptake was achieved by known Ca2+ channel blockers (verapamil and lanthanum). Furthermore Fe and Zn impaired cutaneous Ca2+ transport, indicating that ion transport pathways in the skin modulate in response to these metals.
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Williamson, Ian M. "Investigating plant calcium pumps : an antipeptide antibody approach." Thesis, Oxford Brookes University, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363748.
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Mangano, Enzo, Stefano Brandani, Magdalena M. Lozinska, and Paul A. Wright. "Diffusion of CO 2 in ion-exchanged zeolites Rho studied by the ZLC technique." Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-183844.
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Larsen, Jessica Joline. "Ion Transport in a Commercial ICP-MS." BYU ScholarsArchive, 2017. https://scholarsarchive.byu.edu/etd/6905.
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The performance of an inductively coupled plasma mass spectrometer, ICP-MS, depends on the instrument's ability to transport sample ions through the vacuum interface and focus the ions into a well-defined beam that will eventually reach the mass analyzer. In this study two main experiments were performed on the Perkin Elmer NexION 300S, a commercial ICP-MS. First, planar laser-induced fluorescence images were taken of the ion beam in a working instrument downstream from a unique quadrupole ion deflector. The images showed the ability of the instrument design to focus the ions in the ion beam. Second, laser-induced fluorescence was used to characterize ion flow through the vacuum interface. The interface is unique to the NexION ICP-MS in that there are three extraction cones. The effect of a three-cone interface on ideal skimming is discussed.