Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 338-360).

Made available in DSpace on 2015-05-14T12:59:31Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 3977582 bytes, checksum: 6a5b8750e2be83e492617d4f9954fa26 (MD5) Previous issue date: 2011-02-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Introduction: The Transient Receptor Potential (TRP) superfamily of cation channels is remarkable since it displays greater diversity in activation mechanisms, and are targets for plant-derived compounds. Aim: To investigate the role of TRP channels in the vasorelaxant response of rotundifolone in the superior mesenteric artery from Lyon Normotensive (LN) rats. Methods and Results: Endothelium-denuded artery rings were suspended by platinum hooks for isometric tension recordings. In nominally free-Ca2+ medium, the rings were submitted to successive phenylephrine (Phe) contractions to deplete Ca2+- stores and contracted to CaCl2 (10-2 M). The maximum response (MR) of CaCl2-contractions in presence of nifedipine (10-6 M) (MR = 31.66 ± 2.27 %) were significantly attenuated in the presence of nifedipine plus rotundifolone (3 x 10-4 and 3 x 10-3 M) (MR = 9.30 ± 2.38 and 1.12 ± 0.31 %) or nifedipine plus menthol (10-4 and 10-3 M) (MR = 10.96 ± 1.34 and 1.52 ± 0.82 %). Rotundifolone caused relaxation of vessels pre-contracted with Phe (MR = 100.32 ± 3.88 %; pD2 = 3.59 ± 0.04, n = 6). The vasorelaxant effect induced by rotundifolone was significantly atenuated in the presence of Gd3+ (10-4 M) (MR = 83.74 ± 5.71 %; pD2 = 3.15 ± 0.06); Gd3+ (2.25 x 10-5 or 2 x 10- 6 M) (pD2 = 3.18 ± 0.06 and 3.32 ± 0.03 %) or BCTC (MR = 76.30 ± 2.15 %; pD2 = 3.46 ± 0.04), but no in the presence of ruthenium red, La3+ or Mg2+, nor after TRPV1 desensitization with capsaicin. Menthol caused relaxation of vessels pre-contracted with Phe (MR = 105.07 ± 3.07 %; pD2 = 3.72 ± 0.02). The vasorelaxant effect induced by menthol was significantly potentiated in the presence of ruthenium red (10-5 M), a non-selective TRP channels blocker (pD2 = 4.12 ± 0,04, n = 6). Also, the vasorelaxant response of menthol was significantly attenuated in the presence of La3+ (8 x 10-5 M), non-selective TRP channels blocker (MR = 89.05 ± 1.61 %); Mg2+ (2.25 x 10-3 M), TRPM3, 6 and 7 selective blocker (MR = 90.76 ± 2.94 %); Gd3+ (10-4 M), TRPV4, TRPC1, 3 and 6, TRPM3 and 4 channels blocker (MR = 73.82 ± 5.44 %); Gd3+ (2.25 x 10-5 M), TRPC3 and 6, TRPV4 channels blocker (MR = 88.04 ± 2.33 %); Gd3+ (2 x 10- 6 M), TRPC6 selective blocker (MR = 89,30 ± 3,61 %) or BCTC (2 x 10-6 M), TRPM8 and TRPV1 channels blocker (MR = 66.77 ± 6.05 %), and after TRPV1 desensitization with capsaicin (10-5 M) (RM = 88.96 ± 4.50). The basal tension was reduced by change in the thermostat temperature from 37 ºC to 25ºC and 18ºC (MR = 21.15 ± 0.78 and 28.84 ± 1.03 %). This response was significantly potentiated by rotundifolone (3 x 10-3 M) (MR = 28.01 ± 1.81 and 38.45 ± 1.98 %) or menthol (10-3 M) (MR = 29.87 ± 1.25 and 43.03 ± 2.22 %). In the way similar to menthol, the effects induced by rotundifolone were attenuated in free-Ca2+ medium plus EGTA (MR = 20.42 ± 1.97 and 30.90 ± 2.58 %) or in the presence of BCTC (MR = 17.05 ± 1.94 and 26.48 ± 3.39 %), but not when the vessels were pre-treated with ruthenium red or capsaicin. The RNAm and the protein of the TRPM8 channel are expressed in the superior mesenteric artery from LN rats. Conclusions: These data suggest that rotundifolone