As fosfodiesterases do tipo 2A (PDE2A) hidrolisam os nucleotídeos cíclicos cAMP e cGMP e por isso desempenham papel importante na sinalização intracelular. PDE2A é composta por uma região N-terminal, que ao contrário de outras famílias de PDEs, não possui função conhecida, dois domínios regulatórios, GAF A e GAF B, e um domínio catalítico localizado na porção C-terminal. Sabe-se que a hidrólise dos nucleotídeos cíclicos, pela PDE2A, é ativada pela ligação de cGMP ao domínio regulatório GAF B. Em uma triagem dois híbridos, identificamos XAP2 como a principal proteína interatora de PDE2A. XAP2 é um componente crucial do complexo multiprotéico do receptor Aril hidrocarboneto (Ahr), o principal fator de transcrição que controla a expressão de múltiplos genes envolvidos em detoxificação. Neste trabalho, foi determinado que XAP2 liga o domínio GAF B da enzima PDE2A. Ensaios de atividade fosfodiesterásica, utilizando proteínas purificadas, mostram que a ligação de XAP2 não interfere na atividade enzimática de PDE2A. Para analisar se PDE2A afeta a função de XAP2, foi investigado o deslocamento de Ahr para o núcleo. Sabe-se que a regulação da expressão dos genes alvos de Ah é iniciada pela ligação de tetraclorodibenzodioxina (TCDD) e por uma via, ainda pouco entendida, dependente de cAMP. Verificamos que a ligação de PDE2A à XAP2 inibe o deslocamento de Ahr para o núcleo, induzido por TCDD e por cAMP em células hepáticas Hepa1c1c7 em cultura. Em conclusão, mostramos neste trabalho que XAP2 recruta PDE2A para o complexo Ahr, causando a inibição da mobilidade de Ahr, possivelmente pela redução local da concentração de cAMP. Esses dados suportam o papel de cAMP no controle da função de Ahr. / Phosphodiesterase type 2A (PDE2A) hydrolyzes cyclic nucleotides cAMP and cGMP thus efficiently controlling cNMP-dependent signaling pathways. PDE2A is composed of an N-terminal region, two regulatory GAF domains and a catalytic domain. Cyclic nucleotide hydrolysis is known to be activated by cGMP binding to GAF-B, however, other mechanisms may operate to fine-tune local cyclic nucleotide levels. In a yeast-two-hybrid screening we identified XAP2, a crucial component of the aryl hydrocarbon receptor (AhR) complex, as a major PDE2A-interacting protein. We mapped the XAP2 binding site to the GAF-B domain of PDE2A. PDE assays with purified proteins showed that XAP2 binding does not change the enzymatic activity of PDE2A. To analyze if PDE2A could affect the function of XAP2 we studied nuclear translocation of AhR, i.e. the master transcription factor controlling the expression of multiple detoxification genes. Notably, regulation of AhR target gene expression is initiated by tetrachlorodibenzodioxin (TCDD) binding to AhR and by a poorly understood cAMP-dependent pathway, followed by the translocation of AhR from the cytosol into the nucleus. Binding of PDE2A to XAP2 inhibited TCDD- and cAMP-induced nuclear translocation of AhR in Hepa1c1c7 hepatocytes. We conclude that XAP2 targets PDE2A to the AhR complex thereby restricting AhR mobility, possibly by a local reduction of cAMP levels. Our results provide first insights into the elusive cAMP-dependent regulation of AhR.

