[EN] The plasma membrane H+-ATPase (Pma1) is essential for yeast growth and is activated by glucose metabolism by an unknown mechanism involving double phosphorylation of a regulatory site at the C-terminus (Ser911 Thr912). In this thesis we have investigated in Saccharomyces cerevisiae the role of two protein phosphatases, type 1 Glc7 and type 2A Sit4, and of an essential atypical protein kinase, TORC1, in the activation of Pma1 by glucose. The regulatory site of activated Pma1 can be dephosphorylated "in vitro" by recombinant Glc7 and Sit4, but inhibition "in vivo" of these phosphatases does not activate Pma1. Inhibition of Glc7 by regulated expression of a dominant-negative truncated form (the null mutant is not viable) had no effect on Pma1 activity while deletion of SIT4 gene decreased both Pma1 activity and double phosphorylation of the regulatory site. Inhibition of TORC1 protein kinase by treatment of yeast cells with the drug rapamycin or by exposure to non-permissive temperature of a temperature-sensitive mutant (tor1¿ tor2ts) inhibited Pma1 and decreased double phosphorylation of the regulatory site. We conclude that Sit4 and TORC1 are required for full activation of Pma1 by glucose while Glc7 either does not participate or is redundant with other phosphatases. / [ES] La H+-ATPasa de la membrana plasmática (Pma1) es esencial para el crecimiento de la levadura y se activa por metabolismo de glucosa por un mecanismo desconocido que lleva consigo la doble fosforilación de un sitio regulador en el extremo C-terminal (Ser911 Thr912). En la presente tesis hemos investigado en Saccharomyces cerevisiae la participación de dos proteína fosfatasas, Glc7 de tipo 1 y Sit4 de tipo 2A, y de una proteína kinasa atípica esencial, TORC1, en la activación de Pma1 por glucosa. El sitio regulador de Pma1 en su estado activo puede defosforilarse "in vitro" por Glc7 y Sit4 recombinantes pero la inhibición "in vivo" de estas fosfatasas no activa Pma1. La inhibición de Glc7 mediante la expresión regulada de una forma truncada que actúa como dominante-negativa (el mutante nulo no es viable) no tiene efecto en la actividad de Pma1 mientras que la deleción del gen SIT4 disminuye tanto la actividad de Pma1 como la doble fosforilación del sitio regulador. Inhibición de la proteína kinasa TORC1 mediante tratamiento de las células de levadura con el fármaco rapamicina o exponiéndolas a temperatura no permisiva en el caso de un mutante termosensible (tor1¿ tor2ts) resulta en inhibición de Pma1 y disminución de la doble fosforilación del sitio regulador. Estos resultados indican que Sit4 y TORC1 son necesarias para la máxima activación de Pma1 por glucosa mientras que Glc7 podría no participar o hacerlo de forma redundante con otras fosfatasas. / [CAT] L'H+-ATPasa de la membrana plasmàtica (Pma1) és essencial per al creixement dels llevats i s'activa gràcies al metabolisme de glucosa per un mecanisme desconegut que porta associat la doble fosforilació d'una regió reguladora a l'extrem C-terminal (Ser911 Thr912). En aquesta tesi hem investigat en Saccharomyces cerevisiae la participació de dos proteïnes fosfatases, Glc7 de tipus 1 i Sit4 de tipus 2A, i d'una proteïna quinasa essencial atípica, TORC1, en l'activació de Pma1 per glucosa. La regió reguladora de Pma1, en seu estat activat, pot desfosforar-se "in vitro" per Glc7 i Sit4 recombinants, però la inhibició "in vivo" d'aquestes fosfatases no activa Pma1. La inhibició de Glc7 mitjançant l'expressió regulada d'una forma truncada que actua com a dominant-negativa (el mutant nul no és viable) no té cap efecte en l'activitat de Pma1 mentre que la deleció del gen SIT4 disminueix tant l'activitat de Pma1 com la doble fosforilació de la regió reguladora. La inhibició de la proteïna quinasa TORC mitjançant un tractament de cèl·lules de llevat amb el fàrmac rapamicina o la seua exposició a temperatures no permissives en el cas d'un mutant termosensible (tor1¿ tor2ts) resulta en la inhibició de Pma1 i la disminució de la doble fosforilació de la regió reguladora. Aquests resultats indiquen que Sit4 i TORC1 són necessàries per a l'activació màxima de Pma1 per glucosa, mentre que Glc7 podria no participar o fer-ho d'una forma redundant amb altres fosfatases. / Mahmoud Ali Ibrahim Hamouda, S. (2015). Protein kinases and phosphatases regulating the yeast proton pump [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/54131 / TESIS