induces concentration-dependent relaxation in the mesenteric artery due to inhibition of ROC and SOC channels (probably TRPC1 and TRPC6) and activation of TRPM8 channels. / Introdução: A superfamília Potencial Receptor Transiente (TRP) de canais catiônicos se destaca por exibir uma grande diversidade de mecanismos de ativação, e são alvos de compostos derivados de plantas. Objetivo: Investigar o papel de canais TRP na resposta vasorrelaxante de rotundifolona em artéria mesentérica superior de ratos Normotenso de Lyon (LN). Métodos e Resultados: Anéis de artéria sem endotélio foram suspensos em hastes metálicas para registro de tensão isométrica. Em meio nominalmente sem Ca2+, os anéis foram submetidos a contrações sucessivas com FEN para depleção dos estoques de Ca2+ e contraídos com CaCl2 (10-2 M). O efeito máximo (Emáx) das contrações com CaCl2 na presença de nifedipino (10-6 M) (Emáx = 31,66 ± 2,27 %) foi significativamente atenuado na presença de nifedipino mais rotundifolona (3 x 10-4 e 3 x 10-3 M) (Emáx = 9,30 ± 2,38 e 1,12 ± 0,31 %) e nifedipino mais mentol (10-4 e 10-3 M) (Emáx = 10,96 ± 1,34 and 1,52 ± 0,82 %). Rotundifolona causou relaxamento de vasos pré-contraídos com FEN (Emáx = 100,32 ± 3,88 %; pD2 = 3,59 ± 0,04, n = 6). O efeito vasorrelaxante induzido por rotundifolona foi signigficativamente atenuado na presença de Gd3+ (10-4M) (Emáx = 83,74 ± 5,71 %; pD2 = 3,15 ± 0,06); Gd3+ (2,25 x 10-5 ou 2 x 10-6 M) (pD2 = 3,18 ± 0,06 e 3,32 ± 0,03 %) ou BCTC (Emáx = 76,30 ± 2,15 %; pD2 = 3,46 ± 0,04), mas não na presença de vermeho de rutênio, La3+ or Mg2+, nem após dessensibilização do TRPV1 com capsaicina. Mentol também causou o relaxamento de vasos pré-contraídos com FEN (Emáx = 105,07 ± 3,07 %; pD2 = 3,72 ± 0,02). O efeito vasorrelaxante induzido por mentol foi significativamente potencializado na presença de vermelho de rutênio (10-5 M), um bloqueador não seletivo de canais TRP (pD2 = 4,12 ± 0,04, n = 6) e significativamente atenuada na presença de La3+ (8 x 10-5 M), bloqueador não seletivo de canais TRP (Emáx = 89,05 ± 1,61 %); Mg2+ (2,25 x 10-3 M), bloqueador seletivo dos canais TRPM3, 6 e 7 (Emáx = 90,76 ± 2,94 %); Gd3+ (10-4 M), bloqueador de canais TRPV4, TRPC1, 3 and 6, TRPM3 and 4 (Emáx = 73,82 ± 5,44 %); Gd3+ (2,25 x 10-5 M), bloqueador de canais TRPC3 and 6, TRPV4 (Emáx = 88,04 ± 2,33 %); Gd3+ (2 x 10-6 M), bloqueador seletivo do TRPC6 (Emáx = 89,30 ± 3,61 %) ou BCTC (2 x 10-6 M), bloqueador dos TRPM8 e TRPV1 (Emáx = 66,77 ± 6,05 %), e após a dessensibilização do TRPV1 com capsaicina (10-5 M) (Emáx = 88,96 ± 4,50). A tensão basal foi reduzida por mudança na temperature do banho de 37 ºC para 25ºC e 18ºC (Emáx = 21,15 ± 0,78 e 28,84 ± 1,03 %). Essa resposta foi significativamente potencializada por rotundifolona (3 x 10-3 M) (Emáx = 28,01 ± 1,81 e 38,45 ± 1,98 %) ou mentol (10-3 M) (Emáx = 29,87 ± 1,25 e 43,03 ± 2,22 %). Semelhante ao mentol, os efeitos induzidos por rotundifolona foram atenuados em meio sem Ca2+ mais EGTA (Emáx = 20,42 ± 1,97 e 30,90 ± 2,58 %) ou na presença de BCTC (Emáx = 17,05 ± 1,94 e 26,48 ± 3,39 %), mas não quando os vasos foram pré-tratados com vermelho de rutênio ou capsaicina. O RNAm e a proteína do canal TRPM8 são expressos em artéria mesentérica de ratos LN. Conclusões: Esses dados sugerem que rotundifolona induz relaxamento dependente de concentração em artéria mesentérica devido à inibição de canais ROC e SOC (provavelmente TRPC1 e TRPC6) e ativação de canais TRPM8.