Em todo o mundo, menos de 5% dos doentes com tuberculose (TB), sejam casos novos ou previamente tratados, tem a avaliação dos isolados quanto ao perfil de sensibilidade aos antibioticos. No Rio Grande do Sul, estado localizado no sul do Brasil, cerca de 4700 casos novos de TB são registrados a cada ano, com uma taxa de cura de 68,9%, e uma taxa de abandono de 7,5%. A identificação rápida da resistência às drogas, em isolados clínicos de M. tuberculosis é importante para o estabelecimento de uma quimioterapia eficaz bem como para evitar a propagação de cepas resistentes. Os objetivos deste estudo foram caracterizar os pacientes de TB com maior risco de possuir TB-­‐MDR, analisando o perfil de resistência às drogas dos isolados e o perfil epidemiológico desses pacientes. Além disso utilizou-­‐se as amostras clínicas para avaliar o teste comercial (GenoType® MTBDRplus) e para desenvolver e padronizar um novo teste (Detect-­‐TBMR) para detectar as mutações mais frequentes associadas a resistência à INH e RIF. Uma proporção significativamente maior (75% versus 20%, p = 0,009) de pacientes do gênero masculino foi encontrada entre os casos resistentes às drogas do que entre os casos suscetíveis. 43,8% dos pacientes demoraram mais de 30 dias para procurar assistência médica e no grupo TB MDR, 25% dos casos não tinha sido submetido a qualquer tratamento prévio anti-­‐TB. Em nossas amostras, encontramos uma proporção de 48,3% de TB-­‐ MDR. A família T foi a família de spoligotipo mais frequente. Comparado com o método da proporções, a sensibilidade e especificidade do ensaio MTBDRplus foram 82% e 94% para a resistência à RIF, 60% e 94% para resistência à INH. Comparado com sequenciamento, a sensibilidade e especificidade do ensaio MTBDRplus foi 92% e 97% para a resistência à RIF e 100% e 100% para a resistência à INH, respectivamente. Para detectar resistência à RIF e INH, o ensaio Detect-­‐TBMDR mostrou sensibilidade e especificidade de 79,3% e 77,0% e 100% e 65%, respectivamente, em comparação com o método da proporções. Comparado com o sequenciamento, a sensibilidade e especificidade do ensaio Detect-­‐TBMDR foi de 81,2% e 94,7% e 100% e 96,2%, para detectar e resistência à RIF e INH, respectivamente. Ainda existem discordâncias entre o método das proporções e a abordagem molecular, particularmente em relação a resistência à INH. Contudo, estes métodos são muito importantes para o manejo mais rápido e correto dos pacientes, auxiliando na escolha do melhor esquema terapêutico. / In most parts of the world, less than 5% of new and previously treated tuberculosis (TB) patients are tested for multidrug resistance (MDR) TB. In Rio Grande do Sul state, the southern most Brazilian state; approximately 4700 new cases of TB are recorded each year, with a cure rate of 68.9%, and a noncompliance rate of 7.5%. Rapid identification of drug resistance in clinical isolates of Mycobacterium tuberculosis is important to facilitate rapid and adequate chemotherapy of TB, and to prevent the spread of resistant strains. The aim of this study was to characterize TB patients at higher risk of having MDR-TB, to analyze the drug resistance and epidemiological profile of these patients. Use the clinical samples to assess the commercial test (GenoType® MTBDRplus) and develop and standardize a new test (Detect-MDRTB) for detecting the most frequent mutations associated with resistance to INH and RIF. A significantly higher proportion (75% versus 20%, p = 0.009) of males were found among drug-resistant cases than drug susceptible cases. 43.8% of patients took longer than 30 days to seek medical care and in the MDR group 25% of the cases did not undergo any previous anti-TB treatment. In our samples we found a proportion of 48.3% of MDR-TB. The T family was the most frequent spoligotype family. Compared with the proportion method, the sensitivity and specificity of the MTBDRplus assay were 82% and 94% for RIF-resistance, 60% and 94% for INH resistance. Compared with sequencing, the sensitivity and specificity of the MTBDRplus assay were 92% and 97% for RIF-resistance, 100% and 100% for INHresistance. To detect RIF and INH-resistance, the Detect-TBMDR assay showed a sensitivity and specificity of 79.3% and 77.0%, and 100% and 65%, respectively, compared to proportion method. When compared with sequencing, Detect-TBMDR assay, to detect RIF and INH-resistance, showed a sensitivity and specificity of 81.2% and 94.7% and to 100% and 96.2%, respectively. Discordances still exist between the proportion method and molecular approach, particularly regarding INH-resistance. However, these methods are very important for the management faster and correct patient, helping to choose the best treatment regimen.

Tese de mestrado. Engenharia Biomédica. Faculdade de Engenharia. Universidade do Porto, Faculdade de Medicina. Universidade do Porto. 2009

