Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Sept. 16, 2008). Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 169-208) Also available online.

Thesis (Ph. D.)--Brigham Young University. Dept. of Chemical Engineering, 2001. / Includes bibliographical references.

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 134-139.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Le travaux présenté dans cette thèse concerne le rôle des canaux ioniques de la membrane plasmique en réponse à des stress salins et non salins ainsi qu’aux interactions possibles avec d’autres événements de signalisation conduisant à la mort cellulaire programmée (PCD). Nous avons montré que les réponses cellulaires précoces: tels que l`augmentation du calcium cytosolique et la production de ROS, classiquement impliqués lors de la PCD, ne semblaient pas être impliqué dans la mort cellulaire induite par les stress hyperosmotiques chez les cellules en culture de tabacco BY2 ou d’A. thaliana. Nous avons montré que, dans les cas de stress salin chez les cellules de BY2 un influx précoce de sodium à travers des canaux cationiques non spécifiques participe au développement de la PCD en entraînant un disfonctionement mitochondrial et la production de O2• - par des NADPH oxydases. Dans le cas de stress hyperosmotique non-ionique, nous avons observé une diminaution précoce de l’intensité des courants anioniques. Afin de poursuivre l’étude du rôle des canaux anioniques lors du stress hyperosmotique non salin, nous avons utilisé des cellules A.thaliana nous permettant de travailler avec le mutant de canal anionique SLAC1. Nous avons constaté que l’activation retardée des canaux SLAC1 participait au développement de la PCD induite par un stress hyperosmotique non salin. La réduction précoce de l'activité des canaux anioniques pourrait participer à la signalisation ou l'ajustement osmotique permettant l'adaptation et la survie cellulaire alors que des évènements retardés, à savoir la production d'anion superoxyde (O2• -) par les NADPH-oxydases et l'activation des canaux anioniques pourraient participer au développement de la PCD d'une partie de la population cellulaire. Nous avons aussi étudié le rôle potentiel des petits peptides appartenant à la famille des peptides FMRFamide décrite chez les métazoaires à l'osmorégulation chez des cellules d’A. thaliana. Des génes susceptibles de coder de tels peptides sont en effet présent dans le génome d’A. thaliana. En utilisant des peptides synthétiques, nous avons montré que ces FLPS putatifs pourraient participer aux réponses induites losr de stress hyperosmotique chez les plantes. Ce travail illustre la complexité et l'importance de la régulation des canaux ioniques dans les voies de signalisation et les processus conduisant à la PCD / The work presented in the present thesis relates to the role of ion channels in response to (ionic and non-ionic) hyperosmotic stresses and their interactions with signaling events leading to PCD in plant. Early cell responses such as cytosolic calcium increase and ROS production classically involved in PCD process, seems not to be involved in hyperosmotic-induced cell death in BY2 tobacco and A. thaliana cultured cells. When BY2 tobacco cells were subjected to hyperosmotic stress, an early influx of sodium through non-selective cation channels participates in the development of PCD through mitochondrial dysfunction and NADPH-oxidase-dependent O2•– generation. On the contrary, non-ionic hyperosmotic stress resulted in an early decrease in anion currents. To further investigate the role of anion channels in non-ionic hyperosmotic stress further experiments were conducted by using A.thaliana cells of the anion channel mutant SLAC1. Results showed that the delayed activation of SLAC1 channels was involved in the non-ionic hyperosmotic stress induced pathway leading to cell death. Interestingly, the early anion channel activity decrease could participate to signalisation or osmotic adjustment allowing cell adaptation and survival, when a second set of events, namely superoxide anion (O2•-) generation by NADPH-oxidase and anion channel activation could participate in PCD development of a part of the cell population. In addition, the potential role of small peptides belonging to the FMRFamide-like peptide (FLP) family described in metazoan in osmoregulation in A. thaliana was investigated. By using synthetic peptides, based on FLPs homolog genes existing in A. thaliana, it was possible to demonstrate that these putative FLPs are involved in hyperosmotic stress response. Overall, the present work shed light on the importance and the complexity of ion channels regulation in the signaling pathways and the processes leading to PCD