In this work, we studied two yeast ABC transporters, Pdr10p and Pdr15p. At the time of assignment of this thesis, it was believed that these proteins contribute to the yeast MDR phenotype (PDR) on the grounds of their high homology to another yeast MDR protein, Pdr5p. In order to study these pumps, two sets of isogenic null-mutant strains were prepared with all possible combinations of gene deletions. We report that both of the studied proteins are very important in sus- taining the normal plasma membrane microenvironment for the most abundant, and essential, yeast plasma membrane protein, H+ -ATPase and so influence the membrane potential. Pdr10p and Pdr15p thus play an as yet unknown role in reg- ulation of the activity of this enzyme. Furthermore, we report that deletion of the genes coding for these proteins severely reduces the ability of the H+ -ATPase to be activated by the protonophore CCCP which is a weak acid. Studies performed with immunosuppressant FK506 further show that this compound reduces the viability of S. cerevisiae mutant strain PLY643 lacking genes coding for Pdr5p, Snq2p and Yor1p. Further deletion of Pdr10p and Pdr15p does not increase the lethality of this compound. Neither CCCP nor FK506 are substrates of the stud- ied pumps. 1

Vor zehn Jahren wurde der (Pro)Renin-Rezeptor [(P)RR] entdeckt und als neuer Bestandteil des Renin-Angiotensin-Systems beschrieben. Neuere Studien ergaben, dass der (P)RR mit der vakuolären H+-ATPase (V-ATPase) assoziiert sein kann, weshalb er auch V-ATPase associated protein 2 (ATP6ap2) genannt wird. In Osteoklasten befinden sich V-ATPase hauptsächlich an der zur Knochenoberfläche gerichteten Plasmamembran und transportieren Protonen in den extrazellulären Raum. Mäuse mit genetischer Deletion verschiedener V-ATPase-Untereinheiten charakterisiert durch einen Anstieg von Knochenmasse (Osteopetrose). In der vorliegenden Arbeit fanden wir heraus, dass (P)RR stark in reifen Osteoklasten in vitro und in vivo exprimiert wird. Mäuse mit genetischer Deletion des (P)RR in Osteoklasten wurden durch einen komplexen Knochen-Phänotyp mit reduzierter Knochendichte charakterisiert. (P)RR-defiziten Osteoklasten wiesen vermehrte Differenzierung und/oder Aktivität in vitro und in vivo auf. Wir postulieren deshalb, dass der (P)RR die in der Plasmamembran lokalisierten V-ATPase nicht direkt reguliert, sondern mit der physiologischen Aktivität der Osteoklasten durch andere Mechanismen interferiert. Macrophagen sind speziell auf die Immunabwehr ausgerichtete Fresszellen (Phagozyten). Phagozytose ist ein wesentlicher Zellprozess der die V-ATPase in Lysosomen braucht um die eingeschlossenen Pathogen zu zerstören. Wir generierten transgene Ratten mit konditionellen knockdown von (P)RR unter Nutzung eines Doxyzyclin-induzierten shRNA-Expressionssystems. Eine effiziente (P)RR-Depletion in Makrophagen wurde durch Behandlung mit Doxyzyclin in vivo im Trinkwasser und in vitro im Kulturmedium erreicht. Die vorliegende Arbeit zeigt, dass die Verschiebung des vesikulären pHs erst ziemlich spät nach (P)RR-Depletion auftritt. Wir fanden heraus, dass (P)RR-Depletion weder Phagozytose noch Endozytose beeinträchtigte, sondern für das Recycling des Transferrin-Rezeptors zur Plasmamembran wichtig ist. / A decade ago, the (pro)renin receptor [(P)RR] was discovered and depicted as a new component of the renin-angiotensin system. However, recent studies have put in evidence that the (P)RR associate with and regulate the vacuolar H+-ATPase (V-ATPase), hence its other name vacuolar H+-ATPase associated protein 2 (ATP6ap2). In osteoclasts, V-ATPases are mainly located at the plasma membrane facing the bone surface and extrude protons into the extracellular space. Mice with genetic deletion of various V-ATPase subunits are characterized by an increase of bone mass (osteopetrosis). In this work, we found that the (P)RR is highly expressed in mature osteoclasts in vitro and in vivo. Mice with genetic deletion of the (P)RR in osteoclasts developed a complex bone phenotype characterized by a reduced bone density. Osteoclasts lacking (P)RR displayed increased differentiation and/or activity in vitro and in vivo. We therefore suggest that the (P)RR does not directly regulate V-ATPases located at the plasma membrane but rather interferes with osteoclast physiology through other mechanisms. Macrophages are professionalized phagocytes crucial for immune response. Phagocytosis is an essential cellular process, which requires lysosomal V-ATPases for degradation of engulfed pathogens. We generated transgenic rats with a conditional depletion of the (P)RR with the use of a doxycycline-induced shRNA expression system. Efficient (P)RR depletion in macrophages was accomplished by doxycycline treatment in vivo in drinking water and in vitro in culture medium. In this work, we found that the impairment of vesicular pH occurs lately after (P)RR deletion. Also, we found that (P)RR deletion did not impair neither phagocytosis nor endocytosis but rather perturbed the recycling of the transferrin receptor to the plasma membrane.