Orientador: Jesus Aparecido Ferro / Resumo: O cancro cítrico tem como agente causal a bactéria Xanthomonas citri subsp. citri (Xac), que afeta todas as espécies de citros economicamente importantes. O sequenciamento do genoma do isolado 306 desta bactéria (Xac 306) revelou que 37,4 % dos genes não tinham uma função predita. Assim, a genômica funcional da interação Xac-hospedeiro tem um papel importante na elucidação da função dos genes e dos mecanismos envolvidos na patogenicidade e virulência dessa bactéria. Os genes XAC3981, XAC3982 e XAC3983 fazem parte deste contexto de genes codificadores de proteínas sem função conhecida (hipotéticas). As proteínas codificadas por estes três genes são conservadas em bactérias da família Xanthomonadaceae. Em um estudo prévio, o gene XAC3981 da Xac 306 foi interrompido por inserção do transposon Tn5 e esse mutante apresentou ausência de sintomas quando inoculado em plantas cítricas, sugerindo que possui um papel importante na patogenicidade de Xac. No presente estudo demonstrou-se que os genes XAC3981-3983, juntamente com o gene htrA (XAC3980), constituem um operon funcional de Xac 306. O presente estudo avaliou também os efeitos de mutações por deleção em algumas regiões do operon htrA-3983. Um mutante foi produzido por deleção de 84% da sequência codificadora do gene XAC3982 (ΔXAC3982) e outros três foram obtidos pela deleção das regiões promotoras desse operon. O mutante ΔXAC3982, quando comparado com a estirpe selvagem, alterou o fenótipo em genótipos resistentes e suscetíveis ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The bacterium Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus canker, a disease that affects all economically important citrus species. The genome sequencing of the 306 isolate of this bacterium (Xac 306) revealed that 37.4% of the genes had no predicted function. Thus, the functional genomics of the Xac-host interaction play an important role in the elucidation of the function of the genes and in the mechanisms involved in the pathogenicity and virulence of this bacterium. The genes XAC3981, XAC3982 and XAC3983 are part of this context of protein coding genes with unknown function (hypothetical). The proteins encoded by these three genes are conserved and present exclusively in bacteria of the family Xanthomonadaceae. In a previous study, the ORF XAC3981 from Xac306 was disrupted by insertion of the Tn5 transposon and the mutant showed no symptoms when inoculated in citrus plants, suggesting that it plays an important role in Xac pathogenicity. In the present study, genes XAC3981-3983, together with gene htrA (XAC3980), have been shown to constitute a functional operon of Xac 306. The present study also evaluated the effects of deletion mutations in some regions of XAC3980-3983 operon. One mutant was produced by deleting 84% of the coding sequence of the gene XAC3982(ΔXAC3982) and another three were obtained by deleting the promoter regions of that operon. The ΔXAC3982 mutant, when compared to the wild type strain, altered the phenotype in resistant citrus... (Complete abstract click electronic access below) / Doutor

La vía de señalización celular MEK5-ERK5 juega un papel importante en sistema nervioso, dado que se activa frente a neurotrofinas, frente al estrés oxidativo producido por reactive oxygen species (ROS), tras la isquemia cerebral y tiene un papel en la especificación de fenotipo neuronal. Sin embargo, poco se conoce sobre los sustratos y proteínas que interaccionan con ERK5. En este trabajo se han identificado proteínas que interaccionan con ERK5 en células de neuroblastoma SH-SY5Y mediante el método TAP (Tandem-Affinity Purification). Se han obtenido diversas proteínas que incluyen proteínas de citoesqueleto, chaperonas y proteínas de metabolismo celular. Entre las proteínas identificadas destacan la proteína chaperona Hsp90β y la piruvato kinasa PKM2. Por otro lado, se ha determinado el papel que juega ERK5 en la isquemia cerebral, utilizando el modelo de privación de oxígeno y glucosa (OGD) en cultivos mixtos de neuronas corticales. ERK5 se degrada rápidamente en respuesta a la OGD en cultivos mixtos de neuronas corticales. Esta degradación es mediada por calpaína. ERK5 también se degrada rápidamente in vivo (4 horas) en córtex de cerebros de ratas Sprague-Dawley sometidas a isquemia por oclusión de la arteria cerebral media. En este trabajo se establece que tras la isquemia, la entrada masiva de calcio a través de los receptores de NMDA y la consiguiente activación de calpaína conlleva la degradación de ERK5, produciéndose así una disminución de los niveles totales de esta kinasa. / MEK5-ERK5 pathway plays an important role in nervous system. This pathway is activated in response to neutrophins, oxidative stress induced by reactive oxygen species (ROS), after cerebral ischemia and plays an important role in neuronal fate determination. However, little is known about substrates and proteins that interact with ERK5. This work describes the search for proteins which interact with ERK5 in the cell line SH-SY5Y using Tandem-Affinity Purification (TAP). Using this technique, we obtained different proteins that include citosqueleton components, chaperons and cell metabolism proteins, among which it is worth highlighting Hsp90β and piruvate kinase PKM2.On the other hand, we have established the role of ERK5 in cerebral ischemia, using oxygen and glucose deprivation model (OGD) in rat mixed neuronal cortical cultures. Using this model we observed that ERK5 is quickly degraded in response to OGD, and that calpain is responsible for ERK5 degradation. ERK5 is also quickly (4 hours) degraded in vivo in cerebral cortex from Sprague-Dawley rats subjected to middle cerebral artery occlusion. On this work we establish that after cerebral ischemia, massive income of calcium mediated by NMDA receptors and resulting activation of calpain entails ERK5 degradation, producing a decrease in total levels of this kinase.