The kynurenine pathway produces NAD+ from L-tryptophan. Metabolites known as the kynurenines are produced within the pathway. The effects of the kynurenines have been associated with a number of diseases including cancer, Alzheimer’s disease, Huntington’s disease, and acute pancreatitis. Kynurenine monooxygenase (KMO) is an enzyme that catalyses the conversion of L-kynurenine to 3-hydroxy-L-kynurenine, the downstream product of which is the neurotoxic quinolinic acid. L-kynurenine is positioned at a branching point within the pathway. Metabolism via KMO leads to quinolinic acid production whereas conversion via kynurenine aminotransferase (KAT) produces the neuroprotective kynurenic acid. Inhibition of KMO leads to an increase in kynurenic acid concentration. This has also been shown to ameliorate the symptoms of neurological diseases in a number of animal models as well as to protect against multiple organ dysfunction caused by acute pancreatitis in rodent models. These findings present KMO as a promising drug target. Due to the hydrophobic nature of human KMO (hKMO) it has been necessary to utilise other forms of KMO as models. Past studies have produced crystal structures of a truncated Saccharomyces cerevisiae KMO and of Pseudomonas fluorescens KMO (PfKMO). Previous work in this research group has resulted in the structure of variants of PfKMO bound to either inhibitor molecules or substrate. These structures identified residues involved in substrate binding and the presence of a highly mobile section of the C-terminus, giving rise to open and closed conformations. It was surmised the movement of the C-terminus was dependent upon the presence of substrate and an interactive network between the C-terminus and the rest of the protein. Using improved crystallising conditions high-resolution structures of PfKMO have been produced that allow for further study of residues involved in substrate binding and the interactive network within the C-terminus. The mutants R84K and Y404F showed severely decreased enzyme activity. Crystal structures of these proteins showed disrupted interactions between substrate and active site. These findings underline the importance of residues R84 and Y404 in substrate binding. An H320F mutation gives an analogous active site to hKMO. Crystallographic and kinetic study of this mutant proved very similar to PfKMO, supporting the use of PfKMO as a model for hKMO. Throughout the work each structure had a P21221 space group with two molecules in the asymmetric unit. The presence of an open and closed molecule within each structure, including substrate-free molecules refuted the connection between C-terminus and substrate. R386K and E372T mutations were separately introduced in order to interrupt the interactive network. The presence of both open and closed conformations in the structures of R386K and E372T refutes the necessity for the interactive network in C-terminus movement. The data analysed throughout the project suggest simple mobility and thermal motion as the cause of the movement of the C-terminus. This work, in conjunction with kinetic data from the thesis of Helen Bell, presents structural data to characterise the role of binding residues within the active site of KMO as well as the mechanistic role of the C-terminus. It also highlights the importance of certain binding residues and countered the previously held hypotheses surrounding the significance of the C-terminus. The mechanistic role of the C-terminus therefore remains unclear and requires further study.