Fosfolipases A2 miotóxicas (Lys49, enzimaticamente inativa, e Asp49, com atividade) isoladas do veneno de Bothrops asper, induzem hipernocicepção. Assim, avaliamos os mecanismos estruturais, moleculares e mediadores centrais envolvidos neste efeito. A injeção intraplantar das FLA2s acarretou hiperalgesia, enquanto que apenas a FLA2-Asp49 induziu alodinia. A região C-terminal é a responsável pelo efeito da FLA2-Lys49, enquanto que a atividade catalítica da FLA2-Asp49 parece ser responsável pela indução de hipernocicepção. Canais de Ca2+ e Na+ participam deste efeito. Na medula espinhal, receptores NK1 e para CGRP, receptores ionotrópicos para glutamato, NO, IL-1, prostanóides e adenosina participam da hiperalgesia induzida pelas FLA2s. Adicionalmente, receptores metabotrópicos para glutamato e o TNF?, estão envolvidos na hiperalgesia induzida pela FLA2-Asp49. Receptores NK1 e NK2 e para CGRP, receptores para glutamato, TNF? e prostanóides medeiam a alodinia. A ativação de astrócitos e microglia, na medula espinhal, contribui para a gênese do efeito hipernociceptivo. / Phospholipase A2 (Lys49, catalytically-inactive and Asp49, catalytically active), isolated from Bothrops asper snake venom, induce pain. The present studies examined the molecular, structural and central mechanisms involved in hypernociception induced by both PLA2s. These PLA2s induced mechanical hyperalgesia, whereas only PLA2-Lys49 evoked allodynia. The C-terminal region of the PLA2-Lys49 seems to be responsible for hyperalgesia, whereas the enzymatic activity of PLA2-Asp49 contributes to such an effect. Calcium and sodium channels are involved in PLA2s-induced hyperalgesia. In the spinal cord, NK1 and CGRP receptors, glutamate ionotropic receptors, NO, IL-1, prostanoids and adenosine contribute to hyperalgesia caused by PLA2s. Additionally, metabotropic glutamate receptors and TNF are involved in hyperalgesia induced by PLA2-Asp49. NK1, NK2 and CGRP receptors, glutamate receptors, TNF and prostanoids mediate allodynia. Activation of spinal astrocytes and microglia contribute to the generation of hyperalgesia and allodynia induced by both toxins.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-04-01T12:26:27Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Doutorado. Tese de Lindalva Vieira. Ano 2015..pdf: 1577129 bytes, checksum: 08d435d57b1dfb816c8e6a8dcd551f50 (MD5) / Made available in DSpace on 2016-04-01T12:26:27Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Doutorado. Tese de Lindalva Vieira. Ano 2015..pdf: 1577129 bytes, checksum: 08d435d57b1dfb816c8e6a8dcd551f50 (MD5) Previous issue date: 2015-07-02 / CAPEs / A Diabetes Mellitus (DM) é um grave problema de saúde pública, sendo uma das maiores causas de morbimortalidade na grande maioria dos países. Diferentes órgãos são acometidos pela DM; contudo, as doenças cardiovasculares são as maiores responsáveis pela elevada mortalidade vinculada à diabetes. A metformina é atualmente o principal fármaco utilizado para o tratamento da DM do tipo 2 (DM2), embora os riscos-benefícios cardiovasculares dessa droga não estejam totalmente elucidados. O presente estudo teve como objetivo avaliar o potencial arritmogênico da metformina em corações e cardiomiócitos de ratos saudáveis. No tratamento por 10 semanas com metformina os parâmetros biométricos e a glicemia plasmática não foram modificados, mas foi constatado um prolongamento dos intervalos PR, QT e QTc no eletrocardiograma, e uma redução da corrente transitória de saída de K+, Ito.Quando os cardiomiócitos foram incubados por 24 h com metformina, a amplitude da corrente Ito foi reduzida, enquanto aumentou a duração do potencial de ação