Durant les últimes dècades, l'agregació de proteïnes ha esdevingut un tema d’investigació molt dinàmic que s'estén transversalment per diferents camps de recerca, incloent la bioquímica, la biotecnologia, la biomedicina i la nanotecnologia. D'una banda, l'acumulació de proteïnes en dipòsits amiloids insolubles constitueix una característica comuna de molts trastorns humans, coneguts com a malalties conformacionals. D'altra banda, des d'un punt de vista biotecnològic, l’agregació proteica representa un obstacle habitual en la producció de proteïnes recombinants, que en general s'acumulen en forma de cossos d’inclusió intracel·lulars. Malgrat que els cossos d’inclusió han estat tradicionalment considerats partícules desestructurades i amb molt poc interès, nombroses evidències indiquen que aquests agregats contenen estructura de tipus amiloid, la qual cosa aplana el camí per a emprar-los en l'estudi de l’agregació amiloid. La tesi que aquí es presenta recapitula la feina pertanyent a una sèrie de publicacions relatives a l’agregació amiloid de proteïnes en l'espai intracel·lular. En tres d'aquests treballs, s'utilitzen tres models cel·lulars diferents filogenèticament distants (bacteris, llevats i plantes) per abordar l’estudi de la formació de dipòsits proteics amb l'objectiu de caracteritzar-los i analitzar-ne el seu impacte en el metabolisme cel·lular. En una publicació complementària, explotem els agregats bacterians per tal de desenvolupar un assaig de screening in vitro per trobar moduladors de l’agregació amiloid. Finalment, també s'inclouen dues obres de revisió sobre la deposició de proteïnes en bacteris i el paper d’aquest organisme com a model per a l'estudi de l'agregació amiloid. Les dades obtingudes de tots aquests estudis indiquen que l'agregació en conformació amiloid és una propietat genèrica dels polipèptids i un fenomen omnipresent a la natura. No obstant, l'aparició d'aquests dipòsits en l'entorn cel·lular pot anar acompanyada d'un cert grau de toxicitat. Aquí, analitzem l'efecte d'envelliment promogut pels cossos d'inclusió intracel·lulars en cèl·lules bacterianes. A més, descrivim com les cèl·lules procariotes i eucariotes estan dotades d'una poderosa maquinària de qualitat proteica que permet fer front a aquesta situació perjudicial. Addicionalment, la caracterització en profunditat dels agregats proteics en bacteris i del seu procés de formació permet utilitzar-los com a eina en la recerca d'inhibidors de l'agregació amiloid amb potencial interès biomèdic i farmacèutic. En general doncs, aquesta tesi aprofundeix en l'estudi de l'agregació amiloid de proteïnes i amplia el coneixement per a emprar organismes simples com models cel·lulars rellevants. / During the last decades, protein aggregation has become a dynamic research topic extending across distinct investigation fields, including biochemistry, biotechnology, biomedicine and nanotechnology. On one side, the accumulation of proteins into insoluble amyloid deposits constitutes a common hallmark of many human disorders, known as conformational diseases. On the other side, from a biotechnological point of view, protein deposition is regarded as a usual hindrance in the production of recombinant proteins, which generally assemble into intracellular inclusion bodies. Although inclusion bodies were traditionally considered unstructured particles lacking of interest, the increasing number of evidences indicating that these aggregates contain amyloid-like structure pave the way for employing them in the study of amyloid deposition. The thesis presented here recapitulates the work belonging to a set of publications concerning amyloid protein aggregation in the intracellular space. In three of these works, we use three distinct cellular models phylogenetically distant (bacteria, yeast and plants) to address the formation of protein deposits with the aim to characterize them and to analyze their impact in the cellular metabolism. In a complementary publication, we exploit bacterial aggregates to develop an in vitro screening assay for amyloid modulators. Finally, we also include two revision works about protein deposition in bacteria and its role as model in the study of amyloid aggregation. The data collected from these studies indicate that aggregation into amyloid structures is a general property of polypeptides and a ubiquitous phenomenon in Nature. However, the apparition of these deposits in the cellular environment can be accompanied by a certain degree of toxicity. Here, we analyze the aging effect promoted by intracellular inclusion bodies in bacterial cells. In addition, we report how both prokaryotic and eukaryotic cells are endowed with a powerful protein quality machinery to challenge this damaging scenario. Moreover, the deep characterization of bacterial protein aggregates and their formation process permits their use as a tool in the searching for amyloid aggregation inhibitors with putative biomedical and pharmaceutical interest. Overall, this thesis delves into the study of amyloid protein aggregation and adds insights to employ simple organisms as relevant cellular models.

