Les canaux potassiques à deux domaines P (K2P) contrôlent l’excitabilité cellulaire et jouent un rôle central dans l’établissement et le maintien du potentiel de repos membranaire dans la majorité des cellules animales. Depuis leur identification dans les années 90, ces canaux ont été impliqués dans de nombreuses fonctions comme la modulation de l’activité neuronale, l’activité du muscle cardiaque ou encore la physiologie rénale. Malgré l’importance de ces canaux, peu de données existent sur les processus cellulaires qui contrôlent leur fonction in vivo. Au cours de ma thèse, j’ai utilisé des approches génétiques, d’imagerie et d’électrophysiologie pour comprendre comment l’expression, la distribution et l’activité du canal K2P UNC-58 sont contrôlés chez le nématode modèle C. elegans.Pour cela, j’ai effectué un crible suppresseur du phénotype locomoteur du mutant gain de fonction unc-58(e665). J’ai ainsi obtenu 133 mutants présentant une large gamme de niveaux de suppression, suggérant l’implication de plusieurs gènes dans les processus de régulation du canal. En utilisant les technologies de reséquençage complet de génome, j’ai pu cloner six nouveaux gènes requis pour la fonction d’unc 58.J’ai ensuite caractérisé en détail le rôle d’unc-44/ankyrine dans le contrôle du trafic d’unc 58. Ce travail a conduit à 4 conclusions majeures : (1) UNC-58, malgré sa structure de canal potassique, possède en réalité une sélectivité ionique altérée favorisant le passage des ions sodium, (2) l’addition à UNC 58 de protéine fluorescente par approche CRISPR/Cas9 nous a permis pour la première fois d’observer directement la distribution du canal UNC-58 in vivo, (3) l’ankyrine est nécessaire à l’adressage du canal UNC-58 à la surface des muscles et dans les axones des neurones mécanosenseurs ALM. Cette fonction fait intervenir une poche d’interaction lipidique localisée au sein du module Zu5N-Zu5C-UPA d’UNC-44, (4) ce mécanisme est hautement sélectif puisqu’il n’est pas requis pour l’adressage de 6 autres canaux potassiques musculaires. Mon crible a également identifié une interaction génétique entre unc-70/ß-spectrine et unc-44/ankyrine. Toutefois, la nature moléculaire de cette interaction reste encore à préciser / Two-pore potassium channels (K2P) control cell excitability and play a central role in the establishment and the maintenance of the resting membrane potential of almost all animal cells. Since their identification in the late 90s, these channels have been implicated in a large number of functions ranging from neuronal and cardiac activity to kidney physiology. Despite the crucial functions of these channels, comparatively little is known about the cellular processes controlling their function in vivo. During my PhD, I used a wide range of strategies including genetics, microscopy and electrophysiology to understand how the expression, the distribution and the activity of the K2P channel UNC-58 are controlled in the model nematode C. elegans. I have first performed a genetic suppressor screen targeting the locomotion phenotype of the gain of function mutant unc-58(e665). The screen yielded 133 mutants, displaying a wide range of suppression level, suggesting that several genes may be implicated in the channel regulation process. By using whole genome sequencing technologies, I’ve been able to clone six new genes required for the function of UNC-58.Then, I’ve characterized in detail the role of unc-44/ankyrin in the trafficking of UNC 58. This project led to 4 main conclusions : (1) UNC-58, despite its potassium channel structure, has an altered ionic selectivity, allowing preferably sodium ions to pass through the channel (2) the addition of a fluorescent protein to UNC-58 by CRISPR/Cas9 approaches allowed us for the first time to directly observe the addressing of the UNC-58 channel to the muscle surface and axons of ALM mechanosensory neurons. This function involves a lipid binding pocket located within the Zu5N-Zu5C-UPA module of UNC-44, (4) this mechanism is highly selective since it is not required for the addressing of 6 other muscular channels.My screen also identified a genetic interaction between unc-70/ß-spectrin and unc-44/ankyrin. However, the exact molecular nature of this interaction remains to be elucidated