cardíaco (DPA). A exposição de cardiomiócitos isolados à metformina por um período de 30 min, não reduziu a amplitude da corrente Ito, comprovando que a redução da corrente Ito em cardiomiócitos tratados por 10 semanas e incubados por 24 h com metformina, não ocorreu por uma ligação direta da droga ao canal. A corrente de cálcio tipo L (ICa-L) e a dependência de voltagem da inativação e recuperação da inativação da corrente Ito não foram afetadas em ambas as situações experimentais. Os níveis de RNAm que codifica as subunidades KV4.2, KV4.3 e KChIP2 do canal Ito não foram alterados, sugerindo que a redução da corrente Ito não ocorreria por uma menor síntese proteica das subunidades que compõem o canal Ito. Propõe-se que, esta redução seria resultante de uma acelerada degradação dos canais ou de uma deficiência funcional destes na membrana celular. A partir dos resultados obtidos na presente tese, conclui-se que a metformina apresenta potenciais efeitos arritmogênicos sobre o coração de animais saudáveis, capazes de levar eventualmente à morte súbita. / Diabetes Mellitus (DM) is a major public health problem, and is one of the biggest causes of morbidity and mortality in most countries. Many different human organs are affected by the DM, but cardiovascular diseases are the leading causes of mortality in patients with DM. Even today, the leading drug used to treat type-2 diabetes is metformin. However, its cardiovascular risk-benefits are not entirely clear. This study aimed to determine the potential arrhythmogenic effect of metformin in cardiomyocytes from healthy animals. Therefore, a 10 week treatment with metformin was performed, and it was proved that biometric and metabolic parameters were not affected. However, a lengthening of the PR, QT and QTc interval was found on the electrocardiogram, as well as a reduction in the output of the transitory potassium Ito current. When cardiomyocytes were incubated for 24 h with metformin, the amplitude of the Ito current was reduced and duration of cardiac action potential (APD) increased. Exposure of cardiomyocytes treated with metformin over a period of 30 min did not reduce the amplitude of the current Ito, demonstrating that the reduction in Ito current cardiomyocytes treated for 10 weeks and incubated for 24 h with metformin, does not occur by direct binding of the drug to the canal. The L-type calcium current (ICa-L) and the kinetic properties of voltage-dependent inactivation and recovery from inactivation of Ito current remained unchanged in both experimental conditions. RNAm levels of KV4.2, KChIP2 4.3 and Kv subunits remained unchanged. This suggests that the reduction of the Ito current may not be caused by a decrease in the protein synthesis subunit which is part of the Ito channel.Therefore, this reduction may be caused by a main degradation of the channels or by a worse operating performance in the cell membrane. Experiments accomplished during this thesis try to demonstrate that metformin causes arrhythmogenic effects in the heart of healthy animals.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Com o objetivo de verificar a interferÃncia da broncoprovocaÃÃo antigÃnica sobre a contratilidade traqueal, ratos machos (250 -350 g) foram sensibilizados à ovalbumina (OVA) e, 13 a 14 dias depois, desafiados em intervalos de 15 minutos atravÃs da inalaÃÃo do antÃgeno sensibilizante (OVA, 1 mg/ml, seguida de 5 mg/ml). O sacrifÃcio dos animais foi feito imediatamente (SD0) ou 24 horas (SD24) apÃs o desafio antigÃnico atravÃs de anestesia com hidrato de cloral (0,4 g/Kg). Alguns animais foram tratados com metilsergida (1 mg/Kg) 40 minutos antes do desafio. Os animais controle (CONT) inalaram apenas o veÃculo (NaCl 0,9 %). A traquÃia foi removida e montada em cuba para orgÃo isolado contendo 10 ml de soluÃÃo de Krebs-Henseleit modificada (mantida a 37Â0,5 oC) e aerada com mistura carbogÃnica (O2/CO2â95:5). Foram confeccionadas curvas concentraÃÃo-efeito (CCE) para cloreto de potÃssio (KCl), acetilcolina (ACh) ou serotonina (5-HT). Em outros experimentos foram realizadas CCE ao Ca2+ em preparaÃÃes mantidas previamente em soluÃÃo sem Ca2+ (contendo 10-5 M de EDTA, nifedipina e indometacina, 