The embryo in grasses, at grain maturity, comprises the embryonic axis and the scutellum. The scutellum is supposed to be the single cotyledon in the monocotyledoneus embryos and is attached to the embryo axis in the scutelar node. The embryo has the highest concentration of lipid and lipid soluble vitamins in cereal grains. The embryo in grasses, at grain maturity, comprises the embryonic axis and the scutellum. The scutellum is supposed to be the single cotyledon in the monocotyledoneus embryos and is attached to the embryo axis in the scutelar node. The embryo has the highest concentration of lipid and lipid soluble vitamins in cereal grains. The embryonic axis originates the root, leaves and stem of the new plant. In the mature seed, the embryo axis is formed by the primary root, protected by the coleorhiza, and the stem tip with five or six short internodes and leaf primordia which, as a whole, form the plumule that is surrounded by the coleoptile. The name scutellum (small shield, in latin) derives from its shield-like shape and it liesbetween the embryonic axis and endosperm. Dissected scutellum constitutes 11% or the kernel mass, and about 90% of the embryo. During germination, scutellar epithelial cells suffer an elongation that increases the contact surface between the endosperm and the scutellum and facilitates the transport of the nutrients from the endosperm to the embryo. Scutellar cell elongation is inhibited by ABA and salicylic acid, basic and acid pH and high concentrations of sorbitol. Exogenous gibberellins stimulate elongation, but a reduction in gibberellin synthesis or perception does not inhibit it. Elongation is inhibited by sucrose, but not glucose. Transcription and translation inhibitors reduce scutellar cell elongation, indicating that transcription and translation are necessary for the elongation process. Scutellar epithelium cells play specific roles during germination different to parenchymal cells. So, we expect some differences in the gene expression pattern of this tissue. That’s why we construct a cDNA library using RNA extracted from scutellar epithelial cells 1 day after imbibition and selected them using array hybridization comparing the mRNA accumulated in epithelial cells with the mRNA accumulated in the other scutellar tissues. We identified 30 genes up-regulated in the epithelium. A high proportion of these genes are involved in metabolic processes, the production of energy or in the transport of peptides into the embryo. The roles of 43% of these genes remains undetermined, 27% of them are involved in metabolic processes, 13% in protein synthesis or processing and 7% in cell structure. One of the identified genes from the macroarrays is a peptide transporter: ZmPTR1. It encodes a non-characterized maize peptide transporter protein which has 587 amino acids with a calculated molecular mass of 64.52 kDa. This maize transporter is predominantly expressed in the scutellar epithelium during germination. ZmPTR1 is also expressed to a less extent in the radicle and the hypocotyl. ZmPTR1 is located in the tonoplast and has high sequence similarity with tonoplast di- and tripeptide transporters AtPTR2, AtPTR4 and AtPTR6 from Arabidopsis thaliana. ZmPTR1 is able to transport at least Ala-Ala dipeptide across the membrane and could have a role in the intracellular transport of di- and tripeptides. Seed germination is a complex process that requires cell division, expansion and differentiation. It involves the activation of many metabolic pathways and signal transduction processes, which require a great quantity of energy and stored materials which are provided by seed reserves (oil, storage proteins and starch) and the synthesis and/or activation of many proteins. Plant seeds store triacylglycerols (TAGs) into oil bodies (OBs), specialized organelles which serve as an energy reserve during germination and post-germinative growth. Protein composition analysis of oil bodies from maize embryos during germination identified, in addition to the previously characterized OB-associated proteins, other proteins of diverse function: an embryonic protein DC-8, a globulin 2, 4 proteins with enzymatic activity (protein disulfide isomerase, xylose isomerase, strictosidine synthase and precursor and ATP synthase beta chain), a protein similar to karyopherin- beta-3 (Kap) and a stress induced membrane pore protein involved in membrane transport. Quantitative subproteomic analysis of germinating related changes in the oil bodies of maize scutellum between dry seeds and 2 dai seeds allowed the identification of new proteins interacting with oil bodies in dry seeds or in germinating seeds. In dry seeds: oleosins, cupins, disulfide isomerases, a nucleoside phosphate kinase, a class IV heat shock protein, an embryonic protein DC-8, a 60S acidic ribosomal protein P0 and a rubber elongation factor protein. In germinating seeds: oleosins, mitochondrial protein Tim17, prohibitin-2 and a manganese superoxide dismutase (Mn-SOD). / ESTRUCTURA Y FUNCIÓN DEL ESCUTELO DE MAÍZ DURANTE LAS PRIMERAS ETAPAS DE LA GERMINACIÓN Durante la germinación, las células epiteliales del escutelo sufren una elongación que aumenta la superficie de contacto entre el endospermo y el embrión y facilita el transporte de los nutrientes del endospermo al embrión. La elongación de las células del escutelo es inhibida por ABA y ácido salicílico, pH básico y ácido y altas concentraciones de sorbitol. Las giberelinas exógenas estimulan la elongación, pero una reducción en la síntesis de giberelinas o percepción no la inhiben. La elongación es inhibida por la sacarosa, pero no la glucosa. Los inhibidores de la transcripción y traducción reducen el alargamiento de las células escutelares, lo que indica que la transcripción y la traducción son necesarias para el proceso de alargamiento. Se identificaron 30 genes que muestran una expresión diferencial significativa en las células epiteliales del escutelo de un día después de la imbibición. Las funciones de un 43% de estos genes no se conoce, el 27% de ellos están involucrados en los procesos metabólicos, el 13% en la síntesis o la transformación de proteínas y el 7% en la estructura celular. ZmPTR1 codifica un transportador de maíz péptido no ha sido previamente caracterizado, se expresa predominantemente en el epitelio escutelar durante la germinación. ZmPTR1 se expresa también en menor medida en la radícula y del hipocotilo. La proteína ZmPTR1 se localiza en el tonoplasto y es homóloga a las otras proteínas transportadoras de di-y tripéptidos AtPTR2, AtPTR4 y AtPTR6 de Arabidopsis thaliana. ZmPTR1 es capaz de transportar al menos el dipéptido Ala-Ala. El análisis proteómico de las proteínas asociadas a los cuerpos lipídicos en escutelo de maíz durante la germinación ha permitido la identificación de, además de proteínas previamente caracterizadas asociadas OB, otras proteínas de funciones diversas. El Análisis cuantitativo subproteómico de los cambios relacionados con los cuerpos lipídicos en el escutelo de maíz entre semillas secas y semillas 2 dias después de la germinación permitieron la identificación de nuevas proteínas que interactúan con los cuerpos lipídicos en las semillas secas o en la germinación de las semillas.

Les plantes disposen d’una sèrie de fotoreceptors que informen de manera contínua sobre les característiques lumíniques ambientals, permetent optimitzar el seu creixement i desenvolupament. Els fitocroms són els fotoreceptors millor caracteritzats, que absorbeixen en la regió del roig (R) i del roig llunyà (FR). De tots els processos que regulen, la síndrome de fugida de l’ombra o SAS (de l’anglès shade avoidance syndrome) és probablement el més important en plantes crescudes en llum. La SAS es refereix al conjunt de respostes induïdes en plantes creixent en alta densitat o sota una coberta vegetal. Malgrat la complexitat d’aquestes respostes, la SAS s’indueix per un sol senyal ambiental, que és la disminució de la raó R:FR, la percepció de la qual pels fitocroms inicia una xarxa transcripcional. En el nostre laboratori ens hem centrat en un grup de gens l’expressió dels quals està ràpida i directament alterada per llum de baixa raó R:FR o ombra simulada, anomenant-los gens PAR (de l’anglès phytochrome rapidly regulated). En la present tesi s’ha aprofundit en l’estudi de la cadena de transducció de la senyal en la SAS en Arabidopsis thaliana, emfatitzant en els punts de connexió amb les rutes transcripcionals hormonals. Resultats d’aquesta tesi, que es complementen amb d’altres obtinguts anteriorment en el grup, demostren que un dels gens PAR, ATHB4, que codifica per un factor de transcripció del tipus Homeo-Domain Leucine-Zipper (HD-ZIP subfamília II), té un paper en la senyalització de la SAS com a un modulador complex en aquestes respostes, involucrant diferents hormones en la seva acció. Per dur-ho a terme es van fer aproximacions amb mutants de guany de funció (de sobreexpressió constitutiva amb activitat induïble) i de pèrdua de funció. Particularment s’ha caracteritzat fisiològica, molecular i histològicament el doble mutant athb4hat3, resultats que mostren una disminució de la resposta a diferents estímuls (hormonals i de llum) d’aquest doble mutant. També hem investigat el paper d’ATHB4 com a regulador del desenvolupament vegetal coactuant amb HAT3 a través d’estudis histològics i moleculars del doble mutant ahb4hat3, el qual mostra fortes alteracions morfològiques relacionades amb la polaritat en el desenvolupament. Per aprofundir, hem investigat el paper dels quatre membres més propers (pertanyents a HD-Zip II) en les respostes SAS i al control hormonal a nivell molecular i/o fisiològic utilitzant línies de sobreexpressió i de pèrdua de funció, englobant els resultats en un possible mòdul regulador de determinades respostes de la planta. Aquesta hipòtesi ve recolzada pels resultats que els identifiquen com a gens reguladors complexos d’aquesta resposta. En paral•lel, hem estudiat l’expressió de SAUR15, un gen de resposta a auxines, BRs i ombra simulada; el qual està regulada per ATHB4 de manera directa. S’han identificat elements E-box i AREs, i s’ha estudiat el seu paper en les respostes a ombra i a hormones. Els nostres resultats mostren que aquests elements no són imprescindibles per a la correcta resposta del promotor als estímuls aplicats. Una altra aproximació fou l’estudi de la resposta SAS de mutants específics de brassinosteroides i auxines (els quals estan relacionats amb la modulació de l’expressió de SAUR15), on resultats de la tesi suggereixen una connexió entre BRs i la resposta a ombra simulada. Els resultats obtinguts en quant a components moleculars involucrats en les respostes SAS provenen de l’anàlisi de tota la plàntula sencera. Donat que els llocs de percepció de la llum i de l’acció poden estar físicament separats a la planta, vam estudiar la importància de la comunicació a llarga distància per entendre com la planta creix i es desenvolupa regulada per la llum durant la SAS, obtenint com a resultat que tant els cotilèdons com el meristem apical són importants, però no imprescindibles, en aquesta resposta. / "Hormonal and genetic control of shade avoidance syndrome in Arabidopsis thaliana " Plants sense the presence of competing neighboring vegetation as a change in light quality: i.e. they sense the reduced ratio of red light to far-red light. The responses to shade are generally referred to as the shade avoidance syndrome (SAS), and involve various developmental changes intended to outgrow or outcompete the neighboring plants. In this thesis we analyzed the function of ATHB4, a gene encoding a homeodomain-leucine zipper (HD-Zip) class-II transcription factor from Arabidopsis thaliana, the expression of which is rapidly and directly upregulated after proximity perception by the phytochrome photoreceptors. Our results, altogether with previous studies in my group, suggest that some members of this small gene subfamily can modulate SAS responses by controlling auxin, brassinosteroid and gibberellin molecular and/or physiological responsiveness. In particular, we propose ATHB4 as a new shade signaling component that participates in integrating shade perception and hormone-mediated growth. To get this conclusion we perform experiments with overexpression and loss-of-function lines. We specially characterized double mutant athb4hat3, which has strong morphological alterations, and which let us find a connection point between light and development concerning polarity.