Submitted by ROBERTO PAULO CORREIA DE ARAÚJO (ppgorgsistem@ufba.br) on 2016-10-18T14:55:15Z No. of bitstreams: 1 Milena Ramos Reis.pdf: 2152638 bytes, checksum: 876fac844a4f4a8b22cb82e9cdeb5f3b (MD5) / Made available in DSpace on 2016-10-18T14:55:15Z (GMT). No. of bitstreams: 1 Milena Ramos Reis.pdf: 2152638 bytes, checksum: 876fac844a4f4a8b22cb82e9cdeb5f3b (MD5) / O carvacrol, um monoterpeno fenólico encontrado nos óleos essenciais de diversas plantas do gênero Origanum, já demonstrou causar hipotensão e vasodilatação em diferentes leitos vasculares de ratos normotensos, porém, seu efeito em ratos hipertensos ainda não foi elucidado. O objetivo deste estudo foi investigar os efeitos cardiovasculares do carvacrol em ratos espontaneamente hipertensos (SHR) e comparar com normotensos Wistar, utilizando ensaios farmacológicos in vitro (estudos funcionais e celulares) e in vivo. Nos ensaios funcionais in vitro, anéis de artéria mesentérica superior isolada de animais hipertensos e normotensos foram précontraídos com FEN (1μM) e o efeito de carvacrol (10-8-10-3M) foi observado. Em SHR, este monoterpeno induziu vasodilatação dependente de concentração (pD2=5,13 ± 0,05; Emáx=115,14 ± 5,46%; N=8) e, após a remoção do endotélio funcional, a potência da droga foi alterada significantemente (pD2=4,91 ± 0,05 N=9; p<0,01), sugerindo que a resposta vasodilatadora induzida por carvacrol, provavelmente, envolve uma via dependente e outra independente do endotélio vascular, porém, esta última parece ser a majoritária e, por isso, os ensaios seguintes foram realizados na ausência do endotélio vascular. Interessantemente, quando comparada com animais normotensos, a potência farmacológica de carvacrol foi reduzida significantemente (pD2=4,91 ± 0,05; N=9; p<0,05). Em anéis de ratos hipertensos, carvacrol reduziu o influxo de Ca2+ por canais Cav tipo-L, SOC e ROC, estes resultados foram semelhantes aos obtidos em ratos normotensos. Em ratos hipertensos, mas não em normotensos, a potência farmacológica do carvacrol em anéis pré-contraídos com FEN e na presença de diferentes inibidores de canais TRP (íon Gd3+, 10-5M; 2-APB, 10-6M ou 10-5M; BCTC, 2μM; 9-fenantrol, 10-5M; ou HC03003-1, 10-5M), foi reduzida em relação ao controle na ausência destes bloqueadores, sugerindo que os canais sensíveis à estes bloqueadores (TRPC1-7, TRPM2, M4 e TRPM8, TRPV1 e TRPA1), provavelmente, estão participando dos efeitos vasculares mediados por carvacrol e podem estar envolvidos no processo hipertensivo. Em estudos de patch-clamp em células de artéria mesentérica dispersas de ratos hipertensos, carvacrol (300μM) reduziu as correntes de entrada de Ba2+ por Cav tipo-L e este efeito foi semelhante em ratos normotensos. Além disso, em células de ratos hipertensos, o Mg2+ (2,5mM), bloqueador do TRPM6 e TRPM7, reduziu as densidades de ITRPM de entrada e saída, assim como carvacrol (100μM e 300μM), na ausência ou presença do 2-APB (100μM), bloqueador de TRPM7. A presença do 2-APB provocou inibição adicional nas densidades de ITRPM pelo carvacrol (100μM, mas não 300μM). Altas concentrações intracelulares de Mg2+ reduziram the magnitude of ITRPM7. Foi evidenciado que a ITRPM no controle é menor em ratos hipertensos que em normotensos. Estes dados obtidos e os relatados na literatura são sugestivos para provável inibição de ITRPM7 por carvacrol em células mesentéricas nativas. O efeito anti-hipertensivo do carvacrol foi avaliado por administração via orogástrica (50mg/kg/dia) durante 20 dias foi capaz de reduzir a pressão arterial média dos animais SHR tratados, no 20º dia do tratamento. O tratamento subcrônico com carvacrol não alterou os pesos cardíaco e corpóreo, nem a reatividade vascular. Em conclusão, esses dados sugerem que carvacrol possui atividade anti-hipertensiva em animais SHR, que pode ser devido ao seu efeito vasodilatador em anéis de artéria mesentérica superior isolada, provavelmente, por inibição do influxo de Ca2+ por Cav tipo-L, ROC, SOC e/ou canais TRPC1, 3 ou 6, além da inibição de correntes tipo-TRPM7 em miócitos mesentéricos.