Este estudo investigou o efeito da glicose sobre a atividade e expressão da isoforma 1 do trocador Na+/H+ (NHE1) e da H+-ATPase do tipo vacuolar, em células MDCK (Mardin Darby Canine Kidney), linhagem derivada de rim de cão, que apresenta características similares às células principais e intercalares das porções distais do néfron. Por microscopia de fluorescência, se avaliou a velocidade de recuperação do pHi (dpHi/dt) e a capacidade tamponante (<font face=\"symbol\">bi). A partir desses parâmetros, se calculou o efluxo de H+ (JH+). Por Western blot, se avaliou a expressão de NHE1 e da subunidade E da H+-ATPase do tipo vacuolar. Resultados: Na condição controle o efluxo de H+ foi de 6.27 ± 0.51 mM/min (n = 9). O tratamento agudo com glicose (25 mM) aumentou o efluxo de H+ via NHE1, o qual foi modulado pela PI3 cinase. Na mesma condição, não se observou alterações na atividade da H+-ATPase. O tratamento crônico com glicose (25 mM) induziu significante aumento do efluxo de H+, via NHE1 e H+-ATPase. O efeito estimulador da glicose sobre a atividade de NHE1 e H+-ATPase foi dependente da atividade da p38 MAP cinase. Além disso, o tratamento crônico com glicose (25 mM) induziu fosforilação do sistema ezrin/radixin/moesin (ERM) e Akt. Conclusões: Nossos resultados indicam que no tratamento agudo com glicose (25 mM), o NHE1 foi modulado pela PI3 cinase. Contudo, no tratamento crônico com glicose (25 mM), a atividade do NHE1 foi modulada pelo sistema ERM/Akt e a atividade da H+-ATPase foi modulada pela p38 MAP cinase. / This study investigated the effect of glucose on the activity and expression of Na+/H+ exchanger isoform 1 (NHE1) and vacuolar H+-ATPase, in Mardin Darby Canine Kidney (MDCK) cells from dog kidney, with similar characteristics to principal and intercalated cells of the distal nephron. The pHi recovery rate (dpHi/dt) and the buffering capacity (<font face=\"symbol\">bi) was evaluated through fluorescence microscopy. From these parameters the H+ efflux (JH+) was calculated. By Western blot, the NHE1 and H+-ATPase (E subunit) expression was evaluated. Results: In the control situation the H+ efflux was 6.27 ± 0.51 mM/pH units (n = 9). Acute treatment with glucose (25 mM) increased the H+ efflux via NHE1, which was modulated by PI3 kinase. In the same condition, the H+-ATPase activity did not change. Chronic treatment with glucose (25 mM) induced significant increase in H+ efflux via NHE1 and H+-ATPase. The stimulatory effect of glucose on the NHE1 and H+-ATPase activity was dependent on p38 MAP kinase activity. Furthermore, chronic treatment with glucose (25 mM) induced Ezrin/radixin/moesin (ERM) and Akt phosphorylation. Conclusions: Our results indicate that during the acute treatment with glucose (25 mM), the NHE1 is modulated by PI3 kinase. However, during chronic treatment with glucose (25 mM), NHE1 activity was modulated by the ERM/Akt system and of H+-ATPase activity was modulated by p38 MAP Kinase.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / O presente trabalho teve por objetivo estudar as respostas fisiolÃgicas e bioquÃmicas de clones de cajueiro anÃo-precoce (Anacardium occidentale L.) ao estresse salino. Os experimentos foram conduzidos em casa de vegetaÃÃo, sendo as plÃntulas cultivadas em vasos plÃsticos contendo vermiculita. No primeiro experimento, cinco clones de cajueiro anÃo-precoce foram submetidos aos tratamentos com NaCl a 0 (controle), 8 e 16 dS.m-1 de condutividade elÃtrica e objetivou selecionar clones com tolerÃncias diferenciadas ao estresse salino. Para isso, foram estudados os efeitos da salinidade no crescimento, nas trocas gasosas, no teor de Ãgua, na suculÃncia foliar, no potencial osmÃtico, nas concentraÃÃes de prolina, N-aminossolÃveis e carboidratos solÃveis e nos teores dos Ãons inorgÃnicos (Na+, Cl- e K+). A salinidade reduziu o crescimento das plÃntulas de todos os clones estudados. Os efeitos inibitÃrios do NaCl foram mais conspÃcuos na parte aÃrea do que nas raÃzes. O clone CCP 06 foi aquele que apresentou maior