La majoria dels cultius alimentaris, i en particular els cereals com el blat de moro són deficients en vitamines claus. Les plantes transgèniques podrien ser una solució per mitigar les deficiències de vitamines. No obstant això, en les plantes són limitats els coneixements dels mecanismes que regulen l’acumulació de determinades vitamines, com la vitamina C (àcid ascòrbic, AsA).Per aquesta raó, he investigat el contingut en vitamina C i el mecanisme genètic de les rutes metaboliques que controlen l’acumulació de vitamina C en les plantes de panís i he generat blat de moro transgènic que conté diferents combinacions de gens de la ruta metabòlica de la vitamina C i així, trobar una estratègia per augmentar el contingut d`aquesta vitamina en el endosperm de la llavor del blat de moro. A més a més, com a primer pas, he generat plantes de panís transgèniques que contenen múltiples gens derivats de la bactèria Bacillus thuringiensis amb activitat insecticida. Aquest després serán creaudes amb blat de moro trangénic que conté grans quantitats de vitamines. / Most staple food crops, particularly the major cereals, such as maize are deficient in key vitamins. Transgenic approaches hold great promising promise in alleviating, to a major extend such vitamins deficiencies. However, there is a limited understanding of the regulatory mechanisms that control the accumulation of specific vitamins in plants, such as vitamin C (Ascorbic acid, AsA). For that reason, I analyzed the AsA metabolism and the genetic mechanism of L-galactose biosynthesis pathway controlling ascorbic acid accumulation in corn plants and I also established a combinatorial transgenic corn population comprising different combinations of genes related to AsA metabolism, aiming to provide insight into the interactions among alternative AsA biosynthetic pathways and help in the development of strategies to boost the accumulation of AsA in the endosperm. In addition, I generated transgenic corn plants engineered with multiple insecticidal genes derived from the bacterium Bacillus thuringiensis as a first step in developing corn plants with durable resistance to insect pests. / La mayoría de los cultivos alimentarios, y en particular los cereals como el maíz son deficientes en las vitaminas básicas. Las plantas transgénicas pueden ser una solución muy prometedora para mitigar las deficiencias de vitaminas. Sin embargo, el conocimiento de los mecanismos que regulan la acumulación de determinadas vitaminas en las plantas, es limitado, como por ejemplo la vitamina C (ácido ascórbico, AsA).Para aumentar los conocimientos de la ruta metabolica de la vitamina C en maíz, he analizado el contenido de vitamina C y el mecanismo genético de las rutas metabólicas que controlan la acumulación de está en las plantas de maíz y he generado maíz transgénico que contiene diferentes genes de la ruta metabólica de la vitamina C ya que nos proporcionará una estrategia para augmentar el contenido de esta vitamina en el endospermo de maíz. Además, he generado plantas transgénicas que contienen multiples genes con actividad insecticida derivados de la bacteria Bacillus thuringiensis como primer paso, para obtener plantas resistentes a plagas que más tarde serán cruzadas con maíz nutricionalmente modificado.