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-09-19T11:49:34Z No. of bitstreams: 1 2011 - Michel Jos? Sales A. Helayel.pdf: 11350186 bytes, checksum: ab1a4bf6f150ee6e971e58207f995bff (MD5) / Made available in DSpace on 2016-09-19T11:49:34Z (GMT). No. of bitstreams: 1 2011 - Michel Jos? Sales A. Helayel.pdf: 11350186 bytes, checksum: ab1a4bf6f150ee6e971e58207f995bff (MD5) Previous issue date: 2011-03-16 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / This study aimed to evaluate the protective effect of acetamid in experimental poisoning by Pseudocalymma elegans in sheep, goats and rabbits, in order to prove indirectly that monofluoroacetate (MF) is responsible for the clinical signs and death of animals that ingested the plant. Experiments were performed to determine for sheep and goats the lethal dose of P. elegans collected in Rio Bonito, RJ, in different seasons, and to adjust the dose of acetamid to be administered. In the first experiment, four animals received 1.0g/kg of fresh P. elegans, and two others were pretreated with 2.0g/kg of acetamid. None of the animals showed clinical signs or died. Possibly, the plant could be less toxic, since it was collected at the end of the rainy season. In the second experiment, two sheep and two goats received 0.67 and 1.0g/kg of the dried plant, after pretreatment with 2.0 and 3.0g/kg of acetamid, respectively. All animals died, as the administered doses of P. elegans were very high. In the third experiment, two sheep and two goats received 0.333g/kg of dried P. elegans after previous administration of 2.0g/kg of acetamid; a week later, the protocol above was repeated, but without the antidote. In experiments with rabbits, doses of 0.5 and 1.0g/kg of dried P. elegans were given after administration of 3.0g/kg of acetamid; seven days later, the same protocol was repeated, except the administration of acetamide. This procedure, when acetamid was administered before, prevented the appearance of clinical signs and death of sheep, goats and rabbits. But the animals not treated with acetamid showed symptoms of poisoning and died. Clinically, the sheep and goats had tachycardia, engorged jugular vein, positive venous pulse, lateral recumbence, and muscle tremors. In the "dramatic phase?, the animals fell into lateral position, stretched the limbs, were paddling and died within minutes. The rabbits showed apathy, muscle tremors, vocalization and lateral decumbence minutes before death. At postmortem examination, the sheep and goats had engorged jugular veins and atria, dilated Vena cava cranialis and caudalis, as well as pulmonary edema, hepatic congestion and edema of the gallbladder subserosa. In rabbits, the main macroscopic alterations were dilated atria, engorged Vena cava cranialis and caudalis, and congested liver and diaphragm vessels. Histopathology revealed, in two sheep and one goat, vacuolar-hydropic degeneration of the distal convoluted kidney tubules, together with caryopicnosis. In the rabbits, the liver showed severe congestion with numerous shock corpuscles. The experimental results show indirectly that MF is to be held responsible for death of the animals that ingested P. elegans; since "acetate donor" compounds, such as acetamid, are capable to reduce the competitive inhibition of MF for the same active site (Coenzyme A) which prevents the formation of fluorocitrate, its active metabolite, formed in the body through the socalled "lethal synthesis". / O presente trabalho teve como objetivo avaliar o efeito protetor da acetamida nas intoxica??es experimentais por Pseudocalymma elegans (Bignoniaceae) em ovinos, caprinos e coelhos, com a finalidade de comprovar indiretamente que o monofluoroacetato ? respons?vel pela sintomatologia e morte dos animais que ingerem essa planta. Foram realizados experimentos para determinar a dose letal da planta coletada em Rio Bonito, RJ, em diferentes ?pocas do ano para ovinos e caprinos e ajustar a dose de acetamida a ser administrada. No primeiro experimento, dois ovinos e dois caprinos receberam 1,0 g/kg de P. elegans fresca e um animal de cada esp?cie foi tratado previamente com 2,0 g/kg de acetamida. Nenhum animal apresentou altera??es cl?nicas ou morreu. Ao que tudo indica a planta poderia estar menos t?xica, j? que foi coletada no fim da esta??o das ?guas. No segundo experimento, dois ovinos e dois caprinos receberam 0,67 e 1,0 g/kg da planta dessecada, ap?s tratamento pr?vio, com 2,0 e 3,0 g/kg de acetamida, respectivamente. Todos os animais morreram, pois administramos doses muito altas de P. elegans. No terceiro experimento, dois ovinos e dois caprinos receberam, 0,333 g/kg de P. elegans dessecada, ap?s administra??o pr?via de 2,0 g/kg de acetamida. Uma semana depois, o protocolo acima foi repetido, por?m sem o ant?doto. Nos experimentos com coelhos, foram administradas doses de 0,5 e 1,0 g/kg de P. elegans dessecada ap?s a administra??o de 3,0 g/kg de acetamida. Sete dias depois, o mesmo protocolo foi repetido, com exce??o da administra??o de acetamida. Esta, quando administrada previamente, evitou o aparecimento dos sinais cl?nicos e a morte dos ovinos, caprinos e coelhos, j? os animais n?o tratados com acetamida apresentaram sintomatologia e morreram. Clinicamente, os ovinos e caprinos manifestaram taquicardia, jugulares ingurgitadas, pulso venoso positivo, dec?bito esternal e tremores musculares. Na ?fase dram?tica?, os animais ca?am em dec?bito lateral, esticavam os membros, faziam movimentos de pedalagem e morriam em poucos minutos. Nos coelhos observaram-se apatia, tremores musculares, dec?bito lateral e vocaliza??o minutos antes da morte. A avalia??o macrosc?pica revelou, nos ovinos e caprinos, jugulares ingurgitadas, aur?culas, veia cava caudal e cranial dilatadas, al?m de edema pulmonar, congest?o hep?tica e edema na subserosa da ves?cula biliar. Nos coelhos as principais altera??es observadas foram aur?culas dilatadas, veia cava caudal e cranial ingurgitadas, f?gado e vasos do diafragma congestos. O exame histopatol?gico revelou, em dois ovinos e um caprino, degenera??o hidr?pico-vacuolar dos t?bulos urin?feros contornados distais associada ? cariopicnose. Nos coelhos havia congest?o hep?tica acentuada com numerosos corp?sculos de choque. Nossos resultados comprovam, de forma indireta, que o MF ? respons?vel pela morte dos animais que ingerem essa planta, uma vez que compostos ?doadores de acetato? como a acetamida, s?o capazes de reduzir a inibi??o competitiva do MF pelo mesmo s?tio ativo (Coenzima A), o que impede a forma??o do fluorocitrato, seu metab?lito ativo, formado no organismo por meio da denominada ?s?ntese letal?.