reduÃÃo no crescimento foliar, enquanto os clones BRS 189 e CCP 09 foram os que apresentaram as menores reduÃÃes. A salinidade inibiu a mobilizaÃÃo das reservas cotiledonÃrias, principalmente, na dose mais elevada de sal. A reduÃÃo no crescimento, pela salinidade, correlacionou-se com a reduÃÃo na taxa de fotossÃntese lÃquida. Os clones CCP 06 e BRS 189 apresentaram, respectivamente, a maior e a menor reduÃÃo na taxa fotossintÃtica a 8 dS.m-1. Embora a salinidade tenha reduzido a condutÃncia estomÃtica dos clones de cajueiro anÃo-precoce, essa reduÃÃo nÃo foi acompanhada por mudanÃas nas concentraÃÃes internas de CO2. Os clones estudados nÃo apresentaram alteraÃÃes, em funÃÃo da salinidade, no estado hÃdrico das folhas e raÃzes, porÃm, apresentaram reduÃÃes no potencial osmÃtico, favorecendo o ajustamento osmÃtico e, consequentemente, a manutenÃÃo da turgescÃncia dos tecidos. Sob condiÃÃes de estresse salino, os clones BRS 189 e CCP 09 foram os mais eficientes na regulaÃÃo do transporte do Ãon Na+ para a parte aÃrea da plÃntula, acumulando-o nas raÃzes. Em relaÃÃo ao Cl-, o clone CCP 09 mostrou-se o mais eficiente no controle do transporte desse Ãon. PorÃm, CCP 06 foi o clone que mais acumulou ambos os Ãons tÃxicos na parte aÃrea da planta. Com o aumento da salinidade, os teores de potÃssio dos clones estudados tiveram seus valores reduzidos apenas nas raÃzes. Na dose de 8 dS.m-1, o BRS 189 foi o clone que mais aumento suas concentraÃÃes de N-aminosolÃveis e prolina no suco radicular. Nesse mesmo nÃvel de sal, a salinidade aumentou a concentraÃÃo de carboidratos apenas nos clones CCP 06 e BRS 189. De posse destes resultados, o segundo experimento foi realizado com os clones CCP 06 e BRS 189 que foram os que se mostraram, respectivamente, o menos e o mais tolerante à salinidade. Esse experimento teve por objetivo estudar os efeitos da salinidade (NaCl a 8 dS.m-1) na atividade da H+-ATPase e na composiÃÃo e peroxidaÃÃo dos lipÃdios de membrana plasmÃtica isoladas de raÃzes das plÃntulas dos dois clones contrastantes. A salinidade estimulou a atividade da H+-ATPase apenas no clone tolerante, o BRS 189, sendo esse clone o que apresentou maior conteÃdo de esterÃis totais e menor relaÃÃo fosfolipÃdios totais (PLt)/ esterÃis totais (Et), tanto em condiÃÃes controle como de estresse. Esses resultados foram concordantes com o fato de ter sido o BRS 189 o clone que melhor excluiu o Na+ da parte aÃrea. Nesse clone nÃo foram observadas alteraÃÃes nos teores de malondialdeÃdo, diferentemente do que ocorreu com o CCP 06, cujos teores aumentaram com o estresse salino. A maior proteÃÃo da membrana plasmÃtica do clone BRS 189 ao dano oxidativo està de acordo com os maiores acÃmulos de prolina e N-aminossolÃveis observados nesse clone. Os principais fosfolipÃdios da membrana plasmÃtica isolada de raÃzes do clone BRS 189 foram fosfatilglicerol (PG), fosfatidiletalonamina (PE) e fosfatilserina (PS). A salinidade provocou alteraÃÃes nas proporÃÃes relativas dos fosfolipÃdios, sendo PE e fosfatidilinositol (PI) os que apresentaram maiores aumentos em relaÃÃo ao total, enquanto que fosfatidilglicerol (PG) e Ãcido fosfatÃdico (PA) foram os que apresentaram maiores reduÃÃes. A percentagem de PS, em relaÃÃo ao total, nÃo foi afetada pela salinidade. No entanto, a relaÃÃo entre essas mudanÃas na composiÃÃo lipÃdica do BRS 189 pela salinidade e o aumento na atividade da H+-ATPase necessita ser melhor investigada. / Early-dwarf cashew seedlings (Anacardium occidentale L.) were used in order to investigate the physiological and biochemical changes induced by salt stress. The seeds (nuts) were sown in plastics pots containing vermiculite moistened with either distilled water (control treatment) or NaCl solutions at 8 and 16 dS.m-1 of electrical conductivity (saline treatment), and kept in greenhouse throughout the experimental period. Uniform 28-day-old seedlings were used for the analyses. The first experiment aimed to select, among five clones (CCP 06, CCP 09, CCP 76, Embrapa 51 and BRS 189), the ones