Tesi realitzada al Centre de Recerca en Agrigenómica (CRAG) / Los estudios realizados sobre la proteína Arv1 en levaduras y animales indican que estaría involucrada en el mantenimiento de la homeostasis lipídica intracelular, aunque se desconoce su función bioquímica concreta. A. thaliana posee los genes ARV AtARV1 y AtARV2, que codifican las proteínas funcionales AtArv1 y AtArv2, respectivamente, ancladas a la membrana del RE. Partiendo de la hipótesis de que las proteínas AtArv interaccionan con otras proteínas para poder ejercer su función biológica, este trabajo se inició con el cribado de un banco de cDNA de plántulas de Arabidopsis empleando AtArv1 como cebo y la técnica de doble híbrido en levadura (MbYTH), donde se identificó que la proteína maMYB interacciona con AtArv1. La proteína maMYB, codificada por un único gen en Arabidopsis, tiene características de un MTTF de la familia MYB, puesto que contiene un dominio citosólico R2R3-MYB en la región C-terminal y dos DTMs en la región N-terminal. En este sentido, se determinó que maMYB se localiza en las membranas del RE y presenta ambos extremos proyectados hacia el citosol. Además, se demostró que la versión truncada maMYB91-309, que contiene el dominio citosólico R2R3-MYB, se localiza en el núcleo y preferentemente en el nucléolo. El análisis del patrón de expresión de la proteína maMYB reveló que lo hace mayoritariamente en raíz y parte aérea de plántulas de Arabidopsis. Empleando una estrategia de silenciamiento inducible basada en el uso de microRNAs artificiales (amiRNA), se obtuvieron líneas mutantes de maMYB condicionales en donde se observó una reducción pronunciada en los niveles de mRNA y proteína maMYB y una alteración severa del desarrollo de las plántulas de Arabidopsis, que se caracterizó por una reducción del tamaño general de la plántula, una disminución de la longitud de la raíz primaria, una inhibición de la formación de raíces laterales y pelos radiculares, una alteración en la forma, el tamaño y el número de las células de las diferentes capas de la raíz, una desestructuración de las células epiteliales de los cotiledones y una alteración en los niveles de los productos finales de la ruta de esteroles y de sus precursores inmediatos. Mediante el estudio de la expresión génica global con la tecnología RNA‐seq se determinó que el silenciamiento de maMYB provoca la desregulación de grupos de genes involucrados en el desarrollo y el crecimiento de la planta, que son coherentes con el fenotipo observado. Tanto en la raíz como en la parte aérea se identificaron genes desregulados involucrados en la elongación celular y relacionados con la pared celular. En la raíz se identificaron genes desregulados relacionados con la síntesis de chalconas, el transporte de auxinas y la morfogénesis de los pelos radiculares. En la parte aérea se identificaron genes desregulados implicados en la síntesis de fenilpropanoides y auxinas y la respuesta a auxinas. Además, los genes desregulados como consecuencia del silenciamiento de maMYB presentan un alto enriquecimiento de genes regulados epigenéticamente a través de la trimetilación de la lisina 27 de la histona H3 (H3K27me3), lo que sugiere que maMYB podría estar relacionado con este proceso necesario para el correcto desarrollo de la planta. Por otro lado, se observó que la sobreexpresión de maMYB91-309 revierte (en mayor o menor medida) el fenotipo causado por el silenciamiento de maMYB, lo cual avala la funcionalidad in vivo del dominio citosólico y el papel de maMYB como MTTF, cuyo dominio R2R3-MYB citosólico necesitaría liberarse por acción de proteasas intramembrana y trasladarse al núcleo donde ejercería su función. Las alteraciones observadas en el desarrollo de los mutantes de silenciamiento de maMYB sugieren que éste sea un MTTF que se libere en respuesta a estímulos de desarrollo. / In yeast and animals, Arv1 protein is involved in the intracellular lipid homeostasis, although its particular biochemistry function remains unknown. A. thaliana has the ARV genes AtARV1 and AtARV2, which codify the functional proteins AtArv1 and AtArv2, respectively, tethered to the ER membrane. Assuming that AtArv proteins need to interact with other proteins to carry out its biological function, this work was initiated with a screening of a cDNA library from Arabidopsis seedlings using AtArv1 as a bait and the yeast two hybrid version MbYTH, where we identified maMYB as an interactor of AtArv1. maMYB protein, codified by a unique gene in Arabidopsis, has characteristics of MYB family MTTF, since it contains a citosolic R2R3-MYB domain at the C-terminal region and two TMDs at the N-terminal region. In this way, we demonstrated that maMYB is localized in the ER membrane facing both extremes to the citosol, and the truncated form maMYB91-309, which contains the R2R3-MYB domain, is localized in the nucleus and preferentially in the nucleolus. The analysis of the expression pattern of maMYB protein revealed that the highest levels were found in the root and the shoot of Arabidopsis seedlings. Using an inducible silencing strategy based on the artificial microRNAs (amiRNAs) technology, we obtained maMYB inducible silencing mutants that showed a strong alteration in the root and shoot development of Arabidopsis seedlings. Through a global expression study using RNA-seq technology, we determined that maMYB silencing causes the deregulation of gene groups involved in plant development and growth, which is coherent with the observed phenotype. Interestingly, we observed that the maMYB91-309 overexpression revert (at more or less extent) the phenotype caused by maMYB silencing, which support the functionality of the citosolic R2R3-MYB domain in vivo and the role of maMYB as a MTTF, whose R2R3-MYB domain may be released by the action of intramembrane proteases and be transported to the nucleus where it may develop its function. The developmental alterations observed in the maMYB silencing mutants suggest that maMYB might be a MTTF released in respond to developmental cues.