My thesis research comprises two projects looking into physiological functions of Drosophila ion channels: first, contribution of several T ransient R eceptor P otential (TRP) channels to gravity sensing; second, regulation of metabolic homeostasis by a D egenerin/ E pithelial Na + C hannel (DEG/ENaC). Many animal species sense gravity for spatial orientation. In humans recurrent vertigo and dizziness are often attributable to impairment of gravity sensing in the vestibular organs. However, the molecular bases for gravity sensing and its disruption in vestibular disease remain uncertain. Here I studied gravity sensing in the model organism Drosophila melanogaster, with a combination of genetic, behavioral and electrophysiological methods. My results show that gravity sensing requires Johnston’s organ, a mechanosensory structure located in the antenna that also mediates hearing. Johnston’s organ neurons fire action potentials in a phasic manner in response to body rotations in the gravitational field. Furthermore, gravity sensing and hearing require different TRP channels with distinct anatomical localizations, implying separate neural mechanisms underlying gravity sensing and hearing. These findings set the stage for understanding how TRP channels contribute to the sensory transduction of gravity. Drosophila melanogaster has over 20 genes belonging to the DEG/ENaC family, which are collectively referred to as pickpockets (ppks) . Genetic analyses have implicated ppk genes in salt taste, tracheal liquid clearance, pheromone detection, and developmental timing. These results, together with the conserved presence of DEG/ENaC genes through evolution, suggest that further studies on fly ppk genes may help gain insights to a number of physiological processes. Here I report that the ppk11 gene regulates metabolic homeostasis. A ppk11 enhancer/promoter fragment labels the fat body, the lipid storage organ of Drosophila. ppk11 mutants are lean — they store less triacylglyceride (TAG), possess smaller lipid droplets and are sensitive to starvation compared to wild–type flies. ppk11 mutants also show signs of enhanced insulin sensitivity — they store more glycogen and maintain a lower level of circulating carbohydrate (trehalose). Moreover, the mutants have extended life span, suggesting ppk11–dependent activities of the fat body have systematic and long–term effects on the fly body. Understanding the cellular function of ppk11 may offer new insights into mechanisms that regulate metabolic homeostasis.

No description available.

We have studied the modulation of gating properties of the Ca2+-permeable, cation channel TRPV4 transiently expressed in HEK293 cells. The phorbol ester 4αPDD transiently activated a current through TRPV4 in the presence of extracellular Ca2+. Increasing the concentration of extracellular Ca2+ ([Ca2+]e) reduced the current amplitude and accelerated its decay. This decay was dramatically delayed in the absence of [Ca2+]e. It was also much slower in the presence of [Ca2+]e in a mutant channel, obtained by a point mutation in the 6th transmembrane domain, F707A. Mutant channels, containing a single mutation in the C-terminus of TRPV4 (E797 , were constitutively open. In conclusion, gating of the 4αPDD-activated TRPV4 channel depends on both extra- and intracellular Ca2+, and is modulated by mutations of single amino acid residues in the 6th transmembrane domain and the C-terminus of the TRPV4 protein.

Nociception is the sensory nervous system that detects harmful stimuli including excessive heat, cold, toxic chemicals, and noxious mechanical stimulations. Transient receptor potential (TRP) channels are a group of evolutionarily conserved ion channels consisting of 4 subunits, each with 6 transmembrane spans, and detect a variety of external and internal nociceptive stimuli. Due to their critical roles in nociception, it is essential to understand the mechanisms that regulate TRP channels and subsequent nociception. Here, I investigated two distinct types of regulation of Drosophila transient receptor potential cation channel A1 (TrpA1): regulation via the expression of different TrpA1 isoforms, and via its binding with associated proteins. I found that one of the TrpA1 isoforms, TrpA1(E), inhibits the thermal responses of other TrpA1 isoforms in vitro. I also identified potential TrpA1 binding partners through Co- immunoprecipitation (Co-IP) and mass spectrometry analysis. These binding partners need further validation and characterization through biochemical, cellular, and behavioral assays to illustrate their roles in nociception, and may serve as potential drug targets for chronic pain.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19753号 / 工博第4208号 / 新制||工||1649(附属図書館) / 32789 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 濵地 格, 教授 梅田 眞郷 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20064号 / 工博第4252号 / 新制||工||1658(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 跡見 晴幸, 教授 梅田 眞郷 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM