Master of Science / Department of Entomology / Kristopher S. Silver / Kun Yan Zhu / The yellow fever mosquito, Aedes aegypti, is a significant vector of several viral diseases, including Zika, dengue fever, yellow fever, and chikungunya. Since vaccines are not currently available for these viruses, control of the disease vectors by using insecticides is the most common practice for preventing disease. As a result, Ae. aegypti has developed resistance against many of the most commonly used insecticides, including organophosphates and pyrethroids. The rise in resistance in vector mosquitoes requires the search for new control strategies, such as RNA interference (RNAi), to manage mosquito populations. Vacuolar H[sup plus]+-ATPase (V-ATPase), a multi-subunit enzyme involved in many cellular processes, including membrane energization, acidification of organelles, and entry of dengue virus into the cytoplasm, is a potential target for RNAi, though little is known about its genetic structure or expression patterns in Ae. aegypti. In this study, I performed genome-wide surveys to identify the genes encoding different subunits of the V-ATPase protein complex, partially characterized the molecular properties and expression patterns of selected V-ATPase subunit genes, and tested the feasibility of using oral-based delivery of nanoparticles formed from double-stranded RNA (dsRNA) and chitosan to suppress the expression of selected V-ATPase subunit genes in Ae. aegypti. My genome-wide surveys revealed that Ae. aegypti V-ATPase consists of 13 different subunits (A, B, C, D, E, F, G, H, a, c, c”, d, e) encoded by 14 genes. Analysis of exon-intron arrangements for each gene demonstrated that each V-ATPase subunit gene has between one (subunit c) and 12 (subunit C) exons, with most genes (11) having 3 to 6 exons. Subsequent phylogenetic analysis of the deduced amino acid sequences of each subunit showed that V-ATPase subunits A, B, C, F, G, H, and a exhibited high levels of conservation among all the examined species, but subunits D, E, c, c”, d, and e showed high conservation only among dipteran species. Analysis of the expression profiles in different tissues and developmental stages of three specific V-ATPase subunits (A, D, and H) showed that whereas the expression of these genes varied between tissues and developmental stages, the patterns of expression of subunits A, D, and H were very similar. The highest mRNA expression level was observed in Malpighian tubules in fourth-instar larvae. Interestingly, expression of subunits A, D, or H in different tissues of adults was highest in male hindgut versus Malpighian tubules in females. Feeding mosquito larvae with chitosan nanoparticles made with dsRNA complementary to subunits A, D, or H resulted in significant suppression of mRNA transcript levels of each of these subunits. Peak suppression of V-ATPase A, D, or H transcripts occurred on the fifth day, where the gene transcript level was suppressed by 66.0, 27.3, or 70.4%, respectively, as compared with those of the control. Additionally, feeding of dsRNA/chitosan nanoparticles targeting subunit D caused mortality starting on day 3, with cumulative larval mortality reaching 14.8% on the sixth day. These results suggest that oral delivery of dsRNA/chitosan nanoparticles can substantially suppress target gene expression in Ae. aegypti larvae. However, increasing RNAi efficiency in targeting V-ATPase subunit genes in mosquito larvae appears to be necessary in order to obtain higher larval mortality using oral delivery of dsRNA/chitosan nanoparticles.

<div>Ebolavirus, a genus of filoviruses, are responsible for outbreaks that cause up to 90% fatality, including the recent outbreak in West Africa that has resulted in over 28,603 reported cases and 11,301 deaths according to the WHO. Inhibitors of Vacuolar-ATPase (V-ATPase), a key protein complex that is responsible for endosomal acidification and represents a unique method to block this common viral pathway. V-ATPase inhibitors have previously been explored as therapies for many diseases but have failed due to high toxicity. Diphyllin is a natural, arylnaphthalene lignan that represents a novel structural class of V-ATPase inhibitors with a greater selectivity index than previous V-ATPase inhibitors. Diphyllin has shown promising anti-tumor and anti-osteoclast activity, as well as strong anti-viral activity against Influenza and Dengue viruses. </div><div>Herein, novel modifications of the lactone and phenol functional groups of diphyllin were explored for the ability to enhance the potency or therapeutic selectivity of the diphyllin core. Four initial sets of derivatives were synthesized and assayed for activity against ebolavirus infection, inhibition of cellular endosomal acidification, cytotoxicity and biochemical inhibition of isolated V-ATPase. Modification of diphyllin’s lactone functional group reduced both activity and selectivity, while alkylation of the phenol groups significantly enhanced activity. The incorporation of basic heterocycles to the alkyl group created an alkylamino series of derivatives that exhibited significantly improved therapeutic selectivity compared to diphyllin. Further investigation of the alkylamino class indicated that they retained activity against Marburgvirus infection, a filovirus related to Ebolavirus. Alkylamino derivatives inhibited ebolavirus infection of human macrophages at low micromolar levels with no apparent cytotoxicity.</div><div>Further investigation of the alkylamino class of diphyllin derivatives was conducted to determine if potency and/or therapeutic selectivity could be optimized. The addition of a 1-methylpiperazine moiety to the end of the alkyl chain improved potency 1260-fold over diphyllin, though therapeutic selectivity was not improved. The modification of the alkylamino linker to an acetamide eliminated cytotoxicity but decreased derivative activity against V-ATPase activity. To evaluate if the cytotoxicity evidenced by the alkylamino derivatives was evidenced in organisms, the derivative toxicity was assessed in zebrafish and mouse models. Derivatives displayed toxicity in a zebrafish developmental model but were all at least 10-fold less toxic than the known V-ATPase inhibitor bafilomycin A1. Three derivatives were well tolerated in CD-1 mice when administered at therapeutically relevant concentrations and caused no abnormal changes in their blood chemistry. Overall, these results demonstrate that the alkylamino and acetamide diphyllin phenol derivatives should be further studied as therapies for ebolavirus infection in addition to other V-ATPase mediated diseases.</div><div><br></div>

In this dissertation I am using M. truncatula as a model legume that forms indeterminate nodules with rhizobia under limited nitrogen conditions. I take advantage of an M. truncatula Tnt1 mutant population that provides a useful resource to uncover and characterize novel genes. Here, I focused on several objectives. First, I carried out forward and reverse genetic screening of M. truncatula Tnt1 mutant populations to uncover novel genes involved in symbiotic nitrogen fixation. Second, I focused on reverse genetic screening of two genes, identified as encoding blue copper proteins, and characterization of their mutants' potential phenotypes. Third, I further characterized a nodule essential gene, M. truncatula vacuolar iron transporter like 8 (MtVTL8), which encodes a nodule specific iron transporter. I characterized the expression pattern, expression localization and function of MtVTL8. Additionally, I characterized several residues predicted to be essential to function using a model based on the known crystal structure of Eucalyptus grandis vacuolar iron transporter 1 (EgVIT1), a homologous protein to MtVTL8. I identified several potential essential residues of the MtVTL8 protein, mutagenized them, and through complementation experiments in planta and in yeast assessed functionality of the resulting protein. This helped us to better understand the potential mechanism by which MtVTL8 functions.

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.

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.

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Sugarcane is a crop that is farmed commercially due to the high amounts of sucrose that is stored within the mature internodes of the stem. Numerous studies have been done to understand sugar metabolism in this crop as well as to enhance sucrose yields. Until now sugarcane improvement strategies have been implemented through either breeding programs or transgenic manipulation. Public mistrust and regulatory hurdles, however, have made the commercialisation of transgenic crops difficult, expensive and timeconsuming. In this thesis two projects will address issues relating to the above. The first will address an effort to increase sucrose accumulation within the sugarcane culm. This was attempted via the expression of an Arabidopsis thaliana vacuolar pyrophosphatase (AtV-PPase) gene, linked to the maize ubiquitin promoter, in sugarcane callus. It was anticipated that increased activity of the tonoplast-bound AtV-PPase will result in increased sucrose accumulation in the vacuole. Transgenic sugarcane callus lines were tested for soluble sugar content which suggested no significant increase in sucrose content. However, this may change upon further assessment of sugarcane suspension cultures and glasshouse plants. The second project was concerned with the development of a novel sugarcane transformation technology that utilises only sugarcane sequences. This ‘cisgenic’ approach to sugarcane transformation will require a native sugarcane promoter, terminator, vector backbone and selection marker. It was attempted to first isolate a functional promoter as well as developing a selection system based on an endogenous selection marker. A promoter was amplified from sugarcane, using primers designed on a sorghum template, and its expression assessed using a GFP reporter gene. Unfortunately expression could not be confirmed in transgenic sugarcane callus. Currently, an alternative approach is followed by using short fragments of constitutively expressed genes to screen sugarcane Bacterial Artificial Chromosome (BAC) libraries to isolate their corresponding promoters. Lastly, it was attempted to develop a selection system for transgenic sugarcane based on resistance to the herbicide chlorosulfuron. A mutant acetolactate synthase (alsb) gene from tobacco, which has shown to confer resistance to the tobacco, was transformed into sugarcane callus. It was anticipated that this gene will confer chlorosulfuron resistance to transgenic sugarcane. If resistance is achieved, the corresponding sugarcane gene will be mutated via site-directed mutagenesis and checked if it also confers resistance to sugarcane. Results showed that although transgenic lines were generated, resistance development is still inconclusive. / AFRIKAANSE OPSOMMING: Suikerriet is ‘n kommersiële gewas wat verbou word as gevolg van die hoë hoeveelhede sukrose wat gestoor word in die volwasse tussenknope van die stam. Verskeie studies is al gedoen om suiker metabolisme in die gewas te ondersoek, sowel as om die sukrose opbrengs te verhoog. Huidige strategieë vir suikerriet verbetering word beywer deur middel van teel-programme of transgeniese manipulasie. Die kommersialiseëring van transgeniese gewasse word egter bemoeilik deur publieke wanpersepsies, sowel as regulatoriese uitdagings. Hierdie tesis beoog om boenoemde kwessies aan te spreek, deur middel van twee projekte. Die eerste projek poog om sukrose akkumulasie in sukerriet te verhoog. Dit was onderneem om die Arabidopsis thaliana vakuolere pirofosfatase (AtV-PPase) geen, wat verbind is met die mielie ubiquitien promoter, uit te druk in suikerriet kallus. Daar was verwag dat die verhoogde aktiwiteit van die tonoplast-gebonde AtV-PPase sal veroorsaak dat meer sukrose in die vakuool akkumuleer. Oplosbare suiker inhoud was getoets in transgeniese suikerriet kallus lyne, maar geen merkbare verhoging in sukrose inhoud was waargeneem nie. Hierdie mag egter verander met verdere ondersoeke in suikerriet suspensie-kulture en glashuis-plante. Die tweede projek het beywer om ‘n nuwe suikerriet transformasie tegnologie te ontwikkel, wat slegs van suikerriet genetiese materiaal gebruik maak. Hierdie ‘cisgeniese’ benadering tot suikerriet transformasie sal ‘n inheemse suikerriet promoter, terminator, vektor ruggraat en seleksie-merker, benodig. Dit was eers beoog om ‘n funksionele promoter te isoleer, sowel as om ‘n seleksie sisteem, gebasseer op ‘n inheemse seleksie merker, te ontwikkel. Deur gebruik te maak van primers wat op ‘n sorghum templaat gebasseer is, was ‘n promotor geisoleer vanuit suikerriet; die uitdrukking hiervan is bepaal deur gebruik te maak van ‘n GFP verklikker geen. Ongelukkig kon uitdrukking nie bevestig word in transgeniese suikerriet kallus nie. Tans word suikerriet Kunsmatige Bakterieële Chromosoom (KBC) biblioteke geskandeer, deur gebruik te maak van geen-fragmente van globaal-uitgedrukte gene, om ooreenstemmende suikerriet promoters te isoleer. Die tweede deel van die cisgeniese projek het beoog om ‘n seleksie sisteem vir transgeniese suikerriet te ontwikkel, wat gebasseer is op weerstand teen die plantdoder chlorosulfuron. Suikerriet kallus was getranformeer met ‘n mutante tabak geen – asektolaktaat sintase (alsb) – wat chlorosulfuron weerstand in tabak meebring. Daar was verwag dat die geen chlorosulfuron weerstand aan transgeniese suikerriet sou oordra. Indien weerstand ontwikkel, sal die ooreenstemende suikerriet geen deur gerigte mutagenese gemuteer word; dan sal dit kan bepaal word of weerstand ook oorgedra word aan suikerriet. Daar is bevind dat alhoewel transgeniese lyne gegenereer is, daar steeds nie ‘n konklusiewe bevestiging van weerstand ontwikkeling is nie.

In plant cells, actin filaments are nucleated in two different ways: The growth of single filaments or their bundles is enabled by various types of formins, whereas branched meshworks emerge due to Arp2/3 complex activity. Mutations in genes of these nucleators lead to various phenotypic traits. This thesis deals in the first place with influence of Arp2/3 complex subunits' dysfunction on intracellular motility (cytoplasmic streaming, stop-and-go movements of Golgi apparatus cisternae), since it had not been extensively studied before, and also attempts to quantify the already known impacts of mutations in genes for ARP2 and ARPC5 subunits on the vacuolar morphogenesis. For comparison, a few experiments with plants which carried a mutation in gene for FH1 formin were also realised when measuring the cytoplasmic streaming. The experiments were conducted with a model plant Arabidopsis thaliana. The methods particularly included transformation with fluorescent markers by Agrobacterium tumefaciens (or usage of a fluorescent dye), microscopy (both standard and confocal) and subsequent evaluation of the acquired data using a computer. During the cytoplasmic streaming research, effects of cytoskeletal drug latrunculin B were studied, too. The outputs did not prove that the Arp2/3 complex defects would manifest...

No description available.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O acÃmulo de Na+ no vacÃolo central representa um importante mecanismo de defesa de plantas contra o estresse salino. A regulaÃÃo dos volumes e conteÃdos dos vacÃolos de cÃlulas vegetais depende da atividade de transportadores e canais localizados no tonoplasto (membrana vacuolar). A membrana vacuolar possui duas distintas bombas de prÃtons (V-ATPase e V-PPase), aquoporinas e vÃrios sistemas de transportes ativos e/ou secundÃrios, como os contra-transportadores Na+/H+ vacuolares. As duas bombas de prÃtons transmembranares funcionam como sistemas de transporte primÃrio nas cÃlulas vegetais e ambas as enzimas geram uma diferenÃa de potencial eletroquÃmico de prÃtons atravÃs da membrana vacuolar. Os contra-transportadores vacuolares Na+/H+ utilizam o gradiente eletroquÃmico de prÃtons gerado pelos transportadores primÃrios para transportar Na+ para dentro do vacÃolo. No presente trabalho inicialmente foram determinados os conteÃdos de Ãons Na+ e K+ em raÃzes, hipocÃtilos e folhas e em seguida a anÃlise da expressÃo dos genes das bombas de prÃtons (VHA-A, VHA-E e HVP) e dos contra-transportadores vacuolares (NHX2 e NHX6) em plÃntulas de Vigna unguiculata (L.) Walp cv. Vita 5 submetidas a estresse salino e osmÃtico. As plÃntulas foram crescidas em meio nutritivo na ausÃncia de NaCl e PEG (controle), na presenÃa de 100 mM de NaCl (estresse salino) ou na presenÃa de 200,67 g/L de PEG (estresse osmÃtico). O conteÃdo de Ãons Na+ aumentou em todos os tecidos da planta quando submetidos ao estresse salino (NaCl 100 mM) enquanto que o conteÃdo de Ãons K+ diminuiu na mesma condiÃÃo. A expressÃo dos genes das bombas de prÃtons e dos contra-transportadores vacuolares de folhas e de raÃzes no estresse salino aumentou em todas as condiÃÃes estudadas, porÃm o aumento foi mais expressivo para os genes da V-PPase, NHX2 e NHX6 sugerindo uma regulaÃÃo paralela entre esses genes. JÃ no estresse osmÃtico, os resultados para as folhas mostraram que a expressÃo dos genes VHA-A e VHA-E aumentaram enquanto que os outros genes nÃo sofreram mudanÃas significativas. Nossos resultados sugerem que o estresse salino e o estresse osmÃtico induziram uma regulaÃÃo diferenciada em todos os genes sendo o contra-transportador Na+/H+ importante na homeostase celular quando as plantas foram submetidas ao estresse salino e