showing contrasting salt-tolerance. The effect of salinity on the growth, gas exchange, water content, leaf succulence, osmotic potential and inorganic (Na+, Cl-, K+) and organic (proline, soluble carbohydrates, quaternary ammonium compounds) solute concentration for both salt-sensitive and salt-tolerant clones was studied. Salinity inhibited the growth of all clones studied, being the inhibitory effect on shoot growth more conspicuous than in root growth. Clone CCP 06 leaf area was the most inhibited by salt stress, while clones BRS 189 and CCP 09 leaf areas were the least affected by salinity. Salt stress caused a great decrease in the cotyledon reserve mobilization especially at 16 dS.m-1. Growth reduction was correlated to the reduction in net photosynthetic rate. CCP 06 and BRS 189 showed the greatest and the lowest reduction in photosynthetic rate at 8 dS.m-1, respectively. Although, salinity reduced stomatal conductance, this reduction was not followed by changes in CO2 internal concentration. The water status, expressed as water content in relation to dry mass, was not changed by salt-stress. Salinity induced the lowering of osmotic potential both in leaves and roots of all clones studied. This osmotic adjustment might have lead to turgor maintenance of those tissues. The concentrations of Cl- and Na+ increased with increasing salt stress. Clones BRS 189 and CCP 09 accumulated more Na+ in the roots, and this could explain their efficiency in maintaining a lower ion concentration in shoots, i.e. they regulated more efficiently the transport of Na+ from roots to shoots. The regulation of Cl- transport to shoots was more efficient in clone CCP 09 than in the others. Salinity did not induce significant changes in leaves and stems K+ concentration, but it induced a reduction of K+ concentration in roots. Salinity also induced increases of quaternary ammonium compounds and proline concentration in BRS 189 root at 8 dS.m-1. In addition, this level of salinity increased soluble carbohydrates in the root sap especially in clones BRS 189 and CCP 06. During the second experiment, the effect of salt stress (NaCl at 8 dS.m-1) on the activity of H+-ATPase, lipid composition and peroxidation of root plasma membrane of both salt-tolerant (BRS 189) and salt- sensitive (CCP 06) clones were studied. The vanadate-sensitive H+-ATPase activity was studied in plasma membrane-enriched vesicles isolated by discontinuous sucrose gradient centrifugation from roots. ATP hidrolizing activity in this fraction was mostly inhibited by vanadate and scarcely, by azide and molybdate, indicating that it was essentially enriched in plasma membrane vesicles. Salinity induced a 1.3-fold increase in the H+-ATPase specific activity in roots of BRS 189 seedlings. Salinity had no appreciable effect on the hydrolytic activity of this enzyme during the growth of CCP 06 seedlings. Likewise, clone BRS 189 roots plasma membrane showed higher sterol content and lower phospholipids/total sterol ratio than clone CCP 06. Both properties could contribute to the decrease in Na+ influx or increase in Na+ efflux or âexclusionâ from roots. This could result in less Na+ being transported to the shoot, and thus explaining the higher salt-tolerance of clone BRS 189. The higher degree of root plasma membrane lipid peroxidation of clone, and the lower proline and ammonium quaternary compounds contents of CCP 06 when compared to BRS 189 could also explain the differences in salt-tolerance between the two clones. These organic solutes could protect and stabilize plasma membrane against oxidative stress. Phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and phosphatidylserine (PS) were the major phospholipids in the plasma membrane from BRS 189 roots. Salinity induced increases in the relative proportions of PE and phosphatidylinositol (PI), while PG and PA were reduced. No changes were detected in PS in relation to control plant. The importance of lipid composition changes on H+-ATPase activity must be more studied.

PIZARRO, Juan Carlos Alvarez. Caracteres fisiológicos e bioquímicos da tolerância à salinidade em clones de cajueiro anão precoce. 2006. 135 f. Dissertação (Mestrado em Bioquímica) - Universidade Federal do Ceará, Fortaleza-CE, 2006. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-05-30T14:16:32Z No. of bitstreams: 1 2006_dis_jcapizarro.pdf: 935692 bytes, checksum: c709b8b0747f8a929112611a9c584729 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-07-12T23:10:54Z (GMT) No. of bitstreams: 1 2006_dis_jcapizarro.pdf: 935692 bytes, checksum: c709b8b0747f8a929112611a9c584729 (MD5) / Made available in DSpace on 2016-07-12T23:10:54Z (GMT). No. of bitstreams: 1 2006_dis_jcapizarro.pdf: 935692 bytes, checksum: c709b8b0747f8a929112611a9c584729 (MD5) Previous issue date: 2006 / Early-dwarf cashew seedlings (Anacardium occidentale L.) were used in order to investigate the physiological and biochemical changes induced by salt stress. The seeds (nuts) were sown in plastics pots containing vermiculite moistened with either distilled water (control treatment) or NaCl solutions at 8 and 16 dS.m-1 of electrical conductivity (saline treatment), and kept in greenhouse throughout the experimental period. Uniform 28-day-old seedlings were used for the analyses. The first experiment aimed to select, among five clones (CCP 06, CCP 09, CCP 76, Embrapa 51 and BRS 189), the ones showing contrasting salt-tolerance. The effect of salinity on the growth, gas exchange, water content, leaf succulence, osmotic potential and inorganic (Na+, Cl-, K+) and organic (proline, soluble carbohydrates, quaternary ammonium compounds) solute concentration for both salt-sensitive and salt-tolerant clones was studied. Salinity inhibited the growth of all clones studied, being the inhibitory effect on shoot growth more conspicuous than in root growth. Clone CCP 06 leaf area was the most inhibited by salt stress, while clones BRS 189 and CCP 09 leaf areas were the least affected by salinity. Salt stress caused a great decrease in the cotyledon reserve mobilization especially at 16 dS.m-1. Growth reduction was correlated to the reduction in net photosynthetic rate. CCP 06 and BRS 189 showed the greatest and the lowest reduction in photosynthetic rate at 8 dS.m-1, respectively. Although, salinity reduced stomatal conductance, this reduction was not followed by changes in CO2 internal concentration. The water status, expressed as water content in relation to dry mass, was not changed by salt-stress. Salinity induced the lowering of osmotic potential both in leaves and roots of all clones studied. This osmotic adjustment might have lead to turgor maintenance of those tissues. The concentrations of Cl- and Na+ increased with increasing salt stress. Clones BRS 189 and CCP 09 accumulated more Na+ in the roots, and this could explain their efficiency in maintaining a lower ion concentration in shoots, i.e. they regulated more efficiently the transport of Na+ from roots to shoots. The regulation of Cl- transport to shoots was more efficient in clone CCP 09 than in the others. Salinity did not induce significant changes in leaves and stems K+ concentration, but it induced a reduction of K+ concentration in roots. Salinity also induced increases of quaternary ammonium compounds and proline concentration in BRS 189 root at 8 dS.m-1. In addition, this level of salinity increased soluble carbohydrates in the root sap especially in clones BRS 189 and CCP 06. During the second experiment, the effect of salt stress (NaCl at 8 dS.m-1) on the activity of H+-ATPase, lipid composition and peroxidation of root plasma membrane of both salt-tolerant (BRS 189) and salt- sensitive (CCP 06) clones were studied. The vanadate-sensitive H+-ATPase activity was studied in plasma membrane-enriched vesicles isolated by discontinuous sucrose gradient centrifugation from roots. ATP hidrolizing activity in this fraction was mostly inhibited by vanadate and scarcely, by azide and molybdate, indicating that it was essentially enriched in plasma membrane vesicles. Salinity induced a 1.3-fold increase in the H+-ATPase specific activity in roots of BRS 189 seedlings. Salinity had no appreciable effect on the hydrolytic activity of this enzyme during the growth of CCP 06 seedlings. Likewise, clone BRS 189 roots plasma membrane showed higher sterol content and lower phospholipids/total sterol ratio than clone CCP 06. Both properties could contribute to the decrease in Na+ influx or increase in Na+ efflux or “exclusion” from roots. This could result in less Na+ being transported to the shoot, and thus explaining the higher salt-tolerance of clone BRS 189. The higher degree of root plasma membrane lipid peroxidation of clone, and the lower proline and ammonium quaternary compounds contents of CCP 06 when compared to BRS 189 could also explain the differences in salt-tolerance between the two clones. These organic solutes could protect and