10-6 M para ambas) e estimuladas por KCl, ACh ou 5-HT. A sensibilizaÃÃo e o posterior desafio antigÃnico (SD) promoveram um significativo aumento da resposta mÃxima (RM) das CCE ao KCl (forÃa em grama, mÃdia  E.P.M.: CONT = 0,52  0,01; SD24 = 1,22  0,03; n = 06; p < 0,01), à ACh (CONT = 2,11  0,10; SD24 = 3,53  0,03; n = 06; p < 0,01) ou à 5-HT (CONT = 0,60  0,03; SD24 = 1,48  0,09; n = 06; p < 0,01). As traquÃias de animais sensibilizados e desafiados apresentaram aumento da RM ao Ca2+ apenas quando prÃ-contraÃdas com KCl (CONT = 0,84  0,08; SD24 = 1,98  0,05; n = 6; p < 0,01) ou ACh (CONT = 0,98  0,09; SD24 = 1,54  0,15; n = 6; p < 0,05). Quando o agonista utilizado foi a 5-HT esta diferenÃa nÃo ocorreu, sendo observada uma maior RM somente na ausÃncia de nifedipina (CONT = 0,81  0,06; SD24 sem nifedipina = 1,23  0,08; n = 05; p < 0,01). Foram comparados os animais apenas sensibilizados e nÃo desafiados (SENS) com os SD na presenÃa de Ãcido niflÃmico (AN). O AN inibiu a hiperreatividade para a 5-HT induzida pelo desafio antigÃnico (SENS = 1,28  0,09; SD24 + AN = 1,01  0,08; n = 05), enquanto que foi ineficaz em inibir a hiperreatividade para a ACh (SENS = 1,44  0,01; SD24 + NA = 1,67  0,03; n = 05; p < 0,01). Portanto, os resultados mostram que hà envolvimento de canais de Ca2+, tanto dependentes de voltagem (VOC) como operados por receptor (ROC), na hiperreatividade do mÃsculo liso respiratÃrio, induzida pela reapresentaÃÃo do antÃgeno a animais previamente sensibilizados e que o desenvolvimento da hiperreatividade para a 5-HT està relacionado à abertura de canais de Cl- ativados por Ca2+ / In order to verify the interference of the antigenic challenge on traqueal contractility in vitro, male rats (250 -350 g) were ovalbumine (OVA)-sensitized and, 13 to 14 days later, they were challenged through the sensitizing antigen inhalation (OVA, 1 mg/ml, followed by 5 mg/ml). Animals were sacrificed immediately (SD0) or 24 hours (SD24) after the antigenic challenge through chloral hydrate anaesthesia (0,4 g/Kg). Some animals were treated with metilsergide (1 mg/Kg) 40 minutes before the challenge. Control animals (CONT) received only the vehicle (NaCl 0,9 %) by inhalation. Tracheal rings were carefully removed and mounted in a 10 ml isolated bath chamber with modified Krebs-Henseleit solution (at 37Â0,5 oC) bubbled with a mixture of 5% of CO2 in 95% of O2. Concentration-effect curves (CCE) were constructed for potassium chloride (KCl), acetycholine (ACh) or serotonin (5-HT). In another experiments, CCE for Ca2+ addition were constructed under Ca2+-free conditions (with 10-5 M EDTA, nifedipine and indomethacin, 10-6 M each) in the presence of KCl, ACh or 5-HT. Sensitization and subsequent antigenic challenge (SD) promoted a significant increase of the maximal response (RM) of the CCE to KCl (force in grams, mean  S.E.M.: CONT = 0,52  0,01; SD24 = 1,22  0,03; n = 06; p < 0.01), ACh (CONT = 2.11  0,10; SD24 = 3,53  0,03; n = 06; p < 0.01) or to 5-HT (CONT = 0,60  0,03; SD24 = 1,48  0,09; n = 06; p < 0.01). Tracheal rings of sensitized and challenged animals showed a rise in RM for Ca2+ only when they were pre-contracted with KCl (CONT = 0,84  0,08; SD24 = 1,98  0,05; n = 6; p < 0.01) or ACh (CONT = 0,98  0,09; SD24 = 1,54  0,15; n = 6; p < 0,05). When 5-HT was used as agonist this hyperresponsiveness did not occur, being observed only in the nifedipine absence (CONT = 0.81  0,06; SD24 without nifedipine = 1,23  0,08; n = 05; p < 0.01). The sensitized but not challenged animals (SENS) were compared to SD animals in the presence of niflumic acid (AN). AN inhibited the antigenic shock-induced hyperresponsiveness for 5-HT (SENS = 1,28  0,09; SD24 + AN = 1.01  0,08; n = 05), while it was ineffective in inhibit that for the ACh (SENS = 1,44  0,01; SD24 + AN = 1,67  0,03; n = 05; p < 0.01). Therefore, the results show that there is a participation of Ca2+ channels, both voltage- and receptor-operated channels, in the hyperresponsiveness of the respiratory smooth muscle, induced by the representation of the antigen to previously sensitized animals and, in addition, the Cl- channels Ca2+-activated have an important role on 5-HT hyperresponsiveness development