The field of neuroscience has recently seen optogenetics emerge as a highly utilized and powerful method of non-invasive neural activation and inhibition. This thesis seeks to enhance the optogenetic toolbox through the design, construction, and evaluation of a number of hardware and software modules for research in Caenorhabditis elegans neuroscience. In the first aim, we combine optogenetics, microfluidics, and automated image processing, to create a system capable of high-throughput analysis of synaptic function. In the second aim, we develop a multi-modal illumination system for the manipulation of optogenetic reagents. The system is capable of multi-spectral illumination in definable patterns, with the ability to dynamically alter the intensity, color, and shape of the illumination. The illumination system is controlled by a set of software programs introduced in aim three, and is demonstrated through a set of experiments in aim four where we selectively activate and inhibit specific neural nodes expressing optogenetic reagents in freely moving C. elegans. With the ability to target specific nodes in a freely moving animal, we can correlate specific neural states to behaviors allowing for the dissection of neural circuits. Taken together, the developed technologies for optogenetic researchers will allow for experimentation with previously unattainable speed, precision and flexibility.

Lipids carry out important structural as well as signaling functions in the cell. In recent years, enzymes that metabolize lipids have been emerging as key regulators of basic cellular functions and developmental processes. In order to study metabolism of lipids, we have focused our research on a class of proteins: the cytochrome P450s (CYPs), which are involved in lipid production in many organisms. We have used C. elegans, a classical genetic model system, to investigate lipid metabolism because this nematode offers several technical advantages that render it suitable for our investigations. The aim of our project was to identify and characterize essential lipids for the development of worms. We have performed RNAi (RNA interference) against C. elegans CYP31A, and found that silencing of this enzyme leads to the arrest of embryonic development. Further characterization of this embryonic lethal phenotype revealed that it is caused by problems in establishment of polarity and failure in the extrusion of a polar body. Moreover, we found that embryos depleted of CYP31A are osmotic sensitive and their eggs are permeable to dyes (hoechst, FM 4-64 etc.). The defects described above are common to a class of mutants that received the denomination of POD (for Polarity and Osmotic Defects). Analysis by electron microscopy demonstrated that cyp31A(RNAi) embryos exhibit an improperly constructed eggshell. Further functional studies have demonstrated that the defects observed in cyp31A(RNAi) embryos can be ascribed to the malfunctioning of one of the three layers of the eggshell: the lipid-rich layer, but additional problems in the assembling of the other two layers are also present. In order to identify the product of CYP31A, we set up a bioassay in which we tested the capability of lipidic extract from wild type embryos to rescue the embryonic lethality. The bioassay provided a method to track the activity and allowed us to enrich the metabolic product of CYP31A by the fractionation of the total lipid extract. Another POD gene, emb-8, codes for an NADPH CYP reductase. This 4 protein supplies electrons to the CYPs for their metabolic reactions. A mutant of emb-8 (emb-8(hc69)), gives a similar phenotype as the knockdown CYP31A. With the aim to test if EMB-8 and CYP31A act in the same pathway we extracted lipids from emb-8TS mutants. We tested in the bioassay if extracts from emb-8(hc69) mutants, containing the metabolic product of CYP31A, can rescue cyp31A(RNAi) phenotype. The results obtained suggest that EMB-8 and CYP31A work in the same metabolic pathway. Conclusively, CYP31A and EMB-8 cooperate to produce a class of lipids that are required for the construction of a functional eggshell. A defective eggshell causes failure in polarity establishment, extrusion of the polar bodies, osmotic sensitivity and permeability and eventually it leads to the arrest of the development of C. elegans embryos.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Feb. 19, 2010). Includes bibliographical references.

Thesis (Ph.D.). / Written for the Dept. of Experimental Surgery. Title from title page of PDF (viewed 2008/01/12). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [84]-100).

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / "July 2004" Typescript. Vita. Includes bibliographical references (l. 123-133). Also issued on the Internet.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 75-81).

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 217-230). Also available on the Internet.