osmÃtico. / The acummulation of Na+ in the central vacuole represents an important mechanism for plants to cope with salt stress. The vacuolar content and the regulations of their volumes in vegetable cells depend on the activity of transporters and channels located in the tonoplast (vacuolar membrane). The vacuolar membrane possesses two different proton pumps (V-ATPase and V-PPase), aquoporine, and systems of primary and secondary transporters like the vacuolar Na+/H+ antiporter (NHX). The two transmembrane proton pumps work as systems of primary transport in vegetable cells and both enzymes generate a difference of proton electrochemical potential through the vacuolar membrane which can provide energy to antiport system, H+/substrate. The vacuolar Na+/H+ antiporter, uses the electrochemical gradient generated by the primary transporters to pump Na+ ions inward the vacuole. In the present work were first determined the Na+ and K+ content followed by the gene expression of the vacuolar proton pumps (VHA-A, VHA-E and HVP) and the vacuolar antiporters (NHX2 and NHX6) from seedlings of Vigna unguiculata subjected to salt and osmotic stress. The seedlings were grown on nutritive medium in the absence of NaCl and PEG (control condition), presence of NaCl 100 mM (salt stress) or in the presence of PEG 6000 200,67g.L-1 (osmotic stress). The ion Na+ content essay showed an increase in all plant tissues when submitted to salt stress, while the K+ ions decreased in the same condition. The gene expression of the vacuolar proton pumps and the Na+ antiporter from roots and leaves showed an increase in all studied conditions being more expressive to V-PPase, NHX2 and NHX6 suggesting a coordinated regulation of these genes. The results from leaves showed that VHA-A and VHA-E were increased, while the others genes tend to remain constant in the osmotic stress. These results suggest that salt and osmotic stress induced a differential regulation of all studied genes, being the vacuolar Na+ antiporters an important part on keep the cellular homeostasis when the plants were submitted to salt stress

SOBREIRA, Alana Cecília de Menezes. Estudo da expressão dos genes das bombas de prótons (V-ATPase e V-PPase) e dos contra-transportadores vacuolares (NHX) de Vigna unguiculata (L.) Walp submetidos a estresses abióticos.2009 f. Dissertação (Mestrado em Bioquímica) - Centro de Ciências, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, 2009. / Submitted by JOANA BEZERRA (joanabib@yahoo.com.br) on 2012-11-09T21:07:31Z No. of bitstreams: 1 2009_tese_acmsobreira.pdf: 1938746 bytes, checksum: 064fea08dd8578e2f6b67de140140d86 (MD5) / Approved for entry into archive by Érica Barros(ericabarros@ufc.br) on 2012-11-12T22:49:16Z (GMT) No. of bitstreams: 1 2009_tese_acmsobreira.pdf: 1938746 bytes, checksum: 064fea08dd8578e2f6b67de140140d86 (MD5) / Made available in DSpace on 2012-11-12T22:49:16Z (GMT). No. of bitstreams: 1 2009_tese_acmsobreira.pdf: 1938746 bytes, checksum: 064fea08dd8578e2f6b67de140140d86 (MD5) / The acummulation of Na+ in the central vacuole represents an important mechanism for plants to cope with salt stress. The vacuolar content and the regulations of their volumes in vegetable cells depend on the activity of transporters and channels located in the tonoplast (vacuolar membrane). The vacuolar membrane possesses two different proton pumps (V-ATPase and V-PPase), aquoporine, and systems of primary and secondary transporters like the vacuolar Na+/H+ antiporter (NHX). The two transmembrane proton pumps work as systems of primary transport in vegetable cells and both enzymes generate a difference of proton electrochemical potential through the vacuolar membrane which can provide energy to antiport system, H+/substrate. The vacuolar Na+/H+ antiporter, uses the electrochemical gradient generated by the primary transporters to pump Na+ ions inward the vacuole. In the present work were first determined the Na+ and K+ content followed by the gene expression of the vacuolar proton pumps (VHA-A, VHA-E and HVP) and the vacuolar antiporters (NHX2 and NHX6) from seedlings of Vigna unguiculata subjected to salt and osmotic stress. The seedlings were grown on nutritive medium in the absence of NaCl and PEG (control condition), presence of NaCl 100 mM (salt stress) or in the presence of PEG 6000 200,67g.L-1 (osmotic stress). The ion Na+ content essay showed an increase in all plant tissues when submitted to salt stress, while the K+ ions decreased in the same condition. The gene expression of the vacuolar proton pumps and the Na+ antiporter from roots and leaves showed an increase in all studied conditions being more expressive to V-PPase, NHX2 and NHX6 suggesting a coordinated regulation of these genes. The results from leaves showed that VHA-A and VHA-E were increased, while the others genes tend to remain constant in the osmotic stress. These results suggest that salt and osmotic stress induced a differential regulation of all studied genes, being the vacuolar Na+ antiporters an important part on keep the cellular homeostasis when the plants were submitted to salt stress. / O acúmulo de Na+ no vacúolo central representa um importante mecanismo de defesa de plantas contra o estresse salino. A regulação dos volumes e conteúdos dos vacúolos de células vegetais depende da atividade de transportadores e canais localizados no tonoplasto (membrana vacuolar). A membrana vacuolar possui duas distintas bombas de prótons (V-ATPase e V-PPase), aquoporinas e vários sistemas de transportes ativos e/ou secundários, como os contra-transportadores Na+/H+ vacuolares. As duas bombas de prótons transmembranares funcionam como sistemas de transporte primário nas células vegetais e ambas as enzimas geram uma diferença de potencial eletroquímico de prótons através da membrana vacuolar. Os contra-transportadores vacuolares Na+/H+ utilizam o gradiente eletroquímico de prótons gerado pelos transportadores primários para transportar Na+ para dentro do vacúolo. No presente trabalho inicialmente foram determinados os conteúdos de íons Na+ e K+ em raízes, hipocótilos e folhas e em seguida a análise da expressão dos genes das bombas de prótons (VHA-A, VHA-E e HVP) e dos contra-transportadores vacuolares (NHX2 e NHX6) em plântulas de Vigna unguiculata (L.) Walp cv. Vita 5 submetidas a estresse salino e osmótico. As plântulas foram crescidas em meio nutritivo na ausência de NaCl e PEG (controle), na presença de 100 mM de NaCl (estresse salino) ou na presença de 200,67 g/L de PEG (estresse osmótico). O conteúdo de íons Na+ aumentou em todos os tecidos da planta quando submetidos ao estresse salino (NaCl 100 mM) enquanto que o conteúdo de íons K+ diminuiu na mesma condição. A expressão dos genes das bombas de prótons e dos contra-transportadores vacuolares de folhas e de raízes no estresse salino aumentou em todas as condições estudadas, porém o aumento foi mais expressivo para os genes da V-PPase, NHX2 e NHX6 sugerindo uma regulação paralela entre esses genes. Já no estresse osmótico, os resultados para as folhas mostraram que a expressão dos genes VHA-A e VHA-E aumentaram enquanto que os outros genes não sofreram mudanças significativas. Nossos resultados sugerem que o estresse salino e o estresse osmótico induziram uma regulação diferenciada em todos os genes sendo o contra-transportador Na+/H+ importante na homeostase celular quando as plantas foram submetidas ao estresse salino e osmótico.