stabilize plasma membrane against oxidative stress. Phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and phosphatidylserine (PS) were the major phospholipids in the plasma membrane from BRS 189 roots. Salinity induced increases in the relative proportions of PE and phosphatidylinositol (PI), while PG and PA were reduced. No changes were detected in PS in relation to control plant. The importance of lipid composition changes on H+-ATPase activity must be more studied. / O presente trabalho teve por objetivo estudar as respostas fisiológicas e bioquímicas de clones de cajueiro anão-precoce (Anacardium occidentale L.) ao estresse salino. Os experimentos foram conduzidos em casa de vegetação, sendo as plântulas cultivadas em vasos plásticos contendo vermiculita. No primeiro experimento, cinco clones de cajueiro anão-precoce foram submetidos aos tratamentos com NaCl a 0 (controle), 8 e 16 dS.m-1 de condutividade elétrica e objetivou selecionar clones com tolerâncias diferenciadas ao estresse salino. Para isso, foram estudados os efeitos da salinidade no crescimento, nas trocas gasosas, no teor de água, na suculência foliar, no potencial osmótico, nas concentrações de prolina, N-aminossolúveis e carboidratos solúveis e nos teores dos íons inorgânicos (Na+, Cl- e K+). A salinidade reduziu o crescimento das plântulas de todos os clones estudados. Os efeitos inibitórios do NaCl foram mais conspícuos na parte aérea do que nas raízes. O clone CCP 06 foi aquele que apresentou maior redução no crescimento foliar, enquanto os clones BRS 189 e CCP 09 foram os que apresentaram as menores reduções. A salinidade inibiu a mobilização das reservas cotiledonárias, principalmente, na dose mais elevada de sal. A redução no crescimento, pela salinidade, correlacionou-se com a redução na taxa de fotossíntese líquida. Os clones CCP 06 e BRS 189 apresentaram, respectivamente, a maior e a menor redução na taxa fotossintética a 8 dS.m-1. Embora a salinidade tenha reduzido a condutância estomática dos clones de cajueiro anão-precoce, essa redução não foi acompanhada por mudanças nas concentrações internas de CO2. Os clones estudados não apresentaram alterações, em função da salinidade, no estado hídrico das folhas e raízes, porém, apresentaram reduções no potencial osmótico, favorecendo o ajustamento osmótico e, consequentemente, a manutenção da turgescência dos tecidos. Sob condições de estresse salino, os clones BRS 189 e CCP 09 foram os mais eficientes na regulação do transporte do íon Na+ para a parte aérea da plântula, acumulando-o nas raízes. Em relação ao Cl-, o clone CCP 09 mostrou-se o mais eficiente no controle do transporte desse íon. Porém, CCP 06 foi o clone que mais acumulou ambos os íons tóxicos na parte aérea da planta. Com o aumento da salinidade, os teores de potássio dos clones estudados tiveram seus valores reduzidos apenas nas raízes. Na dose de 8 dS.m-1, o BRS 189 foi o clone que mais aumento suas concentrações de N-aminosolúveis e prolina no suco radicular. Nesse mesmo nível de sal, a salinidade aumentou a concentração de carboidratos apenas nos clones CCP 06 e BRS 189. De posse destes resultados, o segundo experimento foi realizado com os clones CCP 06 e BRS 189 que foram os que se mostraram, respectivamente, o menos e o mais tolerante à salinidade. Esse experimento teve por objetivo estudar os efeitos da salinidade (NaCl a 8 dS.m-1) na atividade da H+-ATPase e na composição e peroxidação dos lipídios de membrana plasmática isoladas de raízes das plântulas dos dois clones contrastantes. A salinidade estimulou a atividade da H+-ATPase apenas no clone tolerante, o BRS 189, sendo esse clone o que apresentou maior conteúdo de esteróis totais e menor relação fosfolipídios totais (PLt)/ esteróis totais (Et), tanto em condições controle como de estresse. Esses resultados foram concordantes com o fato de ter sido o BRS 189 o clone que melhor excluiu o Na+ da parte aérea. Nesse clone não foram observadas alterações nos teores de malondialdeído, diferentemente do que ocorreu com o CCP 06, cujos teores aumentaram com o estresse salino. A maior proteção da membrana plasmática do clone BRS 189 ao dano oxidativo está de acordo com os maiores acúmulos de prolina e N-aminossolúveis observados nesse clone. Os principais fosfolipídios da membrana plasmática isolada de raízes do clone BRS 189 foram fosfatilglicerol (PG), fosfatidiletalonamina (PE) e fosfatilserina (PS). A salinidade provocou alterações nas proporções relativas dos fosfolipídios, sendo PE e fosfatidilinositol (PI) os que apresentaram maiores aumentos em relação ao total, enquanto que fosfatidilglicerol (PG) e ácido fosfatídico (PA) foram os que apresentaram maiores reduções. A percentagem de PS, em relação ao total, não foi afetada pela salinidade. No entanto, a relação entre essas mudanças na composição lipídica do BRS 189 pela salinidade e o aumento na atividade da H+-ATPase necessita ser melhor investigada.

Este estudo investigou o efeito da glicose sobre a atividade e expressão da isoforma 1 do trocador Na+/H+ (NHE1) e da H+-ATPase do tipo vacuolar, em células MDCK (Mardin Darby Canine Kidney), linhagem derivada de rim de cão, que apresenta características similares às células principais e intercalares das porções distais do néfron. Por microscopia de fluorescência, se avaliou a velocidade de recuperação do pHi (dpHi/dt) e a capacidade tamponante (<font face=\"symbol\">bi). A partir desses parâmetros, se calculou o efluxo de H+ (JH+). Por Western blot, se avaliou a expressão de NHE1 e da subunidade E da H+-ATPase do tipo vacuolar. Resultados: Na condição controle o efluxo de H+ foi de 6.27 ± 0.51 mM/min (n = 9). O tratamento agudo com glicose (25 mM) aumentou o efluxo de H+ via NHE1, o qual foi modulado pela PI3 cinase. Na mesma condição, não se observou alterações na atividade da H+-ATPase. O tratamento crônico com glicose (25 mM) induziu significante aumento do efluxo de H+, via NHE1 e H+-ATPase. O efeito estimulador da glicose sobre a atividade de NHE1 e H+-ATPase foi dependente da atividade da p38 MAP cinase. Além disso, o tratamento crônico com glicose (25 mM) induziu fosforilação do sistema ezrin/radixin/moesin (ERM) e Akt. Conclusões: Nossos resultados indicam que no tratamento agudo com glicose (25 mM), o NHE1 foi modulado pela PI3 cinase. Contudo, no tratamento crônico com glicose (25 mM), a atividade do NHE1 foi modulada pelo sistema ERM/Akt e a atividade da H+-ATPase foi modulada pela p38 MAP cinase. / This study investigated the effect of glucose on the activity and expression of Na+/H+ exchanger isoform 1 (NHE1) and vacuolar H+-ATPase, in Mardin Darby Canine Kidney (MDCK) cells from dog kidney, with similar characteristics to principal and intercalated cells of the distal nephron. The pHi recovery rate (dpHi/dt) and the buffering capacity (<font face=\"symbol\">bi) was evaluated through fluorescence microscopy. From these parameters the H+ efflux (JH+) was calculated. By Western blot, the NHE1 and H+-ATPase (E subunit) expression was evaluated. Results: In the control situation the H+ efflux was 6.27 ± 0.51 mM/pH units (n = 9). Acute treatment with glucose (25 mM) increased the H+ efflux via NHE1, which was modulated by PI3 kinase. In the same condition, the H+-ATPase activity did not change. Chronic treatment with glucose (25 mM) induced significant increase in H+ efflux via NHE1 and H+-ATPase. The stimulatory effect of glucose on the NHE1 and H+-ATPase activity was dependent on p38 MAP kinase activity. Furthermore, chronic treatment with glucose (25 mM) induced Ezrin/radixin/moesin (ERM) and Akt phosphorylation. Conclusions: Our results indicate that during the acute treatment with glucose (25 mM), the NHE1 is modulated by PI3 kinase. However, during chronic treatment with glucose (25 mM), NHE1 activity was modulated by the ERM/Akt system and of H+-ATPase activity was modulated by p38 MAP Kinase.

Teleost fishes incorporate renal ammonia excretion as part of a greater acid-base regulatory system. However, the transport mechanisms employed by the renal epithelium to excrete ammonia are relatively unknown. I hypothesized that, under metabolic acidosis, increased renal ammonia excretion would be the product of tubular secretion and involve a Na+/NH4+ exchange metabolon mediated through Rhesus (Rh) glycoproteins. To induce metabolic acidosis, goldfish (Carassius auratus) were exposed to a low pH environment (pH 4.0; 48-h). There was a clear signal of metabolic acidosis: a reduction in both plasma [HCO3-] and blood pH with no influence on plasma PCO2. Goldfish demonstrated an elevation in total plasma [ammonia] with a reduction in PNH3 under acidosis. Metabolic acidosis induced higher rates of urinary excretion of acidic equivalents in the form of both NH4+ and titratable acidity-HCO3- (TA-HCO3-) excretion. Urinary Na+ excretion was not affected by acidosis and urine [Na+] did not correlate with urinary [ammonia]. Alanine aminotransferase activity in the kidney was higher in acidotic goldfish. Glomerular filtration rate and urine flow rate were not affected by acidosis. Increased renal NH4+ excretion was due to increased secretion, and not increased filtration, of ammonia. There was a corresponding elevation in Rhcg1b mRNA expression but no change in renal Na+ reabsorption. My data support a secretion-based mechanism of teleost renal ammonia transport. This system is Na+ independent and is likely mediated by Rh glycoproteins and H+ ATPase, involving a parallel H+/NH3 secretion mechanism. To investigate effects of metabolic acidosis on elasmobranch fish, Pacific spiny dogfish (Squalus acanthias suckleyi) were infused with an acidic saline (125 mM HCl/375 mM NaCl; 3 ml/kg/h; 24-h). The results are preliminary, with no marked effects of HCl infusion on plasma acid-base or N-status, but increased branchial NHE2 and lower renal NHE3 protein expressions. These data are summarized in an Appendix. / Thesis / Master of Science (MSc)